STI571 F8

Genetic toxicity: in vitro

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:

2003

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 022401

Method

Target gene:

The actual batch of the strains was tested for ampicillin res.stance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in September 2000.

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 .

Metabolic activation:

with and without

Metabolic activation system:

The microsomal fraction of homogenised livers of female Sprague Dawley rats treated once with 500 mg/kg of Aroclor 1254 was used (S9 mix).

Test concentrations with justification for top dose:

The concentrations for the first experiment were set according to a preliminary toxicity test.

The test substance was only slightly toxic at 5000 µg/plate. Therefore this concentration was chosen as highest concentration, which is the limit concentration according to the guidelines. Each of the other 4 concentrations was 1/3 of the preceding one.
The number and intervals of the concentrations are in accordance with the guidelines .

Vehicle / solvent:

Solvent: Dimethylsulfoxide.

Details on test system and experimental conditions:

The exposure was performed according to the 'Plate lncor oration Assay', in which bacteria, test substance (and microsomes) are in contact on the plat13 without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 108 cells per ml.


For each sample the following solutions were combined:

• 0.1 ml of the overnight culture of the bacteria,

• 0.5 ml of S9-mix (or phosphate buffered saline for samples without metabolic activation),

• 0.1 ml of the appropriate test- or reference substance solution and

• 2 ml of top agar.

The combined solutions were mixed and spread over a pla1e with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
The plates with less than about 50 revertant colonies, i.e. tile plates of TA98 and TA1535 with the exception of the positive controls, were counted vi ;ually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.

Evaluation criteria:

The criteria for a positive result are:

A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.

• For the strains with a high spontaneous revertant rate i.n. TA97a, TA100 and TA102: The
1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test

Statistics:

Means and standard deviations were calculated for the number of mutants in every concentration group.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA97a, TA98, TA100, TA102 and TA1:535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight toxicity was noted only in the 5000 µg/plate samples without metabolisation, resulting in a reduced bacterial background lawn.

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA97a, TA98, TA100, TA102 and TA1:535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
In none of the concentrations and in none of the strains
used a relevant increase of the mutation frequency was
observed. Metabolic activation did not change this result.

Results were reproduced in an independent experiment.

Remarks on result:

other: preliminary test

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

According to these results, "STl571 F8" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.
The test result was negative with metabolic activation and negative without metabolic activation