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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:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, compliant with OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: "Testing Methods for New chemical Substances", November 21, 2003 (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2 and Kanpokihatsu No. 031121002).
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(1E)-2-(2,4-diamino-6-hydroxypyrimidin-5-yl)diazen-1-yl]-5-methylbenzene-1-sulfonic acid
EC Number:
700-002-8
Cas Number:
1333483-07-0
Molecular formula:
C11H12N6SO4
IUPAC Name:
2-[(1E)-2-(2,4-diamino-6-hydroxypyrimidin-5-yl)diazen-1-yl]-5-methylbenzene-1-sulfonic acid
Details on test material:
Physical State / Colour: solid / yellow
Storage: at room temperature
Expiry Date: 31.12.2009

Method

Target gene:
Bacterial reverse mutation assays use amino-acid requiring strains of Salmonella typhimurium (S. typhimurium) and Escherichia coli (E. coli) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of these bacterial reversion assays is that they detect mutations which functionally reverse mutations present in the tester strains and restore the capability to synthesise an essential amino acid.
The purpose of this study is to establish the potential of the test item to induce gene mutations in bacteria by means of two independent S. typhimurium and E. coli reverse mutation assays. For the confirmatory experiment modifications are carried out which include the performance of the liquid pre-incubation assay with uninduced hamster liver S9 according to Prival et al. Under these conditions reduction of azo compounds to their constituent aromatic amines occurs to allow an evaluation of their mutagenic potential.
The S. typhimurium histidine (his) reversion system and the E. coli tryptophan (trp) reversion system measures his" -» his+ reversions and trp" -» trp+ . The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535) and fiameshift (TA 98, TA 1537) mutations. The E. coli strain WP2 uvrA detects only base substitution mutagens. These assays directly measure heritable DNA mutations of a type which is associated with adverse effects. Point mutations are the cause of many human genetic diseases and there is substantial evidence that somatic cell point mutations in oncogens and tumor suppressor genes are involved in cancer in humans and experimental systems.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa"; uvrB"; R-factor: frame shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa"; uvrB"; R-factor: base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa"; uvrB": base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: his C 3076; rfa"; uvrB": frame shift mutations
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: base-pair substitutions and others
Metabolic activation:
with and without
Metabolic activation system:
Mammalian microsome enzyme activation mixture (liver S9 of induced male Wistar rats)
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
The test item was suspended in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide, 4-nitro-o-phenylene-diamine, Methyl methane sulfonate, 2-aminoanthracene.
Remarks:
For each strain was used a specific positive control substance.
Details on test system and experimental conditions:
The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment IT) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA, Experiment II was performed with the modification according to Prival to allow for azo reduction.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic ctivation. The concentrations, including the controls, were tested in triplicate.
Evaluation criteria:
Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of them background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.

Evaluation of Mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

According to the OECD guidelines, the biological relevance of the result is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
A test is considered acceptable if for each strain;

the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
the control plates without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range
-S9
TA98: 18-54
TA 100; 75-167
TA 1535: 5 -29
TA 1537: 5 -30
E. coli WP2 uvrA 35-92

- corresponding background growth on both negative control and test plates is observed,
- the positive controls show a distinct enhancement of revertant rates over the control plate.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimnrium strains TA98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. Experiment II was performed with the modification according to Prival to allow for azo reduction. In two independent experiments several concentrations of the test item
were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 ug/plate
Precipitation of the test item was observed in all tester strains used in experiment I and II with and without metabolic activation. In experiment I
precipitation of the test item was found at doses of 1000 (ug/plate and higher (with and without metabolic activation), in experiment II
precipitation of the test item was found at doses of 2500 ug/plate and higher (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic
activation in experiment I and II. The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment I in tester strain TA 1535 at a dose of 100 (ug/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response
relationship
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

hi conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.