(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental dates: 16 February 2011 to 17 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 437 guidelines

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

EC Number:

700-729-0

Cas Number:

1369500-14-0

Molecular formula:

C12H12Cl2O2

IUPAC Name:

(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

Details on test material:

- Molecular formula (if other than submission substance): C12H12Cl2O2
- Molecular weight (if other than submission substance): 259.1
- Physical state: Brown powder
- Storage condition of test material: <30°C

Test animals / tissue source

Species:

other: Bovine corneas

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

No test animals were used.
Bovine corneas supplied by A Traves and Son Ltd., Abattoirs, York, United Kingdom. Following slaughter the eyes were removed, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility. Upon arrival at the test facility the corneas were excised from the eyes and loaded onto specifically designed holders, filled with pre-warmed Eagle's Minimal Essential Medium (EMEM) and incubated at 32°C for 1 hour.

Test system

Vehicle:

other: No vehicle used

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg
- Concentration (if solution): undiluted test material

Duration of treatment / exposure:

4 hour incubation at 32°C
Further 1.5 hours at 32°C to achieve the permeability end point

Number of animals or in vitro replicates:

No animals were used in this test.
Three corneas were used per test substance, positive and negative control

Details on study design:

APPLICATION OF TEST SUBSTANCE AND CONTROL MATERIALS:
The test article did not produce a suitable suspension or solution at a concentration of 20% w/v in 0.9% sodium chloride solution, therefore the solid test article was used as supplied. 750 mg of test article was applied to the anterior surface of each of three corneas followed by a four hour incubation at 32°C ± 1°C.
A volume of 750 µL of the negative (0.9% sodium chloride) or positive control (20% w/v Imidazole) was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.
REMOVAL OF TEST SUBSTANCE
- Washing: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red, the opacities measured and the anterior chamber emptied
- Time after start of exposure: 4 hours

SCORING SYSTEM:
In vitro Irritation Score (IVIS)

TOOL USED TO ASSESS SCORE:
For the permeability endpoint, 1 mL of sodium fluorescein (5 mg/mL solution) was added into the anterior chamber and the corneas were incubated at 32°C ± 1°C for 1.5 hours ± 5 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were transferred to a 96 well plate and analysed for optical density at 490 nanometers (OD490).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

16.37

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The In Vitro Irritation Score (IVIS) for the test article is calculated as follows:
IVIS = mean opacity value + (15 x mean permeability value)
The test article will be concluded as a corrosive or severe irritant if it causes damage to the cornea such that the IVIS is >55.1.

The following mean in vitro irritation scores (IVIS) were calculated. The test article produced an IVIS of 16.37 and the positive control article produced an IVIS of 84.89 (within two Standard Deviations of the historical mean).
These results indicate that the IVIS for the test article was not ≥55.1 and therefore the test article may be considered not to be a corrosive or severe irritant, in compliance with the test guideline. The positive control showed an appropriate response in the assay system. The assay acceptance criteria were achieved. No other test substance related effects were observed.

Any other information on results incl. tables

Corneal Opacity

Test chemical	Cornea number	Initial opacity reading	Post incubation opacity	Change in opacity	Average change in opacity (negative control)	Corrected opacity	Mean opacity
Negative control	4	2	4	2	2.00	0.0	0.0
	1	2	4	2		0.0
	22	2	4	2		0.0
Positive control	20	3	73	70	N/A	68.0	60.0
	6	1	59	58		56.0
	10	1	59	58		56.0
Test article	24	3	20	17	N/A	15.0	16.0
	21	1	18	17		15.0
	11	2	22	20		18.0

Permeability

Test chemical	Cornea number		OD490reading	Corrected OD490	Mean negative control corrected OD490	Final corrected OD490	Average group corrected OD490
Negative control		4	0.050	0.005	0.005	-0.001	0.000
		1	0.049	0.003		-0.002
		22	0.053	0.008		0.002
Positive control		20	1.212	1.166	N/A	1.161	1.659
		6	1.966	1.921		1.916
		10	1.952	1.907		1.901
Test article		24	0.081	0.036	N/A	0.030	0.025
		21	0.073	0.028		0.022
		11	0.072	0.026		0.021
Mean blank			0.046

Calculated IVIS

Test chemical	Mean opacity	Mean permeability x 15	IVIS (mean opacity + (15 x mean permeability))
Negative control	0	0	0
Positive control	60	24.89	84.89
Test article	16	0.37	16.37

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive or a severe irritant

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

The test subtance was considered not to cause corrosion or severe irritation to the eye under the conditions of the assay.

Executive summary:

The result indicates that the substance should be non-corrosive to the eye and not a severe eye irritant. Without furtherin vivo data the result has to be considered as inconclusive regarding classification.