Dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

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:

1993-05-06 - 1993-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta - S. typhimurium: The Salmonella typhimurium reverse mutation assay,

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

EC Number:

222-860-8

EC Name:

Dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

Cas Number:

3637-26-1

Molecular formula:

C11H21NO5S

IUPAC Name:

dimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl](3-sulphopropyl)ammonium hydroxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): N,N-Dimethyl-N-methacryloxyethyl-N (3-sulfopropy1)-anunonium-betaine (SPE)
- Physical state: white powder
- Expiration date of the lot/batch: Stated to be stable for more that 2 years, taken as from Date of Manufacture in 1992
- Storage condition of test material: stored at room temperature, in the dark.

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:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

liver fraction (S-9 mix) prepared from rats previously treated with a compound (Aroclor 1254)

Test concentrations with justification for top dose:

Preliminary toxicity test : 5, 50, 500, 5000 µg/plate
Mutation tests : 39.06, 79.13, 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test material was assessed. SPE was found to be soluble at 50 mg/mL in water. Therefore water was the chosen solvent for the subsequent tests.

Controls

Untreated negative controls:

other: solvent control (water)

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST:
Four concentrations of test substance were assessed for toxicity using the six tester strains. The highest concentration was 50 mg/mL of test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent, water, was used as the negative control.
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S-9 mix or 0.5 mL 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediately by 2 mL of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. A single petri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined. Revertant colonies were counted using a Seescan Automatic Colony Counter.
Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration used in the main tests is the same as that used in the preliminary toxicity test. If toxic effects are observed a lower concentration may be chosen for the main assays. Ideally the concentrations chosen for the mutation tests should include a minimum of four non-toxic concentrations.

MUTATION TEST PROCEDURE.
The test substance was added to cultures of the six tester strains at five concentrations separated by 2-fold dilutions. The highest concentration of SPE used was 5000 µg/plate. The negative control was the chosen solvent, water. The positive control compounds were also included. Additional dose levels were included with TA 1538 and TA 98 in the presence of S-9 mix only, at 156.25, 78.125 and 39.0625 µg/plate.
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S-9 mix or 0.5 mL 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the test solution was added, followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter.
At a later date the main test was repeated using the procedures described above with the same concentrations of test substance. Additional dose levels with TA 1538 and TA 98 were not included.

Evaluation criteria:

ASSESSMENT OF RESULTS
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test material is assessed by applying the following criteria:

(a) If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the material's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the material is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989).

Results and discussion

Test results

Test results 1

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

The test substance was not toxic towards the tester strains. Contamination was observed in the presence of S-9 mix with TA 1538 and TA 98 at 5000 µg/plate. 5000 µg/plate was chosen as the top dose level in the mutation tests. Additional dose levels were included with strains TA 1538 and TA 98, in the presence of S-9 mix only, at 156.25, 78.125 and 39.0625 µg/plate.

Applicant's summary and conclusion

Conclusions:

The study was performed according to the OECD Guideline 471 according to the principles of the good laboratory practice and therefore considered to be of highest quality (reliability Klimisch 1). No evidence of mutagenic activity was seen at any dose level of the test material in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in water, SPE was not mutagenic in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of N,N-dimethyl-N-methacryloxyethyl-N-(3- sulfopropy1)-ammonium-betaine (SPE), histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test material, diluted in water which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. Contamination was observed in the presence of S-9 mix with TA 1538 and TA 98 at 5000 µg/plate. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, 312.5, 156.25, 78.125, 39.0625 µg/plate. No evidence of mutagenic activity was seen at any dose level of SPE in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in water, SPE was not mutagenic in this bacterial system.