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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 to 19 Jan 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 437 (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
migrated information: powder
Specific details on test material used for the study:
Batch No.: E010016692Purity: 99.3%

Test animals / tissue source

other: cattle
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

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 315.6 to 365.5 mg
Duration of treatment / exposure:
240±10 minutes
Duration of post- treatment incubation (in vitro):
1 hour
Number of animals or in vitro replicates:
Three eyes each group
Details on study design:
SELECTION AND PREPARATION OF CORNEASThe eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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.QUALITY CHECK OF THE ISOLATED CORNEASAfter 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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. NUMBER OF REPLICATESThree corneas were selected at random for each treatment group.NEGATIVE CONTROL USEDphysiological salineSOLVENT CONTROL USED (if applicable)POSITIVE CONTROL USED20% (w/v) ImidazoleAPPLICATION DOSE AND EXPOSURE TIMEThe 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. Test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (315.6 to 365.5 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.TREATMENT METHOD: [closed chamber / open chamber]POST-INCUBATION PERIOD: no.REMOVAL OF TEST SUBSTANCE- Number of washing steps after exposure period: 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.- POST-EXPOSURE INCUBATION:METHODS FOR MEASURED ENDPOINTS: - Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)- Others: Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.SCORING SYSTEM: In Vitro Irritancy Score (IVIS)DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
-0.2 - 1.3
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
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 -0.3 to 1.2 and permeability values ranging from 0.003 to 0.022. The corneas were clear after the 240 minutes of treatment with test substance. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.2 to 1.3 after 240 minutes of treatment with test substance.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.8 after 240 minutes of treatment.Since test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.