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Toxicological information

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:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four tester strains tested, no tester strain to detect cross-linking mutagens was included
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
December 29, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
July 13, 1987
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 21016/C
- Physical state: violet powder
- Analytical purity: 96 %
- Lot/batch No.: 400047.32
- Expiration date of the lot/batch: December 1998
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 400047.32
- Expiration date of the lot/batch: December 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:
Histidine auxotrophic Salmonella strains
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
5000, 1666.67, 555.56, 185.19 and 61.73 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylformamide
- Justification for choice of solvent/vehicle: Dimethylformamide was found to be the best suited vehicle for the formation of a suspension of the test material at room temperature.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA100, TA 98, TA 1537 with Metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
TA 1535 with Metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without Metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without Metabolic activation
Details on test system and experimental conditions:
Setting up of the test plates:
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.

Preliminary range finding test:
Six concentrations of FAT 21016/C (Irganol Brillant violett base FLS trocken) ranging from 20.6 to 5000 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.The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity test:
The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Colony counting and scoring of the plates:
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated.
Evaluation criteria:
Assay acceptance 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 will be considered to be positive in the test system if the following condition is met:
At least a reproducible meaningful increase of the mean number of revertants 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 results
Species / strain:
other: TA 98, TA 100, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None

Any other information on results incl. tables

Range finding test:

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 µg/plate with and without metabolic activation.

Mutagenicity test:

In the original experiments performed with and without metabolic activation, with one exception, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with FAT 21016/C (Irganol Bnllantviolettbase FLS trocken) did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control. In the experiment with activation carried out on strain TA 1537, a marginally increased number of revertant counts was registered at the highest concentration. Since no such effect occurred in the repeat experiment with activation on this strain, the increase in the number of back-mutants is attributed to fluctuations of the spontaneous rate of back-mutants and not due to action of the test substance.

In the confirmatory experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with FAT 21016/C (Irganol Brillantviolettbase FLS trocken) no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control.

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

Applicant's summary and conclusion

Conclusions:
FAT 21016/C did not induce gene mutations in the Salmonella reverse mutation assay.
Executive summary:

FAT 21016/C was assesed for mutagenic activity in bacterial test systems in the presence and absence of a rat liver metabolic activation system as per the OECD guideline 471, under Good Laboratory Practice conditions.

The test was conducted using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. 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 suspended in dimethylformamide and tested at five concentrations in the range of 61.7 to 5000.0 ug/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 the same concentrations. 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 21016/C led to a biologically relevant increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Acid violet 109 and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.