5-Thiazolecarboxamide, N-(2-chloro-6-methylphenyl)-2-[(6-chloro-2-methyl-4-pyrimidinyl)amino]-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 13th, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study completed according to GLP and OECD methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The test substance, was tested using the Bovine Corneal Opacity & Permeability Assay (BCOP) which was developed by Pierre Gautheron 1992 as an in vitro alternative to the in vivo Draize Eye Irritation test. The assay uses isolated bovine corneas as a means of assessing the ocular irritancy potential of test substances in vitro.

GLP compliance:

yes

Remarks:

OECD ENV/MC/CHEM(98)17 AND EC 2004/10/EC

Test material

Test material information

Constituent 1

Reference substance name:

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide

EC Number:

695-686-7

Cas Number:

302964-08-5

Molecular formula:

C16 H13 C12 N5 O S

IUPAC Name:

2-[(6-chloro-2-methylpyrimidin-4-yl)amino]-N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

The test article was administered to the test system as a 20% (w/v) suspension in saline. The test article suspension was prepared by weighing out approximately 1400 mg of test article into a prelabeled conical tube. Saline was added until a 20% (w/v) suspension was achieved and the vial was vortexed for approximately 1 minute prior to application.

Test animals / tissue source

Species:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised and then placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The corneas were used within 24 hours of receipt.

The eyes were grossly examined for damage and those exhibiting defects were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the 0-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were
tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, supplemented with 1% fetal bovine serum (Complete MEM). Each corneal holder was uniquely identified with a number written in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1°C for a minimum of 1 hour.

The test article was administered to the test system as a 20% (w/v) suspension in saline. The test article suspension was prepared by weighing out approximately 1400 mg of test article into a prelabeled conical tube. Saline was added until a 20% (w/v) suspension was achieved and the vial was vortexed for approximately 1 minute prior to application

The pH of the test article was determined using pH paper (EM Science). Initially, the test article was added to 0-14 pH paper with 1 pH unit increments to approximate a narrow pH range. Next, the test article was added to 0-6 pH paper with 0.5 pH unit increments, to obtain a more precise pH value. The pH value obtained from the narrower range pH paper is recorded in
Table 1.

Test system

Vehicle:

other: 20% (w/v) suspension in saline

Controls:

other: positive and negative control substances

Amount / concentration applied:

The solid test article, BMS 540268, was tested as 20% (w/v) suspension in saline. Due to the viscous nature of the test article suspension, 750 µl of the test article was uniformly administered directly onto the cornea. An aliquot of 750 µl of either the positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea.

Duration of treatment / exposure:

The corneas were incubated in the presence of
either the test article, positive control or negative control at 32 ± 1°C for approximately 4 hours.

After the 4-hour exposure period, the test article treatment was initially removed from the exposed corneas. The control treatment corneas were also removed after the 4-hour exposure period. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test articles. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the
posterior chambers were refilled with fresh Complete MEM and the final measure of opacity was performed immediately (with no further post-exposure incubation).

Observation period (in vivo):

After the final opacity measurement was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 ml of a 5 mg/ml fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1°C. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered
corresponding to chamber number. Aliquots of 360 µl from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. Ifthe OD490 value of a control or test article sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM. A 360 µl sample of each 1:5 dilution was transferred to its specified well on the 96-well plate

Number of animals or in vitro replicates:

Corneas were treated in triplicate with either the test substance (20% w/v saline suspension), positive control compound (20% w/v oflmidazole suspension) or negative control (0.9% USP Saline).

Details on study design:

After the tratment of the corneas, described above, the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.

The next stage is to perform the permeability assay:

After the final opacity measurement was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 ml of a 5 mg/ml fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1°C. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered
corresponding to chamber number. Aliquots of 360 µl from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. Ifthe OD490 value of a control or test article sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM. A 360 µl sample of each 1:5 dilution was transferred to its specified well on the 96-well plate.
The plate was read again and the final reading was saved to a designated print file

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: opacity

Basis:

mean

Time point:

other: 4 hours

Score:

20.8

Results 2

Irritation parameter:

other: In vitro Irritancy score

Basis:

mean

Remarks:

in vitro score

Time point:

other: 4 hrs

Score:

20.8

Results 3

Irritation parameter:

other: Corneal Permeability (OD490)

Basis:

mean

Time point:

other: 4 hours

Score:

-0.003

Irritant / corrosive response data:

Positive control results:
Opacity value: 62.0
Permeability: 2.115
In vitro irritancy: 93.7
The results of the positive control fell within two standard deviations of the historical mean (within a range of 71.4 to 137.7), therefore the assay was considered valid.

Any other information on results incl. tables

The pH of the test sample formulation, measured using universal pH sticks, was approximately 5.5.

Opacity Measurement: The change in opacity for each cornea (including negative control corneas) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition.

Permeability Measurement: The corrected OD490 was calculated by subtracting the mean OD490 of the negative control corneas from the OD490 value of each treated cornea. The mean OD490 value of each treatment group was calculated by averaging the corrected OD490 values of the treated corneas for that treatment condition.

The following formula was used to determine the in vitro score: In Vitro Score = Mean Opacity Value + (15 x Mean OD490 Value).

In Vitro Score:

• from 0 to 25 = mild irritant

• from 25.1 to 55 = moderate irritant
• from 55.1 and above = severe irritant

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance elicited an In Vitro Irritancy Score of 20.8

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance. Imidazole was tested in parallel as a positive control.

The assay uses isolated bovine corneas as a means of assessing the ocular corrosivity or severe irritancy potential of test substances in vit1ro. The isolated corneas are obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, are measured and combined to give an In Vitro Irritancy Score which can be used to classify and rank test

substances as potential eye irritants according to the Classification criteria (OECD 437)

The test substance, BMS 540268, gave an In Vitro Irritancy Score of 20.8.