Benzenemethanaminium, N,N,N-trimethyl-, 2-hydroxy-2-methylpropanoate (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

Study period:

20 April 2012 to 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

benzyltrimethylazanium 2-hydroxy-2-methylpropanoate

EC Number:

805-580-6

Cas Number:

1431696-36-4

Molecular formula:

C14 H23 O3 N

IUPAC Name:

benzyltrimethylazanium 2-hydroxy-2-methylpropanoate

Test material form:

other: solid

Details on test material:

- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 31 Jan 2013 as indicated by the sponsor, and the sponsor holds this responsibility.
- Physical state, appearance: Solid, beige
- Storage conditions: Room temperature (protected from moisture; under N2 conditions)

Method

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:

Liver S9 mix from induced rats (ßNF (orally) and Phenobarbital (i.p.): each 80 mg/kg bw/d for 3d)

Test concentrations with justification for top dose:

0; 33; 100; 333; 1 000; 2 800 and 5 600 μg/plate (SPT)
0; 33; 100; 333; 1 000; 2 800 and 5 600 μg/plate (PIT)

Vehicle / solvent:

Due to the good solubility of the test substance in ultrapure water, ultrapure water was used as vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: without S9: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD); with S9: 2-aminoanthracene;

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) standard plate test and pre- incubation test

DURATION
- Preincubation: Preincubation test: 20 min
- Exposure duration: 48-72h

NUMBER OF PLATES: 3 plates per dose per test

SELECTION AGENT (mutation assays): Salmonella typhimurium: 0.5 mM histidine + 0.5 mM biotin, E.coli: 0.5 mM tryptophan

DETERMINATION OF BACTERIOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer

Evaluation criteria:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10E+09 cells per mL were used. For approval the titer of viable bacteria was ≥ 10E+08 colonies per mL.

Assessment criteria
The test substance 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 S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least 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:

In the standard plate test only with the strain TA 98 without S9 mix at the top dose of 5 600 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Precipitation: No test substance precipitation was found with and without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not mutagenic in bacteria.