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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
Study period:
March 15, 2013 - March 21, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
- Storage condition of test material: At room temperature in the dark

Test system

Vehicle:
physiological saline
Amount / concentration applied:
TEST MATERIAL
A 20% (w/w) suspension of the test substance in physiological saline (Merck, Germany)
Duration of treatment / exposure:
4 hours
Details on study design:
SELECTION AND 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 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 (75 minutes).

QUALITY CHECK OF THE ISOLATED CORNEAS
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 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
750 µL of physiological saline per cornea

POSITIVE CONTROL USED
750 µL of a 20% (w/v) Imidazole solution in physiological saline per cornea

EXPOSURE TIME
4 hours (at 32°C)

TREATMENT METHOD
The medium from the anterior compartment was removed and the control solutions and test substance suspension were introduced onto the epithelium of the cornea.
The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions/suspension over the entire cornea.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation)

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: 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 post-treatment reading.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated, after another incubation period of 85 minutes at 32°C. 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).
- Others: Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM
In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA
- A test substance that induces a mean IVIS >55 is classified with Eye Irr. Cat. 1 in accordance with the CLP Regulation
- A test substance that induces a mean IVIS ≤ 3 is not classified for Eye irritation/corrosion in accordance with the CLP Regulation
- For a test substance that induces a mean IVIS of >3 - ≤55, no prediction on the classification can be made. More information is needed.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 3
Value:
4
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores:

Treatment

Mean opacity

Mean permeability

Mean in vitro irritation score

Negative control

0

0.000

0.0

Positive control

98

1.614

122

Test substance

4

-0.003

4.0

 

Individual in vitro irritancy scores:

Eye

In vitro Irritancy Score

Negative control

1

0.1

2

0.0

3

0.9

Positive control

4

108

5

116

6

142

Test substance

16

4.9

17

4.0

18

3.9

The corneas were clear after the 240 minutes of treatment with the test substance.

No pH effect of the test substance was observed on the rinsing medium.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on a mean in vitro irritancy score of 4.0 in a Bovine Corneal Opacity and Permeability test, it is concluded that no prediction can be made whether the substance needs to be classified for eye irritation/corrosion. For this, more information is needed.
Executive summary:

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. A 20% (w/w) suspension of the test substance in physiological saline was applied on the corneas. Adequate negative and positive controls were included. The mean in vitro irritancy score was 4.0 after 240 minutes of treatment. Since the substance induced a mean IVIS >3 and ≤ 55, no prediction can be made whether the substance needs to be classified for eye irritation/corrosion. For this, more information is needed.