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Diss Factsheets

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:
20.05.2020 - 29.05.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(2R,2'R)-3,3'-disulfanediylbis(2-((S)-2-aminopropanamido)propanoic acid)
Cas Number:
115888-13-6
Molecular formula:
C12H22N4O6S2
IUPAC Name:
(2R,2'R)-3,3'-disulfanediylbis(2-((S)-2-aminopropanamido)propanoic acid)
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- EpiDerm Skin Model (EPI-SIT, Lot no.: 30869 Kit Q)
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TREATMENT
Before the assay was started the tissues were transferred to new 6-well plates containing 0.9 mL Assay medium per well. At least 25 mg solid was added into the 6-well plates on top of the skin tissues. Test items tissues were moistened before application of the test item with DPBS (25 µL), to ensure close contact to the tissue. Three tissues were treated with 30 µL DPBS (negative control) and 3 tissues with 30 µL 5% SDS (positive control) respectively.
After the exposure period with the test item (35 ± 1.0 minutes at 37.0 ± 1.0°C and the remaining period of the 60 ± 1 minutes test item exposure at room temperature ), the tissues were thoroughly rinsed with Dulbecco’s phosphate buffered saline (DPBS) to remove residual test item. If necessary cotton wool swabs were used to remove any remaining test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 0.9 mL pre-warmed assay medium until all tissues were dosed and rinsed. Subsequently the skin tissues are incubated for 24 ± 2 hours at 37°C. The tissues were transferred to 0.9 mL fresh Assay medium and placed back for a post-incubation period of 18 ± 2 hours at 37°C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 3 hours ± 5 minutes at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. After incubation the tissues were washed with DPBS and formazan was extracted with 2 mL isopropanol for at least 2 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 60 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 60 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
32.8 to 38.7 mg
Duration of treatment / exposure:
60 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
Value:
92
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20245940). Because the solutions did not turn blue / purple and/or a blue / purple precipitate was observed and/or the OD for the test item solution was ≤0.08, therefore it was concluded that the test item did not interfere with the MTT endpoint.
The relative mean tissue viability obtained after 60 ± 1 minutes treatment with the test item compared to the negative control tissues was 92%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.
The positive control had a mean cell viability of 3.9% after 60 ± 1 minutes exposure. The absolute mean OD570 of the negative control tissues was slightly above the laboratory historical control data range, but well within the acceptance limits of OECD439 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8). As the laboratory historical control data is limited and the OECD439 range is leading, this is acceptable. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 is non-irritant in the in vitro skin irritation test under the experimental conditions described.