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Toxicological information

Genetic toxicity: in vitro

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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:
2013-03-05 to 2013-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimentelle Toxikologie und Ökologie, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: COD-001701
- Expiration date of the lot/batch: 2010-02-01
- Purity test date: 2012-10-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (typically +25°C) or cooler
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed until 01 Nov 2014 as indicated by the sponsor.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Cofactor-supplemented postmitochondrial fraction (S9) obtained from livers of rats treated with an enzyme-inducing agent (phenobarbital/beta-naphthoflavone)
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2600, 5200 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: historical control data which demonstrated that DMSO is suitable
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
Remarks:
see details in results tables
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
see details in results tables
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
see details in results tables
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
see details in results tables
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h to 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
OECD 471 Guideline study test conditions
Evaluation criteria:
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.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.
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.
Statistics:
Calculation of standard deviation, mean

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none occured
- Water solubility: none occured
- Precipitation: none occured
- Definition of acceptable cells for analysis: Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.
- Other confounding effects: none occured

RANGE-FINDING/SCREENING STUDIES: 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.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 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
- Negative (solvent/vehicle) historical control data: The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: detected by decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer

Any other information on results incl. tables

Table 1: Standard Plate test

Concentration
µg/plate

TA 1535

TA 1535

TA 100

TA 100

TA 1537

TA 1537

TA 98

TA 98

E.coli WP2 uvrA

E.coli WP2 uvrA

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Solvent control

9

11

39

51

9

11

15

28

85

93

33 µg

8

10

37

60

9

9

16

27

73

83

100 µg

9

12

42

47

6

10

15

30

78

80

333 µg

8

9

47

47

10

13

12

21

78

90

1000 µg

11

13

47

51

4

9

15

35

83

88

2600 µg

10

15

26

35

5

7

16

23

68

79

5200 µg

7 (R*)

12 (R*)

20 (R*)

25 (R*)

4 (R*)

7 (R*)

12 (R*)

15 (R*)

63 (R*)

46 (R*)

MNNG

1071

-

923

-

-

-

-

-

-

-

2-AA

-

411

-

1464

-

224

-

1291

-

372

AAC

-

-

-

-

545

-

-

-

-

-

NOPD

-

-

-

-

-

-

837

-

-

-

4-NQO

-

-

-

-

-

-

-

-

740

-

R*: Reduced Background Growth

MNNG:N-methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

AAC: 9-aminoacridine

NOPD: 4-nitro-o-phenylendiamine

4-NQO: 4-Nitroquinoline-N-oxide

 

Table 2: Preincubation Test

Concentration
µg/plate

TA 1535

TA 1535

TA 100

TA 100

TA 1537

TA 1537

TA 98

TA 98

E.coli WP2 uvrA

E.coli WP2 uvrA

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Solvent control

11

10

38

39

5

7

20

30

52

26

33 µg

10

12

39

40

5

7

19

27

55

28

100 µg

9

8

43

41

6

6

21

31

49

27

333 µg

9

10

37

42

7

4

21

32

54

21

1000 µg

14

8

40

45

7

5

15

23

58

25

2600 µg

6 (R*)

11 (R*)

20 (R*)

23 (R*)

3 (R*)

3 (R*)

8 (R*)

13 (R*)

18 (R*)

16 (R*)

5200 µg

R*

R*

16

R*

R*

R*

R*

R*

R*

8 (R*)

MNNG

1288

-

1278

-

-

-

-

-

-

-

2-AA

-

192

-

1092

-

201

-

1153

-

252

AAC

-

-

-

-

429

-

-

-

-

-

NOPD

-

-

-

-

-

-

893

-

-

-

4-NQO

-

-

-

-

-

-

-

-

516

-

R*: Reduced Background Growth

MNNG:N-methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

AAC: 9-aminoacridine

NOPD: 4-nitro-o-phenylendiamine

4-NQO: 4-Nitroquinoline-N-oxide

Applicant's summary and conclusion