Betaines, coco alkyldimethyl(3-sulfopropyl)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983-01-25 - 1983-02-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

TA1538 used instead of TA102 or E.coli WP2 uvrA

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Reference 2

Reference Type:

other: study report amendment

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Draft

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Guideline B.14 (Draft)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Betaines, coco alkyldimethyl(3-sulfopropyl)

EC Number:

269-225-1

EC Name:

Betaines, coco alkyldimethyl(3-sulfopropyl)

Cas Number:

68201-55-8

Molecular formula:

C7H17NO3S·[CH2]6-16

IUPAC Name:

[3-(dodecyldimethylazaniumyl)propyl]trioxo-λ⁶-sulfanuide

Test material form:

liquid

Remarks:

clear

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: from B.N. Ames, University of California Berkeley CA, 94720, USA, May 1981.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
from B.N. Ames, University of California Berkeley CA, 94720, USA, May 1981.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate, as required by the guideline, evaluated up to 158 µg/plate due to toxic effects

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Controls

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);
- Cell density at seeding (if applicable): 100 µl of a 2 x 10 exp.5 diluted bacteria suspension

DURATION
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): his-minimal agar

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
To estimate the toxicity of the test material, prototrophic bacteria (spontaneous revertants of TA 1537 = RTA) were added as an internal standard to the selective agar plates, together with TA 1537. The difference in the number of colonies on plates with and without added prototrophic bacteria at each dose level of the test material was compared to the number of colonies obtained with the negative control. The ratio of the two values was expressed as relative survival rate with the negative control at a 100 % survival rate.
Additionally, the toxicity of the test material can be shown in the mutagenicity assay, by a reduction in the number of revertants or by a clearing of the back ground lawn.

Rationale for test conditions:

The test procedure was according to Ames et. al.. The microbial assay is based on reverse mutation of Salmonella typhimurium from auxotrophism (histidine dependant) to prototrophism (histidine independant). When mutated Salmonella typhimurium are exposed to a mutagen, mutation to the histidine independent form takes place in a proportion of the bacterial population. These are readily detectable due to their ability to grow on a histidine deficient medium.
Using the strains of Salmonella typhimurium, revertants may be produced after exposure to a chemical mutagen which have arisen as a result of a base-pair substitution in the genetic material or as a frameshift mutation in which genetic material is either added, deleted or miscoded.
Since many compounds do not exert their mutagenic activity until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria mere incubated both in the absence and in the presence of liver microsomal fraction taken from rats previously treated with a compound known to induce enzyme activity.
To validate the test, reference mutagens were tested parallel to the test material.

Evaluation criteria:

A material is identified as a mutagen in this test system if there is a reproducible demonstration of a dose effect relation with a 2fold increase in the number of revertants over the controls in a minimum of one strain. With the strain TA 100, a 1.5fold increase is the criterion for a positive result.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The study was performed according to the OECD Guideline 471 with deviations (strain TA 1538 instead of TA 102) according to the principles of the good laboratory practice and therefore considered to be of high quality (reliability Klimisch 2). Strain TA 1538 is sensitive to frameshift mutagens, at or near GCGCGCGC (Delta GpC frameshift), and would hence serve as a surrogate for e.g. strain TA98, which was used anyway, but not for the recently recommended strains E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. The latter detects transitions and transversions including A:T to G:C (base substitution at A:T basepair), which allows hence only the assessment Klimisch 2 instead of 1. However, since the strains E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.
The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material did not induce significant increases in the frequency of revertant colonies in any of the bacterial strains. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material, when evaluated up to the limit of toxicity, caused neither basepair substitutions nor frameshift mutations. Therefore, the test results with the test item revealed no indication of potential gene mutagenic activity.

Executive summary:

This study was performed according to OECD TG 471 (Draft version 1983) under GLP to investigate the potential gene mutagenic activity of the test item, according to the plate incorporation test of Ames et. al. using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538.

The test urns per formed with and without liver microsomal activation. The test material was tested at the following concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate. Each concentration, including the controls, was tested in triplicate .

The test material showed a toxic effect in the experiment without S9 mix at the concentrations starting from 500 µg per plate and in the experiment with S9 mix at the concentration of 5000 µg per plate. Therefore, an evaluation of the potential mutagenic activity of the test material at these concentrations was prevented.

Up to the highest evaluated dose 158 µg per plate , no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain when compared with the corresponding controls. The presence of microsomal activation did not influence these findings.

In conclusion, it can be stated that during this in vitro Salmonella/mammalian microsome assay with the test item, no gene mutagenic activity could be demonstrated in the concentrations evaluated, under the experimental conditions reported.