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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 (OECD 471)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(July 21, 1997)
Deviations:
yes
Remarks:
(2-aminoanthracene as sole indicator for the function of S9 mix)
GLP compliance:
yes (incl. QA statement)
Remarks:
(BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Oxirane, [(4-nonylphenoxy)methyl]-, reaction product with ethylene glycol
EC Number:
807-586-4
Cas Number:
634602-80-5
Molecular formula:
Unspecified
IUPAC Name:
Oxirane, [(4-nonylphenoxy)methyl]-, reaction product with ethylene glycol

Method

Target gene:
his- / trp-gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix obtained from phenobarbital and β-naphthoflavone induced rat livers
Test concentrations with justification for top dose:
1st Experiment (standard plate test): 33, 100, 333, 1000, 2500 and 5000 μg/plate
2nd Experiment (preincubation test): 3.3, 10, 33, 100, 333 and 1000 μg/plate (Salmonella strains); 33, 100, 333, 1000, 2500 and 5000 μg/plate (E. coli WP2 uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data were available.
Controls
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
other: 2-aminoanthracene, N-methyl-N'nitrosoguanidine, 4-nitro-o-phenylenediamine
Remarks:
+ S9: 2-AA (TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate; E. coli WP2 uvrA: 60 µg/plate); -S9: MNNG (TA 1535, TA 100: 5 μg/plate); NOPD (TA 98: 10 µg/plate); AAC (TA 1537: 100 µg/plate); 4-NQO (E. coli WP2 uvrA: 5 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: about 20 min (preincubation test)
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 plates per dose or control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp-background growth), reduction in the titer

OTHER:
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.
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.

Assessment 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 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:
cytotoxicity
Remarks:
(from about 333 μg/plate onward depending on the strain and test conditions)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(from about 333 μg/plate onward depending on the strain and test conditions)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect (reduced his-background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 333 μg/plate onward. In the preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed depending on the strain and test conditions from about 333 μg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

0

56 ± 7

10 ± 2

69 ± 8

18 ± 2

6 ± 1

33

66 ± 6

11 ± 3

70 ± 7

17 ± 3

5 ± 2

100

53 ± 8

10 ± 2

72 ± 5

15 ± 2

5 ± 3

333

43 ± 3P

8 ± 1P

63 ± 2P

14 ± 3P

5 ± 2P

1000

11 ± 3P

3 ± 1P

59 ± 5P

8 ± 2P

4 ± 1P

2500

0B/P

0B/P

56 ± 2P

0B/P

0B/P

5000

0B/P

0B/P

28 ± 3P

0B/P

0B/P

Positive controls, –S9

Name

MNNG

MNNG

4-NQO

NOPD

AAC

Concentrations (μg/plate)

5

5

5

10

100

Mean No. of colonies/plate (average of 3 ± SD)

705 ± 61

923 ± 57

806 ± 30

490 ± 69

464 ± 51

+

0

85 ± 8

11 ± 2

68 ± 3

26 ± 7

7 ± 1

+

33

87 ± 11

13 ± 3

69 ± 5

25 ± 3

7 ± 2

+

100

79 ± 9

14 ± 2

68 ± 2

31 ± 8

6 ± 1

+

333

68 ± 8P

12 ± 2P

75 ± 6P

21 ± 3P

5 ± 1P

+

1000

44 ± 10P

8 ± 2P

73 ± 8P

22 ± 3P

4 ± 1P

+

2500

16 ± 6B/P

3 ± 1B/P

62 ± 2P

17 ± 3B/P

2 ± 1B/P

+

5000

0B/P

0B/P

45 ± 5P

0B/P

0B/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)

792 ± 39

138 ± 9

158 ± 4

659 ± 20

161 ± 27

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

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

(μg/plate)

(average of 3 plates ± Standard deviation)

 

TA 100

TA 1535

TA 98

TA 1537

 

E. coli WP2 uvrA

0

53 ± 2

11 ± 2

20 ± 3

7 ± 1

0

60 ± 6

3.3

53 ± 3

10 ± 3

19 ± 4

7 ± 1

33

56 ± 15

10

56 ± 8

11 ± 1

21 ± 4

7 ± 2

100

68 ± 6

33

57 ± 3

11 ± 2

21 ± 3

6 ± 1

333

50 ± 11P

100

50 ± 5

10 ± 2

19 ± 2

7 ± 3

1000

64 ± 3P

333

43 ± 5P

7 ± 1

14 ± 2P

5 ± 2P

2500

44 ± 9P

1000

8 ± 3P

3 ± 1

8 ± 2P

1 ± 1P

5000

23 ± 3P

Positive controls, –S9

Name

MNNG

MNNG

NOPD

AAC

Name

4-NQO

Concentrations (μg/plate)

5

5

10

100

Concentrations (μg/plate)

5

Mean No. of colonies/plate (average of 3 ± SD)

763 ± 33

757 ± 24

584 ± 23

324 ± 22

Mean No. of colonies/plate (average of 3 ± SD)

738 ± 34

+

0

72 ± 6

12 ± 2

24 ± 4

7 ± 2

0

63 ± 3

+

3.3

74 ± 8

11 ± 2

25 ± 3

6 ± 2

33

61 ± 8

+

10

71 ± 8

12 ± 2

25 ± 4

6 ± 1

100

71 ± 7

+

33

72 ± 6

10 ± 1

28 ± 3

6 ± 2

333

61 ± 8P

+

100

67 ± 6

11 ± 2

23 ± 1

6 ± 3

1000

57 ± 7P

+

333

61 ± 3P

9 ± 1P

18 ± 4P

5 ± 1P

2500

46 ± 4P

+

1000

34 ± 6P

4 ± 2P

8 ± 1P

3 ± 0P

5000

30 ± 2P

Positive controls, –S9

Name

2AA

2AA

2AA

2AA

Name

2AA

Concentrations (μg/plate)

2.5

2.5

2.5

2.5

Concentrations (μg/plate)

60

Mean No. of colonies/plate (average of 3 ± SD)

861 ± 55

172 ± 5

602 ± 39

142 ± 12

Mean No. of colonies/plate (average of 3 ± SD)

159 ± 5

P: precipitation

B: reduced backgrownd growth

2-AA: 2-aminoanthracene

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

NOPD: 4-nitro-o-phenylenediamine

AAC: 9-aminoacridine

4-NQO: 4-nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative