1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Endpoint summary

Description of key information

The skin sensitizing potential of the test substance was assessed using an in vitro OECD guideline testing strategy comprising the following assays:

- Direct Peptide Reactivity Assay (DPRA),

- Keratinocyte Activation Assay (LuSens), and

- Dendritic Cell Line Activation Assay (h-CLAT).

Each test was conducted under GLP according to the respective OECD guideline.

The results were as follows:

- DPRA: negative

- LuSens: negative

- h-CLAT: positive

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

DPRA:

The reactivity of 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile 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 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 100 mM in propanol. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for Kcontaining peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a coelution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm. The test substance was dissolved in propanol 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. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 1.70%. The mean K-peptide depletion, caused by the test substance was determined to be 1.04%. Thus, the mean peptide depletion was calculated to be 1.37%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2 -oxonicotinonitrile shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

LuSens:

The keratinocyte activating potential of test substance 1-butyl-1,2-dihydro-6 -hydroxy-4-methyl-2-oxonicotinonitrile was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. No cytotoxicity was observed. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

In summary, after 48 hours of exposure to test substance 1-butyl-1,2-dihydro-6-hydroxy-4- methyl-2-oxonicotinonitrile luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile does not have a keratinocyte activating potential.

h-CLAT:

The potential of test substance 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2 -oxonicotinonitrile 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 (non-GLP) was performed. Cells were exposed to several 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 2262 μg/mL (corresponding to test substance as provided by the sponsor). 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 experiments were performed. At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations) and in 0.2% DMSO in culture medium (final concentrations). No precipitates were noticed in any concentration after 24 hours.

In summary, after 24 hours of exposure to test substance 1-butyl-1,2-dihydro-6-hydroxy-4 -methyl-2-oxonicotinonitrile 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 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile induces dendritic cell activation.

Test Method	Test Result	Test Evaluation
Direct Peptide Reactivity Assay (DPRA)	1.37% mean peptide depletion (1.70% cysteine-peptide depletion; 1.04% lysinepeptide depletion).	Negative
Kerstinocyte Activation Assay – LuSens	In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.a	Negative
Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)	In at least two independent experiments an induction of the expression of CD54 (above 200%) was observed at sufficiently non-cytotoxic (cell viabilit≥ 50%) concentration.	Positive

Based on the results of all three tests and applying the evaluation criteria, 1-butyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile is not peptide reactive, does not activate keratinocytes and activates dendritic cells. The test item is predicted not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item was not classified and labelled accordingto Regulation (EC) No 1272/2008 (CLP).