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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-06-09 to 2011-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant 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 (September, 2009).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2009-03-30

Test material

Constituent 1
Reference substance name:
Neodecanoic acid, cobalt salt
EC Number:
248-373-0
EC Name:
Neodecanoic acid, cobalt salt
Cas Number:
27253-31-2
IUPAC Name:
Neodecanoic acid, cobalt salt
Details on test material:
- Name of test material (as cited in study report): Cobalt neodecanoate
- Storage: At room temperature

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were 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 was added to the medium.

Test system

Vehicle:
other: saline and unchanged
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
Prior to the application the test item was tried to suspend in saline (20% (w/v)) by stirring heavily and additionally treating in an ultrasonic bath. Since the test item could not be suspended sufficiently, 0.75 mL of the insufficient suspension was placed directly onto the corneae. Additionally, the stamp of a syringe was dipped into the test item. A part of the amount sticking on the stamp was applied carefully to the corneae in a manner, that the corneae were covered by the test item evenly.
Duration of treatment / exposure:
240 minutes (± 5 minutes)
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.

Prior to the application the test item was tried to suspend in saline (20% (w/v)) by stirring heavily and additionally treating in an ultrasonic bath. Since the test item could not be suspended sufficiently, 0.75 mL of the insufficient suspension was placed directly onto the corneae. Additionally, the stamp of a syringe was dipped into the test item. A part of the amount sticking on the stamp was applied carefully to the corneae in a manner, that the corneae were covered by the test item evenly. The positive control was 10% (w/v) Benzalkonium chloride in saline. 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, 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 vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
cornea 1
Run / experiment:
240 min
Value:
-1.02
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
cornea 2
Run / experiment:
240 min
Value:
-0.78
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
cornea 3
Run / experiment:
240 min
Value:
-6.99
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- With the negative control neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.86).
- The positive control showed clear opacity of the corneae (mean in vitro irritation score 305.30) corresponding to a classification as corrosive to the eye (CLP/EPA/GHS (class I)).
- Relative to the negative control, the test item cobalt neodecanoate did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritation score was 0.00 and therefore the test item was classified as non eye irritant.

Any other information on results incl. tables

In vitro irritation score calculation:

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

0 - 3

Non eye irritant

3.1 – 25

Mild eye irritant

25.1 – 55

Moderate eye irritant

 ≥ 55.1

Severe eye irritant

EU (R41) or EPA/GHS

(Category 1, H318)

Applicant's summary and conclusion

Interpretation of results:
other: not severely eye irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item cobalt neodecanoate is not corrosive to the eye according to regulation (EC) No.: 1272/2008 and directive 67/548/EC.