4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

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:

4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

EC Number:

947-879-5

Cas Number:

2475232-73-4

Molecular formula:

C23H18Cl2F4N2O4

IUPAC Name:

4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Test material form:

solid

Details on test material:

Off-white

Method

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:

The S9 Microsomal fraction (Sprague-Dawley) was purchased from Moltox and stored at approximately -196 °C in a liquid nitrogen freezer; Lot No. 4123 was used in this study and the protein level was adjusted to 20 mg/mL.

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation test): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (highest concentration is the maximum recommended concentration level)
Experiment 2 (pre-incubation test): 15, 50, 150, 1500 and 5000 μg/plate

Vehicle / solvent:

Dimethyl sulphoxide (supplied by ThermoFisher Scientific, >99.7% purity)

Controls

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

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

Without S9-mix: N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitroquinoline-1-oxide
With S9-mix: 2-aminoanthracene, benzo[a]pyrene

Details on test system and experimental conditions:

Media: Top agar was prepared using 0.6% w/v Bacto agar (lot number 8255817 07/2023) and 0.5% w/v sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 51673 09/2019 and 51956 10/2019).
Precultures: A culture of each of the bacterial strains was prepared by inoculating nutrient broth with the appropriate coded stock culture and incubated, with shaking, for approximately 10 hours at 37 ± 3 °C. The bacterial cell count for each culture was determined by viable count analysis on nutrient agar plates on the day of test. The cell number after the pre-culture period in experiments 1 and 2 was 3.1E09 and 1.9E09 for TA100, 1.3E09 and 3.6E09 for TA1535, 2.4E09 and 3.3E09 for WP2uvrA pKM101, 2.2E09 and 1.4E09 for TA98 and 1.7E09 and 2.1E09 for TA1537, respectively.
Experiment 1: plate incorporation test
Eight concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method. For the study without metabolic activation, 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates. For the study with metabolic activation, the same procedure was used except that 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. 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 (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation.
Experiment 2: pre-incubation method
Six test item concentration levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic concentration levels and the maximum recommended concentration following the change in test methodology from plate incorporation to pre-incubation. For the test without metabolic activation, 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 30 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate. The procedure for the study with metabolic activation was the same except that 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 30 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate. 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 (toxicity). Manual counts were performed because of test item precipitation from 150 and 500 μg/plate in the absence and presence of S9, respectively.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix; Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Rationale for test conditions:

The maximum concentration of the test item in the first experiment was selected as the maximum recommended concentration of 5000 μg/plate.

Evaluation criteria:

If exposure to a test item produces a reproducible increase, in one or more concentration, in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship in at least one strain with or without metabolic activation system, it will be considered to exhibit mutagenic activity in this test system (Mortelmans and Zeiger 2000). No statistical analysis was performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice those of the concurrent vehicle controls (as described above) will not be considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director will use his/her scientific judgment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn in any of the five tester strains either with or without S9 mix following exposure to the test substance at any test concentration in both experiments. A test substance precipitate was noted from 500 and 150 μg/plate in both the presence and 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 concentration of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method). A minor increase was noted in Experiment 2 (TA1535 at 15 μg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the relevant strain, did not exceed the threshold of twice the concurrent control and was, therefore considered of no biological relevance.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All counts were within the min-max range of historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, consistent with the laboratory’s positive historical control data, thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium and Escherichia coli strains used.

Executive summary:

The ability of the substance to induce reverse mutations in bacteria, either directly or after metabolic activation, in the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2), using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strain WP2uvrApKM101 was tested under GLP to OECD TG 471 (1997).
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.
The maximum concentration of the test item in the first experiment was selected as the maximum recommended concentration of 5000 μg/plate.
In the first mutation test (plate incorporation method), there was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn, in any of the five tester strains either in the presence or absence of metabolic activation (S9 mix) following exposure to the substance. Consequently, the same maximum concentration of 5000 μg/plate was used as the maximum concentration in the second mutation test. Similarly, there was no toxicity in either the presence or absence of metabolic activation (S9-mix), at any test item concentration in the second mutation test (pre-incubation method).
A test item precipitate (particulate in appearance) was noted from 500 and 150 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method).
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies which were consistent with the laboratory’s historical positive control data, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Based on the results it was found that the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial tester strains used.