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

Study period:

03 May 2016 to 15 May 2016

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

248-387-7

EC Name:

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

Cas Number:

27287-73-6

Molecular formula:

C6H7N5O

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

-Storage conditions: room temperature in the dark
-Colour: beige

Method

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

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:

Phenobarbitone / 5,6 benzoflavone

Test concentrations with justification for top dose:

Strains TA100, TA1535, TA1537 and WP2 uvrA:
Experiment 1: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Strain TA98:
Experiment 1: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 : 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test item was determined in a number of solvents and found to be most soluble in DMSO.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) for experiment 1 and preincubation for experiment 2.

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72hrs

NUMBER OF REPLICATIONS: Triplicate for each treatment

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed since additionally this is not required by the guideline. If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence of S9 mix.

Any other information on results incl. tables

Experiment 1 (Plate Incorporation Assay)

In the first experiment, toxicity, observed as reduction in the number of revertant colonies, was obtained in strains following exposure to the test item in strain TA98 at 150 μg/plate and above in the absence of S9 mix and in strain TA1535 at 1500 μg/plate in the absence of S9 mix. No precipitate was observed on plates following exposure to the test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second experiment.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Strain TA98, in the absence of S9 mix, did not fulfil the criteria for a valid experiment as set out in the study plan as only 3 non-toxic concentrations were obtained, therefore an additional experiment was performed.

 

Additional Plate Incorporation Assay

No evidence of toxicity was obtained following exposure to the test item in strain TA98. Previously toxic concentrations (150 μg/plate and above) produced 0.6 fold revertants of the vehicle control culture. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second experiment. No substantial increases in revertant colony numbers over control counts were obtained with strain TA98 following exposure to the test item at any concentration up to and including5000 μg/plate in the absence of S9 mix.

Experiment 2 (Pre-incubation Assay)

Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA98 following exposure to the test item at 500 ug/plate and above in the absence and presence of S9 mix. Thinning of the background lawn was observed at 1500 μg/plate and above in both the absence and presence of S9 mix in strain TA98.

Toxicity, observed as reduction in the number of revertant colonies, was obtained in strain TA1535 following exposure to the test item at 5, 500 and 5000 ug/plate in the absence of S9 mix and 5000 μg/plate in the presence of S9 mix. The reduction to 0.5 fold at 5 ug/plate in the absence of S9 mix is considered to be an anomaly and therefore considered not to be of significance.

Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA1537 following exposure to the test item at 500 and 5000 μg/plate in the absence of S9 mix and 50 μg/plate in the presence of S9 mix.

Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain WP2 uvrA (pKM101) following exposure to the test item at 5000 μg/plate in the presence of S9 mix.

No precipitate was observed on plates following exposure to the test item. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

The mutagenic potential of the test item was assessed according to the OECD Test Guideline 471. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

The mutagenic potential of the test item to 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 assessed according to the OECD Guidelines for the Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix).

The first experiment was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of the test item up to 5000 μg/plate were tested. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. In the first experiment, toxicity, observed as reduction in the number of revertant colonies, was obtained in strains following exposure to the test item in strain TA98 at 150 μg/plate and above in the absence of S9 mix and in strain TA1535 at 1500 μg/plate in the absence of S9 mix. No precipitate was observed on plates following exposure to test item. Strain TA98, in the absence of S9 mix, did not fulfil the criteria for a valid experiment as set out in the study plan, therefore an additional experiment was performed. No evidence of toxicity was obtained in the additional experiment following exposure to the test item in strain TA98. Previously toxic concentrations (150 μg/plate and above) produced 0.6 fold revertants of the vehicle control culture.

In the second experiment, toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA98 following exposure to the test item at 500 ug/plate and above in the absence and presence of S9 mix. Thinning of the background lawn was observed at 1500 μg/plate and above in both the absence and presence of S9 mix in strain TA98. Toxicity, observed as reduction in the number of revertant colonies, was obtained in strain TA1535 following exposure to the test item at 5, 500 and 5000 ug/plate in the absence of S9 mix and 5000 μg/plate in the presence of S9 mix. The reduction to 0.5 fold at 5 ug/plate in the absence of S9 mix is considered to be an anomaly and therefore considered not to be of significance. Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain TA1537 following exposure to the test item at 500 and 5000 μg/plate in the absence of S9 mix and 50 μg/plate in the presence of S9 mix. Toxicity, observed as a reduction in the number of revertant colonies, was obtained in strain WP2 uvrA (pKM101) following exposure to the test item at 5000 μg/plate in the presence of S9 mix. No evidence of mutagenic activity was seen at any concentration of the test item in either experiment.

The concurrent positive controls verified the sensitivity of the assay and the metabolising 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.