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, 2017

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2017

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:
2016
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:
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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2015; signature: September 2015
Type of assay:
bacterial reverse mutation assay
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:
Experiment 1 (plate incorporation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1.

Salmonella strain TA100 and E.coli strain WP2uvrA (presence and absence of S9 mix) and Salmonella strain TA98 (presence of S9-mix only):
0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strains TA1535 and TA1537 (presence and absence of S9-mix) and Salmonella strain TA98 (absence of S9-mix only):
0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.
- Other: Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test substance.
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 (pre-incubation)

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). A number of manual counts were performed, predominantly due to colonies spreading, thus distorting the actual plate count.

Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

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).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
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
Remarks:
Migrated from field 'Test system'.

Table 1 : Test Results: Experiment 1 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

(DMSO)

108

108

85

(100)

13.3#

23

23

25

(24)

1.2

34

25

26

(28)

4.9

22

21

29

(24)

4.4

8

12

21

(14)

6.7

1.5 µg

92

97

119

(103)

14.4

14

23

23

(20)

5.2

33

22

31

(29)

5.9

22

30

26

(26)

4.0

10

14

8

(11)

3.1

5 µg

97

113

102

(104)

8.2

11

27

15

(18)

8.3

20

33

31

(28)

7.0

22

12

18

(17)

5.0

7

13

7

(9)

3.5

15 µg

111

85

98

(98)

13.0

23

18

17

(19)

3.2

18

18

18

(18)

0.0

18

25

30

(24)

6.0

16

9

21

(15)

6.0

50 µg

92

128

119

(113)

18.7

13

21

19

(18)

4.2

27

31

22

(27)

4.5

22

21

16

(20)

3.2

12

16

16

(15)

2.3

150 µg

80

93

111

(95)

15.6

17

18

20

(18)

1.5

27

22

16

(22)

5.5

27

31

30

(29)

2.1

10

10

21

(14)

6.4

500 µg

61

100

78

(80)

19.6

0 V

0 V

0 V

(0)

0.0

18

20

16

(18)

2.0

16 S

17 S

17 S

(17)

0.6

0 V

0 V

0 V

(0)

0.0

1500 µg

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

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

448

341

393

(394)

53.5

293

289

314

(299)

13.4

720

815

740

(758)

50.1

134

171

194

(166)

30.3

354

349

344

(349)

5.0

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

120

167

95

(127)

36.6#

22

9

21

(17)

7.2

21

33

38

(31)

8.7

23

21

17

(20)

3.1

8

8

17

(11)

5.2

1.5 µg

114

92

88

(98)

14.0

12

23

17

(17)

5.5

31

33

21

(28)

6.4

26

25

20

(24)

3.2

8

10

9

(9)

1.0

5 µg

84

80

87

(84)

3.5

21

12

13

(15)

4.9

38

25

22

(28)

8.5

19

21

19

(20)

1.2

10

14

7

(10)

3.5

15 µg

84

69

75

(76)

7.5

18

17

16

(17)

1.0

30

27

36

(31)

4.6

21

16

24

(20)

4.0

20

8

5

(11)

7.9

50 µg

87

81

97

(88)

8.1

22

14

13

(16)

4.9

26

29

20

(25)

4.6

22

16

19

(19)

3.0

9

9

17

(12)

4.6

150 µg

99

91

75

(88)

12.2

15

9

9

(11)

3.5

29

22

17

(23)

6.0

17

14

21

(17)

3.5

12

14

8

(11)

3.1

500 µg

87

76

78

(80)

5.9

9 S

9 S

14 S

(11)

2.9

27

18

13

(19)

7.1

21

14

17

(17)

3.5

7 S

4 S

10 S

(7)

3.0

1500 µg

68 S

67 S

25 S

(53)

24.5

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

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

432

363

445

(413)

44.1

296

280

332

(303)

26.6

228

406

357

(330)

91.9

159

189

220

(189)

30.5

434

367

393

(398)

33.8

 

Table 2 : Test Results: Experiment 2 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

(DMSO)

78

90

68

(79)

11.0#

22

13

21

(19)

4.9

21

15

24

(20)

4.6

18

24

20

(21)

3.1

12

6

12

(10)

3.5

0.15 µg

N/T

 

21

13

16

(17)

4.0

N/T

 

18

18

31

(22)

7.5

9

4

10

(8)

3.2

0.5 µg

68

77

62

(69)

7.5

22

20

8

(17)

7.6

17

21

16

(18)

2.6

21

24

19

(21)

2.5

3

4

4

(4)

0.6

1.5 µg

73

78

71

(74)

3.6

8

11

11

(10)

1.7

16

24

16

(19)

4.6

31

15

16

(21)

9.0

7

7

7

(7)

0.0

5 µg

83

75

75

(78)

4.6

9

20

22

(17)

7.0

14

15

27

(19)

7.2

12

30

19

(20)

9.1

16

20

5

(14)

7.8

15 µg

65

71

89

(75)

12.5

24

8

11

(14)

8.5

24

19

25

(23)

3.2

26

11

13

(17)

8.1

9

12

4

(8)

4.0

50 µg

72

85

67

(75)

9.3

12

13

13

(13)

0.6

13

14

14

(14)

0.6

18

23

28

(23)

5.0

11

8

21

(13)

6.8

150 µg

71

72

80

(74)

4.9

12 S

8 S

6 S

(9)

3.1

19

9

9

(12)

5.8

23

23

27

(24)

2.3

8 S

4 S

4 S

(5)

2.3

500 µg

34 S

50 S

28 S

(37)

11.4

7 S

8 S

4 S

(6)

2.1

12 S

13 S

13 S

(13)

0.6

13 S

7 S

7 S

(9)

3.5

0 V

0 V

0 V

(0)

0.0

1500 µg

0 T

0 T

0 T

(0)

0.0

N/T

 

0 T

0 T

0 T

(0)

0.0

N/T

 

N/T

 

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

229

277

427

(311)

103.3

99

94

132

(108)

20.6

351

402

345

(366)

31.3

149

127

187

(154)

30.4

122

240

197

(186)

59.7

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

74

92

117

(94)

21.6#

11

15

11

(12)

2.3

24

21

25

(23)

2.1

21

21

22

(21)

0.6

13

7

12

(11)

3.2

0.15 µg

N/T

 

11

11

11

(11)

0.0

N/T

 

N/T

 

12

11

7

(10)

2.6

0.5 µg

109

82

86

(92)

14.6

24

11

11

(15)

7.5

15

24

14

(18)

5.5

17

22

18

(19)

2.6

16

15

10

(14)

3.2

1.5 µg

76

100

108

(95)

16.7

16

22

18

(19)

3.1

18

23

24

(22)

3.2

17

29

22

(23)

6.0

6

12

10

(9)

3.1

5 µg

86

96

101

(94)

7.6

10

9

13

(11)

2.1

17

18

15

(17)

1.5

16

26

20

(21)

5.0

22

8

19

(16)

7.4

15 µg

105

101

77

(94)

15.1

13

13

9

(12)

2.3

21

27

19

(22)

4.2

26

20

20

(22)

3.5

6

12

12

(10)

3.5

50 µg

107

84

102

(98)

12.1

11

17

16

(15)

3.2

19

25

30

(25)

5.5

13

19

17

(16)

3.1

9

5

6

(7)

2.1

150 µg

83

106

107

(99)

13.6

9

17

8

(11)

4.9

20

25

21

(22)

2.6

25

18

17

(20)

4.4

17

15

9

(14)

4.2

500 µg

98

99

86

(94)

7.2

0 V

0 V

0 V

(0)

0.0

15 S

10 S

12 S

(12)

2.5

17 S

9 S

6 S

(11)

5.7

5 S

5 S

5 S

(5)

0.0

1500 µg

47 S

24 S

48 S

(40)

13.6

N/T

 

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

N/T

 

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

440

490

520

(483)

40.4

151

171

175

(166)

12.9

87

138

247

(157)

81.7

92

111

111

(105)

11.0

128

135

110

(124)

12.9

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: Sparse bacterial background lawn

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

#: Standard deviation

Conclusions:
Interpretation of results:
Negative

Under the conditions of this study the test material 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 B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance 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 substance using both the Ames plate incorporation and pre incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test substance. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (dimethyl sulphoxide) 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. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix. Weakened bacterial background lawns were initially noted in the absence of S9-mix from 500 µg/plate (TA1535, TA98 and TA1537) and 1500 µg/plate (TA100 and WP2uvrA). In the presence S9-mix weakened bacterial background lawns were again initially noted from 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA100, WP2uvrA and TA98). Consequently for the second mutation test the toxic limit was employed as the maximum dose concentration. Results from the second mutation test (pre-incubation method) confirmed the toxicity previously noted with weakened bacterial background lawns observed in the absence of S9-mix from 150 µg/plate (TA1535 and TA1537) and 500 µg/plate (WP2uvrA, TA100 and TA98). In the presence of S9-mix, weakened bacterial background lawns were noted from 500 µg/plate (TA1535, TA1537, TA98 and WP2uvrA) and 1500 µg/plate (TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). It was concluded that, under the conditions of this assay, the test substance 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:
2016
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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2016; signature: October 2016
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 non smoking volunteer (aged 18-35) 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 over 20 years 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 16 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 23/08/15
Test concentrations with justification for top dose:
The maximum dose level was 1923 µg/mL, the 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 1923 μg/mL range (full results recorded in the full study report).
The purity of the test item was accounted for in the test item formulations (see 'confidential details on test material').

I. Preliminary toxicity test: 0 (control) , 7.51, 15.02, 30.05, 60.09, 120.19, 240.38, 480.75, 961.5 and 1923 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%), 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:
4(20)-hour without S9: 0*, 7.5, 15*, 30*, 60*, 120*, 160, 200, 240, MMC 0.2*
4(20)-hour with S9: 0*, 7.5, 15, 30*, 60*, 120*, 160*, 200, 240, CP 2*
24-hour without S9: 0*, 7.5*, 15*, 30*, 60*, 120, 160, MMC 0.1*
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in Minimal Essential Medium (MEM) at 19.3 mg/mL but was soluble in DMSO at 192.3 mg/mL in solubility checks performed. The test item formulations were corrected for purity by an appropriate correction factor. The maximum dose level (determined prior to the test based on molecular weight) was 1923 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 1923 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
Untreated negative controls:
other: Vehicle control served as the negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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, 10% (FBS); 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 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment. 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. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was reported in the full study report.
- Other: Scoring: Where possible, 300 consecutive well-spread metaphases from each concentration (150 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), ISCN (1985). Where the analysis of the slide resulted in a large frequency of aberrant cells then the analysis was terminated after a total of 15 metaphases with aberrations (excluding gaps) were recorded. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Evaluation criteria:
Positive response criteria
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.

Negative response criteria
A test item can be classified as non-genotoxic if:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment.

Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first. Statistical methods are used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
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: Greasy/oily Precipitate observed in the blood-free cultures at the end of exposure, at 480.75 µg/mL in the 4(20)-hour exposure groups in the absence of S9 and a Cloudy Precipitate observed in the blood-free cultures at the end of exposure in the 4(20)-hour in the presence of S9. A Greasy/oily and cloudy precipitate was seen at 480.75 µg/mL in the continuous exposure 24-hour in the absence of S9, respectively.
Main test: Precipitate observations were made at the end of exposure in blood-free cultures and no precipitate was observed in all three exposure groups (up to 240 µg/mL in the 4(20)-hour groups and/or 160 µg/mL in the 24-hour exposure groups, respectively.
- Other confounding effects: In the preliminary test and the main test: Haemolysis was observed in the 4(20)-hour exposure groups, at and above 120 µg/mL in the absence of S9 and at and above 60 µg/mL in the presence of S9. Haemolysis was observed in the 24-hour exposure group, at and above 120 µg/mL. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1923 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 (DMSO) 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
Remarks:
Migrated from field 'Test system'.

1. Chromosome Aberration Test – Main Test

The dose levels of the controls and the test item are given below:

4(20)-hour without S9: 0*, 7.5, 15*, 30*, 60*, 120*, 160, 200, 240, MMC 0.2*

4(20)-hour with S9: 0*, 7.5, 15, 30*, 60*, 120*, 160*, 200, 240, CP 2*

24-hour without S9: 0*, 7.5*, 15*, 30*, 60*, 120, 160, MMC 0.1*

where: * = dose levels selected for metaphase analysis ; MMC= Mitomycin C and CP = Cyclophosphamide

 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test. In the presence of metabolic activation (S9), the maximum dose level of the test item with metaphases suitable for scoring was 160 µg/mL. In the absence of metabolic activation (S9) the maximum dose level of the test item with metaphases suitable for scoring was 120 µg/mL. In the 24-hour exposure group, the maximum dose level of the test item with metaphases suitable for scoring was 60 µg/mL. Precipitate observations were made at the end of exposure in blood-free cultures and no precipitate was observed in all three exposure groups.

Qualitative observations indicated that a dose-related inhibition of mitotic index was observed only in the absence of S9, although all three exposure groups were tested to toxic dose levels. The steepness of the toxicity curve made achieving optimum toxicity difficult. In the 4(20)-hour exposure group in the absence of S9, 31% mitotic inhibition was achieved at 120 µg/mL. An inhibition of mitotic index of 37% was noted at 60 µg/mL in the 24-hour continuous exposure group. It should be noted that all surviving dose levels in the 24-hour continuous exposure group exhibited mitotic inhibition, and dose selection was based on the maximum surviving dose level. In the presence of S9, the toxicity curve was extremely steep with no toxicity demonstrated at 160 µg/mL and no metaphases available for scoring at the higher dose level of 200 µg/mL.

The maximum dose level selected for metaphase analysis was 120 µg/mL for the 4(20)-hour exposure in the absence of S9, 160 µg/mL in the presence of S9 and 60 µg/mL for the 24-hour exposure group. Cloudy precipitate was only observed at 961.5 µg/mL in the preliminary cell growth inhibition test. No precipitate was observed in the main test.

 

- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.

- 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 either in the absence or presence of metabolic activation.

- The test item did not induce any statistically significant increase in the numbers of polyploid cells or endoreduplicated chromosomes in any of the three exposure groups.

 

2. Additional comments:

- In the presence of metabolic activation the test item demonstrated no apparent marked toxicity based on the MI values of the surviving dose levels. However, observations made during slide assessment indicate toxicity was a factor. The numbers of cells present on the slides were reduced when compared to the vehicle control, with the most marked effect observed at the maximum dose with metaphases present, 160 µg/mL. Whilst the overall cell numbers had reduced the mitotic index values remained high and this is considered to be due to toxicity driven cell cycle delay occurring. Whereby additional time for DNA repair process results in additional cells going through to metaphase. As the concentration of the test item increased, reduction in cell numbers is observed due to apoptosis as demonstrated in the preliminary test.

- In the absence of metabolic activation, the toxicity achieved at the maximum dose level scored for aberrations in both the 4(20)-hour exposure and the 24-hour exposure was less than optimum due to a steep toxicity curve. Reduced cell numbers were observed on the slides of the 4(20)-hour exposure group at 120 µg/mL and of the 24-hour exposure group at 60 µg/mL. Indicating that toxicity was the limiting factor. It was therefore considered, that the test item had been adequately tested since it was tested to cytotoxic dose levels. Toxicity was demonstrated by a reduction in cell numbers or by a reduction in mitotic index in all three exposure conditions.

Conclusions:
Interpretation of results:
Negative

Under the conditions of this study, the test substance 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 Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, 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 four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 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, 4 hours exposure in the absence of metabolic activation (S9) with 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 precipitate. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%): 0, 7.5, 15, 30, 60, 120, 160, 200, 240 μg/mL and 24-hour without S9: 0, 7.5, 15, 30, 60, 120, 160 μg/mL, respectively. All vehicle (DMSO) 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 exposure groups in the absence and presence of S9, or the 4(20)-hour exposure group in the absence of S9. The 4(20)-hour exposure group in the absence of S9 was tested to toxic dose levels with the maximum dose level scored for aberrations achieving 31% mitotic inhibition. In the 4(20)-hour exposure group in the presence of S9 scoring was limited due to toxic dose levels. The 24-hour exposure group in the absence of S9 was tested to toxic dose levels with the maximum dose level scored for aberrations achieving 37% mitotic inhibition. Under the conditions of the test, and due to reduced cell numbers observed in the slides of the 4(20)-hour exposure group at 120 µg/mL and of the 24-hour exposure group at 60 µg/mL, that the test item had been adequately tested since it was tested to cytotoxic dose levels. Toxicity was demonstrated by the reduction in cell numbers or by a reduction in the mitotic index in all three exposure conditions. 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

Description of key information

negative up to 1000 mg/kg bw/day, in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus, OECD TG 407 modified to OECD TG 474, 2017

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to relevant guidelines performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
The study was conducted as part of a sub-chronic, 28-day repeated-dose toxicity study under OECD TG 407 and EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP. The study was modified according to the relevant listed regulatory guidelines in order to incorporate micronucleus assessment following exposure to the test item in a manner compatible with OECD TG 474 (2014) and listed guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2015; signature: September 2015
Type of assay:
mammalian germ cell cytogenetic assay
Species:
rat
Strain:
other: Crl:CD(SD) IGS BR
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 guideline and consistent with the OECD TG 474 guideline.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 to 8 weeks
- Weight at study initiation: males 205 - 311 g and females 139 - 215 g; individuals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Fasting period before study: None
- Housing: Polypropylene body with a stainless steel mesh lid with softwood flake bedding, changed at appropriate intervals; group housed (5 per group) by sex. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. Cage distribution within the holding rack was randomized.
- Diet (e.g. ad libitum): Rodent 2014C, Global Certified Diet, ad libitum
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 5 days.
DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent 2014C, Global Certified Diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2016-03-04 To: 2016-04-15
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Arachis oil BP was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable for at least eleven days when stored refrigerated. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored at approximately 4 ºC in the dark.
- Concentration of test material in vehicle: Samples of the test item formulations were taken on two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ± 4% of the nominal concentration. Arachis oil formulations was assessed and confirmed at nominal concentrations of 3.75 mg/mL and 250 mg/mL, during refrigerated storage.
- Amount of vehicle (if gavage or dermal): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group; positive control was 10 mL/kg. For further information see 'Doses / concentrations'.
- Type and concentration of dispersant aid (if powder): Not applicable.
- Other: Dose-formulations were analysed during the study and were reported as within ± 10% applied limits.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period (treatment free) for the OECD 407 part of the study. The last dose was administered on Day 28. Micronucleus assessment proceeded after in non-recovery groups thereafter.
Frequency of treatment:
Once daily at approximately the same time each day.
Post exposure period:
24-hours following final dosing in relevant treatment groups only
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control - Group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High - Group
No. of animals per sex per dose:
10 per sex per dose (5 male / 5 female)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): The choice of positive control was made in accordance with the OECD TG 474 guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 1 dose; 25 mg/kg bw
- Other: five (5) males - single dose were used for micronucleus test, treated with cyclophosphamide at 25 mg/kg bw/day - dosed on Day 28 only.
Tissues and cell types examined:
Bone marrow was extracted and smear preparations were made and stained Polychromatic (PCE) and nomtochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCEINCE ratio was calculated as an indicator for toxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The test item dose levels used in the main study were 100, 350 and 1000 mg/kg bw/day. The maximum dose level was considered to be more than adequate to investigate the genotoxic endpoint in this study (confirmed as part of investigations in 7-d range finder and 28-day repeated dose toxicity testing).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Males/females were treated daily for 28-days at the specified dose levels. 24-hours following the last treatment (day 28) the treatment groups were terminated and then bone marrow was extracted from the left femur as part of sampling. Smear preparations were subsequently made and stained.

DETAILS OF SLIDE PREPARATION:
24-hours following last treatment, left femurs were extracted, aspirated with foetal bovine serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald / Giemsa. Then allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
Stained smears were coded and examined blind using microscopy (x1000) magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with the appropriate group mean values and standard deviations.

OTHER: Not applicable.
Evaluation criteria:
1. Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group:
- A positive response would be demonstrated when: there is a statistically significant dose response, toxicologically relevant increases in the number of micronucleated polychromatic erythrocytes observed at the 24-hour termination time compared with the vehicle control group
- A negative response would be fulfilled if the positive response criteria were not fulfile (no statistically significant dose responses, no toxicologically relevant increases in micronucleated polychromatic erythrocytes.

2. A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically lower than the vehicle control group.

Data is subject to statistical analysis, where appropriate.
Statistics:
Wherea appropriate, data is subject to statistical analysis using appropriate methods as recommended in UKEMS Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No statistically significant PCE/NCE decreases at up to 1000 mg/kg bw/day observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: positive control male (number 65) slide was disregarded due to excessive bone marrow toxicity. The loss of one individual from the PC scoring data was considered to have no impact on the outcome of the study.
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not applicable.
- Induction of micronuclei (for Micronucleus assay):
Vehicle Control, 0 mg/kg bw/day: Group mean (SD) = 12.0 (10.6) PCE with micronuclei per 4000 PCE
Low dose, 100 mg/kg bw/day: Group mean (SD) = 8.5 (8.9) PCE with micronuclei per 4000 PCE
Intermediate dose, 350 mg/kg bw/day: Group mean (SD) = 6.8 (4.5) PCE with micronuclei per 4000 PCE
High dose, 1000 mg/kg bw/day: Group mean (SD) = 8.8 (5.0) PCE with micronuclei per 4000 PCE
Positive Control, 25 mg/kg bw/day: Group mean (SD) = 83.8 ** (71.7) PCE with micronuclei per 4000 PCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01
- Ratio of PCE/NCE (for Micronucleus assay):
Vehicle Control, 0 mg/kg bw/day: Group mean (SD) = 1.29 (0.39) PCE/NCE
Low dose, 100 mg/kg bw/day: Group mean (SD) = 1.22 (0.62) PCE/NCE
Intermediate dose, 350 mg/kg bw/day: Group mean (SD) = 0.90 (0.52) PCE/NCE
High dose, 1000 mg/kg bw/day: Group mean (SD) = 1.01 (0.39) PCE/NCE
Positive Control, 25 mg/kg bw/day: Group mean (SD) = 0.50 ** (0.06) PCE/NCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01
- Appropriateness of dose levels and route: The highest dose was set in accordance with the findings of the 7-day Range Finding study and the OECD TG 407 study. The appropriateness of the dose and route was confirmed during the study (recorded in the full study report). Applicant assessment: The maximum dose 1000 mg/kg bw/day is in accordance with the OECD TG 474 guideline for a period of administration greater than 14-days and three dose levels with a separation factor less than 4.0.
- Statistical evaluation: No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw/day relative to the concurrent vehicle control.
- Other: positive control male (number 65) slide was disregarded due to excessive bone marrow toxicity. The loss of one individual from the PC scoring data was considered to have no impact on the outcome of the study. A statistically significant (P < 0.01) response was observed in PCE with micronuclei per 4000 PCE therefore confirming the sensitivity of the test system under the conditions of the test.

Table 1.0 – Table of results

Treatment Group

Number PCE with micronuclei per 4000 PCE

PCE/NCE Ratio

 

Group mean

SD

Group mean

SD

Vehicle Control, 0 mg/kg bw/day

12.0

10.6

1.29

0.39

Low dose, 100 mg/kg bw/day

8.5

8.9

1.22

0.62

 

Intermediate dose, 350 mg/kg bw/day

6.8

4.5

0.90

0.52

High dose, 1000 mg/kg bw/day

8.8

5.0

1.01

0.39

 

 

 

 

 

Positive Control, 25 mg/kg bw/day

83.8**

71.7

0.50**

0.06

Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; ** P < 0.01

0.01

Conclusions:
Under the conditions of this study, the test item was considered to be non-genotoxic.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Additional parameters covering an in vivo, micronucleus assessment was included under the following guidelines OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 as part of the study. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) IGS BR rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 100, 350 or 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Furthermore, as part of micronucleus assessment a concurrent positive control group of five males was exposed to 25 mg/kg bw/day cyclophosphamide on day 28 only. Within the micronucleus assessment, there was no indications of genotoxicity. The vehicle control group frequency of micronucleated PCE and PCE/NCE was within the normal range. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item groups when compared with the vehicle control group. There were no statistically significant decreases in the PCE/NCE ratio in any of the test item groups when compared with the vehicle control group. Additionally, the vehicle control groups were within the normal range for the frequency of micronucleated PCEs and for PCE/NCE ratio. The positive control group showed a marked increase in the incidence of mononucleated polychromatic erythrocytes. Confirming sensitivity of the test system to the known mutagenicity of the cyclophosphamide under the conditions of the test. Under the conditions of this study, the test item would not be considered as genotoxic.

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

Additional information

Key study: OECD TG 471, 2017 : The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance 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 substance using both the Ames plate incorporation and pre incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test substance. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. The vehicle (dimethyl sulphoxide) 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. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). It was concluded that, under the conditions of this assay, the test substance 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, 2017 : The study was performed to the requirements of OECD TG 473 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, 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 four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 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, 4 hours exposure in the absence of metabolic activation (S9) with 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 precipitate. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%): 0, 7.5, 15, 30, 60, 120, 160, 200, 240 μg/mL and 24-hour without S9: 0, 7.5, 15, 30, 60, 120, 160 μg/mL, respectively. All vehicle (DMSO) 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 exposure groups in the absence and presence of S9, or the 4(20)-hour exposure group in the absence of S9. The 4(20)-hour exposure group in the absence of S9 was tested to toxic dose levels with the maximum dose level scored for aberrations achieving 31% mitotic inhibition. In the 4(20)-hour exposure group in the presence of S9 scoring was limited due to toxic dose levels. The 24-hour exposure group in the absence of S9 was tested to toxic dose levels with the maximum dose level scored for aberrations achieving 37% mitotic inhibition. Under the conditions of the test, and due to reduced cell numbers observed in the slides of the 4(20)-hour exposure group at 120 µg/mL and of the 24-hour exposure group at 60 µg/mL, that the test item had been adequately tested since it was tested to cytotoxic dose levels. Toxicity was demonstrated by the reduction in cell numbers or by a reduction in the mitotic index in all three exposure conditions. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Repeated dose – Oral – modified with micronucleus assessment parameters:

Key study: OECD TG 407, 2017 modified eq. to OECD TG 474 : The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Additional parameters covering an in vivo, micronucleus assessment was included under the following guidelines OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 as part of the study. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) IGS BR rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 100, 350 or 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Furthermore, as part of micronucleus assessment a concurrent positive control group of five males was exposed to 25 mg/kg bw/day cyclophosphamide on day 28 only. Within the micronucleus assessment, there was no indications of genotoxicity. The vehicle control group frequency of micronucleated PCE and PCE/NCE was within the normal range. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item groups when compared with the vehicle control group. There were no statistically significant decreases in the PCE/NCE ratio in any of the test item groups when compared with the vehicle control group. Additionally, the vehicle control groups were within the normal range for the frequency of micronucleated PCEs and for PCE/NCE ratio. The positive control group showed a marked increase in the incidence of mononucleated polychromatic erythrocytes. Confirming sensitivity of the test system to the known mutagenicity of the cyclophosphamide under the conditions of the test. Under the conditions of this study, the test item would not be considered as genotoxic.

Justification for classification or non-classification

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