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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The assay was performed according to OECD guideline 471 and EU Method B.13/14 under GLP-conditions in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Because of contamination, which led to irregular background growth, data evaluation was not possible in experiment I in strain TA 98 with and without S9 mix. Therefore, this part of experiment was repeated as a plate incorporation assay at the following concentrations (reported as experiment Ia):

Experiment Ia:

Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test sample at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase in revertant colony number, not reaching the threshold of twice the number of the corresponding solvent control, was observed in experiment Ia at 5000 μg/plate.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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:
Oct 2018 - Noc 2018
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:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
liver microsomal activation (Phenobarbital/β-naphthoflavone induced rat liver S9)
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment Ia:
Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
deionized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
For each strain and dose level, including the controls, three plates were used.
Experiment I and Ia (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. 3.4.3 Precultures),
2000 μL Overlay agar
Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. 3.4.3 Precultures),
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
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 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, but tested up to precipitating 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
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
Additional information on results:
This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I and Ia) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Because of contamination, which led to irregular background growth, data evaluation was not possible in experiment I in strain TA 98 with and without S9 mix. Therefore, this part of experiment was repeated as a plate incorporation assay at the following concentrations (reported as experiment Ia):
Experiment Ia:
Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the sample at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase in revertant colony number, not reaching the threshold of twice the number of the corresponding solvent control, was observed in experiment Ia at 5000 μg/plate. Since no relevant increase in the number of revertant was observed in experiment II, which was performed as pre-incubation assay, this effect can be judged as biologically irrelevant.

Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The assay was performed according to OECD guideline 471 and EU Method B.13/14 under GLP-conditions in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Because of contamination, which led to irregular background growth, data evaluation was not possible in experiment I in strain TA 98 with and without S9 mix. Therefore, this part of experiment was repeated as a plate incorporation assay at the following concentrations (reported as experiment Ia):

Experiment Ia:

Strains TA 98: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used, except of strain TA 98 with and without S9 mix, in which reduced background growth was observed in experiment Ia at 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A minor increase in revertant colony number, not reaching the threshold of twice the number of the corresponding solvent control, was observed in experiment Ia at 5000 μg/plate.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Additional information

Justification for classification or non-classification

Based on these results the substance is not classified according to Regulation (EC) No 1272/2008.