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Toxicological information

Genetic toxicity: in vitro

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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:
Study initiated 11 August 2009. Experimental phase 17 to 28 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): cloquintocet-mexyl
- Physical state: beige solidified melt
- Analytical purity: 95.4% w/w
- Purity test date: 09 July 2009
- Reanalysis date: end of July 2011
- Storage condition of test material: <30°C

Method

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:
Rat liver S9 [phenobarbital (80 mg/kg) i.p. and β-naphthoflavone p.o. induced]
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (active ingredient)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: because of its solubility properties and its relative non-toxicity to the bacteria
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9, 10 µg/plate, TA1535 and TA100
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9, 10 µg/plate, TA1537 and TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9, 3 µL/plate, WP2 uvrA pKM101 and WP2 pKM101
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9, 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100) and 10 µg/plate (WP2 uvrA pKM 101, WP2 pKM 101)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar; plate incorporation (experiment 1) and preincubation (experiment II)

DURATION
- Preincubation: 60 minutes
- Incubation period: at least 48 hours (at 37°C in the dark)

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose 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.
Statistics:
Not required

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see table 1
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see table 1
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli, other: WP2 pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see table 1
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations in strain TA 1535 without metabolic activation and in strains TA 1535, TA 98, and TA 100 with metabolic activation in experiment I. In experiment II, toxic effects were observed in strain TA 1537 without metabolic activation and in strains TA 100, WP2 uvrA pKM101, and WP2 pKM101 with metabolic activation.

Table 1: Toxic effects observed at the following concentrations (µg/plate)

Strains

Experiment I

Experiment II

 

With S9 mix

Without S9 mix

With S9 mix

Without S9 mix

TA 1535

5000

2500, 5000

/

/

TA 1537

/

/

1000

/

TA 98

/

5000

/

/

TA 100

/

2500, 5000

/

5000

WP2 uvrA pKM101

/

/

/

2500, 5000

WP2 pKM101

/

/

/

2500, 5000

/ = no toxic effects observed

Table 2: Summary of Results Experiment I

Test Group

Dose Level µg/ plate

Revertant Colony Counts (Mean ± SD)

Without Activation

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA pkm 101

WP2pKm 101

DMSO

 

13 ± 4

14 ± 2

29 ± 8

138 ± 14

569 ± 26

238 ± 15

Untreated

 

15 ± 5

11 ± 2

34 ± 3

152 ± 18

539 ± 36

226 ± 3

Cloquintocet-

mexyl

3

16 ± 4

11 ± 1

32 ± 9

135 ± 12

600 ± 25

215 ± 2

10

14 ± 2

13 ± 6

28 ± 5

144 ± 12

630 ± 37

206 ± 11

33

14 ± 1

14 ± 6

29 ± 2

135 ± 13

589 ± 39

207 ± 15

100

14 ± 4

15 ± 1

28 ± 6

132 ± 9

604 ± 18

211 ± 14

333

13 ± 4P

17 ± 2P

26 ± 2P

111 ± 6P

542 ± 35P

223 ± 14P

1000

11 ± 3P M

15 ± 1P

28 ± 5P

122 ± 8P

564 ± 23P

197 ± 7P

2500

8 ± 1P M

13 ± 1P M

27 ± 3P M

72 ± 4P M

525 ± 22P M

153 ± 13P M

5000

4 ± 2P M

14 ± 4P M

20 ± 2P M

71 ± 7P M

502 ± 17P M

129 ± 8P M

NaN3

10

1343 ± 733

 

 

1564 ± 417

 

 

4-NOPD

10

 

 

329 ± 13

 

 

 

4-NOPD

50

 

89 ± 2

 

 

 

 

MMS

3.0 µL

 

 

 

 

2871 ± 453

2797 ± 455

With Activation

 

 

 

 

 

 

 

 

DMSO

 

17 ± 2

20 ± 4

36 ± 7

154 ± 8

627 ± 34

224 ± 15

Untreated

 

19 ± 2

23 ± 4

39 ± 9

181 ± 7

645 ± 46

224 ± 20

Cloquintocet-

mexyl

3

16 ± 4

20 ± 3

35 ± 9

159 ± 9

580 ± 51

218 ± 7

10

17 ± 5

20 ± 8

35 ± 8

154 ± 5

624 ± 17

212 ± 8

33

16 ± 3

21 ± 10

39 ± 4

147 ± 21

640 ± 69

181 ± 22

100

17 ± 3

24 ± 5

39 ± 10

141 ± 16

595 ± 45

207 ± 6

333

17 ± 2

21 ± 8

36 ± 6

155 ± 11

473 ± 42

226 ± 22

1000

13 ± 3P

19 ± 6P

29 ± 8P

145 ± 7P

426 ± 8P

199 ± 2P

2500

6 ± 3P M

12 ± 2P M

28 ± 4P M

65 ± 4P M

434 ± 20P M

188 ± 19P M

5000

3 ± 2P M

18 ± 3P M

15 ± 3P M

51 ± 4P M

400 ± 12P M

181 ± 11P M

2-AA

2.5

324 ± 17

259 ± 12

1578 ± 109

2744 ± 155

 

 

2-AA

10.0

 

 

 

 

1828 ± 75

2428 ± 145

P = Precipitate M = Manual count

 

Table 3: Summary of Results Experiment II

Test Group

Dose Level µg/plate

Revertant Colony Counts (Mean ±SD)

Without Activation

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA pkm 101

WP2pKm 101

DMSO

 

22 ± 6

11 ± 2

35 ± 6

115 ± 15

410 ± 29

198 ± 13

Untreated

 

20 ± 6

11 ± 1

40 ± 2

146 ± 5

431 ± 7

221 ± 6

Cloquintocet-

mexyl

3

20 ± 6

12 ± 2

36 ± 12

122 ± 12

397 ± 19

180 ± 6

10

16 ± 1

13 ± 4

37 ± 5

117 ± 2

413 ± 32

198 ± 15

33

23 ± 1

15 ± 4

34 ± 3

126 ± 8

400 ± 23

158 ± 4

100

18 ± 2

11 ± 4

25 ± 3

101 ± 4

427 ± 41

174 ± 4

333

19 ± 4P

8 ± 3P

34 ± 11P

90 ± 12P

406 ± 8P

171 ± 7P

1000

16 ± 5P

5 ± 3P

29 ± 1P

93 ± 8P

409 ± 42P

177 ± 1P

2500

20 ± 6P

9 ± 2P

24 ± 10P

97 ± 14P

388 ± 10P

156 ± 7P

5000

19 ± 5P M

10 ± 3P M

25 ± 7P M

73 ± 11P M

361 ± 14P M

131 ± 15P M

NaN3

10

1551 ± 119

 

 

1570 ± 44

 

 

4-NOPD

10

 

 

420 ± 5

 

 

 

4-NOPD

50

 

99 ± 8

 

 

 

 

MMS

3.0 µL

 

 

 

 

2036 ± 48

2437 ± 98

With Activation

 

 

 

 

 

 

 

DMSO

 

22 ± 4

19 ± 4

52 ± 7

137 ± 9

423 ± 21

203 ± 24

Untreated

 

23 ± 11

19 ± 3

50 ± 12

175 ± 16

513 ± 16

230 ± 5

Cloquintocet-

mexyl

3

24 ± 3

15 ± 5

50 ± 8

148 ± 34

437 ± 5

186 ± 14

10

22 ± 6

20 ± 4

60 ± 7

144 ± 5

451 ± 14

207 ± 15

33

20 ± 8

23 ± 1

43 ± 7

146 ± 4

459 ± 20

211 ± 18

100

29 ± 4

19 ± 8

49 ± 2

160 ± 11

423 ± 36

193 ± 15

333

20 ± 3

23 ± 7

43 ± 2

147 ± 14

364 ± 7

197 ± 13

1000

20 ± 1P

21 ± 2P

43 ± 4P

123 ± 2P

272 ± 13P

170 ± 13P

2500

12 ± 8P

14 ± 4P

31 ± 4P

63 ± 11P

91 ± 18P

76 ± 38P

5000

19 ± 9P M

20 ± 5P M

27 ± 4P M

38 ± 8P M

19 ± 6P M

20 ± 11P M

2-AA

2.5

334 ± 43

216 ± 22

1054 ± 24

1378 ± 222

 

 

2-AA

10.0

 

 

 

 

1633 ± 47

2136 ± 515

P = Precipitate M = Manual count

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Cloquintocet-mexyl did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The potential of cloquintocet-mexyl to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 uvrA pKM 101 and WP2 pKM 101 was investigated.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with cloquintocet-mexyl at any dose level, in the presence or absence of metabolic activation (S9 mix). There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Positive control chemicals showed appropriate responses in the relevant strains.