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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency, Method: HG-Gene Muta - S. ryphimurium: The Salmonella ryphimurium reverse mutation assay, 1984.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ametryn
EC Number:
212-634-7
EC Name:
Ametryn
Cas Number:
834-12-8
Molecular formula:
C9H17N5S
IUPAC Name:
N2-ethyl-6-(methylsulfanyl)-N4-(propan-2-yl)-1,3,5-triazine-2,4-diamine
Details on test material:
Identity: Ametryn technical
Chemical name: Ametryn (iso)
Batch number: 825
Expiry: 24 March 1996
Purity: 100.40 %
Appearance: White powder
Storage conditions: Room temperature in the dark
Date received: 16 May 1994

Method

Target gene:
In the In vitro technique described by Ames and his co-workers, (Ames, McCann and Yamasaki 1975, Maron and Ames 1983) mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test substance. Normally S. typhimurium is capable of synthesising the amino acid histidine it requires for growth but the mutant strains used in this test are incapable of this function. When large populations of these strains are exposed to a mutagen, reverse mutation to the original histidine independent form takes place in a proportion of the bacterial population. These colonies (revertant) are readily detectable due to their ability to grow on a histidine deficient medium.

The following strains of S. typhimurium were used in the test:
S. typhimurium TA 1535 hisG46 rfa uvrB
S. typhimurium TA 1537 hisC3076 rfa uvrB
S. typhimurium TA 98 hisD3052 rfa uvrB pKM101
S. typhimurium TA 100 hisG46 rfa uvrB pKM101
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
In the preliminary dose range finding study with dose levels of up to 5000 µg/plate.
A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.
Vehicle / solvent:
dimethyl sulphoxide
Controls
Untreated negative controls:
yes
Remarks:
dimethyl sulphoxide
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Ethyl-N-nitro-N-nitrosoguanidine, 9-Aminoacridine, 2-Nitrofluorene
Details on test system and experimental conditions:
In this in vitro assessment of the mutagenic potential of Ametryn technical, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control.
Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.
Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear positive or negative response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substances mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear positive response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et at.
(1989).
Statistics:
see above

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.
No evidence of mutagenic activity was seen at any dose level of Ametryn technical in either mutation test.
The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.
It is concluded that, when tested in dimethyl sulphoxide, Ametryn technical was not mutagenic in this bacterial system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative