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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from J-check, 2017

Data source

Reference
Reference Type:
other: J-check
Title:
Reverse Mutation Test of chlorocyclohexane on Bacteria
Author:
National Institute of Technology and Evaluation
Year:
2017
Bibliographic source:
Japan Chemicals Collaborative Knowledge Database

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Bacterial reverse mutation test was performed to determine the mutagenic nature of Chlorocyclohexane
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Chlorocyclohexane
- Molecular formula: C6H11Cl
- Molecular weight: 118.606 g/mol
- Substance type: Organic
Specific details on test material used for the study:
- Name of test material: Chlorocyclohexane
- Molecular formula: C6H11Cl
- Molecular weight: 118.606 g/mol
- Substance type: Organic
- Physical state: Slightly pale yellow from colorless liquid
- Purity: 99.7%
- Impurities (identity and concentrations): 0.3%

Method

Target gene:
Histidine for Salmonella typhimurium and tryptophan for E. coli
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
SD male rat liver, induced by phenobarbital and 5,6-benzoflavone
Species / strain:
E. coli WP2 uvr A pKM 101
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
SD male rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA1535 strains),
9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA98, TA1537, WP2uvrA/pKM101 strains)

+S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100, TA1535, TA98, TA1537 strains),
9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (WP2uvrA/pKM101 strain)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Negative controls:
not specified
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA 100, TA98 and WP2 uvrA/pKM101); +S9 mix: 2-aminoanthracene (all strains)
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
In any strain(s) tested with or without S9 mix, when the mean number of revertant colonies per plate increased twice more than that of the negative control and when the increase was shown to be dose-related and reproducible, the chemical was judged mutagenic.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 1535, TA 1537, TA 98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic effect were observed.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic effect were observed.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Concentration: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 μg/plate with and without S9

COMPARISON WITH HISTORICAL CONTROL DATA: No data

Any other information on results incl. tables

 Table: Mutagenic nature of Chlorocyclohexane

Metabolic activation

Dose (μg/plate)

TA100

TA1535

Wp2uvrA

TA98

TA1537

S9 mix (-)

DMSO

123

15

65

17

16

1.22

118

16

69

26

21

4.88

111

15

82

16

16

19.5

100

19

64

24

22

78.1

94*

16*

68

18

18

313

0*

0*

59*

20*

8*

1250

0*

0*

0*

0*

0*

5000

0*

0*

0*

0*

0*

 

 

 

S9 (+)

DMSO

133

16

104

24

19

1.22

117

10

104

24

25

4.88

107

14

98

24

16

19.5

112

16

92

31

25

78.1

88

17

84

31

22

313

90*

7*

77*

24*

12*

1250

0*

0*

56*

0*

0*

5000

0*

0*

0*

0*

0*

S9 (-)

 

AF-2

NaN3

AF-2

AF-2

9-AA

 

Dose

0.01

0.5

0.0005

0.1

80

 

Revertants/plate

623

560

640

837

389

S9 (+)

 

2-AA

2-AA

2-AA

2-AA

2-AA

Dose

1

2

2

0.5

2

Revertants/plate

959

212

600

423

154

 

Metabolic activation

Dose (μg/plate)

TA100

TA1535

Wp2uvrA

TA98

Ta1537

S9 mix (-)

DMSO

103

91

100

9

12

13

94

86

85

22

16

22

11

13

13

1.22

108

102

92

6

9

8

-

-

-

4.88

129

93

88

11

10

11

-

-

-

9.77

116

96

95

6

11

7

84

74

98

28

22

28

11

11

15

19.5

92

95

94

8

9

9

117

106

93

17

24

16

12

8

17

39.1

94

106

104

11

6

8

109

88

84

17

16

23

13

14

12

78.1

108*

96*

111*

13*

10*

7*

102

87

81

22

22

24

16

13

13

156

112*

94*

107*

9*

10*

8*

101

93

70

17

29

25

18

16

16

313

-

-

59*

79*

51*

17*

15*

22*

9*

10*

14*

 

 

 

S9 (+)

DMSO

96

103

113

10

10

13

116

130

103

24

25

30

15

13

11

9.77

81

115

133

13

8

11

108

117

108

25

18

29

13

14

15

19.5

108

95

108

9

8

6

115

110

108

19

25

27

23

19

11

39.1

103

120

120

7

7

14

100

100

93

26

26

30

19

22

17

78.1

109

116

98

11

14

9

117

110

105

29

29

22

19

21

11

156

119*

112*

95*

8*

11*

11*

93

109

108

28

24

24

19

15

15

313

106*

87*

100*

8*

6*

3*

81*

114*

76*

22*

24*

27*

15*

10*

15*

625

-

-

49*

50*

69*

-

-

S9 (-)

 

AF-2

NaN3

AF-2

AF-2

9-AA

Dose

0.01

0.5

0.0005

0.1

80

Revertants/plate

692

587

644

570

635

630

1226

1219

1038

907

894

986

444

381

452

S9 (+)

 

2-AA

2-AA

2-AA

2-AA

2-AA

Dose

1

2

2

0.5

2

Revertants/plate

1446

1280

1324

230

193

222

880

674

604

441

461

396

205

226

175

 

Metabolic activation

Dose (μg/plate)

TA100

TA1535

Wp2uvrA

TA98

Ta1537

S9 mix (-)

DMSO

95

113

105

10

8

10

62

75

75

19

15

13

18

13

9

1.22

103

97

102

10

10

10

-

-

-

4.88

112

102

94

14

11

10

-

-

-

9.77

110

104

114

8

9

9

88

59

76

18

23

19

16

9

8

19.5

113

104

108

8

10

14

75

86

73

19

18

13

9

11

13

39.1

110

117

99

14

9

14

67

78

74

19

18

16

10

12

19

78.1

85*

86*

82*

7*

13*

6*

84

73

80

24

21

16

9

15

13

156

85*

88*

87*

7*

7*

9*

80

61

72

15

18

15

10

11

15

313

-

-

68*

78*

59*

13*

17*

3*

9*

7*

10*

 

 

 

S9 (+)

DMSO

101

111

121

13

14

11

83

110

114

25

18

25

19

17

17

9.77

111

115

117

9

15

13

87

9

101

22

23

32

17

17

12

19.5

123

111

118

9

9

16

99

89

92

24

24

23

17

16

15

39.1

114

129

101

9

15

11

90

115

84

23

20

32

15

18

18

78.1

129

126

121

11

15

15

93

84

82

18

25

26

19

19

11

156

119*

110*

110*

11*

11*

9*

93

108

102

27

22

24

22

13

16

313

86*

91*

84*

7*

13*

8*

99*

97*

8*

24*

24*

24*

11*

15*

18*

625

-

-

58*

68*

44*

-

-

S9 (-)

 

AF-2

NaN3

AF-2

AF-2

9-AA

Dose

0.01

0.5

0.0005

0.1

80

Revertants/plate

688

672

726

554

597

568

1230

976

853

984

805

767

328

310

350

S9 (+)

 

2-AA

2-AA

2-AA

2-AA

2-AA

Dose

1

2

2

0.5

2

Revertants/plate

1669

1290

1398

200

248

293

876

714

667

379

372

430

188

189

190

 

 

Applicant's summary and conclusion

Conclusions:
Chlorocyclohexane did not induce gene mutation in S. typhimurium TA 1535, TA 1537, TA 98, TA100 and E. coli WP2 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Bacterial reverse mutation test was performed to determine the mutagenic nature of Chlorocyclohexane. The study was performed as per the preincubation protocol using S. typhimurium TA 1535, TA 1537, TA 98, TA100 and E. coli WP2 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA1535 strains), 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA98, TA1537, WP2uvrA/pKM101 strains) without S9 and 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100, TA1535, TA98, TA1537 strains), 9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (WP2uvrA/pKM101 strain) with S9. The plates were preincubated for 20 mins at 37°C and the exposure duration was 48 hrs. The plates were observed for dose-related and reproducible doubling of the mean number of revertant colonies per plate than that of the negative control. Chlorocyclohexane did not induce gene mutation in S. typhimurium TA 1535, TA 1537, TA 98, TA100 and E. coli WP2 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.