Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2020-03-10 to 2020-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
440/2008/EC, 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998-08
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use
Version / remarks:
2011-11
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
his/trp
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 (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
16.0, 50.0, 160.0, 500.0, 1600.0, 5000.0 μg/plate, recommended maximum test concentration
Vehicle / solvent:
- Solvent used: ultrapure water (ASTM Type 1)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
without S9 mix, 4 μg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Remarks without S9 mix, 2 μg/plate for TA100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, 50 μg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, 2 μL/plate for E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
with S9 mix, 2 μg/plate for all TA strains, 50 μg/plate for E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: nutrient agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development
Evaluation criteria:
Evaluation of Experimental Data
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = (Mean revertants at the test item (or control*) treatments) / mean revertants of vehicle control
* untreated, vehicle or positive control

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.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1:Summary Table of the Results of the Initial Mutation Test (Plate Incorporation Test)

Concentrations (μg/plate)

Salmonella typhimuriumtester strains

Escherichia coli WP2 uvrA

 TA98

TA100 

TA1535 

TA1537 

-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

21.0

0.89

22.7

0.84

61.7

0.88

79.7

0.94

11.0

1.03

10.0

1.07

6.0

1.06

5.0

1.00

27.3

0.89

42.3

0.86

DMSO Control

26.3

1.00

23.3

1.00

99.3

1.00

10.0

1.00

5.0

1.00

4.7

1.00

41.7

1.00

Ultrapure Water Control

23.7

1.00

27.0

1.00

70.0

1.00

84.3

1.00

10.7

1.00

9.3

1.00

5.7

1.00

5.0

1.00

30.7

1.00

49.3

1.00

5000

26.3

1.11

27.0

1.00

57.0

0.81

89.0

1.06

10.7

1.00

9.0

0.96

6.7

1.18

6.0

1.20

38.3

1.25

36.3

0.74

1600

27.0

1.14

28.3

1.05

55.3

0.79

90.0

1.07

8.7

0.81

12.3

1.32

6.3

1.12

8.0

1.60

42.0

1.37

51.7

1.05

500

25.3

1.07

18.7

0.69

56.0

0.80

88.7

1.05

8.0

0.75

10.7

1.14

4.7

0.82

4.7

0.93

45.3

1.48

49.7

1.01

160

27.0

1.14

25.3

0.94

70.3

1.00

78.7

0.93

8.0

0.75

10.7

1.14

4.3

0.76

6.0

1.20

38.3

1.25

46.3

0.94

50

20.7

0.87

27.0

1.00

61.0

0.87

88.7

1.05

9.7

0.91

10.3

1.11

4.3

0.76

5.7

1.13

35.3

1.15

40.7

0.82

16

23.3

0.99

24.3

0.90

69.7

1.00

90.3

1.07

8.0

0.75

9.7

1.04

8.0

1.41

5.7

1.13

37.3

1.22

44.7

0.91

NPD (4 μg)

377.7

14.34

SAZ (2 μg)

654.3

9.35

956.0

89.63

9AA (50 μg)

204.0

40.80

MMS (2 μL)

629.7

20.53

2AA (2 μg)

1293.3

55.43

1130.7

11.38

141.7

14.17

98.3

21.07

2AA (50 μg)

167.3

4.02

Table 2: Summary Table of the Results of the Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (μg/plate)

Salmonella typhimuriumtester strains

Escherichia coli 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

24.3

0.91

28.0

0.70

80.7

0.97

90.3

0.85

6.3

0.70

10.7

1.00

6.7

1.05

5.3

1.14

19.3

0.69

27.3

0.79

DMSO Control

20.0

1.00

26.0

1.00

88.3

1.00

10.7

1.00

6.0

1.00

6.7

1.00

45.0

1.00

Ultrapure Water Control

26.7

1.00

40.0

1.00

83.3

1.00

106.0

1.00

9.0

1.00

10.7

1.00

6.3

1.00

4.7

1.00

28.0

1.00

34.7

1.00

5000

9.7

0.36

9.7

0.24

32.3

0.39

57.7

0.54

9.7

1.07

10.0

0.94

2.3

0.37

6.3

1.36

14.0

0.50

19.0

0.55

1600

7.7

0.29

32.7

0.82

36.7

0.44

114.0

1.08

8.3

0.93

9.3

0.88

2.3

0.37

6.3

1.36

27.3

0.98

45.7

1.32

500

17.7

0.66

28.0

0.70

55.0

0.66

120.3

1.14

10.7

1.19

12.0

1.13

7.7

1.21

3.7

0.79

41.3

1.48

50.0

1.44

160

23.7

0.89

29.7

0.74

83.7

1.00

113.0

1.07

7.0

0.78

8.7

0.81

8.3

1.32

3.7

0.79

42.3

1.51

53.0

1.53

50

25.3

0.95

29.0

0.73

83.3

1.00

108.3

1.02

10.7

1.19

10.7

1.00

5.3

0.84

6.7

1.43

39.0

1.39

36.3

1.05

16

27.7

1.04

23.0

0.58

85.3

1.02

117.0

1.10

8.3

0.93

11.0

1.03

5.3

0.84

6.3

1.36

45.0

1.61

61.3

1.77

NPD (4 μg)

281.3

14.07

SAZ (2 μg)

552.0

6.62

777.3

86.37

9AA (50 μg)

799.3

133.22

MMS (2 μL)

1312.0

46.86

2AA (2 μg)

2044.0

78.62

1375.3

15.57

110.0

10.31

153.3

23.00

2AA (50 μg)

211.0

4.69

Applicant's summary and conclusion

Conclusions:
In an bacterial reverese mutation assay (AMES) according to OECD Guideline 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.
Executive summary:

The mutagenic potential of the test item was determined in an in vitro bacterial reverse mutation assay (AMES) according to OECD Guideline 471. Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (16, 50, 160, 500, 1600, 5000 µg/plate), including the controls, were tested in triplicate (positive and negative controls were run concurrently). Negative, vehicle and positive controls were valid. No cytotoxicity was observed up to the max. concentration. No precipitation was observed throughout the study. No substantial increases were observed in revertant colony numbers of any of the five tester strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay shows, 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.