N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

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:

2007-03-20 to 2007-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

June 8, 2000

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

EC Number:

264-761-2

EC Name:

N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

Cas Number:

64265-45-8

Molecular formula:

C15H30N2O4

IUPAC Name:

N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Details on test material:

- Analytical purity: 50.6%
- Physical state: liquid

Method

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (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: additional rfa, gal, chl, bio and uvrB mutations in S. typhimurium strains and uvrA mutation in E.coli strain

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
First test: 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Second test: 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

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

Controls

Controls 1

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

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: (without metabolic activation)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replicates, 2 independent repeats

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, increase in the size of the microcolonies, reduction of revertant colonies

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

With the exception of tester strain TA 100 in the absence of S9-mix, where a moderate reduction of the revertant colonies compared to the solvent control was observed at the highest tested concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. With the exception of tester strain TA 100 in the absence of S9-mix, where a moderate reduction of the revertant colonies compared to the solvent control was observed at the highest tested concentration.
- Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
negative and strain-specific positive control values were within laboratory historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY (DEFINITIVE TEST):
In tester strain TA 100 in the absence of S9-mix, a moderate reduction of the revertant colonies was observed at the highest tested concentration, however the reduction was not below the historical control data range. In the presence of S9-mix, fluctuations in the number of revertant colonies just below or around the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance.
In all other tester strains both in the first and second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Amphopropionate C8 (50%) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (July, 1997) and EU Method B. 13/14 (June, 2000) strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed in two independent experiments to Amphopropionate C8 (50 %) at concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (first test) and 100, 333, 1000, 3330, 5000 µg/plate (second test) in the absence and presence of mammalian metabolic activation using the plate incorporation method.

 

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in both tester strains.

In tester strain TA 100 in the absence of S9-mix, a moderate reduction of the revertant colonies was observed at the highest tested concentration, however the reduction was not below the historical control data range. In all other tester strains both in the first and second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C8 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C8 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  This study is classified as acceptable. It satisfies the requirements for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.