Registration Dossier

Administrative data

Description of key information

Skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

ARE-Nrf2 Luciferase LuSens Test Method

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential)by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay based on the OECD 442D Guideline.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (1000 µM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMEM (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and p-Phenylenediamine (80 µM) as positive control.

No substantial and reproducible dose-dependent increase in luciferase induction equal or above 1.5 fold was observed in both experiments up to the maximal tested concentration of the test item.

Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2transcription factor (no sensitizing potential).

In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

In Vitro Skin Sensitisation assays associated with the process of activation of monocytes and dendritic cells This in vitro study was performed to assess the sensitising potential of the test item by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells, according to the OECD guideline 442E.

Two valid experiments with a treatment period of 24 hours were performed.

In the experiments, the highest nominal applied concentration (5000 µg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared and tested. Precipitation of the test item was not visible in any of the experiments.

As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium. As positive control, 2,4-dinitrochlorobenzene was used. Prior to the study, the cells used in the experiments were checked in a reactivity check and were found to be suitable for the experiments.

All acceptance criteria were met and therefore, the study was considered valid.

In both experiments (I and II) no cytotoxicity occurred at any tested concentration.

In both experiments (I and II) the RFI of CD54 was ≥ 200 % at any tested concentration with cell viability ≥ 50 %.

In experiment I and the RFI of CD86 was ≥ 150 % in the following concentrations (1395.4, 1674.5, 2009.4 and 2411.3 µg/mL). The EC 200 for CD54 is 329.25 µg/mL.

In experiment II the RFI of CD86 was not ≥ 150 % in any tested concentration.

In accordance with the classification criteria, the result of this study is positive and the substance is considered to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the Guidance on the application of CLP criteria (ECHA, 2017): ''Validated in vitro/in chemico methods exist with the aim to identify a sensitising potential of a chemical. These include OECD TG442C (Peptide/protein binding), TG442D (keratinocyte response) and TG 442E (monocytic/dendritic cell response). The in vitro/in chemico tests are not regarded as stand alone tests and the result from such a test should be used together with other data in an overall assessment. Further, at present there is no agreed strategy on how to use in vitro/in chemico methods for direct estimation of sensitising potency, but data from such tests can be used together with other data in order to assess skin sensitisation potency. See also the Guidance on IR&CSA, especially Section R.7.3.4.1.''

Based on the results obtained in the two in vitro tests above described, the test substance has been classified as skin sensitizer Category 1.