Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

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:

2015-07-16 to 2015-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

May 30, 2008

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Cas Number:

93820-52-1

Molecular formula:

C12 fatty acid based: C19H37N2NaO4 - C18 fatty acids based: C23H45N2NaO4

IUPAC Name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Aqueous solution of beta-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-cocoacyl derivs., monosodium salts

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

Details on mammalian cell type (if applicable):

Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates is performed

Additional strain / cell type characteristics:

other: Salmonella strains: rfa-, uvrB-; E. coli strain: uvrA-

Metabolic activation:

with and without

Metabolic activation system:

rat liver 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: 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate

Vehicle / solvent:

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

Controls

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); 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION:plate incorporation (experiment I) and preincubation (experiment II)

DURATION
- Preincubation period: 60 min (only experiment II)
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): all Salmonella typhimurium strains: histidine; E. coli strain: tryptophane

NUMBER OF REPLICATES: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants; reduction of the bacterial background lawn

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:

not mandatory

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:

cytotoxicity

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: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA: In experiment I the number of colonies did not quite reach the lower limit of the laboratory's historical control data in the negative control of strain TA 1535 with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Any other information on results incl. tables

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000-5000	1000-5000	1000-2500	2500
TA 1537	1000-5000	1000-5000	333-2500	1000-2500
TA 98	1000-5000	1000-5000	333-2500	1000-2500
TA 100	333-5000	333-5000	333-2500	1000-2500
WP2 uvrA	1000-5000	1000-5000	2500	2500

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000-5000	2500-5000	1000-2500	2500
TA 1537	1000-5000	2500-5000	1000-2500	2500
TA 98	1000-5000	1000-5000	333-2500	1000-2500
TA 100	33; 333-5000	333-5000	33-2500	333-2500
WP2 uvrA	2500-5000	2500-5000	2500	2500

Applicant's summary and conclusion

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, no substantial increase in revertant colony numbers of any of the five tester strains was observed 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. Therefore, Amphopropionate C12-18 is considered to be non-mutagenic in this Salmonella typhimurium and 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 (2008) 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 C12 -18 (ca. 40% a.i.) at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation method and at concentrations of 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate using the reincubation method both in the absence and presence of mammalian metabolic activation.

 

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. Precipitation of Amphopropionate C12 -18 did not occur up to the highest investigated dose. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C12 -18 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C12 -18 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.