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### REPORT

T he B ovine C orneal O pacity and P ermeability A ssay (BCOP)

HLS s tudy number: CEF0005

Version ID: Audited Draft V 2

Issue date: 1 August 2013Huntingdon Life Sciences
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## Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambridgesh i re PE28 4HS

UKHuntingdon Life Sciences
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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 s olution

............ 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 Permeabil ity 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 Permeabilit y (OD 490 )

................................ ................................ ............ 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 r ecords

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

................................ ................................ ................................ ................. 19Huntingdon Life Sciences
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## List of Appendices

Appendix 1 BCOP data

................................ ................................ ................................ ... 20



## List of Annexes

Annex 1 Huntingdon Research Centre GLP compliance statement 2012

................... 22Huntingdon Life Sciences
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## Compliance with Good Laboratory Practice

The Bovine Co rneal Opacit y 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 SciencesHuntingdon Life Sciences
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## Quality Assurance Statement

The Bovine Corneal Opacity and Permeability A ssay (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 2 7 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

B ovine C orneal O pacity 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.Huntingdon Life Sciences
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## Contributing Scientists

The Bovine Corneal Opacity and Perm eability A ssay (BCOP)



### Study m anagement

Joanne Hubbard, HNC (Applied Biology) Study Director

In Vitro Safety Assessment Department of In Vitro Technologies

Leslie C. Akhurst, BSc (Hons) MSc CBiol M SB

Head of In Vitro Safety Assessment Department of In Vitro TechnologiesHuntingdon Life Sciences
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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

, Imidazo le was tested in parallel as a positive control.

The assay use d isolated bovine corneas to assess the ocular corrosivity or severe irritancy potential of the test substance in vitro . The isolated corneas we re obtained as a by -product of the meat production industry. Two endpoints, corneal opacity and permeability, we re 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-s evere eye irritant

.Huntingdon Life Sciences
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## 1. Introduction

1.1 Objective

The objective of the test was to evaluate the ocular corrosivity/severe ir ritancy 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 test ing 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 accept ance criteria for both negative and positive controls.Huntingdon Life Sciences
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## 2. Experimental Procedure

2.1 Study s chedule

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 s ubsta nce d etails

2.2.1 Test s ubstance

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 r eagents

2.3.1 Reagents

Minimal Essential Medium Eagles without phenol r ed ( E MEM)

Minimal Essential Medium Eagles with phenol r ed ( E MEM with phenol red) L-Glutamine (200 mM Solution) Hanks Balanced Salt Solution (HBSS) containing Ca ++ and Mg ++ without phenol r ed

Foetal c alf s erum
Penicillin - Streptomycin (10000 IU/ mL - 10000 μ g/ mL ) Solution

0.9% Sodium chloride solutionHuntingdon Life Sciences
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11 2.3.2 Preparation of complete Minimal Essential Medium (cMEM)

To a 500 mL bottle of E MEM the following were add ed:

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 S8776 6)

Lot number: RNBC6652 Expiry: 12 February 2015

The posit ive control material was i midazole ( Sigma, I0125 )

Lot number: BCBD9321V Expiry: February 2014 Purity: 99.6%

2.3.4 Preparation and q uality c ontrol of s odium f luorescein s olution

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

Quality Con trol

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

a. 50 μ L of Sodium Fluorescein stock + 950 μ L of cMEM

b. 40 μ L of Dilution a. + 960 μ L of cMEM

The Optical Density (OD) of Dilution b was measured on a spectrophotomet er 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 1.796 ).

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

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

2.3.5 Calibration of o pacitometer

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 cali bration blocks were inserted in to 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 14 3

- 15 8 (value = 154 ) and Calibrator No. 3 fell in the range of 21 4

- 23 6 (value = 225 ). Once calibrated, the unit remained switched on for the duration of the assay.Huntingdon Life Sciences
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12 2.3.6 Bovine e yes

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 ).

In structions were given to avoid damaging the corneas during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in suffic ient HBSS, containing 1% (v/v) p enicillin/ s treptomycin 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 c orneas

All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmen tation, 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 scl era present around the cornea. The isolated corneas were stored i n a petri dish containing HBSS plus 1% p enicillin/ s treptomycin solution until all the corneas had been dissected. Once all the corneas had been dissected, they wer e rinsed in fresh HBSS plus 1%

p enicillin/ s treptomycin solution prior to mounting.

The corne as 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 o f 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 tem perature (approximately 20°C) the HBSS plus 1% penicillin/streptomycin was removed from both chambers . Both chambers were filled with

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

were then incubated in the u pright 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

ante rior 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 compartm ent was then plugged and the basal opacity measurements performed.

2.4.2 Opacity m easurement

The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each c ornea 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 opacityHuntingdon Life Sciences
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13 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 g roups

Corneas were treated in trip licate 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 c orneas

The test substance, a white crystalline powder

, and the positive control material (i midazole ) 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 μ L (750 μ L) of test substance, negative control (0.9% sodium chloride solution) or positive control material (i midazole 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 was hed, 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 attach ed to a vacuum pump. The corneas were gently rinsed with

the wash medium until the medium was clea r 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.

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

2.4.6 Permeability d etermination

The sodium fluorescein stock solution (5 mg/ mL ) was thawed prior to commencing the permeability incubation.Huntingdon Life Sciences
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14 Following the final opacity measurem ent, 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 sid e 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 (OD 490 ) and a sam ple of cMEM read (OD = 0.067 ) . The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution giving an

OD 490 value above 1.8 was diluted 1 in 5 with cMEM.

2.4.7 Criteria for d etermination of a v alid te st

T he posi tive control should elicit an In Vitro Irritancy Score that f a ll s within two standard deviations of the historical mean for the laboratory

.

The negative control mean opacity change v alue should be ≤3 .0 and the permeability mean
value ≤0.1.



### 2.5 Assessment of r e sults

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 t he 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 Permea bility (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 value of each

cornea.

The mean corrected permeability value of each treatment group wa s calculated from the individual corrected permeability values of the treated corneas.Huntingdon Life Sciences
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15 2.5.3 In V itro Irritancy s core c alculation

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

In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected 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 IV I S of each individual cornea in the treatment group.

2.5.4 Classification cr iteria

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 l abelling 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 Maintenanc e 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 wil l 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.

Sam ples 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.Huntingdon Life Sciences
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16 

### 2.8 Deviations from p rotocol

Correction to typographical error

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

follows: The negative control (0.9% saline) mean opacity c hange value should be less than or equal to 3.0 and the permeability mean value should be less than or equal to 0.1.Huntingdon Life Sciences
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## 3. Results

3.1 Assay v alidity

The positive control, imidazole

, elicited an In Vitro Irritancy Score of 174.5. This value was within the histori cal 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 d etermination

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



### 3.3 BCOP r esults

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 n egative 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 N ot applicableHuntingdon Life Sciences
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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

.Huntingdon Life Sciences
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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 As say 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

Irrita nts.Huntingdon Life Sciences
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20 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.Huntingdon Life Sciences
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21 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 u sed for calculations by Excel spreadsheet.Huntingdon Life Sciences
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22 Annex 1 Huntingdon Research Centre GLP compliance s tatement 2 0 1 2