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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
- Source: The experiment was carried out on the reconstructed human epidermis model epiCS® (CellSystems, Troisdorf, Germany).
The tissue equivalents were shipped in 24 well cell culture plates on agarose supplemented with maintenance medium (Kit contents epiCS® CellSystems, Cat.-No.CS-1001). Inserts were of 0.6cm2 size.
Justification for test system used:
The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.
The viability of the living cells in the model must be sufficiently high to discriminate well between the positive and negative control substances. Cell viability is measured by the amount of MTT reduction, i.e. an OD value, following exposure to the negative control substance or the test item.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The experiment was carried out in vitro using the commercially available test method epiCS

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 2°C
- Temperature of post-treatment incubation (if applicable): 37 ± 2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure to the test item the inserts were washed carefully in PBS
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: EL808, Bio-Tek; 96 well format, 200 µL
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 1 experiment, 3 replicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Killed control and Color control not required.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure and post-treatment incubation is less than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
20 min
Duration of post-treatment incubation (if applicable):
42 h and 3 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
92.85
Vehicle controls validity:
not examined
Remarks:
no vehicle used
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none stated
- Direct-MTT reduction: no
- Colour interference with MTT: no

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:
GHS criteria not met
Conclusions:
Testing was performed via a GLP OECD 439 guideline study on the registered substance itself.
The validity criteria are met, making the results sufficiently reliable to assess the irritating potential of the test item to the skin. The present in vitro method allows the identification of irritating chemical substances and mixtures.
The cell viability was 92.85 % for the test item. Thus the test item requires no classification (Category 2 or Category 1) under the experimental conditions of the test.
Executive summary:

A GLP study was performed according to OECD 439 and EU Method B.46 for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.  

Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%

The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The following value of cell viability was recorded for the test item: 92.85 %

In conclusion the results of the assay used show no skin irritant properties of the test item and thus, the test item requires no classification (Category 2 or Category 1) here.