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Eye irritation

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eye irritation
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
other: OECD Guideline for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September, 2009).
GLP compliance:
yes (incl. certificate)
signed, 2009-03-30

Test material

Details on test material:
- Name of test material (as cited in study report): Cobalt (II) oxide
- Storage condition of test material: At room temperature

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Freshly isolated bovine eyes of at least 9 month old donor cattle will be collected from the abattoir. Excess tissue will be removed from the excised eyes. The isolated eyes will be transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae will be isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran will be added to the medium.

Test system

physiological saline
yes, concurrent vehicle
Amount / concentration applied:
- Concentration (in vehicle): 20%
- Amount(s) applied:0.75mL
Duration of treatment / exposure:
240min (+-5min)
Observation period (in vivo):
measurement for opacity directly after incubation
Number of animals or in vitro replicates:
Number of Corneae: 3
Details on study design:
Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item or negative or positive control at a volume of 0.75 mL each on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath in a horizontal position.
The test item was tested as a 20% suspension (w/v) in saline. The positive control was 10% (w/v) Benzalkonium chloride. Saline was used as negative control item.
The incubation time lasted 240 minutes (± 5 minutes).
After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added in both compartments and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. Fresh complete medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5% (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

Results and discussion

In vivo

Irritation parameter:
other: in vitro score
Time point:
other: 240 min

Any other information on results incl. tables

The following formula was used to determine the in vitro irritation score of the negative control:

In vitro Irritation Score = opacity value + (15 x OD490value)

The following formula was used to determine the in vitro irritation score of the positive control and the test item:

In vitro Irritation Score = (opacity value – opacity valuemean negative control) + (15 x corrected OD490value)

The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro score irritation value of each treated group was calculated from the individual in vitro irritation score values. Depending on the score obtained, the test item was classified into one of the following categories:

In vitro Irritation Score

Proposed in vitro Irritation Scale (according to the INVITTOX (UK) protocol no. 98)

0 - 3

Non eye irritant

3.1 – 25

Mild eye irritant

25.1 – 55

Moderate eye irritant


Severe eye irritant

CLP/EPA/GHS (class I)

Applicant's summary and conclusion

Interpretation of results:
other: not severely eye irritating
Criteria used for interpretation of results: EU
The test item cobalt oxide had a calculated mean in vitro score of 7.15 and therefore, the test item was classified as mild eye irritant. No classification according to regulation (EC) 1272/2008 or directive 67/548/EC required.