Benzyltriethylammonium 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

Study period:

no data to 1984-10-25

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study report

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only two strains evaluated (Salmonella typhimurium TA98 and TA100), limited data on the test item and the methodology

Principles of method if other than guideline:

Ames assay was performed to determine the mutagenic potential of test substance.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltriethylammonium chloride

EC Number:

200-270-1

EC Name:

Benzyltriethylammonium chloride

Cas Number:

56-37-1

Molecular formula:

C13H22N.Cl

IUPAC Name:

N-benzyl-N,N-diethylethanaminium chloride

Details on test material:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: 0000008753- Expiration date of the lot/batch: 2008-03-01- Purity test date: no data STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature, light protected- Stability under test conditions:Not indicated by the sponsor- Solubility and stability of the test substance in the solvent/vehicle: The final concentration of deionised water in the culture medium was 10 % (v/v). No data on stability is provided..- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

Specific details on test material used for the study:

- Name of test material (as cited in study report): T779
- IUPAC name: N-Benzyl-N,N-diethylethanaminium chloride
- Substance type: white powder
- Physical state: solid

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: other: See below

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

With and without metabolic activation, main and repeat experiments: 0, 312.5, 625, 1250, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation at 5 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: at 1 µg/plate for TA100

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation: for both strains at 1 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Tests were performed by mixing test article dilutions, or appropriate controls, 1-6 x E8 bacteria from an overnight culture in nutrient broth (0.1 mL /plate +/- 0.5 mL of S9 mix in histidine-biotin supplemented top agar in petri dishes.
After incubation of the plates for 48 hours at 37°C in the dark, the number of macroscopic colonies (his+) revertants were counted.

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
- The viability of the strain was also verfied by plating appropriate dilutions of the bacterial suspension onto histidine rich agar plates.

Rationale for test conditions:

No data

Evaluation criteria:

The results were considered as positive if there was a dose-related increase in the number of revertant colonies with at least a twofold increase over the controls.
Results that did not meet the criteria for determining a positive result were considered to be negative.

Statistics:

no data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 98 and TA 100

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES:
- Before conducting the mutagenicity study the test substance was assessed for bacterial cytotoxicity using one of the bacterial strains. The Salmonella strain was inoculated from cultures on master plates into nutrient broth and grown overnight at 37°C. The viability of the strain was tested by plating appropriate (10E-6) dilutions of the bacterial suspension onto histidine rich agar plates. Appropriate dilutions of test substance, bacterial strain (10E-6) and top agar were mixed and poured on agar plates. The number of surviving colonies were counted and the toxic dose level was determined. The test substance was tested for bacterial cytotoxicity at doses up to 5000 µg/plate. At doses of 100, 500, 1000 and 5000 µg/plate the cell viability of the bacteria was only slightly reduced. 5000 µg/plate was selected as the highest dose for the plate incorporation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive controls, 5 µg 2-nitrofluorene/plate, 1 µg sodiumazide/plate and 1 µg 2-aminoanthracene/plate, induced a significant increase in the number of revertant colonies

ADDITIONAL INFORMATION ON CYTOTOXICITY: See range finding/screening study data

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

Test substance did not induce a significant increase in the number of revertant colonies beyond the negative control incidence in any of the tested strains either with or without a metabolic activation system.

Executive summary:

Ames assay was performed to determine the mutagenic potential of test substance. The test chemical was dissolved in water and used at dose levels of 0, 312.5, 625, 1250, 2500, and 5000 µg/plate in the presence and absence of S9 metabolic activation system in the main and repeat experiments performed. After incubation of the plates for 48 hours at 37°C in the dark, the number of macroscopic colonies (his+) revertants were counted. Concurrent solvent and positive control chemicals were also included in the study. The results were considered as positive if there was a dose-related increase in the number of revertant colonies with at least a twofold increase over the controls. Results that did not meet the criteria for determining a positive result were considered to be negative. Test substance did not induce a significant increase in the number of revertant colonies beyond the negative control incidence in any of the tested strains either with or without a metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.