Registration Dossier

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:
07.11.2000 - 14.03.2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was performed according to GLP and the methods applied are fully compliant with OECD TG 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Method

Target gene:
HIS operon (S. thyphimurium)TRY operon (E. coli)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Species / strain:
S. typhimurium TA 1537
Details on mammalian cell lines (if applicable):
his C 3076, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 102
Details on mammalian cell lines (if applicable):
his G 428, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
E. coli WP2
Details on mammalian cell lines (if applicable):
his C 3076, uvrB, rfa
Additional strain characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan: 1. Series: 5, 33, 100, 333, 1000, 2500, 5000 µg/plate2. Series: 5, 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle:
DMSO (> 99 %)
Controls
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and conditions:
Acceptability of the AssayThe 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 frequenciesEvaluation of ResultsA test substance is considered positive if either a dose related and reproducible, biologically relevant increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.A test substance producing neither a dose related and reproducible, biologically relevant increase in the number of revertants, nor a biologically relevant and reproducibly increase at any one of the test points is considered non-mutagenic in this system.A biologically relevant, test substance-related increase occurs if in the strains TA 98, TA 100, TA 102 and WP2 uvrA the number of reversions is at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate Method:This study was performed to investigate the potential the test material to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II).
Evaluation criteria:
cf. details in results
Statistics:
n.a.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Precipitation of the test substance on the minimal agar plates was observed at 1000 µg/plate and above with activation and at 5000 µg/without metabolic activation in experiment I, and at 2500 µg/plate with metabolic activation in experiment II.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activationWith and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

This study was performed to investigate the potential the test material to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA

100, and TA 102 and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Results

No toxic effects, evident as a reduction in the number of revertants, were observed in experiment with and without metabolic activation in both experiments. Furthermore, the plates incubated with the test substance showed normal background growth up to

5000 µg/plate with and without metabolic activation in both independent experiments.

Precipitation of the test substance on the minimal agar plates was observed at 1000

IJg/plate and above with activation and at 5000 µg/without metabolic activation in experiment I, and at 2500 µg/plate with metabolic activation in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of S9 mix.

Appropriate reference mutagens were used as positive controls which showed distinct increases of induced revertant colonies.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.