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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-11-24 - 2021-07-29
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)
GLP compliance:
yes (incl. QA statement)
Type of study:
ARE-Nrf2 luciferase KeratinoSens™ test method
Justification for non-LLNA method:
The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line (e.g. KeratinoSens™) derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Test material

Chemical structure
Reference substance name:
By-product from Guanidinoacetic acid manufacturing
EC Number:
Molecular formula:
- Hydantoic Acid Sodium salt: C3H5N2O3Na - Cyanoguanidine: C2H4N4 - Sodium acetate: C3H3O2Na - Glycine: C2H5NO2 - N,N'-Guanidinodiacetic acid: C5H9N3O4 - N,N-Guanidinodiacetic acid: C5H9N3O4 - Guanidinoacetic acid:C3H7N3O2 - Urea: CH4N2O - Water: H2O - Biguanidinoacetic acid: C4H10N6O2 - Sodium formate: CHO2Na - Glycylglycine: C4H8N2O3
By-product from Guanidinoacetic acid manufacturing
Test material form:
Specific details on test material used for the study:
201009MA2 solid

In vitro test system

Details of test system:
Keratinoses transgenic cell line [442D]
Vehicle / solvent control:

Results and discussion

In vitro / in chemico

Key result
test chemical
Run / experiment:
Imax [442D]
1.07 %
Vehicle controls validity:
not specified
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and

In the present study By-product from Guanidinoacetic acid manufacturing was dissolved in dist. water.

Based on a calculated molecular weight of 77.20 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.53 was determined at a test item concentration of 2000 µM, but increase was not statistically significant (p <0.05) compared to the negative control. The corresponding cell viability was 71.9%. At the next lower concentration of 1000 µM, a statistically significant luciferase activity of 1.52 was determined. The corresponding cell viability was 82.1%. No further induction about the threshold of 1.5 was observable. Microscopically, slight cytotoxic effects were observed at the two highest test item concentrations. The calculated EC1.5 was <1000 µM (850.55). Due to the microscopically observed cytotoxic effects, the not statistically significant Imax and the missing dose-response relationship, the test material is considered to be a non-sensitiser in the first experiment.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range and no EC1.5 value could be calculated. Therefore, the test material is considered to be a non-sensitiser in the second experiment.

Under the condition of this study the test item is therefore considered as non-sensitiser

The controls confirmed the validity of the study.