Registration Dossier

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:
From 25th of January to March 1, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted according to internationally accepted testing guidelines.

Data source

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

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
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Principles of method if other than guideline:
Internal method based on SOP 30 50 01
GLP compliance:
yes (incl. certificate)
Type of assay:
other: bacterial/mammalian microsome mutagenicity test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: red power
- Stability: stable
- Storage: room temperature
- Submitted by: CIBA-GEIGY limited

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 and TA 1535): obtained from Prof. B. Ames, Berkeley, USA (1983)
Strain TA 100: obtained from Dr. M. Schüpbach Hoffmann-La Roche Limited, Basel, Switzerland (1986)
Strain TA 1537: was obtained from Dr. S. Albertini, Hoffmann-La Roche Limited, Basel, Switzerland (1994).
Test concentrations with justification for top dose:
RANGE FINDING TEST: 20.6 to 5000.0 ug/plate
ORIGINAL EXPERIMENT: 61.7 to 5000.0 ug/plate
CONFIRMATORY EXPERIMENT: 61.7 to 5000.0 ug/plate
Vehicle / solvent:
- Dimethylsulfoxide (DMSO)
SOLUBILIZATION OF THE TEST SUBSTANCE
The test substance was dissolved in DMSO at room temperature (solubility up to the concentration of 16.66 mg/ml). Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. Due to insufficient solubility at higher concentrations, the concentrations of 5000.0 and 1666.6 ug/plate were weighed separately.
At the concentration of 50 mg/ml, some precipitates were noted. However, after mixing the suspension with top agar, a clear solution appeared. No precipitates of the test material occurred on the agar plates.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-amminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
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
- Strain: TA 100 with and without metabolic activation
- Concentration: 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.
Incubation: 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
- Strains: Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation according to Standard Operating
Procedures of Genetic Toxicology.
- Concentratios: 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.
- Incubation: 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.

NUMBER OF CELLS EVALUATED:
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.

DETERMINATION OF CYTOTOXICITY
The test substance will be considered to be positive in the test system if 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.

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.
Evaluation criteria:
When the Salmonella strains are exposed to a mutagen, some of the bacteria in the treated population, through chemical interaction with the compound or its metabolites, undergo genetic changes which cause them to revert to a non-histidine-requiring state and thus to grow in the absence of exogenous histidine. Mutagenic effects of the test substance are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone reverse-mutation to histidine prototrophism. Different tester strains are used because of differing sensitivities to known mutagens. The following bacterial strains have been used in this study:
Strain: type of mutation
S. typhimurium TA 100: base-pair substitution
S. typhimurium TA 1535: base-pair substitution
S. typhimurium TA 98: frame-shift
S. typhimurium TA 1537: frame-shift
Statistics:
Not performed.

Results and discussion

Test results
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:
no cytotoxicity
Untreated negative controls validity:
valid
Additional information on results:
RANGE FINDING STUDY
The highest concentration suitable for the mutagenicity test was selected to be 5000.0 ug/plate with and without metabolic activation.

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

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 did not increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control.

In the original and confirmatory mutagenicity tests normal background growth was observed with all strains at all concentrations. In the experiments with or without metabolic activation signs of toxic effects, expressed as a reduction of the numbers of revertant colonies, were observed occasionally
with strains TA 1535, TA 98 and TA 1537 at the higher concentrations.
Remarks on result:
other: other: histidine-prototrophic mutants
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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.
Executive summary:

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 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.

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.

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.