Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-06 to 2017-04-06

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

EC Number:

236-860-0

EC Name:

Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Cas Number:

13518-93-9

Molecular formula:

C3H6N6.1/2H4O7P2

IUPAC Name:

diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Test material form:

solid

Method

Target gene:

His operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

microsomal preparation derived from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160 or 5000 μg dimelamine pyrophosphate per plate

Dimelamine pyrophosphate was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. Hence, 5000 μg dimelamine pyrophosphate/plate was chosen as the top concentration for the main study in the plate incorporation test and in the preincubation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Because of the small solubility of the test item in all solvents recommended, dimelamine pyrophosphate was suspended in highly purified water.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes for the preincubation method
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Evaluation criteria:

A test item is considered to show a positive response if:

- the number of revertants was significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) was observed;
- positive results were reproducible and the histidine independence of the revertants was confirmed by streaking random samples on histidine-free agar plates.

A test item for which the results does not meet the above mentioned criteria was considered as non-mutagenic in the AMES test.

Statistics:

Statistical methods were not reported.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test item precipitation was noted at all concentrations in all test strains.

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, dimelamine pyrophosphate tested up to a concentration of 5000 μg/plate, that led to test item precipitation, caused no mutagenic effect in the Salmonella typhimurium strains T A98, TA 100, TA 102, TA 1535, and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.