Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 4 to July 22, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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)
Principles of method if other than guideline:
The purpose of the study is to evaluate the potential ocular irritancy/toxicity of a test article as measured by the test article's ability to induce opacity and permeability to fluorescein in an isolated bovine cornea.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
white powder; stored at room temperature in the dark

Test animals / tissue source

Species:
other: bovine corneas
Details on test animals or tissues and environmental conditions:
Bovine eyes were obtained as by-product of freshly slaughtered animals, transported in Hank's Balanced Salt Solution supplemented with penicillin/streptomycin and sent on ice packs. The corneas are used within 24 hour of receipt. The tissue surrounding the eyeball were carefully pulled away and the corneas were excised such that a 2 to 3 mm rim of sclera was present around the cornea. The corneas are mounted in corneal folders with the endothelial side against the O-ring of the chamber and the anterior chamber secured on top. The chambers were filled with Minimum Essential Medium Eagle (MEM) without phenol red, supplemented with 1 % fetal bovine serum and incubated at 32 +- 1 C for a minimum of 1 hour.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent no treatment
Amount / concentration applied:
An aliquot of 750ug was administered as 20 % w/v ( 200 mg/ml) suspension in saline.
Duration of treatment / exposure:
Corneas were exposed to the test material suspension for approximately four hours and then the corneas are removed from the incubator. The epithelial side of each cornea was rinsed three times with complete MEM containing phenol red and a final rinse with complete MEM without phenol red. Then the solution is replaced with fresh complete MEM, supplemented with 1 % fetal bovine serum.
Observation period (in vivo):
Opacity measurements performed immediately after exposure period ends.
Number of animals or in vitro replicates:
Five corneas are tested.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
after 4 hrs
Value:
1.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
pH6

Any other information on results incl. tables

The bovine corneal opacity and permeability assay uses an opacity measurement and a permeability measurement to determine a final in vitro score. The opacity measurement is determined by the change in opacity of each cornea by subtracting the calculated pre-treatment opacity reading from the final opacity reading. The corrected opacity value of each cornea was calculated by subtracting the average change in opacity of the negative control corneas from that of the treated corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity value of the treated corneas from each treatment condition. The permeability measurements: the corrected OD 490 is determined by subtracting the negative controls mean OD 490 from each of the treated cornea OD 490 value. The mean OD 490 of each treatment group was calculated by averaging the corrected OD 490 values. The formula used is: in vitro score = mean opacity value + ( 15 x mean OD 490 Value). The in vitro score is then compared to a classification system established based on studies with a wide range of test materials. The classification provides a good initial guide to interpret these in vitro data: an in vitro score from 0 to 25 is mild irritant, from 25.1 to 55 is a moderate irritant and 55.1 and above is a severe irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on an in vitro score of 1.4 and valid positive controls, the test material was classified according to this assay's classification scheme as a mild irritant.