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Toxicological information

Genetic toxicity: in vitro

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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

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998
Reference Type:
other: Amendment
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Controlsopen allclose all
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
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
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
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
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 resultsopen allclose all
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
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
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
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
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.