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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
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 resultsopen allclose all
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
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 ± 2P

11 ± 1P

22 ± 5P

112 ± 26P

246 ± 8P

234 ± 16P

 

 

1000 µg

9 ± 2PM

6 ± 3PM

23 ± 4PM

130 ± 16P

232 ± 4P

148 ± 2PM

 

 

2500 µg

10 ± 2PM

7 ± 2PM

17 ± 6PM

86 ± 7PM

159 ± 20PM

154 ± 9PM

 

 

5000 µg

7 ± 2PM

7 ± 2PM

14 ± 3PM

86 ± 2PM

134 ± 25PM

126 ± 14PM

 

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 ± 3P

10 ± 3P

41 ± 6P

133 ± 11P

261 ± 11P

453 ± 22P

 

 

1000 µg

10 ± 3PM

9 ± 1PM

27 ± 2PM

117 ± 7P

243 ± 34P

263 ± 10PM

 

 

2500 µg

10 ± 2PM

9 ± 3PM

33 ± 4PM

87 ± 8PM

232 ± 22PM

224 ± 8PM

 

 

5000 µg

7 ± 2PM

7 ± 3PM

26 ± 5PM

91 ± 10PM

183 ± 30PM

153 ± 17PM

 

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 ± 2P

27 ± 6P

116 ± 8P

251 ± 17P

270 ± 13P

 

 

1000 µg

n.r.*

8 ± 2P

19 ± 1PM

106 ± 5PM

243 ± 3P

285 ± 14PM

 

 

2500 µg

n.r.*

7 ± 2PM

17 ± 3PM

81 ± 9PM

184 ± 11PM

261 ± 4PM

 

 

5000 µg

n.r.*

7 ± 1PM

14 ± 4PM

81 ± 16PM

143 ± 9PM

183 ± 6PM

 

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 ± 2P

10 ± 2P

31 ± 6P

123 ± 13P

293 ± 14P

309 ± 18P

 

 

1000 µg

10 ± 0P

9 ± 3PM

37 ± 5P

125 ± 1P

275 ± 22P

434 ± 11PM

 

 

2500 µg

8 ± 1PM

8 ± 2PM

24 ± 7PM

129 ± 5PM

238 ± 13PM

348 ± 11PM

 

 

5000 µg

6 ± 2PM

6 ± 2PM

23 ± 5PM

117 ± 6PM

188 ± 18PM

272 ± 26PM

 

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.