Benzenemethanaminium, N,N,N-trimethyl-, 2-hydroxy-2-methylpropanoate (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation (OECD 471, GLP).

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

Study period:

20 April 2012 to 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

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

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:

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.

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.

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

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'.

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not mutagenic in bacteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test substance 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.

 

Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

Dose range: 33 μg - 5 600 μg/plate (SPT); 33 μg - 5 600 μg/plate (PIT)

Test conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

Solubility: No precipitation of the test substance was found with and without S9 mix.

Toxicity: A weak bacteriotoxic effect was observed on a single tester strain at 5 600 μg/plate, only.

 

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

Justification for selection of genetic toxicity endpoint
Only study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available study is considered reliable, but in the absence of information on clastogenicity, insufficient data for classification purposes under 67/548/EEC is available. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable. In the absence of information on clastogenicity, insufficient data for classification purposes under Regulation 1272/2008 is available. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).