Sodium 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazinecarboxamidate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-04-13 to 2017-04-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazinecarboxamidate

EC Number:

283-029-3

EC Name:

Sodium 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazinecarboxamidate

Cas Number:

84522-34-9

Molecular formula:

C14H16N4NaO3S

IUPAC Name:

5-methyl-N-[2-(4-sulfamoylphenyl)ethyl]pyrazine-2-carboxamide;sodium

Specific details on test material used for the study:

Batch: 16024R046
Purity: 100.3%

In chemico test system

Details on the study design:

Test system
- Test system: Synthetic peptides containing cysteine (SPCC) or synthetic peptides containing lysine (SPCL).
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH

Experimental Design
- Test Item Preparation: 52.96 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1542 μL MQ/ACN (1:1, v/v), to obtain a 100 mM solution.
- Preparation of Solutions for Cysteine Reactivity Assay:
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
- Preparation of Solutions for Lysine Reactivity Assay:
Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
- Sample Incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 26 and 27 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- HPLC-PDA Analysis: SPCC and SPCL peak areas in the samples were measured by HPLC-PDA.
- Other measurement: Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

Results and discussion

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 73.6% ± 0.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD<14.9%).
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 58.3% ±3.3%.. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD<11.6%).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC depletion%

Positive controls validity:

valid

Remarks on result:

other: The test item clearly co-eluted with SPCC and calculation of the peptide depletion was not possible.

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL depletion%

Value:

6.5

Positive controls validity:

valid

Other effects / acceptance of results:

- Precipitation: Cysteine Reactivity Assay: Upon preparation no precipitate was observed in any of the test item samples. However, after incubation a precipitate was observed in the co-elution control (CC) and test item samples; Lysine Reactivity Assay: Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

Any other information on results incl. tables

In the cysteine reactivity assay the test item clearly co-eluted with SPCC and calculation of the peptide depletion was not possible. In the lysine reactivity assay the test item showed 6.5% SPCL depletion. However, after incubation precipitation of the test item was observed. Consequently, SPCL depletion may be underestimated. Since no DPRA prediction and reactivity classification can be made based on SPCL data alone, the DPRA results obtained for test item are inconclusive.

Applicant's summary and conclusion

Interpretation of results:

other: The DPRA results for test item are inconclusive.

Conclusions:

The DPRA results obtained for test item are inconclusive.

Executive summary:

The reactivity of test item towards model synthetic peptides containing either cysteine (SPCC) or lysine(SPCL) was determined according to OECD guideline 442C.

 

After incubation of test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution andphotodiodearray (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values werecalculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

 

In the cysteine reactivity assay the test item clearly co-eluted with SPCC and calculation of the peptide depletion was not possible. In the lysine reactivity assay the test item showed 6.5% SPCL depletion. However, after incubation precipitation of the test item was observed. Consequently, SPCL depletion may be underestimated. Since no DPRA prediction and reactivity classification can be made based on SPCL data alone, the DPRA results obtained for test item are inconclusive.

 

In the cysteine reactivity assay the test item clearly co-eluted with SPCC and calculation of the peptide depletion was not possible. In the lysine reactivity assay the test item showed 6.5% SPCL depletion. However, after incubation precipitation of the test item was observed. Consequently, SPCL depletion may be underestimated. Since no DPRA prediction and reactivity classification can be made based on SPCL data alone, the DPRA results obtained for test item are inconclusive.