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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:
weight of evidence
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study has been properly conducted, in accordance with recommended guidelines.

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sample 10.058461.01 was tested.

Method

Target gene:
Salmonella typhimurium TA 98, TA100, TA1535, TA1537 strains are marked by a defect in the histidine gene (His-). S. typhimurium strains have GC base pairs at the primary reversion site.
E. coli WP2 strain is marked by a defect in the tryptophan gene (Trp-). E. coli WP2 strain has an AT base pair at the primary reversion site.
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
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Test concentrations were:
C1 5 mg/plate;
C2 1.6 mg/plate;
C3 0.5 mg/plate;
C4 0.16 mg/plate;
C5 0.05 mg/plate.
Vehicle / solvent:
Sample was diluted in Acetone and all concentrations were obtained in Acetone.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, 9-Aminoacridine, 4-Nitroquinoline 1-oxide, 4-Nitro 1, 2 phenylene diamine, Nitro Furantoin in absence of S9. 2-Aminoantracene, Benzo(a)pyrene in presence of S9.
Details on test system and experimental conditions:
Mutagenicity study without metabolic activation system:
In a sterile tube 1.5 mL of Minimal Soft Agar and 0.9 mL of distilled water were mixed. The medium was mixed and kept at 45°C. Then to the tube were added:
- Negative control: 100 μL of distilled sterile water
- Negative control: 100 μL of acetone (solvent used for the sample preparation);
- Positive control: 100 μL of the positive controls solutions, specific for each strain;
- Test substance: 100 μL of opportune sample’s dilution.
- The tube was removed from the 45°C thermostatic bath; then 100 μL of the working culture were added immediately. The solution was mixed and then poured in a Vogel-Bonner plate. When the medium was solidified, the plates were incubated at 37°C for 72 hours.
The test was carried out in triplicate for the negative control and in triplicate for the positive control and the test substance.

Mutagenity study in the presence of metabolic activation system:
In a sterile tube 1 mL of Minimal Soft Agar and 0.9 mL of distilled water were mixed. The
tube was then kept at 45°C.
Then to the tube were added:
- Negative control: 100 μL of sterile distilled water (diluent used for the sample preparation);
- Positive control: 100 μL of positive controls solutions, specific for each strain;
- Test substance: 100 μL of opportune sample’s dilution.
- The tube was removed from the 45°C thermostatic bath; then immediately 100 μL of the working culture and 0.5 mL of S9 mix were added. The suspension was mixed and poured in a Vogel-Bonner plate. After solidification, the plates were incubated at 37°C for 72 hours.
The negative control test, the positive control and the test substance was carried out in triplicate.
Evaluation criteria:
Tester Strains TA98, TA 100 and WP2uvrA:
according to OECD 471 there are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system Statistical methods may be used as an aid in evaluating the test results.
According to the indications above, the test substance is considered positive when, in at least one of these tester strains, it produces at least a 2-fold rise of the increase value over the increase value in the appropriate vehicle control. This rise in the increase value must be accompanied by a positive Dunnett’s test (alpha = 0.05 1-sided) and/or a dose response when the concentrations of the test substance is increased.

Tester Strains TA1535, TA1537:
according to OECD 471 there are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system Statistical methods may be used as an aid in evaluating the test results. According to the indications above, the test substance is considered positive when, in at least one of these tester strains, it produces at least a 3-fold rise of the increase value over the increase value in the appropriate vehicle control. This rise in the increase value must be accompanied by a positive test (alpha = 0.05 1-sided) and/or a dose response when the concentrations of the test substance is increased.

Results and discussion

Test resultsopen allclose all
Key result
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 examined
Positive controls validity:
valid
Key result
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 examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

Under the test conditions, barium hexaferrite is not mutagen in Salmonella and E.coli tester strains.
Executive summary:

Barium hexaferrite was tested in Salmonella and E. coli strains for its mutagenicity according to bacterial reverse mutation test (Ames test): the substance did not show mutagen activity.