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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:
weight of evidence
Study period:
from 2017-05-05 to 2017-06-30
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Demanded by the OECD Guideline 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT, EpiDerm™ tissue (MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue Lot number: 25825
- Delivery date: 2017-06-27
- Date of initiation of testing: 2017-06-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: 35 min at 37 ± 1.5 °C, follwed by 25 min at room temperature
- Temperature of post-treatment incubation: ~ 41 h at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h at 37 ± 1.5 °C
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
30 μL of the test item were added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. Since the test item did not dye water an additional test (step 2) with viable tissues (but without MTT addition) was not necessary to be performed.

The test item was further evaluated for its potential to interfere with the MTT assay. To test if a test item directly reduces MTT, 30 μL of the test item was added to 1 mL of the MTTsolution (1 mg/mL) and the mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control. Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

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 irritant to skin if the mean relative tissue viability of three individual tissues is reduced ≤ 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
41 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
11.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No
- 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

Any other information on results incl. tables

 

Mean absorbance

Mean rel. Absorbance (%)

Rel. S.D (%)

Negative Control

1.670

100.0

4.6

Positive Control

0.055

3.3

0.6

Test substance

0.192

11.5

6.4

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In an in vitro skin irritation study (RHE) according to OECD Guideline 439, a cell viability of 11.5 % was determined. The test item showed skin irritating properties.
Executive summary:

In an in vitro study (RHE) according to OECD Guideline 439, the skin irritating potential of the test item was assessed. 30 µL of the neat test item, the positive control (5 % SDS solution), or the negative control (DPBS) were dispensed directly atop the EpiDerm™ tissue (triplicates) and incubated for 1 h at 37 ± 1.5 °C. After the incubation phase, all tissues were each rinsed from the test item. Further, the tissues were incubated for another 41 h at 37 ± 1.5 °C. After the post-incubation phase, cell viability was determined via MTT-assay. Concurrent positive and negative controls were valid. A cell viability of 11.5 % was determined after incubation with the test item. Based on the prediction model, the test item has to be considered as skin irritating.