Tributylethylammonium ethyl sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: "Testing Methods for New Chemical substances"

Version / remarks:

November 2003

GLP compliance:

yes (incl. QA statement)

Remarks:

date certificate 21.07.2004

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tributylethylammonium ethyl sulphate

EC Number:

268-329-4

EC Name:

Tributylethylammonium ethyl sulphate

Cas Number:

68052-51-7

Molecular formula:

C14H32N . C2H5O4S (or C16H37NO4S)

IUPAC Name:

tributyl(ethyl)azanium ethyl sulfate

Test material form:

solid

Remarks:

, slight yellow

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1 and 2
Without and with S9-mix: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Water
- Justification for choice of solvent:
Test compound was stable in water and water has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 10 µg/plate for TA100 and TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

without S9; 10 µg/plate for TA98; 40 µg/plate for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 1 µL/plate for WP2 uvrA

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, 2-AA

Remarks:

with S9; 2.5 µg/plate in DMSO for TA98, TA100, TA1535 and TA 1537; 10 µg/plate in DMSO for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the number of revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

Evaluation od Cytotoxicity
Cytotoxicity can be detected by a diminution of the background lawn or a reduction on the number of revertans dow to a mutation factor ≤ 0.5 in relation to the solvent control.

Criteria of validity
a test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100)
- the spontaneous revesion frequencies of control plates without S9 are within the historical control data range.
- corresponding background growth on both the negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plates.

Evaluation of mutagenicity
Mutation factor: mean revertant counts divided by mean values of the solvent control

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertans occurs and/or
- a biologically relevent positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA100 and WP2 uvrA the number of reversions is at least twice as high
- is in tester strains TA1535, TA1537 and TA98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the OECD guidelines a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 1000 µg/plate and above and with S9: 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: No toxicity observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: 2500 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: 2500 µg/plate and above and with S9: No toxicity observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Eperiment 1:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Experiment 2:
TA1535: without S9: 2500 µg/plate and above and with S9: 5000 µg/plate
TA1537: without S9: 1000 µg/plate and above and with S9: 5000 µg/plate
TA98: without S9: 2500 µg/plate and above and with S9: No toxicity observed
TA100: without S9: 2500 µg/plate and above and with S9: 2500 µg/plate and above
WP2uvrA: without S9: 2500 µg/plate and above and with S9: No toxicity observed

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD/EC guideline and GLP principles, TBEAES was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD/EC guideline and in accordance with GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No precipitation of the test substance was observed. No toxic efffects of the test substance were noted in any of the five tester strains with and without metabolic activation in experiment 1. Toxic effects of the test substance were noted in all tester strains used in experiment 2 at concentrations of 1000 ug/plate and higher (without metabolic activation) and at concentrations of 2500 ug/plate and higher (with metabolic activation), depending on the tester strain.

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.

Based on the results of this study it is concluded that TBEAES is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.