2,4,6-trimethylpyridine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in the bacterial 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:

2020-01-16 to 2020-04-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair substitution (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537).

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 rats induced with phenobarbital/ß-naphthoflavone)

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: sodium azide (10 µg/plate), strains TA1535, TA100 4-nitro-o-phenylene-diamine (10 µg/plate), strains TA1537, TA98 methyl methane sulfonate (2 µg/plate), WP2 uvrA with S9 mix 2-aminoanthracene (10 µg/plate) all strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Experiment I: Plate Incorporation
- Experiment II: Pre-Incubation

DURATION
- Pre-incubation period: 60 min at 37°C (pre-incubation test)
- Expression time (cells in growth medium): 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

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.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced background lawn in Experiment 2 at the highest concentration and reduction in the number of revertants observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced background lawn in Experiment 2 at the highest concentration and reduction in the number of revertants (with S9 mix) observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Reduced background lawn in Experiment 2 at the highest concentration but no reduction in the number of revertants observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced background lawn in Experiment 2 at the highest concentration and reduction in the number of revertants (without metabolic activation) observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Reduced background lawn in Experiment 2 at the highest concentration but no reduction in the number of revertants observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Please refer to the attached document.

Conclusions:

The test substance induced no genetic damage (considered not mutagenic) in the Ames test under the described experimental conditions.

Executive summary:

The Ames test was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) 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. Experiments I and II were performed with and without liver microsomal activation, experiment Ia without liver microsomal activation only. 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

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

No precipitation of the test item occurred up to the highest investigated dose. In experiment I 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. In experiment II reduced background growth was observed in all strains with and without S9 mix at 5000 μg/plate. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment II in strains TA 1537 without S9 mix, TA 100 with S9 mix and WP2 uvrA with and without S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The Ames test was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) 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. Experiments I and II were performed with and without liver microsomal activation, experiment Ia without liver microsomal activation only. 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

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

No precipitation of the test item occurred up to the highest investigated dose. In experiment I 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. In experiment II reduced background growth was observed in all strains with and without S9 mix at 5000 μg/plate. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment II in strains TA 1537 without S9 mix, TA 100 with S9 mix and WP2 uvrA with and without S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Justification for classification or non-classification

Based on the results obtained the test item was not classified for genetic toxicity according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.