2-[(5A,7A)-17-(CYCLOPROPYLMETHYL)-3,6-DIMETHOXY-18,19-DIHYDRO-4,5-EPOXY-6,14-ETHENOMORPHINAN-7-YL]-3,3-DIMETHYLBUTAN-2-OL

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-06-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented study performed according to following guideline: INVITTOX Protocol no. 98 an d BCO-P SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol n. 98 "Bovine Corneal Opacity and Permeability Assay" (dated Feb 1994)

Deviations:

no

Test guideline 2

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:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Study was performed equivalent to the OECD437 guideline

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[(1S,2R,6S,14R,15R,16R)-3-(cyclopropylmethyl)-11,15-dimethoxy-13-oxa-3-azahexacyclo[13.2.2.1²,⁸.0¹,⁶.0⁶,¹⁴.0⁷,¹²]icosa-7,9,11-trien-16-yl]-3,3-dimethylbutan-2-ol

Cas Number:

16524-65-5

Molecular formula:

C30H43NO4

IUPAC Name:

(2S)-2-[(1S,2R,6S,14R,15R,16R)-3-(cyclopropylmethyl)-11,15-dimethoxy-13-oxa-3-azahexacyclo[13.2.2.1²,⁸.0¹,⁶.0⁶,¹⁴.0⁷,¹²]icosa-7,9,11-trien-16-yl]-3,3-dimethylbutan-2-ol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-18163678-AAA (T002713)
- 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: 00477910 RT002713G4A051
- Expiration date of the lot/batch: 2006-06-30
- Purity: 100%
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, light protected
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

FORM AS APPLIED IN THE TEST : solution of 20% in saline

Test animals / tissue source

Species:

other: bovine corneas

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 used immediately after delivery in the laboratory and within four hours after slaughtering.

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 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 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) 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 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 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.

Test system

Vehicle:

physiological saline

Remarks:

natrium chloratum 0.9%

Controls:

other: negative control (saline); positive control (imidazole)

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20%
Until administration, the solution was stirred with a magnetic stirrer

VEHICLE / NEGATIVE CONTROL
- Amount applied: 0.75 mL

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 20%

Duration of treatment / exposure:

240 minutes in a water-bath at 32°C +/- 2°C

Duration of post- treatment incubation (in vitro):

about 90 minutes in a water-bath at 32°C +/-2 °C

Number of animals or in vitro replicates:

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

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the test item was rinsed off from the application side by changing "complete minimum essential medium" (cMEM) several times until precipitates of the test item could be observed no longer, fresh cMEM was replaced in both compartments.
- Time after start of exposure: 240 minutes

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 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 item and was incubated in a horizontal position in 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 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

Value:

4.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 3.1 to 6.4

Results 2

Irritation parameter:

cornea opacity score

Value:

5.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 3.7 to 6.7

Results 3

Irritation parameter:

other: permeability score

Value:

-0.028

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: -0.039 to -0.019

Other effects / acceptance of results:

Treatment of the corneas with the test item resulted in a mean in vitro score of 4.9 +/- 1.7 after 240 minutes incubation, ranging from 3.1 to 6.4. The net value of the opacity score ranged from 3.7 to 6.7, the mean value was 5.3 +/- 1.5. The mean corrected permeability value of the corneas was -0.028 +/- 0.010, ranging from -0.039 to -0.019.
The in vitro score of saline , used as negative control was 2.7 +/- 1.1 (1.4 to 3.5) with the mean opacity value of 1.3 +/- 1.2 (0 to 2) and the mean permeability value of 0.092 +/- 0.008 (0.083 to 0.098).
The in vitro score of the positive control (imidazole, 20% dissolved in saline) was 95.7 +/- 4.7 (92.2 to 101.0) confirming the validity of the study. The corrected mean value of the opacity was 67.7 +/- 3.5, ranging from 65.7 to 71.7. The corrected mean value of the permeability was 1.867 +/-0.448, ranging from 1.447 to 2.357.

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 1.721.
According to the results obtained in this experiment, the test was considered acceptable.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The in vitro score of the test item was found to be 4.9 +/- 1.7. According to the in vitro irritation scale stated in the INVITTOX Protocol, under the given test conditions the test item T002713 is considered to be mild eye irritant.