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Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator 4°C in the dark, under nitrogen
- Other: clear colourless

Method

Species / strainopen allclose all
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.
Controls
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).

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.