Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sept 2017 to 05 Dec 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 437, adopted September 7, 2009.
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder
Details on test material:
White powder

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
Test System
The bovine eyes were received from Spear Products on 04 Oct 2017 and transported to MB Research in Hank’s Balanced Salt Solution (HBSS) in a refrigerated container.

Test system

other: Minimal Essential Medium (MEM)
yes, concurrent vehicle
Amount / concentration applied:
0.75 ml of the test article mixture was applied to the epithelium of each of the five treated corneas.
Duration of treatment / exposure:
4 hours (+/- 10 minutes)
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
5 bovine eyes
Details on study design:
Pretest Procedures
Fresh assay solutions were prepared prior to use. Minimum Essential Media (MEM) solution was prepared by combining together one jar of MEM powder (sufficient to make one liter), 2.2 g Sodium Bicarbonate, 0.292 g L-Glutamine, 10 ml of Fetal Bovine Serum (FBS) and distilled water was added to a total volume of 1000 ml. The MEM solution was kept in an incubator for the duration of testing. HBSS was prepared by combining together HBSS powder (sufficient to make one liter) and 0.35 g Sodium Bicarbonate; the solution was brought to a final volume of 1000 ml with distilled water. HBSS was maintained at room temperature.
The eyes were examined and any eye with a cornea exhibiting evidence of vascularization, pigmentation,
opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.
The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with (MEM) solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder with the cornea was then placed in a 32 (±2)°C incubator and allowed to equilibrate for
at least one hour, but not longer than two hours.
Following the equilibration, the holders containing the corneas were removed from the incubator. The MEM solution was removed from both chambers and the chambers refilled with fresh MEM solution. At this time, five corneas were selected for dosing with the test article and two were selected as controls.
A pre-exposure determination of opacity was made for each control by measuring each against the blanks supplied by the opacitometer. A pre-exposure determination of opacity was made for each test cornea by measuring against each control cornea (a total of 10 determinations).

Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber and 0.75 ml of the test article mixture was applied to the epithelium of each of the five treated corneas.
The holders and corneas were then placed in the 32 (±2)°C incubator in a horizontal position to ensure contact of the test article with the corneas. After four hours (±10 minutes), the test article (or MEM solution in the controls) was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution and opacity measurements were made taken with each treated cornea compared to each of the two control corneas. Opacity measurement of the cornea was made using an OP-KIT opacitometer produced by Electro-Design Corporation of Riom, France.
Immediately following the four hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco's Phosphate Buffered Saline (DPBS). Each holder was then returned to the 32 (±2)°C incubator in a horizontal position ensuring contact of the fluorescein with the cornea.
After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye that passed through the cornea was measured as the optical density at 490 nm by spectrophotometric analysis.

Results and discussion

In vitro

Irritation parameter:
cornea opacity score
Run / experiment:
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Other effects / acceptance of results:
The corrected mean optical density (permeability) score was 1.492.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
The in vitro score was calculated as 125.08 and is classified as a severe irritant. (Southee, 1998).