Imidodicarbonic acid, 2-[5-bromo-3-[(1R)-1-[2-[[[(5-cyano-1-methyl-1H-pyrazol-3-yl)methyl]methylamino]carbonyl]-5-fluorophenyl]ethoxy]-2-pyridinyl]-, 1,3-bis(1,1-dimethylethyl) ester, compd. with 2-methyl-2-butanol (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 to 19 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-methylbutan-2-ol; tert-butyl N-{5-bromo-3-[(1R)-1-(2-{[(5-cyano-1-methyl-1H-pyrazol-3-yl)methyl](methyl)carbamoyl}-5-fluorophenyl)ethoxy]pyridin-2-yl}-N-[(tert-butoxy)carbonyl]carbamate

EC Number:

812-621-1

Cas Number:

1910113-99-3

Molecular formula:

C31H36BrFN6O6•C5H12O

IUPAC Name:

2-methylbutan-2-ol; tert-butyl N-{5-bromo-3-[(1R)-1-(2-{[(5-cyano-1-methyl-1H-pyrazol-3-yl)methyl](methyl)carbamoyl}-5-fluorophenyl)ethoxy]pyridin-2-yl}-N-[(tert-butoxy)carbonyl]carbamate

Constituent 2

Reference substance name:

di-tert-butyl {5-bromo-3-[(1R)-1-(2-{[(5-cyano-1-methyl-1H-pyrazol-3-yl)methyl](methyl)carbamoyl}-5- fluorophenyl)ethoxy]pyridin-2-yl}imidodicarbonate t-amyl alcohol solvate

IUPAC Name:

di-tert-butyl {5-bromo-3-[(1R)-1-(2-{[(5-cyano-1-methyl-1H-pyrazol-3-yl)methyl](methyl)carbamoyl}-5- fluorophenyl)ethoxy]pyridin-2-yl}imidodicarbonate t-amyl alcohol solvate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch No.: E010016713
Purity: not specified

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

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.
-Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 311.9 to 340.6 mg

Duration of treatment / exposure:

240±10 minutes

Number of animals or in vitro replicates:

Three eyes each group

Details on study design:

Removal of test substance
- Washing (if done): 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.
- Time after start of exposure: 240±10 minutes
Reference items
- Negative control: A negative control, physiological saline was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
- Positive control: 20% (w/v) Imidazole solution prepared in physiological saline.
Opacity measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
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 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.
Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
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. 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.
Permeability determinations
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. 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 the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vivo

Results

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 240 minutes

Score:

1.9

Max. score:

2.6

Remarks on result:

other: Scoring based upon IVIS value

Irritant / corrosive response data:

The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.9. The individual positive control in vitro irritancy scores ranged from 101 to 154. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test substance showed opacity values ranging from 1.3 to 2.1 and permeability values ranging from 0.009 to 0.031. The corneas were clear after the 240 minutes of treatment with test substance. Hence, the in vitro irritancy scores ranged from 1.4 to 2.6 after 240 minutes of treatment with test substance.

Other effects:

No pH effect of the test item was observed on the rinsing medium.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.9 after 240 minutes of treatment.
Since test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.