1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-10 (even numbered) acyl derivs., hydroxides, inner salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

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:

2-[(3-decanamidopropyl)dimethylazaniumyl]acetate 2-[dimethyl(3-octanamidopropyl)azaniumyl]acetate

EC Number:

944-170-2

Molecular formula:

not applicable, UVCB substance

IUPAC Name:

2-[(3-decanamidopropyl)dimethylazaniumyl]acetate 2-[dimethyl(3-octanamidopropyl)azaniumyl]acetate

Test material form:

solid - liquid: aqueous solution

Method

Target gene:

his-operon (Salmonella strains); trp (E.coli strain)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: uvrB- (Salmonella); uvrA- (E. coli)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate
(all concentrations adjusted to purity)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to solubility properties and relative nontoxicity to the bacteria

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes. at 37°C (only Experiment II)
- Exposure duration: least 48 hours at 37 °C in the dark

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: background growth; reduction in the number of revertants below the indication factor of 0.5

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

only in strain TA 100 toxic effects were observed at 5000 μg/plate without S9 mix in experiment I and with and without S9 mix in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I without S9 mix, the data in the solvent control of strain TA 100 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Applicant's summary and conclusion

Conclusions:

C8-10 Alkylamidopropyl betaine was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of rat liver S9 mix. Under the conditions of the study, the test substance was negative for mutagenic potential.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to C8-10 Alkylamidopropyl betaine (36% a.i. in aqueous solution) in deionized water at concentrations of 0 (control), 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in the first experiment (plate incorporation assay) and 0 (control), 33, 100, 333, 1000, 2500 and 5000 μg/plate in the second experiment (preincubation assay) in the presence and absence of mammalian metabolic activation (rat liver S9 mix).. All concentrations were adjusted to purity.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were absent in nearly all strains, only in strain TA 100 toxic effects were observed at 5000μg/plate without S9 mix in experiment I and with and without S9 mix in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In experiment I without S9 mix, the data in the solvent control of strain TA 100 were slightly above the historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, C8-10 Alkylamidopropyl betaine did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.