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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

254-296-3

EC Name:

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

Cas Number:

39108-47-9

Molecular formula:

C11H14N2O2

IUPAC Name:

1-butyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch No.: 162050203

In vitro test system

Details on the study design:

TEST SYSTEM
- Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol); Lysine-(K-)containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA and/or JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K­ containing peptide). The peptide stock solution were used for preparing the calibration samples, the test-substance and control samples.

CONTROLS
- Negative control (NC): vehicle control = propanol
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in propanol.
Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide.

TEST-SUBSTANCE PRPARATION
The test substance was prepared as a 100 mM preparation in propanol (considering a molecular weight of 206.24 g/mol and a purity/contents of 98.7%). After short stirring the test
substance was soluble in the vehicle.
Vehicle: propanol
Reason for the vehicle: The test substance was soluble in propanol.

EXPERIMENTAL PROCEDURE
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

TEST SUBSTANCE SOLUBILITY
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

PREPARATION OF PEPTIDE STOCK SOLUTIONS
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.

PREPARATION OF THE TEST-SUBSTANCE SAMPLES
The samples were prepared in suitable tubes and capped tightly and were incubated in the dark for 24 ± 2 hours. They should be incubated at 25°C ± 2.5°C. Due to a technical error of the incubator the actual incubation temperature was 29.6°C. Hence, the incubation temperature given in the OECD TG 442C was exceeded by ca. 2°C. However, this deviation does not influence the result of the DPRA as was demonstrated in an additional study.
Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

PREPARATION OF THE VEHICLE CONTROLS
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (propanol) instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

PREPARATION OF THE CO-ELUTION CONTROL
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Results and discussion

In vitro / in chemico

Results

Parameter:

other: mean peptide depletion [%]

Value:

1.37

Other effects / acceptance of results:

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%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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.

Executive summary:

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 following results were obtained in the DPRA:

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 cited in chapter 3.10 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.