Amines, bis(C11-14-branched and linear alkyl), O,O-di-iso-Pr phosphorodithioates

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing two major steps of the sensitization process: protein reactivity and activation of dendritic cells has been proposed in a strategy to assess the sensitizing potential. To that end, the test substance has been tested in the Direct Pepitde Reactivity Assay (DPRA) and the Dendritic Cell Activation Assay Myeloid U937 Skin Sensitization Test (MUSST). In addition, a third in vitro experiement (Antioxidant response element (ARE) reporter assay) is currently ongoing.

Direct Peptide Reactivity Assay (DPRA)

Chemical reactivity has been shown to be well associated with allergenic potency (Gerberick et al., 2007). Within this context measuring the amount of proteins with nucleophilic side chains such as cysteine or lysine residues after incubation with putative allergens may serve as surrogate markers. The reactivity of the test article 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 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the negative control samples and expressed as relative peptide depletion.

The test substance did not cause depletion of the cysteine-containing peptide. However, depletion of the lysine-containing peptide between 19.4% and 26.5% was observed. In addition co-elution of the test substance with the lysine-containing peptide was noticed. Nevertheless the results are considered to be valid as the three test runs performed with different analytical methods produced comparable values for peptide depletion. Additionally peak purity, determined in the 3rdtest run, demonstrated that the peptide peaks could be integrated correctly.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a low chemical reactivity in the DPRA under the test conditions chosen.

Dendritic Cell Activation Assay Myeloid U937 Skin Sensitization Test (MUSST)

The myeloid U937 skin sensitization test (MUSST) is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane marker CD86 measured by flow cytometry after 48 hours of test substance exposure was determined.

In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance (0.5 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test, test substance was used at five final concentrations determined with regard to the CV75 value: CV75 x 2, CV75, CV75/2, CV75/4, CV75/8. A test substance was predicted to have a dendritic cell activating potential when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently oncytotoxic (cell viability ≥ 70%) concentration in at least two independent experiments.

After 48 hours of exposure to test substance CD 86 expression was induced in U937 cells at concentration 24.5 μg/mL affording at least 70% viability (marker induction value was 1.47 in experiment 1 and 1.30 in experiment 3). From this it has to be concluded that test substance does induce dendritic cell activation.

Antioxidant response element (ARE) reporter assay

This assay is currently ongoing and the registration dossier will be updated accordingly upon completion.

Summary

In two in vitro tests the test article gave an indication for a sensitizing potential. Therefore, the test substance has to be classified for skin sensitization.

Migrated from Short description of key information:
The results of an in vitro test battery (DPRA and MUSST assay) suggest a skin sensitization potential for the test article.

Justification for classification or non-classification

The rsults of two in vitro tests give indications for a skin sensitizing potential. Therefore, the test article is classified for skin sensitization in accordance with Directive 67/548/EEC and Regulation (EC) No 1272/2008.