ACETIC ACID, 2-(3-CHLORO-5-CYANOPHENOXY)-, ETHYL ESTER

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th September 2013 - 12th November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

ethyl 2-(3-chloro-5-cyanophenoxy)acetate

EC Number:

815-596-5

Cas Number:

1613307-27-9

Molecular formula:

C11H11CINO3

IUPAC Name:

ethyl 2-(3-chloro-5-cyanophenoxy)acetate

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine corneas were mounted in special holders and exposed to the test articles.
Bovine eyes were obtained from Spear Products and transported to MB Research in
Hank's Balanced Salt Solution (HBSS) 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 HBSS.

Test system

Vehicle:

other: Minimum Essential Media (MEM)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.75 ml of a 20% suspension of L-005183690-000F003.
2 g of test article were brought to a total volume of 10 ml with MEM and mixed prior to dosing (Off-white particles suspended in clear liquid).

Duration of treatment / exposure:

Five corneas were incubated in a horizontal position at 32°C for approximately 4 hours. Two corneas were incubated in the control at 32ºC.

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

The holders and corneas were then placed in the 32°C incubator for four hours in a horizontal position to insure contact of the test article with the corneas.

Number of animals or in vitro replicates:

Five corneas for the test article and two corneas for the control.

Details on study design:

Preparation of 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. 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 insure there were no defects. The entire holder with the cornea was then placed in a 32°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. 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).

Test Item Preparation:
2 g of test article were brought to a total volume of 10 ml with MEM and mixed prior to dosing (Off-white particles suspended in clear liquid).

Treatment of Corneas and Opacity Measurements:
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°C incubator in a horizontal position to insure contact of the test article with the corneas. After four hours, 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:
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.

Permeability Determinations and application of sodium fluorescein:
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°C incubator in a horizontal position insuring 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

Results 1

Irritation parameter:

cornea opacity score

Value:

-1.6

Results 2

Irritation parameter:

in vitro irritation score

Value:

-0.9

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro score was calculated as -0.90 and is classified as a non-irritant (Southee. 1998).

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. In Vitro classification based on Southee JA, 1998. Evaluation of the Prevalidation Process, Part 2, final report, Volume 2, The Bovine Corneal Opacity and Permeability (BCOP) Assay. European Community Contract No. 11279-95-10F lED ISP GB and included an analysis based on OECD Guideline for the Testing of Chemicals #437, adopted September 7,2009.

Method Synopsis: Five corneas were dosed with 0.75 ml of a 20% suspension of L-005183690-000F003. Opacity measurements and sodium fluorescein permeability were determined.

Summary: The corrected mean opacity score was -1.6. The corrected mean optical density (permeability) score was 0.047.

Conclusion: The in vitro score was calculated as -0.90 and is classifled as a non-irritant (Southee, 1998).