2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting and completion date: 2005-06-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants

Deviations:

yes

Remarks:

: 1)The estimated age and/or weight of the animals providing the corneas was not provided

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay", dated February 1994.

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Deviations:

not specified

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

EC Number:

618-043-4

Cas Number:

876666-07-8

Molecular formula:

C29H31F2NO4

IUPAC Name:

2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-17303806-AAA (T001586)
- Physical state: solid, powder
- Appearance: white, beige

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT001586G1B121
- Expiration date of the lot/batch: 31-12-2005
- 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:
Solubility in water: 0.01 g/L
Solubility in ethanol: 6.9 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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 magnetic stirrer.

Test animals / tissue source

Species:

other: bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Test system: freshly isolated bovine cornea
Source: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland

Collection of bovine eyes:
Freshly isolated bovine eyes were collected from the abattoir. 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 preparation. The eyes were delivered the day before treatment and the isolated corneas were stored over night in a preservation medium in a refrigerator.

Preparation of the 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 finally with a view box for the defects listed before.
Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium over night in a refrigerator at about 4°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) o
f 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 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°C +/- 2°C in a waterbath.
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.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- amount applied: 0.75 mL
- Concentration: 20% suspension in saline
Until administration, the solution was stirred with a magnetic stirrer.

NEGATIVE CONTROL
- amount applied: 0.75 mL

POSITIVE CONTROL
- amount applied: 0.75 mL
- Concentration: 20% in saline

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

9 bovine eyes in total (3 test item replicates, 3 negative controls, 3 positive controls)

Details on study design:

PREPARATION OF THE TEST ITEM SOLUTION
The test item was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in a solution. Until administration, the solution was stirred with a magnetic stirrer.

OUTLINE OF STUDY
Fresh cMEM was filled into the posterior compartment, while the anterior compartment received test item solution or negative or positive control evenly distributed on the surface of the corneas. During the whole experiment, cornea holders and medium were maintained in a water-bath ar 32 +/- 2°C. After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test item could be observed no longer, fresh cMEM was replaced in both compartments and opacity was measured (t240min). To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step.

OPACITY MEASUREMENT:
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 item, the negative and positive controls, respectively.
Medium was completely removed from the anterior compartment and replaced by the test item, positive or negative controls. 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 item and will be incubated in a horizontal positioning a water-bath at 32°C +/- 2°C.

PERMEABILITY DETERMINATION:
Following the opacity readings after treatment, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32°C +/-2°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.

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 item cornea:
in vitro score = corrected opacity value + (15 x corrected OD490 value)

Depending on the score obtained, the test item was classified into one of the following categories:
in vitro score 0 - 3: non eye irritant
in vitro score 3.1 - 25: mild eye irritant
in vitro score 25.1 - 55: moderate eye irritant
in vitro score 55.1 - 80: severe eye irritant
in vitro score > 80.1: very severe eye irritant

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

test item after 240 minutes of treatment

Value:

2.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Test item IVIS range: 0.7 to 4.6

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

test item after 240 minutes of treatment

Value:

2.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item opacity score range: 0.7 to 4.7

Results 3

Irritation parameter:

other: permeability value

Run / experiment:

test item after 240 minutes of treatment

Value:

-0.004

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item permeability range: -0.008 to 0.004

Other effects / acceptance of results:

The in vitro score of saline, used as negative control was -0.3 +/- 0.7 (-0.8 to 0.4) with the mean opacity value of -0.7 +/- 0.6 (-1 to 0) and the mean permeability value of 0.022 +/- 0.007 (0.016 to 0.029).
The in vitro score of the positive control (imidazole, 20% dissolved in saline) was 100.6 +/- 18.7 (79.1 to 113.1) 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 2.087 +/- 0.532, ranging from 1.565 to 2.628.

Any other information on results incl. tables

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 μg/mL showed an optical density (OD490) of 1.610 to 1.910. The value found by spectroscopy was 1.737.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions, the test item is considered to be non eye irritant.