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, 2014

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2015

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:
29-01-2014 to 04-03-2014
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: July 2012 ; signature: November 2012
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 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 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. The dose levels were selected based on the results of Experiment 1.

All Salmonella strains (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 500 μg/plate.
Salmonella strain TA100 and TA1537 (with S9-mix) and E.coli strain WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strain TA98 and TA1537 (with S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
E.coli strain WP2uvrA (with S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 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 (if required) adjusted by an appropriate factor to allow for the stated purity of the test item.
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 (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer (or S9-activation mix, as applicable) and 0.1 mL of the test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. After setting, the plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure. All testing for this experiment was performed in triplicate. Concurrent negative controls were dosed using the standard plate incorporation method. All of the plates were incubated at 37 °C± 3 °C 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. The procedure for incubation and duration was the same as in Experiment 1.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
In accordance with the relevant guidelines.
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

Note: The experimental procedure for Experiment 1 tested up to 5000 µg/plate was repeated with limited dose range due to excessive toxicity in the original test. The data for the Salmonella strains given in Experiment 1, refer to the repeat experiment only. Original Experiment 1 data for the Salmonella strains was discarded as there were less than four non-toxic doses.

 

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)

78          (75)

82          8.9#

65

15          (13)

73          4.9

16

25          (21)

17          4.0

20

9             (16)

20          6.4

20

20          (19)

24          5.6

13

 

0.015 µg

53         (65)

79          13.1

64          

8           (12)

12          2.1

11          

N/T

20         (23)

25          2.5

23          

13         (14)

17          3.1

11          

 

0.05 µg

56          (66)

71         9.0

72          

11          (11)

12         1.5

9             

N/T

23          (15)

11         6.9

11          

9             (18)

25         8.2

20          

 

0.15 µg

83         (82)

82          1.5

80          

1           (7)

9             5.3

11          

N/T

25         (18)

16          6.2

13          

21         (15)

9             6.0

15

 

0.5 µg

69         (66)

63          3.1

67          

3           (8)

5             6.4

15          

N/T

20         (19)

17          1.5

19          

15         (15)

17          2.5

12

 

1.5 µg

74         (89)

100        13.3

92          

11         (12)

12          1.0

13          

23         (19)

12          6.4

23          

16         (17)

13          4.0

21          

11         (16)

15          6.1

23          

 

5 µg

61         (63)

75          11.6

52          

8           (8)

8             0.6

9             

23         (16)

16          7.5

8             

24         (25)

27          2.1

23          

12         (15)

19          3.5

15          

 

15 µg

45 S      (54)

57 S       7.9

60 S       

12 S      (9)

3 S         4.9

11 S       

12         (15)

15          2.5

17          

20 S      (18)

13 S       4.4

21 S       

3 S        (3)

0 S         3.5

7 S         

 

50 µg

79 S       (72)

88 S       21.0

48 S

11 S       (12)

13 S       1.0

12 S

24          (15)

16          9.5

5

19 S       (13)

12 S       5.1

9 S

3 S         (6)

9 S         3.1

5 S

 

150 µg

N/T

N/T

11          (15)

20          4.5

15

N/T

N/T

 

500 µg

N/T

N/T

23 S      (24)

31 S       7.0

17 S

N/T

N/T

 

1500 µg

N/T

N/T

15 S      (17)

24 S       6.2

12 S       

N/T

N/T

 

5000 µg

N/T

N/T

12 S       (15)

13 S      4.4

20 S       

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

788       (842)

913        64.2

825

1576     (1649)

1613      96.8

1759      

520       (388)

414        146.2

231        

128       (143)

154        13.5

147        

3702      (3014)

2663      595.6

2678

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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)

75          (83)

94          10.0#

79

11          (15)

25          8.7

9

32          (35)

36          2.6

37

13          (23)

28          8.7

28

5             (8)

11          3.0

8

 

1.5 µg

76         (69)

68          6.1

64          

23         (15)

12          7.4

9             

19         (25)

23          6.7

32          

24         (24)

23          0.6

24          

15          (12)

9             3.1

11

 

5 µg

63         (77)

68          19.5

99          

12         (13)

8             5.6

19          

27         (27)

35          8.0

19          

19         (19)

19          0.6

20          

9             (10)

13          2.6

8

 

15 µg

87         (84)

95          13.3

69          

11         (10)

9             1.2

11          

21         (25)

36          10.0

17          

24         (22)

27          5.7

16          

5           (9)

12          3.5

9             

 

50 µg

75         (86)

96          10.5

87          

8           (9)

11          1.5

9             

24         (23)

21          1.5

23          

23         (21)

21          2.0

19          

5           (7)

11          3.5

5             

 

150 µg

80         (77)

67          8.5

83          

15 S      (10)

8 S         4.0

8 S         

25         (22)

21          2.6

20          

24 S      (25)

25 S       1.5

27 S       

9           (9)

11          2.0

7             

 

500 µg

82 S      (79)

102 S    24.6

53 S       

9 S        (8)

7 S         1.2

9 S         

17         (19)

12          8.2

28          

17 S      (19)

19 S       1.5

20 S       

5 S         (8)

13 S      4.6

5 S         

 

1500 µg

68 V     (65)

68 V      5.2

59 V      

4 V        (7)

9 V        2.6

8 V        

20 S      (21)

20 S       1.7

23 S       

15 S      (18)

21 S       3.1

17 S       

7 S        (5)

1 S         3.8

8 S         

 

5000 µg

74 V     (73)

74 V      1.2

72 V      

1 V        (3)

5 V        2.0

3 V        

24 S      (20)

20 S       4.5

15 S       

13 S      (9)

8 S         3.2

7 S         

7 S         (5)

4 S         1.7

4 S

 

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

548        (588)

547        69.6

668

155        (138)

124        15.7

135

182        (153)

140        25.5

136

122        (117)

127        13.2

102

132        (134)

120        15.6

151

 

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)

68          (77)

87          9.6#

75

15          (17)

19          2.0

17

25          (20)

11          7.8

24

8             (16)

19          6.7

20

12          (13)

17          4.0

9

 

0.05 µg

72         (81)

84          7.6

86          

12         (10)

9             1.7

9             

 

N/T

25         (18)

17          6.6

12          

8           (10)

15          4.0

8             

 

0.15 µg

87          (81)

72         7.8

83          

9             (9)

9           0.6

8             

 

N/T

17          (15)

15         2.5

12          

12          (11)

5           5.6

16          

 

0.5 µg

72         (69)

61          7.4

75          

9           (8)

8             0.6

8             

15         (18)

19          2.6

20          

13         (13)

11          2.0

15          

13         (8)

3             5.0

7             

 

1.5 µg

72          (67)

61          5.7

69

13          (12)

13          1.2

11

12          (12)

16          3.5

9

19          (17)

17          2.0

15

7             (10)

7             5.8

17

 

5 µg

60          (64)

69          4.5

64

11          (10)

11          1.2

9

15          (16)

15          1.2

17

12          (14)

15          1.7

15

5             (8)

9             2.3

9

 

15 µg

60 S      (69)

71 S       7.8

75 S       

7 S        (9)

13 S       3.5

7 S         

16         (16)

9             7.5

24          

20         (17)

21          5.5

11          

11 S      (9)

11 S       3.5

5 S         

 

50 µg

74 S      (66)

61 S       7.0

63 S       

7 S        (6)

5 S         1.2

7 S         

21         (15)

12          4.9

13          

11 S      (9)

8 S         1.7

8 S         

1 S        (3)

4 S         2.1

5 S         

 

150 µg

 

N/T

 

N/T

25          (19)

17         5.3

15          

 

N/T

 

N/T

 

500 µg

 

N/T

 

N/T

15 S       (14)

17 S      3.1

11 S       

 

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

746       (802)

859        56.5

802        

450       (435)

370        59.4

486        

1056     (913)

877        128.4

807        

202       (189)

162        23.1

202        

568        (559)

492       62.5

616        

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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)

72          (71)

74          3.1#

68

23          (19)

15          4.0

19

24          (26)

27          1.7

27

29          (23)

19          5.5

20

12          (12)

12          0.0

12

 

0.15 µg

 

N/T

13         (12)

13          1.2

11          

 

N/T

16         (19)

20          2.3

20          

 

N/T

 

0.5 µg

83          (80)

80         3.5

76          

19          (17)

19         4.0

12          

 

N/T

28          (25)

24         2.3

24          

4             (7)

5           4.4

12          

 

1.5 µg

64         (64)

67          3.0

61          

16         (15)

16          1.7

13          

25         (27)

24          3.8

31          

16         (17)

17          0.6

17          

9           (8)

9             1.2

7             

 

5 µg

67          (75)

84          8.5

74

8             (15)

24          8.2

13

16          (23)

28          6.2

25

21          (23)

27          3.8

20

13          (11)

12          2.1

9

 

15 µg

67          (70)

72          2.9

72

15          (16)

16          0.6

16

29          (28)

23          5.0

33

20          (21)

25          3.2

19

5             (11)

7             8.1

20

 

50 µg

75         (79)

83          4.0

78          

24         (17)

15          6.2

12          

23         (25)

24          2.6

28          

12         (17)

17          4.5

21          

3           (9)

8             6.6

16          

 

150 µg

78         (78)

90          12.5

65          

20 S      (14)

11 S       4.9

12 S       

17         (17)

15          2.5

20          

21 S      (24)

29 S       4.6

21 S       

5           (10)

7             6.4

17          

 

500 µg

59 S       (52)

48 S      6.4

48 S       

 

N/T

27          (18)

13         8.1

13          

 

N/T

9 S         (5)

3 S        3.2

4 S         

 

1500 µg

 

N/T

 

N/T

16 S       (15)

13 S      1.5

15 S       

 

N/T

 

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

746       (802)

859        56.5

802        

450       (435)

370        59.4

486        

1056     (913)

877        128.4

807        

202       (189)

162        23.1

202        

568        (559)

492       62.5

616        

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 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 B13/14, 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 using the Ames pre incubation method 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 item.The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. However, due to excessive toxicity, a repeat experiment had to be performed (Salmonella strains dosed in the absence of S9-mix only) employing a dose range of 0.015 to 50 μg/plate. The second experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Seven test item dose levels were 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. The dose range was amended following the results of Experiment 1 and ranged between 0.05 and 1500 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. However the Salmonella strains dosed in the absence of S9-mix had to be repeated and were tested up the toxic limit (i.e. 50 μg/plate based on the first experiment). In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all the tester strains in both the presence and absence of S9-mix. 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. 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. 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). 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:
27-01-2014 to 28-05-2014
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:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according 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: March 2014; signature: May 2014
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 (typically 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. 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 which 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: Lot Nos. PB/βNF S9 20/10/13 and 15/12/13
Test concentrations with justification for top dose:
The maximum dose level was 2684 µg/mL, or 10 mM concentration, the maximum recommended dose level. The test item was insoluble in culture medium (MEM) at 26.84 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 268.4 mg/mL in solubility checks. 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 2684 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, if required (see 'confidential details on test material').

I. Preliminary toxicity test: 0 (control), 10.48, 20.97, 41.94, 83.88, 167.75, 335.5, 671, 1342 and 2684 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-mix 0*, 10, 20, 30*, 40*, 60*, 80*, MMC 0.4* μg/mL
4(20)-hour with S9 (2%) 0*, 10, 20, 40, 50*, 100*, 120*, 180*, CP 5* μg/mL
24-hour without S9-mix 0*, 10, 20*, 30*, 40*, 60*, 80, MMC 0.2* μg/mL
4(20)-hour with S9 (1%) 0*, 10, 20, 40, 50*, 100*, 120*, 180*, CP 5* μg/mL
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 culture medium (MEM) at 26.84 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 268.4 mg/mL in solubility checks. 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 2684 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).
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% or 1%, as applicable 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, 200 consecutive well-spread metaphases from each concentration (100 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 (30 to 50%) then the analysis was terminated after a total of 50 metaphases with aberrations 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 and the observed increase in the frequency of cells with structural aberrations is considered to be dose-related. Marked increases observed in only one dose level are assessed case by case.

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.

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: There 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: Cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 335.5 μg/mL, in the 4(20)-hour exposure groups and at and above 671 μg/mL in the continuous exposure group. Greasy/oily precipitate of the test item was also observed in the parallel blood-free cultures at the end of the exposure, at and above 83.88 μg/mL, in the 4(20)-hour exposure group in the absence of S9-mix and continuous exposure group. In the 4(20)-hour exposure group in the presence of S9-mix greasy/oily precipitate was observed at and above 1342 μg/mL.
Main test: No precipitate was observed at the end of exposure, in any of the four exposure groups.

- Other confounding effects: In the preliminary test and the main test: Haemolysis was observed following exposure to the test item at harvesting at and above 41.94 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, and at and above 167.75 μg/mL in the 24-hour continuous exposure group, reduced haemolysis was observed in both these exposure groups at 2684 μg/mL. In the 4(20)-hour exposure group in the presence of S9-mix haemolysis was observed from 167.75 μg/mL to 1342 μg/mL, it was not observed at the top dose of 2684 μg/mL. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Main test: Haemolysis was observed following exposure to the test item at and above 10 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, and at and above 40 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix (2%). Haemolysis was observed following exposure to the test item at and above 60 μg/mL in the 24 hour continuous exposure group and at and above 100 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix (1%).

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 2684 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

Chromosome Aberration Test – Main Test

4(20)-hour without S9: 0*, 4.06, 8.13, 16.25*, 32.5*, 48.75*, 65, MMC 0.4*

4(20)-hour with S9: 0*, 8.13, 16.25, 32.5, 43.5*, 54*, 65*, 130, CP 5*

24-hour without S9: 0*, 4.06, 8.13, 16.25, 32.5, 43.5*, 54*, 65*, MMC 0.2*

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 and that there were metaphases suitable for scoring at 65 μg/mL in both the 4(20)-hour exposure groups in the absence and presence of metabolic activation (S9-mix) and also in the 24-hour continuous exposure group in the absence of metabolic activation (S9-mix). No precipitate was observed at the end of exposure, in any of the three exposure groups.

The mitotic index data confirm a dose-related inhibition of mitotic index:

4(20)-hour without S9: 49% and 38% mitotic inhibition was achieved at 48.75 μg/mL and 65 μg/mL respectively in the absence of S9-mix. Even though the mitotic index at 65 μg/mL was 38% this slightly exceeded the optimum toxicity and the metaphases were overall very poor and was therefore not selected for scoring.

4(20)-hour with S9: A dose-related inhibition of mitotic index was observed and was 42% at 65 μg/mL.

24-hour without S9: A dose-related inhibition of mitotic index was observed and was 45% at 65 μg/mL.

 

- 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.

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, EU Method B.10, and Japan METI 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 four dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; in experiment 1: 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. In experiment 2: 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 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 and cytotoxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with S9-Mix (2%): 0, 10, 20, 30, 40, 60, 80 μg/mL, 4(20)-hour without S9 Mix: 0, 10, 20, 40, 50, 100, 120, 180 μg/mL, 4(20)-hour with S9-Mix (1%): 0, 10, 20, 40, 50, 100, 120, 180 μg/mL and 24-hour without S9: 0, 10, 20, 30, 40, 60, 80 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 at dose levels that achieved near optimum toxicity (approximately 50% 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, 2014 : 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 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 using the Ames pre incubation method 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 item.The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. However, due to excessive toxicity, a repeat experiment had to be performed (Salmonella strains dosed in the absence of S9-mix only) employing a dose range of 0.015 to 50 μg/plate. The second experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Seven test item dose levels were 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. The dose range was amended following the results of Experiment 1 and ranged between 0.05 and 1500 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. However the Salmonella strains dosed in the absence of S9-mix had to be repeated and were tested up the toxic limit (i.e. 50 μg/plate based on the first experiment). In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all the tester strains in both the presence and absence of S9-mix. 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. 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. 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). 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, 2015 : The study was performed to the requirements of OECD TG 473, EU Method B.10, and Japan METI 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 four dose levels, together with vehicle and positive controls. In this study, four exposure conditions were investigated; in experiment 1: 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. In experiment 2: 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 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 and cytotoxicity. The dose levels selected for the Main Test were as follows: 4(20)-hour with S9-Mix (2%): 0, 10, 20, 30, 40, 60, 80 μg/mL, 4(20)-hour without S9 Mix: 0, 10, 20, 40, 50, 100, 120, 180 μg/mL, 4(20)-hour with S9-Mix (1%): 0, 10, 20, 40, 50, 100, 120, 180 μg/mL and 24-hour without S9: 0, 10, 20, 30, 40, 60, 80 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 at dose levels that achieved near optimum toxicity (approximately 50% 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