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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
Study period:
1994-03-24 to 1994-04-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The restrictions were that duplicate plates were used, not triplicate, and 2-amino anthracene was the only control with metabolic activation. An authorised translation was available to the reviewer.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only duplicate plates; 2-AA only control +MA
Principles of method if other than guideline:
E coli Reverse mutation assay
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
histidine operon (Salmonella strains); tryptophan operon (E. coli)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
50 to 10000 µg/plate (range-finding);
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone (dehydrated)
- Justification for choice of solvent/vehicle: insoluble in water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
AF-2: TA98, TA 100 and E. coli WP2 uvrA without metabolic activation: 0.1 µg/plate (TA 98); 0.01 µg/plate (TA 100 and E. coli)
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
NaN3: TA 1535 without metabolic activation: 0.5 µg/plate
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-methoxy-6-chloro-9(3-(2-chloroethyl)-aminopropylamino) acridine 1µg/plate
Remarks:
ICR-191: TA 1537 without metabolic activation
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: µg/plate: 0.5 (TA 98), 1 (TA 100), 2 (TA1535 and 1537) and 1 (E.coli)
Remarks:
2AA: all strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: duplicate (triplicate for solvent controls); range-finding and main experiments evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn; reduction in number of revertants

OTHER: ACTIVATION: S9 mix included glucose-6-phosphate, NADP and NADPH as cofactors, and 10% S9. 1.5 ml S9 mix was added to 0.3 ml of bacteria and 0.15 ml test substance -concentration of S9 was therefore approximately 7.5% during pre-incubation.
Evaluation criteria:
The test substance was judged positive if the number of revertants was more than twice the negative control, and the response was dose-dependent and reproducible.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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

Any other information on results incl. tables

Table 1a Dose range finding test revertants per plate (mean of 2 plates)

Concentration µg/plate

TA 100

TA 1535

E. coli WP2 uvrA

-MA

+MA

Cytotoxic

-MA

+MA

Cytotoxic

-MA

+MA

Cytotoxic

0*

135

141

no

15

15

no

42

48

no

50

123

129

no

11

16

no

43

46

no

100

121

127

no

15

19

no

43

50

no

200

132

125

no

10

16

no

47

56

no

500

127

121

no

14

16

no

44

51

no

1000

118

117

no

13

19

no

47

53

no

2000

123

127

no

13

19

no

46

50

no

5000

129

108

no

9

19

no

39

49

no

10000

123

123

no

13

20

no

52

53

no

Positive control

539

1026

-

420

306

-

508

457

-

* Solvent control with acetone, mean of three plates

Table 1b Dose range finding test revertants per plate (mean of 2 plates)

Concentration µg/plate

TA 98

TA 1537

-MA

+MA

Cytotoxic

-MA

+MA

Cytotoxic

0*

27

31

no

11

19

no

50

26

29

no

8

18

no

100

28

31

no

8

16

no

200

27

33

no

10

19

no

500

33

27

no

6

19

no

1000

29

31

no

10

17

no

2000

24

32

no

11

13

no

5000

31

33

no

11

20

no

10000

32

32

no

9

12

no

Positive control

621

301

-

1581

203

-

* Solvent control with acetone, mean of three plates

Table 2a Main test revertants per plate (mean of 2 plates)

Concentration µg/plate

TA 100

TA 1535

E. coli WP2 uvrA

-MA

+MA

Cytotoxic

-MA

+MA

Cytotoxic

-MA

+MA

Cytotoxic

0*

128

133

no

16

17

no

46

52

no

313

122

128

no

14

11

no

50

46

no

625

129

129

no

13

12

no

43

48

no

1250

140

125

no

16

15

no

42

48

no

2500

124

121

no

11

13

no

38

49

no

5000

137

127

no

13

14

no

41

51

no

10000

144

133

no

13

16

no

40

50

no

Positive control

645

1042

-

445

273

-

547

492

-

* Solvent control with acetone, mean of three plates

Table 2b Main test revertants per plate (mean of 2 plates)

Concentration µg/plate

TA 98

TA 1537

-MA

+MA

Cytotoxic

-MA

+MA

Cytotoxic

0*

25

35

no

11

17

no

313

24

33

no

10

13

no

625

24

32

no

10

15

no

1250

26

31

no

12

14

no

2500

30

34

no

10

15

no

5000

22

40

no

10

10

no

10000

29

40

no

13

14

no

Positive control

627

318

-

1303

233

-

* Solvent control with acetone, mean of three plates

Applicant's summary and conclusion

Conclusions:
Hexamethyldisiloxane has been tested according to a Japanese guideline that is similar to OECD 471 and under GLP, using the pre-incubation method. No evidence of test-substance induced increase in the number of revertants was observed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 or E. coli WP2 uvrA, with or without metabolic activation, up a concentration exceeding current limit concentrations in either the dose finding test or the main assay. Appropriate solvent, positive and sterility controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test substance.