Cyclopenta[c]cyclopropa[g][1,6]diazacyclotetradecine-12a(1H)-carboxylic acid, 2,3,3a,4,5,6,7,8,9,11a,12,13,14,14a-tetradecahydro-2-[[7-methoxy-8-methyl-2-[4-(1-methylethyl)-2-thiazolyl]-4-quinolinyl]oxy]-5-methyl-4,14-dioxo-, ethyl ester, (2R,3aR,10Z,11aS,12aR,14aR)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-01 to 2015-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Principles of method if other than guideline:

The study procedures described in the study are also in compliance with the following documents:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P, Dukic M, Alix D and Sina J F, Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (1R,4R,6S,7Z,15R,17R)-17-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)-13-methyl-2,14-dioxo-3,13-diazatricyclo[13.3.0.0^{4,6}]octadec-7-ene-4-carboxylate

EC Number:

618-843-3

Cas Number:

923604-57-3

Molecular formula:

C37H46N4O6S

IUPAC Name:

ethyl (1R,4R,6S,7Z,15R,17R)-17-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)-13-methyl-2,14-dioxo-3,13-diazatricyclo[13.3.0.0^{4,6}]octadec-7-ene-4-carboxylate

Test material form:

solid

Details on test material:

- Name of test material (as cited in study reports): JNJ-38970191-AAA (T003009)
- Physical state: solid
- Appearance white solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15AB0148
- Expiration date of the lot/batch: 21 January 2017 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed, no homogeneous suspension could be obtained, despite treatment with vortex and ultrasonic waves.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
A 20% (w/w) non-homogeneous suspension with lumps (treated with vortex and ultrasonic waves) of the test item was prepared in physiological saline (Eurovet Animal Health, Bladel, The Netherlands), in the first and third test as requested by the sponsor.
In the second test, since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Test animals / tissue source

Species:

other: bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

physiological saline

Remarks:

first and third test

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl (first and second test); 324.7 to 360 mg, applied directly on the corneas in such a way that the cornea was completely covered (second test).
- Concentration (if solution): 20% (w/w) (first and third test)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
- Lot/batch no. (if required): no data
- Purity: no data

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20% (w/v)

Duration of treatment / exposure:

Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light.
Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and
1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the
O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

NEGATIVE CONTROL USED
physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

TREATMENT METHOD:
In the first and third test, the medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/w) test item was introduced onto the epithelium of the cornea. In the second test, the medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (324.7 to 360.0 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol
red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The
medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against
an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Nafluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the
sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Read
er). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less
than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490
before the dilution factor was applied to the readings.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

-1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

-1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -2 - 0

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -0.3 to 2.7

Results 5

Irritation parameter:

other: permeability value

Run / experiment:

2

Value:

0.001

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -0.002 - 0.007

Results 6

Irritation parameter:

other: permeability value

Run / experiment:

3

Value:

0.003

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: -0.008 - 0.015

Other effects / acceptance of results:

2nd test:
mean in vitro irritancy score (range):
negative control: 0.0
positive control: 133.6 (116.2 - 156.2)
mean opacity scores (range):
negative control: 0.0
positive control: 104.3 (75.0 - 135.0)
mean permeability scores (range):
negative control: 0.000
positive control: 1.951 (1.412 - 2.746)

3rd test:
mean in vitro irritancy score (range):
negative control: 0.0 (-0.4 - 0.5)
positive control: 153.9 (151.3 - 158.8)
mean opacity scores (range):
negative control: 0.0
positive control: 106.0 (92.7 - 115.7)
mean permeability scores (range):
negative control: 0.000
positive control: 3.195 (2.373 - 3.935)

Interpretation:
The IVIS of all replicates was within one category

Results:
first test: The invidual opacity scores for the negative controls ranged from 4 to 8 which is above the historical range, therefore the test was cancelled.
second test:
The individual in vitro irritancy scores for the negative controls were 0.0. The individual positive control in vitro irritancy scores ranged from 116.2 to 156.2. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test item showed opacity values ranging from -2.0 to 0.0 and permeability values ranging from -0.002 to 0.007. The corneas were clear after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.9 to 0.0 after 240 minutes of treatment with the test item.
third test:
The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 0.5. The individual positive control in vitro irritancy scores ranged from 151.3 to 158.8. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test item showed opacity values ranging from -0.3 to 2.7 and permeability values ranging from -0.008 to 0.015. The corneas were clear after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.3 to 2.5 after 240 minutes of treatment with the test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since the test item induced an IVIS ≤ 3 in the second and third experiment, no classification is required for eye irritation or serious eye damage.