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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:
2009-12-01 to 2010-01-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Propanesulfonamide, N-[3-[[5-bromo-1-(2,6-dichlorobenzoyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-
EC Number:
806-756-5
Cas Number:
1262985-24-9
Molecular formula:
C24 H16 Br Cl2 F2 N3 O4 S
IUPAC Name:
1-Propanesulfonamide, N-[3-[[5-bromo-1-(2,6-dichlorobenzoyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl]-2,4-difluorophenyl]-
Test material form:
not specified
Details on test material:
Purity: >99 % (w/w)

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rats induced with Phenobarbitone/beta-naphthoflavone
Test concentrations with justification for top dose:
Preliminary test and Experiment I : 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate
Experiment II : 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate
The test item precipitated in the overlay agar in the test tubes from 1000 ug/plate up to 5000 ug/plate in both experiments. Precipitation on the incubated agar plates was observed from 333 ug/plate up to 5000 ug/plate in experiment I without S9 mix and from 1000 ug/plate up to 5000 ug/plate in experiment I with S9 mix and in experiment II with and without S9 mix.
The undissolved particles had no influence on the data recording.
Vehicle / solvent:
Dimethyl sulphoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene 2-AA, 4-nitro-o-phenylene-diamine 4-NOPD
Details on test system and experimental conditions:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
In the pre-experiment the concentration range of the test item was 3 - 5000 ug/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 ug/plate were chosen as maximal concentration. Based on the observed precipitation seven concentrations were tested
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
10; 33; 100; 333; 1000; 2500; and 5000 ug/plate.
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 uL Test solution at each dose level (solvent or reference mutagen solution (positive control)) ,
500 uL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 uL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 uL Overlay agar
In the pre-incubation assay 100 uL test solution (solvent or reference mutagen solution(positive control)), 500 uL S9 mix / S9 mix substitution buffer and 100 uL bacterial suspension were mixed in a test tube and shaken at 37 C for 60 minutes. After preincubation 2.0 mL overlay agar (45 C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 C in the dark
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 ug/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test
Executive summary:

Introduction. This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

Methods. The assay was performed in two independent experiments both with and without liver microsomal activation.

Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experimen/Experiment I 3; 10; 33; 100; 333; 1000; 2500; and 5000 ug/plate.

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 ug/plate

The plates incubated with the test item showed normal background growth in all strains with and without metabolic activation in both experiments.

Results. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion. The test item was considered to be non-mutagenic under the conditions of this test.