Reaction products of 2,4-bis(2-chlorophenyl)-5-(3,4-dimethoxy phenyl)-1H-imidazole and 2-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline: Draft Test Guideline for the Bovine Corneal Opacity and Permeability (BCOP) Test Method for Identifying Ocular Corrosives and Severe Irritants

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 100% as a reaction product

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine Corneas
Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

Preparation of Corneas
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 O-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, containing 1% fetal bovine serum and 2 mM L-glutamine (Complete MEM). The corneal holders were incubated at 32 ± 1 ºC for a minimum of 1 hour.

Test system

Controls:

yes

Amount / concentration applied:

Test Substance Preparation
The test substance was administered to the test system as a 20% (w/v) dilution in sterile, deionized water. The test substance dilution was prepared by weighing approximately 1,200 mg of the test substance into a prelabeled conical tube. Sterile, deionized water was added until a 20% (wlv) dilution was achieved and the conical was vortexed for approximately 1 minute prior to application. The resulting dilution was described as a cloudy yellow non-viscous suspension. For the remainder of this report, the test substance dilution is referred to as the test substance.

Test Substance pH Determination
0-14 pH and 0-6 pH paper were used to obtain a pH value. The test substance discolored the pH paper and the pH could not be determined.

Controls
Positive: Imidazole. 20% (w/v) solution in complete MEM without phenol red
Negative: sterile, deionized water

Duration of treatment / exposure:

4 hours

Details on study design:

The Bovine Corneal Opacity and Permeability Assay (BCOP) is used to assess the potential ocular irritancy of the test substance as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas. Based on changes in corneal opacity and permeability (relative to the control corneas), an in vitro score is determined.

Opacity Measurement: The change in opacity for each cornea (including the 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 the treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition.

Permeability Measurement: The mean OD490 value for the blank wells was calculated. The mean blank OD490 value was then subtracted from the raw OD490 values of each well (corrected OD490). Any dilutions that were made to bring the OD490 readings into the linear range of the plate reader (OD490 should be less than 1,500), had each diluted OD490 reading multiplied by the dilution factor. The final corrected OD490 values of the test substance and the positive control were then calculated by subtracting the average corrected OD490 value of the negative control corneas from the corrected OD490 value of each treated cornea:

Final Corrected OD490 = (raw OD490 - mean blank OD490) - average corrected negative control OD490

The mean OD490 value of the treatment group was calculated by averaging the final 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)

The following classification system was established by Sina et al (Fundamental and Applied Toxicology 26:20-31) based on studies with a wide range of test materials. While this classification system provides a good initial guide to interpretation of these in vitro data, these specific ranges may not be applicable to all classes of materials.

In Vitro Score:
0 to 25 = mild irritant
25.1 to 55 = moderate irritant
55.1 and above = severe irritant

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

Mean Opacity Value + (15 x Mean OD490 Value)

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Imidazole had a mean opacity of 63.7 and a mean OD490 of 1.726. Giving an in vitro score of 89. Since the results of the positive control fell within two standard deviations of the historical mean (within a range of 68.3 to 131.4), the assay was considered valid.
The test substance had a mean opacity of 0.0 and a mean OD490 (Permeability) of 0.003. Giving an in vitro score of 0.0. Based on the classification, the test substance would be considered a mild irritant.

Criteria for Determination of a Valid Test
The BCOP assay was accepted when the positive control (Imidazole) caused an in vitro score that fell within two standard deviations of the historical mean, updated every three months.

Any other information on results incl. tables

Opacity Results

Treatment	Initial	Final	Change	Corrected	Avg	Std Dev
Test Substance	2	5	3	1.0	0.0	1.0
	2	3	1	-1.0
	1	3	2	0.0
	2	5	3	1.0
	3	4	1	-1.0
Negative Control (sterile, Dl water)	4	7	3	NA	2.0
	3	5	2	NA
	2	3	1	NA
Positive Control (Imidazole)	1	64	63	61.0	63.7	8.3
	1	60	59	57.0
	2	77	75	73.0

x

Permeability Results

Treatment	Initial OD490	Dilution Factor	Corrected OD490	Avg	Std Dev
Test Substance	0.003	1	0.000	0.003	0.002
	0.007	1	0.004
	0.005	1	0.002
	0.004	1	0.001
	0.007	1	0.004
Negative Control (sterile, Dl water)	0.007	NA	0.007	0.003
	-0.004	NA	-0.004
	0.005	NA	0.005
Positive Control (Imidazole)	0.342	5	1.707	1.726	0.119
	0.324	5	1.617
	0.371	5	1.852

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Mild irritant

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test substance to isolated bovine corneas. Bovine corneas, obtained as a by-product from freshly slaughtered animals, were mounted in special holders and exposed to the test substance. An in vitro score was determined for the test substance based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. Based on the classification scheme, the test substance would be considered a mild irritant.