Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 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:
- Name of test substance (as cited in study report): Cyanethylimidazol
- Analytical purity: > 70% - < 80% (18% - 28% Water)
- Impurities: Acrylnitril > 2% - < 4%; Imidazole 3%
- Test substance No.: 11/0432-1
- Batch No.: 000STD77L0 of C1310
- Homogeneity: the tets substance was homogenous by visual inspection.
- Storage stability: the stability under storage conditions over the study period was guaranteed
- pH-value: ca. 8 (undiluted test substance)

Test animals / tissue source

Species:
other: in vitro test on isolated bovine cornea
Strain:
other: not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
750 µL of the undiluted test substance
Duration of treatment / exposure:
10 min followed by a 2-hours post-incubation period
Observation period (in vivo):
not applicable (in vitro test)
Number of animals or in vitro replicates:
not applicable (in vitro test); each treatment group consisted of 3 corneas
Details on study design:
The test system (target system) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consist of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles's MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any cornea that showed macroscopic tissue damage or an opacity value < 566 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups.
Before application the medium in the anterior chamber was removed using a syringe. 750 µL of the undiluted liquid test substance was applied into the anterior chamber using a pipette. Control tissues concurrently received 750 µL of highly de-ionized water (NC) or 750 µL of 1% (w/v) solution of sodium hydroxide solution in highly de-ionized water (positive control; PC) in the anterior chamber using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approx. 10 min. The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red). Both chambers were then refilled with fresh Eagle's MEM (without phenol red).
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle's MEM. Final corneal opacity readings were taken for each cornea with an opacitometer. For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
1

In vivo

Irritant / corrosive response data:
IVIS NC: 0.7
IVIS PS: 61.5

Any other information on results incl. tables

Table 1: Opacity score

Test substance

Cornea No.

Initial Opacity

Final Opacity

Opacity Change

Corrected Opacity Change

Mean

SD

Test substance

13

5.9

6.7

0.9

0.1

0.6

0.6

14

1.1

3.2

2.1

1.3

15

0.8

2.1

1.3

0.5

NC

1

4.8

5.7

0.9

NA

0.8

0.3

2

2.5

3.5

1.0

NA

3

-0.3

0.2

0.5

NA

PC

4

3.3

22.2

18.9

18.1

17.7

3.0

5

3.9

19.2

15.3

14.5

6

3.4

24.7

21.2

20.5

 

Table 2: Permeability score

Test substance

Cornea No.

Mean OD490

Dilution Factor

Mean Corrected OD490

Mean

SD

Test substance

13

0.030

1

0.033

0.027

0.008

14

0.016

1

0.018

15

0.029

1

0.031

NC

1

-0.006

1

NA

-0.002

0.003

2

-0.001

1

NA

3

0.000

1

NA

PC

4

0.479

5

2.397

2.923

0.846

5

0.494

5

2.472

6

0.779

5

3.899

 

Table 3: In Vitro Irritancy Score (IVIS)

Test substance

Cornea No.

Opacity per cornea

Permeability per cornea

IVIS

Per cornea

Per group

mean

SD

Test substance

13

0.1

0.033

0.6

1.0

0.5

14

1.3

0.018

1.6

15

0.5

0.031

0.9

NC

1

0.9

-0.006

0.8

0.7

0.3

2

1.0

-0.001

1.0

3

0.5

0.000

0.4

PC

4

18.1

2.397

54.1

61.5

15.1

5

14.5

2.472

51.6

6

20.5

3.899

78.9

NC = negative control

PC = positive control

 

In this study a 0.4% instead of a 1% solution of sodium hydroxide in highly de-ionized water was used as positive control. However, the results are in line with previously obtained results for 0.4% NaOH (mean IVIS = 64.1; SD = 16.4). Thus the 0.4% NaOH is considered to be sufficient to demonstrate the sensitivity of the test.

Applicant's summary and conclusion

Interpretation of results:
other: no serious eye damage