Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Endpoint summary

Description of key information

Skin sensitisation: Based on the results of an in chemico/in vitro test strategy the test item is peptide reactive (DPRA) and activates dendritic cells (h-CLAT). Therefore, the substance is predicted to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A combination of three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of the in vitro Skin Sensitization Turnkey Testing Strategy to access the skin sensitization potential of the test item. However, in the current case the results derived with DPRA and h-CLAT were sufficient for a final assessment. Therefore, further testing in LuSens was waived.

DPRA

The reactivity of the test item towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (=VC) were incubated with the peptides.

Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. The mean C-peptide depletion, caused by the test substance was determined to be 100%. The mean K-peptide depletion could not be determined due to interference using the standard analytical procedure.

Due to the unambiguous result of the C-peptide reaction, an evaluable K-peptide reaction cannot lead to another result of the study, as evenwhen no K-peptide depletion was present, the prediction would still be the same and hence no repetition of the K-peptide reaction using another analytic method was performed.

Based on the observed results and applying the cysteine 1:10 prediction the test item shows a high chemical reactivity in the DPRA under the test conditions chosen.

h-CLAT

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 7 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 50 μg/mL. In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid and evaluable experiments were performed.

At concentrations used in the main experiment the test substance was soluble in DMSO and in 0.2% DMSO in culture medium. No precipitates were noticed in any concentration after 24 hours.

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 and an EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable. In summary, after 24 hours of exposure to test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance induces dendritic cell activation.

Conclusion

Taken together, the test item is peptide reactive and activates dendritic cells. Applying the evaluation criteria, the test item is predicted to be a skin sensitizer (BASF, 2017).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes. As a result the substance is considered to be classified for skin sensitisation Cat 1 (H317: "May cause an allergic skin reaction") under Regulation (EC) No 1272/2008,as amended for the tenth time in Regulation (EU) No 2017/776.