Reaction mass of cobalt and copper and iron

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-04-14 to 2011-04-14

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 1152/2010: B. 47. Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (Official Journal of the European Union, L 324, 9.12.2010).

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed according to the INVITTOX (UK) protocol no. 98 “The Bovine Corneal Opacity and Permeability Assay”, 1996.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed, 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt

EC Number:

231-158-0

EC Name:

Cobalt

Cas Number:

7440-48-4

Molecular formula:

Co

IUPAC Name:

cobalt(2+)

Details on test material:

- Name of test material (as cited in study report): Cobalt Powder
- Storage: In a closed container at room temperature
- Median Particle Diameter (D50): 2.3 µm

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:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL/VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item

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 suspended in saline (20% (w/v)). 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 andopacity 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 vitro

Results

Irritation parameter:

in vitro irritation score

Value:

1.79

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 1.22).
- The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean in vitro score 250.64) corresponding to a classification as corrosive to the eye (CLP/EPA/GHS (class I)).
- Relative to the negative control, the test item cobalt powder did not cause any increase of the corneal opacity or permeability. The calculated mean in vitro score was 1.79. According to the INVITTOX (UK) protocol no. 98 the test item is classified as non eye irritant.

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 value_{mean 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
≥55.1	Severe eye irritant CLP/EPA/GHS (class I)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item cobalt powder is not corrosive to the eye. Corresponding to the INVITTOX (UK) protocol no. 98 classification cobalt powder is non eye irritant.
No classification according to regulation (EC) 1272/2008 required.