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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-09-07 to 2018-12-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use,
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Initial and confirmatory test I
±S9: 5000; 1600; 500; 160; 50 and 16 μg/plate

Confirmatory test II
-S9: 16, 5, 1.6, 0.5, 0.16 and 0.05 µg/plate
Vehicle / solvent:
- Vehicle/solvent used:
test item: acetone
positive controls: water or DMSO
- Justification for choice of solvent/vehicle: The test item solutions were prepared in acetone and diluted prior to treatment. This vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary solubility test.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine (NPD) 4 µg/plate, -S9, TA98; 2-aminoanthracene (2AA) 2 or 50 µg/plate for all salmonella strains or E.coli, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h in the dark

SELECTION AGENT: his and trp gene

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background lawn
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1 summary of signs of cytotoxicity

Confirmatory Mutation Test

Concentrations

(µg/plate)

Salmonella typhimurium

Escherichia coli WP2 uvrA

TA98

TA100

TA1535

TA1537

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

5000

B0

SB <<

B0

B <<

B0

B

A

B <<

B <<

1600

B0

SB <

B0

B <<

B0

SB

B0

B <<

B <<

500

B <<

SB <<

B <<

SB <<

B0

SB

B0

B

B <<

160

B <<

B <<

< *

SB <<

SB0

SB <<

50

B <<

B <<

< *

SB <<

SB0

<< 

16

B <<

B <<

< *

SB <<

SB0

<< 

Complementary Pre-Incubation Test

Concentrations

(µg/plate)

Salmonella typhimurium

Escherichia coli WP2 uvrA

TA98

TA100

TA1535

TA1537

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

‑S9

+S9

16

B <<

 

B <<

 

B <<

 

B0

 

SB <<

 

5

<< 

 

SB <

 

 

B <<

 

 

1.6

 

< *

 

 

 

 

0.5

 

 

 

 

 

0.16

 

 

 

 

 

0.05

 

 

 

 

 

A:           No bacterial growth: absent revertant colonies and absent background lawn;

B0:        Absent revertant colonies and reduced background lawn development;

B:           Reduced background lawn development;

SB:         Slightly reduced background lawn development;

SB0:      Absent revertant colonies and slightly reduced background lawn development;

<< :      Revertant colony numbers below the vehicle and historical control data ranges;

< :          Revertant colony numbers within of the vehicle control range but below the historical control data ranges;

< * :      Revertant colony numbers within of the vehicle control range but below the historical control data ranges; however considered as not inhibition, but being within the biological variability range of the applied test system;

:       Revertant colony numbers lower than the revertant colony number of the vehicle control but within the historical control data ranges;

‑:            No inhibition.

Cells with dark grey filling: The concentration was not tested for the corresponding strain.

 


Table 2 Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

12.7

0.97

21.0

1.21

72.3

0.84

95.0

0.97

10.0

1.07

12.3

1.28

6.3

1.06

6.7

1.18

35.0

1.14

42.3

1.15

DMSO Control

17.0

1.00

22.7

1.00

91.3

1.00

11.0

1.00

6.0

1.00

6.7

1.00

43.0

1.00

Ultrapure Water Control

74.0

1.00

16.7

1.00

39.0

1.00

Acetone Control

13.0

1.00

17.3

1.00

85.7

1.00

98.0

1.00

9.3

1.00

9.7

1.00

6.0

1.00

5.7

1.00

30.7

1.00

36.7

1.00

5000

35.7

2.74

19.3

1.12

75.3

0.88

70.7

0.72

11.7

1.25

11.7

1.21

9.0

1.50

8.0

1.41

31.7

1.03

38.7

1.05

1600

18.7

1.44

19.7

1.13

78.7

0.92

110.0

1.12

7.7

0.82

9.7

1.00

8.3

1.39

8.7

1.53

32.7

1.07

39.7

1.08

500

13.7

1.05

16.0

0.92

67.3

0.79

88.0

0.90

8.3

0.89

8.3

0.86

7.3

1.22

9.0

1.59

39.7

1.29

41.0

1.12

160

12.7

0.97

18.7

1.08

64.7

0.75

90.0

0.92

11.3

1.21

12.0

1.24

6.7

1.11

5.3

0.94

32.0

1.04

39.0

1.06

50

14.0

1.08

16.3

0.94

62.0

0.72

88.7

0.90

10.3

1.11

12.0

1.24

7.0

1.17

5.3

0.94

31.3

1.02

39.7

1.08

16

18.3

1.41

22.7

1.31

65.3

0.76

96.3

0.98

10.3

1.11

10.3

1.07

6.3

1.06

6.3

1.12

40.0

1.30

37.0

1.01

NPD (4mg/plate)

498.0

29.29

SAZ (2mg/plate)

1397.3

18.88

965.3

57.92

9AA (50mg/plate)

736.7

122.78

MMS (2mL/plate)

1157.3

29.68

2AA (2mg/plate)

1154.7

50.94

1306.7

14.31

157.0

14.27

123.7

18.55

2AA (50mg/plate)

253.0

5.88

 

 

 

Table 3 Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

16.0

0.91

29.0

1.78

68.3

0.87

87.3

0.94

14.7

1.05

9.0

0.87

7.7

1.28

9.7

0.91

24.0

0.81

39.7

0.97

DMSO Control

16.7

1.00

17.0

1.00

85.7

1.00

10.0

1.00

6.3

1.00

8.7

1.00

43.3

1.00

Ultrapure Water Control

75.7

1.00

11.3

1.00

32.3

1.00

Acetone Control

17.7

1.00

16.3

1.00

78.3

1.00

93.3

1.00

14.0

1.00

10.3

1.00

6.0

1.00

10.7

1.00

29.7

1.00

41.0

1.00

5000

0.0

0.00

11.3

0.69

0.0

0.00

11.3

0.12

0.0

0.00

7.0

0.68

0.0

0.00

2.7

0.25

10.3

0.35

41.3

1.01

1600

0.0

0.00

13.7

0.84

0.0

0.00

35.3

0.38

0.0

0.00

5.3

0.52

0.0

0.00

4.3

0.41

8.0

0.27

37.7

0.92

500

1.7

0.09

12.0

0.73

7.0

0.09

69.0

0.74

0.0

0.00

8.7

0.84

0.0

0.00

5.7

0.53

8.0

0.27

42.7

1.04

160

0.7

0.04

17.0

1.04

2.0

0.03

77.3

0.83

4.0

0.29

8.7

0.84

0.0

0.00

10.3

0.97

10.0

0.34

34.7

0.85

50

0.7

0.04

25.3

1.55

1.0

0.01

88.7

0.95

2.7

0.19

9.3

0.90

0.0

0.00

8.7

0.81

12.3

0.42

40.0

0.98

16

1.3

0.08

18.3

1.12

27.0

0.34

81.3

0.87

4.3

0.31

11.3

1.10

0.0

0.00

8.7

0.81

13.7

0.46

40.7

0.99

NPD (4mg/plate)

546.7

32.80

SAZ (2mg/plate)

840.0

11.10

845.3

74.59

9AA (50mg/plate)

404.0

63.79

MMS (2mL/plate)

824.0

25.48

2AA (2mg/plate)

1253.3

73.73

1237.3

14.44

119.0

11.90

121.7

14.04

2AA (50mg/plate)

342.7

7.91

 

Table 4 Summary Table of the Results of the Complementary Pre-Incubation Test

Complementary Pre-Incubation Test

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

12.0

0.86

67.0

1.03

7.7

0.88

5.7

1.06

30.7

1.00

DMSO Control

12.0

1.00

6.3

1.00

Ultrapure Water Control

 

 

73.7

1.00

8.7

1.00

29.3

1.00

Acetone Control

14.0

1.00

65.0

1.00

8.7

1.00

5.3

1.00

30.7

1.00

16

4.7

0.33

44.0

0.68

2.7

0.31

0.0

0.00

15.7

0.51

5

10.3

0.74

57.0

0.88

6.7

0.77

3.3

0.63

25.7

0.84

1.6

16.7

1.19

72.0

1.11

8.7

1.00

5.7

1.06

32.0

1.04

0.5

17.7

1.26

77.0

1.18

8.3

0.96

4.7

0.88

24.7

0.80

0.16

14.0

1.00

75.3

1.16

9.7

1.12

6.0

1.13

29.7

0.97

0.05

13.0

0.93

75.7

1.16

7.3

0.85

5.0

0.94

21.3

0.70

NPD (4mg/plate)

526.7

43.89

SAZ (2mg/plate)

846.7

11.49

1072.0

123.69

9AA (50mg/plate)

473.3

74.74

MMS (2mL/plate)

1173.3

40.00

 

 

MR: Mutation Rate;      NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarcs: Acetone was applied as vehicle for the test item, ultrapure water was applied as vehicle of the positive control substances SAZ and MMS and DMSO was applied as vehicle of NPD, 9AA and 2AA. The mutation rate of the test item and the untreated control is given referring to the acetone, the mutation rate of SAZ and MMS is given referring to ultrapure water and the mutation rate of NPD, 9AA and 2AA is given referring to the DMSO.

 

Applicant's summary and conclusion

Conclusions:
Results of this mutagenicity assay show that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

An in vitro bacteria reverse mutation assay according to OECD 471 was conducted with the test item. The test item was dissolved in acetone. In the initial and confirmatory mutation tests the following concentrations were examined: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate. Because of the noticed strong inhibition obtained in the confirmatory mutation test an additional, complementary pre-incubation test was carried out in the absence of exogenous metabolic activation and the following concentration levels were investigated: -S9: 16, 5, 1.6, 0.5, 0.16 and 0.05 µg/plate. In the initial, confirmatory mutation and complementary pre-incubation tests Salmonella typhimurium TA98, TA100, TA1535, TA1537 strains and Escherichia coli WP2 uvrA were investigated.
Five bacterial strains were used to investigate the mutagenic potential of the test item in independent experiments, in a plate incorporation test (experiment I, initial mutation test), in a pre-incubation test (experiment II, confirmatory mutation test) and because of the strong inhibitory effect obtained in the confirmatory mutation test, in an additional, complementary pre-incubation test. The initial and confirmatory mutation tests were conducted with and without metabolic activation (±S9), the complementary pre-incubation test was performed in absence of metabolic activation (-S9), only. In the performed experiments the concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently).
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (±S9) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the initial mutation test inhibitory effects of the test item were not observed. In the confirmatory mutation test and as well as in the completing complementary pre-incubation test inhibitory effect of the test item was noticed in all strains examined. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn) and/or decreased revertant colony counts (absent revertants or revertants below historical control data and/or below the corresponding vehicle data ranges).

In the initial mutation test following plate incorporation procedure microdrops (colloid-chemical phenomenon) were noticed in all strains at the highest examined concentration of 5000 µg/plate, without and with addition of exogenous metabolic activation (±S9). The obtained microdrops did not interfere with the scoring of the colonies and evaluation of background lawn development in any case.

No microdrops or precipitate of the test item were observed on the plates in the examined bacterial strains at any examined concentration level (±S9) following the pre-incubation procedures.

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.