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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
dopted 29 July 2016
Deviations:
not specified
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone
EC Number:
947-565-8
Molecular formula:
C30 H42 O4
IUPAC Name:
2-acetyl-3-(4-hexadecoxyphenyl)-1,4-benzoquinone

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm Skin Model (EPI-200, EPI-212)
- Tissue batch number(s):28831 kit O&P
- Shipping date: 11 July 2018
- Date of initiation of testing: One day after receipt

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out at 37.0 ± 1.0°C (actual range 36.7 -37.3°C).


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 2
- Observable damage in the tissue due to washing: /
- Modifications to validated SOP: /

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 3 hours
- Spectrophotometer: The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No interference with MTT

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%. In addition, a test item considered non-corrosive (viability  50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
- The test substance is considered to be non-corrosive to skin if the relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 34.8 to 48.8 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL / POSITIVE CONTROL
2 tissues were treated with 50 μl Milli-Q water (negative control) and 2 tissues were treated with 50 μl 8N KOH (positive control)
Duration of treatment / exposure:
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.4%.
In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was<9%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, V134434 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate V134434 for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of V134434 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD 431 and EC guidelines (B.40 BIS).

Batch v134434/CA of V134434 was an orange-red powder. Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of V134434 was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7.4% after the 1-hour exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit£2.8) andthe laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 9%,indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with V134434 compared to the negative control tissues was 96% and 98%, respectively. Because the mean relative tissue viability for V134434 was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment, V134434 is considered to be not corrosive to the skin.

In conclusion, V134434 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.