Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Gelb LD 6259 (No 08/0646-1)
- Physical state: solid/ sun yellow
- Analytical purity: Main component: 87.6 area% or 89.1 area% (depending on the method; see analytical report, study code 08L00346)
- batch No.: 0817 VP 01
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: room temperature; avoid temperatures > 60°C
- Other: the test substance was homogeneous by visual inspection

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction is prepared according to Ames et al. from male Wistar rats [Crl:WI(Han)] (200 - 300 g) treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally
Test concentrations with justification for top dose:
1st Experiment (standard plate test): 0, 20, 100, 500, 2500 and 5000 μg/plate;
2nd experiment (preincubation test): 0, 312.5, 625, 1250, 2500 and 5 000 μg/plate;
3rd experiment (standard plate test):0, 23, 115, 575, 2875 and 5750 μg/plate;
4th experiment (preincubation test): 0, 23, 115, 575, 2875 and 5750 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: to achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly.
All test substance formulations were prepared immediately before administration.
Controls
Untreated negative controls:
yes
Remarks:
sterile controls were also done
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: All dissolved in DMSO; with S9 mix: 2-aminoanthracene (2-AA; 2.5 μg/plate, for TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate for E. coli WP2 uvrA). Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 5 μg/plate for TA 1535, TA 100), see below
Remarks:
positive control without S-9 mix, continue: 4-nitro-o-phenylenediamine (NOPD; 10 μg/plate for TA 98), 9-aminoacridine (AAC; 100 μg/plate for TA 1537 and 4-nitroquinoline-N-oxide (4-NQO; 5 μg/plate for E. coli WP2 uvrA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Expression duration: 48-72 hours in the dark at 37°C

NUMBER OF REPLICATIONS: 4 experiments in triplicates

DETERMINATION OF CYTOTOXICITY
- Method: colonie count. The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: after incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
Evaluation criteria:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was > 108/mL.
Assessment criteria
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
not applicable

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of his+ or trp+ revertants neither in standard plate test nor in preincubation test (see Table 1&2)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test and the preincubation assay depending on the strain and test conditions from about 2 875 μg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No test substance precipitation was found with and without S9 mix
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in four experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Table 1: Standard Plate Test; Maximum revertants per plate and corresponding concentration

 

Strain

Experiment

Dose level (µg/plate)

Maximum revertants per plate (mean of triplicate) at the corresponding dose

Without S-9 mix

With S-9 mix

Mean

SD

FAC

Mean

SD

FAC

 

 

 

TA 1535

 

First

Negative control

16

3

1.0

17

1

1.0

20, 500

17

2

1.1

 

 

 

100

 

 

 

16

2

0.9

Positive Control

586

105

35.9

119

2

6.9

 

Second

Negative control

14

3

1.0

16

2

1.0

115

 

 

 

20

1

1.2

575

20

7

1.4

 

 

 

Positive Control

532

23

37.1

149

40

9.1

 

 

 

TA 100

 

First

Negative control

95

9

1.0

100

16

1.0

20

107

17

1.1

 

 

 

100

 

 

 

99

11

1.0

Positive Control

586

32

6.2

662

95

6.6

 

Second

Negative control

14

6

1.0

16

2

1.0

115

 

 

 

20

1

1.2

575

20

7

1.4

 

 

 

Positive Control

532

23

37.1

149

40

9.1

 

 

 

TA 98

 

First

Negative control

28

2

1.0

34

5

1.0

2500

 

 

 

32

10

1.0

5000

26

3

0.9

 

 

 

Positive Control

462

41

16.3

534

21

15.9

 

Second

Negative control

32

7

1.0

38

4

1.0

23

28

5

1.0

34

6

1.0

Positive Control

437

30

13.8

787

25

20.5

 

 

 

TA 1537

 

First

Negative control

7

1

1.0

8

2

1.0

100

 

 

 

13

2

1.5

5000

10

2

1.3

 

 

 

Positive Control

350

20

47.7

86

12

10.3

 

Second

Negative control

8

2

1.0

8

1

1.0

23

 

 

 

10

1

1.0

5750

10

4

1.3

 

 

 

Positive Control

344

45

43.0

152

37

18.2

 

 

E. coli WP2 uvrA

 

First

Negative control

35

4

1.0

55

2

1.0

2500

39

3

1.1

 

 

 

5000

 

 

 

53

5

1.0

Positive Control

872

18

24.7

252

10

4.6

 

Second

Negative control

34

5

1.0

38

7

1.0

23

39

5

1.1

 

 

 

44

 

 

 

44

4

1.2

Positive Control

649

124

20.3

223

5

5.9

FAC: factor

 

Table 2: Preincubation Test; Maximum revertants per plate and corresponding concentration

 

Strain

Experiment

Dose level (µg/plate)

Maximum revertants per plate (mean of triplicate) at the corresponding dose

Without S-9 mix

With S-9 mix

Mean

SD

FAC

Mean

SD

FAC

 

 

 

TA 1535

 

First

Negative control

16

5

1.0

17

2

1.0

312.5

19

3

1.1

 

 

 

5000

 

 

 

16

2

0.9

Positive Control

572

15

35.0

148

16

8.9

 

Second

Negative control

15

2

1.0

16

3

1.0

23

 

 

 

15

3

0.9

5750

16

4

1.1

 

 

 

Positive Control

1076

23

73.4

127

15

7.8

 

 

 

TA 100

 

First

Negative control

98

4

1.0

98

3

1.0

625

103

6

1.0

104

5

1.1

Positive Control

864

25

8.8

817

60

8.3

 

Second

Negative control

128

9

1.0

112

19

1.0

115

 

 

 

148

4

1.3

575

135

17

1.1

 

 

 

Positive Control

1199

8

1.1

809

53

7.2

 

 

 

TA 98

 

First

Negative control

30

3

1.0

34

5

1.0

312.5

 

 

 

39

2

1.2

2500

31

3

1.0

 

 

 

Positive Control

553

20

18.6

502

164

14.9

 

Second

Negative control

30

4

1.0

29

5

1.0

23

32

6

1.1

34

2

1.2

Positive Control

477

67

15.9

621

36

21.4

 

 

 

TA 1537

 

First

Negative control

8

1

1.0

8

3

1.0

312.5

 

 

 

9

3

1.2

5000

10

5

1.3

 

 

 

Positive Control

393

32

51.2

140

16

18.2

 

Second

Negative control

7

3

1.0

9

2

1.0

115

 

 

 

39

2

1.2

2500

31

3

1.0

 

 

 

Positive Control

553

20

18.6

502

164

14.9

 

 

E. coli WP2 uvrA

 

First

Negative control

30

2

1.0

45

5

1.0

312.5

38

8

1.3

48

9

1.1

Positive Control

576

23

19.0

226

21

5.0

 

Second

Negative control

41

3

1.0

53

8

1.0

2875

52

5

1.3

50

8

0.9

Positive Control

535

45

13.1

242

22

4.6

FAC: factor

 

Applicant's summary and conclusion