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Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 05 December 2016 and 10 February 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium glyoxylate
EC Number:
EC Name:
Sodium glyoxylate
Cas Number:
Molecular formula:
sodium oxoacetate
Specific details on test material used for the study:
Identification: Safelink SPM-01
Batch: G150201
Purity: 99.9% HPLC
Appearance: White, crystalline
Expiry Date: 17 November 2017
Stability in Solvent: Not indicated by the Sponsor
Storage Conditions: At room temperature

In vitro test system

Test system:
human skin model
Source species:
Cell type:
other: Normal Human-Derived Epidermal Keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Justification for test system used:
Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .
unchanged (no vehicle)
Details on test system:
EpiSkin™ Kit Components Needed for the Assay

EpiSkin™ Kit Lot No.: 17-EKIN-006
1 Sealed 12-well plate: Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate: For MTT viability assay
1 bottle Assay Medium: Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium: Basic medium for incubations

The MTT-solution was prepared freshly on day of use with DMEM or assay medium (concentration: 0.3 mg/mL).

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 07 February 2017. On the day of receipt the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (approximately 10 mg) was mixed with 90 μL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed. An additional test with viable tissues (without MTT addition) was not necessary. For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 10 mg of the test item were added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. If the MTT solution colour turns blue/purple, the test item is presumed to have reduced the MTT. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues was not necessary.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount(s) applied (volume or weight with unit): 10 mg (5 μL of deionised water were first topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis)

NEGATIVE CONTROL (Deionised water)
- Amount(s) applied (volume or weight): 10 μL

- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% in deionised water
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:

Test system

Details on study design:
Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for 22.6 hours.

The test item tissues were wetted with 5 μL of deionised water. The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after report finalization.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the wells were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for nearly 3 hours at room temperature with gentle agitation.
Per tissue sample 2 × 200 μL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease (119.3%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Any other information on results incl. tables

Results after treatment with Safelink SPM-01 and controls

Test Group

Absor-bance 570 nm
Tissue 1*

Absor-bance 570 nm
Tissue 2*

Absor-bance 570 nm
Tissue 3*

Mean Absor-bance of 3 Tissues

Relative Absor-bance [%] Tissue 1, 2 + 3**

Relative Standard Deviation [%]

Rel. Absor-bance

[% of Negative Control]***

Negative Control








Positive Control








Test Item








*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]

***     relative absorbance per treatment group [rounded values]

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, it can be stated that in this study and under the experimental conditions reported, Safelink SPM-01 is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of Safelink SPM-01 by means of the Human Skin Model Test according to OECD TG 439.

The white test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with 6eionized water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (6eionized water) or the positive control (5% Sodium lauryl sulfate) for 15 minutes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value did not decrease (119.3%). This value does not affect the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the Safelink SPM-01 is not irritant to skin according to UN GHS and EU CLP regulation.