Registration Dossier

Administrative data

Description of key information

Not a skin sensitiser

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The in vitro 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 WoE assessment. Two in vitro tests were performed for Key Event 2-Keratinocyte response- and Key Event 3-Dendritic cell response.

The first in vitro study was performed using the LuSens cell line. The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (2015). The assay differs in some points from the OECD guideline. The assay was performed in 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 (25 µg/ml) was chosen with regard to the cytotoxic reaction in the CRFT. Furthermore, a geometric series (factor 1.2) of eleven dilutions was prepared. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as medium control. Furthermore, Lactic acid (5000 µM) was used as negative control and EGDMA 2 (120 µM) as positive control.

No substantial and reproducible, dose-dependent increase in luciferase induction above 1.5 fold was observed in either experiment up to the maximal concentration of the test item. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess sensitising potential in accordance to the BASF protocol.

The second in vitro study was performed according to the OECD Guideline 442E (2017). The test is based on the 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. For this purpose the human monocytic leukaemia cell line THP-1 was used and treated with the test item for 24 h before evaluation.

Two valid experiments with a treatment period of 24 hours were performed. For the experiments, the highest nominal applied concentration (2000 µg/mL) was chosen based on the results obtained in the pre-tests. A geometric series (factor 1.2) of 7 dilutions was prepared. 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 (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used.

All acceptance criteria were met and therefore, the study was considered valid. In both experiments the RFI of CD86 was not ≥ 150 % as well as the RFI of CD54 was not ≥ 200 % at any tested concentration with cell viability ≥ 50 %.

Since the results of both individual runs is negative, the test item is considered as “negative” in the h-CLAT test. Therefore, the test item is considered to have no potential to activate dendritic cells and is not a sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In the CLP Regulation (EC 1272/2008), a skin sensitiser is defined as a substance that will lead to an allergic response following skin contact. Validated in vitro/in chemico methods exist with the aim to identify a sensitising potential of a chemical. The subject of in vitro testing for skin sensitisation is discussed in the Guidance on IR&CSA, Section R.7.3.4. There are several validated test methods for the assessment of skin sensitisation potential in vitro and, for some of them, EU/OECD- adopted test guidelines are available. These test methods have been developed with the purpose of using several in chemico/in vitro methods together, as described in section 8.3.1 of Annex VII to the REACH Regulation. Annex VII to the REACH Regulation specifies that when new data need to be generated to fulfil the standard information requirement for skin sensitisation, as a first step in chemico/in vitro studies assessing three key events of skin sensitisation should be performed, unless data from fewer key events already allows classification and risk assessment, as specified in Annex VII, section 8.3, column 2. Indicators of potency such as the level of peptide depletion and concentration-responses can be obtained from the existing in chemico and in vitro tests, respectively. Data from the tests: Direct Peptide Reactivity Assay (DPRA) for Key Event 1 Peptide/protein binding,  ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) for Key Event 2 Keratinocyte response and Human Cell Line Activation Test (h-CLAT) for Key Event 3 Monocytic /Dendritic cell response may be accepted to fulfil Annex VII requirement when used in combination with each other. These test methods were developed to address specific events of the skin sensitisation AOP (OECD, 2012). The AOP for skin sensitisation describes the current understanding of key events linked to skin sensitisation. As each of the test methods only addresses a specific key event of skin sensitisation, currently they should not be used in isolation to identify a skin sensitizer or a non-sensitizer.

In particular for the test item the following results were obtained: no potential to activate The Keap1-Nrf2-ARE pathway under the conditions of the LuSens test in accordance to the BASF protocol and no potential to activate dendritic cells under the conditions of the h-CLAT test in accordance to the OECD 442E.

Based on the available experimental data regarding the skin sensitisation potential on the test item, no classification as a skin sensitiser is warranted according to the CLP Regulation (EC) No. 1272/2008.