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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
Study period:
January - February 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. No relevant toxic effects were observed. 5000 µg/plate was chosen as maximal concentration.
The following concentrations were tested: 33, 100, 333, 1000, 2500 and 5000 µg/plate.
Vehicle:
DMSO
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, without S9, TA1537, TA98. 2-aminoanthracene : with S9, all strains
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) = experiment 1; preincubation = experiment 2

Pre-experiment for toxicity :
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA98 and TA100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in the pre-experiment were the same as described for the experiment 1 (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants of a clearing of the bacterial background lawn.
Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

For each strain and dose level, including the controls, three plates were used.

In the preincubation assay, test solution, S9 mix and bacterial suspension were mixed and incubated at 37°C for 60 minutes. After pre-incubation agar was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Acceptability of the assay :
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the arnge of the historical control data
-the psitive control substances should produce a significant increase in mutant colony frequencies
Evaluation criteria:
A test item is considered positive if either a dose related increase in the number of revertants of a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any dose of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as : A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate instrains TA98, TA 100 and TA 102, or trice in strains TA1535, TA1537. Also a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.
Statistics:
no

Results and discussion

Test results
Key result
Species / strain:
other: TA98, TA100, TA102, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
No precipitation of the test item occurred up to the highest investigated dose.
No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Ames tables

Pre-experiment

Test group

Concentration per plate (µg)

Revertants per plate (mean of 3 plates)

TA98

TA98

TA 100

TA 100

- S9

+ S9

- S9

+ S9

Negative control

-

28

38

166

138

Solvent control

-

20

32

166

155

4-NOPD

10

223

-

-

-

Sodium azide

10

-

-

690

-

2-AA

2.5

-

223

-

742

Test item

3

22

29

143

126

Test item

10

29

28

139

132

Test item

33

30

29

143

128

Test item

100

27

32

143

126

Test item

333

27

30

133

118

Test item

1000

18

3

 

132

130

Test item

2500

24

23

131

133

Test item

5000

24

22

124

130

 

Experiment 1

TA1535 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

8

7

11

9

2.1

 

Solvent control

8

13

8

10

2.9

1.0

Positive control

615

664

664

648

28.3

67.0

33

13

10

7

10

3.0

1.0

100

9

9

10

9

0.6

1.0

333

10

7

8

8

1.5

0.9

1000

6

11

5

7

3.2

0.8

2500

9

5

9

8

2.3

0.8

5000

6

10

8

8

2.0

0.8

 

TA1535 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

8

8

11

9

1.7

 

Solvent control

15

15

9

13

3.5

1.0

Positive control

73

86

64

74

11.1

5.7

33

8

11

17

12

4.6

0.9

100

8

8

12

9

2.3

0.7

333

7

12

10

10

2.5

0.7

1000

11

11

12

11

0.6

0.9

2500

10

7

5

7

2.5

0.6

5000

6

9

7

7

1.5

0.6

 

TA1537 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

6

5

6

6

0.6

 

Solvent control

10

7

4

7

3.0

1.0

Positive control

52

51

52

52

0.6

7.4

33

8

5

7

7

1.5

1.0

100

8

5

4

6

2.1

0.8

333

5

8

5

6

1.7

0.9

1000

5

7

8

7

1.5

1.0

2500

6

4

4

5

1.2

0.7

5000

4

5

5

5

0.6

0.7

 

TA1537 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

16

11

7

11

4.5

 

Solvent control

5

7

5

6

1.2

1.0

Positive control

107

96

84

96

11.5

16.9

33

12

7

7

9

2.9

1.5

100

8

8

6

7

1.2

1.3

333

6

6

6

6

0

1.1

1000

8

7

4

6

2.1

1.1

2500

5

5

5

5

0

0.9

5000

8

4

4

5

2.3

0.9

 

 

TA98 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

27

27

31

28

2.3

 

Solvent control

23

20

18

20

2.5

1.0

Positive control

241

212

217

223

15.5

11.0

33

29

32

30

30

1.5

1.5

100

28

29

25

27

2.1

1.3

333

24

29

27

27

2.5

1.3

1000

17

18

19

18

1.0

0.9

2500

25

22

26

24

2.1

1.2

5000

23

24

26

24

1.5

1.2

 

TA98 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

38

39

38

38

0.6

 

Solvent control

34

32

29

32

2.5

1.0

Positive control

379

430

402

404

25.5

12.7

33

29

27

30

29

1.5

0.9

100

31

31

34

32

1.7

1.0

333

29

32

28

30

2.1

0.9

1000

21

23

25

23

2.0

0.7

2500

22

23

24

23

1.0

0.7

5000

19

23

24

22

2.6

0.7

 

TA100 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

156

161

180

166

12.7

 

Solvent control

166

160

171

166

5.5

1.0

Positive control

721

702

648

690

37.9

4.2

33

143

148

138

143

5.0

0.9

100

143

142

145

143

1.5

0.9

333

132

135

131

133

2.1

0.8

1000

139

128

130

132

5.9

0.8

2500

130

133

129

131

2.1

0.8

5000

125

121

127

124

3.1

0.8

 

TA100 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

147

132

135

138

7.9

 

Solvent control

158

157

149

155

4.9

1.0

Positive control

713

757

756

742

25.1

4.8

33

130

129

124

128

3.2

0.8

100

127

121

131

126

5.0

0.8

333

122

118

115

118

3.5

0.8

1000

125

129

135

130

5.0

0.8

2500

132

129

139

133

5.1

0.9

5000

132

128

131

130

2.1

0.8

 

TA102 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

207

194

194

198

7.5

 

Solvent control

195

182

201

193

9.7

1.0

Positive control

973

915

1026

971

55.5

5.0

33

233

198

205

212

18.5

1.1

100

229

195

178

201

26.0

1.0

333

210

210

183

201

15.6

1.0

1000

190

190

177

186

7.5

1.0

2500

193

205

177

192

14.0

1.0

5000

217

203

185

202

16.0

1.0

 

TA102 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

192

149

149

163

24.8

 

Solvent control

161

194

238

198

38.6

1.0

Positive control

749

785

806

780

28.8

3.9

33

184

198

237

206

27.5

1.0

100

196

196

167

186

16.7

0.9

333

147

165

181

164

17.0

0.8

1000

172

182

198

184

13.1

0.9

2500

156

139

117

137

19.6

0.7

5000

116

107

117

113

5.5

0.6

 

 

Experiment 2

TA1535 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

9

10

8

9

1.0

 

Solvent control

13

11

9

11

2.0

1.0

Positive control

738

754

677

723

40.6

65.7

33

13

11

7

10

3.1

0.9

100

14

10

13

12

2.1

1.1

333

10

10

11

10

0.6

0.9

1000

8

10

10

9

1.2

0.8

2500

10

7

7

8

1.7

0.7

5000

6

8

8

7

1.2

0.7

 

TA1535 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

10

13

13

12

1.7

 

Solvent control

13

10

9

11

2.1

1.0

Positive control

120

128

119

122

4.9

11.5

33

10

11

11

11

0.6

1.0

100

13

9

8

10

2.6

0.9

333

8

7

9

8

1.0

0.8

1000

5

13

6

8

4.4

0.8

2500

11

12

8

10

2.1

1.0

5000

13

9

10

11

2.1

1.0

 

TA1537 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

7

5

7

6

1.2

 

Solvent control

5

7

6

6

1.0

1.0

Positive control

47

49

65

54

9.9

8.9

33

6

6

8

7

1.2

1.1

100

6

6

6

6

0.0

1.0

333

5

7

7

6

1.2

1.1

1000

8

6

6

7

1.2

1.1

2500

4

7

5

5

1.5

0.9

5000

5

5

4

5

0.6

0.8

 

TA1537 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

12

11

11

11

0.6

 

Solvent control

12

8

9

10

2.1

1.0

Positive control

57

55

55

56

1.2

5.8

33

7

9

10

9

1.5

0.9

100

12

9

12

11

1.7

1.1

333

9

8

13

10

2.6

1.0

1000

9

11

10

10

1.0

1.0

2500

9

0

10

6

5.5

0.7

5000

11

11

11

11

0.0

1.1

 

 

TA98 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

25

24

32

27

4.4

 

Solvent control

31

21

23

25

5.3

1.0

Positive control

220

203

234

219

15.5

8.8

33

34

26

23

28

5.7

1.1

100

30

30

21

27

5.2

1.1

333

19

25

30

25

5.5

1.0

1000

21

30

23

25

4.7

1.0

2500

27

27

21

25

3.5

1.0

5000

19

24

26

23

3.6

0.9

 

TA98 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

30

44

40

38

7.2

 

Solvent control

45

44

34

41

6.1

1.0

Positive control

586

591

548

575

23.5

14.0

33

52

38

46

45

7.0

1.1

100

47

47

42

45

2.9

1.1

333

44

38

46

43

4.2

1.0

1000

35

33

36

35

1.5

0.8

2500

40

30

32

34

5.3

0.8

5000

37

38

34

36

2.1

0.9

 

TA100 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

97

112

129

113

16.0

 

Solvent control

129

107

100

112

15.1

1.0

Positive control

832

902

856

863

35.6

7.7

33

99

100

106

102

3.8

0.9

100

101

107

113

107

6.0

1.0

333

91

102

100

98

5.9

0.9

1000

116

109

101

109

7.5

1.0

2500

90

97

104

97

7.0

0.9

5000

62

84

66

71

11.7

0.6

 

TA100 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

81

78

113

91

19.4

 

Solvent control

132

125

131

129

3.8

1.0

Positive control

691

711

698

700

10.1

5.4

33

119

111

124

118

6.6

0.9

100

104

104

114

107

5.8

0.8

333

98

83

84

88

8.4

0.7

1000

83

100

95

93

8.7

0.7

2500

100

82

84

89

9.9

0.7

5000

89

90

78

86

6.7

0.7

 

TA102 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

210

220

212

214

5.3

 

Solvent control

199

157

189

182

21.9

1.0

Positive control

1040

984

1090

1038

53.0

5.7

33

212

214

183

203

17.3

1.1

100

228

214

207

216

10.7

1.2

333

205

215

198

206

8.5

1.1

1000

184

196

182

187

7.6

1.0

2500

196

199

166

187

18.2

1.0

5000

174

156

163

164

9.1

0.9

 

TA102 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

275

191

191

219

48.5

 

Solvent control

191

201

229

207

19.7

1.0

Positive control

805

723

822

783

52.9

3.8

33

218

234

214

222

10.6

1.1

100

262

251

217

243

23.5

1.2

333

230

224

218

224

6.0

1.1

1000

219

215

202

212

8.9

1.0

2500

225

214

218

219

5.6

1.1

5000

218

228

223

223

5.0

1.1

 

 

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.
The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) sing the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 500 µg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.

The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.