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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
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:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
yes
Remarks:
The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible
Qualifier:
according to
Guideline:
other: MAFF Japan 59-Nousan-4200, Testing Guidelines for Toxicity Studies
Deviations:
yes
Remarks:
The stability of the test substance formulations has not been determined at room temperature for the period of dosing. However, the impact to the study is negligible
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT Assay)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Purity: 93.6%

Method

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
Species / strain
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Assay: Nine concentrations of the test substance ranging from 0.5 to 3340 μg/mL (approximately 10 mM) in the presence and absence of S9 reaction mixture.
The test substance formed clear solutions in DMSO from 0.05 to 334 mg/mL. Visible precipitate was observed in the treatment medium at concentrations ≥ 1500 μg/mL at the beginning and end of treatment. Cloning efficiency relative to the solvent control (relative cloning efficiency) at 3340 μg/mL was 98% in the absence of S9 activation and 101% in the presence of S9 activation. Selection of concentrations for the mutagenesis assay was based on the precipitate profile observed in the preliminary toxicity assay.
Mutagenesis Assays: Based on the preliminary toxicity assay, the concentrations chosen for the mutagenesis assay ranged from 250 to 1500 μg/mL for both the non-activated and S9-activated cultures.
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent of choice based on solubility information provided by the Sponsor and compatibility with the target cells. The Sponsor indicated that the test substance is soluble in DMSO at a concentration of 334 mg/mL.
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Remarks:
BaP for S9-activated test system. EMS for non-activated test system.
Details on test system and conditions:
METHOD OF APPLICATION: The mutagenesis assay was performed according to a protocol developed from published methodologies (Hsie et al., 1981 and O'Neill et al., 1977). Treatment flasks were identified with the test substance number and a code to designate the treatment condition and test phase. Exponentially growing CHO-K1-BH4 cells were seeded in F12FBS5+Hx at a density of 1x1000000 cells/25 cm2 flask and were incubated at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air for 18-24 hours. F12FBS5+Hx is Ham's F12 medium with hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 μg streptomycin/mL and 2 mM L-glutamine/mL
- Cell density at seeding (if applicable): 1x1000000 cells/25 cm²

DURATION
- Preincubation period: 18-24 hours
- Exposure duration: 5±0.5 hours at 37±1ºC
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 7 to 10 days

SELECTION AGENT (mutation assays): For selection of the 6-thioguanine (TG, 2-amino-6-mercaptopurine)-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2x100000 cells/100 mm dish in F12FBS5 without Hypoxanthine (F12FBS5-Hx) containing 10 μM TG.

NUMBER OF REPLICATIONS: Mutagenesis assay: Duplicates, Cytotoxicity: Triplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were fixed with methanol, stained with 0.05% to 0.06% Crystal Violet, counted and cloning efficiency determined

NUMBER OF CELLS EVALUATED: 2X100000 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: Relative cloning efficiency
- Any supplementary information relevant to cytotoxicity: The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2x1000000 cells: 10 plates at 2x100000 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 1000000 clonable cells.

References:
Hsie, A.W., D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. O'Neill, and B.L.Whitfield (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. A report of the Gene-Tox Program, Mutation Research 86:193-214.
O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch, and A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Research 45:91-101.
Evaluation criteria:
All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data:
The test material was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies of >40 mutants per 1000000 clonable cells.
If a single point above 40 mutants per 1000000 clonable cells was observed at the highest dose, the assay was considered equivocal.
If no culture exhibited a mutant frequency of >40 mutants per 1000000 clonable cells, the test substance was considered negative.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: No positive responses

Any other information on results incl. tables

Table 1: Preliminary Toxicity Assay using IN-QEU76

Treatment (µg/mL)

Non-Activated

S9-Activated

Plate counts

Cloning Efficiency (%)

Relative Cloning Efficiency (%)

Plate counts

Cloning Efficiency (%)

Relative Cloning Efficiency (%)

1

2

3

1

2

3

Solvent

126

118

117

60

100

149

139

140

71

100

0.5

132

164

144

73

122

149

132

153

72

101

1.5

94

91

83

45

74

129

118

90

56

79

5

124

110

123

60

99

137

134

145

69

97

15

118

103

113

56

93

139

149

165

76

106

50

125

124

134

64

106

184

179

147

85

119

150

114

140

132

64

107

153

178

167

83

116

500

98

112

112

54

89

147

152

138

73

102

1500 P

173

144

154

79

130

150

167

152

78

110

3340 P

121

116

117

59

98

139

156

137

72

101

Solvent = DMSO, P – Precipitating concentration

Cloning efficiency = average colonies divided by 200 cells per dish X 100
Relative cloning efficiency = cloning efficiency of treatment group divided by cloning efficiency of solvent group X 100

Table 2: Concurrent Cytotoxicity Test using IN-QEU76

Treatment (µg/mL)

Plate counts

Cloning Efficiency (%)

Relative Cloning Efficiency (%)

1

2

3

Non-Activated Cultures

Solvent A

160

143

166

78

100

Solvent B

*

156

156

250 A

151

157

160

73

93

250 B

131

131

144

500 A

124

157

132

72

93

500 B

144

156

154

750 A

151

156

156

74

95

750 B

140

141

147

1000 A

123

143

129

67

86

1000 B

146

126

138

1500 A (P)

143

149

139

74

94

1500 B (P)

154

152

146

EMS (0.2μL/mL) A

106

72

88

45

58

EMS (0.2μL/mL) B

100

86

87

Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration, * - Plate lost due to contamination

Cloning efficiency = average colonies divided by 200 cells per dish X 100
Relative cloning efficiency = cloning efficiency of treatment group divided by cloning efficiency of solvent group X 100

Table 2 Cont.: Concurrent Cytotoxicity Test using IN-QEU76

Treatment (µg/mL)

Plate counts

Cloning Efficiency (%)

Relative Cloning Efficiency (%)

1

2

3

S9-Activated Cultures

Solvent A

137

150

138

82

100

Solvent B

170

198

193

250 A

172

184

147

83

102

250 B

169

177

152

500 A

156

158

160

84

102

500 B

184

183

163

750 A

169

185

186

86

104

750 B

157

164

166

1000 A

162

167

183

82

100

1000 B

160

167

145

1500 A (P)

179

196

172

86

104

1500 B (P)

146

171

165

B(a)P (4μL/mL) A

94

98

107

45

55

B(a)P (4μL/mL) B

81

88

74

Solvent = DMSO, A and B are duplicate cultures,
P – Precipitating concentration

Cloning efficiency = average colonies divided by 200 cells per dish X 100
Relative cloning efficiency = cloning efficiency of treatment group divided by cloning efficiency of solvent group X 100

Table 3: Non-activated (-S9) Study using IN-QEU76

Treatment
(μg/mL)

Cloning Efficiency Plates

Cloning
Efficiency

Selection (Mutation) Plates

Average Colonies

Mutants/106
Clonable
Cells

Relative
Cloning
Efficiency (%)

Plate Counts

Average
Colonies

Plate Counts

1

2

3

1

2

3

4

5

Solvent A

131

154

129

139.8

0.7

0

0

0

0

0

0.1

0.7

100

Solvent B

149

150

126

0

1

0

0

0

250 A

165

165

153

152.5

0.76

0

0

0

0

0

0.1

0.7

93

250 B

136

145

151

0

0

1

0

0

500 A

143

116

157

142.7

0.71

0

0

1

0

0

0.5

3.5

93

500 B

146

149

145

0

2

0

0

2

750 A

162

139

171

150.8

0.75

0

0

0

0

0

0

0

95

750 B

154

136

143

0

0

0

0

0

1000 A

156

159

130

157.7

0.79

2

0

0

0

0

0.3

1.9

86

1000 B

170

165

166

0

0

0

0

1

1500 A (P)

124

150

132

136.2

0.68

0

0

0

0

0

0.1

0.7

94

1500 B (P)

136

132

143

1

0

0

0

0

EMS (0.2μL/mL) A

105

99

108

108.5

0.54

50

39

52

62

44

45.5

419.4

58

EMS (0.2μL/mL) B

121

113

105

31

44

39

37

57

Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration

Cloning efficiency = average colonies divided by 200 cells per dish X 100
Mutants/106 clonable cells = average mutant colonies divided by cloning efficiency x 2 x 105 cells X 106

Table 4: Activated (+S9) Study using IN-QEU76

Treatment
(μg/mL)

Cloning Efficiency Plates

Cloning
Efficiency

Selection (Mutation) Plates

Average Colonies

Mutants/106
Clonable
Cells

Relative
Cloning
Efficiency (%)

Plate Counts

Average
Colonies

Plate Counts

1

2

3

1

2

3

4

5

Solvent A

132

162

191

156.2

0.78

0

1

1

0

0

1.1

7.0

100

Solvent B

139

153

160

5

3

0

1

0

250 A

180

186

187

186.8

0.93

0

0

0

0

0

0.5

2.7

102

250 B

204

210

154

2

0

0

2

1

500 A

176

163

175

174.8

0.87

2

0

1

2

0

0.6

3.4

102

500 B

166

171

198

0

0

0

1

0

750 A

175

192

160

172.5

0.86

0

2

2

5

5

2.0

11.6

104

750 B

166

171

171

2

1

3

0

0

1000 A

146

167

178

156.3

0.78

1

3

2

2

1

0.9

5.8

100

1000 B

144

141

162

0

0

0

0

0

1500 A (P)

177

156

191

169.2

0.85

1

1

0

0

1

0.4

2.4

104

1500 B (P)

149

167

175

0

0

1

0

0

B(a)P
(4μg/mL) A

136

149

123

123.7

0.62

40

33

54

29

37

35.7

288.7

55

B(a)P
(4μg/mL) B

90

121

125

29

36

28

40

31

Solvent = DMSO, A and B are duplicate cultures, P – Precipitating concentration

Cloning efficiency = average colonies divided by 200 cells per dish X 100
Mutants/106 clonable cells = average mutant colonies divided by cloning efficiency x 2 x 105 cells X 106

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test material did not cause a dose-responsive increase in mutant frequency in the non-activated or S9-activated test systems in the CHO/HGPRT Mutation Assay. No positive responses were observed in either the presence or absence of S9 activation. Therefore, the test substance was concluded to be negative in this assay.
Executive summary:

The test material was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the dose range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test substance. Dosing formulations were adjusted to compensate for the purity of the test substance (93.6%) using a correction factor of 1.07. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice.

The maximum concentration of the test substance in treatment medium was 3340 μg/mL (approximately 10 mM) in the preliminary toxicity assay. The test substance formed clear solutions in DMSO from 0.05 to 334 mg/mL. Visible precipitate was observed in the treatment medium at concentrations ≥ 1500 μg/mL at the beginning and end of treatment. No substantial toxicity (i.e., relative cloning efficiency) was observed at any concentration in the presence or absence of S9 activation.

The concentrations chosen for the mutagenesis assay ranged from 250 to 1500 μg/mL for both the non-activated and S9-activated cultures. Visible precipitate was observed in the treatment medium at a concentration of 1500 μg/mL at the beginning and end of treatment. No positive responses, i.e., treated cultures with mutant frequencies > 40 mutants per 106 clonable cells, were observed in either the presence or absence of S9 activation. No substantial toxicity (i.e., relative cloning efficiency) was observed at any concentration in the presence or absence of S9 activation.

All criteria for a valid study were met. Under the conditions of this study, the test material did not cause a dose-responsive increase in mutant frequency in the non-activated or S9-activated test systems in the CHO/HGPRT Mutation Assay. No positive responses, i.e., treated cultures with mutant frequencies > 40 mutants per 106 clonable cells, were observed in either the presence or absence of S9 activation. Therefore, the test substance was concluded to be negative in this assay.