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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD 471, RCC Cytotest Cell Research GmbH 2000
Link to relevant study records
Reference
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:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP; minor deviation is not considered to to affect the reliability of the study.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The bacterial cultures were incubated in a shaking water bath for 3 hours at 37° C since the bacterial density had been reached at that incubation time.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
see above.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected February 1998; signature: March 1998
Type of assay:
bacterial reverse mutation assay
Target gene:
The histidine dependent strains are derived from S. typhimurium strain L T2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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 test: 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate (with strains TA98 and TA100)
Experiment 1: 33, 100, 333, 1000, 2500, 5000 ug/plate
Experiment 2: 33, 100, 333, 1000, 2500, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene,
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

DURATION
- Preincubation period: 60 minutes
- Expression time (cells in growth medium): 48 h at 37 degrees C in the dark

NUMBER OF REPLICATIONS: 3 plates per dose
Evaluation criteria:
- A test item is considered positive if either a dose related increase in the number of revertants or 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 one of the test points is considered non-mutagenic in this system.
- A biologically relevant response is described as follows:
A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains TA 1535 and TA 1537. Also, a dose-dependent and reproducible 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:
A statistical analysis of the data is not required.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no toxicity below 5000 μg/plate limit level in TA100 strain
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
An isolated reduction of the colony count in strain TA 98 at 100 μg/plate in the first experiment without metabolic activation was judged as an artefact since the colony count was back to normal at higher concentrations. 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 and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). However, a slight increase in revertant colony numbers close to or reaching the threshold of twice the colony count of the corresponding solvent control was observed in experiment I at 2500 and 5000 μg/plate in strain TA 100 without metabolic activation. This increase was judged as biologically irrelevant since it is, based upon a solvent control colony count close to the lower limit of the laboratory historical range. The absolute numbers of colonies remained well within the range of negative and solvent controls. Furthermore, this increase was not reproduced in the second experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Summary of revertants

 +/- S9 mix

Test material concentration (μg/plate)

TA100

TA1535

TA1537

TA98

TA102

 

Revertants

 

Revertants

 

Revertants

 

Revertants

 

Revertants

Replicate

Experiment 1

Experiment 2

Replicate

Experiment 1

Experiment 2

Replicate

Experiment 1

Experiment 2

Replicate

Experiment 1

Experiment 2

Replicate

Experiment 1

Experiment 2

- S9 Mix

Negative control

1

78

85

1

9

7

1

5

9

1

16

15

1

227

267

2

77

100

2

9

9

2

6

7

2

14

19

2

216

290

3

83

96

3

11

15

3

4

11

3

17

12

3

220

265

Mean

79

94

Mean

10

10

Mean

5

9

Mean

16

15

Mean

221

274

Solvent control

1

94

84

1

7

8

1

8

8

1

19

16

1

186

229

2

82

54

2

11

12

2

5

7

2

15

12

2

198

213

3

73

93

3

9

8

3

6

9

3

13

18

3

180

255

Mean

83

77

Mean

9

9

Mean

6

8

Mean

16

15

Mean

188

232

Positive control

1

1292

715

1

213

268

1

57

67

1

96

89

1

991

904

2

1361

820

2

367

412

2

41

65

2

99

86

2

972

867

3

1321

931

3

509

468

3

44

56

3

92

92

3

1154

1017

Mean

1325

822

Mean

363

383

Mean

47

63

Mean

96

89

Mean

1039

929

33

1

59

58

1

8

9

1

3

5

1

7

11

1

192

216

2

70

65

2

7

5

2

5

7

2

12

12

2

205

225

3

63

55

3

6

3

3

3

7

3

4

18

3

223

268

Mean

64

59

Mean

7

6

Mean

4

6

Mean

8

14

Mean

207

236

100

1

68

61

1

5

8

1

6

5

1

3

13

1

184

234

2

66

62

2

6

7

2

5

6

2

2

12

2

159

202

3

66

69

3

9

3

3

2

4

3

2

13

3

171

214

Mean

67

64

Mean

7

6

Mean

4

5

Mean

2

13

Mean

171

217

333

1

105

75

1

10

9

1

4

6

1

15

11

1

177

195

2

114

82

2

4

3

2

2

4

2

8

15

2

164

176

3

83

77

3

5

7

3

3

3

3

5

10

3

210

237

Mean

101

78

Mean

6

6

Mean

3

4

Mean

9

12

Mean

184

203

1000

1

132

112

1

3

2

1

6

3

1

9

7

1

170

225

2

133

119

2

5

2

2

7

2

2

8

7

2

185

260

3

122

102

3

1

3

3

5

1

3

19

10

3

207

263

Mean

129

111

Mean

3

2

Mean

6

2

Mean

12

8

Mean

187

249

2500

1

149

84

1

6

6

1

3

5

1

9

11

1

212

262

2

170

93

2

1

17

2

4

7

2

13

12

2

204

150

3

167

72

3

1

5

3

2

5

3

11

25

3

214

218

Mean

162

83

Mean

3

9

Mean

3

6

Mean

11

16

Mean

210

210

5000

1

158

96

1

5

13

1

3

4

1

3

13

1

222

188

2

182

94

2

2

15

2

6

12

2

3

11

2

223

193

3

125

68

3

3

14

3

8

6

3

5

3

3

144

197

Mean

155

86

Mean

3

14

Mean

6

7

Mean

4

9

Mean

196

193

+ S9 Mix

Negative control

1

133

144

1

4

8

1

10

8

1

14

18

1

253

170

2

127

122

2

15

9

2

4

7

2

15

17

2

263

144

3

126

116

3

11

7

3

4

8

3

18

21

3

250

159

Mean

129

127

Mean

10

8

Mean

6

8

Mean

16

19

Mean

255

158

Solvent control

1

122

104

1

9

6

1

6

14

1

17

19

1

233

147

2

110

98

2

5

5

2

5

7

2

15

23

2

258

135

3

127

94

3

16

9

3

9

8

3

11

23

3

230

167

Mean

120

99

Mean

10

7

Mean

7

10

Mean

14

22

Mean

240

150

Positive control

1

636

454

1

74

81

1

55

42

1

123

167

1

409

1255

2

569

546

2

75

74

2

50

46

2

114

179

2

1132

1127

3

608

622

3

86

65

3

52

49

3

100

152

3

1144

1036

Mean

604

541

Mean

78

73

Mean

52

46

Mean

112

166

Mean

895

1139

33

1

112

88

1

8

6

1

9

7

1

17

14

1

191

136

2

111

110

2

5

10

2

9

10

2

15

9

2

202

157

3

106

115

3

9

4

3

5

7

3

16

15

3

202

164

Mean

110

104

Mean

7

7

Mean

8

8

Mean

16

13

Mean

198

152

100

1

125

101

1

6

5

1

7

6

1

9

17

1

192

159

2

121

113

2

4

12

2

6

6

2

10

22

2

209

154

3

132

90

3

7

4

3

6

7

3

12

22

3

195

160

Mean

126

101

Mean

6

7

Mean

6

6

Mean

10

20

Mean

199

158

333

1

122

89

1

9

8

1

6

5

1

21

9

1

215

187

2

136

95

2

4

6

2

6

6

2

15

17

2

222

205

3

145

136

3

4

7

3

10

11

3

20

19

3

246

170

Mean

134

107

Mean

6

7

Mean

7

7

Mean

19

15

Mean

228

187

1000

1

120

111

1

6

5

1

11

10

1

6

11

1

179

131

2

122

98

2

10

5

2

8

8

2

3

22

2

189

171

 

3

133

100

3

3

10

3

13

7

3

14

15

3

154

169

 

Mean

125

103

Mean

6

7

Mean

11

8

Mean

8

16

Mean

174

157

 

2500

1

123

93

1

8

3

1

8

10

1

9

10

1

169

130

 

2

126

110

2

4

10

2

10

6

2

13

16

2

182

140

 

3

132

90

3

6

6

3

9

5

3

7

11

3

161

125

 

Mean

127

98

Mean

6

6

Mean

9

7

Mean

10

12

Mean

171

132

 

5000

1

107

87

1

7

6

1

6

3

1

5

15

1

91

102

 

2

113

81

2

3

5

2

2

5

2

8

18

2

139

94

 

3

105

96

3

5

3

3

9

3

3

5

6

3

141

100

 

Mean

108

88

Mean

5

5

Mean

6

4

Mean

6

13

Mean

124

99

Conclusions:
Interpretation of results (migrated information):
negative

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

The study was performed to guideline OECD 471 and EU Method B.14 under GLP to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, TA1537 in both the presence and absence of S-9 mix. A preliminary test was performed to determine the test concentrations for main study. In the main study two experiments (one using the plate incorporation method and the other with the pre-incubation method) were performed and the test substance was evaluated at a concentration of up to 5000 µg/plate in DMSO vehicle. Positive controls appropriate for each strain, in the presence and absence of S9 -mix were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, 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 the positive control substances. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of S-9 mix.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

OECD 471, 2000 -The study was performed to guideline OECD 471 and EU Method B.14 under GLP to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, TA1537 in both the presence and absence of S-9 mix. A preliminary test was performed to determine the test concentrations for main study. In the main study two experiments (one using the plate incorporation method and the other with the pre-incubation method) were performed and the test substance was evaluated at a concentration of up to 5000 µg/plate in DMSO vehicle. Positive controls appropriate for each strain, in the presence and absence of S9 -mix were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, 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 the positive control substances. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of S-9 mix.


Justification for selection of genetic toxicity endpoint
Study selected is an in vitro study (Klimisch 1)

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for mutagenicity.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity.