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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:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPP 84-2
Deviations:
no
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
Qualifier:
according to guideline
Guideline:
other: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methomyl
EC Number:
240-815-0
EC Name:
Methomyl
Cas Number:
16752-77-5
Molecular formula:
C5H10N2O2S
IUPAC Name:
(E)-[1-(methylsulfanyl)ethylidene]amino N-methylcarbamate
Test material form:
solid: crystalline
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-394
- Purity: 98.35%

Method

Target gene:
his, trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA100, TA1535, TA97a, and TA98
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Male SD rat liver homogenate activation system (S9 Mix)
Test concentrations with justification for top dose:
Trial I: 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for TA100 and WP2 uvrA (pKM101)
10, 50, 100, 500, 1000, 2500, and 5000 μg/plate for TA97a, TA98, and TA1535
Highest concentration is the guideline limit dose for this test system
Trial II: 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate for all strains
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance appeared completely soluble at 50 mg/mL producing a solution immediately upon mixing. Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene, ICR 191 Acridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)
- Cell density at seeding: At least 1E+8 bacteria

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Overnight culture

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the background lawn
Evaluation criteria:
A test substance was classified as positive when: (1) the average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the negative control; and (2) there was a positive doseresponse relationship in that same strain.

A test substance was classified as negative when either: (1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control; and (2) there was no positive doseresponse relationship.

Results not meeting these criteria for positive or negative assessments were evaluated using scientific judgment.
Statistics:
Data for each tester strain were evaluated independently. For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA100, TA1535, TA97a, and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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: Mutagenic activity of the test substance in strain TA100 in Trail 2

Without activation

 

 

 

Concentration of test substance (µg/plate)

Revertants

Average

SD

Plate 1

Plate 2

Plate 3

0.0

99

104

107

103

4

10.0

86

96

113

98

14

50.0

108

118

113

113

5

100.0

110

107

92

103

10

500.0

94

92

99

95

4

1000.0

92

105

99

99

7

2500.0

118

91

104

104

14

5000.0

103

86

84

91

10

Sodium azide (2 µg/plate)

891

985

994

957

57

With activation

 

 

 

 

 

0.0

93

99

107

100

7

10.0

95

115

107

106

10

50.0

108

108

93

103

9

100.0

97

101

97

98

2

500.0

103

95

99

99

4

1000.0

107

111

93

104

9

2500.0

89

92

101

94

6

5000.0

98

80

91

90

9

2AA (1 µg/plate)

984

920

902

935

43

Table-2: Mutagenic activity of the test substance in strain TA1535 in Trail 2

Without activation

 

 

 

Concentration of test substance (µg/plate)

Revertants

Average

SD

Plate 1

Plate 2

Plate 3

0.0

21

18

23

21

3

10.0

17

20

14

17

3

50.0

18

13

11

14

4

100.0

14

22

15

17

4

500.0

13

12

15

13

2

1000.0

13

19

19

17

3

2500.0

19

13

22

18

5

5000.0

17

16

17

17

1

Sodium azide (2 µg/plate)

673

799

793

755

71

With activation

 

 

 

 

 

0.0

17

18

17

17

1

10.0

19

12

14

15

4

50.0

14

15

15

15

1

100.0

18

14

10

14

4

500.0

12

17

13

14

3

1000.0

14

11

17

14

3

2500.0

19

14

16

16

3

5000.0

17

15

11

14

3

2AA (1 µg/plate)

314

332

328

325

9

Table-3: Mutagenic activity of the test substance in strain TA97a in Trail 2

Without activation

 

 

 

Concentration of test substance (µg/plate)

Revertants

Average

SD

Plate 1

Plate 2

Plate 3

0.0

125

131

138

131

7

10.0

121

121

125

122

2

50.0

143

135

128

135

8

100.0

132

125

124

127

4

500.0

119

120

112

117

4

1000.0

116

133

137

129

11

2500.0

125

118

117

120

4

5000.0

118

114

126

119

6

ICR 191 (2 µg/plate)

1912

1745

1914

1857

97

With activation

 

 

 

 

 

0.0

140

124

137

134

9

10.0

121

127

125

124

3

50.0

127

123

126

125

2

100.0

133

131

130

131

2

500.0

133

136

143

137

5

1000.0

120

137

129

129

9

2500.0

132

135

126

131

5

5000.0

108

106

101

105

4

2AA (1 µg/plate)

1057

1050

1048

1052

5

Table-4: Mutagenic activity of the test substance in strain TA98 in Trail 2

Without activation

 

 

 

Concentration of test substance (µg/plate)

Revertants

Average

SD

Plate 1

Plate 2

Plate 3

0.0

29

29

28

29

1

10.0

25

28

21

25

4

50.0

22

23

26

24

2

100.0

31

26

36

31

5

500.0

32

34

35

34

2

1000.0

27

21

26

25

3

2500.0

40

33

27

33

7

5000.0

29

28

38

32

6

2NF (25 µg/plate)

1327

1570

1439

1445

122

With activation

 

 

 

 

 

0.0

34

31

35

33

2

10.0

39

36

38

38

2

50.0

41

37

41

40

2

100.0

44

37

43

41

4

500.0

44

39

44

42

3

1000.0

34

36

31

34

3

2500.0

36

41

27

35

7

5000.0

34

28

37

33

5

2AA (2 µg/plate)

1838

1590

1789

1739

131

Table-5: Mutagenic activity of the test substance in strain WP2 uvrA (PKM101) in Trail 2

Without activation

 

 

 

Concentration of test substance (µg/plate)

Revertants

Average

SD

Plate 1

Plate 2

Plate 3

0.0

192

189

178

186

7

10.0

204

182

190

192

11

50.0

182

188

187

186

3

100.0

210

187

190

196

13

500.0

218

205

190

204

14

1000.0

202

188

185

192

9

2500.0

203

213

205

207

5

5000.0

217

204

194

205

12

MMS (1000 µg/plate)

1985

1834

2183

2001

175

With activation

 

 

 

 

 

0.0

205

175

187

189

15

10.0

165

208

208

194

25

50.0

206

194

186

195

10

100.0

213

179

197

196

17

500.0

200

189

206

198

9

1000.0

192

196

195

194

2

2500.0

201

201

208

203

4

5000.0

196

171

194

187

14

2AA (25 µg/plate)

1878

1907

2141

1975

144

Applicant's summary and conclusion

Conclusions:
Negative in Salmonella typhimurium strains and in Escherichia coli strain with and without an exogenous metabolic activation system (S9)
Executive summary:

The test substance, was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). The study was conducted according to OECD guidelines 471 and 472, U.S. EPA 84-2.

Concentrations of 10, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate were initially evaluated using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). In the absence of any notable bacterial toxicity or test substance precipitation, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were subsequently tested in Salmonella typhimurium strains TA97a, TA98, and TA1535 to complete the first trial.

In a second confirmatory trial, concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate were tested in comparison to negative (solvent) controls in all strains.

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. The test substance was negative.