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Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: validated "in vitro" test method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-26 to 2010-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
other: OECD 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original guideline adopted 22 July 2010)
Deviations:
no
Qualifier:
according to
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:
other: Commission Regulation (EC) No. 440/2008 B.46
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Details on test animals 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): About 11 mg of the test item were applied to each of triplicate tissues, spread evenly over the surface of the tissues, and wetted with 15 µL deionised water.No further information 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-040) are 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. 031110); 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. Additionally, the test item tissues were each wetted with 15 µL of deionised water. The 12-well plates were placed into the incubator for 15 ± 1 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 approx. 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 is 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 for 68-69 hours in the refrigerator.Per each tissue sample 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm 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 100For 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:
88
Max. score:
90.5
Reversibility:
no data
Irritant / corrosive response data:
After treatment with the test item cobalt 2-ethylhexanoate the relative absorbance values decreased to 88.0%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with cobalt 2-ethylhexanoate:

 

Dose group

Treatment Interval

Absorbance 570 nm Tissue 1

Absorbance 570 nm Tissue 2

Absorbance 570 nm Tissue 3

Mean Absorbance of 3 Tissues

Relative Absorbance [%] Tissue 1, 2 + 3

StandardDeviation [%]

Mean Rel. Absorbance

[% of Negative Control]

Negative Control

15 min

1.006

0.898

0.979

0.961

104.7

93.5

101.9

5.8

100.0

Positive Control

15 min

0.195

0.363

0.323

0.294

20.3

37.8

33.7

9.1

30.6

Test Item

15 min

0.870

0.802

0.864

0.845

90.5

83.4

89.9

3.9

88.0

 

 

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 irritating
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item cobalt 2-ethylhexanoate is not irritant to the skin and therefore, 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
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-17 to 2011-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2002-04-24
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 weeks (male); 14 - 15 weeks (females)- Weight at study initiation: 2649 g (male); 3140 - 3334 g (females)- Housing: individually in stainless steel cages- Diet (ad libitum): pelleted standard Teklad Global High Fiber Rabbit Diet 2031C (batch no. 80/10, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland); a piece of wood (batch no. 122201, imported by Indulab AG, Gams / Switzerland from ABEDD® - LAB & VET GmbH, 1160 Vienna / Austria) and a haystick 4642 (batch no. 37/10, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) were also provided for environmental enrichment.- Water (ad libitum): community tap water from Füllinsdorf- Acclimation period: five days (male rabbit) or 6 days (female rabbits) under laboratory conditions, after health examination. Only animals without any visual signs of illness were used for the study.ENVIRONMENTAL CONDITIONS- Temperature: 17-23 °C- Relative humidity: 30-70%- Air changes: 10-15 air changes per hour- Photoperiod (hrs dark / hrs light): 12/12 (music played during the daytime light period)
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): the test item was ground using a mortar and a pestle and was applied as a weight of 0.1 g/animal. The test item was applied 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.
Duration of treatment / exposure:
not applicable
Observation period (in vivo):
approximately 1, 24, 48 and 72 hours as well as 7, 10 and 14 days after administration
Number of animals or in vitro replicates:
1 male rabbit / 2 female rabbits(a single animal (the male) 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.)
Details on study design:
The eyes of the animals were examined one day prior to test item administration.SCORING SYSTEM: according to the Draize scaleScleral reddening and ocular discharge were also assessed (scale for assessment can be seen in the field "Any other information on materials and methods incl. tables" below).TOOL USED TO ASSESS SCORE: Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).OBSERVATIONS- Viability / Mortality: daily from acclimatization of the animals to the end of the observation period.- Clinical Signs (systemic): daily from acclimatization of the animals to the end of the observation period.- Body Weights: at start of acclimatization, on the day of application and at the end of the observation period.- Pathology: no necropsy was performed on the animals sacrificed at termination of observation.The animals were sacrificed by intravenous injection of 1.0 mL/kg body weight of a solution of 162 mg/mL sodium pentobarbitone into the ear vein and discarded.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
2.33
Max. score:
3
Reversibility:
fully reversible within: 10 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
The instillation of cobalt 2-ethylhexanoate into the eye resulted in slight opacity of the cornea in the two female animals 24 hours after treatment and persisted until 72 hours after treatment. Marked reddening of the conjunctivae was observed in the two female animals 1 hour after treatment and slight reddening was observed in the male animal 24 hours after treatment. Conjunctival reddening persisted as slight to marked until 72 hours (male), 7 days (one female) or 10 days (second female) after treatment. Slight to moderate chemosis of the conjunctivae was observed in the two female animals 1 hour after treatment and persisted until 72 hours after treatment. Moderate reddening of the sclerae was observed in the two female animals 1 hour after treatment and persisted as slight to marked reddening until 7 days after treatment. Slight to moderate ocular discharge was observed in the two female animals 1 hour after treatment and persisted until 72 hours after treatment. All eye reactions were reversible and were no longer evident 7 days (male), 10 days (one female) or 14 days (second female) after treatment. No abnormal findings were observed for the iris light reflex. No corrosion was observed. No staining of the treated eyes by the test item was observed. Test item remnants were observed in the treated eyes of all animals 1 hour after treatment and persisted in one animal (male) until 24 hours after treatment.Thus, the test item did not induce significant or irreversible damage to the rabbit eye.
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 weights were within the range commonly recorded for this strain and age.- Pathology: no necropsy was performed at the end of the study.
Interpretation of results:
Category II
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
Cobalt bis(2-ethylhexanoate) is considered to be an eye irritant.According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as eye irritant.According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is classified as Category 2.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

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

Justification for selection of eye irritation endpoint:
Key study

Effects on eye irritation: irritating

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 in an in vitro human skin model test (according to OECD 431) are as follows:

Relative viability 3 min after treatment: 106.9 %

Relative viability 60 min after treatment: 80.7 %

The classification criteria according to regulation (EC) 1272/2008 as corrosive to skin are not met since the relative viability after 3 and 60 minutes exposure was above 50 % and 15% respectively, hence cobalt bis(2-ethylhexanoate) will not be classified as skin corrosive.

 

Skin irritation

Reference Heppenheimer (2011) 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 88.0%.

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 (2011) and Mallaun (2011) are considered as the key studies for severe eye irritation and will be used for classification.

According to Heppenheimer (2011) the mean in vitro score after 240 minutes incubation results of the in vitro bovine corneal opacity and permeability assay (BCOP, according to OECD TG 437) was 0. 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 Mallaun (2011) the overall irritation results 24, 48 and 72 hours after application per animal are as follows:

- Corneal opacity = 0.00, 1, 1

- Iris light reflex = 0.00 for all three animals

- Conjunctival redness = 1.00, 3.00, 2.33

- Conjunctival oedema = 0.00, 1.33, 1.33

The classification criteria acc. to regulation (EC) 1272/2008 as irritating to eyes are met, since corneal opacity was above the threshold of

≥ 1 and conjunctival redness was above the threshold of ≥ 2 as well as the effect were fully reversible within 14 days. Thus, cobalt bis(2 -ethylhexanoate will be classified as eye irritant category 2 (H319).

Respiratory irritation

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