Trimethylamine, N-oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-MAR-2022 - 11-AUG-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

L142, 31 May 2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997, Corrected 26 June 2020

GLP compliance:

yes (incl. QA statement)

Remarks:

Deviation from GLP: Concentration, stability, and homogeneity of test material formulations were not determined in this study. However, this did not impact the overall integrity of the study or the interpretation of the study results and conclusions.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Trimethylamine N-oxide Dihydrate

Cas Number:

62637-93-8

Molecular formula:

(CH3)3NO·2H2O

IUPAC Name:

Trimethylamine N-oxide Dihydrate

Specific details on test material used for the study:

Dihydrate form of the registered substance Trimethylamine N-oxide (CAS 1184-78-7)

Lot: BCCF8795
Clear to white powder
Purity: ≥ 98%

Method

Target gene:

Point mutation

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver metabolic activation system

Test concentrations with justification for top dose:

Range finder: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Direct plate: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Pre-incubation: 52, 164, 512, 1600 and 5000 µg/plate
No precipitation observed at top dose; cytoxicity, as evidenced by a reduction of the bacterial background lawn, was only observed in pre-incubation test in tester strains TA1535, TA1537 and TA100 in the absence of S9-mix at the highest tested concentration.

Vehicle / solvent:

Milli-Q water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 6-Chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride

Details on test system and experimental conditions:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate The highest concentration of the test material used in the subsequent mutation assays was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain in the absence and presence of S9-mix.
The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or
microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn observed in the absence of S9-mix at the highest tested concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn in the absence of S9-mix at the highest tested concentration in pre-incubation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn was observed in the absence of S9-mix in pre-incubation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All bacterial strains showed negative responses over the entire dose-range, i.e. no biologically
relevant dose-related increase in the number of revertants in two independently repeated
experiments.
The negative and strain-specific positive control values were within the laboratory historical
control data ranges indicating that the test conditions were adequate and that the metabolic
activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose-range, i.e. no biologically relevant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Executive summary:

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test material was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test material was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. No biologically relevant reduction of the bacterial background lawn or decrease in the number of revertants was observed. In the second mutation experiment, the test material was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was only observed in tester strains TA1535, TA1537 and TA100 in the absence of S9-mix at the highest tested concentration. In conclusion, based on the results of this study it is concluded that Trimethylamine N-oxide dihydrate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

The study was conducted in compliance with EC B.13/14 (2008) and OECD 471 and is GLP compliant with a Quality Assurance statement. Based on the test methods used and the level of detail in the study report, there are no concerns regarding deficiencies.