Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2014 - 15 Jan 2014
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
other: cattle
Strain:
other: animals freshly slaughtered
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: A. Moksel AG, Buchloe, Germany
- Time interval prior to initiating testing: Cornea preparation was initiated immediately after arrival of the eyes.
- 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 are tightened with screws.
- Test medium used in the cornea holder: RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI)
- Equilibration time: 1 h at 32±1 °C
- Quality check of the equilibrated corneas: corneas with an initial opacity above 7 in the opacitometer were discarded.

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

Test system

Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
other: number of eyes for the negative control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount applied (volume or weight with unit): 750 µL
- Concentration: 20% in vehicle

VEHICLE
- Substance: physiol. saline
- Concentration: 0.9% NaCl solution
- Source: Diprom
- Lot/batch no.: 13001-4
- Expiry date: 12/2015

POSITIVE SUBSTANCE
- Substance: Imidazole
- Concentration: 20% imidazole solution in physiol. saline
- Amount applied in the test: 750 µL
- Source: Sigma,
- Lot/batch no.: SLBC7446V
- Expiry date: 11/2014
Duration of treatment / exposure:
4 hours±5 minutes incubation at 32±1 °C
Observation period (in vivo):
Not applicable.
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: 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). Corneas were exposed for 4 h at 32±1 °C with the test substance or the controls.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed and the epithelium washed at least three times.
- Medium for washing the corneas: EMEM containing phenol red
- Medium for final rinsing: complete 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: After refilling complete RPMI in the anterior chamber the final opacity was measured.

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 complete RPMI. 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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
280.15
Vehicle controls validity:
valid
Remarks:
1.30
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
172.12

Any other information on results incl. tables

Tab. 1: Opacity values

Parameter

Cornea No.

Initial opacity

Final opacity

Change of opacity value

Corrected opacity value

Negative control

1

3

4

1

 

2

3

2

-1

3

3

2

-1

Mean

3.00

2.67

-0.33

Positive control

1

4

137

133

133.3

2

4

151

147

147.33

3

4

153

149

149.33

Mean

4.00

147.00

143.00

143.33

Test substance

1

3

330

327

327.33

2

3

251

248

248.33

3

3

268

265

265.33

Mean

3.00

283.00

280.00

280.33

 

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

Parameter

Cornea No.

OD490

Corrected OD490 value

Negative control

1

0.104

 

2

0.120

3

0.1102

Mean

0.109

Positive control

1

2.048

1.939

2

2.036

1.927

3

1.999

1.890

Mean

2.028

1.919

Test substance

1

0.134

0.025

2

0.110

0.001

3

0.046

-0.063

Mean

0.097

-0.012

 

Tab. 3: In-Vitro Irritancy Score (IVIS) values

Parameter

Cornea No.

Change of opacity value

Corrected OD490 value

IVIS

Negative control

1

1.00

0.104

1.30

2

-1.00

0.120

3

-1.00

0.1102

Mean

-0.33

0.109

Positive control

1

133.33

1.939

172.12

2

147.33

1.927

3

149.33

1.890

Mean

143.33

1.919

Test substance

1

327.33

0.025

280.15

2

248.33

0.001

3

265.33

-0.063

Mean

280.33

-0.012

 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
CLP: Cat. 1, H318