Registration Dossier

Administrative data

Description of key information

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.

The myeloid U937 skin sensitization test (MUSST) is a dendritic cell activation test to predict skin sensitizing potential (BASF SE 65V0788/11A479, 2012). 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 CD 86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold in relation to vehicle control in at least two independent experiments.

Test substance GSID 3056-1 was tested in a concentration range of 2.74 to 43.80 µg/mL. No decrease in cell viability below 70% was observed. ln experiments 1 and 2 an induction of the expression of CD 86 was observed at sufficiently non-cytotoxic (cell viability > 70%) concentration.

ln summary, after 48 hours of exposure to test item GSID 3056 -1, CD 86 expression was induced in U937 cells at concentration between 2.74 and 10.95 µg/mL affording at least 70% viability. From this it has to be concluded that test substance GSID 3056-1 does induce dendritic cell activation and can be considered to be sensitizing.

The second in vitro assay performed is the Direct Peptide Reactivity Assay (DPRA) (BASF SE 64V0788/11A48, 2012). 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. In the Direct Peptide Reactivity Assay (DPRA) the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The peptides are custom material containing phenylalanine to facilitate UV-detection and either cysteine or lysine as the reactive center. The test substance was solved at a 100 mM concentration in propanol. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

The test item showed a reactivity of 61.8 % towards cysteine-peptide and of 0 (-3.6) to lysine-peptide. The mean reactivity is therefore calculated to be 30.9 %. Hence, the test substance is considered to be a moderate sensitizer.

Considering both positve results by reference to the paper by Bauch et al., 2012, the test item should be classified as sensitizing.

References:

- Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP. Quantification of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2): 417-427, 2007

- Bauch C., Kolle SN., Ramirez T., Eltze T., Fabian E., Mehling A., Teubner W., van Ravenzwaay B., Landsiedel R. Putting the Parts together: Combining in vitro methods to test for skin sensitizing potentials. Regulatory Toxicology and Pharmacology 63(3): 489 -504, 2012.


Migrated from Short description of key information:
Myeloid U937 Skin Sensitization Test MUSST (BASF SE 65V0788/11A479, 2012): indication for sensitizing potential
Direct Peptide Reactivity Assay DPRA (BASF SE 64V0788/11A486, 2012): indication to be a moderate sensitizer

Justification for selection of skin sensitisation endpoint:
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 (Bauch et al., 2012, Regulatory Toxicology and Pharmacology, 63 (3), 489-504).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Two acceptable in vitro Assays (MUSST, DPRA) in pre-validation give indications for the test item GSID 3056 -1 to be a skin sensitizer. Thus, the classification of GSID 3056 -1 as R43 according to Directive 67/548/EEC and in Category 1 according to the CLP Regulation (EC) Nn 1272 -2008 is proposed.