Registration Dossier

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 437 of July 2013: Bovine Corneal Opacity and Permeability test method for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: in vitro test
Strain:
other: bovine eyes

Test system

Vehicle:
physiological saline
Amount / concentration applied:
750 µl of test substance, 20% in physiol. saline, positive control solution (20% w/w imidazole in physiol. saline) or negative control solution (physiol. saline) was introduced into the anterior part of each cornea holder. Following application, turning of the holder into horizontal position and its slight rotation ensured uniform distribution of the test substance over the surface of the cornea.
Duration of treatment / exposure:
4 hours (± 5 min.).
Each cornea holder was incubated in a horizontal position at 32°C ± 1°C in a waterbath. Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed, at least three times or until the wash medium (EMEM* with phenol red) was clear and there was no discolouration.

* EMEM, Eagles Minimal Essential Medium
Details on study design:
Each treatment group (test substance, negative/positive controls) comprised 3 corneas from bovine eyes. Each cornea surrounded by ca. 2 to 3 mm of sclera was mounted to a holder. Eyes were maintained and transported to the laboratory and isolated corneas stored in sufficient HBSS* containing 1% penicilin/streptomycin solution until being used. The eyes were used within 4 hours of slaughter. Adequate exposure of the epithelial surface of the cornea to the test substance was provided. Opacity of the cornea was determined by measurement of light transmission through the centre of each mounted cornea using a calibrated opacitometer. Permeability of the cornea was determined by spectophotometric measurement at 490 nm (OD490) of medium from the posterior compartment of the cornea holder after addition of 1 ml of sodium fluorescein solution (4 mg/ml) to the anterior compartment, thus determining the degree of penetration of sodium fluorescein through all corneal cell layers. Prior to the spectophotometric measurement, incubation with fluorescein solution had happened in horizontal position of the cornea holder at 32°C ± 1°C for 90 ± 5 minutes in a waterbath. Then the medium in the posterior compartment was mixed by drawing approximately 2.5 ml gently up and down a 5 ml syringe, with a needle attached, three times. Any solution giving an OD490 value above 1.8 was diluted 1 in 5 with cMEM.

Effects to the cornea (corresponding to possible eye damage) were measured as opacity and permeability, which when combined gave an In Vitro Irritancy Score (IVIS) for each treatment group. A substance that induces an IVIS greater than 55 is defined as a corrosive or severe irritant.

*HBSS Hanks Balanced Salt Solution containing Ca++ and Mg++ without phenol red

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
replicate 1
Value:
0.333
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
replicate 2
Value:
1.333
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
replicate 3
Value:
1.333
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
replicate 1
Value:
0.483
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
replicate 2
Value:
1.063
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
replicate 3
Value:
1.078
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met