Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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

Constituent 1
Chemical structure
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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.
Controlsopen allclose all
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
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
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
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
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
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
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
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
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
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 resultsopen allclose all
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
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
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
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
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.