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Working for a better future

REPORT 

The Bovine Corneal Opacity and Permeability Assay (BCOP)

HLS study number: CEF0005

Version ID: Audited Draft V2 

Issue date: 1 August 2013
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Sponsor and Test Facility Details 

Sponsor

Test facility Huntingdon Life Sciences

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 Regulatory compliance ..................................................................................... 9

1.3 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 complete Minimal Essential Medium (cMEM)............... 11

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.................................................................. 13

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

2.6 Computer systems.......................................................................................... 15

2.7 Maintenance of records .................................................................................. 15

2.8 Deviations from protocol ................................................................................. 16

3. Results ....................................................................................................................... 17

3.1 Assay validity.................................................................................................. 17

3.2 pH determination ............................................................................................ 17

3.3 BCOP results.................................................................................................. 17

4. Conclusion ................................................................................................................. 18

5. References................................................................................................................. 19
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List of Appendices

Appendix 1 BCOP data...................................................................................................20

List of Annexes

Annex 1 Huntingdon Research Centre GLP compliance statement 2012...................22
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Compliance with Good Laboratory Practice 

The Bovine Corneal Opacity and Permeability Assay (BCOP) 

The study described in this report was conducted in compliance with the following Good 

Laboratory Practice standards, with the exception noted below, and I consider the data 

generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 

3106, as amended by Statutory Instrument 2004 No. 994). 

OECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MC/CHEM (98) 17. 

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No L 

50/44). 

These principles of Good Laboratory Practice are accepted by the regulatory authorities of 

the United States of America and Japan on the basis of intergovernmental agreements.

Chemical analysis of the formulated test substance for determination of stability, 

homogeneity and concentration was not undertaken. 

___________________________________________ ____________________ 

Joanne Hubbard, HNC (Applied Biology) Date

Study Director

Huntingdon Life Sciences 
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Quality Assurance Statement

The Bovine Corneal Opacity and Permeability Assay (BCOP) 

The following inspections and audits have been carried out in relation to this study: 

Study Phase Date(s) of Inspection Date of Reporting to Study 

Director and Management

Protocol Audit 14 May 2013 15 May 2013

Report Audit 27 Jun 2013 27 Jun 2013

Process based inspections: At or about the time this study was in progress inspections of 

procedures employed on this type of study were carried out. These were conducted and 

reported to appropriate Company Management as indicated below: 

Process Based Inspections Date(s) of Inspection Date of Reporting to

Management

Bovine Corneal Opacity and 

Permeability Assay

06 Feb 2013-08 Feb 2013 11 Feb 2013

Study Management and 

Conduct

31 May 2013 03 Jun 2013

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were reported to Company Management. 

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

Allan T Drummond, MRQA, Date

Lead Auditor, 

Department of Quality Assurance,

Huntingdon Life Sciences. 
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Contributing Scientists

The Bovine Corneal Opacity and Permeability Assay (BCOP) 

Study management

Joanne Hubbard, HNC (Applied Biology) 

Study Director

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, Imidazole was tested in 

parallel as a positive control.

The assay used isolated bovine corneas to assess the ocular corrosivity or severe irritancy 

potential of the test substance 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 was used to classify the test 

substance as potential eye irritants according to OECD guideline 437 (OECD 437). 

The test substance, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was 

predicted to be a non-corrosive/non-severe eye irritant. 
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1. Introduction

1.1 Objective

The objective of the test was to evaluate the ocular corrosivity/severe 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 Regulatory compliance

The test was conducted in accordance with the following testing guideline:

OECD 437. OECD Guideline for the Testing of Chemicals: Bovine Corneal Opacity and 

Permeability (BCOP) Test method for Identifying Corrosives and Severe Irritants, 7 

September 2009. 

Ten proficiency chemicals, listed in the guideline OECD 437, had previously been tested in 

this laboratory, to validate the test.

1.3 Test system

The test substance, 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 rabbit Draize Eye Irritation test. The BCOP assay uses isolated bovine corneas as 

a means of assessing the ocular corrosivity or severe irritancy potential of test substances in 

vitro. The test includes acceptance criteria for both negative and positive controls. 
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2. Experimental Procedure 

2.1 Study schedule

The protocol was approved by the Sponsor and Huntingdon Life Sciences Management on 13 

May 2013 and 14 May 2013, respectively.

Study initiation:

(protocol signed by Study Director)

14 May 2013 

Experimental start date: 14 May 2013 

Experimental completion date: 16 May 2013 

2.2 Test substance details

2.2.1 Test substance

Identity:

Alternative name:

Intended use: 

Lot number: 12342 

Expiry: Considered to be suitable for use (expiry 

date will be documented in raw data and 

final report)

Appearance: White crystalline powder

Storage conditions: Room temperature(ca 20°C), in the dark 

Purity: Purity will be documented in the raw data 

and final report 

2.3 Materials and reagents

2.3.1 Reagents

Minimal Essential Medium Eagles without phenol red (EMEM)

Minimal Essential Medium Eagles with phenol red (EMEM 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 
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2.3.2 Preparation of complete Minimal Essential Medium (cMEM)

To a 500 mL bottle of EMEM the following were added:

5 mL of L-Glutamine (200 mM solution)

5 mL of Foetal calf serum

2.3.3 Controls

The negative control was sodium chloride solution, 0.9% (Sigma, Sigma S87766)

Lot number: RNBC6652

Expiry: 12 February 2015

The positive control material was imidazole (Sigma, I0125)

Lot number: BCBD9321V

Expiry: February 2014

Purity: 99.6%

2.3.4 Preparation and quality control of sodium fluorescein solution

A stock solution of Sodium Fluorescein (5 mg/mL) was prepared using cMEM.

Quality Control

Two aliquots of this solution were diluted 1:500 in cMEM, in two steps, as follows:

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

b. 40 μL of Dilution a. + 960 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 1.796).

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=75). The other two calibrators were checked in the right hand calibration block. 

Calibrator No. 2 fell within the range of 143-158 (value = 154) and Calibrator No. 3 fell in 

the range of 214-236 (value = 225). Once calibrated, the unit remained switched on for the 

duration of the assay.
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2.3.6 Bovine eyes

The bovine eyes, supplied by Joseph Morris Abattoir, were excised by an abattoir employee 

and collected as soon after slaughter as possible (excised at 12.40 hours, 14 May 2013). 

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 14.41 hours, 14 May 2013).

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 HBSS plus 1% penicillin/streptomycin, 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, overnight at room temperature (approximately 20°C). 

After overnight incubation at room temperature (approximately 20°C) the HBSS plus 1% 

penicillin/streptomycin was removed from both chambers. Both chambers were filled with 

cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. The corneas 

were then incubated in the 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 (posterior chamber first). 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. 

Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity 
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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.

2.4.3 Test substance pH

An estimate of the pH of the test substance (20% w/w) 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 (imidazole) 

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, a white crystalline powder, and the positive control material 

(imidazole) were tested at 20% (w/w) in 0.9% sodium chloride solution. The test substance 

was mixed during preparation using a mortar and pestle. The 0.9% sodium chloride solution 

was added to achieve the desired concentration.

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, 

negative control (0.9% sodium chloride solution) or positive control material (imidazole 

20% w/w solution) was introduced in to the anterior part of the holder. The test substance at 

20% w/w formed a white suspension and was added to the anterior compartment using a 

1 mL syringe via the top port. Following application the anterior compartment was plugged. 

The holder was slightly rotated to ensure uniform distribution of the test substance over the 

surface of the cornea. Each holder was incubated in a horizontal position at 32°C ± 1°C for 

4 hours ± 5 minutes 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 

(EMEM 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 corneas were gently rinsed with 

the wash medium until the medium was clear and the colour unchanged. The corneas treated 

with the test substance required 7 washes. Following completion of the wash step, the 

medium was removed from both compartments and re-filled 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. 

2.4.6 Permeability determination

The sodium fluorescein stock solution (5 mg/mL) was thawed prior to commencing the 

permeability incubation.
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Following the final opacity measurement, the medium was removed from the anterior 

compartment of the holder. One mL (1 mL) 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 

490 nm (OD490) and a sample of cMEM read (OD = 0.067). 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 ≤3.0 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/Non-Severe irritant

2.6 Computer systems

The computer system used in this study was as follows:

Pristima: pharmacy test substance management

2.7 Maintenance of records

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. This will include but may not be limited 

to the Study Protocol, raw data, test material and other samples and specimens generated 

during the course of this study. If requested, Huntingdon Life Sciences will continue to 

retain the materials at additional cost.

Samples and specimens that no longer afford evaluation will be discarded in accordance with 

Standard Operating Procedures. 

Huntingdon Life Sciences will retain in its archive the Quality Assurance records relevant to 

this study for a period of 20 years and a copy of the final report indefinitely.
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2.8 Deviations from protocol

Correction to typographical error 

Protocol, Section 3.15, Criteria for determination of a valid test, 2nd paragraph should read as 

follows:

The negative control (0.9% saline) mean opacity change value should be less than or equal to 

3.0 and the permeability mean value should be less than or equal to 0.1.
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3. Results 

3.1 Assay validity

The positive control, imidazole, elicited an In Vitro Irritancy Score of 174.5. This value was 

within the historical range (mean ± 2 x SD 126.0 – 185.4) for the assays performed to date. 

The negative control, 0.9% saline, opacity mean change value was 0.000 which was below 

the maximum acceptance value of 3.0. The permeability mean of the negative control was 

0.078 which was below the maximum acceptance value of 0.1. 

3.2 pH determination

The pH of the test substance, measured using pH sticks, was approximately 6.0. 

3.3 BCOP results

The study data are shown in Appendix 1. 

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

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

corneas treated with the positive control, imidazole, 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

1.000 ± 1.000 0.025 ± 0.038 1.4 ± 1.5 Non-Corrosive/ 

Non-Severe irritant

Imidazole 126.333 ± 24.583 3.212 ± 0.563 174.5 ± 17.6 Corrosive/Severe irritant

0.9% Saline 0.000 ± 0.000 0.078 ± 0.004 Not applicable Not applicable
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4. Conclusion

The test substance, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was 

predicted to be a non-corrosive/non-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:

0.9% Sodium chloride solution (Negative control)

Opacity Permeability

Cornea Pre Post Change OD 490

6 4 4 0 0.073

7 4 4 0 0.080

8 4 4 0 0.081

Mean change 0.000 Mean 0.078

SD 0.000 SD 0.004

Sample:

Opacity Corrected Permeability

Cornea Pre Post Change change OD 490 Corrected

9 5 7 2 2.000 0.146 0.068

10 4 5 1 1.000 0.077 -0.001

11 4 4 0 0.000 0.086 0.008

Mean corrected change 1.000 Mean corrected OD 490 0.025

SD 1.000 SD 0.038

(Opacity + (15 x Corrected OD 490))

Cornea Opacity 15xOD490 In Vitro irritancy score

9 2.000 1.020 3.020

10 1.000 -0.015 0.985

11 0.000 0.120 0.120

Mean in vitro irritancy score 1.4

SD 1.5

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: 20% w/w Imidazole (positive control)

0.9% Sodium chloride solution (Negative control)

Opacity Permeability

Cornea Pre Post Change OD 490

6 4 4 0 0.073

7 4 4 0 0.080

8 4 4 0 0.081

Mean change 0.000 Mean 0.078

SD 0.000 SD 0.004

Sample: 20% w/w Imidazole (positive control)

Opacity Corrected Permeability

Cornea Pre Post Change change OD 490 Corrected

3 5 129 124 124.000 3.640 3.562

4 3 155 152 152.000 2.640 2.562

5 5 108 103 103.000 3.590 3.512

Mean corrected change 126.333 Mean corrected OD 490 3.212

SD 24.583 SD 0.563

20% w/w Imidazole (positive control)

(Opacity + (15 x Corrected OD 490))

Cornea Opacity 15xOD490 In Vitro irritancy score

3 124.000 53.430 177.430

4 152.000 38.430 190.430

5 103.000 52.680 155.680

Mean in vitro irritancy score 174.5

SD 17.6

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 2012