Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
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. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cobalt
- Molecular formula: Co
- Physical state: Solid
- Storage condition of test material: In a closed container at room temperature
No further information on the test material was stated.

Test animals

Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

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.

Results and discussion

In vivo

Results
Irritation parameter:
other: relative viability (%)
Basis:
mean
Time point:
other: after 15 min incubation
Score:
95.1
Max. score:
104.8
Reversibility:
no data
Irritant / corrosive response data:
After treatment with the test item cobalt 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.

Any other information on results incl. tables

Results after treatment with cobalt powder:

 

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

1.009

0.852

0.885

0.916

104.8

88.5

91.9

8.6

95.1

 

 

 

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

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item cobalt powder 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.