2-[1-(ethylsulfonyl)azetidin-3-ylidene]acetonitrile

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

Version / remarks:

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

Deviations:

no

Principles of method if other than guideline:

Study based on the original reference

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[1-(ethylsulfonyl)azetidin-3-ylidene]acetonitrile

EC Number:

691-838-1

Cas Number:

1187595-85-2

Molecular formula:

C7H10N2O2S

IUPAC Name:

2-[1-(ethylsulfonyl)azetidin-3-ylidene]acetonitrile

Test material form:

solid: particulate/powder

Details on test material:

Lot no: 4500566148-0003

Specific details on test material used for the study:

Purity/Composition: 100%. Off-white powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Spear Products on 25 Jan 2012 and transported to the test facility in Hank’s Balanced Salt Solution in a refrigerated container

Test system

Vehicle:

other: 2 g of test article was mixed with Minimum Essential Media (MEM) to 10 ml (Tan suspension) to give a 20% solution

Controls:

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75ml

VEHICLE
- Amount(s) applied (volume or weight with unit): MEM
- Concentration (if solution): 20%
- Purity: 100%

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 2°C.

Number of animals or in vitro replicates:

Five corneas were selected

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
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).
The dissected corneas were mounted in specially designed holders (Electro-Design Corporation of Riom, France) 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).

QUALITY CHECK OF THE ISOLATED CORNEAS:
The eyes were examined within one hour after receipt. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.

NUMBER OF REPLICATES; 5

NEGATIVE CONTROL USED: MEM medium

SOLVENT CONTROL USED (if applicable): NO

POSITIVE CONTROL USED: NO

APPLICATION DOSE AND EXPOSURE TIME: 20% for 4 hours

TREATMENT METHOD: [closed chamber / open chamber]: Not specified

REMOVAL OF TEST SUBSTANCE
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
produced by Electro-Design Corporation of Riom, France.

- POST-EXPOSURE INCUBATION: 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 32o
C (± 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 pectrophotometric analysis.

METHODS FOR MEASURED ENDPOINTS:
The Corrected Mean Opacity Score was calculated using the control and treated cornea opacity values as determined from the OP-KIT. The corrected Mean Optical Density Score was calculated using the control and treated Optical Density values from the fluorescein permeability analysis. The in vitro score was calculated as follows:
In Vitro Score = Corrected Mean Opacity Score + 15 (Corrected Mean Optical Density Score)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

ca. 2.46

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

ca. 1.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

ca. 0.084

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since 3017431 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage. The corrected mean opacity score was 1.2 and the corrected mean optical density (permeability) score was 0.084. The in vitro score was calculated as 2.46, therefore Compound 3017431 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 3017431, (Tan suspension). Opacity measurements and sodium fluorescein permeability were determined.

Summary: The corrected mean opacity score was 1.2. The corrected mean optical density (permeability) score was 0.084.