4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(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

Study period:

07 Mar 2019 to 08 Apr 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Cas Number:

2061933-85-3

Molecular formula:

C23H19Cl2F4N3O4

IUPAC Name:

4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid: particulate/powder

Method

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 3

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/ß-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared from male Wistar rats (RjHan:WI; weight approx. 220 – 320 g) induced by peroral administration of 80 mg/kg b. phenobarbital and by peroral administrations of ß-naphthoflavone each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. The protein concentration in the S9 preparation was 34.4 mg/mL in the pre-experiment / Experiment I and 33.3 mg/mL in experiment II. Experiment IIa was performed without S9 mix only.
- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10 % v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.

Test concentrations with justification for top dose:

3; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

PRE-EXPERIMENT FOR CYTOTOXICITY
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test). Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the Assay were met.

EXPERIMENTAL PERFORMANCE
For each strain and concentration including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each concentration, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer* (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains; OD = 0.9 - 1.2; wavelength = 500 nm; approx. 8x108 cells/mL),
2000 µL Overlay agar
For the pre-incubation method test solution (100 µL) (solvent or reference mutagen solution (positive control)), S9 mix / S9 mix substitution buffer* (500 µL) and bacteria suspension (100 µL) were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre- incubation overlay agar (2.0 mL, 45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark, plates were then stored at 4 °C until counted.
In parallel to each test a sterile control of the test substance was performed and documented in the raw data. Therefore, stock solution (100 μL) and S9 mix / S9 mix substitution buffer* (500 µL) were mixed with overlay agar (2.0 mL) and poured on minimal agar plates.
* Substitution buffer: 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M.

DATA RECORDING
The colonies were counted using a Petri Viewer with the software program Ames Study Manager. The evaluation unit was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). The print outs are kept with the raw data. Due precipitation of the test substance some test groups were scored manually.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce an increase in mutant colony frequencies of at least 2-fold concurrent control
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

EVALUATION OF RESULTS
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above the spontaneous mutation rate of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration (6).
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

E. coli, other: WP2 uvrA pKM101 and WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment the concentration range of the test substance was 3 - 5000 µg/plate. The pre-experiment is reported as Experiment I. Since no relevant cytotoxic effects were observed 5000 µg/plate was chosen as the maximal concentration in Experiment II. Since no bacterial colony growth occurred in the positive control of strain TA1535 without S9 mix in Experiment II, this part was repeated under the same conditions as Experiment II (reported as Experiment IIa). Experiments I and II were performed with and without liver microsomal activation, Experiment IIa only without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The concentration range included two logarithmic decades. The test substance was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiments II and IIa: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

STUDY RESULTS
Ames test:
- Signs of toxicity: The plates incubated with the test substance showed normal background growth up to the maximal dose of 5000 µg/plate with and without S9 mix in all strains used. Minor cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in Experiment I in strain WP2 uvrA (pKM101) at 5000 µg/plate with and without S9 mix. No further cytotoxic effects were observed, neither in the remaining test groups of Experiment I, nor in Experiments II and IIa.
- Mean number of revertant colonies per plate and standard deviation: No relevant increase in revertant colony numbers in any of the six tester strains was observed following treatment with test substance 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. Appropriate reference mutagens were used as positive controls. They showed a distinct in- crease in induced revertant colonies.

Any other information on results incl. tables

Table 1. Summary of Results Pre-Experiment/Experiment I

Metabolic Activation	Test Group	Concen tration (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO		9 ± 1	8 ± 3	21 ± 6	111 ± 17	263 ± 19	318 ± 83
	Untreated		13 ± 3	13 ± 7	24 ± 8	125 ± 12	276 ± 32	282 ± 5
	test substance	3 µg	10 ± 5	11 ± 4	25 ± 8	102 ± 6	227 ± 20	370 ± 54
	.	10 µg	12 ± 4	9 ± 2	25 ± 7	132 ± 5	249 ± 7	274 ± 29
		33 µg	9 ± 0	12 ± 1	23 ± 3	120 ± 13	246 ± 5	237 ± 7
		100 µg	9 ± 4	9 ± 3	23 ± 3	122 ± 15	248 ± 19	198 ± 16
		333 µg	12 ± 2^P	11 ± 1^P	22 ± 5^P	112 ± 26^P	246 ± 8^P	234 ± 16^P
		1000 µg	9 ± 2^PM	6 ± 3^PM	23 ± 4^PM	130 ± 16^P	232 ± 4^P	148 ± 2^PM
		2500 µg	10 ± 2^PM	7 ± 2^PM	17 ± 6^PM	86 ± 7^PM	159 ± 20^PM	154 ± 9^PM
		5000 µg	7 ± 2^PM	7 ± 2^PM	14 ± 3^PM	86 ± 2^PM	134 ± 25^PM	126 ± 14^PM
	NaN3	10 µg	927 ± 29			1679 ± 73
	4-NOPD	10 µg			449 ± 40
	4-NOPD	50 µg		68 ± 10
	MMS	2.0 µL					3641 ± 308	3096 ± 193
With Activation	DMSO		10 ± 4	11 ± 3	40 ± 7	125 ± 27	280 ± 8	364 ± 76
	Untreated		10 ± 1	14 ± 7	42 ± 7	131 ± 11	300 ± 33	375 ± 51
	test substance	3 µg	12 ± 4	11 ± 6	42 ± 6	119 ± 7	289 ± 36	505 ± 53
		10 µg	9 ± 3	10 ± 1	46 ± 5	114 ± 13	281 ± 53	479 ± 113
		33 µg	10 ± 4	11 ± 5	48 ± 6	139 ± 12	278 ± 19	264 ± 26
		100 µg	10 ± 4	12 ± 4	33 ± 6	122 ± 4	280 ± 25	255 ± 13
		333 µg	9 ± 3^P	10 ± 3^P	41 ± 6^P	133 ± 11^P	261 ± 11^P	453 ± 22^P
		1000 µg	10 ± 3^PM	9 ± 1^PM	27 ± 2^PM	117 ± 7^P	243 ± 34^P	263 ± 10^PM
		2500 µg	10 ± 2^PM	9 ± 3^PM	33 ± 4^PM	87 ± 8^PM	232 ± 22^PM	224 ± 8^PM
		5000 µg	7 ± 2^PM	7 ± 3^PM	26 ± 5^PM	91 ± 10^PM	183 ± 30^PM	153 ± 17^PM
	2-AA	2.5 µg	423 ± 17	233 ± 45	3416 ± 163	3824 ± 210
	2-AA	10.0 µg					1105 ± 106	1945 ± 223

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3	sodium azide	P	Precipitate
2-AA	2-aminoanthracene	M	Manual count
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

Table 2: Summary of Results Experiment II

Metabolic Activation	Test Group	Concen tration  (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO		n.r.*	13 ± 2	24 ± 7	110 ± 5	277 ± 6	299 ± 15
	Untreated		n.r.*	14 ± 4	27 ± 4	132 ± 14	289 ± 20	343 ± 10
	test substance	33 µg	n.r.*	11 ± 1	25 ± 5	113 ± 14	262 ± 16	324 ± 28
		100 µg	n.r.*	12 ± 2	24 ± 7	116 ± 26	248 ± 25	302 ± 34
		333 µg	n.r.*	14 ± 2^P	27 ± 6^P	116 ± 8^P	251 ± 17^P	270 ± 13^P
		1000 µg	n.r.*	8 ± 2^P	19 ± 1^PM	106 ± 5^PM	243 ± 3^P	285 ± 14^PM
		2500 µg	n.r.*	7 ± 2^PM	17 ± 3^PM	81 ± 9^PM	184 ± 11^PM	261 ± 4^PM
		5000 µg	n.r.*	7 ± 1^PM	14 ± 4^PM	81 ± 16^PM	143 ± 9^PM	183 ± 6^PM
	NaN3	10 µg	Not valid			1821 ± 30
	4-NOPD	10 µg			450 ± 15
	4-NOPD	50 µg		87 ± 14
	MMS	2.0 µL					3558 ± 89	3185 ± 107
With Activation	DMSO		10 ± 4	11 ± 2	34 ± 8	158 ± 26	294 ± 25	365 ± 27
	Untreated		13 ± 2	13 ± 2	41 ± 6	139 ± 21	334 ± 18	387 ± 20
	test substance	33 µg	11 ± 4	10 ± 2	31 ± 7	143 ± 2	307 ± 25	390 ± 32
		100 µg	12 ± 0	9 ± 2	35 ± 6	142 ± 16	283 ± 16	341 ± 11
		333 µg	11 ± 2^P	10 ± 2^P	31 ± 6^P	123 ± 13^P	293 ± 14^P	309 ± 18^P
		1000 µg	10 ± 0^P	9 ± 3^PM	37 ± 5^P	125 ± 1^P	275 ± 22^P	434 ± 11^PM
		2500 µg	8 ± 1^PM	8 ± 2^PM	24 ± 7^PM	129 ± 5^PM	238 ± 13^PM	348 ± 11^PM
		5000 µg	6 ± 2^PM	6 ± 2^PM	23 ± 5^PM	117 ± 6^PM	188 ± 18^PM	272 ± 26^PM
	2-AA	2.5 µg	436 ± 21	112 ± 11	3389 ± 446	4087 ± 331
	2-AA	10.0 µg					976 ± 49	1820 ± 30

  

Key to Positive Controls		Key to Plate Postfix Codes
NaN3	sodium azide	P	Precipitate
2-AA	2-aminoanthracene	M	Manual count
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test sunstance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This GLP compliant OECD 471 study was performed to investigate the potential of test substance to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II and IIa) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA (pKM101) and WP2 (pKM101).

The plates incubated with the test item showed normal background growth up to the maximal dose of 5000 µg/plate with and without S9 mix in all strains used. Minor cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in Experiment I in strain WP2 uvrA (pKM101) at 5000 µg/plate with and without S9 mix. No further cytotoxic effects were observed, neither in the remaining test groups of Experiment I, nor in Experiments II and IIa. No relevant increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).  There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls, which showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.