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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
according to guideline
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder

Test animals / tissue source

other: Bovine Eyes
Details on test animals or tissues and environmental conditions:
Preparation of corneas:
All eyes were carefully examined for defects by holding the eyes to the light after submerging in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded. The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)).The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) 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.

Test system

physiological saline
yes, concurrent vehicle
Amount / concentration applied:
324.2 to 333.9 mg (applied directly onto corneas in such a way that the corneas were completely covered).
Duration of treatment / exposure:
240 ± 10 minutes at 32 ± 1°C
Number of animals or in vitro replicates:
Three eyes for the test substance, negative control and positive control substance.
Details on study design:
Details on 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) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (324.2 to 333.9 mg).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 phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). 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 measurements:
The opacitometer determined the difference in the light transmission between each control or test substance 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 post-treatment 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 changein 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.

Permeability determination:
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 ul 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before thedilution factor was applied to the readings.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
3 times
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls were -1 to 0.0. The individual positive control in vitro irritancy scores ranged from 107 to 161. 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 of -1 and permeability values ranging from 0.002 to 0.047. The corneas were clear after the 240 minutes of treatment. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.0 to -0.3 after 240 minutes of treatment.

Any other information on results incl. tables

Treatment Mean Opacity Mean Permeability Mean In Vitro Irritation Score
Negative Control 0 0.000 0.0
Positive Control 92 2.986 137
Test substance -1 0.018 -0.7

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage