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Genetic toxicity: in vitro

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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:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer chapter 13 for detailed read across justification.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
None

Method

Target gene:
The experiments were performed to detect any properties of the test material or its metabolites to induce gene mutations in histidine-requiring strains of Salmonella typhimurium.
Species / strainopen allclose all
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
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 fraction
Test concentrations with justification for top dose:
- Range in the cytotoxity test: 20.6 - 5000 µg/plate.*
- Range in the original mutagenicity test: 61.7 - 5000 µg/plate.*
- Range in the confirmatory mutagenicity test: 88.95 - 7205 µg/plate.**

*The purity of the tested batch is 69.4%. Related to 100% purity the highest concentration in these parts of the assay was 3470 jug/plate.
** Related to 100% purity the highest concentration in this part of the assay was 5000 jug/plate.

Vehicle / solvent:
Dimethylsulfoxyde.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
mitomycin C
other: Sodium azide and 9 (5) - aminoantharcene
Remarks:
Without microsomal activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-aminoanthracene
Remarks:
With microsomal activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48 at 37°C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the 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 judgment of the Study Director.

Criteria for a positive response
The test substance is considered to be mutagenic in this test system if the following conditions are 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 following strains: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.
Statistics:
In deviation to the OECD guideline (Ref. 1) a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
slight increase of the number of revertant colonies at the concentration of 5000 and 7205 µg/mL in the original and in the confirmatory experiment.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA 1535, TA 98 TA 100 and TA 102
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Original and confirmatory experiment.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA 98 and TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
a slight increase in the number of back-mutants occured at the concentration of 5000 µg/Ll.(original experiment)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA 1537; TA 100 and TA 102
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Original experiment
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Confirmatory experiment
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA 98, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
a slight increase in the number of back-mutants at the concentration of 7205 µg/ml (Confirmatory experiment)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity test/Range finding test

Six concentrations of Terasil Violett BL roh feucht (FAT 3 6038/F) ranging from 20.6 to 5000 /ng/plate were tested with strain

S. typhimurium TA 100 determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies were not reduced. Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000 /xg/plate without and with activation.

Mutagenicity test, original experiment

(concentration range: 61.7 to 5000 microg/plate)

In the experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F) led to a slight increase in the number of revertant colonies at the concentration of 5000 microg/ml. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the

number of back-mutants occurred on strains TA 98 and TA 1535 at the concentration of 5000 microgr/ml. No effects were observed with the other strains.

Mutagenicity test, confirmatory experiment

(concentration range: 88.95 to 7205 /microg/plate)

In the experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F), led to a slight increase in the number of revertant colonies at the concentration of 7205 /Ltg/ml. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98, TA 1535 and TA 1537 at the concentration of 7205 /microg/ml. No effects were observed with the other strains.

Owing to a toxic effect of the test material in the mutagenicity tests without metabolic activation on strain TA 102, a slight decline in the number of revertant colonies was observed at the upper concentrations. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

 

Applicant's summary and conclusion

Conclusions:
Based on the results of these experiments and on standard evaluation criteria, it is concluded that Terasil Violet BL roh feucht (FAT 36038/F) exerted a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535. The metabolites of the test material were weakly mutagenic with strain TA 1537.
Executive summary:

A key study was performed to determine the detection of gene mutations induced by the test material FAT 36038/F or its metabolites in histidine-requiring strains of Salmonella typhimurium.

The FAT 36038/F) were tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 jug/plate.

In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed.

In the original experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F) led to a slight increase in the number of revertant colonies at the highest concentration. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98 and TA 1535 at the highest concentration. No effects were observed with the other strains.

In the confirmatory experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F), led to a slight increase in the number of revertant colonies at the highest concentration. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98, TA 1535 and TA 1537 at the highest concentration. No effects were observed with the other strains.

In the mutagenicity tests without metabolic activation performed on strain TA 102, a slight decline in the number of revertant colonies was registered. The test substance exerted a weak inhibiting effect on the growth of this bacterial strain.

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Terasil Violett BL roh feucht (FAT 36038/F) exerted a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535. The metabolites of the test material were weakly mutagenic with strain TA 1537.