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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:
experimental study
Adequacy of study:
key study
Study period:
19 Jul - 9 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Messungen und Naturschutz, Karlsruhe, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihexyl adipate
EC Number:
203-757-7
EC Name:
Dihexyl adipate
Cas Number:
110-33-8
Molecular formula:
C18H34O4
IUPAC Name:
dihexyl adipate
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Chemical name: Dihexyl adipate
- Physical state: colourless liquid
- Analytical purity: ≥ 97.5% (97.7% active substance content)
- Purity test date: 2012-06-01
- Lot/batch No.: 12027013
- Expiration date of the lot/batch: 2013-05-30
- Storage condition of test material: normal conditions, keep container tightly closed, and store at dry and well ventilated place

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I: 16, 50, 158, 500, 1581 and 5000 µg/plate* (with and without metabolic activation)
Experiment II: 480, 860, 1540, 2780 and 5000 µg/plate* (with and without metabolic activation)
* corresponding to an active substance content of 97.7%
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (0.05 mL per plate)
- Justification for choice of solvent/vehicle: The test substance is poorly soluble in water (0.3 g/L).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
-S9: 2-NF (1.5 µg/plate, TA 98), SA (0.25-0.5 µg/plate, TA 100 and TA 1535), CHP (0.15 µL/plate, TA 102), 9-AA: 9-aminoacridine (49.5 µg/plate, TA 1537); +S9: BaP: (1.5 µg/plate, TA 98), 2-AA: (2 µg/plate, TA 98; 2.5-5 µL/plate, all other strains)
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
cumene hydroperoxide
other: 2-aminoanthracene
Remarks:
2-NF: 2-nitrofluorene; SA: sodium azide; CHP: cumene hydroperoxide; 9-AA: 9-aminoacridine; BaP: benzo(a)pyrene; 2-AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
Evaluation criteria:
According to OECD guideline 471, a sample was considered mutagenic if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without the metabolic activation system. Statistical methods were used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive result. For the evaluation of the results achieved, the increase of revertants in a sample was expressed as the induction rate [IR], (IR = number of revertants in the sample/number of revertants in the solvent control). In the case of a reproducible increase of the number of revertants and if additionally a statistically significant difference between the mean values was demonstrated the sample was evaluated as positive. The sample was evaluated as positive, if at least a doubling of revertant colonies with the sample compared to the control was observed (Induction rate [IR] ≥ 2).
Statistics:
For statistical evaluation the software SigmaStat Version 3.5 (Systat Software Inc., Point Richmond, USA) was applied. First, the data were checked for normality, then for homogeneity of variance. If variance was not equal for Ames test data, an ANOVA on the ranks applying the Kruskal-Wallis Test was performed followed by a pairwise multiple comparison (Dunn’s test) of the different treatments. In the case of variance homogeneity, one-way ANOVA was performed followed by multiple comparisons versus control group (Dunnett’s test). Statistics were only performed if the induction rates were ≥ 1.5. (Kim and Margolin, 1999).

REFERENCE:
Kim, B.S., Margolin, B.H. (1999): Statistical methods for the Ames Salmonella assay: A Review, Mutat. Res., 436, 113-122

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants in the negative control was within the historical ranges of each tester strain and the positive controls induced at least a doubling of the revertants in comparison to the negative controls. The number of mean revertants per plate after treatment with the positive control substance sodium azide in TA 1535 was significantly different in experiment I compared to experiment II (4644 in experiment I vs. 188 in experiment II). However, the positive control sodium azide fulfilled the criteria for the validity of the assay.

Any other information on results incl. tables

Table 1. Test results of first experiment

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± RSD)

EXPERIMENT I (plate incorporation)

S9-Mix

 

Without

 

Concentration (per plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC (water)

26 ± 28

100 ± 16

824 ± 5

11 ± 13

8 ± 16

SC (DMSO)

19 ± 36

92 ± 7

756 ± 8

10 ± 29

6 ± 46

Test item

 

 

 

 

 

16 µg

26 ± 19

107 ± 15

957 ± 14

12 ± 40

6 ± 44

50 µg

26 ± 45

120 ± 8

748 ± 10

8 ± 15

5 ± 40

158 µg

19 ± 16

100 ± 6

691 ± 6

12 ± 46

5 ± 40

500 µg

21 ± 34

93 ± 5

817 ± 19

16 ± 26

4 ± 48

1581 µg

19 ± 11

96 ± 14

825 ± 16

8 ± 25

6 ± 29

5000 µg

19 ± 6

98 ± 4

797 ± 7

12 ± 25

7 ± 42

PC

 

 

 

 

 

2-NF (1.5 µg)

147 ± 3

-

-

-

-

SA (0.25 µg)

-

-

-

4644 ± 10

-

SA (0.5 µg)

-

6755 ± 19

-

-

-

CHP (0.15 µL)

-

-

1647 ± 10

-

-

9-AA (49.5 µg)

-

-

-

-

1201 ± 19

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC (water)

24 ± 10

113 ± 7

706 ± 7

13 ± 44

8 ± 26

SC (DMSO)

22 ± 34

110 ± 10

565 ± 8

14 ± 12

8 ± 22

Test item

 

 

 

 

 

16 µg

22 ± 23

112 ± 9

716 ± 2

15 ± 33

2 ± 50

50 µg

22 ± 10

108 ± 16

684 ± 20

21 ± 26

9 ± 22

158 µg

23 ± 18

122 ± 7

612 ± 3

21 ± 15

9 ± 24

500 µg

23 ± 22

110 ± 21

599 ± 12

14 ± 21

9 ± 29

1581 µg

20 ± 12

120 ± 12

643 ± 8

24 ± 17

7 ± 53

5000 µg

23 ± 16

103 ± 14

633 ± 2

18 ± 11

9 ± 53

PC

 

 

 

 

 

BP (1.5 µg)

100 ± 12

-

-

-

-

2-AA (2.0 µg)

280 ± 7

-

-

-

-

2-AA (2.5 µL)

-

944 ± 21

1776 ± 2

93 ± 30

-

2-AA (5.0 µL)

-

-

-

-

162 ± 17

NC = negative control; SC = Solvent control; PC = Positive control substances; RSD = relative standard deviation;

2-NF: 2-nitrofluorene; SA: sodium azide; CHP: cumene hydroperoxide; 9-AA: 9-aminoacridine; BaP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Table 2. Test results of second experiment

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± RSD)

EXPERIMENT II (plate incorporation)

S9-Mix

 

Without

 

Concentration (per plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC (water)

20 ± 26

132 ± 5

810 ± 6

16 ± 3

7 ± 31

SC (DMSO)

21 ± 25

110 ± 9

618 ± 7

16 ± 15

7 ± 45

Test item

 

 

 

 

 

480 µg

20 ± 26

96 ± 13

911 ± 7

14 ± 12

3 ± 58

860 µg

26 ± 25

113 ± 4

885 ± 11

12 ± 9

7 ± 8

1540 µg

19 ± 5

119 ± 11

729 ± 12

9 ± 34

5 ± 20

2780 µg

22 ± 25

104 ± 5

876 ± 12

15 ± 28

4 ± 71

5000 µg

17 ± 12

106 ± 13

876 ± 5

12 ± 37

5 ± 60

PC

 

 

 

 

 

2-NF (1.5 µg)

119 ± 18

-

-

-

-

SA (0.25 µg)

-

-

-

188 ± 11

-

SA (0.5 µg)

-

476 ± 33

-

-

-

CHP (0.15 µL)

-

-

2745 ± 9

-

-

9-AA (49.5 µg)

-

-

-

-

1205 ± 20

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC (water)

24 ± 26

112 ± 15

878 ± 4

15 ± 25

8 ± 25

SC (DMSO)

22 ± 23

123 ± 7

620 ± 18

12 ± 16

7 ± 42

Test item

 

 

 

 

 

480 µg

22 ± 28

120 ± 6

576 ± 13

10 ± 40

7 ± 52

860 µg

21 ± 19

113 ± 12

591 ± 27

15 ± 14

5 ± 66

1540 µg

19 ± 21

129 ± 10

644 ± 16

16 ± 4

7 ± 25

2780 µg

18 ± 30

127 ± 1

683 ± 13

15 ± 37

6 ± 17

5000 µg

20 ± 27

120 ± 8

711 ± 14

12 ± 20

8 ± 7

PC

 

 

 

 

 

BP (1.5 µg)

239 ± 21

-

-

-

-

2-AA (2.0 µg)

281 ± 22

-

-

-

-

2-AA (2.5 µL)

-

760 ± 21

1733 ± 28

74 ± 5

-

2-AA (5.0 µL)

-

-

-

-

127 ± 4

NC = negative control; SC = Solvent control; PC = Positive control substances; RSD = relative standard deviation;

2-NF: 2-nitrofluorene; SA: sodium azide; CHP: cumene hydroperoxide; 9-AA: 9-aminoacridine; BaP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative