Registration Dossier

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 May 20 to June 05, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

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):
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
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:
RANGE FINDING TEST: 20.6 to 5000.0 µg/plate
ORIGINAL EXPERIMENT: 15.8 to 1581.3 µg/plate (without metabolic activation)
50 to 5000.0 µg/plate (with metabolic activation)
CONFIRMATORY EXPERIMENT: 50 to 5000.0 µg/plate (with and without activation)
Vehicle / solvent:
- Vehicle/solvent used: Dimethylsulfoxide (DMSO)
- Solubilisation of the test substance: the test substance was dissolved in dimethylsulfoxide at a concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with dimethylsulfoxide. No precipitates or aggregates were noted
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:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without 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:
cyclophosphamide
other: 2-aminoanthracene
Remarks:
with 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 substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 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. It was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water.

PRELIMIMNARY TOXICITY/RANGE-FINDING TEST
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with metabolic activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. 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, as well as each negative control was used.

MUTAGENICITY TEST
The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with 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 as well as each positive and negative control with each tester strain. The highest concentration applied in the original experiment was 1581.3 µg/plate without metabolic activation (because of weak toxicity in the range finding test) and 5000 µg/plate in the experiment with activation. In the confirmatory experiment the highest concentration used was 5000 µg/plate without and with activaton. The four lower concentrations were each decreased by a factor of V10 (3.162). 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 with an Artek counter. 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 and standard deviations for all mutagenicity assays were calculated by a previously validated computer program and included in the Results section.

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 judgement of the Study Director.
Evaluation criteria:
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 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.
Statistics:
No statistical method available.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
TOXICITY TEST/RANGE FINDING TEST:
In this test a toxic effect of the test substance by 50% or more on the growth of revertant colonies was observed in the experiment without metabolic activation at the concentrations of 555.6 to 5000 µg/plate.

MUTAGENICITY TEST, ORIGINAL EXPERIMENT
In the original experiment performed without and with metabolic activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Due to toxicity of the test substance the numbers of revertant colonies were occasionally reduced by 50% or more with strains TA 98, TA 100 and TA 1537 at the concentration of 5000 µg/plate in the experiments with metabolic activation.

MUTAGENICITY TEST, CONFIRMATORY EXPERIMENT
In the confirmatory experiment performed with and without metabolic activation, again, the tested concentrations did not lead to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiments with metabolic activation a reduction in the number of revertant colonies by 50% or more was observed with strains TA 98 and TA 100 at the concentration of 5000 µg/plate and with strain TA 1537 at the concentrations of 1581.3 and 5000 µg/plate due to toxicity of the test substance. Similar results occurred in the experiments without metabolic activation on strain TA 100 at the concentrations of 1581.3 and 5000 µg/plate and on strain TA 1537 at the concentration of 5000 µg/plate .
Remarks on result:
other: other: histidine-prototrophic mutants
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Non genotoxic
Executive summary:

Method

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum. The following strains of Salmonella typhimunum were used: TA 98, TA 100, TA 1535, TA 1537, and TA 1538.

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 50 to 5000.0 µg/plate in the presence of a metabolic activation system and 15.8 to 1581.3 µg/plate in 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 50 to 5000.0 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.

Observation

In both experiments, performed with and without metabolic activation, none of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Conclusion

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