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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
2013-08-29 to 2013-09-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
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:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
5 mg/plate or 5 μL/plate were generally selected as maximum test dose.
1st experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
2nd experiment: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and 4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
Standard Plate Test and Preincubation Test both with and without metabolic activation
METHOD OF APPLICATION: Standard plate test based on the method of Ames et al. + Preincubation test based on the method described by Yahagi et al. and Matsushima et al.

Conditions
- Preincubation period: 20 minutes using a shaker
- Exposure duration: 48 - 72 hours
- Temperatur: Incubation at 37 °C

Titer determination
- 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.

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer was recorded for all test groups both with and without S9 mix in all experiments and indicated
in the tables.
Evaluation 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 S-9 mix or after adding a metabolizing system. A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Standard plate test

Preincubation test

TA1535

TA1535

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

11

11

DMSO

11

11

 

33

13

12

33

10

11

 

100

11

11

100

11

13

 

333

12

11

333

10

12

 

1000

12

11

1000

12

9

 

2500

11

10

2500

11

10

 

5000

12

11

5000

10

11

 

 

MNNG

1130

MNNG

1198

 

2-AA

362

2-AA

453

 

 

 

TA100

TA100

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

48

55

DMSO

59

63

 

33

48

52

33

59

67

 

100

49

47

100

56

65

 

333

51

52

333

58

64

 

1000

50

50

1000

59

63

 

2500

52

48

2500

61

64

 

5000

50

50

5000

59

62

 

 

MNNG

1201

MNNG

1477

 

2-AA

1339

2-AA

1606

 

 

 

TA1537

TA1537

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

5

7

DMSO

6

7

 

33

6

6

33

6

7

 

100

7

7

100

6

7

 

333

6

5

333

6

6

 

1000

6

6

1000

6

7

 

2500

6

7

2500

6

6

 

5000

5

8

5000

7

7

 

 

AAC

559

AAC

547

 

2-AA

234

2-AA

229

 

 

 

TA98

TA98

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

18

22

DMSO

16

18

 

33

20

24

33

16

19

 

100

17

24

100

14

17

 

333

19

27

333

15

19

 

1000

16

28

1000

16

17

 

2500

15

21

2500

15

19

 

5000

20

22

5000

15

17

 

 

NOPD

510

NOPD

864

 

2-AA

1239

2-AA

1153

 

 

 

E.coli

E.coli

 

Dose (µg)

Without S9 Mix

With S9 Mix

Dose (µg)

Without S9 Mix

With S9 Mix

 

DMSO

74

63

DMSO

34

32

 

33

66

65

33

34

29

 

100

65

66

100

35

32

 

333

64

70

333

34

33

 

1000

80

68

1000

34

31

 

2500

65

67

2500

36

30

 

5000

69

66

5000

35

29

 

 

4-NQO

824

4-NQO

1278

 

2-AA

554

2-AA

434

 

Applicant's summary and conclusion