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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
of 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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:
other: essential amino acid requiring strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: essential amino acid requiring strain
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.
Test concentrations with justification for top dose:
Experiment 1: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate
Experiment 2: 0; 50; 150; 500; 1500 and 5000 μg/plate
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Justification for choice of solvent/vehicle:
DMSO was a suitable vehicle for exposure to the test substance up to the maximum guideline recommended test substance concentration of 5000 μg/plate.
Controlsopen allclose all
Untreated negative controls:
other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide; 9-aminoacridine; 2-nitrofluorene; 4-nitroquinoline-1-oxide.
Remarks:
Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.
Untreated negative controls:
other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; Benzo[a]pyrene
Remarks:
Positive control substances for tests with metabolic activation (S9 mix). All of them are well established reference mutagens.
Details on test system and experimental conditions:
Standard Plate Incorporation Tests were performed in Experiment 1 and pre-incubation tests in Experiment 2. Both experiments were conducted without and with metabolic activation (S9 mix). The proportion of S9 fraction in the S9 mix was 10% v/v.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

Sodium azide (CAS No. 26628-22-8):
- 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100

9-Aminoacridine (CAS No. 90-45-9):
- 50 μg/plate, dissolved in DMSO: - strain: TA 1537

2-Nitrofluorene (CAS No. 607-57-8):
- 2 μg/plate, dissolved in DMSO: - strain: TA 98

4-Nitroquinoline-1-oxide (CAS No. 56-57-5):
- 2 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

With metabolic activation (S9 mix):

2-Aminoanthracene (CAS No. 613-13-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100
- 10 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

Benzo[a]pyrene (CAS No. 50-32-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1537, TA 98

Evaluation criteria:
The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
A reproducible increase in revertant colony number, (i.e. at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 the concurrent vehicle controls), with some evidence of a positive dose-response relationship. Such positive response in at least one tester strain without or with metabolic activation (S9 mix.) is sufficient for concluding mutagenic activity.

A test substance is considered non-mutagenic in this test if:
Exposure to a test substance does not produce a reproducible increase in revertant colony numbers.
Statistics:
The data were not statistically analysed. The study result was unequivocal.

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
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to the recommended maximum concentration of 5000 µg/plate
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
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to the recommended maximum concentration of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All sterility control plates were colony free. Hence the absence of microbial contamination of the S9 mix, buffer and test substance formulation was confirmed. Viability counts were satisfactory meeting the acceptance criteria.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without and with metabolic activation (S9 mix)