[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-12-10 to 2016-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 26, 2013

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)

Version / remarks:

09 December 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OTWG of the ICCVAM and the 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

Version / remarks:

March 2006

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay

Version / remarks:

2006

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: 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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide

Cas Number:

1950587-20-8

Molecular formula:

C20H17F4N5O2

IUPAC Name:

4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch (Lot) No.of test material: I15DD1622
- Expiration date of the lot/batch: 2017-04-15 (retest date)
- Physical Description: Brown crystalline powder with some small clots
- Purity: 103.0%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

OTHER SPECIFICS:
- Correction factor: 1

Test animals / tissue source

Species:

cattle

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 after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.

- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 334.6 to 366.4 mg (first test) and 350.8 to 379.5 mg (repeat test)
- Concentration (if solution):

NEGATIVE CONTROL (physiological saline)
- Amount(s) applied (volume or weight with unit): 750 µL

POSITIVE CONTROL (Imidazole solution)
- 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

Duration of post- treatment incubation (in vitro):

After 240 ± 10 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed following the opacity measurement after the incubation period of 90 minutes ± 5 minutes in 1 ml of 5 mg Na-fluorescein/ml cMEM solution.

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 Duratec Analysentechnik GmbH (Hockenheim, Germany) 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.

TREATMENT METHOD:
In both the first and repeat 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 (334.6 to 366.4 mg, first test and 350.8 to 379.5 mg, repeat test). 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 (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
opacity = ((I0/I)-0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change of 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 treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control 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 Na-fluorescein/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 Reader). 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 has been performed, the OD490 of each reading of the positive control and test item 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:

Experiment 1

Value:

-2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -4.7 to -0.4

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1

Value:

-2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -4.6 to -0.1

Results 3

Irritation parameter:

other: Permeability value

Run / experiment:

Experiment 1

Value:

-0.004

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -0.022 to 0.015

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Repeat test

Value:

-1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -2.7 to 1.5

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Repeat test

Value:

-1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -3.1 to 1.5

Results 6

Irritation parameter:

other: permeability value

Run / experiment:

Repeat test

Value:

0.009

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -0.016 to 0.042

Other effects / acceptance of results:

Experiment 1
mean in vitro irritancy score (range):
negative control: 3.4 (1.5 to 5.3)
positive control: 116.3 (110 to 121)

mean opacity scores (range):
negative control: 2.8 (1.3 to 5.2)
positive control: 98.3 (88.3 to 105.2)

mean permeability scores (range):
negative control: 0.039 (0.008 to 0.097)
positive control: 1.203 (0.941 to 1.462)


Repeat test
mean in vitro irritancy score (range):
negative control: 2.9 (0.8 to 4.8)
positive control: 165.0 (134.0 to 211.4)

mean opacity scores (range):
negative control: 2.5 (0.6 to 3.9)
positive control: 138.7 (105.0 to 179.5)

mean permeability scores (range):
negative control: 0.027 (0.011 to 0.057)
positive control: 1.753 (1.201 to 2.125)

Turbidity and pH effect
In the first test, the corneas treated with the test item 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 -4.7 to -0.4 after 240 minutes of treatment with the test item.
In the repeat test, the corneas treated with the test item 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 -2.7 to 1.5 after 240 minutes of treatment with the test item.

Interpretation:
Since in both tests the results were highly comparable and the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Discussion:
The mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. Due to equivocal results of the negative control eyes resulting in a mean IVIS >3 in the first test, this test was repeated.
The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 116 (110 to 121) in the first test and 165 (134 to 211) in the repeat test which was within the historical positive control data range. Furthermore the mean opacity and permeability values of the positive control were within two standard deviations of the current historical mean. One of the eyes treated with the positive control showed an opacity value which was above the mean plus 2 standard deviations, but since this is an even more positive response the results of the study are not affected. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
In the first test, the test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -2.7 (-4.7 to 0.4) after 240 minutes of treatment. In the repeat test, the test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.2 (-2.7 to 1.5) after 240 minutes of treatment.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since in both tests the results were highly comparable and the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.