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Diss Factsheets

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:
11 Jul - 17 Aug 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Didocosyl sebacate
EC Number:
255-730-4
EC Name:
Didocosyl sebacate
Cas Number:
42233-75-0
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
Decanedioic acid, diesters with Fatty alcohols C20-22 (even numbered)
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: white solid
- Storage condition of test material: room temperature in the dark
- Expiration date of the lot/batch: 30 Jun 2013

Method

Target gene:
S. typhimurium strains: his operon
E. coli: trp operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvr B) and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens.
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: The E.coli tester strain contains an uvr A DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented postmitochondrial fraction (S9-mix), prepared from the livers of rats induced with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Experiment I:
- 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Experiment II:
- 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: Tetrahydrofuran was selected as the solvent of choice based on insolubility in water and limited solubility in DMSO, dimethyl formamide, acetonitrile and acetone.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N´-nitro-N-nitrosoguanidine (ENNG), 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA), Benzo(a)pyrene (BP)
Remarks:
other:-S9-mix: ENNG: 2, 3, 5 µg/plate (E. coli, TA 100, TA 1535); 9AA: 80 µg/plate (TA 1537); 4NQO: 0.2 µg/plate (TA 98); +S9-mix: 2AA: 1, 2, 10 µg/plate (TA 100, TA 1535 + TA 1537, E.coli); BP: 5 µg/plate (TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- A cytotoxicity pre-experiment was carried out with the tester strains TA 100 and E. coli WP2 uvr A to determine the non-toxic concentrations for the main genotoxicity experiments.
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity (relative total growth) and precipitate by using a dissecting microscope.
Evaluation criteria:
A test system is considered as mutagenic if
- there is a clear and dose-related increase in the number of revertants and/or a
- a reproducible increase at one or more concentrations
- biological relevance against in-house historical control ranges
- statistical analysis of data as determined by UKEMS
- fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
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:
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
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data.

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: Precipitation of the test material was recorded at concentrations equal or greater than 500 µg/plate.

RANGEFINDING/SCREENING STUDIES: In the preliminary toxicity assay, the maximum dose tested was 5000 µg/plate. The test item was non-toxic to the strains of bacteria used (TA 100 and E.coli WP2 uvr A). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material did not induce cytotoxicity in any of the tested strains at any tested concentration.
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 I (plate incorporation)

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

TA 1535

WP2 uvr A

TA 98

TA 1537

0

116 ±9.9

22 ± 3.1

40 ± 5.0

29 ± 3.2

14 ± 3.5

50

122 ± 3.8

21 ± 1.2

35 ± 3.7

25 ± 4.0

13 ± 1.5

150

115 ± 4.5

24 ± 3.2

28 ± 4.7

26 ± 2.0

15 ± 1.5

500 P

114 ± 7.0

18 ± 4.4

42 ± 3.6

25 ± 5.9

14 ± 2.1

1500 P

117 ± 4.9

22 ± 1.5

37 ± 3.6

28 ± 6.1

17 ± 2.0

5000 P

112 ± 7.6

21 ± 3.5

42 ± 3.0

30 ± 2.0

15 ± 4.2

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

710 ± 45.4

557 ± 20.3

1056 ± 164.5

201 ± 15.7

1207 ± 507.6

+

0

110 ± 2.1

23 ± 3.0

49 ± 5.3

32 ± 6.1

15 ± 2.5

+

50

116 ± 5.0

21 ± 2.5

48 ± 8.0

31 ± 4.4

12 ± 3.5

+

150

103 ± 3.8

22 ± 5.9

41 ± 6.0

30 ± 4.0

15 ± 4.4

+

500 P

112 ± 14.8

21 ± 2.6

47 ± 6.0

33 ± 0.6

14 ± 1.5

+

1500 P

112 ± 11.0

19 ± 1.5

45 ± 3.8

33 ± 1.0

13 ± 3.1

+

5000 P

113 ± 6.1

21 ± 0.6

50 ± 2.6

33 ± 1.7

14 ± 2.3

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1171 ± 45.9

207 ± 28.0

377 ± 46.3

300 ± 48.3

151 ± 9.0

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Table 2: Test results of experiment II (plate incorporation). 

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

TA 1535

WP2 uvr A

TA 98

TA 1537

0

126 ± 7.0

13 ± 6.1

20 ± 5.1

18 ± 5.5

14 ± 5.6

50

120 ± 12.2

11 ± 2.1

19 ± 3.5

20 ± 5.7

12 ± 6.6

150

117 ± 14.4

13 ± 3.1

22 ± 7.5

19 ± 6.5

17 ± 1.2

500 P

117 ± 5.2

13 v 0.0

21 ± 3.2

18 ± 2.1

16 ± 3.5

1500 P

110 ± 21.2

17 ± 5.0

24 ± 3.2

21 ± 1.2

15 ± 4.9

5000 P

104 ± 14.8

12 ± 0.6

23 ± 3.2

17 ± 1.2

12 ± 2.1

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

710 ± 49.7

415 ± 28.6

506 ± 8.3

147 ± 21.6

457 ± 166.5

+

0

113 ± 13.5

14 ± 1.0

24 ± 4.2

25 ± 2.1

12 ± 1.5

+

50

109 ± 8.9

12 ± 2.3

21 ± 4.6

23 ± 5.9

13 ± 2.9

+

150

111 ± 9.3

13 ± 2.0

25 ± 3.5

17 ± 6.7

10 ± 0.6

+

500 P

111 ± 9.1

14 ± 1.7

23 ± 4.4

23 ± 5.3

11 ± 2.6

+

1500 P

104 ± 12.4

11 ± 0.6

27 ± 2.0

25 ± 1.0

13 ± 1.5

+

5000 P

98 ± 2.0

13 ± 1.2

20 ± 2.0

23 ± 5.1

14 ± 1.5

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1109 ± 68.7

458 ± 17.0

254 ± 16.1

275 ± 15.3

187 ± 13.7

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of the study the test material is considered not to be mutagenic in this bacterial mutagenicity test in vitro.