Naphthenic acids, cobalt salts

Endpoint summary

Description of key information

Skin irritation: not irritating to the skin
Eye irritation: not irritating to the eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-15 to 2010-04-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable wihtout restrictions

Qualifier:

according to guideline

Guideline:

other: OECD 431: In vitro skin corrosion: Human Skin Model Test (Original guideline adopted 2004-04-13

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

water

Amount / concentration applied:

The test material was not crushed or ground in a mortar and a pestle since this did not improve the consistency.
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): About 25 mg of the test material were applied to the tissues and wetted with 25 μL deionised water. The test item was spread to cover the surface of the tissue.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

Exposure periods of 3 and 60 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE:
EST-1000™ kits (Lot No.: EST 100329-001) were purchased from CellSystems® Biotechnologievertrieb GmbH (53562 St. Katharinen; Germany). The EST-1000™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EST-1000™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅).
EST-1000™ kits were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt EST-1000™ tissues were kept in the refrigerator at 2 - 8 °C in the refrigerator.

The Human Skin Model supplier ensured and demonstrated that each batch of the Human Skin Model used met defined production release criteria, e.g. viability, barrier function, no bacterial and mycoplasma contamination and morphology.

PRE-WARMING OF EST-1000TM TISSUES:
At least one hour before dosing the EST-1000™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed maintenance medium. Two 24-well plates were prepared as holding plates, each well containing 300 μL maintenance medium per well. The holding plates were pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.

EXPOSURE:
Duplicate EST-1000™ tissues were exposed to the test item, positive control (potassium hydroxide as 8.0 N ready-made solution (Cat. No. 17-8, Sigma 82024 Taufkirchen, Lot No.096K6091); Volume 50 µL) or negative control (deionised water (Lot no. 23.3.10, produced in-house); Volume 50 µL) for each of two different exposure periods: 3 minutes and 1 hour.
After the pre-incubation of the EST-1000™ tissues was completed (1 hour 30 minutes for the 1 hour exposure and 2 hours 10 minutes for the 3 minutes exposure) the medium in each well was replaced by 1.0 mL fresh maintenance medium per well. The negative control (50 μL deionised water) was added to the surface of duplicate EST-1000™ tissues. Subsequently, the remaining tissues were exposed to the test item and the positive control in the same manner. The 6-well plates were then placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper.

MTT ASSAY:
After the exposure procedure was completed for all tissues of each time point, the cell culture inserts were transferred from the holding plates to the plates containing 300 µL MTT solution (MTT solution with a final concentration of 1 mg/mL). After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. The inserts were transferred into new 24-well plates. The inserts were immersed in extractant solution by gently pipetting 2 mL of extractant solution (isopropanol) into each insert ensuring that the tissue was completely covered. The 24-well plate was sealed to minimise isopropanol evaporation. The formazan salt was extracted for about 18 hours while shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for approx. 15 minutes until the solution was homogeneous in colour.
3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The optical density (OD) was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue insert.

TEST FOR DIRECT MTT REDUCTION BY TEST ITEM:
To check MTT reducing capability of the test item, a solution of MTT in DMEM (1.0 mg/mL) was prepared and each about 50mg of the test item were added to 1 mL MTT medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumed to have reduced the MTT. No colour change could be observed in the present study. Ones each about 25 mg and a second time each about 50 mg of the test item were added to 1 mL MTT medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumed to have reduced the MTT.

INTERPRETATION OF RESULTS:
The mean OD value obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%.
According to EU CLP Cat.1 and DSD (67/548/EEC):
The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

ACCEPTABILITY OF THE ASSAY:
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability met the acceptance criterion if the mean OD570 of the two tissues in both treatment intervals was ≥ 0.8.
The assay met the acceptance criterion if mean relative tissue viability of the positive control was ≤ 30%.

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

95.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 3 minute treatment. Reversibility: no data. (migrated information)

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

74

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 1 hour treatment. Reversibility: no data. (migrated information)

Irritant / corrosive response data:

After exposure to the test item cobalt naphthenate the relative absorbance values were decreased to 95.6% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 74.0%. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

Results

Results after treatment with cobalt naphthenate

Dose group	Exposure interval	Absorbance 570 nm tissue 1	Absorbance 570 nm tissue 2	Mean absorbance of 2 tissues	Rel. absorbance (% of negative control)
Negative control	3 min	1.266	1.116	1.191	100.0
Positive control	3 min	0.109	0.064	0.086	7.2
Cobalt naphthenate	3 min	1.164	1.113	1.139	95.6
Negative control	1 hour	1.260	1.195	1.227	100.0
Positive control	1 hour	0.009	0.009	0.009	0.7
Cobalt naphthenate	1 hour	0.911	0.906	0.909	74.0

The optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

Historical data:

3 minutes treatment:

Positive control		Negative control
Number of studies	59	Number of studies	59
Period	March 2009 – May 2010	Period	March 2009 – May 2010
Mean viability	12.0 %	Mean OD	1.278
Standard deviation	7.06 %	Standard deviation	0.241
Range of viabilities	33 % - 5 %	Range of ODs	0.9 – 2.0

60 minutes treatment:

Positive control		Negative control
Number of studies	59	Number of studies	59
Period	March 2009 – May 2010	Period	March 2009 – May 2010
Mean viability	2.19 %	Mean OD	1.235
Standard deviation	3.43 %	Standard deviation	0.233
Range of viabilities	16% - 0.5 %	Range of ODs	0.8 – 1.8

Interpretation of results:

other: Test item was not classified as corrosive to the skin.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item Cobalt naphthenate was non corrosive to skin according to Regulation (EC) No.: 1272/2008 and Directive 67/548/EEC.

Reference 2

Endpoint:

skin irritation / corrosion

Remarks:

other: validated "in vitro" test method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-28 to 2010-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 B.46

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original guideline adopted 22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approx. 10 mg of the test item were applied to each of triplicate tissues, spread to match the tissue size, and wetted with 15 µL deionised water.
No further informatrion on the amount/concentration applied was stated.

Duration of treatment / exposure:

15 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE:
EpiSkin™ kit ( Lot No.: 10-EKIN-028) was purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

The RhE (Reconstructed human Epidermis) model supplier ensured and demonstrated that each batch of the RhE model used met defined production release criteria, e.g. viability, barrier function, no bacterial and mycoplasma contamination and histological scoring.

TREATMENT:
The negative control (deionised water (Lot no. 230710, produced in-house); Volume 10 µL) and positive control (5% SLS (Sodium lauryl sulphate, lot no. 1353471 51508322, Fluka, Sigma-Aldrich, 89555 Steinheim, Germany) solution in deionised water, prepared freshly prior to the performance of the experiment; Volume 10 µL), and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues each. The 12-well plates were placed into the incubator for 15 min at 37 ± 1.5 °C, 5 ± 0.5% CO2.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552). The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and can be used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS).
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted about 67 hours in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] X 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.

TEST FOR DIRECT MTT REDUCTION:
It was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 25 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
No colour change could be observed in the present study.
No further information on the study design was stated.

Irritation parameter:

other: relative viability (%)

Basis:

mean

Time point:

other: after 15 min incubation

Score:

76.9

Max. score:

78

Reversibility:

no data

Irritant / corrosive response data:

After treatment with the test item cobalt naphthenate the relative absorbance values decreased to 76.9%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with cobalt naphthenate

 

Dose group	Treat-ment Interval	Absor-bance 570 nm Tissue 1	Absor-bance 570 nm Tissue 2	Absor-bance 570 nm Tissue 3	Mean Absor-bance of 3 Tissues	Absorbance [%] Tissue 1, 2 + 3	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]
Negative Control	15 min	0.924	0.801	0.775	0.834	110.8 96.1 93.0	9.5	100.0
Positive Control	15 min	0.388	0.326	0.280	0.331	46.6 39.1 33.5	6.5	39.7*
Test Item	15 min	0.650	0.650	0.623	0.641	78.0 78.0 74.7	1.9	76.9

*The viability is out of the historical range; nevertheless, since the positive control met the laboratories internal acceptance criteria (viability of ≤40%) and since it showed a positive effect, it can still be considered as valid.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control		Negative Control
Number of Studies	85	Number of Studies	85
Period	July 2007 – June 2010	Period	July 2007 – June 2010
Mean Viability	17.8%	Mean OD	1.051
Standard Deviation	10.7%	Standard Deviation	0.185
Range of Viabilities	3% - 34%	Range of ODs	0.7 – 1.5

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item cobalt naphthenate is not irritating to the skin and therefore, the test item should not be classified and labelled as skin irritant according to Directive 67/548/EEC.
Also, the test item should not be classified and labelled as skin irritant according to Regulation (EC) No.: 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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.

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).

Irritation parameter:

other: in vitro score

Basis:

other: cornea 1

Time point:

other: 120 min

Score:

1.14

Irritation parameter:

other: in vitro score

Basis:

other: cornea 2

Time point:

other: 120 min

Score:

1.11

Irritation parameter:

other: in vitro score

Basis:

other: cornea 3

Time point:

other: 120 min

Score:

1.07

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.

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)

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.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-11-04 to 2010-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted April 24, 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Hillcrest, Dodgeford Lane, Belton, Loughborough, Leics, LE12 9TE, UK
- Age at study initiation: 15 - 16 weeks
- Weight at study initiation: 2535 - 2616 g
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet (ad libitum): Pelleted standard Teklad Global High Fiber Rabbit Diet 2031C (batch no. 25/10, Provimi Kliba AG, 4303 Kaiseraugst, Switzerland)
- Water (ad libitum): Community tap water from Füllinsdorf.
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): Air-conditioned with 10-15 air changes
- Photoperiod (hrs dark / hrs light): Automatically controlled light cycle of 12 hours light and 12 hours dark

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- The test item was ground to a fine powder with a mortar and pestle. The test item was applied as a weight of 0.1 g/animal, the dose specified in the test guidelines for solid test items.
- On the day of treatment, the test item was applied with an eye glass to the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control.
- A single animal was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was completed using the two remaining animals.

Duration of treatment / exposure:

not applicable

Observation period (in vivo):

approximately 1, 24, 48 and 72 hours after administration

Number of animals or in vitro replicates:

3 (male rabbits)

Details on study design:

The eyes of the animals were examined one day prior to test item administration.

Observation and Scoring:
- The eye reactions were assessed according to the Draize scale. Scleral reddening and ocular discharge were also assessed according to following scale (See "Any other information on materials and methods incl. tables"). Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).
- Viability / Mortality: Daily from acclimatization of the animals to termination of the test.
- Clinical signs (systemic): Daily from acclimatization of the animals to termination of the test.
- Body weights: At start of acclimatization, on the day of application and at termination of observation.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

(animal #1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

mean

Remarks:

(animal #1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

(animal #1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

(animal #1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

(animal #2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

mean

Remarks:

(animal #2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

(animal #2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

(animal #2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

(animal #3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

mean

Remarks:

(animal #3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

(animal #3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

(animal #3)

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

- The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity, iris light reflex, conjunctival chemosis, as well as conjunctival redness were 0.00 for all three animals.
- Slight reddening of the conjunctivae and slight reddening of the sclera were noted in one animal (animal #2) one hour after instillation of the test item.
- No abnormal findings were observed in the treated eyes of any animal 24 hours after treatment.
- No staining produced by the test item was observed in the treated eyes.
- No corrosion of the cornea was observed at any of the reading times.
- Black test item remnants were observed in the treated eyes of all animals at the 1-hour reading.

Other effects:

- Viability / Mortality: No intercurrent deaths occurred during the course of the study.
- Clinical signs: No clinical signs were recorded throughout the entire observation period.
- Body weights: The body weight of the animals was within the range commonly recorded for this strain and age.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to 67/548/EEC and subsequent regulations, the test item cobalt naphthenate is not classified as an eye irritant.
According to Regulation (EC) No. 1272/2008 and subsequent regulations, the test item cobalt naphthenate is not classified as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Key study.

Justification for selection of eye irritation endpoint:
Key study.

Justification for classification or non-classification

Skin corrosion

Reference Heppenheimer (2010) is considered as the key study for skin corrosion and will be used for classification. The overall irritation results are as follows:

Relative viability 3 min after treatment: 95.6 %

Relative viability 60 min after treatment: 74 %

The classification criteria according to regulation (EC) 1272/2008 as corrosive to skin are not met since the relative viability after 3 and 60 min was above 50 % and 15 % respectively, hence no classification required.

 

Skin irritation

Reference Heppenheimer (2010) is considered as the key study for skin irritation and will be used for classification. The mean relative absorbance (% of the negative control, correlating with mean tissue viability) after 15 minutes incubation in the in vitro human skin model test (EpiSkin, according to OECD TG 439) was 76.9%.

The classification criteria according to regulation (EC) 1272/2008 as skin irritant are not met, since the mean tissue viability was above the threshold for skin irritants of 50.0%. No classification required.

 

Eye irritation

The references Heppenheimer (2010) and Sieber (2011) are considered as the key studies for severe eye irritation and will be used for classification.

According to Heppenheimer (2010) the mean in vitro score after 120 minutes incubation results of the in vitro bovine corneal opacity and permeability assay (BCOP, according to OECD TG 437) was 1.11. The value was below the threshold for severe eye irritants of 55.1. The classification criteria according to regulation (EC) 1272/2008 as severe eye irritation are not met, hence no classification required.

According to Sieber (2011) the overall irritation results 24, 48 and 72 hours after application per animal are as follows:

-         Corneal opacity=0.00 for all three animals

-         Iris light reflex=0.00 for all three animals

-         Conjunctival redness=0.00 for all three animals

-         Conjunctival oedema (chemosis)=0.00 for all three animals

The classification criteria acc. to regulation (EC) 1272/2008 as irritating to eyes are not met, hence no classification is required.

 

Respiratory irritation

The justification for classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure: inhalation as given in the acute toxicity section.