Reaction products of diazotized Dodecyl Aniline coupled with 8-acetylamine-1-hydroxynaphthalen-3,6-disulphonic Acid, sodium salts

Endpoint summary

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 (2018). This test is part of a tiered strategy for the evaluation of skin sensitization potential. Therefore, data generated 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 assay included a cytotoxicity range finder test (CRFT) and one experiment, consisting of two independent repetitions (repetition 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 these results, the concentrations for the repetitions were determined. In the experiment (repetition I and II), the highest nominal applied concentration (62.5 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 repetitions. DMSO (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 EGDMA (120 µM) as positive control. No substantial and reproducible dose-dependent increase in luciferase induction equal or above 1.5 fold was observed in both repetitions up to the maximal tested non-cytotoxic concentration of the test item. All the validity criteria were fullfilled. Therefore, the test item was negative in the LuSens assay and is considered not to have the potential to activate the Nrf2 transcription factor under the experimental conditions of this study.

(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. In total two pre-tests and three experiments (experiment I - III) with a treatment period of 24 hours were performed.

For the experiments, the highest nominal applied concentration (250 µg/mL for experiment I and II) was chosen based on the results obtained in the pre-tests. For experiment III, the highest nominal applied concentration (300 µg/mL) was chosen based on the result obtained in experiment II. A geometric series (factor 1.2) of 7 dilutions was prepared.

Sedimentation of the test item was visible in the dilutions of the stock solution in all con-centrations in all experiments. No sediments occurred in the working solutions of the ex-periments and the pre-tests, except the 4 highest concentrations of both pre-tests. The sediments did not have any impact on the result of the study, due to the fact that they were dissolved in the working solutions, no precipitates were visible and the flow cytometry analysis was correct and reproduceable regarding viability as well as sensitization.

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.

Due to cytotoxicity of the test item, too few viable cells (<10000) were left to be measured at the following concentrations: experiment I: 250 µg/mL, experiment III: 208.3 µg/mL, 250.0 µg/mL, 300µg/mL; therefore these concentrations were not taken into account for the analysis. In all experiments the RFI of CD86 was not ≥ 150 % as well as the RFI of CD54 was not ≥ 200 % at any tested, non-cytotoxic concentration.

Since the majority result of the three individual runs is negative, the test item is considered as negative in the h-CLAT under the experimental conditions; therefore the substance is considered not to have the potential to activate 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.