Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

skin irritation / corrosion
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.6.2010 - 26.7.2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
The test substance should be removed from tissues by the PBS solution. Because of the test substance is not miscible with water solutions, it was removed by the means of a cotton swab. This change had no impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Tar, brown-coal, low-temp.
EC Number:
EC Name:
Tar, brown-coal, low-temp.
Cas Number:
Molecular formula:
Not known - the substance is complex of hundreds organic compounds.
Tar, brown-coal, low-temp.
Details on test material:
- Name of test material (as cited in study report): Tar, brown-coal, low-temp.
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Smiles notation: not known - UVCB substance
- InChl: not known - UVCB substance
- Substance type: technical product
- Physical state: viscous liquid
- Lot/batch No.: T201E
- Expiration date of the lot/batch: 31.3.2011
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark

Test system

Details on study design:
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide] into a blue formazan salt, caused by dehydrogenase present in cell mitochondria. The conversion is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with sterile water for injection) is used to predict the skin corrosion potential.
Effect of a substance is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each triplet of tissues as % of the mean of the negative control tissues. Skin corrosion potential of the test material is predicted according to Prediction model II (see bellow).

The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ System is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.

a/ Test substance application:
Test substance (50 uL) was placed directly atop the tissue and it was spread on the tissue surface.
b/ Direct MTT reduction:
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility is performed as follows: 25 mg of the test substance is added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.
The MTT assay is affected only if the test material is present in the tissues when the MTT viability test is performed.
The procedure employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test substance similar to viable tissues. Each MTT reducing chemical is applied to two freeze-killed tissues. In addition, two freeze killed tissues are left untreated (Note: The untreated killed controls will show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay protocol is performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues. Data are then corrected as follows:

True viability = Viability of treated tissue – Interference from test chemical = OD tvt – OD kt = OD tvt - (mean OD tkt - mean OD ukt)
OD tvt = optical density of treated viable tissue
OD kt = optical density of killed tissue
OD tkt = optical density of treated killed tissue
OD ukt = optical density of untreated killed tissue (neg. control treated tissue)

If the interference by the test substance is > 30% of the negative control value, additional steps must be taken into account or the test substance may be considered incompatible with this test system.
If the interference by the test substance is < 30% of the negative control value, the net OD of the test substance treated killed control may be subtracted from the mean OD of the test substance treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

c/ Procedure:
On the day of experiment, EpiDerm tissues are conditioned by 1-hour incubation.
After this pre-incubation, the tissues are topically exposed to the test chemicals for 3 and 60 minutes. In each time interval three tissues are used per test chemical, three for the positive control (PC) and three for negative control (NC). After exposition, the tissues are thoroughly rinsed and blotted to remove the test substance (control substances).
After that, the tissues are transferred to 24-well plates containing MTT medium (1 mg/mL). After a 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 mL/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Isopropanol serves as a blank.

Negative Control:
The absolute OD of the negative control (NC) tissues (treated with sterile water for injection) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.
Positive Control:
An 8N KOH solution is used as positive control (PC) and tested with the test chemicals. Viability of positive control should be within 95±1 % confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
Standard Deviation (SD):
Since in each test skin corrosivity potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD from 3 identically treated tissues is ≤15%. At a difference over the limit rejection of the outlying tissue should be considered.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: relative cell viability
Time point:
other: 3 min.
Max. score:
Remarks on result:
other: The value is not correct, the direct MTT reduction was observed - see bellow
Irritation parameter:
other: relative cell viability
Time point:
other: 60 min.
Max. score:
Remarks on result:
other: The value is not correct, the direct MTT reduction was observed - see bellow

Any other information on results incl. tables


1/ Direct MTT reduction

Before the test itself direct MTT reduction was assayed. After incubation of the test substance with MTT medium, colour was compared with control solution of MTT medium only. The test substance changed colour to dark violet of the MTT medium so it reduced MTT directly.

After 3 min, reduction in treated killed tissues vs. reduction in viable negative control was 91.2% and after 60 min the value was 86.2%. This is more than 30% so correction of results could not be made. It is not possible to discern what part of the reduction was caused by the test substance and what was caused by viable cells.

2/ MTT test

Average viability of affected tissues was 123 % of negative control average value after 3 min treatment and 91.5% of negative control after 60 min.

From results obtained in the test of direct MTT reduction and MTT test it is evident that the reduction caused by rests of the test substance is higher than 30%. Correction of results then should not be made and conclusions cannot be done.

Applicant's summary and conclusion

Interpretation of results:
Criteria used for interpretation of results: EU
Executive summary:

The test substance - Tar, brown-coal, low-temp. - was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to Method B.40: Skin corrosion (in vitro), Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

The test substance (50 µl) was placed on the surface of model tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance, three for positive control and three for negative control.

After rinsing, tissues were incubated with MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide] for three hours and extracted overnight subsequently at room temperature without shaking. Optical density of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. Evaluation is then performed according to Prediction Model 2 (stated in Method B.40).

The test substance directly reduces MTT. Amount of the test substance remaining in tissues is so high, that correction cannot be made. Under the above-described experimental design it is not possible to test the corrosion effects of the test substance in this system.