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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 12 to May 20, 1994
Reliability:
2 (reliable with restrictions)
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(No E. Coli strain used; no statistical analysis)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver metabolic fraction S9
Test concentrations with justification for top dose:
Concentration range in the range finding test: 20.6 to 5000.0 µg/plate
Concentration ranges in the mutagenicity tests: Original experiment 61.7 to 5000.0 ug/plate; Confirmatory experiment 61.7 to 5000.0 ug/plate
Vehicle / solvent:
Dimethylsulfoxide
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without metabolic activation
Details on test system and experimental conditions:
Preparation of the bacterial cultures
Inoculates from frozen master copies were set up monthly. They were grown in liquid nutrient broth medium (NB-medium) overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
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
A range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control according to a Standard Operating Procedure of Genetic Toxicology. The highest concentration applied was 5000 ug/plate. The five 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 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.
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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
other: marginal increased revertant counts
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Applicant's summary and conclusion

Conclusions:
Mutagenic
Executive summary:

Method

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum, according to the OECD Guideline 471. The following strains of Salmonella typhimunum were used: 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 dissolved in DMSO 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 five concentrations in the range of 61.7 to 5000.0 ug/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.

 

Observation:

In both experiments, performed with and without metabolic activation, the test substance and its metabolites led to a very strong increase in the number of back-mutants with strains TA 98, TA 100 and TA 1537. The metabolites of the test substance led to weak increased revenant counts with strain TA 1535.

 

Conclusion:

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the substance and its metabolites induced gene mutations in the strains of Salmonella typhimurium us