Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 10 October 2016 and 24 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 471, in compliance with GLP, without deviations that influence the quality of the results.

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:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

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:

Ethyl 4-methyl-2-oxocyclohexanecarboxylate

EC Number:

236-910-1

EC Name:

Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Cas Number:

13537-82-1

Molecular formula:

C10H16O3

IUPAC Name:

ethyl 4-methyl-2-oxocyclohexane-1-carboxylate

Test material form:

liquid

Details on test material:

Physical appearance: Clear colourless liquid
Storage conditions: In refrigerator (2-8°C) in clear glass bottle

Method

Target gene:

- S. typhimurium: Histidine gene
Escherichia coli (WP2uvrA): tryptophan locus

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 S9-mix

Test concentrations with justification for top dose:

- Experiment 1:
The following dose levels were used: in all strains both with and without S9 - 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (the maximum recommended dose level)

- Experiment 2:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
TA100 (without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
TA1535 (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
WP2uvrA (with and without S9-mix), TA98 and TA1537 (with S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate.
TA100 (with S9-mix), TA98 and TA1537 (without S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Up to eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to
pre-incubation.

Vehicle / solvent:

- Solvent used: DMSO
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

(untreated plates)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: direct plate incorporation method
- Experiment 2: change in test methodology from plate incorporation to pre-incubation.

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain

DETERMINATION OF CYTOTOXICITY
All of the plates were incubated at 37 ± 3”C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

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. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

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:

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:

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 500 µg/plate in the absence of S9-mix and to Salmonella tester strains TA100 and TA1535 initially from 1500 µg/plate in the presence S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA (absence or presence of S9-mix), TA1537 and TA98 (presence of S9-mix). Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. In the second mutation test (pre-incubation method), the test item induced a stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA100), 500 µg/plate (TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA100 and TA98). No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of
S9-mix.

There were no toxicologically significant 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant 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 metabolic activation (S9-mix) in Experiment 2 (pre incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 5000 µg/plate in the first mutation test. This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. A single count for WP2uvrA (vehicle control dosed in the absence of S9-mix after the second mutation test) was just below the minimum historical untreated/vehicle control profile. This count was still considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9 mix. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:

FRET 13-0545 was considered to be non-mutagenic under the conditions of this test in AMES following OECD TG 471.

Executive summary:

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 MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 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 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations.  The dose range was amended following the results of Experiment 1 and ranged between 0.5 to 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Up to eight test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally 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 or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 500 µg/plate in the absence of S9-mix and to Salmonella tester strains TA100 and TA1535 initially from 1500 µg/plate in the presence S9-mix.  No toxicity was noted to Escherichia coli strain WP2uvrA (absence or presence of S9-mix), TA1537 and TA98 (presence of S9-mix).  Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix.  In the second mutation test (pre-incubation method), the test item induced a stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA100), 500 µg/plate (TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA).  In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA100 and TA98).  No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence of S9-mix.  The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.  No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically significant 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).  Similarly, no significant 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 metabolic activation (S9-mix) in Experiment 2 (pre incubation method).  A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 5000 µg/plate in the first mutation test.  This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted.  Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Conclusion

FRET 13-0545 was considered to be non-mutagenic under the conditions of this test.