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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:
2011-07-27 to 2011-07-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, CLP and Guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
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

Test animals

Species:
other: reconstituted human epidermis
Strain:
other: reconstituted human epidermis
Details on test animals and environmental conditions:
EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Source: Skinethic, Nice, France
Batch No.: 11-EKIN-030
Expiry date: 01 August 2011

Test system

Type of coverage:
other: not applicable (reconstituted human epidermis)
Preparation of test site:
other: not applicable (reconstituted human epidermis)
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
The test item was applied in its original form, no formulation was required. 10 mg of the test item was applied to each EpiSkin epidermis units.
Duration of treatment / exposure:
15 min
Observation period:
Not applicable (reconstituted human epidermis)
Number of animals:
Not applicable (reconstituted human epidermis). However, in this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used.
Details on study design:
The following steps were performed under axenic conditions.

Pre-incubation (day [-1]):
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

Application (day 0):
Test Item
Epidermal surface was first moistened with 10 µL deionised water and then 10 mg of the test item was applied evenly on the skin. Test substance was spread gently with a curved flat spatula in order to cover evenly all the epidermal surface.

Positive and negative control
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Exposure (day 0):
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Rinsing (day 0):
After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9%) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

Post-incubation (day 0-2):
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2):
After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37°C in an incubator with 5 % CO2 protected from light.

Formazan extraction (day 2):
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis from each replicate was cut from the unit using a biopsy punch. The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol.

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (day 2):
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at 570 nm using acidified isopropanol solution blank (6×200 µL).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
103
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 h total incubation time. (migrated information)

In vivo

Irritant / corrosive response data:
The results obtained from the in vitro skin irritation test in the EPISKIN model with cesium sulphate indicated a mean viability of 103 % (SD 5.02) after a total 42 h incubation time and therfore the test item is considered to be a non-irritant (NI) [UN GHS: No Category].
Other effects:
none

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this in vitro skin irritation test in the EPISKIN model with test item cesium sulphate the results indicated that the test item is considered to be a non-irritant (NI) [UN GHS: No Category].
Executive summary:

In this in vitro skin irritation test using the EPISKIN model, the test item cesium sulphate did not show a significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50% when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits, thus the experiment was considered to be valid. The results indicated that the test item revealed no skin irritantion potential under the utilised testing conditions. According to the current OECD Guideline No. 439, the test item is considered a non-irritant to skin and is therefore not classified.