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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:
15 - 24 Aug 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Version / remarks:
June 2012
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 9-oxo-9H-thioxanthene-2-carboxylate
EC Number:
280-960-7
EC Name:
Ethyl 9-oxo-9H-thioxanthene-2-carboxylate
Cas Number:
83817-60-1
Molecular formula:
C16H12O3S
IUPAC Name:
ethyl 9-oxo-9H-thioxanthene-2-carboxylate
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material as recommended by supplier: < 25 °C in a tighly closed container, protected from direct sunlight and heat
- Storage condition at the testing facility: at room temperature

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:
Prior to the main experiments, a range-finder study was performed using the TA 98 and TA 100 strains with following test concentrations: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate; testing was done in presence and absence of S9 mix. Precipitation was noticed at both, 1600 and 5000 µg/plate, in absence and presence of S9 mix; however, precipitation did not disturb the scoring at 1600 µg/plate. Therefore, 1600 µg/plate was selected as top concentration to be tested within the main experiments.
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 and its compatibility with the bacteria and the S9 mix activity, DMSO 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, confirmatory 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 10^9 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 is considered to have no mutagenic activity in bacteria 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, but tested up to precipitating 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, but tested up to precipitating 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, but tested up to precipitating 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, but tested up to precipitating 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Precipitation occurred at 500 and 1600 µg/plate without S9 mix and at 1600 µg/plate with S9 Mix in both, the first and the second confirmatory experiment.

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)

-

-

102.7 ± 2.31

7.3 ± 1.15

17.7 ± 3.79

Solvent control (DMSO)

5.3 ± 0.58

19.0 ± 1.73

85.7 ± 6.03

11.3 ± 4.04

17.0 ± 3.00

Untreated control

6.7 ± 2.08

18.0 ± 1.00

104.0 ± 9.17

8.7 ± 5.13

20.0 ± 0.00

5

5.0 ± 1.00

15.7 ± 2.31

87.0 ± 5.57

11.0 ± 1.00

23.0 ± 4.36

16

5.3 ± 4.04

16.0 ± 3.46

84.3 ± 12.06

8.7 ± 4.93

25.0 ± 5.57

50

4.7 ± 0.58

16.3 ± 9.24

84.0 ± 13.00

13.7 ± 2.31

22.3 ± 2.52

160

5.3 ± 2.31

15.7 ± 4.51

81.3 ± 8.50

11.7 ± 2.08

28.7 ± 3.51

500

13.3 ± 1.15 P

12.3 ± 3.21 P

71.0 ± 7.94 P

13.7 ± 0.58 P

22.7 ± 1.53 P

1600

6.7 ± 3.06 P

17.3 ± 3.06 P

77.7 ± 4.62 P

12.3 ± 1.15 P

24.3 ± 7.09 P

Positive controls (unit/plate)

9AA
(50 µg)

NPD
(4 µg)

SA
(2 µg)

SA
(2 µg)

MMS
(2 µL)

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

750.0 ± 84.59

342.7 ± 26.10

1245.3 ± 185.44

672.0 ± 66.81

818.7 ± 118.68

+

Solvent control (DMSO)

7.3 ± 3.06

21.7 ± 3.61

128.0 ± 18.33

14.0 ± 2.56

28.0 ± 2.00

Untreated control

6.3 ± 1.53

21.7 ± 6.11

132.0 ± 11.14

12.3 ± 4.93

29.3 ± 4.16

5

8.3 ± 3.51

23.3 ± 2.89

124.0 ± 2.00

12.3 ± 3.51

26.7 ± 3.21

16

7.7 ± 3.06

27.0 ± 7.94

113.3 ± 3.06

13.3 ± 5.51

33.0 ± 4.58

50

9.0 ± 2.00

29.3 ± 3.51

108.0 ± 5.29

13.0 ± 1.73

28.7 ± 0.58

160

10.0 ± 4.58

24.3 ± 2.52

104.7 ± 6.43

15.0 ± 5.29

29.7 ± 4.16

500

9.0 ± 3.61

28.0 ± 4.36

116.0 ± 5.29

14.3 ± 2.52

24.3 ± 3.51

1600

4.3 ± 2.31 P

30.0 ± 5.29 P

128.7 ± 6.43 P

14.3 ± 2.52 P

34.0 ± 2.65 P

Positive controls (µg/plate)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(50)

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

110.0 ± 17.06

1512.0 ± 246.45

2200.0 ± 381.58

199.3 ± 37.65

234.0 ± 15.62

SD = standard deviation

9AA = 9-aminoacridine

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

SA = sodium azide

MMS = methylmethanesulfonate

2AA = 2-aminoanthracene

P = precipitate

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)

-

-

93.3 ± 6.66

11.7 ± 3.79

28.3 ± 1.15

Solvent control (DMSO)

8.0 ± 4.00

16.7 ± 0.58

78.0 ± 9.64

11.0 ± 3.00

24.3 ± 6.43

Untreated control

8.0 ± 2.65

17.7 ± 9.07

90.3 ± 13.20

9.3 ± 2.52

19.3 ± 2.52

5

6.7 ± 4.51

25.0 ± 10.44

78.7 ± 11.72

9.7 ± 1.15

14.0 ± 4.00

16

8.3 ± 4.93

17.0 ± 2.65

78.7 ± 6.11

8.7 ± 4.73

16.0 ± 3.61

50

8.0 ± 2.65

15.3 ± 3.51

75.3 ± 4.16

11.3 ± 6.43

22.3 ± 6.51

160

8.0 ± 4.58

16.3 ± 3.06

82.0 ± 10.00

15.0 ± 2.00

15.7 ± 3.21

500

7.0 ± 5.20 P

17.3 ± 5.51 P

84.3 ± 10.12 P

10.7 ± 1.53 P

20.0 ± 5.29 P

1600

8.3 ± 4.04 P

18.7 ± 1.53 P

86.7 ± 5.03 P

15.0 ± 1.00 P

31.7 ± 5.51 P

Positive controls (unit/plate)

9AA
(50 µg)

NPD
(4 µg)

SA
(2 µg)

SA
(2 µg)

MMS
(2 µL)

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

356.0 ± 107.63

254.0 ± 40.60

1210.7 ± 122.46

965.3 ± 53.27

962.7 ± 158.86

+

Solvent control (DMSO)

6.3 ± 3.06

27.7 ± 2.89

108.7 ± 17.62

13.3 ± 3.21

27.7 ± 4.04

Untreated control

7.7 ± 4.93

28.7 ± 3.51

129.7 ± 8.96

11.7 ± 0.58

32.3 ± 4.51

5

6.7 ± 2.31

20.7 ± 8.02

100.0 ± 8.72

9.3 ± 2.89

28.7 ± 3.21

16

6.0 ± 2.65

19.7 ± 6.66

100.3 ± 8.14

12.0 ± 3.46

31.3 ± 3.51

50

5.3 ± 3.21

20.3 ± 6.11

100.7 ± 8.33

12.0 ± 4.58

35.0 ± 9.85

160

11.7 ± 1.53

17.7 ± 7.23

94.7 ± 5.03

15.0 ± 1.00

35.7 ± 2.52

500

8.3 ± 3.21

19.7 ± 5.13

108.0 ± 17.09

9.3 ± 4.04

33.3 ± 5.03

1600

5.0 ± 0.00 P

22.3 ± 4.16 P

126.7 ± 11.02 P

14.7 ± 1.15 P

36.7 ± 11.68 P

Positive controls (µg/plate)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(2)

2AA
(50)

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

164.7 ± 9.02

1117.3 ± 120.09

1114.7 ± 360.65

138.0 ± 7.81

170.3 ± 15.57

SD = standard deviation

9AA = 9-aminoacridine

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

SA = sodium azide

MMS = methylmethanesulfonate

2AA = 2-aminoanthracene

P = precipitate

Applicant's summary and conclusion