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Skin irritation / corrosion

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skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16.03.2018 - 29.03.2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted: 29th July 2016
Double volume of test item was used for direct reduction MTT test in tubes because there was a need to filtrate final solution.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Justification for test system used:
non-animal test
Details on test system:
MTT Test
Test item application: 25 mg of the test item was placed directly atop to the moistened tissue and it was spread to match size of the tissue.
Negative control - 50 μL H2O tested with every exposure time
Positive control - 50 μL 8N KOH tested with every exposure time

Procedure: After delivery, tissues were pre-incubated overnight to release transport stress related compounds and debris. After overnight pre-incubation, medium was replaced to fresh one and tissues were topically exposed to the test item for 3 (room temperature) and 60 minutes at culture conditions (37±1°C, 5±1 % CO2, humidified). In each time interval three tissues were used per test item (C1), two tissues for the positive control (PC) and two tissues for negative control (NC). In additional freeze-killed tissues were used. In each time interval two tissues were used for the test item (C1) and two tissues for negative control (NC). After exposition, tissues were thoroughly rinsed and blotted to remove the test item/controls.
After that, tissues were transferred to 24-well plates containing MTT medium (1 mg/mL) and incubated at culture conditions for 3 hours. After MTT incubation, the blue formazan salt (formed by cellular mitochondria) was extracted with 2.0 mL/tissue of isopropyl alcohol for 2 hours at room temperature and shaking. Optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
25 mg
Duration of treatment / exposure:
3 min./ 60 min.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
after 3 min.
Negative controls validity:
Positive controls validity:
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
after 60 min.
Negative controls validity:
Positive controls validity:

Any other information on results incl. tables

Direct MTT reduction - functional check in tubes

50 mg of the test item was addedto 2.0 mL of MTT medium. Suspension was incubated for 1 hour (37±1 °C, 5±1 % CO2, humidified). The test item change colour of MTT medium from red to dark red however it doesn`t mean direct reduction of MTT. The test item did not change colour of MTT medium from red to blue.

The test item does not reduce MTT directly, so correction of the results is not necessary.

Colour interference

Colour of water/isopropyl alcohol change colour to yellow (see Figures 2 a, b), so it was decided to proceed colorant control test. Two viable tissues per exposure time was used. These tissues undergo the entire skin corrosion test but they were incubated with medium without MTT instead of MTT solution during the MTT incubation step. The extracts from colorant control tissues had OD570 <5% . Because this value is far from the classification cut-off (50%), it could not affect the final results significantly, so correction of the results is not necessary.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Under the above-described experimental design, the test item Methylhydroquinone was corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.
Executive summary:

As it is possible to see from the results given above average viability of affected tissues was 37.0% of negative control average value after 3 minutes of treatment and 76.3 % after 60 minutes of treatment. According to evaluation criteria the viability after 3-minute exposure is < 50 % and ≥ 25 %. The test item should be regarded as corrosive, Sub‑category 1B/1C.