Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2005-10-27 to 2005-12-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study was conducted according to INVITTOX protocol no. 98 "Bovine Corneal Opacity and Permeability Assay" and Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK , and in a manner consistent with OECD guideline 437. No information on the age of the source animals. Phenol red not added post incubation.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: INVITTOX protocol no.98 "Bovine Corneal Opacity and Permeability Assay"
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd. UK
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
phenol red not added post incubation
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 report): JNJ-559728-AAA (T001250)
- Physical state: solid (powder)
- Appearance: White powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00443418 RT001250G4A661
- Expiration date of the lot/batch: no data
- 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 preparation of a solid: the test item was tested at a concentration oof 20% in saline. Stron stirring with a magnetic stirrer resulted in a suspension. Until administration, the suspension was stirred with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : 20% suspension in saline

Test animals / tissue source

Species:
other: bovine eyes
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine eyes were collected from the abattoir (Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland).
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): no data
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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 overnight in a preservation medium in a refrigerator.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
other: negative (physiol. saline) and positive (imidazole) eyes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% suspension in saline

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9%

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): no data
- Lot/batch no. (if required): 054063/2
- Purity: > 99.5% (Assay)
Duration of treatment / exposure:
240 min
Duration of post- treatment incubation (in vitro):
90 minutes in a water-bath at 32°C +/- 2°C
Number of animals or in vitro replicates:
3 corneas per test group (test item, negative control and positive control)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- 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 with a view box for defects.
- 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. Shortly before use, Dextran was added.
- 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. Care was taken to assure no air bubbles were present within the compartments.
- For equilibrium, the corneas in the holder were incubated for about one hour at 32 deg. C + / - 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 (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged.

TREATMENT METHOD:
- Following the measurement of the basal corneal opacity, fresh cMEM was filled into the posterior compartment, while the anterior compartment received 0.75 mL of the test substance or negative or positive control, respectively, and was evenly distributed on the surface of the corneas. The test substance was tested as a 20% suspension in saline.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the test substance was rinsed off from the application side by changing cMEM solution several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and the opacity was measured (t240min). The incubation lasted 240 minutes. During the whole experiment, cornea holders and medium were maintained in a water bath of 32 °C +/- 2 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determined changes in the light transmission passing through the corneas and displayed a numerical opacity value. The opacitometer was calibrated with a standardized opaque polyester sheet as described in a manual, and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.
After recording the basal opacity of all corneas, the mean value of all corneas was calculated. No cornea deviated from this by more than + / -3 units and no cornea was discarded. Sets of three corneas were used for treatment with the test substance, the negative and positive controls, respectively.
Medium was completely removed from the anterior compartment and replaced by 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 by positioning in a water bath at 32 +/- 2°C.
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 (t240min-t0min).
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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step. Corneal permeability was quantified by measuring the amount of fluorescein sodium dye diffusing into the medium in the posterior chamber. Fresh cMEM was added to the posterior chamber and 1 mL of the fluorescein sodium dye solution, 0.5% dissolved in Dulbecco's phosphate-buffered saline, was placed in the anterior compartment after removing the medium. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32°C +/- 2°C.The optical density of an aliquot of the mixed medium from the posterior chamber, removed with a 5 mL syringe, was measured by photometry at 490 nm. The dye solution is valid for use if a dilution of the stock solution containing 10 ug/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: In Vitro Irritancy Score (IVIS)
- In Vitro Score Calculation:
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 In Vitro Score 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)

DECISION CRITERIA:
Depending on the score obtained, the test substance was classified into one of the following categories:
In-Vitro Score: (Proposed In-Vitro Irritation Scale)
0-3 (non eye irritant)
3.1-25 (mild eye irritant)
25.1-55 (moderate eye irritant)
55.1 -80 (severe eye irritant)
> 80.1 (very severe eye irritant)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
1.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: +/- 1.2
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: +/- 1.2
Irritation parameter:
other: permeability
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.005
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: +/- 0.002
Other effects / acceptance of results:
mean in vitro irritancy score:
negative control: 0.5 +/- 1.5
positive control: 80.9 +/- 12.5

mean opacity scores (range):
negative control: 0.3 +/- 1.5
positive control: 65.7 +/-6.0

mean permeability scores (range):
negative control: 0.013 +/-0.002
positive control: 1.019 +/-0.455

Treatment of the corneas with the test item resulted in a mean in vitro score of 1.4 +/- 1.2 after 240 minutes of incubation, ranging from 0.7 to 2.7. The net value of the opacity score ranged from 0.7 to 2.7, and the mean value was 1.3 +/- 1.2. The mean corrected permeability value of the cornea was 0.005 +/- 0.002, ranging from 0.002 to -0.007.

The in-vitro score of saline, used as a negative control, was 0.5 +/- 1.5 (-0.8 to 2.2) with the mean opacity value of 0.3 +/- 1.5 (-1 to 2) and the mean permeability value of 0.013 +/- 0.002 (0.012 to 0.015).
The in vitro score of the positive control (imidazole, 20%, dissolved in saline), was 80.9 +/- 12.5, proving the validity of the study. The corrected mean value of the opacity was 65.7 +/- 6.0, ranging from 59.7 to 71.7. The corrected mean value of the permeability was 1.019 +/- 0.455, ranging from 0.722 to 1.543.
Before starting the permeability test, the sodium fluorescein dye solution was checked for its quality. The dye solution is valid for use if the stock solution containing 10 µg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.643.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given test conditions the test item T1250 is not considered to be an eye irritant.