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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:
11 April 2017 - 21 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
other: EpiDerm TM Kit (human-derived epidermal keratinocytes)
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Justification for test system used:
The EpiDerm TM is an in vitro system recommended by the corresponding guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 Kit
- Tissue batch number: 25811
- Date of initiation of testing: 2017-05-10

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 3 minutes; room temperature for 60 minutes

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20 times
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be corrosive to skin if:
- < 50% after 3 minutes exposure
- ≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure
The test substance is considered to be non-corrosive to skin if:
- ≥ 50% after 3 minutes exposure AND ≥ 15% after 60 minutes exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
3 min and 60 min
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
1_Exposure: 3 min
Value:
97.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
2_exposure: 3 min
Value:
94.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Corrected Mean_Exposure: 3 min
Value:
91.5
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
1_Exposure: 1 hour
Value:
21.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
2_Exposure: 1 hour
Value:
24.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Corrected Mean_Exposure: 1 hour
Value:
6
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: yes, an additional test with freeze-killed tissues was performed to determine a correction factor for calculating the true viability in the main experiment
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
The test item is considered to be corrosive to skin according to EU CLP and UN GHS.
Executive summary:

An in vitro study was performed to assess the corrosive potential of the test substance by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the colour interference pre-test. Since it proved to be a MTT reducer in the MTT interference pre-test, an additional test with freeze-killed tissues was necessary.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for 17.6 hours at room temperature.

After exposure to the test item the corrected relative absorbance value decreased to 91.5% after 3 minutes exposure. After 1 hour exposure the corrected relative absorbance value was reduced to 6.0%. The 1-hour-value fell below the threshold for corrosivity which is defined to be 15%.

The required acceptability criteria were met.

The test item is considered to be corrosive.