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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:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant OECD guideline study, available as unpublished report, no restrictions, adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): Eastman TEG-EH
- Description: Eastman(TM) TEG-EH (Triethylene Glycol Bis (2-EthylHexanoate)) Plasticizer
- Physical state: clear colourless liquid
- Specific gravity: 0,968 (20/20C)
- Analytical purity: 99,1% (ester by GC)
- Impurities (identity and concentrations): see confidential details on test material
- Composition of test material, percentage of components: see confidential details on test material
- Purity test date: 2007-07-11
- Lot/batch No.: TD-7022702
- Expiration date of the lot/batch : not provided (production date = 2007-07-11)
- Storage condition of test material: room temperature in the dark

Method

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: uvrB- Salmonella strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was immiscible in water according to the information provided by the Sponsor. However, the test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9-mix
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: approximately 10 hours at 37°C
- Exposure duration: approximately 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test material will be considered mutagenic (positive) in the test system if a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation are observed. Biological relevance of the results will be considered first, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, -tested up to the maximum recommended dose level of 5000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.

Table 1: Test Results: Experiment 1 – Without metabolic activation

Test period

From:16 August 2007

To:19 August 2007

With or without S9-mix

Test substance cc (µg/plate)

Mean number (M) of revertant colonies and the standard deviations (SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

M

SD

M

SD

M

SD

M

SD

M

SD

-

0

101

9.6

18

3.5

20

3.8

20

2.1

16

1.2

-

50

112

58.6

18

3.2

17

1.7

18

4.4

15

5.0

-

150

105

10.0

25

8.4

16

5.3

21

5.5

14

2.9

-

500

98

1.5

22

4.2

18

3.8

19

7.2

15

1.7

-

1500

96

11.5

14

6.0

17

1.5

14

3.0

17

3.8

-

5000

87

18.6

17

4.2

21

2.0

13

3.1

13

4.0

Positive controls

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Cc (µg/plate)

3

5

2

0.2

80

N° colonies per plate

480

88.0

79

16.5

291

47.6

435

43.5

566

72.3

 

Table 2: Test Results: Experiment 1 – With metabolic activation

Test period

From:16 August 2007

To:19 August 2007

With or without S9-mix

Test substance cc (µg/plate)

Mean number (M) of revertant colonies and the standard deviations (SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

M

SD

M

SD

M

SD

M

SD

M

SD

+

0

98

13.6

14

0.0

26

6.8

30

6.1

14

2.6

+

50

83

13.6

11

1.5

19

2.5

28

3.1

17

3.2

+

150

82

12.9

11

2.3

17

6.9

27

3.5

16

2.3

+

500

91

16.3

9

1.2

20

8.1

29

1.5

18

2.1

+

1500

92

7.4

11

2.5

25

5.5

22

4.6

13

1.7

+

5000

86

16.1

14

0.6

26

9.0

27

3.5

13

7.8

Positive controls

+

Name

2AA

2AA

2AA

BP

2AA

Cc (µg/plate)

1

2

10

5

2

N° colonies per plate

559

42.9

206

25.2

332

20.2

252

28.4

331

48.0

 

Table 3: Test Results: Experiment 2 – Without metabolic activation

Test period

From:23 August 2007

To:26 August 2007

With or without S9-mix

Test substance cc (µg/plate)

Mean number (M) of revertant colonies and the standard deviations (SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

M

SD

M

SD

M

SD

M

SD

M

SD

-

0

100

3.2

19

4.0

21

4.9

22

2.5

15

3.5

-

50

91

2.3

16

2.5

18

4.4

22

2.3

14

1.2

-

150

88

2.6

16

1.5

21

4.2

22

0.6

11

2.6

-

500

97

4.0

17

1.2

19

3.5

23

2.5

13

1.5

-

1500

95

9.1

17

4.2

24

4.9

18

2.9

14

0.6

-

5000

74

3.1

17

2.6

21

0.6

23

3.1

14

1.5

Positive controls

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Cc (µg/plate)

3

5

2

0.2

80

N° colonies per plate

662

25.7

303

6.7

614

25.7

367

61.5

653

20.1

 

Table 4: Test Results: Experiment 2 – With metabolic activation

Test period

From:23 August 2007

To:26 August 2007

With or without S9-mix

Test substance cc (µg/plate)

Mean number (M) of revertant colonies and the standard deviations (SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

M

SD

M

SD

M

SD

M

SD

M

SD

+

0

99

2.5

15

0.6

27

2.5

22

4.5

12

5.6

+

50

91

14.6

12

1.5

28

3.8

20

2.6

13

9.2

+

150

87

4.2

15

0.6

24

4.0

22

3.0

7

3.8

+

500

92

12.5

12

3.0

23

6.8

22

4.4

7

3.1

+

1500

88

10.1

13

2.5

22

5.5

21

3.5

10

3.5

+

5000

94

11.7

12

0.6

24

2.1

21

2.6

8

1.5

Positive controls

+

Name

2AA

2AA

2AA

BP

2AA

Cc (µg/plate)

1

2

10

5

2

N° colonies per plate

684

18.0

200

35.0

432

26.6

230

26.1

175

9.5

 

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-l-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella trphimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat

liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment.The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) and positive controls were valid. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.