Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2000

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2006

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2001

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-02-2000 to 21-02-2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable deviations.
Remarks:
The efficacy of the S9-mix was only validated using 2-Anthramine (2-Aminoanthracene CAS 613-13-8) and benzopyrene or dimethylbenzanthracene was not used in each test. This is reflected in the reliability score.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The efficacy of the S9-mix was only validated using 2-Anthramine (2-Aminoanthracene CAS 613-13-8) and benzopyrene or dimethylbenzanthracene was not used in each test. This is reflected in the reliability score.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 09 December 1986
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
see above
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
see above
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 1999 ; signature: December 1999
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test (TA98, TA100 and Wp2urvA): 0, 10, 100, 500, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment 1 (plate incorporation method): All strains: 0, 312.5, 625, 1250, 2500, 5000 µg/plate with and without S9 mix
Experiment 2 (preincubation method): All strains: 0, 312.5, 625, 1250, 2500, 5000 µg/plate with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (batch number and supplier listed in full study report).
- Justification for choice of solvent/vehicle: Ethanol was selected as the vehicle. Test item was dissolved in the vehicle at a concentration of 100 mg/ml for the preliminary toxicity test and for both mutagenicity experiments. The preparations were made immediately before use.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preliminary Toxicity Test: in medium; in agar (plate incorporation), Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Preliminary Toxicity Test: To assess the toxicity of the test substance to the bacteria, at least six dose-levels (one plate/dose-level) will be tested in the TA98, TA100 and WP2uvrA strains, with and without S9 mix. Test item solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
Experiment 1. Test item solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
Experiment 2. Test item solution (0.05 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours incubation at 37 ºC the revertants were scored using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Evidence leading to positive result:
1. Reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level
2. Evidence of dose-relationship, in any strain
Reference to historical data and/or other considerations such as biological relevance would be taken into consideration during the evaluation of data

Applicant assessment indicates: The test item is considered non-mutagenic (negative) in the test system if the above criteria are not met. Instances of data prohibiting definitive judgement about test item activity may be reported as equivocal.

Acceptance Criteria:
The study is considered valid if the following criteria are met:
1. The number of revertants in the vehicle controls is consistent with the historical control data range (information attached to the full study report)
2. The number of revertants in the positive control is higher than the vehicle controls and consistent with the historical control range (information attached to the full study report).
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
See table 1 and 2, without S9 mix activation, slight to moderate cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
See table 1 and 2, without S9 mix activation, slight to moderate cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Table 1 : Test Results: Experiment 1 – Preliminary Toxicity Test : with and without metabolic activation and results of concurrent positive controls

Strains

Doses µg/plate

Without S9 Mix

With S9 Mix

 

 

T

E

Revertants per plate

T

E

Revertants per plate

TA98

0

0

0

24

0

0

29

 

10

0

0

21

0

0

24

 

100

0

0

19

0

0

23

 

500

0

0

14

0

0

17

 

1000

0

0

19

0

0

38

 

2500

0

1

23

0

1

20

 

5000

0

2

10

0

1

20

 

 

 

 

 

 

 

 

TA100

0

0

0

82

0

0

95

 

10

0

0

71

0

0

104

 

100

0

0

61

0

0

84

 

500

0

0

43

0

0

83

 

1000

1

0

55

0

0

82

 

2500

1

1

65

0

1

90

 

5000

1

1

38

0

1

90

 

 

 

 

 

 

 

 

WP2 uvrA

0

0

0

29

0

0

40

 

10

0

0

18

0

0

40

 

100

0

0

22

0

0

30

 

500

0

0

31

0

0

30

 

1000

0

0

25

0

0

22

 

2500

0

1

28

0

1

31

 

5000

0

1

21

0

1

34

 

 

 

 

 

 

 

 

0 = vehicle control (Ethanol)

Where:

T = toxicity : score = 0, none, 1 = slight, 2 = moderate, 3 = considerable to total

E = Emulsion of test item: score = 0, none, 1 = slight, 2 = moderate, 3 = strong

 

Table 2 : Test Results: Experiment 2 – Main Test (pre-incubation method) : with and without metabolic activation and results of concurrent positive controls

With S9 Mix

Strains

Doses µg/plate

T

E

Revertants per plate

Mean

Standard Deviation

 

 

 

 

1

2

3

 

 

TA1535

0

0

0

29

30

26

28

2

 

312.5

0

0

25

13

30

23

9

 

625

0

0

23

24

27

25

2

 

1250

0

0

21

13

41

25

14

 

2500

0

0

20

31

25

25

6

 

5000

0

1

27

29

30

29

2

 

2AM(2)

-

-

258

232

226

239

17

 

 

 

 

 

 

 

 

 

TA1537

0

0

0

6

10

7

8

2

 

312.5

0

0

8

4

8

7

2

 

625

0

0

13

7

7

9

3

 

1250

0

0

4

9

4

6

3

 

2500

0

0

15

8

7

10

4

 

5000

0

1

6

3

5

5

2

 

2AM(2)

-

-

81

75

98

85

12

 

 

 

 

 

 

 

 

 

TA98

0

0

0

19

12

21

17

5

 

312.5

0

0

14

15

12

14

2

 

625

0

0

28

13

16

19

8

 

1250

0

0

21

16

17

18

3

 

2500

0

0

28

13

14

18

8

 

5000

0

1

24

12

18

18

6

 

2AM(2)

-

-

1362

1317

1383

1354

34

 

 

 

 

 

 

 

 

 

TA100

0

0

0

139

132

114

128

13

 

312.5

0

0

92

81

93

89

7

 

625

0

0

93

111

91

98

11

 

1250

0

0

76

95

72

81

12

 

2500

0

0

80

74

77

77

3

 

5000

0

1

65

72

64

67

4

 

2AM(2)

-

-

1087

992

921

1000

83

 

 

 

 

 

 

 

 

 

WP2 uvrA

0

0

0

55

44

37

45

9

 

312.5

0

0

46

35

32

38

7

 

625

0

0

50

40

34

41

8

 

1250

0

0

39

26

44

36

9

 

2500

0

0

49

42

46

46

4

 

5000

0

1

39

32

38

36

4

 

2AM(10)

-

-

233

170

179

194

34

 

 

 

 

 

 

 

 

 

Without S9 Mix

TA1535

0

0

0

6

10

12

9

3

 

312.5

0

0

5

5

5

5

0

 

625

0

0

7

8

6

7

1

 

1250

0

0

7

7

5

6

1

 

2500

0

0

13

7

7

9

3

 

5000

0

1

7

10

8

8

2

 

NaN3(1)

-

-

646

616

703

655

44

 

 

 

 

 

 

 

 

 

TA1537

0

0

0

9

14

13

12

3

 

312.5

1

0

5

5

5

5

0

 

625

1

0

8

10

6

8

2

 

1250

2

0

11

7

8

9

2

 

2500

2

0

11

7

7

8

2

 

5000

2

1

9

6

5

7

2

 

9AA(50)

-

-

332

287

435

351

76

 

 

 

 

 

 

 

 

 

TA98

0

0

0

17

16

15

16

1

 

312.5

0

0

11

13

10

11

2

 

625

0

0

18

10

14

14

4

 

1250

1

0

12

17

17

15

3

 

2500

1

0

22

14

18

18

4

 

5000

2

1

15

20

10

15

5

 

2NF(0.5)

-

-

150

164

147

154

9

 

 

 

 

 

 

 

 

 

TA100

0

0

0

107

97

93

99

7

 

312.5

0

0

94

80

81

85

8

 

625

0

0

124

102

104

110

12

 

1250

0

0

83

77

88

83

6

 

2500

0

0

119

90

96

102

15

 

5000

0

1

106

96

76

93

15

 

NaN3(1)

-

-

793

771

773

779

12

 

 

 

 

 

 

 

 

 

WP2 uvrA

0

0

0

30

20

34

28

7

 

312.5

0

0

27

24

28

26

2

 

625

0

0

31

40

32

34

5

 

1250

0

0

35

27

28

30

4

 

2500

0

0

40

29

27

32

7

 

5000

0

1

27

25

23

25

2

 

4NQQ(2)

-

-

1761

1781

1791

1778

15

 

 

 

 

 

 

 

 

 

0 = vehicle control (Ethanol)

Where:

T = toxicity : score = 0, none, 1 = slight, 2 = moderate, 3 = considerable to total

E = Emulsion of test item: score = 0, none, 1 = slight, 2 = moderate, 3 = strong

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

The study was performed to the requirements of OECD Guideline 471, Japanese guidelines for bacterial mutagenicity testing and EU Method B.14, under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix activation system. The test strains were treated in two independent experiments with the test item, utilising the Ames plate incorporation method and then preincubation method, respectively at up to five dose levels, in triplicate, both with and without the addition of a rat liver microsomal metabolic activation system (10% liver S9). The vehicle was ethanol. The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The Experiment 1 dose levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated using the same dose range by the pre-incubation method in Experiment 2. All of the positive controls used in the test induced expected increases in the frequency of revertant colonies, both with or without metabolic activation. The vehicle controls were considered acceptable. Therefore the assay was considered valid. The maximum dose level of the test item in the Experiment 1 and Experiment 2 was the maximum recommended dose level of 5000 μg/plate in all tester strains. A slight emulsion was observed in the plates when scoring the revertants mainly at the highest dose-level. Without S9 mix, a slight to moderate toxicity was generally induced, depending on the dose-levels and the tester strains. With S9 mix, except for a slight decrease in the number of revertants, noted in the second experiment (preincubation method) in the TA1537 and TA100 strains at the highest dose-level, no noteworthy toxicity was induced in both experiments. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-07-2006 to 29-08-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: August 2005 ; signature: November 2005
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test (TA100 and Wp2urvA): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method): All strains: 0, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (plate incorporation method): All strains: 0, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and DMSO at 50 mg/mL but was fully miscible in acetone at the same concentration in solubility checks performed. Acetone was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Experiment 2. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
Statistics:
Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
cytotoxicity
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Table 1 : Test Results: Experiment 1 – Range finding Test : with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

82

77

86

(82)

4.5#

8

12

19

(13)

5.6

20

23

24

(22)

2.1

20

24

20

(21)

2.3

9

13

16

(13)

3.5

50 µg

78

62

84

(75)

11.4

16

10

19

(15)

4.6

35

30

24

(30)

5.5

15

40

19

(25)

13.4

3

5

7

(5)

2.0

150 µg

69

85

85

(80)

9.2

10

13

22

(15)

6.2

40

20

27

(29)

10.1

19

15

18

(17)

2.1

5

15

15

(12)

5.8

500 µg

70

85

78

(78)

7.5

16

11

16

(14)

2.9

25

26

33

(28)

4.4

19

20

26

(22)

3.8

11

7

10

(9)

2.1

1500 µg

75

78

87

(80)

6.2

16

10

18

(15)

4.2

21

34

33

(29)

7.2

24

26

22

(24)

2.0

19

14

7

(13)

6.0

5000 µg

86 P

87 P

74 P

(82)

7.2

23 P

9 P

12 P

(15)

7.4

22 P

27 P

26 P

(25)

2.6

19 P

20 P

25 P

(21)

3.2

11 P

11 P

7 P

(10)

2.3

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

776

746

647

(723)

67.5

284

316

308

(303)

16.7

1209

1261

1215

(1228)

28.4

263

262

252

(259)

6.1

885

936

1166

(996)

149.7

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

76

104

89

(90)

14.0#

14

7

13

(11)

3.8

36

32

46

(38)

7.2

29

15

21

(22)

7.0

9

13

13

(12)

2.3

50 µg

93

77

100

(90)

11.8

10

8

8

(90)

1.2

33

29

37

(33)

4.0

31

15

21

(22)

8.1

12

8

11

(10)

2.1

150 µg

87

98

90

(92)

5.7

8

7

20

(12)

7.2

38

27

44

(36)

8.6

25

21

13

(20)

6.1

13

10

11

(11)

1.5

500 µg

82

68

58

(69)

12.1

10

13

4

(9)

4.6

27

31

37

(32)

5.0

14

20

29

(21)

7.5

13

11

7

(10)

3.1

1500 µg

60

70

66

(65)

5.0

14

9

8

(10)

3.2

27

42

36

(35)

7.5

12

35

24

(24)

11.5

5

15

8

(9)

5.1

5000 µg

80 P

78 P

74 P

(77)

3.1

12 P

5 P

13 P

(10)

4.4

25 P

38 P

31 P

(31)

6.5

23 P

30 P

24 P

(26)

3.8

1 P

9 P

5 P

(5)

4.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1314

1482

1435

(1410)

86.7

275

287

269

(277)

9.2

331

354

222

(302)

70.5

293

301

267

(287)

17.8

388

376

393

(386)

8.7

 

Table 2 : Test Results: Experiment 2 – Main Test : with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

114

113

97

(108)

9.5#

20

29

20

(23)

5.2

29

26

24

(26)

2.5

16

23

25

(21)

4.7

21

12

14

(16)

4.7

50 µg

89

106

118

(104)

14.6

12

12

15

(13)

1.7

21

19

23

(21)

2.0

31

16

26

(24)

7.6

10

13

15

(13)

2.5

150 µg

107

101

119

(109)

9.2

20

13

14

(16)

3.8

19

20

12

(17)

4.4

12

15

23

(17)

5.7

13

8

13

(11)

2.9

500 µg

95

128

107

(110)

16.7

34

29

16

(26)

9.3

40

24

32

(32)

8.0

23

13

22

(19)

5.5

13

11

9

(11)

2.0

1500 µg

104

133

114

(117)

14.7

22

11

42

(25)

15.7

16

12

19

(16)

3.5

15

19

26

(20)

5.6

20

10

15

(15)

5.0

5000 µg

99 P

96 P

118 P

(104)

11.9

35 P

23 P

23 P

(27)

6.9

24 P

29 P

35 P

(29)

5.5

22 P

24 P

16 P

(21)

4.2

14 P

13 P

12 P

(13)

1.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

456

412

659

(509)

131.8

152

152

159

(154)

4.0

653

565

628

(615)

45.3

158

152

170

(160)

9.2

1464

1056

1204

(1241)

206.5

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

70

77

82

(86)

7.8#

10

18

21

(16)

5.7

32

27

38

(32)

5.5

21

25

26

(24)

2.6

20

21

19

(20)

1.0

50 µg

77

81

78

(92)

7.0

16

13

9

(13)

3.5

41

32

34

(36)

4.7

25

29

15

(23)

7.2

15

18

23

(19)

4.0

150 µg

79

76

74

(96)

4.6

10

16

13

(13)

3.0

23

46

33

(34)

3.0

29

24

30

(28)

3.2

25

20

18

(21)

3.6

500 µg

84

79

79

(82)

1.7

15

7

14

(12)

4.4

33

30

36

(33)

3.0

41

22

27

(30)

9.8

26

20

15

(20)

5.5

1500 µg

73

64

76

(65)

4.6

9

11

9

(10)

1.2

37

38

32

(36)

3.2

37

24

25

(29)

7.2

12

14

18

(15)

3.1

5000 µg

75 P

68 P

78 P

(39)

4.0

16 P

15 P

7 P

(13)

4.9

24 P

30 P

45 P

(33)

10.8

30 P

41 P

25 P

(32)

8.2

21 P

16 P

21 P

(19)

2.9

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1116

1164

1211

(1164)

47.5

174

184

186

(181)

6.4

467

446

536

(483)

47.1

181

186

89

(152)

54.6

330

370

291

(330)

39.5

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

S: partial absence of bacterial background lawn

P : precipitate

#: Standard deviation

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

The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 range finding test was predetermined based on the results of a preliminary toxicity assay. The dose levels were 50 to 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on absence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. A item item (oily in appearance) precipitate was observed on the plates at 5000 μg/plate. This did not prevent scoring of the revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-01-2001 to 06-08-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2000 ; signature: April 2000
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable (chromosome aberration test)
Species / strain / cell type:
lymphocytes: Human lymphocytes
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a screened volunteer. Who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 17 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction: PB/βNF S9 23/09/00
Test concentrations with justification for top dose:
The maximum dose level was 2564 µg/mL or 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 2564 μg/mL range (full results recorded in the full study report).

I. Preliminary toxicity test: 0 (control) , 10.02, 20.04, 40.07, 80.13, 160.25, 320.5, 641, 1282 and 2564 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2% or 1% for PT and Exp 1 or Exp 2, respectively), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

II. Main Test:
Experiment 1:
4(20)-hour without S9: 0*, 5, 10*, 20*, 40*, 60, 80, MMC 0.4* μg/mL
4(20)-hour with S9: 0*, 10*, 20*, 40*, 60, 80, 120, CP 12.5* μg/mL
Experiment 2:
4(20)-hour with S9: 0*, 10.02, 20.04*, 40.07*, 80.13*, 120.19, 160.25, CP 12.5* μg/mL
24-hour without S9: 0*, 5.01, 10.02, 20.04*, 40.07*, 60.10*, 80.13, MMC 0.2* μg/mL
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was soluble in acetone at 2564 µg/mL, in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 2564 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 2564 μg/mL range (full results recorded in the full study report).
Untreated negative controls:
other: Vehicle control served as the negative control
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Full details on the positive controls is reported in the full study report.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 15% (FCS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood

DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL (100 μL) of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 10% or 20% S9-mix (i.e. 1% or 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment 1 or Main Experiment 2, respectively. After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.

II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL (100 μL) of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration (200 per duplicate) were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976), UK EMS (1983). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Evaluation criteria:
The test was evaluated in accordance with the OECD TG 473 guidelines.

Statistical analysis is also performed. The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: here was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: a precipitate was observed in the blood-free cultures at the end of exposure, at or above 160.25 µg/mL in all the 4(20)-exposure groups and 320.5 µg/mL in the 24-hour continuous exposure group. Main test: No precipitate observed.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 2564 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).

ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The study was performed to the requirements of OECD TG 473 and EU Method B.10 guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. Secondly, 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with S9-Mix (1%):0, 10, 20, 40, 60, 80, 120 μg/mLand without S9-Mix: 0, 10, 20, 40, 60, 80, 120μg/mL. The doses in the second Main Test was as follows respectively for 4(20)-hour with S9-Mix (2%):0, 10.02, 20.04, 40.07, 80.13, 120.19, 160.25 μg/mLand 24-hour without S9:0, 5.01, 10.02, 20.04, 40.07, 60.10, 80.13 μg/mL. All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour or 24-hour exposure groups in the presence or absence of S9 activation system which included a dose level that induced mitotic inhibition. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study: OECD TG 471, 2000 : The study was performed to the requirements of OECD Guideline 471, Japanese guidelines for bacterial mutagenicity testing and EU Method B.14, under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix activation system. The test strains were treated in two independent experiments with the test item, utilising the Ames plate incorporation method and then preincubation method, respectively at up to five dose levels, in triplicate, both with and without the addition of a rat liver microsomal metabolic activation system (10% liver S9). The vehicle was ethanol. The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The Experiment 1 dose levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated using the same dose range by the pre-incubation method in Experiment 2. All of the positive controls used in the test induced expected increases in the frequency of revertant colonies, both with or without metabolic activation. The vehicle controls were considered acceptable. Therefore the assay was considered valid. The maximum dose level of the test item in the Experiment 1 and Experiment 2 was the maximum recommended dose level of 5000 μg/plate in all tester strains. A slight emulsion was observed in the plates when scoring the revertants mainly at the highest dose-level. Without S9 mix, a slight to moderate toxicity was generally induced, depending on the dose-levels and the tester strains. With S9 mix, except for a slight decrease in the number of revertants, noted in the second experiment (preincubation method) in the TA1537 and TA100 strains at the highest dose-level, no noteworthy toxicity was induced in both experiments. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Key study: OECD TG 471, 2006 : The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 range finding test was predetermined based on the results of a preliminary toxicity assay. The dose levels were 50 to 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on absence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. A item item (oily in appearance) precipitate was observed on the plates at 5000 μg/plate. This did not prevent scoring of the revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

 

Key study: OECD TG 473, 2001 : The study was performed to the requirements of OECD TG 473 and EU Method B.10 guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. Secondly, 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with S9-Mix (1%):0, 10, 20, 40, 60, 80, 120 μg/mLand without S9-Mix: 0, 10, 20, 40, 60, 80, 120μg/mL. The doses in the second Main Test was as follows respectively for 4(20)-hour with S9-Mix (2%):0, 10.02, 20.04, 40.07, 80.13, 120.19, 160.25 μg/mLand 24-hour without S9:0, 5.01, 10.02, 20.04, 40.07, 60.10, 80.13 μg/mL. All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour or 24-hour exposure groups in the presence or absence of S9 activation system which included a dose level that induced mitotic inhibition. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Justification for classification or non-classification

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