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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
Study period:
feb to june 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
- Storage stability: The stability of the test substance under storage conditions was guaranteed until 27 Sep 2014 as indicated by the sponsor and the sponsor holds this responsibility
- Physical state, appearance: Solid, white
- Storage conditions: Room temperature

Method

Target gene:
HGPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

with S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

2nd Experiment
without S9 mix
0; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

with S9 mix
0; 18.8; 37.5; 75.0; 150.0; 200.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and other commonly used vehicles (e.g. DMSO, ethanol, acetone), tetrahydrofurane (THF) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay. The final concentration of the vehicle THF in the culture medium was 0.5% (v/v).
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
with and without S9 mix
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: methylcholanthrene (with metabolic activation, 20 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- pretreatment: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
- Exposure duration: 20-24h
- Expression time (cells in growth medium): 4 and 24h --> washing and first cytotox determination, 1. passage day 5, days 7-9 2. passage into selection medium (TG medium) and second cytotox determination
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16 drying, fixation, staining and counting of the selected colonies

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: number of colony per flask was counted and recorded

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
pH, osmolarity, solubility, cel morphology
Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 5).
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the solubility properties of the test substance in the most suitable vehicle 2500 μg/mL (approx. 5 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. Higher concentrations could not be formulated in a suitable solvent. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as toxicity indicator for dose selection and various parameters were checked for all or at least for some selected doses. In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In culture medium test substance precipitation occurred at 78.1 μg/mL and above at the end of treatment in the absence and presence of S9 mix. After 4 and 24 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% was observed.

Any other information on results incl. tables

In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 9.68 per 106 cells) were close to the respective vehicle control values (MFcorr.:0.50 – 6.92 per 106 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 15.95 per 106 cells; with S9 mix: MFcorr.: 0.00 – 15.68 per 106 cells). The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 108.87 – 381.88 per 106 cells; with S9 mix: MFcorr.: 48.01 – 72.64 per 106 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 48.83 – 1338.10 per 106 cells; with S9 mix: MFcorr.: 26.29 – 413.54 per 106 cells).

Table 5: Summary of results

Exp.

Exposure period

Test groups

S9 mix

Prec.*

Genotoxicity**

MFcorr.

[per 106cells]

Cytotoxicity***

CE1

[%]

CE2

[%]

1

4 h

Vehicle control

3.13 µg/mL

6.25 µg/mL

12.50 µg/mL

25.00 µg/mL

50.00 µg/mL

100.00 µg/mL

200.00 µg/mL

Positive control

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

-

0.50

n.c.

n.c.

2.12

0.00

1.19

4.44

2.56

108.87

100.0

100.9

94.7

89.9

94.9

91.1

93.8

96.2

96.2

100.0

n.c.

n.c.

117.4

86.4

99.2

95.4

100.3

94.1

2

24 h

Vehicle control

12.5 µg/mL

25.0 µg/mL

50.0 µg/mL

100.0 µg/mL

200.00 µg/mL

Positive control

-

-

-

-

-

-

-

-

-

-

+

+

+

-

1.58

5.84

1.58

2.02

1.54

2.90

381.88

100.0

107.9

101.0

91.4

88.6

92.3

71.7

100.0

85.9

80.2

79.6

82.3

95.7

70.5

1

4 h

Vehicle control

3.13 µg/mL

6.25 µg/mL

12.50 µg/mL

25.00 µg/mL

50.00 µg/mL

100.00 µg/mL

200.00 µg/mL

Positive control

+

+

+

+

+

+

+

+

+

-

-

-

-

-

+

+

+

-

3.46

n.c.

n.c.

5.10

1.84

0.47

1.97

3.17

72.64

100.0

96.5

89.8

97.7

86.9

96.8

95.4

93.3

94.4

100.0

n.c.

n.c.

92.6

93.7

96.9

96.7

90.6

91.2

2

4 h

Vehicle control

18.8 µg/mL

37.5 µg/mL

75.0 µg/mL

150.0 µg/mL

200 µg/mL

Positive control

+

+

+

+

+

+

+

-

-

-

+

+

+

-

6.92

1.44

0.00

0.35

9.68

4.45

48.01

100.0

107.9

99.5

110.7

108.4

117.2

124.3

100.0

98.2

102.0

103.9

95.9

79.5

88.7

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

Table 6: Cytotoxicity data - 1st Experiment without S9 mix; 4 -hour exposure period

Test groups

Cell density

(x 105/mL)

At 1stsub-culture

CE1(survival)

(4 h after treatment; about 200 cells/flask seeded)

CE2(viability)

(at the end of the expression period; about 200 cells/flask seeded)

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Abs.

Rel.

Abs.

Rel.

Vehicle control*

A

3.76

109

125

66.3

100.0

142

137

63.1

100.0

B

6.19

155

141

110

115

3.13 µg/mL

A

5.75

116

142

66.9

100.9

n.c.

n.c.

n.c.

n.c.

B

6.13

132

145

n.c.

n.c.

6.25 µg/mL

A

5.52

115

110

62.8

94.7

n.c.

n.c.

n.c.

n.c.

B

6.26

132

145

n.c.

n.c.

12.50 µg/mL

A

5.56

126

111

59.6

89.9

137

128

74.1

117.4

B

6.46

136

103

138

189

25.00 µg/mL

A

5.35

114

112

62.9

94.9

135

95

54.5

86.4

B

7.95

128

149

110

96

50.00 µg/mL

A

5.23

117

123

60.4

91.1

111

108

62.6

99.2

B

6.94

124

119

149

132

100.00 µg/mL

A

6.43

109

131

62.2

93.8

119

109

60.2

95.4

B

7.72

138

119

125

128

200.00 µg/mL

A

5.18

109

125

63.8

96.2

153

142

63.3

100.3

B

6.86

134

142

114

97

300.00 µg/mL

EMS

A

5.52

120

122

63.8

96.2

116

103

59.4

94.1

B

6.51

133

135

150

106

*THF 0.5 % (v/v)

Table 7: Cytotoxicity data - 1st Experiment with S9 mix; 4 -hour exposure period

Test groups

Cell density

(x 105/mL)

At 1stsub-culture

CE1(survival)

(4 h after treatment; about 200 cells/flask seeded)

CE2(viability)

(at the end of the expression period; about 200 cells/flask seeded)

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Abs.

Rel.

Abs.

Rel.

Vehicle control*

A

4.60

155

114

65.8

100.0

119

140

64.6

100.0

B

5.34

135

122

127

130

3.13 µg/mL

A

5.34

150

90

63.5

96.5

n.c.

n.c.

n.c.

n.c.

B

5.99

133

135

n.c.

n.c.

6.25 µg/mL

A

6.42

112

127

59.1

89.8

n.c.

n.c.

n.c.

n.c.

B

6.31

123

110

n.c.

n.c.

12.50 µg/mL

A

6.50

133

153

64.3

97.7

137

105

59.8

92.6

B

6.33

102

126

117

119

25.00 µg/mL

A

6.13

111

107

57.2

86.9

99

143

60.5

93.7

B

6.00

122

117

103

139

50.00 µg/mL

A

6.69

136

119

63.7

96.8

128

109

62.6

96.9

B

5.90

121

133

159

104

100.00 µg/mL

A

6.99

127

115

62.8

95.4

104

114

62.5

96.7

B

6.89

109

151

136

146

200.00 µg/mL

A

7.06

128

116

61.4

93.3

75

111

58.5

90.6

B

6.44

128

119

146

136

20.00 µg/mL

MCA

A

6.80

129

110

62.1

94.4

98

139

58.9

91.2

B

5.16

125

132

116

118

*THF 0.5 % (v/v)

Table 8: Cytotoxicity data - 2nd Experiment without S9 mix; 24 -hour exposure period

Test groups

Cell density

(x 105/mL)

At 1stsub-culture

CE1(survival)

(24 h after treatment; about 200 cells/flask seeded)

CE2(viability)

(at the end of the expression period; about 200 cells/flask seeded)

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Abs.

Rel.

Abs.

Rel.

Vehicle control*

A

4.67

147

127

76.4

100.0

187

182

90.3

100.0

B

7.38

156

181

177

176

12.5 µg/mL

A

6.46

170

177

82.4

107.9

194

181

77.6

85.9

B

7.55

157

155

125

120

25.0 µg/mL

A

7.20

157

138

77.2

101.0

151

151

72.4

80.2

B

7.61

182

140

127

150

50.0 µg/mL

A

6.57

134

145

69.8

91.4

161

139

71.9

79.6

B

7.60

130

149

145

130

100.0 µg/mL

A

6.94

123

137

67.7

88.6

132

156

74.3

82.3

B

7.94

148

133

163

143

200.0 µg/mL

A

7.51

135

151

70.5

92.3

193

194

86.4

95.7

B

8.25

135

143

174

130

300.00 µg/mL

EMS

A

5.65

129

106

54.8

71.7

125

120

63.7

70.5

B

7.07

107

96

115

149

*THF 0.5 % (v/v)

Table 9: Cytotoxicity data - 2nd Experiment with S9 mix; 4 -hour exposure period

Test groups

Cell density

(x 105/mL)

At 1stsub-culture

CE1(survival)

(4 h after treatment; about 200 cells/flask seeded)

CE2(viability)

(at the end of the expression period; about 200 cells/flask seeded)

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Cells

Flask 1

Cells

Flask 2

Cloning efficiency [%]

Abs.

Rel.

Abs.

Rel.

Vehicle control*

A

6.25

110

134

61.7

100.0

158

149

78.6

100.0

B

5.90

126

123

163

158

18.8 µg/mL

A

6.59

135

132

66.6

107.9

170

150

77.2

98.2

B

6.39

128

137

160

137

37.5 µg/mL

A

6.83

138

108

61.4

99.5

165

183

80.2

102.0

B

6.31

122

123

141

152

75.0 µg/mL

A

6.02

113

124

68.3

110.7

162

166

81.7

103.9

B

6.10

139

170

165

160

150.0 µg/mL

A

6.15

145

112

66.9

108.4

167

150

75.4

95.9

B

5.19

126

152

147

140

200.0 µg/mL

A

6.30

144

149

72.3

117.2

127

123

62.5

79.5

B

6.30

145

140

130

120

20.00 µg/mL

MCA

A

6.86

128

155

76.7

124.3

155

137

69.7

88.7

B

7.02

180

150

130

135

*THF 0.5 % (v/v)

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogeneous metabolic activation). Based on the solubility properties of the test substance and according to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment

without S9 mix (4 -hour exposure period)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

with S9 mix (4 -hour exposure period)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

2nd Experiment

without S9 mix

0; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

with S9 mix

0; 18.8; 37.5; 75.0; 150.0; 200.0 µg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix and / or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.