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Diss Factsheets

Administrative data

Description of key information

Corrosive to skin (GLP, OECD TG 431), mean cell viability = 3.3% after 4 hours of exposure (i.e. <35%) compared to negative controls

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkin supplied by SkinEthic, France, batch 19-EKIN-031, expiry date 05 August 2019
Justification for test system used:
The Episkin model has been validated for corrosivity testing in an international study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 431)
Vehicle:
unchanged (no vehicle)
Details on test system:
Preparation: 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 a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Units: EpiSkinTM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 19-MAIN3-034; Exp. Date: 07 August 2019)
A flask of sterile “Assay Medium” (Batch No.: 19-ESSC-032; Exp. Date: 07 August 2019)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test substance: 20 mg (plus 100 μL physiological saline)
Positive and negative controls: 50 μL
Duration of treatment / exposure:
4 hours
Number of replicates:
2 (substance, positive and negative controls), additional 2 with test substance for non-specific optical density evaluation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
3.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After receipt, the two indicators of fitness for the delivered kit (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay.
The mean OD value of the two negative control tissues was 0.717, which satisfied the acceptability criterion of being in the range of 0.6-1.5.
The positive control treated tissue showed 0.5% cell viability demonstrating the proper performance of the assay.
The difference of cell viability between the two test item-treated tissue samples in the MTT assay was 4.2%.
The difference of cell viability between the two negative control treated tissue samples in the MTT assay was 7.5%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.

The test item did not react with MTT as no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

Table 1: Optical density measurements and calculated relative viability of the samples

 

Optical density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative control: physiological saline

1

0.791

0.743

103.7

2

0.737

0.690

96.3

mean

-

0.717

100.0

Positive control: glacial acetic acid

1

0.051

0.003

0.5

2*

0.047

-0.001

-0.1

mean

-

0.003

0.5

Test substance

1

0.071

0.023

3.3

2

0.072

0.024

3.4

mean

-

0.024

3.3

Mean blank was 0.047; optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places); * negative values of positive control were excluded from the calculation

Validity of the test

After receipt, the two indicators of fitness for the delivered kit (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay.

The mean OD value of the two negative control tissues was 0.717, which satisfied the acceptability criterion of being in the range of 0.6-1.5.

The positive control treated tissue showed 0.5% cell viability demonstrating the proper performance of the assay.

assay was 4.2%.

The difference of cell viability between the two negative control treated tissue samples in the MTT assay was 7.5%.

The mean OD value of the blank samples (acidified isopropanol) was 0.047.

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
The substance was assessed to be corrosive to the skin.
Executive summary:

The skin corrosion potential of the substance was studied under GLP in accordance with OECD TG 431 using a reconstructed human epidermis model (EpiSkin). This model is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (Thiazolyl blue, CAS number 298-93-1) assay. Disks of the dermal model (two units) were treated with the powdered test substance and incubated for 4 hours at room temperature. Exposure of the skin model surface to the test substance was terminated by rinsing the units with phosphate buffered saline solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm.

The negative control epidermis units were treated with physiological saline (0.9% w/v NaCl solution), and the positive control units were treated with glacial acetic acid (two units per control). Two additional disks were used to provide an estimate of colour contribution from the coloured test item. For each treated tissue, viability was expressed as a percentage relative to the negative control. All the validity criteria were fulfilled in this study. As the mean cell viability of the test substance was 3.3% compared to the negative control after 4 hours, and this value is clearly less than the cut-off of 35%, the test substance was considered to be corrosive to skin.

Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The skin corrosion potential of the substance was studied in a valid in vitro test under GLP in accordance with OECD TG 431. The mean cell viability was 3.3% compared to the negative controls after an exposure period of 4 hours. This is clearly less than the cut-off value of 35%. The substance was assessed as being corrosive to skin and is classified as Skin Corr. Cat. 1 according to CLP Regulation (EC) No. 1272/2008.