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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 Jul 1997
Deviations:
yes
Remarks:
2-Aminoanthracene is used as the sole indicator of the efficacy of the S9-mix.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): L4-Ligand
- Physical state: Solid, white
- Analytical purity: 97.0 g/100 g
- Lot/batch No.: 0005473663
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period is guaranteed until 01 Feb 2013
- Storage condition of test material: Room temperature (protected from humidity; N2 conditions)

Method

Target gene:
his and trp operon
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital i.p. and β-naphthoflavone orally.
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500 and 5 000 μg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in ultrapure water, ethanol was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S9 mix for all strains
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
without S9 mix for strains TA 1535, TA 100
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
without S9 mix for strain TA 98
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix for strain 1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix for strain E. coli WP2 uvrA
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
The test substance 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.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: A weak bacteriotoxic effect was occasionally observed in the SPT depending on the strain and test conditions from about 1000 μg/plate onward. In the PIT no bacteriotoxicity was observed up to the highest required concentration.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2 500 μg/plate onward only without S9 mix.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (standard plate test).

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

E. coli

TA98

TA1537

0

78 ± 7

11 ± 1

56 ± 8

31 ± 3

9 ± 3

33

73 ± 21

11 ± 1

54 ± 10

28 ± 7

7 ± 2

100

78 ± 1

11 ± 1

52 ± 3

23 ± 3

10 ± 4

333

80 ± 8

11 ± 1

52 ± 8

30 ± 3

7 ± 2

1000

83 ± 15

11 ± 2

50 ± 7

26 ± 1

5 ± 2

2500

72 ± 7P

11 ± 1P

47 ± 11P

30 ± 9 P

5 ± 4 P

-

5000

76 ± 16P

10 ± 2P

41 ± 3 P

24 ± 5 P

6 ± 3 P

Positive controls, –S9

Name

MNNG

MNNG

4NQO

4NOPD

AAC

Concentrations

(μg/plate)

5

5

5

10

100

Mean No. of colonies/plate

(average of 3 ± SD)

655 ± 57

625 ± 54

940 ± 24

636 ± 67

446 ± 31

+

0

80 ± 10

11 ± 1

49 ± 10

36 ± 8

8 ± 2

+

33

86 ± 17

13 ± 2

43 ± 6

30 ± 3

8 ± 1

+

100

73 ± 4

13 ± 3

53 ± 5

42 ± 3

10 ± 6

+

333

82 ± 13

12 ± 2

47 ± 10

35 ± 8

8 ± 1

+

1000

75 ± 12

12 ± 1

44 ± 7

34 ± 5

7 ± 5

+

2500

89 ± 5P

12 ± 1P

50 ± 7 P

38 ± 6 P

8 ± 2 P

+

5000

82 ± 15P

11 ± 1P

54 ± 8 P

30 ± 3 P

7 ± 1 P

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

60

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1198 ± 125

146 ± 31

255 ± 13

 

968 ± 11

155 ± 7

 MNNG = N-methyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

4NOPD = 4-nitroquinoline-N-oxide

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

P = Precipitate

Table 2: Test results of experiment 2 (preincubation test).

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

E. coli

TA98

TA1537

0

74 ± 7

12 ± 1

37 ± 4

19 ± 1

7 ± 2

33

82 ± 7

12 ± 3

39 ± 6

20 ± 5

7 ± 1

100

76 ± 8

13 ± 2

36 ± 8

20 ± 3

7 ± 3

333

76 ± 7

11 ± 3

35 ± 10

21 ± 5

7 ± 2

1000

76 ± 3

12 ± 2

37 ± 7

19 ± 2

7 ± 1

2500

73 ± 7P

12 ± 1P

37 ± 6P

21 ± 2 P

6 ± 2 P

-

5000

73 ± 3P

12 ± 2P

38 ± 6P

20 ± 4 P

6 ± 2 P

Positive controls, –S9

Name

MNNG

MNNG

4NQO

4NOPD

AAC

Concentrations

(μg/plate)

5

5

5

10

100

Mean No. of colonies/plate

(average of 3 ± SD)

869 ± 36

758 ± 24

648 ± 88

541 ± 29

367 ± 28

+

0

83 ± 11

12 ± 2

44 ± 4

24 ± 3

8 ± 2

+

33

86 ± 9

12 ± 2

48 ± 3

27 ± 4

7 ± 2

+

100

83 ± 6

12 ± 3

44 ± 4

25 ± 4

8 ± 2

+

333

79 ± 5

11 ± 3

40 ± 3

25 ± 4

6 ± 2

+

1000

79 ± 4

14 ± 1

45 ± 8

21 ± 4

8 ± 1

+

2500

71 ± 7P

11 ± 2P

40 ± 6P

23 ± 3 P

7 ± 2 P

+

5000

75 ± 8P

12 ± 3P

41 ± 3P

21 ± 3 P

7 ± 0 P

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

60

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

890 ± 97

154 ± 20

272 ± 14

 

687 ± 44

149 ± 27

 

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

4NOPD = 4-nitroquinoline-N-oxide

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

P = Precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative