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EC number: 208-060-1 | CAS number: 506-93-4
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:
- 1988-05-04 to 1988-06-13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 988
- Report date:
- 1988
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1983-05-26
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Guanidinium nitrate
- EC Number:
- 208-060-1
- EC Name:
- Guanidinium nitrate
- Cas Number:
- 506-93-4
- Molecular formula:
- CH6N4O3
- IUPAC Name:
- amino(imino)methanaminium nitrate
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- water
- Reference substance name:
- Ammonium nitrate
- EC Number:
- 229-347-8
- EC Name:
- Ammonium nitrate
- Cas Number:
- 6484-52-2
- Molecular formula:
- H3N.HNO3
- IUPAC Name:
- Ammonium nitrate
- Reference substance name:
- 4,6-diamino-1,3,5-triazin-2(1H)-one
- EC Number:
- 211-455-1
- EC Name:
- 4,6-diamino-1,3,5-triazin-2(1H)-one
- Cas Number:
- 645-92-1
- Molecular formula:
- C3H5N5O
- IUPAC Name:
- 4,6-diamino-1,3,5-triazin-2(1H)-one
- Reference substance name:
- (4,6-Diamino-1,3,5-triazine-2-yl)guanidine
- Cas Number:
- 4405-08-7
- Molecular formula:
- C4H8N8
- IUPAC Name:
- (4,6-Diamino-1,3,5-triazine-2-yl)guanidine
- Reference substance name:
- Melamine
- EC Number:
- 203-615-4
- EC Name:
- Melamine
- Cas Number:
- 108-78-1
- Molecular formula:
- C3H6N6
- IUPAC Name:
- 1,3,5-triazine-2,4,6-triamine
- Reference substance name:
- Cyanoguanidine
- EC Number:
- 207-312-8
- EC Name:
- Cyanoguanidine
- Cas Number:
- 461-58-5
- Molecular formula:
- C2H4N4
- IUPAC Name:
- 1-cyanoguanidine
- Test material form:
- solid: crystalline
Constituent 1
impurity 1
impurity 2
impurity 3
impurity 4
impurity 5
impurity 6
Method
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: Histidine auxotroph
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- other: Histidine auxotroph
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (fraction of liver of male Wistar rats incuded with Aroclor 1254); prepared by the testing facility.
- Test concentrations with justification for top dose:
- Range finder and first experiment: with and without metabolic activation 8, 40, 200, 1000, 5000 μg/plate
Second experiment: with and without metabolic activation: 1000, 2000, 3000, 4000, 5000 μg/plate - Vehicle / solvent:
- - Vehicle used: sterile distilled water
Controls
- Untreated negative controls:
- yes
- Remarks:
- equal to vehicle control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle is sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: not done
- Exposure duration: at least 2 days
NUMBER OF PLATES EVALUATED: three per dose
NUMBER OF REPLICATIONS: two indipendent experiments were performed
DETERMINATION OF CYTOTOXICITY: by inspection of the background lawn - Evaluation criteria:
- ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
(i) the mean negative control counts fell within the normal range of the test facility
(ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
(iii) no more than 5% of the plates were lost through contamination or some other unforseen event
EVALUATION CRITERIA
A test compound was considered to be mutagenic if:
(i) the assay was valid (see ACCEPTANCE CRITERIA)
(ii) two or three-fold increases (dependent on strain) in revertant numbers, were accompanied by significant F-statistics and dose response correlations
(iii) the positive responses described in (ii) were reproducible - Statistics:
- not performed due to absence of two or three-fold infcreases (dependent on strain) in revertant numbers
Results and discussion
Test resultsopen allclose all
- 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:
- valid
- True negative controls validity:
- not examined
- 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:
- valid
- True negative controls validity:
- not examined
- 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:
- valid
- True negative controls validity:
- not examined
- 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:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Strain TA100 was tested with Guanidine Nitrate at final concentrations of 8, 40, 200, 1000 and 5000 μg/plate in the absence and presence of S9. No signs of toxicity were observed, so the data obtained were considered acceptable as TA100 mutagenicity data for experiment 1. The same dose
range was used to treat the other strains in experiment 1, and the only sign of toxicity seen was a reduction in TA9B revertants at the top dose
in the absence of S9.
For experiment 2 the same top dose was used, and a very slight reduction in TA100 revertants at 5000 μg/plate in the absence of S9 may have
indicated some toxicity. 'The range was narrowed (to 1000-5000 μg/plate) to examine more closely those doses most likely to elicit a mutagenic
response.
COMPARISON WITH HISTORICAL CONTROL DATA:
From the data it can be seen that mean solvent control counts fell within the normal historical range, that the positive control chemicals all induced large increases in revertant numbers in the appropriate strains, and that < 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
An initial toxicity range-finder experiment was carried out in TA100 only, using final concentrations of guanidine nitrate at 8, 40, 200, 1000 and 5000 μg/plate plus a solvent and positive control. No evidence of toxicity was observed in the range-finder experiment. - Remarks on result:
- other: all strains/cell types tested
Any other information on results incl. tables
All Guanidine Nitrate treatments, in both experiments, yielded revertant numbers that were similar to those seen in controls. The two-fold (TA98, TA100) and three~fold (TA1535, TA1537, TA1538) increasesin revertant numbers that would be required for a clear positive response were not achieved. The results, therefore, provide no evidence that Guanidine Nitrate can induce reverse mutation in these strains of bacteria, either in the absence or presence of rat liver S9.
Applicant's summary and conclusion
- Conclusions:
- Guanidine Nitrate is considered to be negative in the reverse gene mutation assay in bacteria (S. typhimurium strains TA 98, TA 100 TA 1535, TA l537 and TA l538), when tested up to the limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD guideline 471, 1983, strains TA 98, TA 100TA 1535, TA l537 and TA l538 of S. typhimurium were exposed toGuanidine Nitrate, (99.3 % a.i. dissolved in sterile water), at concentrations of 8, 40, 200, 1000 and 5000 µg/plate in the first experiment and1000, 2000, 3000, 4000, 5000 µg/plate in the second experiment in the presence and absence of mammalian metabolic activation (rat liver S9-mix) using the plate co-incubation method.
Guanidine Nitratewas tested up the limit concentration of 5000 µg/plate. No cytotoxicity and no increase in the number of revertants were observed in all tester strains, with or without metabolic activation.The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.
There was no evidence of induced mutant colonies over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 (1983) for in vitro mutagenicity (bacterial reverse gene mutation) data.
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