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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: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: NOCOLOK FLUX
Chemical name: Aluminium potassium fluoride
Purity: 99%
Appearance: Fine white crystalline powder
Batch number: BWF91021
CAS Reg. number: 60304-36-1
Molecular Formula: K(1-3)AlF(4-6)
Molecular weight: 150 g/mol
Storage conditions: ambient temperature
Expiry date: 30 November 2011
Supplier: Solvay Fluor GmbH, Hannover, Germany

Method

Target gene:
his
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix)
Test concentrations with justification for top dose:
62, 185, 556, 1667 and 5000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene and N-ethyl-N-nitrosourea; with S9: 2-aminoanthracene and benzo(a)pyrene
Details on test system and experimental conditions:
Set up
The plate-incorporation method with the histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98 and TA 100 and the tryptophan-requiring Escherichia coli mutant WP2 uvrA as indicator strains was applied. A preliminary test to assess the toxicity of the test substance was not performed. Therefore the toxicity test was incorporated in the mutagenicity assay.
One bacterial reverse mutation test was performed. The test substance was suspended in DMSO at a concentration of 50 mg/ml based on a purity of 99%. A homogeneous, turbid, white suspension was obtained. Serial dilutions in DMSO were made. Five concentrations were tested, ranging from 62 to 5000 µg/plate.
Negative controls (DMSO) and positive controls were run simultaneously with the test substance.
The actual concentrations of the test substance in the test solutions were not determined. Therefore, the concentrations reported are nominal concentrations.

Mutation analysis
Fresh bacterial cultures were prepared by inoculation of nutrient broth with a thawed aliquot of the stock culture and subsequent incubation for approximately 10-16 h at 37°C while shaking. Briefly, the mutagenicity assay was carried out as follows. To 2 ml molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin or 0.05 mM tryptophane for the S. typhimurium strains, and E. coli WP2 uvrA strain, respectively), maintained at ca. 46 oC, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the test substance or of the negative control or of the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37 oC for approximately 48-72 hours. Subsequently, the his+ and trp+ revertants were counted. Toxicity is defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control.
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, if no more than 5% of the plates are lost through contamination or other unforeseen events, and if at least 3 doses are non toxic.
A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increased in a concentration-related manner or if a two-fold or greater increase is observed compared to the negative control plates. A clear positive response does not need to be verified. Marginally or weakly positive results should be verified by additional testing.
A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Omission of a second test under these conditions is acceptable as a single test does not, or hardly ever results in false negative conclusions (TNO historical data and Kirkland and Dean, 1994).
Both numerical significance and biological relevance are considered together in the evaluation.
Statistics:
No statistical analysis was performed.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.
In both the absence and presence of S9-mix in all strains, NOCOLOK FLUX did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that NOCOLOK FLUX is not mutagenic under the conditions employed in this study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion