p-benzoquinone

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.03.2018 - 29.03.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted: 29th July 2016

Deviations:

yes

Remarks:

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

Test material information

Constituent 1

Reference substance name:

p-benzoquinone

EC Number:

203-405-2

EC Name:

p-benzoquinone

Cas Number:

106-51-4

Molecular formula:

C6H4O2

IUPAC Name:

1,4-benzoquinone

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

non-animal test

Vehicle:

water

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.
Controls:
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

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 3 min.

Value:

37

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 60 min.

Value:

76.3

Negative controls validity:

valid

Positive controls validity:

valid

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

Conclusions:

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.