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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The 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))
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Distillates (coal tar), light oils
EC Number:
EC Name:
Distillates (coal tar), light oils
Cas Number:
Molecular formula:
Distillates (coal tar), light oils
Details on test material:
- Name of test material (as cited in study report): Carbolic oil
- Substance type: mixture of organic compounds
- Physical state: black liquid
- Composition of test material, percentage of components: Main components (by GC) - Indene 17.27 % (w/w), Phenol 13.94 % (w/w), Indane 12.86 % (w/w), Naphthalene 10.54 % (w/w), m-Cresol 8.79 % (w/w), p-Cresol 4.87 % (w/w), o-Cresol 4.49 % (w/w), m-/p-Xylene 3.03 % (w/w), 1,3,5-Trimethylbenzene 1.52 % (w/w), 3-Ethyltoluene 1.41 % (w/w), Toluene 1.33 % (w/w), Ethylbenzene 1.18 % (w/w), 1,2,4-Trimethylbenzene 1.52 % (w/w), Coumarone 1.57 % (w/w), 28 other hydrocarbons from 0.1 up to 1.0 % (w/w), 14 other hydrocarbons up to 0.1 % (w/w)
- Lot/batch No.: Composite sample No. 3, ATE No. 10438
- Expiration date of the lot/batch: 08/2022
- Stability under test conditions: stable
- Storage condition of test material: The sample will be stored in supplied metal container in dry room at the temperature < 30°C.

Test system

Amount / concentration applied:
Test substance (50 µl) was placed directly atop the tissue and it was spread on the tissue surface.

Duration of treatment / exposure:
3 min, 60 min
Details on study design:
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™ 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.

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 was performed. 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. 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.
If the interference by the test substance is greater than 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.

On the day of experiment, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, 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, tissues are thoroughly rinsed and blotted to remove the test substance (controls).
After that, 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.

OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: OD570
Remarks on result:
Basis: mean. Time point: 3 min. Max. score: 1.839. Remarks: viable tissues. (migrated information)
Irritation / corrosion parameter:
other: other: OD570
Remarks on result:
Basis: mean. Time point: 60 min. Max. score: 0.409. Remarks: viable tissues. (migrated information)
Irritation / corrosion parameter:
other: other: OD570
Remarks on result:
Basis: mean. Time point: 3 min. Max. score: 0.217. Remarks: frozen tissues. (migrated information)
Irritation / corrosion parameter:
other: other: OD570
Remarks on result:
Basis: mean. Time point: 60 min. Max. score: 0.659. Remarks: frozen tissues. (migrated information)

Any other information on results incl. tables



Beside the test itself direct MTT reduction was assayed.

After 60 min incubation of the test substance with MTT medium, colour was compared with control solution of MTT medium only. The test substance changed its colour to purple. The test substance reduced MTT directly.

MTT Test

After 3 min, reduction in treated killed tissues vs reduction in viable negative control was 12.6% what is less than 30%, so correction of result could be made.

After 60 min, reduction in treated killed tissues vs. reduction in viable negative control was 32.9 % so correction of result is not possible. Although correction could not be done, it is clear, that all value of reduction after 60 min could be placed to debit direct reduction of MTT by the test substance.



Negative control: Criteria for negative control were fulfilled.

Positive control: Criteria for positive control were fulfilled.

Standard Deviation (SD): Criteria for standard deviation were not fulfilled for positive control after 3 min and in the case of frozen tisues theated by the test substance for 60 min. No tissue was ommited from evaluation. 


As it is possible to see from the results given above (Tables 2,3), average viability of affected tissues was 104.5 % of negative control average value after 3 min treatment and 23.2 % after 60 min. Althought the reduction after 60 min is higher than 15% it all can be put to account of the direct reduction by the test substance. The substance could be regarded as corrosive.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
Under the above-described experimental design, the test substance Carbolic Oil was corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.
Executive summary:

Test substance Carbolic Oil 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 atop the tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (TS), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and extracted overnight subsequently at room temperature without shaking. OD570 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. 

In the simultaneously performed test of direct reduction it was found, that the test substance reduces MTT directly. Therefore, test of reduction by the test substance only was performed consequently in frozen tissues by the same way as the main experiment.

The test showed, that rests of Carbolic Oil in tissues after rinsing reduced MTT directly. The direct reduction was even higher than in the test with viable tissues. It means that all reduction in viable treated tissues (23.2% of negative control) was caused by the test substance.

In the experiment arrangement given above, the test substance Carbolic Oil was corrosive in EpiDermTM model.