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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 September 2006 - 3 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
white powder
Details on test material:
- Name of test material (as cited in study report): ucb108628-1
- Stability under test conditions: not indicated
- Storage condition of test material: room temperature, in the dark

Method

Target gene:
Histidine gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone (80/100 mg per kg per day) induced rat liver S9 mix
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
First mutation test: 50, 150, 500, 1500 and 5000 μg/plate
Second mutation test: 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Solvent used: sterile distilled water
- Justification for choice of solvent/vehicle: The test material was soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
without S9
Positive controls:
yes
Remarks:
without S9
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 μg/plate for TA100 and 5 μg/plate for TA1535
Positive controls:
yes
Remarks:
without S9
Positive control substance:
9-aminoacridine
Remarks:
80 μg/plate for TA1537
Positive controls:
yes
Remarks:
without S9
Positive control substance:
mitomycin C
Remarks:
0.5 μg/plate for TA102
Positive controls:
yes
Remarks:
without S9
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 μg/plate for TA98
Positive controls:
yes
Remarks:
with S9
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 μg/plate for TA100 and 2 μg/plate for TA1535 and TA1537
Positive controls:
yes
Remarks:
with S9
Positive control substance:
benzo(a)pyrene
Remarks:
5 μg/plate for TA98
Positive controls:
yes
Remarks:
with S9
Positive control substance:
other: 1,8-dihydroxyanthraquinone
Remarks:
10 μg/plate for TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: in triplicate against each tester strain

DETERMINATION OF CYTOTOXICITY
- Method: numbers of revertant colonies and growth of the bacterial background lawn
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100).

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. A small, statistically significant increase (P≤0.05) in revertant colony frequency was observed in tester strain TA1535 (without S9) at 5000 μg/plate in Experiment 1 only. This increase was within the spontaneous mutation range and the historical range expected for this strain, was non-reproducible and exhibited no dose response relationship. Therefore, the increase was considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:
An in vitro gene mutation study (AMES study) was conducted according to OECD/EC guidance and GLP principles. The test substance was found to be non-mutagenic under the conditions of this test.