Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jan - 04 Feb 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK GLP Monitoring Authority
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

Target gene:
his operon for S. typhimurium strains and trp operon for E. coli strain
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100
Species / strain / cell type:
E. coli, other: WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from livers treated with phenobarbitone/ß-naphthoflavone (80/100 mg/kg bw/day)
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
S. typhimurium TA100 and E. coli WP2uvrA-: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 1 (range-finding test) and Experiment 2 (main test), plate incorporation method:
All strains: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, DMSO, acetone, dimethyl formamide and acetonitrile at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks. The test material formed the best doseable suspension in DMSO, therefore, this solvent was selected as the vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene (2AA): +S9, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA­ Benzo(a)pyrene (BP): +S9, 5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION:
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:
Criteria for a positive result: 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.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98 and TA100 and E. coli WP2uvrA-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no, but tested up to limit dose
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A black, powdery precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%):
- Data are attached in separate pdf document. Refer to section 'Attached background material'.

Any other information on results incl. tables

Due to the huge quantity and complexity of the results, data are attached in a separate pdf document. Refer to section 'Attached background material'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative