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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

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:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
EC Number:
601-478-9
Cas Number:
117428-22-5
Molecular formula:
C18H16F3NO4
IUPAC Name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
Test material form:
solid
Details on test material:
Purity: 93.3% w/w

Method

Target gene:
histidine and tryptophan
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
Remarks:
pKM101
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from phenobarbital/ß-naphthaflavone-induced SD rats
Test concentrations with justification for top dose:
100, 200, 500, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
Dimethylsulphoxide
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulphoxide
Positive controls:
yes
Positive control substance:
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: Acridine mutagen ICR191 (TA1537/without activation), 2-Aminoanthracene (all strains/with activation), Daunomycin HCl (TA98/without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Top agar (plate incorporation)
- Cell density at seeding: 0.1 mL aliquots of each bacterial strain

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 3 days
- All plates were counted by an automatic colony counter with the discrimination adjusted appropriately to permit the optimal counting of mutant colonies
Evaluation criteria:
A positive response in a valid individual experiment is achieved when one or both of the following criteria are met: A statistically significant dose-related increase in the mean number of revertant colonies is obtained; a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is statistically significant, is observed at one or more concentrations.

A negative result in a valid individual experiment is achieved when: There is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.
Statistics:
An assessment of statistical significance was carried out using a one-tailed Student's t-test. The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p <0.01 are treated as significant, with values of 0.01≤ p <0.05 being indicative of a possible effect.

Results and discussion

Test results
Key result
Species / strain:
other: all species/strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table-1: Test Data for Experimental Phase – 1

Strain

Metabolic activation

Dose levels (µg/plate)

Mean

Standard deviation

Ratio: Test/control

No Revertants/plate

Plate 1

Plate 2

Plate 3

TA1535

+S9

5000

15.3

1.2

1.2*

16

14

16

2500

10.0

7.8

0.8

6

19

5

1000

13.7

6.7

1.1

18

17

6

500

14.0

7.2

1.1

6

20

16

200

10.0

2.6

0.8

13

9

8

100

13.7

3.8

1.1

11

12

18

TA1535

-S9

5000

6.3

1.5

0.8

8

6

5

2500

12.0

5.3

1.4

10

10

8

1000

9.0

7.9

1.1

6

18

3

500

6.0

4.0

0.7

6

2

10

200

11.7

1.5

1.4*

10

12

13

100

8.3

0.6

1.0

8

8

9

TA1537

+S9

5000

3.3

1.5

1.1

5

2

3

2500

2.3

0.6

0.8

2

2

3

1000

3.0

1.0

1.0

4

2

3

500

5.0

1.0

1.7**

6

5

4

200

5.0

2.6

1.7

8

3

4

100

4.3

1.5

1.4

4

6

3

TA1537

-S9

5000

2.3

0.6

0.6

3

2

2

2500

3.0

1.7

0.8

5

2

2

1000

3.3

1.2

0.8

2

4

4

500

3.7

1.5

0.9

4

5

2

200

3.7

2.1

0.9

2

3

6

100

3.3

0.6

0.8

3

3

4

TA98

+S9

5000

11.7

2.1

0.6

14

11

10

2500

20.7

3.1

1.0

20

18

24

1000

17.3

0.6

0.8

17

18

17

500

18.3

2.5

0.9

21

16

18

200

18.3

2.1

0.9

20

16

19

100

20.3

4.5

1.0

20

25

16

TA98

-S9

5000

17.0

1.0

0.9

18

17

16

2500

15.0

4.6

0.8

19

10

16

1000

14.0

3.0

0.7

11

14

17

500

17.0

1.0

0.9

18

16

17

200

18.7

4.6

1.0

16

16

24

100

20.7

3.8

1.1

18

25

19

TA100

+S9

5000

104.0

12.0

1.2*

104

116

92

2500

77.0

5.3

0.9

73

75

83

1000

85.0

5.3

1.0

89

79

87

500

99.3

7.6

1.1*

91

106

101

200

96.5

4.9

1.1

93

100

C

100

90.7

3.5

1.0

94

91

87

TA100

-S9

5000

80.7

1.5

1.1

82

79

81

2500

81.0

2.0

1.1

79

83

81

1000

85.3

8.4

1.1

95

80

81

500

81.7

8.3

1.1

75

91

79

200

89.3

9.3

1.2*

97

79

92

100

83.7

6.5

1.1

90

84

77

WP2P

+S9

5000

45.7

9.5

35

49

49

53

2500

34.7

0.6

35

35

35

34

1000

48.0

5.3

54

46

46

44

500

47.0

3.5

49

49

49

43

200

46.3

3.8

42

49

49

48

100

45.0

4.4

43

42

42

50

WP2P

-S9

5000

37.3

3.2

35

36

36

41

2500

24.3

4.0

28

20

20

25

1000

33.7

6.5

34

40

40

27

500

33.3

7.1

41

32

32

27

200

34.7

4.9

38

37

37

29

100

32.3

2.1

34

30

30

33

WP2P uvrA

+S9

5000

149.7

22.3

164

151

161

124

2500

109.7

8.4

100

115

115

114

1000

147.3

6.4

140

150

150

152

500

136.3

17.2

124

156

156

129

200

150.3

12.0

151

138

138

162

100

143.7

2.3

145

141

141

145

WP2P uvrA

-S9

5000

93.7

12.4

86

108

108

87

2500

76.0

17.3

57

91

91

80

1000

104.3

3.5

108

104

104

101

500

103.3

9.3

111

93

93

106

200

108.7

23.3

119

125

125

82

100

103.3

4.0

104

99

99

107

*: 0.01≤ p < 0.05, ** p <0.01 [One-sided t-Test assumes Test > Control]

C: Contaminated plate

Table 2: Test Data for Experimental Phase 2 (+S9)

Strain

Dose levels (µg/plate)

Mean

Standard deviation

Ratio: Test/control

No Revertants/plate

Plate 1

Plate 2

Plate 3

TA1535

5000

11.0

2.6

0.9

10

14

9

2500

18.0

1.0

1.4*

19

18

17

1000

14.0

4.0

1.1

14

10

18

500

13.7

2.5

1.1

14

11

16

200

8.7

3.5

0.7

9

5

12

100

18.3

1.2

1.5**

19

19

17

TA1537

5000

3.0

1.7

1.0

5

2

2

2500

3.3

1.2

1.1

4

2

4

1000

3.7

1.2

1.2

3

3

5

500

3.7

1.5

1.2

4

2

5

200

4.3

0.6

1.4*

4

5

4

100

2.3

1.5

0.8

4

2

1

TA98

5000

20.3

4.5

0.8

25

20

16

2500

20.7

2.3

0.8

18

22

22

1000

18.3

3.2

0.7

16

22

17

500

21.7

4.5

0.9

22

17

26

200

19.0

2.6

0.8

22

18

17

100

22.0

4.6

0.9

27

21

18

TA100

5000

76.3

16.5

0.9

60

93

76

2500

82.7

4.0

1.0

82

87

79

1000

80.7

12.0

0.9

80

69

93

500

73.3

5.1

0.8

69

72

79

200

85.7

6.0

1.0

85

80

92

100

84.0

12.0

1.0

96

84

72

WP2P

5000

50.7

11.7

1.0

42

64

46

2500

49.3

3.5

0.9

49

53

46

1000

48.0

10.4

0.9

60

41

43

500

53.0

4.0

1.0

57

49

53

200

56.0

5.2

1.1

59

59

50

100

50.0

4.4

0.9

53

45

52

WP2P uvrA

5000

155.7

8.5

1.1

162

159

146

2500

156.0

4.4

1.1

161

154

153

1000

150.0

4.0

1.0

146

150

154

500

157.3

2.1

1.1*

155

159

158

200

160.0

13.7

1.1

163

172

145

100

147.3

14.0

1.0

148

161

133

*: 0.01≤ p < 0.05, ** p <0.01 [One-sided t-Test assumes Test > Control]

Table 3: Test Data for Experimental Phase 2 (-S9)

Strain

Dose levels (µg/plate)

Mean

Standard deviation

Ratio: Test/control

No Revertants/plate

Plate 1

Plate 2

Plate 3

TA1535

5000

14.0

3.5

1.3

12

12

18

2500

11.7

0.6

1.1

11

12

12

1000

13.0

3.0

1.3

10

16

13

500

9.0

1.7

0.9

11

8

8

200

9.3

1.5

0.9

11

8

9

100

9.0

3.6

0.9

13

8

6

TA1537

5000

2.3

0.6

1.0

3

2

2

2500

2.0

1.0

0.9

3

1

2

1000

2.7

2.9

1.2

6

1

1

500

2.7

1.2

1.2

4

2

2

200

2.3

1.2

1.0

3

1

3

100

2.3

0.6

1.0

2

2

3

TA98

5000

14.7

3.2

0.6

17

11

16

2500

18.7

2.5

0.7

16

21

19

1000

24.3

6.0

0.9

30

18

25

500

23.3

4.9

0.9

21

29

20

200

20.7

6.4

0.8

28

17

17

100

24.7

3.5

0.9

25

28

21

TA100

5000

70.3

5.5

0.9

74

73

64

2500

72.3

3.1

0.9

73

75

69

1000

66.3

5.5

0.9

72

61

66

500

65.3

3.8

0.9

61

68

67

200

66.3

3.8

0.9

68

69

62

100

74.3

8.4

1.0

69

84

70

WP2P

5000

34.7

6.0

1.2

34

29

41

2500

27.3

0.6

1.0

27

28

27

1000

27.7

3.2

1.0

30

29

24

500

30.0

3.5

1.1

34

28

28

200

28.0

7.2

1.0

36

26

22

100

28.3

1.2

1.0

29

27

29

WP2P uvrA

5000

133.7

16.3

1.1

148

116

137

2500

111.3

4.5

0.9

107

111

116

1000

121.7

3.1

1.0

119

121

125

500

108.3

4.5

0.9

108

104

113

200

127.3

9.1

1.0

117

134

131

100

127.7

15.0

1.0

140

132

111

Applicant's summary and conclusion

Conclusions:
The test substance was negative (with and without activation) in all strains tested
Executive summary:

The test substance was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2P and WP2P uvrA) in the presence and absence of a rat liver - derived metabolic activation system (S9-mix), following protocols complying with OECD Guideline Numbers 471 and 472 and with the United Kingdom Department of Health and Guidelines (DOH).

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e., non-mutagenic in S. typhimurium and E. coli strains in both the presence and absence of S9 -mix.