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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (reference 7.6.1-1).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Wistar rat livers.
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 µg/plate (SPT and PIT)
Vehicle / solvent:
- Vehicle used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1, 2).

- 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 in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
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 in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
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. (7) and Matsushima et al. (8).
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.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
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:
no cytotoxicity
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: No bacteriotoxic effect was observed under all test conditions.

MUTAGENICITY: A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test item was examined for its mutagenic activity in a serie of a bacterial reverse mutation assay (OECD 471) employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from Wistar Rat liver was used. Treatments of all tester strains were performed using the test item solved in water in the absence and in the presence of S9 mix, using final concentrations between 33 and 5000 µg/plate, plus negative, positive and untreated controls.

All tested controls were valid. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure under all test conditions.

Under the conditions described, a biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

In conclusion, the test material was considered to be non-mutagenic under the described experimental conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

The test item was examined for its mutagenic activity in a bacterial reverse mutation assay (OECD 471) employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms (reference 7.6.1-1).

The plate incorporation test with and without addition of liver S9 mix from Wistar Rat liver was used. Treatments of all tester strains were performed using the test item solved in water in the absence and in the presence of S9 mix, using final concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate, plus negative, positive and untreated controls.

All tested controls were valid. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 mix. During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure under all test conditions.

Under the conditions described, a biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

In conclusion, the test material was considered to be non-mutagenic under the described experimental conditions.

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not need to be classified as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.