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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-06-05 - 1997-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was performed acc. OECD TGs 471 and 472 of 1983, which are combined equal to the recent version of OECD TG 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Guideline no. 471: "Genetic Toxicology: Salmonella typhimurium Reverse Mutation Assay". (adopted May 26, 1983)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
Guideline no. 472: "Genetic Toxicology: Escherichia coli Reverse Mutation Assay", with strain WP2uvrA only (adopted May 26, 1983)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity;
B.13: "Mutagenicity: Escherichia coli - Reverse Mutation Assay", with strain WP2uvrA only
B.14: "Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay". EEC Publication no. L383 (adopted December, 1992)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6-amino-1,3-dimethyluracil
EC Number:
229-662-0
EC Name:
6-amino-1,3-dimethyluracil
Cas Number:
6642-31-5
Molecular formula:
C6H9N3O2
IUPAC Name:
6-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
Test material form:
solid: particulate/powder
Remarks:
off-white to yellow
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions

Method

Target gene:
his (S. typhimurium); trp (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:
Dr, Bruce N. Ames, University of California at Berkeley, U.S.A. Salmonella typhimurium strains)
TA100 received 18-02-1993, used batch: TA100.250297
TA98 received 21-02-1991, used batch: TA98.250297
TA1535 received 14-12-1994, used batch: TA1535.250297
TA1537 received 14-12-1994, used batch: TA1537.250297
- Suitability of cells: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

MEDIA USED
- Periodically 'cleansed' against high spontaneous background: yes
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Prof. Dr. B.A. Bridges, University of Sussex, Brighton, U.K., Escherichia coli strain received 23-10-1987 used batch: EC.290197
- Suitability of cells: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

MEDIA USED
- Periodically 'cleansed' against high spontaneous background: yes
The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9
Test concentrations with justification for top dose:
Top dose: 5 mg/plate
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin (DM), TA98, 4 µg/plate in saline, -S9; 2-Aminoanthracene (2-AA), 5 µg/plate (WP2uvrA), 2.5 µg/plate (TA1537), 1 µg/plate (TA1535, TA98, TA100) in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10exp8 / plate

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his / trp minimal agar

NUMBER OF REPLICATIONS: The test substance were tested in triplicate in each strain and condition, in two independent experiments.

DETERMINATION OF CYTOTOXICITY
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
Rationale for test conditions:
Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
Evaluation criteria:
ACCEPTABILITY OF ASSAY

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.

Strain S9 mix Minimum value Maximum value
TA1535 +S9 4 21
-S9 4 24
TA1537 +S9 3 22
-S9 3 28
TA98 +S9 12 50
-S9 14 58
TA100 +S9 55 223
-S9 63 247
WP2uvrA +S9 5 26
-S9 5 21

b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.

c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary range-finding test or should extend to 5 mg/plate.

DATA EVALUATION AND STATISTICAL PROCEDURES
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation,
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
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:
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:
E. coli WP2 uvr A
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
- Effects of pH: none stated
- Effects of osmolality: none stated
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES:
6-amino-1,3-dimethyluracil was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Precipitate
The test substance did not precipitate in the top agar.
Precipitation of 6-amino-1,3-dimethyluracil on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.
Toxicity
To determine the toxicity of 6-amino-1,3-dimethyluracil, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.
Based on these data, 6-amino-1,3-dimethyluracil was tested in the mutation assay up to concentrations of 5000 µg/plate in the absence and presence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see acceptance criteria
Remarks on result:
other: consistent in both independent experiments

Applicant's summary and conclusion

Conclusions:
The study was performed acc. OECD TGs 471 and 472 of 1983 being hence equal to the recent OECD TG 471. Positive and negative controls gave the appropriate results. Hence, the results can be considered to be sufficiently reliable to assess the potential of 6-amino-1,3-dimethyluracil to induce gene mutations in bacteria.
In strain TA100, in the presence of S9-mix, 6-amino-1,3-dimethyluracil showed 1.6-fold increases in the number of revertant (His+) colonies in both experiments. Since these increases were within the historical control data range and not two-fold, this test result is not considered to be a positive response. Strain TA100, in the absence of S9-mix, and all other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
Based on the results of this study it is concluded that 6-amino-1,3-dimethyluracil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

6-amino-1,3-dimethyluracil was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli Wp2UvrA in two independent experiments acc. OECD TGs 471 and 472 of 1983.

6-amino-1,3-dimethyluracil was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix.

In strain TA100, in the presence of S9-mix, 6-amino-1,3-dimethyluracil showed 1.6-fold increases in the number of revertant (His+) colonies in both experiments. Since these increases were within the historical control data range and not two-fold, this test result is not considered to be a positive response.

Strain TA100, in the absence of S9-mix, and all other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory background historical ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 6-amino-1,3-dimethyluracil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.