Registration Dossier

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
Study period:
2008-05-12 to 2008-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study with minor deviation to the guideline without effects on results: - following the guideline, one dose level with precipitation should be tested and further dose levels should be chosen below this dose level. In this study only two dose level below the dose level causing precipitation (500 µg/plate) were chosen plus two higher dose level (1500 and 5000 µg/plate) which also causing precipitation. - SD not presented for untreated-negative control

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2000
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-10-15
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical state: solid, whitish to white crystals
- Storage condition of test material: room temperature in the dark

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
TA102: his G 428 (pAQ1)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from livers of male Sprague-Dawley rats orally receiving three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg/day)
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from livers of male Sprague-Dawley rats orally receiving three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg/day)
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 (with and without metabolic activation)
Experiment I and II: 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Vehicle:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: test material was insoluble in water, but fully soluble in DMSO at 50 mg/mL.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Positive control without metabolic activation: concentrations: 3 μg/plate (strain TA100) & 5 μg/plate (strain TA1535)
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Positive control without metabolic activation: concentration: 80 μg/plate (strain TA1537)
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control without metabolic activation: concentration: 0.5 μg/plate (strain TA102)
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Positive control without metabolic activation: concentration: 0.2 μg/plate (strain TA98)
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Positive control with metabolic activation: concentrations: 1 μg/plate (strain TA100) & 2 µg/plate (strains TA1535 & TA1537)
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive control with metabolic activation: concentrations: 5 μg/plate (strain TA98)
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone
Remarks:
Positive control with metabolic activation: concentrations: 10 μg/plate (strain TA102)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (direct plate incorporation method)(preliminary test and main study)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
A preliminary test was carried out to determine the toxicity of the test material. Ten doses of the test material and a vehicle control were tested using TA100. In addition, the sterility of the test material was assessed. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MAIN STUDY (Experiment I and II)
- Exposure duration: approximately 48 hours incubation at 37 °C
- Number of replications: triplicate
- Number of cells evaluated: frequency of revertant colonies was assessed using a Domino colony counter. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.
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, as recommended by the UKEMS 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:
no data

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
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: grease-like precipitation (which appeared fibrous under a microscope) was observed at and above 500 µg/plate (did not prevent the scoring of revertant colonies).

RANGE-FINDING/SCREENING STUDIES:
- test material was non-toxic to the strain used (TA100).

MAIN STUDY
- no 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- test material formulation and the S9-mix were both shown to be sterile.
- test material caused no visible reduction in the growth of the bacterial background lawn at any dose level

Please also refer to the field "Attached background material" below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance tested non-mutagenic under the conditions of the study. According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Executive summary:

A reliable bacterial reverse mutation assay was performed according to the OECD guideline 471 (1997). Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the substance using the plate incorporation method at five dose levels in triplicate with and without metabolic activation. Two experiments were conducted with the following concentrations: 50, 150, 500, 1500 and 5000 µg/plate. The substance did not cause visible reduction in the growth of the bacterial background lawn at any dose level. Significant increases in the frequency of revertant colonies were not recorded for any of the bacterial strains at any dose with or without metabolic activation. Thus, the substance tested non-mutagenic under the conditions of this study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.