2-Cyclopentene-1-acetic acid, 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:

2012-01-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2009-09-07

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

, 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, procedure Details, April 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

EC Number:

605-150-6

Cas Number:

15848-49-4

Molecular formula:

C9H14O2

IUPAC Name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Molecular formula: C9H14O2
- Molecular weight: 154.21 g/mol

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item was applied

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

PREPARATION OF THE INCUBATION MEDIA
The medium MEM (Minimum Essential Medium), supplemented with sodium bicarbonate and L-glutamine was prepared one day prior to the start of the assay, and stored in a refrigerator (2 - 8 °C).
Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS). Afterwards, it was called cMEM (complete medium).
The OECD foresees the use of EMEM (Eagle’s minimal essential medium) which is in composition and osmolarity equivalent to the cMEM, thus cMEM could be used without restriction.

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneas were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

OUTLINE OF STUDY
The anterior compartment received the test item or negative control (saline (produced in-house, lot no. 20.12.11))) or positive control (2-Ethoxyethanol (Sigma, 82024 Taufkirchen, Germany), lot no. BCBD1053V) at a volume of 0.75 mL on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath, while the corneae were in a horizontal position. The test item, positive control and negatice control were tested in triplicate.
The test item was tested undiluted. The positive control 2-Ethoxyethanol was tested neat.
The incubation time lasted ten minutes (± 30 seconds).
After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. The corneae were then incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a 2nd opacity reading (t130).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

OPACITY MEASUREMENT
The basal opacity of all corneae was recorded using an opacitometer. Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test items and the negative and positive controls. After the incubation time of 2 hours with the test item, positive control and negative control the opacity was measured again (t130).

PERMEABILITY DETERMINATION
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the in vitro irritation score of the positive control fell within two standard deviations of the current historical mean, which is updated every three months and if the negative results were below the upper limits for background results.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro score

Basis:

mean

Time point:

other: 10 minutes

Score:

2.6

Irritant / corrosive response data:

Relative to the negative control, the test item caused a very slight increase of the corneal opacity. Permeability effects did not occur. The calculated mean in vitro irritation score was 2.60. According to OECD 437 the test item is classified as not corrosive/not severe irritant to the eye.

Any other information on results incl. tables

Table 1: Results after 10 minutes incubation time

Test group	Opacity value = Difference (t130 – t0) of opacity		Permeability at 490 nm (OD490)		In vitro score	Mean in vitro irritation score	Proposed in vitro irritation scale
		Mean		Mean
Negative control	0	-0.33	0.055	0.054	0.83	0.47	Non corrosive / non severe irritant
	0		0.058		0.87
	-1		0.048		-0.28
Positive control	54.33*		0.306*		58.93	65.41	Corrosive / severe irritant
	60.33*		0.389*		66.17
	66.33*		0.320*		71.14
Test item	2.33*		-0.001*		2.32	2.60	Non corrosive / non severe irritant
	2.33*		-0.010*		2.19
	3.33*		-0.003*		3.29

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 0.47)

-The positive control (2 -Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean in vitro irritation score 65.41) corresponding to a classification as corrosive/severe irritant to the eye (CLP/EPA/GHS (cat 1)).

Table 2: Historical data

	Positive control	Negative control
Mean in vitro Irritation Score	74.26	1.02
Standard Deviation	21.53	0.92

Values of 126 studies with liquid test items performed in 2007 to December 2011

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive/not severe irritant to the eye

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not corrosive to the eye (CLP/EPA/GHS (Cat 1)).