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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-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
Type:
Constituent

Test animals / tissue source

Species:
other: 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 to classify substances as “ocular corrosives and severe irritants”. 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, Munich, Germany
- Date of eye collection: on the test day
- Transport medium and temperature conditions: HBSS containing Pen/Strep (5%), on ice

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: cornea holder (MC2, Clermont, France), not further specified
- 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 without phenol red, supplemented with 1% [v/v] fetal calf serum and 2 mM L-glutamine (complete RPMI); prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects, initial opacities of 2.33 (test substance), 4.0 (negative control) and 5.0 (positive control)

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: opacitometer (MC2, Clermont, France), not further specified

Test system

Vehicle:
other: corn oil and 0.9% physiological saline
Controls:
other: number of eyes for the negative controls: 3 per group; number of eyes for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% test substance suspension in corn oil

VEHICLE
- Amount(s) applied (volume or weight with unit): The test substance in physiological saline did not result in an applicable suspension. Therefore, corn oil was used for the suspension of the test substance and as corresponding negative control. The positive control substance was diluted with physiological saline and physiological saline was used as corresponding negative control, too.

POSITIVE CONTROL SUBSTANCE
- Substance: imidazole
- Concentration (if solution): 20% imidazole solution in 0.9% physiological saline
- Amount(s) applied in the test: 750 µl
Duration of treatment / exposure:
4 h ± 5 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
number of eyes for the test substance: 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 fresh complete RPMI without phenol red in the anterior chamber the final opacity was measured.
- Specification of the device: opacitometer (MC2, Clermont, France), not further specified

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
- Specification of the spectrophotometer: spectrophotometer (Jenway, UK), not further specified

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: IVIS
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
3.68
Reversibility:
other: not applicable
Remarks on result:
other: test substance
Irritation parameter:
other: IVIS
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
1.17
Reversibility:
other: not applicable
Remarks on result:
other: negative control: corn oil
Irritation parameter:
other: IVIS
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
199.76
Reversibility:
other: not applicable
Remarks on result:
other: positive control
Irritation parameter:
other: IVIS
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
1.27
Reversibility:
other: not applicable
Remarks on result:
other: negative control: 0.9% physiological saline
Irritation parameter:
other: opacity
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
2.67
Reversibility:
other: not applicable
Remarks on result:
other: test substance
Irritation parameter:
other: opacity
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
169.33
Reversibility:
other: not applicable
Remarks on result:
other: positive control
Irritation parameter:
other: permeability
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
0.067
Reversibility:
other: not applicable
Remarks on result:
other: test substance
Irritation parameter:
other: permeability
Basis:
mean
Remarks:
out of all 3 eyes
Time point:
other: 4 h
Score:
2.028
Reversibility:
other: not applicable
Remarks on result:
other: positive control
Irritant / corrosive response data:
Historical Control Data:
The result of the positive control fell within the two standard deviations of the current historical mean. Therefore, the assay is considered to be valid.

Any other information on results incl. tables

Table 1. Mean opacity values

 

Initial opacity

Final opacity

Change of opacity value

corrected opacity value

Test substance

2.33

5.67

3.33

2.67

Corn oil

4.0

4.67

0.67

---

Physiolog. saline

4.0

4.33

0.33

---

Positive control

5.0

174.67

169.67

169.33

Table 2. Mean permeability values

 

OD490

corrected OD490 value

Test substance

0.101

0.067

Corn oil

0.033

---

Physiolog. saline

0.062

---

Positive control

2.091

2.028

Table 3. Mean in vitro irritation score (IVIS)

 

corrected opacity value

corrected OD490 value

IVIS

Test substance

2.67

0.067

3.68

Corn oil

0.67

0.033

1.17

Physiolog. saline

0.33

0.062

1.27

Positive control

169.33

2.028

199.76

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered as severe eye irritant (Serious eye damage Category 1/R41) based on a positive result in the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2/R36) and shall therefore be subject to further evaluation.