Registration Dossier

Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: The bovine corneal Opacity and Permeability Assay (BCOP)
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 (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension

Test animals / tissue source

Species:
other: Bovine Corneas
Details on test animals or tissues and environmental conditions:
ovine 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:
20% w/v suspension in 0.9% saline
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Opacity measurements performed immediately after exposure period ends.
Details on study design:
The study design requires determination of an opacity value and a permeability measurement. For opacity determination: prior to treatment with test material pre-treatment opacity measurements are taken. This is performed approximately one hour after incubation, the corneas are removed from the incubator, the medium removed and replaced with fresh complete MEM. The opacity readings are taken with an opacitometer and three negative controls were selected whose opacity is close to the mean opacity of all the corneas. The medium for all corneas is removed and replaced with fresh complete MEM and then the test article suspension, positive or negative control solutions are added. Positive and negative controls are used in the study. The positive controls use a 20 % (w/v) suspension of imidazole in complete MEM. The negative control use a 0.9% USP Injectable Saline. The pH of the test material suspension was determined to be 5.5. The corneas were exposed 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. The anterior adn the posterior chambers were refilled with complete MEM and an opacity measurements performed immediately (with no further post-exposure incubation).
Once opacity measurements are complete the medium was replaced with 1 ml of a 5 mg/ml fluorescein solution and incubated for another 90 minutes at 32 +/- 1oC with the anterior chamber up. After 90 minutes the medium was removed from chambers and 360 ul of medium placed in designated tubes of a 96-well plate. The opacity density at 490 nm (OD 490) was determined using a Molecular Devices Vmax kinetic microplate reader. If an OD 490 value was greater than 1.500 a 1:5 dilution of the sample in complete MEM was made. The dilution samples were then retested and final permeability measurements were made.

Results and discussion

In vivo

Results
Irritation parameter:
other: In-vitro score
Basis:
mean
Time point:
other: 4 hrs
Remarks on result:
other: opacity value of 1.3, OD 490 of 0.01, pH 5.5
Irritant / corrosive response data:
Positive control results: opacity value (57.3), OD 490 (57.3) and in vitro score (87.8). The results of the positive control fell within 2 standard deviations of the historical mean and the assay was considered valid.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: other: Classification scheme published in Sinn et al article " A Collaborative Evaluation of Seven Alternatives to the Draize Eye Irritation Test using Pharmaceutical Intermediates", Fundamental and Applied Toxicology 26:20-31.
Conclusions:
Based on an in vitro score of 1.3 and valid positive controls, the test material was classified according to this assay's classification scheme as not irritating