2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

EC Number:

248-383-5

EC Name:

2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Cas Number:

27277-00-5

Molecular formula:

C9H13N5O

IUPAC Name:

2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Test material form:

solid: particulate/powder

Details on test material:

- Physical state/Appearance: Beige powder
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Histidine revertants (for Salmonella typhimurium strains)
tryptophan revertants (for Escherichia coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/5,6-benzoflavone induced rat liver S9 fraction

Test concentrations with justification for top dose:

EXPERIMENT 1 (PLATE INCORPORATION ASSAY) and EXPERIMENT 2 (PRE-INCUBATION ASSAY)
5, 15, 50, 150, 500, 1500, 5000 µg/plate (concentrations were corrected for the purity)

JUSTIFICATION FOR TOP DOSE
The tope dose in EXPERIMENT 1 (5000 μg/plate) is the standard limit concentration recommended in OECD 471. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals.
In the absence of any toxic effects, the maximum concentration selected for use in the Pre-incubation assay (EXPERIMENT 2) is the same as that used in the Plate incorporation assay. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration.

Vehicle / solvent:

- Solvent used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

Sterility control, plates were prepared without the addition of bacteria.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See Table in 'Any other information on materials and methods incl. tables'

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, EXPERIMENT 1) and preincubation (EXPERIMENT 2)

EXPERIMENTAL DESIGN
- Aliquots of 0.1 mL of the test item solutions, positive control or vehicle control were placed in glass tubes.
- S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar.

DURATION
- Preincubation period: 30 minutes with shaking (EXPERIMENT 2 only, preincubation before the addition of the agar overlay)
- Exposure duration: ca. 72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in mean revertant colony counts; sparse or absent background bacterial lawn

ANALYSIS OF DATA
The mean number and standard deviation of revertant colonies were calculated for all groups. The "fold-increases" relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Evaluation criteria:

See 'Any other information on materials and methods incl. tables'

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98 and TA1537

Remarks:

EXPERIMENT 1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA98 and TA1537

Remarks:

EXPERIMENT 1

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA100 and TA1535

Remarks:

EXPERIMENT 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

EXPERIMENT 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

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

Remarks:

EXPERIMENT 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

EXPERIMENT 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In EXPERIMENT 1 and 2, no precipitate was observed on plates following exposure to the test item.

ACCEPTENCE OF RESULTS
- criteria for the vehicle control were satisfied: The control mean revertants per plate for strains TA98 and TA1537 in the absence of metabolic activation in experiment 2 were just below the minimum HCD value (23.4 vs 24.0 and 7 vs 8) but were considered to be fully acceptable.
- criteria for the positive controls were satisfied
- criteria of analyzable concentrations were satisfied.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strains following exposure to the test substance in the absence of S9 mix at 50 μg/plate in strain TA98, and at 50 and 5000 μg/plate in strain TA1537 in EXPERIMENT 1.

Applicant's summary and conclusion

Conclusions:

In this ames test according to OECD 471, no evidence of mutagenic potential of the test item was found.

Executive summary:

In this GLP compliant in vitro study according to OECD 471, the mutagenic potential of the test item was assessed. Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test item diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of 5, 15, 50, 150, 500, 1500, 5000 µg/plate were tested. In the first experiment, toxicity, observed as a reduction in the number of revertant colonies, was obtained in strains following exposure to the test item in the absence of S9 mix at 50 μg/plate in strain TA98, and at 50 and 5000 μg/plate in strain TA1537. No precipitate was observed on any plates containing test item up to 5000 μg/plate. In the second experiment, no signs of toxicity towards the tester strains were observed in either mutation test following exposure. No precipitate was observed on any plates containing test item up to 5000 μg/plate. No evidence of mutagenic activity was seen at any concentration of test item in either experiment. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.