Methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 November 2010 - 9 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance an OECD guideline by a GLP accredited laboratory. A protocol for the test substance has been developed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 437

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OTWG, ICCVAM and NICEATM, Background Review Document: current status of in vitro test methods for identifying ocular corrosives and severe irritants: BCOP test method, March 2006

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: In vitro Techniques in Toxicology Database (NINVITTOX) protocol 124, Bovine Opacity and Permability Assay-SOP of Microbiological Associates Ltd 1999.

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: Gautheron P. et al., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy, Fundam. Appl. Toxicol. 18:442-449, 1992

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

EC Number:

700-527-2

Cas Number:

1271488-66-4

Molecular formula:

C12H20O3

IUPAC Name:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle (Vitelco, 's-Hertogenbosch, NL) obtained byexcission and used as soon as possible after slaughter. Eyes were collected and transported in physiological saline.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

Amount / concentration applied:

750 µl of the test substance or control was put onto the epithelium of the cornea. Holders were slightly rotated with the cornea maintained in horizontal position.

Duration of treatment / exposure:

The incubation period was 10±1 min at 32±1°C.

Number of animals or in vitro replicates:

9 eyes

Details on study design:

Randomly selected corneas were selected and their opacities measured. If the opacity was >3, the cornea was not used for the test. Corneas were mounted in a MC2 (Clermont, France) corneal holder. The compartments of the corneal holder were filled with cMEM. The corneas were incubated for the minimum of 1 hour at 32+/-1oC. 0.750 ml of the control or test substance was put onto the epithelium of the cornea. After 10 minutes, the cornea was washed with MEM with phenol red (to assess pH effects) and cMEM containing phenol red to evaluate possible pH effects. The anterior and posterior compartments of the cornea holder was filled with fresh cMEM and the corneas were incubated for 120+/-10 minutes at 32+/-1oC. After incubation, the opacity was determined and each cornea was inspected visually for dissimilar opacity patterns.

Opacity measurements were based on light transmission across the cornea. The change in opacity is the difference in transmission between pre and post treatment reading. The corrected opacity is calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of the cornea treated with test material, positive control or solvent control. The mean opacity value is the average of the corrected opacity values of the with test material treated corneas.

In the permeability test, the anterior compartment of the cornea holder was filled with 1 ml of 4 mg sodium-fluorescein/ml cMEM solution and the posterior with cMEM only. Corneas were incubated for a period of 90+/-5 minutes at 32+/-1oC. The optical density (OD) of the medium in the posterior compartment was measured in triplicate using a microplate reader (Multiskan spectrum, Thermo labsystems) at a wavelength of 490 nm.

The irritation potential was assessed on the individual mean opacity and mean permeability values. An in vitro irritancy score was based on the mean opacity and mean permeability values according to the following equation:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

An IVIS > 55.1 means that the test substance should be regarded as a corrosive or severe irritant.

The study is acceptable if IVIS is less than two standard deviations from the historical mean and if the opacity and permeability values of the negative control is less than the upper limit of the historical range (acceptability criteria).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Opacity

Basis:

mean

Time point:

other: 2h.

Score:

0

Results 2

Irritation parameter:

other: Permeability

Basis:

mean

Time point:

other: 1.5h.

Score:

3.54

Results 3

Irritation parameter:

overall irritation score

Remarks:

In vitro irritancy score (IVIS)

Basis:

mean

Score:

0

Remarks on result:

other: If IVIS >= 55.1, then test substance is classified as corrosive or severe irritant.

Irritant / corrosive response data:

TABLE I presents the individual opacity scores, in which the mean responses of the negative control is in compliance with the acceptability criteria. The individual permeability scores uncorrected and corrected for the negative control can be found in TABLE II and III. The mean final negative control is within the historical control range. TABLE IV shows the in vitro irritancy scores (IVIS) that takes both the opacity and permeability scores into account. The positive control benzalkonium chloride solution had a mean in vitro irritancy score within the historical range.

Any other information on results incl. tables

TABLE I Individual opacity scores

Eye	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected final opacity	Mean
negative control
1	0	0	0	0
2	0	0	0	0	0
3	0	0	0	0
positive control
1	0	81	81	81
2	0	76	76	76	80
3	0	83	83	83
Test substance
1	0	0	0	0
2	0	0	0	0	0
3	1	0	-1	-1
^1Final opacity = opacity after treatment - opacity before treatment

TABLE II Individual permaebility scores (uncorrected)

Eye	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD
negative control
1	1	0.011	0.010	0.013	0.011
2	1	0.009	0.012	0.006	0.009
3	1	0.011	0.006	0.005	0.007
positive control
1	6	0.653	0.639	0.631	0.641	3.846
2	6	0.523	0.530	0.517	0.523	3.138
3	6	0.640	0.632	0.626	0.633	3.798
Test substance
1	1	0.010	0.005	0.005	0.007	0.007
2	1	0.014	0.010	0.007	0.010	0.010
3	1	0.009	0.007	0.007	0.008	0.008
^1Final OD = OD x dilution factor
Mean final negative control is 0.009

TABLE III Individual permaebility scores (corrected for the mean final negative control)

Eye	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD^1
negative control
1	1	0.002	0.001	0.004	0.002
2	1	0.000	0.003	-0.003	0.000
3	1	0.002	-0.003	-0.004	-0.002
positive control
1	6	0.644	0.630	0.622	0.632	3.792
2	6	0.514	0.521	0.508	0.514	3.084
3	6	0.631	0.623	0.617	0.624	3.744
Test substance
1	1	0.001	-0.004	-0.004	-0.002	-0.002
2	1	0.005	0.001	-0.002	0.001	0.001
3	1	0.000	-0.002	-0.002	-0.001	-0.001
^1Final OD = OD x dilution factor
Mean final negative control is 0.009

TABLE IV In vitro irritancy score (IVIS)

Eye	Negative control corrected final opacity	Negative control corrected final OD490	IVIS
negative control
1	0	0.002	0.0
2	0	0.000	0.0
3	0	-0.002	0.0
mean	0	0.000	0.0
positive control
1	81	3.792	137.9
2	76	3.084	122.3
3	80	3.744	139.2
mean	80	3.540	133
Test substance
1	0	-0.002	0.0
2	0	0.001	0.0
3	-1	-0.001	0.0
mean	0	-0.001	0

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

The test solution did not induce ocular irritation through both end-points. The mean in vitro irritancy score was 0 after 10 minutes of exposure.

Executive summary:

Eye irritancy of the test substance was screened by the Bovine Corneal Opacity and Permability (BCOP) test. The study included a negative (physiological saline) and a positive control (10% (w/v) benzalkonium chloride in physiological saline). The test substance or controls were topically applied and exposure to the test substance was 10 minutes. The opacity was determined spectrophotometrically at 490nm. The test substance did not have any effect on the opacity. No pH effects were observed as seen by rinsing the eye with a solution containing phenol red. In the permeability test with sodium-fluorescein, no permeability was observed.

The in vitro irritancy score (IVIS) was based on both the opacity and permeability results. The IVIS was also 0 and hence the test substance does not induce ocular irritation and should not be regarded as severe irritant for or corrosive to the eye.