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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:
04 October 2007 - 21 October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) 2,2'-thiobisacetate
EC Number:
246-131-9
EC Name:
Bis(2-ethylhexyl) 2,2'-thiobisacetate
Cas Number:
24293-43-4
Molecular formula:
C20H38O4S
IUPAC Name:
2-ethylhexyl 2-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)acetate
Details on test material:
- Name of test material (as cited in study report): Di (2-ethylhexyl) thiodiglycolate
- Physical state: Clear yellow liquid
- Analytical purity: 97.0 %
- Lot/batch No.: 20705
- Expiration date of the lot/batch: 17 March 2008
- Storage condition of test material: Room temperature in the dark, over silica gel

Method

Target gene:
genes involved in histidine synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal preparation (S9)
Test concentrations with justification for top dose:
Sighting study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Main study: 0, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: Test substance was insoluble in water, but fully soluble in DMSO at 50 mg/mL.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine, Mitomycin C, 4-nitroquinoline-N-oxide, 9-aminoacridine, 2-Aminoanthracene, Benzo(a)pyrene, 1,8-Dihydroxanthraquinone
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (direct plate incorporation);

DURATION
- Exposure duration: 48 hours at 37ºC

NUMBER OF REPLICATIONS:
Experiment 1: Five concentrations of the test material were assayed in triplicate against each tester strain.
Experiment 2: Methodology as for experiment 1, using fresh bacterial cultures, test material and control solutions.

NUMBER OF CELLS EVALUATED: 1 to 9.9 x 10(9) bacteria per mL.

DETERMINATION OF CYTOTOXICITY
growth of bacterial background lawn.

Evaluation criteria:
Several criteria for determining a positive result, such as 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. Statistical significance and biological relevance were considered.

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
of 5000 µg/plate, at which precipitation occured.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
Remarks:
of 5000 µg/plate, at which precipitation occured.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test substance was not soluble in water
- Precipitation: A globular precipitate was observed at 5000 µg/plate. This did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The dose range from 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
S. typhimurium TA 100 was used with and without metabolic activation.
No cytotoxicity was observed up to the limit concentration of 5000 µg/plate in S. typhimurium TA100.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the historical vehicle and positive controls were in the range of the concurrent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The material was non-toxic to the strains of bacteria used up to the limit concentration of 5000 µg/plate as determined by the growth of the background lawn.

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

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

-

0

78

12

219

16

10

-

50

78

12

228

13

11

-

150

76

17

237

14

4

-

500

78

15

226

19

7

-

1500

80

14

231

15

7

-

5000

84 P

14 P

231 P

11 P

7 P

Positive

controls

- S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Concentrations

(μg/plate)

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3)

994

619

944

126

4601

 

 

TA 100

TA1535

TA102

TA98

TA1537

+

0

76

11

269

29

15

+

50

80

12

255

27

8

+

150

82

13

263

27

10

+

500

81

10

256

21

13

+

1500

69

13

246

23

14

+

5000

70 P

13 P

234 P

19 P

7 P

Positive

controls

+ S9

Name

2AA

2AA

DAN

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3)

981

158

931

301

445

Concurrent Negative control

 

0

71

14

264

17

11

 

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

MMC = mitomycin C

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

DAN = 1,8-Dihydroxanthraquinone

P = Precipitate

 

 

 

 

 

 

Table 2: Test Results of Experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA1537

-

0

87

23

258

19

11

-

50

63

26

253

15

11

-

150

74

20

256

19

12

-

500

72

21

228

11

9

-

1500

69

18

254

11

4

-

5000

73 P

20 P

241 P

14 P

6 P

Positive

controls

- S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Concentrations

(μg/plate)

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3)

672

760

1327

125

3573

 

 

TA 100

TA1535

TA102

TA98

TA1537

+

0

70

15

254

22

9

+

50

89

19

256

20

13

+

150

95

22

236

25

12

+

500

73

23

256

23

7

+

1500

73

25

251

27

7

+

5000

85 P

14 P

243 P

28 P

12 P

Positive

controls

+ S9

Name

2AA

2AA

DAN

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3)

757

194

1170

330

405

Concurrent Negative control

 

0

77

22

235

21

5

 

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

MMC = mitomycin C

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

DAN = 1,8-Dihydroxanthraquinone

P = Precipitate

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance was found to be non-mutagenic in bacteria.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material Di-(2-ethylhexyl) thiodiglycolate diluted in DMSO using the Ames plate incorporation method according to OECD Guideline No. 471 at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate for both of two consecutive experiments.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. A globular precipitate was observed at the highest dose, 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.