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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

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

Method

Target gene:
histidine (s. typhimurium) or tryptophan (E. coli) operon
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and Escherichia coli (WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9.
Test concentrations with justification for top dose:
10, 33, 100, 333, and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin
Remarks:
TA 98 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 100 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E coli WP2 uvrA without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: Triplicate plates, in each of two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: reduction in number of revertants and condition of bacterial lawn.

OTHER: Range finding study was conducted as part of first experiment - 8 concentrations of TA 100 and E coli WP2uvrA were tested in triplicate

ACTIVATION: Aroclor induced rat liver S9. S9 mix included co-factors: NADP and glucose-6-phosphate; sodium phosphate buffer and magnesium chloride and potassium chloride. S9 mix included S9 at 5% v/v for experiment I and 10% v/v for experiment II). 0.5 ml S9 mix was added to the top agar (total volume 2.7 ml) giving a final concentration of S9 of approximately 1% in experiment I and approximately 2% in experiment II.
Evaluation criteria:
A reproducible, dose-related increase by at least two fold in the number of revertants relative to the control was considered positive.

Results and discussion

Test results
Key result
Species / strain:
other: Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and Escherichia coli (WP2uvrA)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested to precipitating concentrations in experiment II and in some strains in experiment I.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: occurred at concentrations of > 1000 µg/plate

RANGE-FINDING/SCREENING STUDIES: no toxicity observed

COMPARISON WITH HISTORICAL CONTROL DATA: Results of controls were within the ranges of historical controls

Any other information on results incl. tables

The test substance precipitated on the plates at 1000 µg/plate.  The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a
dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537,
TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence
and presence of S9-metabolic activation.  These results were confirmed in an independently repeated experiment.

Table 1a Experiment II Number of revertants per plate (mean of 3 plates)

Strain

TA 1535

TA 1537

TA 98

Concentration

µg/plate

- S9

+ S9

Cytotoxicity

- S9

+ S9

Cytotoxicity

- S9

+ S9

Cytotoxicity

Positive control

283

354

-

244

372

-

124

895

-

Solvent control

19

16

-

6

10

-

13

16

-

10

15

16

no

8

8

no

15

12

no

33

16

19

no

5

6

no

13

14

no

100

16

19

no

9

7

no

12

14

no

333

17

16

no

7

7

no

12

16

no

1000*

18

22

no

6

5

no

12

14

no

* Precipitate

Table 1b Experiment I Number of revertants per plate (mean of 3 plates)

Concentration

µg/plate

TA 100

E coli WP2uvrA

- S9

+ S9

Cytotoxicity

- S9

+ S9

Cytotoxicity

Positive control

964

1754

-

323

91

-

Solvent control

97

94

-

11

7

-

3

115

111

no

8

7

no

10

120

92

no

8

9

no

33

110

114

no

7

9

no

100

102

106

no

8

5

no

333

88

118

no

6

6

no

1000*

108

126

no

4

8

no

3330*

94

101

no

8

7

no

5000*

92

124

no

8

12

no

* Precipitate

Table 2a Experiment II Number of revertants per plate (mean of 3 plates)

Concentration

µg/plate

TA 1535

TA 1537

TA 98

- S9

+ S9

Cytotoxicity

- S9

+ S9

Cytotoxicity

- S9

+ S9

Cytotoxicity

Positive control

152

277

-

575

247

-

132

924

-

Solvent control

5

10

-

4

4

-

15

21

-

10

11

12

no

3

7

no

14

25

no

33

11

11

no

4

8

no

16

22

no

100

11

12

no

3

4

no

15

26

no

333

10

9

no

4

7

no

16

23

no

1000*

11

9

no

5

6

no

15

28

no

* Precipitate

Table 2b Experiment II Number of revertants per plate (mean of 3 plates)

Concentration

µg/plate

TA 100

E coli WP2uvrA

- S9

+ S9

Cytotoxicity

- S9

+ S9

Cytotoxicity

Positive control

965

1722

-

386

152

-

Solvent control

84

95

-

7

9

-

10

93

109

no

9

10

no

33

85

97

no

9

14

no

100

83

106

no

8

8

no

333

83

109

no

10

12

no

1000*

73

101

no

6

8

no

* Precipitate

Applicant's summary and conclusion

Conclusions:
Dodecamethylcyclohexasiloxane has been tested in a reliable study conducted in accordance with OECD 471 and in compliance with GLP. No evidence of test substance induced increase in the number of revertants was observed when the substance was tested in the presence and absence of metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli WP2uvrA, in either the initial or the independent repeat experiment. Appropriate solvent and positive controls were included and gave expected responses. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.