Tributylmethylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

other: Amendment

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

454-210-6

EC Name:

-

Cas Number:

13106-24-6

Molecular formula:

C13 H30 N . C H3 O4 S

IUPAC Name:

Tributylmethylammoniummethylsulfate

Details on test material:

- Name of test material (as cited in study report): 1-Butanaminium, N,N-dibutyl-N-methyl-, methylsulfate
- Storage condition of test material: Room temperature
- Physical state: White to yellowish, solidified melt

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 601

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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:

0; 20; 100; 500; 2,500 and 5,000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S-9 mix, Strain E. coli WP2 uvrA

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S-9 mix, Strain: TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

Without S-9 mix, Strain: TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

Without S-9 mix, Strains: TA 1535, TA 100

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S-9 mix: All strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 3: preincubation

DURATION
- Preincubation period: 20 min
- exposure period of preincubation test: 48 - 72 hours
- exposure period in agar: 48 - 72 hours

NUMBER OF REPLICATIONS: 3 per experiment

DETERMINATION OF CYTOTOXICITY
- Method: a decrease in the number of revertants, clearing or diminution of the background lawn and reduction in the titer

Evaluation criteria:

ACCEPTANCE CRITERIA
The experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was ≥1E9/mL.

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.
- A test substance is generally considered nonmutagenic if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants) was observed using the Salmonella strains depending on the test conditions from about 2,500 µg/plate onward.

SOLUBILITY
No test substance precipitation was found.

Applicant's summary and conclusion

Conclusions:

A bacterial mutagenicity test was performed with tributylmethylammoniummethylsulfate according to OECD TG 471 and 472 and in comliance with GLP. There was no increase in the number of revertants in any of the S. typhimurium or E. coli tester strains at any dose, neither in the standard plate test nor in the preincubation test, with or without metabolic activation.

Executive summary:

In a GLP compliant bacterial mutagenicity test, performed according to OECD TG 471 and 472, 4Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and E. coli strain WP2 uvr A were used to test the mutagenic potential of tributylmethylammoniummethylsulfate both with and without metabolic activation. One standard plate test and one preincubation test were performed at test concentrations of 0; 20; 100; 500; 2500 and 5000 µg/plate. Three plates per experiment per concentration or control were used. No precipitation of the test substance was found. A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants) was observed using the Salmonella strains depending on the test conditions from about 2500 µg/plate onward. There was no increase in the number of revertants in any of theS. typhimuriumorE. colitester strains at any dose, neither in the standard plate test nor in the preincubation test, with or without metabolic activation. It was therefore concluded that the test substance is not mutagenic under these experimental conditions.