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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: read across from similar substance
Adequacy of study:
key study
Study period:
from March 29 to May 2, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was carried out according to an internationally accepted testing guideline, even if it is an earlier version (ver.1983) that includes only four strains and not five as in the last update (ver.1997).The complete justification for the Read Across approach is reported in the Section 13.

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
ver. 1983
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Similar Substance 01
IUPAC Name:
Similar Substance 01

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 characteristics of the strains were checked monthly. Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction)
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: for original and confirmatory experiments from 61.7 to 5000.0 ug/plate
Vehicle / solvent:
- vehicle: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble up to the concentration of 50 mg/ml.
Controls
Untreated negative controls:
yes
Remarks:
solvent alone
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
other: 2-Aminoanthracene
Remarks:
Sodium azide and cyclophosphamide were dissolved in bidistilled water; the other positive control substances in DMSO.
Details on test system and experimental conditions:
PREPARATION:
Aliquots from frozen stocks were grown in liquid nutrient broth medium (NB-medium) for 8 hours and then used for the experiment.

METHOD OF APPLICATION: in agar (plate incorporation) for original experiment and preincubation for the confimatory experiment
Setting up of the test plates
- Standard plate incorporation assay:
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.
- Preincubation assay:
0.1 ml of the overnight cultures were mixed with 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 incubated for 30 min at 37 °C. Thereafter 2 ml of top agar was added to the mixtures and they were 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.

TEST CONDITIONS:
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 REPLICATIONS: 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

RANGE-FINDING/SCREENING STUDIES:
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 µg/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.

CONCENTRATIONS:
The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three.

COLONY COUNTING:
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.

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.
Evaluation criteria:
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:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the original experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with the test substance 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 the test substance 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 occasionally reduced with all strains at the upper concentrations. The test substance exerted a weak toxic effect on the growth of the bacteria.
There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 1666.7 and 5000.0 µg/plate the test material precipitates on the agar plates.
Remarks on result:
other: all strains/cell types tested
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 test substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used, both with and without metabolic activation.
Executive summary:

The test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium, according to the OECD Guideline 471 (1983).

The following strains of Salmonella typhimurium 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 µg/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.

The original experiment with and without metabolic activation and the confirmatory experiment without activation were performed as standard plate incorporation assay. The confirmatory experiment with metabolic activation was carried out as preincubation assay.

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

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