Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September to December 2002
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Appearance: light tan solid
Storage conditions: at room temp, protected by light

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine eyes, excised by an abattoir employee, were collected as soon after slaughter as possible ( excised at -10:35 hours on 11 September 2002). Instructions were given to avoid damaging the corneas during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing I% (v/v) Penicillin/Streptomycin solution, to cover all the eyes in the receptacle. The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 13:43 hours on 11 September 2002).

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20% (w/w) solution or suspension in 0.9% sodium chloride solution
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 4 hours at 32C
Duration of post- treatment incubation (in vitro):
Following incubation, the test substance was removed and the epithelial surface of the cornea washed, at least 3 times or until the wash medium was clear, with cMEM added via the holes on the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. Following completion of the wash step, the medium was removed from both compartments which were re-filled with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final inVitro Score.
Number of animals or in vitro replicates:
3
Details on study design:
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1 % Penicillin/Streptomycin solution until all the corneas had been dissected. Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% Penicillin/Streptomycin solution prior to mounting.

The corneas were mounted in the cornea holders with the endothelial side against the 0-ring of the posterior half of the holder. Each cornea was gently flattened over the 0-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with cMEM, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders. The holders were then plugged and incubated, in an upright position, for 60 minutes ± 5 minutes at 32°C ± 2°C in a waterbath. The
waterbath temperature remained within the limits of 32°C ± 2°C throughout the experiment.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
31.7
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: Permeability
Value:
0.082
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Value:
32.9
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Applicant's summary and conclusion

Interpretation of results:
Category 2A (irritating to eyes) based on GHS criteria
Conclusions:
The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular initancy potential in vitro of test substance BMS 482204-03 (MSA Salt). Imidazole was tested in parallel as a positive control.
Two endpoints, corneal opacity and permeability, were measured in the assay. These measurements were combined to give an In Vitro Score which was used to determine the potential eye irritancy of the test substance according to the Prediction Model developed by Curren el al (1996). In Vitro Scores up to 25 are classified as negative and scores greater than 25 are classified as positive potential eye irritants.

The test sample, BMS 482204-03 (MSA Salt), elicited an In Vitro Score of 32.9 ± 7.1 and was classified as a positive potential eye irritant.