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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
21 May 2013 - 23 Sep 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Justification for read-across is given in Section 13 of IUCLID.

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:
adopted 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
adopted Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
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): Didodecyl fumarate; fumaric acid, di-dodecyl ester
- Physical state: solid, melt, white
- Analytical purity: 93.8 area-%
- Lot/batch No.: 0008043725
- Expiration date of the lot/batch: Dec 2013
- Stability under test conditions: guaranteed until Dec 2013
- Storage condition of test material: room temperature

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:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/beta-naphtoflavone
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2 700 and 5400 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: due to the insolubility of the test substance in ultrapure water, acetone 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
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
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: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 – 72 h.

NUMBER OF REPLICATIONS: 3 plates in 2 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn.

OTHER: a sterility control was conducted to exclude contamination of the plates.
Evaluation criteria:
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 test substance is considered positive in this assay if 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 is observed.
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:
other: 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, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: the results of the negative and positive controls are within the historical control data.

Any other information on results incl. tables

The revertans per plate for both plate incorporation and pre-incubation tests are presented in the following tables.

Table: Plate incorporation – without metabolic activation

Treatment

Dose

[µg/plate]

Revertants/plate (mean±SD)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

vehicle control#

19±5

47±2

12±2

7±1

78±10

Test item

33

22±1

44±8

14±4

7±1

69±1

100

20±1

42±2

12±3

6±2

70±5

333

21±4

45±3

13±7

6±1

72±7

1000

18±6

46±3

12±3

7±2

73±9

2700

18±1

43±4

13±2

7±2

68±6

5400

11±2

31±3

9±1

4±1

62±8

Positive control##

828±85

939±13

884±40

338±21

638±22

#vehicle control = acetone

##positive control = N-methyl-N'-nitro-N-nitrosoguanidine 5 μg/plate in DMSO (TA 1535, TA 100), 4-nitro-o-phenylenediamine 10 μg/plate in DMSO (TA 98), 9-aminoacridine 100 μg/plate in DMSO (TA 1537), 4-nitroquinoline-N-oxide 5 μg/plate in DMSO (WP2 uvrA)

 

Table: Plate incorporation – with metabolic activation

Treatment

Dose

[µg/plate]

Revertants/plate (mean±SD)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

vehicle control#

26±3

46±3

14±3

7±1

82±1

Test item

33

33±1

45±6

12±2

8±2

88±9

100

31±2

42±2

15±1

8±2

88±2

333

27±3

45±5

13±2

8±3

81±2

1000

27±2

45±7

15±3

6±1

74±11

2700

20±1

42±5

12±2

6±1

62±3

5400

19±4

43±2

9±1

7±1

73±5

Positive control##

992±74

1089±89

384±21

219±20

280±9

#vehicle control = acetone

##positive control = 2-aminoanthracene 2.5 μg/plate in DMSO (TA 1535, TA 100, TA 1537, TA 98) and 60 μg/plate in DMSO (WP2 uvrA)

 

Table: Pre-incubation – without metabolic activation

Treatment

Dose

[µg/plate]

Revertants/plate (mean±SD)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

vehicle control#

27±5

39±4

12±3

7±2

55±6

Test item

33

29±6

40±7

11±4

6±1

63±17

100

25±5

44±12

11±1

8±1

59±4

333

27±6

40±10

11±3

8±3

59±4

1000

24±4

42±9

12±3

7±3

58±5

2700

18±3

33±4

11±3

7±2

50±3

5400

14±2

35±2

9±1

6±1

46±10

Positive control##

664±9

1205±65

1418±18

881±8

695±76

#vehicle control = acetone

##positive control = N-methyl-N'-nitro-N-nitrosoguanidine 5 μg/plate in DMSO (TA 1535, TA 100), 4-nitro-o-phenylenediamine 10 μg/plate in DMSO (TA 98), 9-aminoacridine 100 μg/plate in DMSO (TA 1537), 4-nitroquinoline-N-oxide 5 μg/plate in DMSO (WP2 uvrA)

 

Table: Pre-incubation – with metabolic activation

Treatment

Dose

[µg/plate]

Revertants/plate (mean±SD)

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

vehicle control#

29±5

46±8

18±3

14±2

57±8

Test item

33

32±6

45±9

17±3

15±5

61±7

100

29±6

53±17

21±4

16±2

58±3

333

31±2

51±6

15±2

12±1

58±7

1000

26±2

48±7

18±3

16±3

56±8

2700

27±3

41±7

16±7

12±1

59±5

5400

28±4

37±3

13±2

10±2

58±8

Positive control##

1113±10

778±14

335±10

330±13

172±12

#vehicle control = acetone

##positive control = 2-aminoanthracene 2.5 μg/plate in DMSO (TA 1535, TA 100, TA 1537, TA 98) and 60 μg/plate in DMSO (WP2 uvrA)

Applicant's summary and conclusion

Conclusions:
not mutagenic with and without metabolic activation.
Executive summary:

The potential mutagenicity of the category substance was tested in a bacterial gene mutation assay according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100. In two independent experiments, the S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were exposed to the test substance at six concentrations (33, 100, 333, 1000, 2700 and 5400 µg/plate). Both experiments were performed in the absence or presence of a liver microsomal activation system (S9 mix) using either the plate incorporation or the pre-incubation method (20 min pre-incubation time).

No cytotoxicity was noted in the treated bacteria strains up to precipitating concentrations when compared to controls. Precipitation of the test substance was observed at 333 µg/plate onward with and without S9 mix. The mean number of revertant colonies per plate was not significantly increased at any test concentration. The solvent and the positive control substances proved the validity of the study for each tester strain.

Under the conditions of this assay, the substance was found to be non-mutagenic in the tested strains, both in the presence and absence of metabolic activation.