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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:
2002-08-20 to 2002-09-06
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:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Method

Target gene:
histidine operon (Salmonella strains); tryptophan operon (E.coli strain)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: 3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate. Experiment 2: 100, 333, 1000, 2000, 3330, 5000 μg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 (without activation) 1 μg/plate
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without activation) 60 μg/plate
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycine
Remarks:
TA 98 (without activation) 4 μg/plate
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 100 (without activation) 650 μg/plate
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2 uvrA (without activation) 10 μg/plate
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains (with activation) TA 1537 2.5 μg/plate; TA 1535, TA 98 & TA100 1 μg/plate; WP2 uvrA 5 μg/plate
Details on test system and conditions:
ACTIVATION: S9-mix contained per 10 mL:
- 30 mg NADP
- 15.2 mg g-6-p in 5.5 or 5.0 mL Milli-Q water
- 2 mL 0.5 M sodium phosphate buffer pH 7.4
- 1 mL 0.08 M MgCl2
- 1 mL 0.33 M KCl
In experiment 1, 0.5 mL S9-fraction was added (5% (v/v)) was added to 9.5 mL of S9-mix components. 0.5 ml S9 mix was added to 3.2 ml top agar with test substance and bacterial giving a final concentration of approximately 0.7% S9
In experiment 2, 1.0 mL S9-fraction was added (10% (v/v)) was added to 9.0 mL of S9-mix components. 0.5 ml S9 mix was added to 3.2 ml top agar with test substance and bacterial giving a final concentration of approximately 1.4% S9

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 hours at 37°C

NUMBER OF REPLICATIONS: 3 plates for each test concentration; the experiment was repeated, and a third experiment carried out with TA100 without metabolic activation.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn assessment, revertant colony counts.
Evaluation criteria:
For a test substance to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain.

The increase must be accompanied by a dose response towards increasing concentrations of the test article.

The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
3330 μg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
5000 μg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
3330 μg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
3330 μg/plate
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Any other information on results incl. tables

Experiment 1: Number of revertants (Mean of 3 plates)

Concentration (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

DMSO

14

20

116

127

10

13

9

10

20

22

3

 

 

113

138

 

 

 

 

23

24

10

 

 

99

130

 

 

 

 

25

20

33

17

 

137

109

10

 

5

11

24

27

100

12

17

127

110

11

12

7

7

25

26

333

15

18

139

107

11

7

11

10

21

18

1000

13

19

133

102

10

8

5

10

27

22

3330

7**

17

37**

101

MC****

7

MC****

8

15

26

5000

 

14

0****

86*

 

5

347

435

13*

19

Positive control

588

868

679

804

644

218

 

 

455

100

MC = microcolonies

*Bacterial background lawn slightly reduced

**Bacterial background lawn moderately reduced

***Bacterial background lawn extremely reduced

****Bacterial background lawn absent

Experiment 2: Number of revertants (Mean of 3 plates)

Concentration (µg/plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

DMSO

17

18

119

81

10

6

6

6

17

19

100

14

20

74

84

11

9

7

6

16

19

333

15

19

135

149

14

7

8

8

14

17

1000

16

19

122

68

5

11

5

6

15

19

2000

11

-

104

-

8*

-

5

-

-

-

3330

12*

17

73

80

4*

9

2*

6

14

19

5000

-

18

-

83

-

7

-

6

13

15

Positive control

263

278

715

406

325

64

346

60

288

137

*Bacterial background lawn slightly reduced

Experiment 3: Number of revertants (Mean of 3 plates)

Concentration (µg/plate)

TA 100

-MA

DMSO

101

100

127

333

118

1000

106

3330

71*

5000

MC***

Positive control

433

MC = microcolonies

*Bacterial background lawn slightly reduced

***Bacterial background lawn extremely reduced

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

3-[3-(Triethoxysilyl)propyl]oxolane-2,5-dione has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to cytotoxic or limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.