ethyl 2-acetyl-4-methyltridec-2-enoate

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:

8 to 18 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-acetyl-4-methyltridec-2-enoate

EC Number:

816-455-0

Cas Number:

960253-23-0

Molecular formula:

C18H32O3

IUPAC Name:

ethyl 2-acetyl-4-methyltridec-2-enoate

Test material form:

liquid

Method

Target gene:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100: Histidine gene
E. coli WP2: Tryptophan gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch is characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml:
30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA.,USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

First experiment
GR-86-6599 was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.
Second experiment
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, GR-86-6599 was tested up to concentrations of 5000 μg/plate.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria

Source Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

STUDY DESIGN

First experiment:
Seven concentrations of the test item, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Second experiment:
Based on the results of the first mutation assay, five doses (increasing with approximately half-log steps) of the test item were selected and tested in triplicate in each strain in the second experiment.
The highest concentration of the test item used in the second mutation assay was 5 mg/plate.

Experimental procedure:
The test item was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.

Colony counting:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Rationale Recommended test system in international guidelines (e.g. OECD, EC and MITI).
The initial toxicity-mutation assay was used to establish the doserange for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation.
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to concentrations of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to concentrations of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to concentrations of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to concentrations of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

up to concentrations of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that GR-86-6599 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of GR-86-6599 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

GR-86-6599 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of

Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD, EC and MITI guidelines.

Batch 43 of GR-86-6599 was a yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of

the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any

of the concentrations tested and no decrease in the number of revertants was observed.

GR-86-6599 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and

presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that GR-86-6599 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.