6H-Benzofuro[3a,3,2-ef][2]benzazepin-6-one, 1-bromo-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-, (4aR,8aR)-rel-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-09-04 to 1997-09-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

This non-GLP study was performed according to a method equivalent to the OECD guideline n° 43 7. No deviations were reported, however, few data were given on the characteristics of the substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

439-750-2

EC Name:

-

Cas Number:

183487-56-1

Molecular formula:

Hill formula: C17H19BrClNO3 CAS formula: C17H19BrClNO3

IUPAC Name:

(4aR*,8aR*)-1-bromo-4a,5,9,10,11,12- hexahydro-3-methoxy-11-methyl-6H-ben zofuro[3a,3,2-ef][2]benzazepin-6-one hydrochloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): T002113
- Substance type: no data
- Physical state: no data
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.:RT002113PFP031
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material:at room temperature in closed and labelled containers

Test animals / tissue source

Species:

other: bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE:
bovine eyes: the test utilizes bovine corneas, isolated from eyes freshly collected in a local slaughterhouse (Mol). The eyes were excised by a slaughterhouse veterinarian and collected in a plastic jar containing one liter of Hank's Balanced Salt Solution (HBSS, Sigma). Medium storage and transportation of eyes to the laboratory was performed at room temperature. The eyes were used within three hours after killing the animals.

PREPARATION OF THE CORNEAS:
Before dissection, all eyes were carefully examined and those presenting defects, such as neovascularization, pigmentation or scratches were discarded. during dissection great care was taken to avoid damage of corneal surfaces (epithelial and endothelial). Selected corneas were dissected with a 2-3 mm rim of sclera for easier handling and stored in a petri-dish containing Hank's Balanced salt Solution (HBSS) until use. Corneas were mounted in holders, the endothelial side being applied on the O-ring of the posterior part of the cornea holder. The anterior part of the cornea holder was applied onto the posterior part with cornea and held in place with 3 screws. Compartments were filled (the posterior part first) with Minimal Essential Medium (MEM, Sigma) solution. The corneas were incubated for one hour in a water-bath at 32°C.

Test system

Vehicle:

physiological saline

Remarks:

0.9% NaCl (100%)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20 g/g%

NEGATIVE CONROL
- Amount applied: 0.75 mL
- Concentration: 0.9% NaCl
- Lot/batch no.: no data
- Purity: no data

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75mL
- Concentration (if solution): 20 g/g% imidazole

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

Opacity was measured with the opacitometer at time t240. Permeability was evaluated immediately after measuring the opacity.

Number of animals or in vitro replicates:

The total number of corneas used for this experiment was 27 (9 for the first study, 9 for the first repeat study and 9 for the second repeat study). In each study, 3 corneas were treated per control and per test item.

Details on study design:

BACKGROUND OPACITY MEASUREMENT: After incubation of the corneas for one hour in a water-bath at 32°C , the MEM-solution was removed and both compartments were refilled with fresh medium. After careful removal of the air-bubbles, background opacity of all corneas was measured with an opacitometer. The opacitometer (OP-KIT, Electro Design, Riom, france) determines the light transmission of a cornea, and displays a numerical opacity value (arbitrary units). Results were written on a specially prepared spreadsheet. Corneas were rejected for use if they showed a background opacity grade greater than three.

TREATMENT AND OPACITY MEASUREMENT: after backround opacity measurement at time zero (t0), medium was removed from the anterior compartment. The anterior compartment was then filled with the test compound, negative control or positive control. After closing of the holes with caps, corneas were incubated in a horizontal position for 240 minutes at 32°C in a water-bath.

REMOVAL OF TEST SUBSTANCE
- After incubation, the negative control, the positive control and T002113 were removed from the anterior compartment and the epithelium was washed at least 3 times with approx 4 ml of MEMsolution so that the medium was clear. The medium was then removed from the posterior compartment and both compartments were finally refilled with MEM-solution whereafter opacity was measured with the opacitometer (at time t240). Results were written on a specially prepared spreadsheet.

PERMEABILITY MEASUREMENT
- The permeability of the corneas was evaluated immediately after measuring the opacity. The medium was removed from the anterior compartment and replaced by 1mL of a 0.5% sodium-fluorescein solution (sodium fluorescein was diluted in Dulbecco's phosphate buffered saline, Sigma). Corneas were incubated in a horizontal position for 90 minutes at 32°C in a water-bath. After incubation, medium from the posterior chamber was removed, and its optical density (OD) was determined spectrophotometrically at 490nm. The results from the outprint of the spectrophotometer were entered into a specially prepared spreadsheet.

SCORING SYSTEM:
-negative control: when background opacity (at time t0) was subtracted from the opacity value measured at time t240, the mean (+/- SD) corneal opacity of the negative control corneas was calculated. Also the mean (+/- SD) permeability was determined.
-positive control and test article: individual corneal opacity (at t240-t0) and permeability values were corrected with the mean opacity and permeability scores obtained for the negative control. Then, the mean (+/- SD) was calculateds of the corrected opacity and permeability values. Historical data of the positive control (20g/g% Imidazole) were available and gave the possibility to validate the experimental design of the BCOP assay.
- in vitro score: the in vitro score for the corneal injury was calculated using a formula, which combines results from both opacity and permeability. Individual in vitro scores were used to calculate the mean in vitro score: In vitro score = opacity value + 15 times OD value for permeability. In vitro scores were sorted into five classes:
from 0.0 to 3.0 = non eye irritant
from 3.1 to 25.0 = mild eye irritant
from 25.1 to 55.0 = moderate eye irritant
from 55.1 to 80.0 = severe eye irritant
from 80.1 to above = very severe eye irritant.
TOOL USED TO ASSESS SCORE: fluorescein

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

85.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 8.6

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

83.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 9.0

Results 3

Irritation parameter:

other: Permeability score

Run / experiment:

1

Value:

0.132

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 0.042

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

1 (repeat)

Value:

51.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 4.1

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

1 (repeat)

Value:

50.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 3.5

Results 6

Irritation parameter:

other: permeability score

Run / experiment:

1 (repeat)

Value:

0.084

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 0.052

Results 7

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

62.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 16.4

Results 8

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

60.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 16.5

Results 9

Irritation parameter:

other: permeability score

Run / experiment:

2

Value:

0.125

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: +/- 0.045

Other effects / acceptance of results:

In the first experiment, T002113 induced an increase in opacity (83.3 +/- 9.0) and a very small increase in permeability (0.132 +/- 0.042). Consequently, an in vitro score of 85.3 +/- 8.6 was calculated and classified T002113 as a very severe eye irritant. In the first repeat study, an increase in opacity (50.6 +/- 3.5) and a very small increase in permeability (0.084 +/- 0.052) were observed, resulting in a moderate eye irritant (51.9 +/- 4.1) in vitro classification. These results did not confirm the findings of the first study so a second repeat experiment was performed. In the second repeat study, an increase in opacity (60.7 +/- 16.5) and a very small increase in permeability (0.125 +/- 0.045) were observed, resulting in a severe eye irritant (62.6 +/- 16.4) in vitro classification. Although 3 different eye irritation classifications were calculated, the in vitro scores ranges from the upper region of a moderate classification (51.9 +/-4.1) to the lower region of a very severe classification (85.3 +/- 8.6). Consequently, T002113 can be considered as a severe eye irritant. The observed variability between the 3 studies could be related to the solubility of the test article, since T002113 was not completely soluble and a (homogeneous) suspension had to be used to treat the corneas.

Any other information on results incl. tables

Background opacity: for all examined corneas (n=27), background opacity ranged between 0 and 3, indicating that no corneas had to be excluded from the experimental test design and replaced by another one.

In the first test, first repeat and second repeat, each negative control cornea showed an opacity, a permeability and an in vitro score which fell within the laboratory range of historical data. In all studies, the corneas treated with the positive control showed a marked increase in opacity and permeability. Imidazole (20g/g%) was detected as a very severe eye irritant according to the historical data. Based on the findings in the negative and positive control group, it was concluded that the test conditions were optimal for the evaluation of the potential of T002113 to induce opacity and permeability.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The in vitro BCOP eye irritation test classifies T002113 as a severe eye irritant cat 1 according to CLP regulation when classification is made based on a combination of results obtained for opacity and permeability. This in vitro grade is related to an increase in corneal opacity and a very small increase in permeability.