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:
2009-08-18 to 2009-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study - expiration date missing

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 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2005-10-21
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Physical state: solid, brown, dark
- Storage condition of test material: room temperature

Method

Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-
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 fraction prepared from livers of male Wistar rats orally receiving phenobarbitone/β-naphthoflavone on three consecutive days
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from livers of male Wistar rats orally receiving phenobarbitone/β-naphthoflavone on three consecutive days
Test concentrations with justification for top dose:
Standard plate test (experiment 1): 0, 22, 110, 550, 2750 and 5500 μg/plate (with and without metabolic activation)
Preincubation test (experiment 2): 0, 22, 110, 550, 2750, and 5500 μg/plate (with and without metabolic activation)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test item in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests.
Controlsopen allclose all
Negative controls:
no
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: 2.5 μg/plate (strains TA 1535, TA 100, TA 1537, & TA 98) & 60 μg/plate (strain E. coli WP2 uvrA)
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
Positive control without metabolic activation: concentration: 5 μg/plate (strains TA 1535 & TA 100)
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
Positive control without metabolic activation: concentration: 10 μg/plate (strain TA 98)
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Positive control without metabolic activation: concentration: 100 μg/plate (strain TA 1537)
Negative controls:
no
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: 5 μg/plate (strain E. coli WP2 uvrA)
Details on test system and conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

DURATION
- Preincubation period (experiment 2): about 20 minutes
- Exposure duration (experiments 1 & 2): 48 -72 hours at 37 °C

NUMBER OF REPLICATIONS (experiments 1 & 2): 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments.

Precipitation of the test material is recorded.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test item is generally considered non-mutagenic in this test if:
- the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
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:
yes
Remarks:
please refer to the field "Additional information on results" below
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:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
please refer to the field "Additional information on results" below
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:
STANDARD PLATE TEST (experiment 1):
No increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.

PREINCUBATION TEST (experiment 2):
No increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test (experiment 1) depending on the strain and test conditions at the highest applied concentration (5500 μg/plate).
In the preincubation assay (experiment 2) bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitation was found from 110 μg/plate onward with and without S9 mix.

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 bacterial reverse mutation assay was performed with the substance according to the OECD guideline 471 (1997) using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The study was carried out using the standard plate test (experiment 1) and the preincubation test (experiment 2). Both experiments were conducted with and without metabolic activation and each concentration was plated in triplicate. The substance was tested at the following concentrations in the both experiments: 22, 110, 550, 2750, and 5500 µg/plate. Additionally, a vehicle control and several positive controls were run concurrently.

In both experiments no increase in the number of his+ or trp+ revertants was observed with and without metabolic activation.

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at the highest applied concentration (5500 μg/plate). In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2750 μg/plate onward. Test item precipitation was found from 110 µg/plate onward with and without metabolic activation.

The results of the negative as well as the positive controls performed corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

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.