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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
Study period:
17 August 2015 - 24 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 15-EKIN-033; source: SkinEthic Laboratories, Lyon, France). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which resulted in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TESTS FOR INTERFERENCE WITH MTT ENDPOINT
- The test substance was checked for possible colour interference before the study was started. Approximately 10 mg of the test substance was added to 90 µL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 µL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approx. 10 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hour at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (23.4 to 27.2 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS in PBS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

REMOVAL OF TEST SUBSTANCE
- Washing: PBS
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
42 hours at 37°C

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for 69.5 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

SCORING SYSTEM
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amounts applied: 23.4 to 27.2 mg per skin tissue
- The skin tissues were moistened with 5 µL Milli-Q water before application

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulphate in PBS
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
117
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Mean tissue viability for the test substance was > 50% (117%), therefore the test substance is considered not to be irritant to the skin.

Any other information on results incl. tables

TESTS FOR INTERFERENCE WITH MTT ENDPOINT

As no colour changes were observed in the tests, it was concluded that the test substance did not interact with the MTT endpoint.

Applicant's summary and conclusion

Interpretation of results:
other: the substance does not need to be classified for skin irritation according to GHS and CLP
Conclusions:
An in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), conducted according to OECD 439 and GLP principles, the influence of the substance on the viability of human skin was tested. Reliable positive and negative controls were included. The substance was applied directly to 0.38 cm2 cultured skin (23.4 to 27.2 mg, in presence of 5 μL Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours at 37°C. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 13% whereas the substance showed cell viability of 117%. Since the mean relative tissue viability after exposure to the substance was above 50%, it is concluded that the substance is not irritating in the in vitro skin irritation test. Based on the results of this study, the substance is not classified for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 (CLP Regulation).