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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): not mutagenic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA
- Suitability of cells: recommended in OECD TG 471


Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA
- Suitability of cells: use is in accordance with current scientific recommendation for the conduct of this assay
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital and beta-naphthoflavone-induced rat livers
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2650, and 5300 ug/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
In this study, 5.3 mg/plate was used as top dose in all experiments.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: the test substance was soluble in water.
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test and preincubation test

STANDARD PLATE TEST
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
- Salmonella typhimurium: Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added (test solution or vehicle; fresh bacterial culture; S9 mix or phosphate buffer). After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
- Escherichia coli: Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added (test solution or vehicle; fresh bacterial culture; S9 mix or phosphate buffer). After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

PREINCUBATION TEST
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the preincubation assay a weak bacteriotoxicity was observed with S9 mix at a concentration of 5300 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
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
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
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the data available and the criteria for classification and labeling laid down in Regulation (EC) 1272/2008 (CLP), non-classification of the test substance is warranted.