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Genetic toxicity: in vitro

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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:
07-12-2005 to 03-02-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: August 2005 ; signature: November 2005
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
482-160-5
EC Name:
-
Cas Number:
130786-09-3
Molecular formula:
C12H13N
IUPAC Name:
(2Z)-2-phenylhex-2-enenitrile
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: approximately 4°C in the dark, under nitrogen
- Other: colourless

Method

Target gene:
histidine or tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: All strains: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 (plate incorporation method):
TA100 & TA1535: 5, 15, 50, 150, 500, 1500 µg/plate
TA98 & TA1537: 15, 50, 150, 500, 1500, 5000 µg/plate
WP2uvrA- : 50, 150, 500, 1500, 5000 µg/plate

Experiment 2 (plate incorporation methodd): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 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).

Experiment 2. All of the plates were incubated at 37 ± 3 ºC for approximately 48 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).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
Statistics:
Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

99

97

100

(99)

1.5#

18

21

21

(20)

1.7

29

27

20

(25)

4.7

20

23

23

(22)

1.7

4

9

7

(7)

2.5

5 µg

86

98

96

(93)

6.4

20

16

9

(15)

5.6

NT

 

NT

 

NT

 

15 µg

97

99

112

(103)

8.1

15

14

13

(14)

1.0

NT

 

25

24

21

(23)

2.1

7

10

5

(7)

2.5

50 µg

102

102

109

(104)

4.0

24

14

15

(18)

5.5

30

19

21

(23)

5.9

20

28

22

(23)

4.2

9

9

10

(9)

0.6

150 µg

100

99

80

(93)

11.3

14

26

22

(21)

6.1

19

20

15

(18)

2.6

24

18

19

(20)

3.2

13

7

13

(11)

3.5

500 µg

75 S

82 S

86 S

(81)

5.6

7 S

11 S

8 S

(9)

2.1

24

19

18

(20)

3.2

36

21

29

(29)

7.5

7

8

5

(7)

1.5

1500 µg

0 S

0 S

0 S

(0)

0.0

0 S

0 S

0 S

(0)

0.0

19

20

19

(0)

0.6

21

27

17

(22)

5.0

10

9

6

(8)

2.1

5000 µg

NT

NT

 

20

20

20

(20)

0.0

9

22

8

(13)

7.8

3 S

7 S

4 S

(5)

2.1

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

543

454

488

(495)

44.9

490

302

421

(404)

95.1

957

969

1005

(977)

25.0

302

301

279

(294)

13.0

1094

1271

1561

(1309)

235.8

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

115

111

84

(103)

16.0#

13

14

15

(14)

1.0

32

35

30

(32)

2.5

31

34

32

(32)

1.5

19

24

19

(21)

2.9

5 µg

85

90

114

(96)

15.5

14

10

10

(11)

2.3

NT

 

NT

 

 

 

15 µg

108

104

108

(107)

2.3

11

10

13

(11)

1.5

NT

 

25

25

22

(24)

1.7

30

20

15

(22)

7.6

50 µg

104

104

99

(102)

2.9

12

10

9

(10)

1.5

34

33

20

(29)

7.8

29

28

18

(25)

6.1

19

27

20

(22)

4.4

150 µg

93

78

96

(89)

9.6

12

11

11

(11)

0.6

22

22

20

(21)

1.2

23

29

40

(31)

8.6

20

28

27

(25)

4.4

500 µg

57 S

57 S

43 S

(52)

8.1

15 S

7 S

8 S

(10)

4.4

21

22

26

(23)

2.6

29

32

29

(30)

1.7

19

19

16

(18)

1.7

1500 µg

0 S

0 S

0 S

(0)

0.0

0 S

0 S

0 S

(0)

0.0

19

24

24

(22)

2.9

26

37

31

(31)

5.5

11

11

10

(11)

0.6

5000 µg

 

NT

 

 

NT

 

24

19

27

(23)

4.0

37

19

18

(25)

10.7

8 S

17 S

14 S

(13)

4.6

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

955

1002

913

(957)

44.5

288

296

338

(307)

26.9

394

410

443

(416)

25.0

344

340

302

(329)

23.2

393

464

351

(403)

57.1

 

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

120

102

126

(116)

12.5#

16

16

23

(18)

4.0

26

20

26

(24)

3.5

15

18

18

(17)

1.7

8

10

11

(10)

1.5

5 µg

135

133

132

(133)

1.5

21

24

19

(21)

2.5

NT

 

NT

 

NT

 

15 µg

130

113

123

(122)

8.5

25

19

20

(21)

3.2

NT

 

16

18

21

(18)

2.5

9

18

7

(11)

5.9

50 µg

133

124

130

(129)

4.6

19

27

30

(25)

5.7

29

29

18

(25)

6.4

10

20

26

(19)

8.1

11

11

8

(10)

1.7

150 µg

85

115

134

(111)

24.7

11

20

15

(15)

4.5

18

26

14

(19)

6.1

13

13

20

(15)

4.0

12

16

11

(13)

2.6

500 µg

104 S

104 S

121 S

(110)

9.8

19 S

14 S

11 S

(15)

4.0

21

16

16

(18)

2.9

19

15

5

(13)

7.2

13

13

11

(12)

1.2

1500 µg

0 S

0 S

0 S

(0)

0.0

0 S

0 S

0 S

(0)

0.0

12

12

9

(11)

1.7

18

19

15

(17)

2.1

8

4

7

(6)

2.1

5000 µg

NT

 

NT

 

16

26

24

(22)

5.3

9

15

19

(14)

5.0

0 S

0 S

0 S

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

541

566

585

(564)

22.1

691

431

475

(532)

139.2

817

789

883

(830)

48.3

223

218

216

(219)

3.6

588

999

1518

(1035)

466.0

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

81

95

82

(86)

7.8#

12

10

10

(11)

1.2

35

23

15

(24)

10.1

20

33

24

(26)

6.7

23

15

30

(23)

7.5

5 µg

97

84

95

(92)

7.0

8

7

9

(8)

1.0

NT

 

NT

 

NT

 

15 µg

100

91

97

(96)

4.6

9

9

11

(10)

1.2

NT

 

23

19

31

(24)

6.1

16

23

21

(20)

3.6

50 µg

81

81

84

(82)

1.7

7

4

10

(7)

3.0

44

24

30

(33)

10.3

22

22

29

(24)

4.0

23

21

22

(22)

1.0

150 µg

62

70

62

(65)

4.6

7

11

15

(11)

4.0

30

30

25

(28)

2.9

22

30

24

(25)

4.2

21

24

22

(22)

1.5

500 µg

35 S

38 S

43 S

(39)

4.0

9

5

7

(7)

2.0

22

25

20

(22)

2.5

29

31

23

(28)

4.2

12

20

19

(17)

4.4

1500 µg

0 S

0 S

0 S

(0)

0.0

0 S

0 S

0 S

(0)

0.0

25

21

18

(21)

3.5

24

33

25

(27)

4.9

11

15

16

(14)

2.6

5000 µg

NT

 

NT

 

22

30

35

(26)

4.0

30

21

18

(23)

6.2

10 S

8 S

7 S

(8)

1.5

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

762

837

873

(824)

56.6

306

261

245

(271)

31.6

560

542

637

(580)

50.5

209

163

180

(184)

23.3

578

522

536

(545)

29.1

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

S: partial absence of bacterial background lawn

#: Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item using both the Ames plate incorporation and pre incubation methods at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The dose levels were 5 to 1500 µg/plate for Salmonella strains TA100 and TA1535, 15 to 5000 µg/plate for Salmonella strains TA98 and TA1537 and 50 to 5000 µg/plate for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (dimethyl sulphoxide) 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on cytotoxicity. The test item caused a visible reduction in the growth of the bacterial background lawn, both in the presence and absence of S9, to Salmonella strains TA100 and TA1535 from 500 µg/plate and at 5000 µg/plate to TA1537. The test item caused no visible reduction in the growth of the bacterial background lawn to either Salmonella strain TA98 or Escherichia coli strain WP2uvrA-. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type. The test item was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

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