Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

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:

Experimental start date:08 March 2021. Experimental completion date: 19 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

EC Number:

243-497-1

EC Name:

Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

Cas Number:

20073-13-6

Molecular formula:

C13H20O3

IUPAC Name:

methyl 2-[(1S,2S)-3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl]acetate

Specific details on test material used for the study:

Identification: Methyl Jasmonate
Trade Name: JASMONEIGETM(Methyl Jasmonate)
Chemical Name: Methyl 3-oxo-2-(pent-2-enyl)cyclopentaneacetate
Physical state/Appearance: Clear colourless liquid
CAS Number: 39924-52-2
LOT Number: LOT 0900175
Batch Number: 0900175
Purity: 99.03%
Molecular Weight: 224.3
Expiry Date: 25 March 2023
Storage Conditions: Stored cold at ~ 4 °C; used/formulated at ambient temperature 10 to 30°C

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM) reconstituted with cold, sterile water to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix.

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method
The maximum concentration was 5000 g/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed for each tester strain, using the direct plate incorporation method.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 5, 15, 50, 150, 500, 1500 and 5000 g/plate.

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide (DMSO) at the same concentration in solubility checks performed in house. DMSO was therefore selected as the solvent.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.
With Metabolic Activation
The procedure was the same as described previously except that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). Sporadic manual counts were performed on three individual plates (TA1535 dosed in the absence of S9-mix at 5 µg/plate and WP2uvrA at 15 and 50 µg/plate in the presence of S9-mix) due to light background contamination which prevented an accurate automated count. In these cases manual counts are performed which are considered to be equivalent to the machine counts.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.
With Metabolic Activation
The procedure was the same as described previously except that untreated controls were not performed and, following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

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:

cytotoxicity

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
For the untreated controls (spontaneous mutation rates) and viability a single count for TA100 (after the first mutation test) was below the minimum level of the in-house historical untreated/solvent control minima for the tester strain. This count was considered acceptable as the other solvent and untreated control counts were within the expected range and the tester strain responded very well to the respective positive controls in both the presence and absence of S9 mix.
The number of revertant counts for the solvent (dimethyl sulphoxide) control plates were within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without S9 mix. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation) –
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate as recommended for a soluble and non-toxic substance.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix, although small reductions in colony numbers were noted at 5000 µg/plate for TA100 dosed in the absence of S9-mix and TA1537 dosed in the absence and presence of S9-mix.
No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Experiment 2 (pre-incubation) –
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
The test item exhibited toxicity as weakened bacterial background lawn growth after employing the pre-incubation modification with visible reductions noted for all of the tester strains at and above 1500 µg/plate in both the presence and absence of S9-mix.
No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item, Methyl Jasmonate did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test, Methyl Jasmonate was considered to be non-mutagenic.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MOE, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on the guidance given in OECD TG 471 and was selected as 1.5 to 5000 mg/plate.  As the overall result of
Experiment 1 was Negative, Experiment 2 was performed using the pre-incubation method with fresh cultures of the bacterial strains and fresh test item formulations.  The dose range was modified following the results of Experiment 1 and was 5 to 5000 µg/plate.  Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non‑toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

 

Results

The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were at or close to the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the metabolic activation (S9-mix) were validated.

The dose level of the test item in the first experiment was selected as the OECD TG 471 recommended maximum dose level of 5000 µg/plate.  There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method), although small reductions in colony numbers were noted at 5000 µg/plate for TA100 dosed in the absence of S9-mix and TA1537 dosed in the absence and presence of S9-mix.

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method).  The test item exhibited toxicity as weakened bacterial background lawn growth after employing the pre-incubation

modification with visible reductions noted for all of the tester strains at and above 1500 µg/plate in both the presence and absence of S9-mix. 

No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix in Experiments 1 and 2.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1 (plate incorporation method). 

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre‑incubation method). 

 

Conclusion

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item, Methyl Jasmonate did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix).  Under the conditions of this test, Methyl Jasmonate was considered to be non-mutagenic.