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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
After the incubation the epithelium was washed 3x with MEM containing phenol red. By rinsing the corneas with this medium, possible pH effects of the test substance on the corneas can be observed. This had no effect on the result of the study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name: Carbamic acid, N-(3-(triethoxysilyl)propyl)2-(ethyl-(4-nitrosophenyl) amino) ethyl ester
- Batch 3445-60
- CAS 1195231-94-7
- Purity: 95 %

Test animals / tissue source

Species:
other: Bovine eyes
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
310.3 to 332.2 mg test item
750 µL of the negative and positive control
Duration of treatment / exposure:
4 h
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
0
Details on study design:
PREPARATION OF CORNEAS
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded. The isolated corneas were stored at 32 ± 1 °C in a petridish with cMEM (Eagle's Minimum Essential Medium (lnvitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (lnvitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (lnvitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Ciermont, France) with the endothelial side against the 0-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.

CORNEA SELECTION AND OPACITY READING
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, 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.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) lmidazole solution (positive control) were introduced onto the epithelium of the cornea. Three corneas were covered with 310.3 to 332.2 mg of test item 3445-60. 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. After the incubation the solutions and the test compound were removed
and the epithelium was washed at least three times with MEM with phenolred (Eagle's Minimum Essential Medium). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

OPACITY MEASUREMENT
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 posttreatment reading. The corrected opacity for each positive control or test substance 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 substance 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 (Merck) 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 Iabelled 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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). An 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. lf 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.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Mean in vitro irritation score
Basis:
mean
Time point:
other: 240 min
Score:
10.3
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: test item (maximum score cannot be given)
Irritation parameter:
other: Mean in vitro irritation score
Basis:
mean
Time point:
other: 240 min
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: negative control (aximum score cannot be given)
Irritation parameter:
other: Mean in vitro irritation score
Basis:
mean
Time point:
other: 240 min
Score:
119
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: positive control (maximum score cannot be given)
Irritant / corrosive response data:
SUMMARY OF OPACITY, PERMEABILITY AND IN VITRO SCORES:

- NEGATIVE CONTROL:
Mean Opacity 0
Mean Permeability 0.000
Mean in vitro Irritation Score 0.0

- POSITIVE CONTROL:
Mean Opacity 71
Mean Permeability 3.186
Mean in vitro Irritation Score 119

- TEST ITEM 3445-60:
Mean Opacity 5
Mean Permeability 0.351
Mean in vitro Irritation Score 10.3

Any other information on results incl. tables

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 115 to 124. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with 3445-60 showed opacity values of 5 and permeability values ranging from 0.206 to 0.449. The corneas were coloured green after the 240 minutes of treatment with 3445-60.

No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 8.1 to 11.7 after 240 minutes of treatment with 3445-60.

Applicant's summary and conclusion

Interpretation of results:
other: not severely irritating or corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Finally, it is concluded that this test is valid and that the test item 3445-60 is not severe irritant or corrosive in the Bovine Cornea.
Opacity and Permeability test under the experimental conditions described in this report.
Executive summary:

SUMMARY

Screening for the eye irritancy potential of test item 3445-60 using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of 3445-60 on an isolated bovine cornea. The possible ocular irritancy of 3445-60 was tested through topical application for 240 ± 10 minutes. The study procedures described in this report were based on the most recent OECD guideline.

3445-60 was a green powder with lumps with a purity of 95%. The test substance was applied undiluted (310.3 to 332.2 mg) directly an top of the corneas. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% w/w lmidazole) was 119 and within the historical positive control data range.

It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 3445-60 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 10.3 after 240 minutes of treatment.

Finally, it is concluded that this test is valid and that 3445-60 is not severe irritant or corrosive in the Bovine Cornea. Opacity and Permeability test under the experimental conditions described in this report.