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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 March, 1994 to 22 March, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
Trisodium [μ-[3-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-4,5-dihydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)
EC Number:
278-174-4
EC Name:
Trisodium [μ-[3-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-4,5-dihydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)
Cas Number:
75284-35-4
Molecular formula:
C39H20Cu2N5O15S3.3Na
IUPAC Name:
trisodium [μ-[3-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-4,5-dihydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)
Test material form:
solid: particulate/powder

Method

Target gene:
Histidine auxotrophs
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction)
Test concentrations with justification for top dose:
Six concentrations of FAT 11127/C (Solophenyl Marine BL roh trocken) ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.
Vehicle / solvent:
Dimethyl Sulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-Aminoanthracene: TA 100, TA102, TA 98 and TA 1537 with S9
Details on test system and experimental conditions:
Agar plate incorporation : 0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl and was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+) biotin dissolved in water.

Incubation period : 48 hours at 37±1.5°C in darkness.
Evaluation criteria:
Assay accpetance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response
The test substance is considered to be positive if the following condition is met:
• At least a reproducible meaningful increase of the mean number of revertant per plate above that of the negative control at any concentration for one or more of the strains tested. Generally, a concentration-related effect should be demonstrable.
Statistics:
A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: TA 100, TA 1535, WP2 uvrA, TA 102, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Range finding test :

Six concentrations of FAT 11127/C (Solophenyl Marine BL roh trocken) ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

Mutagenicity test, original experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 11127/C did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment:

Since the purity of the test material is about 80%, in this experimental part the concentration range of 77.2 to 6250.0 µg/plate was used. In the experiments performed with and without metabolic activation for strains TA 100, TA 102, TA 1535 and TA 1537, no increase in the incidence of either histidine-prototrophic mutants was observed in comparison with the negative control. However, in the experiment with strain TA 98 without metabolic activation, a marginal increase in the number of back-mutants was observed at the concentration of 6250 µg/plate. No effect was seen in the corresponding experiment with microsomal activation.

Mutagenicity test, confirmatory experiment 2 :

To confirm the effect observed in the first confirmatory without metabolic activation on strain TA 98, this experiment was repeated. Again, a marginal increase in the number of back-mutants was registered at the highest concentration.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Applicant's summary and conclusion

Conclusions:
FAT 11127/C exerted a marginal mutagenic effect in strain TA 98 without metabolic activation.
Executive summary:

FAT 11127/C was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium viz TA 98, TA 100, TA 102, TA 1535 and TA 1537 according to OECD Guideline 471. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was tested as a suspension in DMSO at five concentrations in the range of 312.5 to 5000.0 µg/plate in the presence and absence of a metabolic activation system.

 

In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 77.2 to 6250.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

 

In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 11127/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control with strains TA 100, TA 102, TA 1535 and TA 1537. With strain TA 98, however, a slight increase in the number of histidine-prototrophic mutants was observed in the experiments without metabolic activation at the concentration of 6250.0 µg/plate. No effect was seen in the corresponding experiment with metabolic activation. In order to confirm the effect observed in the first confirmatory without metabolic activation on strain TA 98, this experiment was repeated. Again a marginal increase in the number of back-mutants was registered at the highest concentration.

Hence it can be concluded that, based on the results of these experiments and on standard evaluation criteria, FAT 11127/C (Solophenyl Marine BL roh trocken) exerted a marginal mutagenic effect in strain TA 98 without metabolic activation.