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Description of key information

not 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 as follows:

(i) Direct Peptide Reactivity Assay (DPRA) for Key Event 1 Peptide/protein binding

DPRA is not applicable to the testing of UVCB substances due to the defined molar ratio of the test substance and peptide. However, the structures of the main constituents of the organic content were modelled in the QSAR Toolbox 4.2 and noProtein binding potency Lys (DPRA 13%) and Protein binding potency Cys (DPRA 13%) alerts were prediced

(ii) ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) for Key Event 2 Keratinocyte response

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor by using the LuSens cell line, according to the OECD guideline 442D (Draft v. 21. Dec. 2017).

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 (250 μg/mL) 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.

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

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

The viability in the positive control was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

The viability of negative control was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

In both experiments none of the tested concentrations induced a cytotoxic effect. But since this deviation was considered as uncritical, the study is considered as valid.

No cytotoxic effect was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.

In all tested concentrations no increase ≥ 1.5 fold in luciferase induction or a concentration-related effect was measured. In addition, in both experiments a negative trend was observed at the higher test item concentrations.

Both experiments are clearly negative. Therefore, the test item was negative in the LuSens assay under the experimental conditions of this study and it is considered not having the potential to activate the Nrf2 transcription factor.

(iii) Human Cell Line Activation Test (h-CLAT) for Key Event 3 Monocytic /Dendritic cell response.

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. One pre-test and two valid experiments with a treatment period of 24 hours were performed.

For the experiments, the highest nominal applied concentration (1000 µg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions was prepared. Precipitation of the test item was not visible in none 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.

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 the cell viability was higher than 90%. The RFI of CD86 was not ≥150 % and the RFI of CD54 was not ≥ 200 % at any tested concentration.

Therefore, both experiments induced a clearly negative result.

Under the experimental conditions of this study, the test item was negative in the h-CLAT and is therefore considered not to have the potential toactivate dendritic cells.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

In the CLP Regulation (EC) no. 1272/2008 a skin sensitizer 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

(i) Direct Peptide Reactivity Assay (DPRA) for Key Event 1 Peptide/protein binding

(ii) ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) for Key Event 2 Keratinocyte response

(iii) 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 substance the following results were obtained:

(i) DPRA is not applicable to the testing of UVCB substances due to the defined molar ratio of the test substance and peptide. However, the structures of the main constituents of the organic content were modelled in the QSAR Toolbox 4.2 and no Protein binding potency Lys (DPRA 13%) and Protein binding potency Cys (DPRA 13%) alerts were prediced

(ii) no potential to activate The Keap1-Nrf2-ARE pathway under the conditions of the LuSens test

(iii)  no potential to activate dendritic cells under the conditions of the h-CLAT test.

Therefore, based on the results of skin sensitisation, no classification for skin sensitization is warranted under the CLP Regulation (EC) no. 1272/2008.