Registration Dossier

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:
The experimental phase of this study was performed between 24 April 2009 and 26 May 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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)
Remarks:
Date of inspection: 19/08/08 Date of Signature: 04/03/09
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification : PDE-28
Description : red powder
Chemical name : 3-(4-chlorophenyl)-6-(biphenyl)-2.5-dihydropyrrole-1,4-dione
Purity : 96.9%
Batch number : 1RF-8017
Date received : 23 March 2009
Storage conditions : Room temperature in the dark

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate.
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in-house. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Negative controls:
yes
Remarks:
Spontaneous mutation rates
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2007 and 2008 in Appendix 2 (please see attachment)
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 2007 and 2008 are presented in Appendix 2.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.
- Precipitation: The precipitate of the test material was observed above 500µg/plate.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). There were no revertant colonies observed for WP2uvrA- at 5000 µg/plate in the absence of S9 only. This observation was not reproduced in either of the main experiments and was, therefore, considered spurious and of no biological relevance. The test material formulation and S9-mix used in this experiment were both shown to be sterile. (See table in any other information and results section for the preliminary test).

COMPARISON WITH HISTORICAL CONTROL DATA:
A history profile of vehicle and positive control values for 2007 and 2008 is presented in Appendix 2.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Any other information on results incl. tables

Preliminary Toxicity Test:

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-)

S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

85

86

92

89

90

92

76

71

92P

77P

93P

+

TA100

91

79

90

70

87

76

76

75

81P

92P

78P

-

WP2uvrA-

26

19

25

20

29

21

24

18

23P

18P

0P

+

WP2uvrA-

37

31

29

31

34

22

37

23

36P

38P

29P

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 S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pink colour with associated precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

100

 

20

 

23

 

29

 

11

 

107

(97)

18

(18)†

27

(25)

16

(22)

19

(14)

85

 

17

 

26

 

20

 

12

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

87

 

19

 

27

 

17

 

5

 

96

(97)

16

(17)†

38

(35)

8

(15)

7

(5)

109

 

15

 

40

 

21

 

4

 


         Experintal procedure perford at later dates (with and without S9) due to excessive contamination in the original tests

 

Table2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 27 April 2009

From: 01 May 2009†

To: 30 April 2009

To: 04 May 2009†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535†

WP2uvrA-

TA98

TA1537

-

0

113

122

120

(118)

4.7#

13

16

26

(18)

6.8

24

21

20

(22)

2.1

19

18

23

(20)

2.6

14

11

14

(13)

1.7

-

50

98

91

125

(105)

18.0

19

20

29

(23)

5.5

20

25

29

(25)

4.5

19

14

19

(17)

2.9

14

9

10

(11)

2.6

-

150

111

97

110

(106)

7.8

27

13

26

(22)

7.8

26

24

23

(24)

1.5

21

18

23

(21)

2.5

11

8

10

(10)

1.5

-

500

90 P

119 P

117 P

(109)

16.2

25 P

18 P

17 P

(20)

4.4

24 P

25 P

18 P

(22)

3.8

16 P

22 P

20 P

(19)

3.1

10 P

9 P

12 P

(10)

1.5

-

1500

99 P

97 P

112 P

(103)

8.1

26 P

19 P

26 P

(24)

4.0

23 P

18 P

24 P

(22)

3.2

19 P

20 P

21 P

(20)

1.0

10 P

13 P

10 P

(11)

1.7

-

5000

101 P

95 P

111 P

(102)

8.1

18 P

11 P

15 P

(15)

3.5

20 P

24 P

24 P

(23)

2.3

21 P

16 P

22 P

(20)

3.2

12 P

8 P

10 P

(10)

2.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

372

404

389

(388)

16.0

178

174

131

(161)

26.1

1402

1296

1518

(1405)

111.0

135

112

143

(130)

16.1

457

539

882

(626)

225.5

 

Table3              Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 27 April 2009

From: 01 May 2009†

To: 30 April 2009

To: 04 May 2009†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535†

WP2uvrA-

TA98

TA1537

+

0

131

106

106

(114)

14.4#

21

9

8

(13)

7.2

37

33

31

(34)

3.1

22

26

25

(24)

2.1

13

14

13

(13)

0.6

+

50

113

114

112

(113)

1.0

11

8

17

(12)

4.6

32

29

33

(31)

2.1

21

24

23

(23)

1.5

13

12

13

(13)

0.6

+

150

111

125

124

(120)

7.8

9

13

11

(11)

2.0

31

33

33

(32)

1.2

24

20

20

(21)

2.3

14

14

11

(13)

1.7

+

500

111 P

114 P

120 P

(115)

4.6

12 P

13 P

10 P

(12)

1.5

25 P

27 P

32 P

(28)

3.6

20 P

18 P

20 P

(19)

1.2

13 P

15 P

14 P

(14)

1.0

+

1500

112 P

115 P

121 P

(116)

4.6

10 P

14 P

13 P

(12)

2.1

31 P

27 P

33 P

(30)

3.1

27 P

22 P

23 P

(24)

2.6

15 P

15 P

10 P

(13)

2.9

+

5000

106 P

115 P

128 P

(116)

11.1

9 P

13 P

8 P

(10)

2.6

27 P

32 P

34 P

(31)

3.6

24 P

20 P

26 P

(23)

3.1

12 P

10 P

13 P

(12)

1.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1225

1487

1525

(1412)

163.3

182

252

229

(221)

35.7

564

517

503

(528)

32.0

272

269

244

(262)

15.4

470

349

362

(394)

66.4

Table 4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 13 May 2009

From: 23 May 2009†

To: 16 May 2009

To: 26 May 2009†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535†

WP2uvrA-

TA98

TA1537

-

0

118

108

116

(114)

5.3#

20

18

18

(19)

1.2

30

28

26

(28)

2.0

25

24

19

(23)

3.2

15

11

11

(12)

2.3

-

50

110

98

118

(109)

10.1

18

15

18

(17)

1.7

26

31

30

(29)

2.6

25

22

22

(23)

1.7

12

12

8

(11)

2.3

-

150

118

102

87

(102)

15.5

16

18

16

(17)

1.2

24

36

36

(32)

6.9

24

17

20

(20)

3.5

8

12

10

(10)

2.0

-

500

108 P

90 P

107 P

(102)

10.1

18 P

18 P

18 P

(18)

0.0

25 P

32 P

24 P

(27)

4.4

20 P

16 P

17 P

(18)

2.1

4 P

11 P

14 P

(10)

5.1

-

1500

95 P

120 P

118 P

(111)

13.9

18 P

20 P

16 P

(18)

2.0

15 P

36 P

36 P

(29)

12.1

19 P

20 P

18 P

(19)

1.0

9 P

10 P

13 P

(11)

2.1

-

5000

98 P

96 P

88 P

(94)

5.3

14 P

16 P

18 P

(16)

2.0

31 P

28 P

31 P

(30)

1.7

16 P

19 P

17 P

(17)

1.5

13 P

10 P

7 P

(10)

3.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

394

386

453

(411)

36.6

134

137

142

(138)

4.0

200

214

132

(182)

43.9

77

94

94

(88)

9.8

319

148

125

(197)

106.0

Table 5              Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 13 May 2009

From: 23 May 2009†

To: 16 May 2009

To: 26 May 2009†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535†

WP2uvrA-

TA98

TA1537

+

0

102

85

83

(90)

10.4#

10

12

9

(10)

1.5

33

35

30

(33)

2.5

24

26

22

(24)

2.0

9

9

9

(9)

0.0

+

50

85

95

95

(92)

5.8

10

12

11

(11)

1.0

35

35

26

(32)

5.2

24

28

21

(24)

3.5

12

10

6

(9)

3.1

+

150

101

102

85

(96)

9.5

11

10

9

(10)

1.0

15

19

14

(16)

2.6

15

29

20

(21)

7.1

9

12

5

(9)

3.5

+

500

68 P

88 P

102 P

(86)

17.1

8 P

9 P

13 P

(10)

2.6

34 P

34 P

25 P

(31)

5.2

14 P

28 P

32 P

(25)

9.5

10 P

12 P

10 P

(11)

1.2

+

1500

102 P

99 P

87 P

(96)

7.9

12 P

10 P

11 P

(11)

1.0

19 P

35 P

25 P

(26)

8.1

17 P

11 P

24 P

(17)

6.5

7 P

9 P

8 P

(8)

1.0

+

5000

81 P

87 P

83 P

(84)

3.1

10 P

11 P

12 P

(11)

1.0

17 P

35 P

30 P

(27)

9.3

29 P

30 P

20 P

(26)

5.5

11 P

7 P

12 P

(10)

2.6

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1770

1880

1777

(1809)

61.6

381

405

404

(397)

13.6

477

535

514

(509)

29.4

131

224

171

(175)

46.7

391

489

359

(413)

67.7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

         Experintal procedure perford at later date due to excessive contamination in the original test

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction:

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirments of 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 (TSCA) OPPTS harmonised guidelines.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. 

Results:

The vehicle (acetone) control plates gave counts of revertant colonies 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.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pink colour with associated precipitate was observed at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion:

The test material was considered to be non-mutagenic under the conditions of this test.