Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Aug - 15 Sep 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in a closed container, protected from light and humidity

Method

Target gene:
his operon, tryp operon
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:
cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats, treated with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Based on a range-finding study (performed in tester strains TA 98 and TA 100; doses applied: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate μg/mL), the following concentrations were used in the main experiments:
First and second experiment (all strains): 16, 50, 160, 500, 1600 and 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in DMSO, it was selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first experiment); preincubation (second experiment)

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and inspection of the bacterial background lawn
Evaluation criteria:
Acceptance criteria
The study was considered valid if:
- the phenotypes of the tester strains could be confirmed
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains
- the tester strain culture titers are in the 109 cells/mL order
- the batch of S9 used in this study shows the appropriate biological activity
- the reference mutagens show an increase of at least: 3-fold in induced revertant colonies over the mean value of the respective vehicle control
- at least five analyzable concentrations were presented in all strains of the main tests (a minimum of three non-toxic dose levels is required to evaluate assay data)

Evaluation criteria
When the test substance shows a biologically relevant and dose-related increase in the number of revertant colonies of more than two times (TA 100) or three times (TA 98, TA 1535, TA 1537 and WP2 uvrA ) compared to that of the solvent control, the response is judged to be positive. Additionally, the positive response should be reproducible for at least one of the dose groups and should occur in at least one strain with or without metabolic activation. The biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is therefore not regarded as necessary.

The test item was considered to have shown no mutagenic activity in this study 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.
Statistics:
Mean values and standard deviations were calculated.

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
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
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
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:
cytotoxicity
Remarks:
number of revertant colonies decreased to almost half compared to solvent control in exp. 2 at 5000 μg/ plate with S9 mix (58 %)
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1:Summary of test results (experiment 1; Initial Mutation Test, Plate Incorporation Method)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates)

Frameshift type

Base-pair substitution type

TA1537

TA98

TA100

TA1535

WP2 uvrA

Solvent control (ultrapure water)

-

-

97.3 ± 6.66

9.3 ± 2.08

22.7 ± 1.53

Solvent control (DMSO)

6.7 ± 2.31

15.3 ± 3.21

92.7 ± 11.72

11.0 ± 4.36

21.7 ± 2.08

Untreated control

5.7 ± 1.15

12.3 ± 1.53

96.0 ± 7.00

11.0 ± 3.00

23.7 ± 3.51

16

6.3 ± 1.15

21.3 ± 6.03

94.3 ± 11.37

13.7 ± 2.08

27.3 ± 8.50

50

6.3 ± 2.08

15.0 ± 7.21

98.7 ± 4.62

10.3 ± 2.08

27.7 ± 6.11

160

9.7 ± 3.79

15.0 ± 2.00

92.3 ± 12.66

15.0 ± 2.65

30.7 ± 7.64

500

6.0 ± 1.00

15.7 ± 1.15

85.0 ± 5.57

9.0 ± 3.61

28.7 ± 10.02

1600

6.7 ± 3.21

14.0 ± 2.00

81.7 ± 5.86

12.7 ± 3.06

24.7 ± 6.66

5000

10.0 ± 6.00

17.7 ± 4.04

94.0 ± 6.24

11.0 ± 2.65

22.7 ± 5.86

Positive controls (unit/plate)

9AA
(50 µg)

4-NPD
(4 µg)

SAZ
(2 µg)

SAZ
(2 µg)

MMS
(2 µL)

Mean No. of colonies/plate (average of 3 plates)

385.3 ± 68.86

318.0 ± 17.09

1450.7 ± 56.76

1069.3 ± 148.02

1162.7 ± 68.97

+

Solvent control (DMSO)

7.0 ± 3.00

20.0 ± 3.61

96.7 ± 8.62

12.7 ± 4.73

28.3 ± 3.21

Untreated control

6.3 ± 2.52

23.7 ± 4.04

129.3 ± 6.43

10.0 ± 2.65

28.7 ± 3.79

16

7.3 ± 2.08

22.7 ± 6.11

121.0 ± 14.42

12.0 ± 4.00

33.7 ± 1.15

50

8.3 ± 4.04

21.7 ± 6.35

125.3 ± 7.57

12.7 ± 1.53

36.3 ± 3.51

160

10.3 ± 4.62

20.3 ± 0.58

117.7 ± 4.16

11.3 ± 3.06

28.7 ± 2.08

500

7.3 ± 2.89

18.0 ± 5.00

122.7 ± 5.51

12.3 ± 0.58

32.3 ± 6.81

1600

7.7 ± 3.21

21.3 ± 8.96

105.7 ± 13.65

14.7 ± 2.52

28.0 ± 7.94

5000

6.3 ± 1.15

18.0 ± 2.65

118.3 ± 8.74

12.0 ± 1.00

30.3 ± 2.52

Positive controls (µg/plate)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(50)

Mean No. of colonies/plate (average of 3 plates)

140.0 ± 14.42

1373.3 ± 124.28

1957.3 ± 367.07

160.0 ± 14.73

154.7 ± 6.11

9AA = 9-aminoacridine

4-NPD = 4-nitro-1,2-phenylene-diamine

SAZ = sodium azide

MMS = methyl-methanesulfonate

2AA = 2-aminoanthracene

  

Table 2: Summary of test results (experiment 2; Confirmatory Mutation Test, Pre-Incubation

Method)

With or without S9-Mix

Test substance (μg/plate)

Mean number of revertant colonies per plate (average of 3 plates)

Frameshift type

Base-pair substitution type

TA1537

TA98

TA100

TA1535

WP2 uvrA

Solvent control (ultrapure water)

-

-

94.7 ± 15.37

13.3 ± 5.51

26.3 ± 3.06

Solvent control (DMSO)

7.7 ± 4.73

18.3 ± 3.51

82.0 ± 6.56

12.7 ± 5.51

20.0 ± 4.36

Untreated control

8.0 ± 5.29

17.3 ± 5.86

93.0 ± 9.17

14.3 ± 5.13

24.7 ± 7.02

16

7.7 ± 0.58

19.7 ± 1.15

90.7 ± 4.62

10.3 ± 4.04

20.0 ± 2.65

50

7.7 ± 5.13

13.7 ± 3.21

86.0 ± 7.21

11.7 ± 3.79

21.3 ± 2.89

160

5.7 ± 2.52

16.3 ± 2.52

92.7 ± 11.02

12.3 ± 2.08

26.3 ± 3.06

500

9.3 ± 7.09

16.3 ± 2.52

81.3 ± 6.11

13.7 ± 2.52

22.0 ± 2.65

1600

8.7 ± 4.73

20.7 ± 4.04

82.0 ± 15.62

11.3 ± 0.58

27.3 ± 4.62

5000

9.0 ± 3.46

16.3 ± 1.53

82.7 ± 7.02

12.3 ± 4.16

22.3 ± 0.58

Positive controls (unit/plate)

9AA
(50 µg)

4-NPD
(4 µg)

SAZ
(2 µg)

SAZ
(2 µg)

MMS
(2 µL)

Mean No. of colonies/plate (average of 3 plates)

818.0 ± 20.30

276.0 ± 28.00

1149.3 ± 160.86

665.3 ± 20.13

976.0 ± 50.12

+

Solvent control (DMSO)

6.3 ± 0.58

21.0 ± 1.00

98.0 ± 21.00

9.7 ± 1.15

35.0 ± 3.61

Untreated control

7.7 ± 0.58

21.0 ± 3.46

104.3 ± 2.08

13.0 ± 2.65

27.7 ± 4.73

16

7.7 ± 4.73

17.0 ± 6.00

106.7 ± 12.22

11.0 ± 2.65

44.0 ± 7.21

50

5.0 ± 3.46

20.3 ± 8.50

100.0 ± 8.72

10.0 ± 1.00

37.0 ± 7.21

160

7.0 ± 1.00

23.0 ± 2.65

104.7 ± 8.08

8.0 ± 1.73

37.7 ± 4.16

500

5.7 ± 4.62

29.0 ± 9.54

108.7 ± 6.11

12.0 ± 5.57

44.0 ± 4.58

1600

5.7 ± 2.31

32.0 ± 6.93

106.3 ± 8.50

13.0 ± 1.73

42.0 ± 1.73

5000

3.7 ± 0.58

16.0 ± 4.00

103.3 ± 4.16

9.3 ± 1.15

44.7 ± 9.07

Positive controls (µg/plate)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(50)

Mean No. of colonies/plate (average of 3 plates)

137.0 ± 19.08

1648.0 ± 85.51

1124.7 ± 127.03

165.0 ± 8.19

165.0 ± 25.12

9AA = 9-aminoacridine

4-NPD = 4-nitro-1,2-phenylene-diamine

SAZ = sodium azide

MMS = methyl-methanesulfonate

2AA = 2-aminoanthracene

Applicant's summary and conclusion