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Description of key information

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:
2010-10-13 to 2010-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
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
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
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 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-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], present 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 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:
99.6
Max. score:
99.9
Reversibility:
no data
Irritant / corrosive response data:
After treatment with the test item cobalt carbonate the relative absorbance values decreased to 95.1%. 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 carbonate:

Dose group

Treatment Interval

Absorbance 570 nm Tissue 1

Absorbance 570 nm Tissue 2

Absorbance 570 nm Tissue 3

Mean Absorbance of 3 Tissues

Absorbance [%] Tissue 1, 2 + 3

Standard Deviation [%]

Rel. Absorbance

[% of Negative Control]

Negative Control

15 min

1.057

0.920

0.912

0.963

109.8

95.5

94.7

8.5

100.0

Positive Control

15 min

0.204

0.185

0.285

0.225

21.2

19.2

29.6

5.5

23.4

Test Item

15 min

0.960

0.956

0.962

0.959

99.7

99.2

99.9

0.3

99.6

 

 

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 information Criteria used for interpretation of results: EU
Conclusions:
The test item cobalt carbonate 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 and 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, other
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented with individual Draize Scoring Scheme Minor deviations from guideline with no effect on the results: - There were no observations made at 1 hour. - It was not stated if effects other than ocular were observed. - The purity and stability of the test item was missing.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
, adopted 1987-02-24
Deviations:
yes
Remarks:
, see "rational for reliability"
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Sweetwater farms
No further information on the test animals was stated.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of the undiluted sample was applied to the right eye of each of six rabbits. The left eye of all rabbits was untreated and served as a cntrol.
No further information on amount/concentration applied was stated.



Duration of treatment / exposure:
In 3 of 6 rabbits the test material was left in the eye for 30 seconds . For the remaining three rabbits no statement regarding duration of treatment/exposure was made.
Observation period (in vivo):
24, 48 and 72 hours after application of the test material
Number of animals or in vitro replicates:
6 rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eyes of three rabbits were irrigated following application with 200 ml of lukewarm tap water. The treated eyes of the three remaining received no further treatment.
- Time after start of exposure: Irrigation after 30 seconds

SCORING SYSTEM: Scoring of irritative effects was according to the method of Draize in which corneal, iris and conjunctival effects are scored separately. In this scoring system, injuries to the cornea and iris may represent as much as 80 percent of the total score. Cornea and iris scores are heavily weighted because of their essential role in vision.
No further information on the study design was stated.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0.67
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
other: 24, 48, and 72 hours
Score:
0
Max. score:
0
Remarks on result:
other: Eyes were not rinsed after application of the test item.
Irritant / corrosive response data:
At the 24 to 48-hour readings following application of cobalt carbonate powder without a rinse, mild to moderate conjunctivitis in all rabbits was noted. All irritative effects subsided by the 72-hour reading (for results see "Overall irritation/ corrosion results" above).
After application of cobalt carbonate powder followed after 30 seconds with a 200 ml rinse, irritative effects at the 24-hour reading included mild conjunctival erythema in two rabbits. No irritative effects were noted at the 48 and 72-hour observation intervals (for results see "Remarks on results including tables and figures"; table 1).

Table 1. Eye irritation scores in New Zealand White rabbits following application of 0.1 gram of cobalt carbonate powder followed in 30 seconds by irrigation with 200 mls of lukewarm tap water

Irritation parameter

Basis

Time point

 

Max. Score

Reversibility

Remarks

Cornea score

Mean animal #4

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Iris score

Mean animal #4

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Conjunctivae score

Mean animal #4

24, 48, and 72 hours

0.33

1

 

Eyes rinsed after 30 seconds

Chemosis score

Mean animal #4

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Cornea score

Mean animal #5

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Iris score

Mean animal #5

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Conjunctivae score

Mean animal #5

24, 48, and 72 hours

0.33

1

 

Eyes rinsed after 30 seconds

Chemosis score

Mean animal #5

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Cornea score

Mean animal #6

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Iris score

Mean animal #6

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Conjunctivae score

Mean animal #6

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Chemosis score

Mean animal #6

24, 48, and 72 hours

0

0

 

Eyes rinsed after 30 seconds

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The following classification is based on the results obtained without rinsing the eyes after application.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is classified as not irritating.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is classified as not irritating.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

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 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 99.6%. 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

Reference Vinegar (1977) is considered as the key study for eye irritation and will be used for classification. The overall irritation results 24, 48 and 72h after application per animal are as follows:

Corneal opacity: mean score=0.0 for all 3 animals

Iritis: mean score=0.0 for all 3 animals

Conjunctival redness: 0.33, 0.67, 1.0

Conjunctival oedema (chemosis): 0.0 for all 3 animals

The classification criteria acc. to regulation (EC) 1272/2008 as irritating to eyes are not met, since none of the 3 animals showed after 24, 48 and 72hours a conjunctival and chemosis score of 2.0 and above, or a cornea and iritis score of 1.0 and above, thus cobalt carbonate will not be classified.

 

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.