[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23rd January 2012 to 28th February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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

Guideline:

other: Bovine Corneal Opacity and Permeability Test: An In-Vitro Assay of Ocular Irritancy, (1992); Gautheron, Pierre; Dukic, Martine; Alix, Danielle and Sina, Joseph F.; Fundamental and Applied Toxicology 18, 442-449.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

{1-(ETHYLSULFONYL)-3-[4-(4,4,5,5-TETRAMETHYL-1,3,2-DIOXABOROLAN-2-YL)-1H-PYRAZOL-1-YL]AZETIDIN-3-YL}ACETONITRILE

Cas Number:

191987-50-5

Molecular formula:

C16H25BN4O4S

IUPAC Name:

{1-(ETHYLSULFONYL)-3-[4-(4,4,5,5-TETRAMETHYL-1,3,2-DIOXABOROLAN-2-YL)-1H-PYRAZOL-1-YL]AZETIDIN-3-YL}ACETONITRILE

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes were received on 25 Jan 2012 and transported in Hank’s Balanced Salt Solution in a refrigerated container.
The eyes were examined within one hour after receipt. 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 Hank’s Balanced Salt Solution (HBSS).

Test system

Vehicle:

other: Minimum Essential Media (MEM) solution

Controls:

yes, concurrent no treatment

Amount / concentration applied:

2 g of test article was mixed with MEM to 10 ml. (Tan suspension).
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

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

5

Details on study design:

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°C (± 2 oC) incubator and allowed to equilibrate for at least one hour, but not longer than 2 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 that 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).
Following the pretest observations, the MEM solution was removed from the anterior chamber and 0.75ml 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 32oC (± 2 oC) incubator in a horizontal position to ensure contact of the test article with the corneas. After four hours, the test substance (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 with each treated cornea compared to each of the two control corneas. Opacity measurement of the cornea was made using an OP-KIT opacitometer. 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% solution of sodium fluorescein in Dulbecco's Phosphate Buffered Saline (DPBS). Each holder was then returned to the 32oC (± 2 oC) 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

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 5 replicates

Value:

-0.01

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The corrected mean opacity score was -0.3 and the corrected mean optical density (permeability) score was 0.019. The in vitro score was calculated as -0.01, therefore Compound 3030577 is considered to be a non-irritant.

Executive summary:

Objective: To determine the potential for ocular irritation using an alternative to the Draize methodology. This protocol is based on the methodology described in Bovine Corneal Opacity and Permeability Test: An In-Vitro Assay of Ocular Irritancy, (1992); Gautheron, Pierre; Dukic, Martine; Alix, Danielle and Sina, Joseph F.; Fundamental and Applied Toxicology 18, 442-449.

Method Synopsis: Five corneas were dosed with 0.75 ml of a 20% suspension of Compound 3030577, (Tan suspension). Opacity measurements and sodium fluorescein permeability were determined.

Summary: The corrected mean opacity score was -0.3. The corrected mean optical density (permeability) score was 0.019.

Conclusion: The corrected mean opacity score was -0.3 and the corrected mean optical density (permeability) score was 0.019. The in vitro score was calculated as -0.01, therefore Compound 3030577 is considered to be a non-irritant.