[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July -- Aug 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

plate incorporation and preincubation test:
50, 160, 500, 1600 and 5000 µg/plate (according to guideline)

repeated preincubation test (based on observed cytotoxicity):
with metabolic activation
160, 300, 500, 750, 1000 and 1600 µg/plate (TA 1535)
500, 800, 1200, 1600, 2500 and 5000 µg/plate (TA 1537)

without metabolic activation
5, 16, 50, 160, 500, 1600 and 5000 µg/plate (all Sallmonella strains)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility/homogeneity

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation) and preincubation; i
- Cell density at seeding (if applicable):
0.1 mL of overnight nutrient broth culture of the bacterial tester strain

DURATION
- Preincubation period: 20 - 30 min
- Exposure duration: approx. 48 h

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxixity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control

Rationale for test conditions:

based on observed cytotoxicity

Evaluation criteria:

Criteria for a positive response:
A test compound is classified as mutagenic if it has either of the following effects:
a. it produced 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. it 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.

The assays 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 innuce increases in the mutation frequency which are significant and within the laboratory's normal range.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Test results 2

Key result

Species / strain:

S. typhimurium 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:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Additional information on results:

- 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 historical control range.

- Solubility and toxicity
The test item was dissolved in DMSO and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 2500, 1600, 1200, 1000, 800, 750, 500, 300, 160, 50, 16 and 5 µg/plate were used in the different mutagenic experiments. Visible precipitation of the test substance on the plates was observed at 160 µg/plate and above. Because of the strong precipitation of the test item the bacterial lawn could not be evaluated at the dose level of 5000 µg/plate in the plate incorporation test. In the palte incorporation test toxicity was not observed either in the presence or absence of metabolic activation.
In the preincubation test the test item proved to be toxic to all Salmonella strains at dose levels of 1600 µg/plate and above and to E. coli WP2uvrA at a dose level of 5000 µg/plate in the presence of metabolic activation. In the absence of metabolic activation toxicity was observed with all Salmonella strains at all concentrations (50 µg/plate and above) and with E. coli WP2uvrA at concentrations of 160 µg/plate and above.
In the repetition of the preincubation test the test compound proved to be toxic to all Salmonella strains at dose levels of 16 µg/plate and above without metabolic activation. In the presence of metabolic activation toxicity was observed at a concentration of 1600 µg/plöate with the tester strain TA 1535 and at concentrations of 1600 µg/plate and above with tester strain TA 1537. Thinning of bacterial lawns and/or a reduction in the number of colonies was observed at these dose levels.

- Mutagenicity
Thr test item did not cause a significant increase in the number of revertant colonies at any dose level with any tester strains either in the absence or in the presence of S9-mix in each mutationtest. No dose-dependent effect was obtained. In the first preincubation test a dose/control ration of 1.8 - 2.0 was obtained with the strains TA 1535 and TA 1537 in the presnece of metabolic activation with individual doses. Although mean revertant values did not differ from the historical solvent control data range and no dose-dependency was observed, the preincubation test with these strains was repeated using a narrower dose range for clarification. No significant or dose-dependent increase in the number of revertants was observed in the repeat test confirming that the test substance is not mutagenic.
All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficicay of the exogeneous metabolic activation system were deminstrated.

Applicant's summary and conclusion

Conclusions:

The results of this Ames test lead to the conclusion that the test substance is not mutagenic either in the absence or in the presence of an exogeneous metabolising system.

Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella thyphimurium and with E. coli WP2uvrA.

Two independent mutagenicity studies were conducted (one plate incorporation and one preincubation test), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. Additionally a repetition of the preincubation test was performed with the strains TA 1535 and TA 1537 in the presence of S9 -mix due to equivocal results and with all Salmonella strains in the absence of S9 -mix due to high toxicity.

For all studies, the test substance was dissolved in DMSO and each bacterial strain exposed to 5 dose levels in the plate incorporation and in the preincubation test. In the repetition of the preincubation test the tester strains were exposed to 6 dose levels with metabolic activation and to 7 dose levels without metabolic activation. Concentrations used for plate incorporation and preincubation test were 50, 160, 500, 1600 and 5000 µg/plate. For the repetition of the preincubation test in the absence of metabolic activation dose levels from 5 to 5000 µg/plate were chosen, due to high toxicity in the first preincubation test. In the presence of metabolic activation dose ranges were variable across the bacterial strains because of equivocal results. For tester strain TA 1535 concentrations of 160, 300, 500, 750, 1000 and 1600 µg/plate and for strain TA 1537 concentrations of 500, 800, 1200, 1600, 2500 and 5000 µg/plate were chosen.

Visible precipitation of the test substance on the plates was obserced at 160 µg/plate and above. Because of the strong precipitation of the test item the bactrial lawn could not be evaluated at the dose level of 5000 µg/plate in the plate incorporation test.

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

Toxicity:

In the plate incorporation test toxicity was not observed either in the presence or in the absence of metabolic actiovation. In the preincubation the test item proved to be toxic to all Salmonella strain at dose levels of 1600 µg/plate and above and to E. coli WP2uvrA at a dose level of 5000 µg/plate in the presence of metabolic activation. Inthe absence of metabolic activation toxicity was observed with all Salmonella strains at all concentrations (50 µg/plate and above) and wit E. coli WP2uvrA at concentrations of 160 µg/plate and above.

In the repetition of the preincubation test the test compound proved to be toxic to all Salmonella strains at dose levels of 16 µg/plate and above without metabolic activation. In the presence of metabolic activation toxicity was observed at a concentration of 1600 µg/plate with the trster strain TA 1535 and at concentrations of 1600 µg/plate and above with tester strain TA 1537.

Thinning of bacterial lawns ansd /or reduction in the number of colonies was observed at these dose levels.

Mutagenicity:

In the presence and in the absence of the metabolic activation system the test item did not result in relevant increases in th number of revertants in any of the bacterial strains. In the first preincubation test a dose/control ratio of 1.8 - 2.0 was obtained with the strains TA 1535 and TA 1537 in the presence of metabolic activation with individual doses. Although mean revertant values did not differ from the historical solvent control data range and no dose-dependency was observed, the preincubation test with these strains was repeated using a narrower dose range for clarification. No significant or dose-dependent increase in the number of revertants was observed in the repetition test confirming that the test sbstance is not mutagenic.