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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Study period:
2010-07-5 to 2010-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to EU-Method B.46 " In vitro Skin Irritation: Reconstructed human epidermis model test" (23. July 2009)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: According to Human Skin Model Test EU-Method B.46 adopted 23. July 2009
Deviations:
no
Principles of method if other than guideline:
The in vitro skin irritation test is used to determine the skin irritancy of chemicals by measuring the cell viability in a reconstructed human epidermis (RhE) model after applying the test substance. The RhE model consists of human-derived epidermal keratinocytes, which have been cultured to form a multilayered model of the human epidermis. Irritant chemicals are identified by their ability to decrease cell viability by quantitatively measuring the enzymatic conversion of the vital dye MTT (3-[4,5-dimethyl thiazole 2-yl] 2,5-diphenyl-tetrazoliumbromide) to a blue formazan salt. Skin irritation potential of the test substance is assessed by reducing the formazan production to less than 50% of the negative control.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Charcoal (Probe 1: C-Fix = 73.3%)
Batch No.: 19062009 1PP
Appearance: Charcoal typic
CAS-No.: 16291-96-6
EINECS-No.: 240-838-3
Purity: C-Fix = 73.3%
Date of expiry: Dec. 2030
Storage conditions: Room temperature 20 ± 5°C

Test animals

Species:
other: not applicable (this is an in vitro test)
Strain:
other: not applicable (this is an in vitro test)
Details on test animals and environmental conditions:
not applicable (this is an in vitro test)

Test system

Type of coverage:
other: not applicable (this is an in vitro test)
Preparation of test site:
other: not applicable (this is an in vitro test)
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
approx. 25 mg
Duration of treatment / exposure:
60 min
Observation period:
not applicable (cell viability was determined after the end of treatment period)
Number of animals:
not applicable (this is an in vitro test)
Details on study design:
The human skin model test was performed with commercially available Epi-200-SIT-Kit. The EpiDermTM tissues were produced from MatTek Corporation in Ashland ,USA. The day of delivery was the 01. July 2010 batch: 13657

Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) without CaCl2 and MgCl2
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer
Positive control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in deionised H2O, concentration 50 g/L
One plate (three tissues) was used as positive control; each tissue was treated with 30 µL SDS-solution
Test item: ground charcoal comprised of particles < 100 µm about 25 mg
One plate (three tissues) was used for treatment with the test item

Experimental performance:
After dosing the tissues with negative control, positive control and test item, they were incubated at 37 °C for 35 min. After 60 min a rinsing step and two incubation periods at 37°C over a total period of 42 h followed.
After incubation, a MTT assay was performed and formazan production was measured by a spectral photometer at 570 nm.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: percentage values of formazan production in comparison to the negative control
Remarks on result:
other:
Remarks:
Basis: other: provided on the above section 'any other information on materials and methods incl. tables'. Time point: 60 min. Reversibility: other: not applicable. Remarks: Result: non - irritant. (migrated information)

In vivo

Irritant / corrosive response data:
The relative absorbance values were reduced to 70.4% after the treatment with charcoal (Probe 1: C-Fix = 73.3%). This value is above the threshold for irritation (50%). Therefore, the test item is considered not irritant. The reduction of the relative absorbance of the positive control was 7.8%.
Other effects:
not applicable

Any other information on results incl. tables

Comparison of formazan production:

For the test item and the positive control the following percentage values of formazan production were calculated in comparison to the negative control:

% Formazan Production

Designation

Charcoal (Probe1: C-Fix=73.3%)

Positive control

% Formazan production (Tissue 1)

69.3%

7.6%

% Formazan production (Tissue 2)

73.8%

8.1%

% Formazan production (Tissue 3)

68.0%

7.9%

% Formazan production Mean

70.4%

7.8%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It can be stated that under the experimental conditions reported, charcoal did not exhibit any skin irritating potential.
Executive summary:

This in-vitro study human skin model test (EU method B.46) was performed in order to evaluate the potential of charcoal (Probe 1: C-Fix = 73.3%) to evoke skin irritation in a human-skin-model. Charcoal was spread on the surface of three sections of an EpiDermTMtissue and incubated for 60 min. The negative control consisting of Dulbecco’s phosphate buffered saline was applied to another three tissue sections. As positive control a SDS-solution (50 g/L) was used for three other tissue sections. After rinsing and two incubation steps of altogether 42 h an enzyme assay to determine cell viability was performed. The tissues were incubated with MTT-reagent for 3 h. The tissues were washed with PBS, dried and transferred to a 24 -well-plate. After incubating with isopropanol at room temperature over night, the developed formazan concentration was measured in a spectral photometer at 570 nm.

The mean percentage values of formazan production were calculated in comparison to the negative control. The relative absorbance values were reduced to 70.4% after the treatment with charcoal. This value is above the threshold for irritation (50%). The positive control showed a reduction of formazan production of 7.8%.

In conclusion, it can be stated that the test item charcoal (Probe 1: C-Fix = 73.3%) is considered as non skin irritant under the experimental conditions used in this study.