Naphthenic acids, cobalt salts

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: validated "in vitro" test method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-08-10 to 2010-08-10

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:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals 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:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids, cobalt salts

EC Number:

263-064-0

EC Name:

Naphthenic acids, cobalt salts

Cas Number:

61789-51-3

IUPAC Name:

λ²-cobalt(2+) bis(3-(3-ethylcyclopentyl)propanoate)

Details on test material:

- Name of test material (as cited in study report): Cobalt naphthenate
- Physical state: blue, solid
- 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 from at least nine month old donor cattle were collected from the abattoir. The donor cows were at least at the age of 9 month. 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:

- Since the test item was of coarse-grained consistency, it was crushed and ground in a mortar with a pestle prior to the test.
Nevertheless, a 20 % (w/v) homogeneous suspension or solution in saline could not be produced. Therefore, the grained suspension in saline (0.9 (w/v) NaCl in deionsied water) was spread equally onto the corneae as demanded by the OECD guideline 437 for these cases.
- Amounts applied: 0.75 mL

Duration of treatment / exposure:

- test item: 120 minutes (± 3 minutes)
- controls: 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.

Due to its coarse-grained consistency, the test item was crushed and ground in a mortar with a pestle prior to the test. Since a 20 % (w/v) homogeneous suspension or solution in saline could not be produced. The suspension was grained. According to the OECD guideline 437 it was spread equally onto the corneae. The positive control was 10% (w/v) Benzalconium chloride. Saline was used as negative control item.

The incubation time lasted 120 minutes (± 3 minutes) for the test item and 240 minutes (± 5 minutes) for the controls.

After the test item or control items, respectively, were rinsed off from the application side with saline at least three times or until no visual evidence of the test item was observed. Fresh cMEM was replaced in both compartments and opacity was measured (t240). Also, each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test substance, non-uniform opacity pattern).

In the second step of the assay, permeability of the cornea possibly caused by the test item, 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

Results

Results 1

Irritation parameter:

other: in vitro score

Basis:

other: cornea 1

Time point:

other: 120 min

Score:

1.14

Results 2

Irritation parameter:

other: in vitro score

Basis:

other: cornea 2

Time point:

other: 120 min

Score:

1.11

Results 3

Irritation parameter:

other: in vitro score

Basis:

other: cornea 3

Time point:

other: 120 min

Score:

1.07

Results 4

Irritation parameter:

other: in vitro score

Basis:

mean

Time point:

other: 120 min

Score:

1.11

Irritant / corrosive response data:

- With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro score was calculated as 0.76.
- The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The mean in vitro score was calculated as 163.40.
- The test item cobalt naphthenate did not cause any permeability or opacity of the corneae compared with the results of the negative control. The calculated mean in vitro score was 1.11 and therefore, the test item was classified as non eye irritant.

Any other information on results incl. tables

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

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

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

In vitro Score = (opacity value – opacity value_{mean negative control}) + (15 x corrected OD₄₉₀value)

The in vitro score was calculated for each individual treatment and positive control cornea. The mean in vitro score value of each treated group was calculated from the individual in vitro 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 naphthenate is not corrosive to the eye according to regulation (EC) No.: 1272/2008 and directive 67/548/EC.