Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 OECD Guideline GLP-compliant studies:

- OECD Guideline 471 (Bacterial Reverse Mutation Assay): negative

- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test): negative

- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 May 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was suspended in dimethylsulfoxide at room temperature. The highest concentration was 50 mg/ml. In the toxicity test the three highest concentrations were weighed out separately and the three lowest concentrations were prepared by appropriate dilution of the third highest concentration with dimethylsulfoxide. In the mutagenicty test all concentrations of the test substance were weighed out separately.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspended in dimethylsulfoxide (DMSO)
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (supplemented with cofactors) derived from Aroclor 1254 (i.p.) induced rat liver
Test concentrations with justification for top dose:
312.5; 625; 1250; 2500; 5000 µg/plate

3 plates per test substance concentration and controls

2 independent experiments were performed
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in all common solvents used for this test system, the solvent dimethylsulfoxide was chosen according to an internal SOP of the test facility.
Untreated negative controls:
other: = solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-aminoanthracene (2.5 - 50 μg/plate, dissolved in DMSO; with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: about 48 h

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)
was recorded for all test groups both with and without S9 mix in all experiments.
Rationale for test conditions:
In a range finding test 6 concentrations of the test substance ranging from 20.6 to 5000 µg/plate were tested with strain Salmonella typhimurium TA100 and strain Escherichia coli WP2uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate with and without metabolic activation.
Evaluation criteria:
Acceptance criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positve response:
The test substance was considered to be positive in the test system if one or both of the following conditions are met:
• At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA.
• A reproducible increase of the mean number of revertants per plate above that of the negative control by at least a factor of 1.5 for any concentration at least for one of the following strains: TA 100 and TA 102.
Generally a concentration-related eflfect should be demonstrable.
Statistics:
A statistical analysis was not performed.
Species / strain:
S. typhimurium TA 1535
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
Untreated negative controls validity:
valid
Remarks:
= vehicle control
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
Untreated negative controls validity:
valid
Remarks:
= vehicle control
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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
Untreated negative controls validity:
valid
Remarks:
= vehicle control
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
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
Untreated negative controls validity:
valid
Remarks:
= vehicle control
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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
Untreated negative controls validity:
valid
Remarks:
= vehicle control
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
Remarks:
= vehicle control
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
There were no known circumstances or occurrences in this study that were considered to have aflfected the quality or integrity of the test data.


Conclusions:
Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.
Executive summary:

The test substance, a solid and white powder, purity > 98%, was tested for mutagenic eflfects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in dimethylsulfoxide and tested at five concentrations in the range of 312.5 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substance led to an increase in the incidence of either histidine- or tryptophanprototrophic mutants by comparison with the negative control.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
yes
Remarks:
lacking of one strain (E.coli)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was suspended in acetone by ultrasonication.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspended in acetone
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (supplemented with cofactors) derived from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
25, 75, 225, 675 and 2025 µg/0.1 ml

3 Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Remarks:
acetone
Negative solvent / vehicle controls:
yes
Remarks:
= negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
cyclophosphamide
other: daunorubicin-HCl (for TA 98; 5 and 10 µg/0.1 ml phosphate buffer); N-methyl-N'-nitro-N-nitrosoguanidine (for TA 1535; 3 and 5 µg/0.1 ml phosphate buffer)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

DETERMINATION OF CYTOTOXICITY
- decrease in the number of histidine-prototrophic mutants
Evaluation criteria:
The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight decrease in the number of histidine-prototrophic mutants with and without activation mix at a conc. of 2025 µg/0.1 ml
Vehicle controls validity:
valid
Untreated negative controls validity:
other: = vehicle control
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight decrease in the number of histidine-prototrophic mutants with and without activation mix at a conc. of 2025 µg/0.1 ml
Vehicle controls validity:
valid
Untreated negative controls validity:
other: = vehicle control
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight decrease in the number of histidine-prototrophic mutants with and without activation mix at a conc. of 2025 µg/0.1 ml
Vehicle controls validity:
valid
Untreated negative controls validity:
other: = vehicle control
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight decrease in the number of histidine-prototrophic mutants with and without activation mix at a conc. of 2025 µg/0.1 ml
Vehicle controls validity:
valid
Untreated negative controls validity:
other: = vehicle control
Positive controls validity:
valid

The slight increase in the number of back-mutant colonies observed in the experiment without microsomal activation on strain TA 1537 is attributed to fluctuations in the rate of spontaneously occurring back-mutants.

 

 

Conclusions:
No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.
Executive summary:

The test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed a slight reduction in the colony count due to a growth-inhibiting effect of the compound at the concentration of 2025 µg/0.1 ml.

No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD Guideline 471 (Bacterial Reverse Mutation Assay):

The test substance was tested for mutagenic eflfects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in dimethylsulfoxide and tested at five concentrations in the range of 312.5 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substance led to an increase in the incidence of either histidine- or tryptophanprototrophic mutants by comparison with the negative control.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.