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Toxicological information

Eye irritation

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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:
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:
other: OECD 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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.