1-Butanaminium, N,N-dibutyl-N-methyl-, methyl sulfate (1:1)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

tributyl(methyl)azanium methyl sulfate

Details on test material:

Batch No.: 601
Date of manufacture: June 1997
Test substance NO.: 97/272
Degree of purity: 101.0 g/100 g (1H-NMR)
Appearance, consistency: White to yellowish, solidified melt
Storage: Room temperature

Method

Target gene:

Salmonella: His
E. coli: Trp

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:

S9-Mix

Test concentrations with justification for top dose:

First experiment: 0; 20; 100; 500; 2500 and 5000 µg/plate
Second experiment: 0; 20; 100; 500; 2500 and 5000 µg/plate

Vehicle / solvent:

water

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-AA, MNNG, NOPD, AAC, ENNG

Details on test system and experimental conditions:

3 test plates per dose or per control were made.

Sterility control: Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains.
Vehicle control: The vehicle control with and without S-9 mix only contains the vehicle used for the test substance at the same concentration and volurne for all tester strains.
Positive controls
The following positive controls are used to check the mutability of the bacteria and the activity of the S-9 mix:
With S-9 mix: 2-aminoanthracene (2-AA)
- 2.5 µg/plate, dissolved in DMSO, strains: TA 1535, TA 100, TA 1537, TA 98
- 60 µg/plate, dissolved in DMS0, strain: Escherichia coli WP2 uvrA
Without S-9 mix: N-methyl-N' -nitro-N-nitrosoguanidine (MNNG)
- 5 µg/plate, dissolved in DMS0, strains: TA 1535, TA 100
4-nitro-o-phenylendiamine (NOPD)
- 10 µg/plate, dissolved in DMS0, strain: TA 98
9-aminoacridine (AAC)
- 100 pg/plate, dissolved in DMS0
- strain: TA 1537
N-ethyl-N' -nitro-N-nitrosoguanidine (ENNG)
- 10 µg/plate, dissolved in DMSO, strain: E. coli WP2 uvrA

Standard plate test:
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli:
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45'C, and the remaining components are added in the following order: 0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 h in the dark, the bacterial colonies (trp+ revertants) are counted.

Preincubation test:
0.1 ml test solutian or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 h in the dark, the bacterial colonies are counted.

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 in this test 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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting with 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see table 1

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1: Schematic presentation of the number of maximum revertants per plate (concentration where revertants were observed) in the solvent control and substance treated strains from Experiment 1 and 2:

Standard plate test:

	Control		Testsubstance
Test strain	-S9	+S9	-S9	+S9
TA1535	20 ± 1	20 ± 3	20 ± 1 (20 µg)	19 ± 2 (20 µg)
TA100	128 ± 8	128 ± 4	135 ± 18 (100 µg)	128 ± 20 (100 µg)
TA1537	11 ± 2	11 ± 2	10 ± 1 (20 µg)	10 ± 1 (20 µg)
TA98	30 ± 2	37 ± 2	24 ± 4 (20 µg)	28 ± 1 (20 µg)
E. coli WP2 uvrA	31 ± 4	38 ± 1	28 ± 2  (20 µg)	33 ± 2  (20 µg)

Preincubation test:

	Control		Testsubstance
Test strain	-S9	+S9	-S9	+S9
TA1535	20 ± 3	20 ± 3	16 ± 3 (20 µg)	19 ± 1 (100 µg)
TA100	128 ± 21	124 ± 12	125 ± 12 (100 µg)	147 ± 16 (100 µg)
TA1537	16 ± 3	16 ± 3	16 ± 4 (100 µg)	15 ± 3 (20 µg)
TA98	23 ± 2	38 ± 3	22 ± 4 (100 µg)	38 ± 2 (2500 µg)
E. coli WP2 uvrA	24 ± 1	43 ± 2	27 ± 4  (500 µg)	40 ± 1  (20 µg)