Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 18 to 20, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Japanese Substance Control Law (JSCL) Test Guideline M.1 Chromosome Aberration Test with bacteria
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Method

Target gene:
The Ames test with Salmonella typhimurium strains investigates the ability of the test compound to induce reverse (back) mutations to histidine prototrophy using histidine auxotrophic mutants.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Source of bacteria
The strains of Salmonella ryphimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. coli was obtained from E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the celi suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored in liquide nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeabili, ampicillin resistance, tetracyclin resistance, crystal violet sensitivi, UV resistance and response to diagnostic mutagens.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver fraction
Test concentrations with justification for top dose:
a - with metabolic activation(10 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate
b - without metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
(Concentration of stock solution: 50 mg/ml)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Stability and homogeneity in the solvent: is guaranteed for 4 hours in DMSO, by HPLC analysis
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ASSAY PROCEDURE
An independent mutation test was performed using the plate incorporation method. When results were negative or equivocal, a second test was conducted. This included a preincubation step if the first test was clearly negative. Preincubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed alter microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM).
The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain 0.1 ml test compound solution (dissolved in DMSO)
In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approx. 30 °C.
After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose).
Alter incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) if induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 1535, TA 1537 and TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

STERILITY CHECKS AND CONTROL PLATES

Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historicalcontrol range.

SOLUBILITY AND TOXICITY

The test substance was dissolved in DMSO and a stock solution of 50 mg/ml was prepared forthe highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutionsof 1600, 500, 160 and 50 µg/plate were used in the plate incorporation test.

Visible precipitation of the test substance on the plates was observed at 160 µg/plate and above.

The test substance proved to be not toxic to the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Mutagenic
Executive summary:

Method

The substance was tested for mutagenic effects according to OECD Guideline 471,with the strains TA 100,TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA.

One mutagenicity study was conducted as the standard plate test with the plate incorporation method. The study was performed in the absence and in the presence of a metabolizing systemderived from a rat liver homogenate. For the study, the compound was dissolved in DMSO, andeach bacterial strain was exposed to 5 dose levels in the plate incorporation test.

Doses for the plate incorporation test ranged from 50 to 5000 µg/plate.

Observation

Control plates without mutagen showed that the number of spontaneous revertant colonies waswithin the laboratory's historical control. All positive controls gave the expected increase in thenumber of revertant colonies.

Visible precipitation of the test substance on the plates was observed at 160 µg/plate andabove.

In the plate incorporation test toxicity was not observed either with or without metabolicactivation.

In the absence of a metabolic activation system, the test compound resulted in relevant and dose-dependent increases in the number of revertant colonies with the bacterialstrains TA 1535, TA 1537 and TA 98. Also in the presence of rat liver S9-mix, treatment of thecells with the test substance resulted in relevant increases in the number of revertant colonieswith the bacterial strains TA 100, TA 1535, TA 1537 and TA 98.

Conclusion

Under these experimental conditions the test substance is mutagenic in the standard plate test (plate incorporation test) in the absence and in the presence of a metabolic activation system.