5-nitro-2,4,6-triaminopyrimidine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

5-nitro-2,4,6-triaminopyrimidine

EC Number:

213-742-7

EC Name:

5-nitro-2,4,6-triaminopyrimidine

Cas Number:

1006-23-1

Molecular formula:

C4 H6 N6 O

IUPAC Name:

5-nitropyrimidine-2,4,6-triamine

Details on test material:

- Name of test material (as cited in study report): 5-Nitroso-2,4,6-triaminopyrimidine
- Analytical purity: 100 % (HPLC at 328 nm)
- Physical state: Solid / pink to red
- Homogeneity: homogeneous by visual inspection
- Storage conditions: Room temperature

In chemico test system

Details on the study design:

synthetic peptides (Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH; Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH)

The test substance was prepared as a 100 mM stock solution in methanol just prior to incubation with the synthetic peptides.

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)
The peptides are custom material (Supplier: RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and
either cysteine or lysine as the reactive center.

Negative control (NC): vehicle control = methanol
Positive control (PC): Ethylene glycol dimethacrylate (CAS-no. 97-90-5)

Because the test substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.

Three samples of the test substance were incubated with each peptide for 24h at room temperature. The incubation tubes were sealed. 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 peptide standards of known concentration, prepared from the respective peptide stock solution used for test substance incubation, were measured in parallel with the same analysis method.

The test substance was incubated with the Cysteine-containing peptide in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance, pH 7.5 phosphate buffer
) and with the Lysine-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance, pH 10.2 ammonium acetate buffer).

The mean peptide depletion of a test substance was calculated as the mean value of Cys containing peptide depletion and Lys-containing peptide depletion.
According to the classification tree model described by Gerberick et al. for substances with known molecular weight highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitizer, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitizer, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitizer.

Results and discussion

Positive control results:

70% of the peptide was depleted by the positive control substance ethylene glycol dimethacrylate using methanol as solvent.

In vitro / in chemico

Results

Run / experiment:

other: Cys-Peptide

Parameter:

other: % peptide depletion

Value:

75

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

The mean Cysteine-peptide depletion, caused by the test substance was determined to be 75.70 %.

The mean Lysine-peptide depletion, caused by the test substance was determined to be -0.77%.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 37.85 %.

No co-elution of test substance and peptides was noticed.

Details are provided in the table.

Applicant's summary and conclusion

Interpretation of results:

other: peptide binding