Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-06-21 to 2005-06-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study performed equivalent/similar to OECD Test Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants) and according to the protocols on which the OECD guideline was based (INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay), dated February 1994 and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: OECD Test Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
: No information on the age of the cattle from which the eyes were obtained (eyes from cattle greater than 60 months old are not typically used).
Qualifier:
according to
Guideline:
other: INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay", dated February 1994.
Deviations:
yes
Remarks:
: No information on the age of the cattle from which the eyes were obtained (eyes from cattle greater than 60 months old are not typically used).
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
Deviations:
yes
Remarks:
: No information on the age of the cattle from which the eyes were obtained (eyes from cattle greater than 60 months old are not typically used).
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-117676-AAA (T000263)
- Physical state: solid (powder)
- Appearance: White, slight beige amorphous, crystalline, micropowder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8693
- Expiration date of the lot/batch: 2005-12-31
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (range of 20 +/- 5°C), light protected
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: the test item was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in a suspension. Until administration, the suspension was stirred with a magentic stirrer

Test animals / tissue source

Species:
cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Freshly isolated bovine corneas were collected from Abattoir Basel (Schlachthofstrasse 55, CH-4055 Basel, Switzerland). After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank's balanced salt solution containing penicillin/streptomycin and then transported for further preparations. The eyes were delivered the day before treatment and the isolated corneas were stored over night in a refrigerator.

Test system

Vehicle:
physiological saline
Remarks:
natrium chloratum 0.9%
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL as a 20% solution in NaCl
- Concentration (if solution): 20% in saline

VEHICLE/NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): charge number 768707/1
- Purity: no data

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20%, dissolved in saline
- Lot/batch no. (if required): 054063/2
- Purity: >99.5% (Assay)
Duration of treatment / exposure:
240 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
three corneas per treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
- The eyes were used immediately after delivery in the laboratory and within four hours after slaughtering. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2 - 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for the defects listed above.
- Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium overnight in a refrigerator at about 4 deg C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Dextran was added shortly before use.
- Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring, but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. The posterior compartment had to be filled first to return the cornea to its natural convex position. Care must be taken to assure no air bubbles were present within the compartments.
- For equilibration, the corneas in the holder were incubated for about one hour at 32 ± 2 deg C in a water-bath.
- At the end of the incubation period, the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (T0).

TREATMENT METHOD:
- Medium was completely removed from the anterior compartment and replaced by 0.75 -mL aliquots of the test substance, positive or negative control. The anterior compartment was plugged. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test substance and was incubated in a horizontal positioning water-bath at 32 ± 2 deg C. Fresh cMEM was filled into the posterior compartment. The test substance was tested as a 20% suspension in saline. The incubation lasted 240 minutes. During the whole experiment, cornea holders and medium were maintained in a water-bath at 32 ± 2 deg C.

REMOVAL OF TEST SUBSTANCE
- After the test substance was rinsed off from the application side by changing cMEM several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and opacity was measured (T240).
- Time after start of exposure: 4 hours

METHODS FOR MEASURED ENDPOINTS:
Opacity:
- The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (T240 - T0).
- The average change in opacity of the negative control corneas was calculated and this value subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- The mean corrected opacity value of each treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition.

Permeability:
- After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution, 0.5% dissolved in Dulbecco's phosphate-buffered saline. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32 ± 2 deg C. Medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and transferred to a cuvette of 10 mm path length and the optical density at 490 nm (OD490) was determined with a spectrophotometer. The dye solution is valid for use, if a dilution of the stock solution containing 10 µg/mL shows an optical density (OD490) of 1.610 to 1.910.
- The corrected OD490 value of each treated cornea or positive control corneas was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
- The mean corrected permeability values of each treatment group were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.

SCORING SYSTEM:
- The following formula was used to determine the In-Vitro Score: In-Vitro Score = opacity value + (15 x OD490 value)
- The In-Vitro Score was calculated for each individual treatment and positive control cornea. The mean value of each treated group was calculated from the individual In-Vitro Score values:
Negative control: In-Vitro Score = opacity value + (15 x OD490 value)
Positive control and test substance cornea: In-Vitro Score = corrected opacity value + (15 x corrected OD490 value)
- See Table 1 for the scoring classification system.

TOOL USED TO ASSESS SCORE:
- An opacitometer was used to determine the changes in light transmission passing through the corneas. The opacitometer was calibrated with a standardized opaque polyester sheet as described in the manual, and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.
- To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
172.07
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD: +/- 6.20
Remarks:
range: 166.5 - 178.8
Irritation parameter:
cornea opacity score
Value:
158
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD: +/- 4.7
Remarks:
range: 152.7 - 161.7
Irritation parameter:
other: permeability score
Value:
0.935
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: SD: +/- 0.199
Remarks:
range: 0.742 - 1.140
Other effects / acceptance of results:
- The in-vitro score of saline, used as the negative control, was -0.4 +/- 0.6 (-0.8 to 0.3) with the mean opacity value of -0.7 +/- 0.6 (-1 to 0) and the mean permeability value of 0.018 +/- 0.004 (0.014 to 0.021).
- The in-vitro score of the positive control (imidazole, 20%, dissolved in saline) was 100.2 +/- 18.6, proving the validity of the study. The corrected mean value of the opacity was 69.3 +/- 12.1, ranging from 55.7 to 78.7. The corrected mean value of the permeability was 2.059 +/- 0.523, ranging from 1.5437 to 2.588.

Before starting the permeability test, the dye solution sodium fluorescein was checked for its quality. The dye solution is valid for use, if a dilution of the stock solution containing 10 ug/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.737.

Any other information on results incl. tables

Opacity, Permeability and In Vitro Irritation Score Results

Samples

Opacity

Permeability [OD]

In-vitro score

Cornea no.

Treatment

T0 min

T240 min

T240 - T0

corrected

490 nm

corrected

1

Negative Control: Saline

2

1

-1

 -

0.021

 -

-0.7

2

2

1

-1

 -

0.014

 -

-0.8

3

3

3

0

 -

0.020

 -

0.3

Mean ± SD

 

 

-0.7 ± 0.6

 -

0.018 ± 0.004

 -

-0.4 ± 0.6

4

Positive control

3

58

55

55.7

1.561

1.543

78.8

5

2

80

78

78.7

2.066

2.047

109.4

6

1

74

73

73.7

2.606

2.588

112.5

Mean ± SD

 

 -

 -

69.3 ± 12.1

 -

2.059 ± 0.523

100.2 ± 18.6

7

Test substance

2

161

159

159.7

0.761

0.742

170.8

8

1

153

152

152.7

0.941

0.922

166.5

9

1

162

161

161.7

1.158

1.140

178.8

Mean ± SD

 -

 -

 -

158 ± 4.7

 -

0.935 ± 0.199

172 ± 6.2

 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the given test conditions, the test item T000263 is considered to be very severe irritant.
The test substance can be classified based on CLP criteria as category 1 (irreversible effects on the eye)