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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:
7 - 8 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
amended 9 Dec 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyl ascorbic acid
- Storage condition of test material: in the freezer (≤ -15 °C)
- Substance type: white to very pale yellow, crystalline, powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Characteristics of donor animals: Bovine eyes from young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v)

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in physiological saline

NEGATIVE CONTROL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
240 ± 10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
in triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the initial opacity was determined. Each cornea with a value of the initial opacity >7 was discarded.

TREATMENT METHOD:
The isolated corneas were mounted in a corneal holder (BASF, Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for a minimum of 1 h at 32 ± 1 °C. At the end of the equilibration period, the medium was removed from the anterior compartment and 0.75 ml of either test substance or control was applied to the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany)
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite M200 Pro Plate Reader) at 490 nm
- Others: After removal of the test substance, possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
13
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: After the incubation period with the test substance the corneas were clear with a spot. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed.

Any other information on results incl. tables

Table 2. Results after 240 min incubation time.

Test group

Opacity value

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

Mean

 

Mean

 

 

Negative

control

-0.7

-0.9

-0.001

0.000

-0.7

-0.9

0.6

-0.003

0.6

-2.7

0.004

-2.6

Positive

control

107.8

0.907

121.4

113.4

81.1

1.851

108.9

87.5

1.501

110.0

Test substance

11.8

0.59

20.7

13.0

1.0

0.219

4.3

5.1

0.586

13.9

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive
Conclusions:
The irritation potential of the test substance was assessed in the BCOP assay. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 13.0. According to OECD Guideline 437 no prediction on the irritation potential can be made but the test substance does not have to be classified as serious eye irritant (Eye Dam. Cat. 1)