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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: No mutagenic effect was detected.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium and E. coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
20 ug - 5000 ug/plate (standard plate test and preincubation test)
Vehicle / solvent:
Due to the limited insolubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoquanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and N-ethyl-N'-nitro-N-nitrosoquanidine
Details on test system and experimental conditions:
Standard plate test
Salmonella typhimurium:
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0 .6 % agar + 0 .6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants : 0 .5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order :
0.1 ml test solution or vehicle; 0.1 ml fresh bacterial culture; 0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosohate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar : 980 ml aqua dest.; 20 ml Vogel-Bonner E medium; 15 g Difco bacto agar; 20 g D-glucose, monohydrate. After incubation at 37°C for 48 - 72 hours in the
dark, the bacterial colonies (his* revertants) are counted.
Escherichia coli:
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0 .6% agar + 0 .6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants : 0 .5 mM tryptophan) are kept in a water bath at 45°C, and the remaining components are added in the following order : 0 .1 ml test solution or vehicle; 0 .1 ml fresh bacterial cultur e
0 .5 ml S-9 mix (in tests with metabolic activation) or 0 .5 ml phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto minimal agar plates within approx . 30 seconds.
The composition of the minimal agar (SAl selective agar) is based on the description of Green, M .H .L . and Muriel, W .J . (5), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar : 300 ml solution B (agar ); 100 ml solution A (saline solution); 8 ml solution C (glucose solution); 10 ml solution D (casein solution). After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp* revertants) are counted.

Preincubation test
0 .1 ml test solution or vehicle, 0 .1 ml bacterial suspension and 0 .5 ml S-9 mix are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
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 S-9 mix or after adding a metabolizing system.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: A bacteriotoxic effect was observed in the standard plate test from about 2500 ug/plate onward.
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:
other: A bacteriotoxic effect was observed in the standard plate test from about 2500 ug/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames

The study tested the mutagenicity of the test substance. The study was conducted according to OECD TG 471 and OECD TG 472. The standard plate and preincubation tests were performed. Both were done with and without the addition of a metabolizing system obtained from rat liver (S-9 mix) using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. A bacteriotoxic effect was observed in the PIT from about 2500 µg/plate onward. All tests and strains showed a negative effect for genotoxicity. Therefore the test substance was considered non mutagenic in this study under the corresponding conditions.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218. As a result the substance is considered to be not classified as mutagenic.