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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-30 to 2010-10-04
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:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 431: In vitro skin corrosion: Human Skin Model Test (Original guideline adopted 2004-04-13)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
, 2008
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 propionate
- Physical state: Solid
- Storage condition of test material: 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:
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.
After the pre-incubation of the EST-1000™ tissues was completed (1 hour 30 minutes for the 1 hour exposure and about 2 hours 25 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 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 of 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 18.5 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.

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%.
No further information on the study design was stated.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: relative viability (%)
Value:
84.4
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:
57.9
Remarks on result:
other:
Remarks:
Basis: mean. Time point: after 1 hour treatment. Reversibility: no data. (migrated information)

In vivo

Irritant / corrosive response data:
After exposure to the test item cobalt oxyhydroxide the relative absorbance values were reduced to 84.4% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 57.9%. 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.

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive to the skin.
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item cobalt propionate was non corrosive to skin according to Regulation (EC) No. 1272/2008 and Directive 67/548/EEC.