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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

point mutation bacteria: Ames, OECD 471, TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without MA: negative (BASF, 1998)

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella: His
E. coli: Trp

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:

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

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.

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

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'.

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)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

There is one reliable and relevant study available investigating the genetic toxicity of 1-Butanaminium, N,N-dibutyl-N-methyl-, methyl sulfate.

Point mutation in bacteria

OECD Guideline Study:

The substance 1-Butanaminium, N,N-dibutyl-N-methyl-, methyl sulfate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD 471 under GLP. Strains used are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with doses range up to 5,000 pg/plate (SPT and PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix) are conducted. No precipitation of the test substance was found and a bacteriotoxic effect (slight decrease) was observed in SPT and PIT. An increase in the number of his* or trp* revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance 1-Butanaminium, N,N-dibutyl- N-methyl-, methyl sulfate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions (BASF, 1998).

As this study is the only avaiable information, but reliable and good documented, this information is integrated as key study. Further according to the integrated testing strategy (ITS) for genetic toxicity no furhter testing in necessary for the applied tonnage band if results from a ames study is negative and no further hints for mutagenicity are observed.

Assessement:

As the available information for 1-Butanaminium, N,N-dibutyl-N-methyl-, methyl sulfate shows no hints for mutagenicity the substance is considered to be not mutagenic (Ames).

Justification for classification or non-classification

Based on the above information and according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, a classification of the substance for mutagenicity is not justified.

Mutagenicity:

-GHS: no classification

-DSD: no classification