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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:
2008-01-28 - 2008-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100 without metabolic activation 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
TA98 - 10 µg/plate, TA1537 - 50 µg/plate without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without metabolic activation 3 µl/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
TA1535, TA1537, TA98, TA100 - 2.5 µg/plate and WP2 uvrA - 10 µg/plate with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: S9 mix contained glucose-6-phophate and NADP as co-factors, the S9 supernatant was 10% v/v in the S9 mix of which 0.5 ml were added to 2 ml top agar, giving a final concentration of approximately 2% S9.

DURATION
- Preincubation period: 60 minutes at 37ºC
- Exposure duration: Experiment 1 - 72 hours at 37ºC, experiment II - 72 hours at 37ºC + a further 72 hours at 4ºC


SELECTION AGENT: histidine deficient agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn



Evaluation criteria:
A result is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and WP2 uvrA) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
Statistics:
No statistical analysis was performed.

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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

COMPARISON WITH HISTORICAL CONTROL DATA: yes, included in report and all within limits

Any other information on results incl. tables

Results of plate incorporation assay Experiment I; revertants per plate (mean of 3 plates)

[Note, the pre-experiment is reported as Experiment 1, as there were evaluable plates at all concentrations in all strains used.]

Dose level (µg/plate)

Metabolic activation

Mean revertant colony counts

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Solvent control**

-

16

13

22

130

54

Negative control***

-

14

12

24

130

47

3

-

11

14

21

138

50

10

-

11

14

29

130

46

33

-

11

12

21

122

51

100

-

9

14

25

120

45

333

-

16

18

21

136

52

1000

-

15

10

24

138

52

2500

-

15

13

22

137

59

5000

-

13

8

26

158

62

Positive control

-

1935

78

281

2196

1314

Solvent control**

+

15

16

33

172

64

Negative control***

+

18

13

37

154

49

3

+

17

16

33

149

64

10

+

17

15

35

165

57

33

+

17

16

34

177

65

100

+

18

17

38

165

61

333

+

18

18

35

161

64

1000

+

12*

15*

29*

157*

55*

2500

+

11*

13*

25*

156*

55*

5000

+

12*

11*

26*

148*

55*

Positive control

+

234

123

998

1334

231

* non-interfering precipitate

** with acetone

*** Untreated

Results of preincubation assay, Experiment 2; revertants per plate (mean of 3 plates)

 

Dose level µg/plate

Metabolic activation

Mean revertant colony counts

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Solvent control**

-

12

12

30

133

37

Negative control***

-

14

17

30

120

44

33

-

13

13

23

136

42

100

-

14

11

23

135

45

333

-

15

13

31

125

46

1000

-

15

12

26

133

43

2500

-

15

11

21

118

39

5000

-

14

10

31

125

38

Positive control

-

2143

99

335

1941

901

Solvent control**

+

19

15

34

121

60

Negative control***

+

16

18

33

156

49

33

+

18

17

31

142

52

100

+

15

19

33

142

52

333

+

16

19

36

137

60

1000

+

18*

16*

30*

129

60

2500

+

15*

16*

30*

135*

56*

5000

+

17*

14*

30*

154*

49*

Positive control

+

307

195

1206

863

349

 

* non-interfering precipitate

** with acetone

*** Untreated

Applicant's summary and conclusion

Conclusions:
1,1,1,3,5,5,5-heptamethyltrisiloxane has been tested for mutagenicity to bacteria in a study which was conducted according to OECD 471 and in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without activation in the initial plate incorporation assay or the repeat experiment using the preincubation method. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.