Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests, an HPRT assay and a chromosome aberration test were performed according GLP and OECD guideline 471, 473 and 476 to evaluate the mutagenic abd clastogenic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. Therefore, the substance is not considered to be mutagenic under the conditions of these tests.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
HGPRT
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).
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.
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.

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)

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa wall mutation, deletion of the uvrB gene, contain the R-factor plasmid, pKM101
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA DNA repair deficiency
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from aroclor induced rat liver
Test concentrations with justification for top dose:
The doses tested were 33.3,100, 333, 1000,3330, and 5000 ug per plate in both the presence and absence of S9 mix.
Vehicle / solvent:
- Vehicle: ethanol
Untreated negative controls:
yes
Remarks:
cells and medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene (TA 100, TA 1535, TA 1537, WP2uvrA, with S9 mix), ICR-191 (TA 1537, without S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

DURATION
When S9 mix was not required, 100 /µL of tester strain and 100 /µL of vehicle or test article dose were added to 2.5 mL of molten selective top agar (maintained at 45 ± 2°C). When S9 mix was required, 500 /µL of S9 mix, 100 /uL of tester strain and 100 /µL of vehicle or test article dose were added to 2.0 mL of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2°C. Positive control articles were plated using a 50 /µL plating aliquot.

NUMBER OF REPLICATIONS: in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Dose Rangefinding Assay
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay.

Design
The dose rangefinding assay was performed using tester strains TAIOO and WP2MvrA both in the presence and absence of S9 mix. Ten doses of test article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of 5 mg per plate.
Evaluation criteria:
Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows:
Tester Strains TA98, TA100, and WP2uvrA. For a test article to be considered positive, it had to produce at least a 2-foId increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated. The results of these calculations are presented in tabular form in the Data Tables section of this report. The historical control data are presented after the data tables.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: see above in study design

COMPARISON WITH HISTORICAL CONTROL DATA: yes, data were presented after data tables

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table 1: Mutagenicity assay results - Summary of mean revertants per plate with standard deviation (SD), Experiment 1

 

Dose/plate

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Background lawn

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Microsomes: rat liver vehicle control

34

3

109

3

16

4

10

2

19

1

Normal

Test article

33.3 µg

100 µg

333 µg

1000 µg

3330 µg

5000 µg

30

34

32

33

31

31

7

3

5

5

1

6

108

104

107

92

91

103

15

6

15

6

2

19

15

12

15

15

17

11

2

2

2

3

4

3

6

7

8

6

7

5

2

1

3

1

1

2

16

15

19

17

15

15

3

1

8

1

5

5

Normal

Normal

Normal

Normal / slight precipitate

Normal / moderate precipitate

Normal / moderate precipitate

Positive control

461

27

1034

218

156

16

187

28

242

26

Normal

Microsomes: none vehicle control

26

3

108

13

11

2

6

2

17

4

Normal

Test article

33.3 µg

100 µg

333 µg

1000 µg

3330 µg

5000 µg

21

18

22

19

10

15

4

4

3

4

2

7

97

105

96

88

91

72

7

10

3

5

6

2

11

11

12

11

13

12

2

2

1

1

6

1

6

8

5

7

3

4

2

2

1

3

2

2

18

20

18

18

20

15

4

6

1

8

3

7

Normal

Normal

Normal / slight precipitate

Normal / moderate precipitate

Normal / moderate precipitate

Normal / moderate precipitate

Positive control

173

13

657

60

509

6

585

12

186

19

Normal

Table 2: Mutagenicity assay results - Summary of mean revertants per plate with standard deviation (SD), Experiment 2

 

Dose/plate

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Background lawn

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Microsomes: rat liver vehicle control

35

8

141

4

15

4

7

3

19

4

Normal

Test article

33.3 µg

100 µg

333 µg

1000 µg

3330 µg

5000 µg

37

33

51

37

34

34

4

6

3

3

8

5

125

132

124

127

114

116

4

8

11

13

8

23

19

15

20

20

17

11

2

1

2

6

1

4

9

7

9

9

10

9

2

3

1

4

3

2

18

19

22

22

18

13

2

6

3

3

6

1

Normal

Normal

Normal

Normal / slight precipitate

Normal / moderate precipitate

Normal / moderate precipitate

Positive control

414

40

982

116

126

7

204

26

267

16

Normal

Microsomes: none vehicle control

18

6

102

7

12

4

8

2

19

2

Normal

Test article

33.3 µg

100 µg

333 µg

1000 µg

3330 µg

5000 µg

NC

NC

NC

NC

NC

NC

-

-

-

-

-

-

98

99

93

98

75

87

13

17

7

5

7

5

9

11

13

17

13

8

4

3

2

6

3

2

4

7

4

9

5

4

2

1

3

3

3

2

19

21

20

19

16

18

6

4

3

2

6

8

Normal

Normal

Normal / slight precipitate

Normal / moderate precipitate

Normal / moderate precipitate

Normal / moderate precipitate

Positive control

16

4

538

76

476

64

498

83

244

43

Normal

Conclusions:
The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9)
Executive summary:

The results of the dose rangefinding study were used to select the doses tested in the mutagenicity assay. The doses tested were 33.3, 100, 333,1000, 3330, and 5000 /µg per plate in both the presence and absence of S9 mix. In the initial mutagenicity assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9 mix. In the confirmatory assay, all data generated with tester strain TA98 in the presence of S9 mix, and with tester strains TA100, TA1535, TA1537, and WP2uvrA in both the presence and absence of S9 mix, were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of these tester strain/activation condition combinations. In this experiment, the mean positive control value for tester strain TA98 in the absence of S9 mix was not acceptable (did not exhibit at least a 3-fold increase over the mean vehicle control value). For this reason, the test article-treated plates were not scored and tester strain TA98 was retested in the absence of S9 mix in Experiment 21285-Dl. In Experiment 21221-Dl, the mean positive control value for tester strain TA98 in the absence of S9 mix was lower than routinely observed in this laboratory, although it did exhibit at least a 3-fold increase over the mean vehicle control for this tester strain. For this reason, tester strain TA98 was retested in the absence of S9 mix in Experiment 21221-D2. In Experiment 21221-D2, all data generated with tester strain TA98 in the absence of S9 mix were acceptable, and no positive increases in the mean number of revertants per plate were observed. All criteria for a valid study were met.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO cell line was derived from an ovarian biopsy of a female Chinese hamster. The Chinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent cell line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells were subsequently subcloned in this laboratory, and stock cultures stored in liquid nitrogen. The CHO-WBL subclone is a permanent cell line with an average cycle time of 12 to 14 hours and a modal chromosome number of 21.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from aroclor induced rat liver
Test concentrations with justification for top dose:
Initial assay
4.19, 5.98, 8.54, 12.2, 17.4, 24.8, 35.4, 50.5, 72.1, 103, 147, 210, and 300 ug/mL for 3.0 hours with and without S9 and harvested at 20.0 hours after the initiation of treatment. 72.1, 103, 147, and 210 [ig/mL without metabohc activation and 50.1, 72.1, 103, and 147 |ag/mL with metabolic activation were analyzed for chromosomal aberrations.

Confirmatory assay
6.25 - 200 ug/ml, 17.7h with/out activation, 75 - 200 ug/ml were evaluated
Vehicle / solvent:
The test article was dissolved in ethanol and treated at 1.0% (10.0 nL/mL). The vehicle control cultures were treated with 10.0 µL/mL of Ethanol.
Solubility was evaluated in water and dimethylsulfoxide, and the test article was not soluble or suspended at an acceptable level in either of these two vehicles.
Untreated negative controls:
yes
Remarks:
cells and medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- after 1 day culture initiation
- test article added: at 0h
- Exposure duration: 3h and 17.7h
- colcemid added: after 18h
- harvest started: after 20h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 (100 per culture)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- test for mycoplasma contamination
- karyotype stability
Evaluation criteria:
Assay Evaluation Criteria
The following factors are taken into account in evaluation of the test article data:
• The number and percentages of aberrant cells excluding gaps (-g).
• The number and percentages of aberrant cells including gaps (+g).
• Evidence for increasing amounts of damage with increasing dose, i.e., a dose related increase in aberrations.
The experimental unit is the cell, and therefore the percentage of cells with structural aberrations was the basis for evaluation.

A test article was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p<0.01) in the number of cells with chromosomal aberrations is observed at one or more concentrations. The linear trend test evaluated the dose responsiveness. If a significant increase is seen at one or more concentrations, a dose-response should be observed.

A test article was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.
Statistics:
Statistical analysis employed a Cochran-Armitage test for linear frend and Fisher's Exact Test (Thakur et a l , 1985) to compare the percentage of cells with aberrations in treated cells to the results obtained for the vehicle controls. Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p <0.01) increases in these events as indicators of possible induction of numerical aberrations; however, the test article was evaluated only for structural aberrations and not for numerical aberrations by this protocol.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Chromosomal Aberrations Assay Without Metabolic Activation

Initial Assay

A precipitate was observed after dosing and prior to washing in the cultures treated with 300 µg/mL, and a slight amount of precipitate was observed prior to harvest in these cultures. A precipitate was observed after dosing and prior to washing in the cultures treated with 210 µg/mL. A slight amount of precipitate was observed after dosing and prior to washing in the cultures treated with 147 µg/mL. No visible signs of toxicity were observed in any of the test cultures. Slight reductions of 15%, 9%, 3%, and 3% were observed in the mitotic indices of the cultures treated with 72.1, 103, 147, and 300 µg/mL as compared with the vehicle control cultures (Table 1). Chromosomal aberrations were analyzed from the cultures treated with 72.1, 103, 147, and 210 µg/mL (Table 2). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. Based on the results from the initial assay, the confirmatory aberrations assay was conducted testing concentrations of 6.25, 12.5, 25.0, 50.0, 75.0, 100, 150, and 200 µg/mL. Treatment

period was for 17.7 hours and cultures were harvested 20.0 hours from the initiation of treatment.

Table 1: Assessment of toxicity for chromosomal aberrations assay, 3h-treatment, 20h-harvest, -S9 mix

Treatment

Confluencea

% Solvent Control

% Mitotic Index

A culture

% Mitotic Index

B culture

Average %

Mitotic Index

% Mitotic Index

Reduction

Negative control

100

11.3

12.3

11.8

-

Vehicle control

Ethanol 10.0 µL/mL

100

12.4

8.9

10.7

0

Test article

72.1 µg/mL

103 µg/mL

147 µg/mLb,d

210 µg/mLc,e

300 µg/mLc,e,f

100

100

100

100

100

8.8

9.3

8.5

9.0

10.0

9.3

10.0

12.2

12.7

10.8

9.1

9.7

10.4

10.9

10.4

15

9

3

0

3

a This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

b Slight precipitate at dose

c Precipitate at dose

d Slight precipitate at wash

e Precipitate at wash

f Slight precipitate at harvest

Table 2: Chromosomal Aberrations, cells fixed 20 hours after treatment, 3 hour treatment, without S9

Trial I: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment
Metabolic Activation: -S9
% mitotoic index reduction # Endoreduplicated cells # Polyploid cells Judgementa Numbers and precentages (%) of cells showing structural chromosomal aberrations Judgementc
Gap Chromatid Type Chromosome Type GT Totalsb
  Dose level Cells scored g ctb cte csb cse -g +g
negative control (McCoy's 5a)   A 100   0 4               0 0  
B 100   0 4           1   1 1  
Total 200                 1   1 1  
Average % --- 0 4           0.5   0.5 0.5  
solvent control (ethanol) 10 µl/ml A 100   0 1               0 0  
B 100   0 5               0 0  
Total 200                     0 0  
Average % 0 0 3               0 0  
Positive Control (MMC) 1.5 µg/ml A 25   0 4   2 4 10     1 11 11  
B 25   0 5     4 7     5 13 13  
Total 50         2 8 17     6 24 24  
Average % --- 0 4.5 - 4 16 34     12 48 48 +
Test article 72.1 µg/ml A 100   0 2               0 0  
B 100   0 1               0 0  
Total 200                     0 0  
Average % 15 0 1.5 -             0 0 -
103 µg/ml A 100   0 2               0 0  
B 100   0 1               0 0  
Total 200                     0 0  
Average % 9 0 1.5 -             0 0 -
147 µg/ml A 100   0 1               0 0  
B 100   0 3               0 0  
Total 200                     0 0  
Average % 3 0 2 -           0 0 -
210 µg/ml A 100   0 1     1         1 1  
B 100   0 4               0 0  
Total 200           1         1 1  
Average % 0 0 2.5 -   0.5         0.5 0.5 -

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

a Significantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

b -g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

c Significantly greater in -g than the vehicle control, p ≤ 0.01

MMC = Mitomycin C

Confirmatory Assay

A slight amount of precipitate was observed after dosing and prior to washing in the cultures treated with 200 µg/mL. A slight amount of precipitate was observed prior to washing in the cultures treated with 150 µg/mL. No visible signs of toxicity were observed in any of the test cultures, except for debris in the cultures treated with 100, 150, and 200 µg/mL. Reductions of 1%, 22%, 40%, and 43% were observed in the mitotic indices of the cultures treated with 25.0, 100, 150, and 200 µg/mL as compared with the vehicle control cultures (Table 3). Chromosomal aberrations were analyzed from the cultures treated with 75.0, 100, 150, and 200 µg/mL (Table 4). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The sensitivity of the cell culture for induction of chromosomal aberrations is shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under nonactivation conditions.

Table 3: Assessment of toxicity for chromosomal aberrations assay, 17.6h-treatment, 20h-harvest, -S9 mix

Treatment

Confluencea

% Solvent Control

% Mitotic Index

A culture

% Mitotic Index

B culture

Average %

Mitotic Index

% Mitotic Index

Reduction

Negative control

100

6.5

7.7

7.1

-

Vehicle control

Ethanol 10.0 µL/mL

100

6.9

6.5

6.7

0

Test article

25.0 µg/mL

50.0 µg/mL

75.0 µg/mL

100 µg/mL

150 µg/mLc

200 µg/mLb,c

100

100

100

100

100

100

6.3

8.1

6.9

5.7

3.2

3.3

6.8

9.5

7.2

4.6

4.8

4.3

6.6

8.8

7.1

5.2

4.0

3.8

1

0

0

22

40

43

aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

bSlight precipitate at dose

cSlight precipitation at wash

Table 4: Chromosomal Aberrations, cells fixed 20 hours after treatment, 17.6 hour treatment, without S9

Trial II: Cells Fixed 20.0 Hours After Initiation of Treatment, 17.6 Hour Treatment
Metabolic Activation: -S9
% mitotoic index reduction # Endoreduplicated cells # Polyploid cells Judgementa Numbers and precentages (%) of cells showing structural chromosomal aberrations Judgementc
Gap Chromatid Type Chromosome Type GT Totalsb
  Dose level Cells scored g ctb cte csb cse -g +g
negative control (McCoy's 5a)   A 100   0 0               0 0  
B 100   0 1               0 0  
Total 200                     0 0  
Average % --- 0 0.5               0 0  
solvent control (ethanol) 10 µl/ml A 100   0 0               0 0  
B 100   0 0               0 0  
Total 200                     0 0  
Average % 0 0 0               0 0  
Positive Control (MMC) 0.4 µg/ml A 25   0 1   1 4 7 2   2 10 10  
B 25   0 2   1 4 9     1 12 12  
Total 50         2 8 16 2   3 22 22  
Average % --- 0 1.5 - 4 16 32 4   6 44 44 +
Test article 75 µg/ml A 100   0 2               0 0  
B 100   0 0               0 0  
Total 200                     0 0  
Average % 0 0 1 -             0 0 -
100 µg/ml A 100   0 4               0 0  
B 100   0 2               0 0  
Total 200                     0 0  
Average % 22 0 3 -             0 0 -
150 µg/ml A 100   0 0               0 0  
B 100   0 0     1         1 1  
Total 200           1         1 1  
Average % 40 0 0 -   0.5         0.5 0.5 -
200 µg/ml A 100   0 1               0 0  
B 100   0 0               0 0  
Total 200                     0 0  
Average % 43 0 0.5 -             0 0 -

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

aSignificantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

b-g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

cSignificantly greater in -g than the vehicle control, p ≤ 0.01

MMC = Mitomycin C

Chromosomal Aberrations Assay With Metabolic Activation

Initial Assay

A precipitate was observed after dosing and prior to harvest of the cultures treated with 210 and 300 µg/mL, and a slight amount of precipitate was observed prior to washing in these cultures. A slight amount of precipitate was observed after dosing, and prior to washing and harvest of the cultures treated with 147 µg/mL. A slight amount of precipitate was observed prior to harvest of the cultures treated with 103 µg/mL. No visible signs of toxicity were observed in any of the test cultures, except for debris in the cultures treated with 50.5, 72.1, 103, 147, 210, and 300 µg/mL. Slight reductions of 26%, 8%, and 12% were observed in the mitotic indices of the cultures treated with 50.1, 72.1, and 300 µg/mL as compared with the vehicle control cultures (Table 5). Chromosomal aberrations were analyzed from the cultures treated with 50.5, 72.1, 103, and 147 µg/mL (Table 6). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. Based on the results from the initial assay, the confirmatory aberrations assay was conducted testing concentrations of 50.0, 75.0, 100, 150, and 200 µg/mL. Treatment period was for 3.0 hours and cultures were harvested 20.0 hours from the initiation of treatment.

Table 5: Assessment of toxicity for chromosomal aberrations assay, 3h-treatment, 20h-harvest, + S9 mix

Treatment

Confluencea

% Solvent Control

% Mitotic Index

A culture

% Mitotic Index

B culture

Average %

Mitotic Index

% Mitotic Index

Reduction

Negative control

100

11.3

12.8

12.1

-

Vehicle control

Ethanol 10.0 µL/mL

100

12.5

14.1

13.3

0

Test article

50.5 µg/mL

72.1 µg/mL

103 µg/mLe

147 µg/mLb,d,e

210 µg/mLc,d,f

300 µg/mLc,d,f

100

100

100

100

100

100

10.0

11.6

15.2

14.1

12.9

11.8

9.7

12.9

11.7

14.5

14.6

11.6

9.9

12.3

13.5

14.3

13.8

11.7

26

8

0

0

0

12

aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

bSlight precipitate at dose

cPrecipitate at dose

dSlight precipitate at wash

eSlight precipitate at harvest

fPrecipitate at harvest

Table 6: Chromosomal Aberrations, cells fixed 20 hours after treatment, 3.0 hour treatment, with S9

Trial I: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment
Metabolic Activation: +S9
% mitotoic index reduction # Endoreduplicated cells # Polyploid cells Judgementa Numbers and precentages (%) of cells showing structural chromosomal aberrations Judgementc
Gap Chromatid Type Chromosome Type GT Totalsb
  Dose level Cells scored g ctb cte csb cse -g +g
negative control (McCoy's 5a)   A 100   0 0   1           0 1  
B 100   1 2           2   2 2  
Total 200         1       2   2 3  
Average % --- 0.5 1   0.5       1   1 1.5  
solvent control (ethanol) 10 µl/ml A 100   0 0       1       1 1  
B 100   0 1               0 0  
Total 200             1       1 1  
Average % 0 0 0.5       0.5       0.5 0.5  
Positive Control (CP) 5.0 µg/ml A 25   0 4     2 8   2   9 9  
B 25   0 2   2 1 6   1   7 8  
Total 50         2 3 14   3   16 17  
Average % --- 0 3 - 4 6 28   6   32 34 +
Test article 50.5 µg/ml A 100   0 2               0 0  
B 100   0 1           1   1 1  
Total 200                 1   1 1  
Average % 26 0 1.5 -         0.5   0.5 0.5 -
72.1 µg/ml A 100   0 2   1       1   1 2  
B 100   0 2               0 0  
Total 200         1       1   1 2  
Average % 8 0 2 - 0.5       0.5   0.5 1 -
103 µg/ml A 100   0 0           1   1 1  
B 100   0 3               0 0  
Total 200                 1   1 1  
Average % 0 0 1.5 -       0.5   0.5 0.5 -
147 µg/ml A 100   0 0   1           0 1  
B 100   1 0               0 0  
Total 200         1           0 1  
Average % 0 0.5 0 - 0.5           0 0.5 -

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

aSignificantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

b-g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

cSignificantly greater in -g than the vehicle control, p ≤ 0.01

CP = Cyclophosphamide

Confirmatory assay

A precipitate was observed after dosing and prior to washing of the cultures treated with 200 µg/mL, and a slight amount of precipitate was observed prior to harvest of these cultures. A precipitate was observed after dosing and prior to washing of the cultures treated with 150 µg/mL. A slight amount of precipitate was observed after dosing and prior to washing of the cultures treated with 100 µg/mL. No visible signs of toxicity were observed in any of the test cultures, except for debris in the cultures treated with 150 and 200 µg/mL. A slight reduction of 4% was observed in the mitotic indices of the cultures treated with 200 µg/mL as compared with the vehicle control cultures (Table 7). Chromosomal aberrations were analyzed from the cultures treated with 50.0, 75.0, 100, and 150 µg/mL (Table 8). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test article was considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under activation conditions.

Table 7: Assessment of toxicity for chromosomal aberrations assay, 3h-treatment, 20h- harvest, + S9 mix

Treatment

Confluencea

% Solvent Control

% Mitotic Index

A culture

% Mitotic Index

B Culture

Average %

Mitotic Index

% Mitotic Index

Reduction

Negative control

100

9.0

10.2

9.6

-

Vehicle control

Ethanol 10.0 µL/mL

100

8.3

9.9

9.1

0

Test article

50.0 µg/mL

75.0 µg/mL

100 µg/mLb,d

150 µg/mLc,e

200 µg/mLc,e,f

100

100

100

100

100

9.8

10.5

12.1

10.8

9.2

8.5

10.4

9.0

8.2

8.1

9.2

10.5

10.6

9.5

8.7

0

0

0

0

4

aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

bSlight precipitate at dose

cPrecipitate at dose

dSlight precipitate at wash

ePrecipitate at wash

fSlight Precipitate at harvest

Table 8: Chromosomal Aberrations, cells fixed 20 hours after treatment, 3.0 hour treatment, with S9

Trial II: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment
Metabolic Activation: +S9
% mitotoic index reduction # Endoreduplicated cells # Polyploid cells Judgementa Numbers and precentages (%) of cells showing structural chromosomal aberrations Judgementc
Gap Chromatid Type Chromosome Type GT Totalsb
  Dose level Cells scored g ctb cte csb cse -g +g
negative control (McCoy's 5a)   A 100   0 0               0 0  
B 100   0 0               0 0  
Total 200                     0 0  
Average % --- 0 0               0 0  
solvent control (ethanol) 10 µl/ml A 100   0 2               0 0  
B 100   0 1               0 0  
Total 200                     0 0  
Average % 0 0 1.5               0 0  
Positive Control (CP) 5.0 µg/ml A 25   1 2     1 12 1 1   13 13  
B 25   1 2     1 10 3     10 10  
Total 50           2 22 4 1   23 23  
Average % --- 1 2 -   4 44 8 2   46 46 +
Test article 50.0 µg/ml A 100   0 2               0 0  
B 100   0 3               0 0  
Total 200                     0 0  
Average % 0 0 2.5 -             0 0 -
75.0 µg/ml A 100   0 0               0 0  
B 100   0 2         1 1   2 2  
Total 200               1 1   2 2  
Average % 0 0 1 -       0.5 0.5   1 1 -
100 µg/ml A 100   0 0               0 0  
B 100   0 0               0 0  
Total 200                     0 0  
Average % 0 0 0 -           0 0 -
150 µg/ml A 100   0 0           1   1 1  
B 100   0 0               0 0  
Total 200                 1   1 1  
Average % 0 0 0 -         0.5   0.5 0.5 -

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

aSignificantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

b-g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

cSignificantly greater in -g than the vehicle control, p ≤ 0.01

CP = Cyclophosphamide

Conclusions:
The test substance was considered to be negative for inducing chromosomal aberrations in CHO cells with and without metabolic activation.
Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation.

The test article was dissolved in ethanol and treated at 1.0 % (10.0 µL/mL). A stock solution of the test substance was prepared at a concentration of 30.0 mg/mL for the assay. The vehicle control cultures were treated with 10.0 µL/mL of Ethanol. The high dose tested, 300 µg/mL, was above the solubility limit in the test system.

In the initial trial of the chromosome aberrations assay, replicate cultures of CHO cells were incubated with 4.19, 5.98, 8.54, 12.2, 17.4, 24.8, 35.4, 50.5, 72.1, 103, 147, 210, and 300 µg/mL for 3.0 hours with and without S9 and harvested at 20.0 hours after the initiation of treatment. Cultures treated with 72.1, 103, 147, and 210 µg/mL without metabolic activation and 50.1, 72.1, 103, and 147 µg/mL with metabolic activation were analyzed for chromosomal aberrations. No increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

In the confirmatory trial, replicate cultures of CHO cells were incubated with 5.25, 12.5, 25.0, 50.0, 75.0, 100, 150, and 200 µg/mL without S9 and 50.0, 75.0, 100, and 150 µg/mL with S9. Treatment periods of 17.7 and 3.0 hours were used without and with S9, respectively, and cultures harvested at 20.0 hours after the initiation of treatment. Cultures treated with 75.0, 100, 150, and 200 µg/mL without S9 and 50.0, 75.0, 100, and 150 µg/mL with S9 were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

The test substance was considered negative for inducing chromocomal aberrations in CHO cells with and without metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test

An Ames test was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100, TA 1538 and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without S9 mix from aroclor induced rat liver and non-induced Syrian hamster liver. The test item was tested at concentrations of 5000, 2500, 1250, 625 and 312.5 µg/plate. No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation.  Another GLP-conform Ames test according OECD guideline 471 was performed. The tester strains used in this mutagenicity assay were S. typhimurium tester strains TA98, TA100, TA1535, TA1537, and E. coli tester strain WP2uvrA. The assay was conducted with seven doses of test article, dissolved in acetone, in the presence of S9 mix and nine doses of test article in the absence of S9 mix along with concurrent vehicle and positive controls. The doses tested were 5000, 3330, 1000, 333, 100, 33.3, and 10.0 /µg per plate in the presence of S9 mix and 5000, 3330, 1000, 333, 100, 33.3, 10.0, 3.33, and 1.00 /µg per plate in the absence of S9 mix. The test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of metabolic activation.

Chromosome Aberration

In order to evaluate the ability of the test substance to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation a chromosomal aberration test was performed. The test article was dissolved in ethanol and treated at 1.0% (10.0 µl/ml). The high dose tested, 300 µg/ml, was above the solubility limit in the test system. In the initial trial of the chromosome aberrations assay, replicate cultures of CHO cells were incubated with 4.19 to 300 µg/ml for 3.0 hours with and without S9 and harvested at 20.0 hours after the initiation of treatment. In the confirmatory trial, replicate cultures of CHO cells were incubated with 6.25 to 200 µg/ml without S9 and 50.0 to 200 µg/ml with S9. Treatment periods of 17.7 and 3.0 hours were used without and with S9, respectively, and cultures harvested at 20.0 hours after the initiation of treatment. No increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

HPRT

The test material 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. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses from 3 -200 µg/ml were tested in presence and absence of a metabolic activation system. 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

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.