Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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)
Version / remarks:
adopted in 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Attenberger Fleisch GmbH&Co. KG, München, Germany
- Donor animals: no details given
- Date and time of eye collection: directly before the start of the experiment
- Time interval prior to initiating testing: no details given
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) on ice with penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: MC2 (Clermont, France)
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: RPMI with and without phenol red, supplemented with 1% [v/v] fetal calf serum and 2 mM L-glutamine; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: initial opacity of 2 (corneas with an initial opacity above 7 in the opacitometer or with any macroscopic defects were not used)

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer and was read against an air-filled chamber.
- Specification of the device: MC2, Clermont, France

Test system

Vehicle:
physiological saline
Controls:
other: number of eyes for the negative control: 3; number of eyes for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20%

VEHICLE
- Substance: physiol. saline
- Concentration: 0.9% NaCl solution in deionised water
- Amount applied: 750 µL

POSITIVE SUBSTANCE
- Substance: imidazole in physiol. saline
- Concentration: 20% imidazole in 0.9% NaCl solution in deionised water
- Amount applied: 750 µL
Duration of treatment / exposure:
4 h at 32 ± 1 °C
Number of animals or in vitro replicates:
Number of eyes for the test item: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: closed-chamber method

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed at least three times.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: RPMI without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: Directly after refilling complete RPMI without phenol red in the anterior chamber the final opacity was measured.
- Specification of the device: MC2, Clermont, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: OD490 of a 1 mL aliquot was determined.
- Dilution of the medium: undiluted
- Specification of the spectrophotometer: Jenway, UK

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean out of all 3 eyes
Run / experiment:
4 h exposure
Value:
235.99
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
An IVIS of 235.99 was observed for the test substance indicating that the test susbstance has corrosive or severe irritant properties.

Any other information on results incl. tables

Table 1: Opacity values

Parameter

Initial opacity

Final opacity

Opacity change

Mean opacity change of NC

Corrected opacity change

Mean opacity value

Negative control

2

1

-1

-0.67

-

-

2

1

-1

 

2

2

0

 

Test substance

1

216

215

-

215.67

236

2

266

264

264.67

2

229

227

227.67

Positive control

3

162

159

-

159.67

158

3

194

191

191.67

3

125

122

122.67

 

 

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter

OD490

Mean OD490 value

Corrected OD490 values

Mean Corrected OD490 values

Negative control

0.010

0.010

-

-

0.007

0.012

Test substance

0.003

0.009

-0.007

-0.001

0.012

0.002

0.012

0.002

Positive control

1.702

1.651

1.692

1.641

1.511

1.501

1.740

1.730

 

 

Table 3: In-Vitro Irritancy Score (IVIS) values

 

Mean IVIS

 

Test substance

235.99

Positive control

182.62

 

 

 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 1 according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: Cat. 1, H318