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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:
2010 -06-07 to 2010-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E. coli
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: better than other
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
TA 1535, TA 100
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide, NaN3
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
TA 1537, TA 98
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
WP2 uvrA
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With metabolic activation
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) 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.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item in the overlay agar was observed on the incubated agar plates at 5000 µg/plate in both experiments in the absence of metabolic activation and at 2500 µg/plate and 5000 µg/plate in the presence of metabolic activation. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No reduced background growth was observed in all strains in both experiments with and without metabolic activation.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed in all strains with and without S9 mix in either experiment.

Any other information on results incl. tables

Summary Tables

  Summary of Results Experiment I Plate incorporation (mean of 3 plates)

Study Name: 1323706

Study Code: Harlan CCR 1323706

Experiment: 1323706 VV

Date Plated: 07/06/2010

Assay Conditions:

Date Counted: 10/06/2010

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

Ethanol

11 ± 3

10 ± 3

34 ± 1

130 ± 7

50 ± 7

Untreated

7 ± 1

7 ± 1

25 ± 7

131 ± 3

51 ± 4

2,4,6,8-tetramethylcyclo

3 µg

14 ± 2

13 ± 1

31 ± 6

126 ± 6

51 ± 6

Tetrasiloxane

10 µg

12 ± 1

10 ± 1

33 ± 4

135 ± 21

49 ± 5

33 µg

11 ± 3

13 ± 1

32 ± 4

135 ± 21

53 ± 1

100 µg

11 ± 4

8 ± 4

36 ± 2

147± 10

50 ± 12

333 µg

12 ± 4

11 ± 6

31 ± 3

142 ± 8

47 ± 10

1000 µg

13 ± 2

12 ± 1

34 ± 1

160 ± 16

53 ± 7

2500 µg

7 ± 2

7 ± 2

32 ± 5

159 ± 10

52 ± 8

5000 µg

7 ± 1P

7 ± 1P

37 ± 1P

155 ± 1P

59 ± 2P

NaN3

10 µg

1711 ± 94

1814 ± 211

4-NOPD

10 µg

256 ± 7

4-NOPD

50 µg

1714 ± 109

MMS

3.0 µL

1068 ± 28

With Activation

Ethanol

17 ± 5

18 ± 2

46 ± 3

174 ± 20

60 ± 4

Untreated

20 ± 4

20 ± 2

42 ± 7

159 ± 29

51 ± 6

2,4,6,8 -Tetramethylcyclo

3 µg

20 ± 2

18 ± 3

45 ± 4

154 ± 16

57 ± 12

tetrasiloxane

10 µg

21 ± 1

18 ± 3

36 ± 2

149 ± 10

65 ± 11

33 µg

19 ± 5

16 ± 6

43 ± 12

169 ± 17

68 ± 3

100 µg

19 ± 5

17 ± 2

47 ± 5

169 ± 11

72 ± 6

333 µg

17 ± 4

15 ± 1

35 ± 8

135 ± 12

63 ± 11

1000 µg

19 ± 2

17 ± 2

46 ± 4

142 ± 5

58 ± 10

2500 µg

22 ± 4P

19 ± 2P

41 ± 1P

170 ± 7P

66 ± 12P

5000 µg

20 ± 4P M

18 ± 4P M

37 ± 4P M

154 ± 27P

60 ± 5P M

2-AA

2.5 µg

393 ± 6

392 ± 4

3105 ± 154

3852 ± 240

2-AA

10.0 µg

289 ± 5

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

Summary of Results Experiment II Preincubation (mean of 3 plates)

Study Name: 1323706

Study Code: Harlan CCR 1323706

Experiment: 1323706 HV2 Pre

Date Plated: 18/06/2010

Assay Conditions:

Date Counted: 21/06/2010

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

Ethanol

11 ± 4

10 ± 2

33 ± 10

129 ± 9

59 ± 11

Untreated

16 ± 3

17 ± 5

28 ± 5

132 ± 12

46 ± 7

2,4,6,8 -Tetramethylcyclo

33 µg

10 ± 2

9 ± 2

33 ± 10

118 ± 10

55 ± 8

tetrasiloxane

100 µg

15 ± 2

10 ± 4

31 ± 3

135 ± 15

56 ± 8

333 µg

12 ± 3

11 ± 3

33 ± 4

127 ± 5

57 ± 7

1000 µg

13 ± 4

11 ± 3

31 ± 2

141 ± 26

56 ± 5

2500 µg

12 ± 3

10 ± 2

36 ± 6

143 ± 16

59 ± 4

5000 µg

11 ± 5P

11 ± 3P

27 ± 3P

136 ± 13P

62 ± 3P

NaN3

10 µg

1906 ± 2

2200 ± 55

4-NOPD

10 µg

344 ± 4

4-NOPD

50 µg

86 ± 2

MMS

3.0 µL

208 ± 3

With Activation

Ethanol

17 ± 4

13 ± 3

39 ± 5

155 ± 6

57 ± 2

Untreated

19 ± 5

15 ± 4

42 ± 3

173 ± 8

64 ± 3

2,4,6,8 -Tetramethylcyclo

33 µg

21 ± 2

12 ± 2

38 ± 5

148 ± 18

61 ± 3

tetrasiloxane

100 µg

21 ± 5

12 ± 4

47 ± 4

153 ± 9

55 ± 4

333 µg

15 ± 5

12 ± 6

45 ± 8

150 ± 5

69 ± 8

1000 µg

21 ± 2

12 ± 4

43 ± 8

126 ± 8

49 ± 11

2500 µg

21 ± 2P

11 ± 5P

37 ± 8P

138 ± 5P

63 ± 6P

5000 µg

16 ± 6P

9 ± 1P

33 ± 9P

139 ± 10P

69 ± 3P

2-AA

2.5 µg

425 ± 67

499 ± 12

2985 ± 288

3981 ± 123

2-AA

10.0 µg

359 ± 30

Key to Positive Controls

Key to Plate Postfix Codes

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

Precipitate

Applicant's summary and conclusion

Conclusions:
2,4,6,8-Tetramethylcyclotetrasiloxane has been tested according to OECD 476 and under GLP. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of any of the strains used, with and without activation, in either the initial plate incorporation or the repeat preincubation assays. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Executive summary:

SUMMARY OF RESULTS

This study was performed to investigate the potential of 2,4,6,8 -tetramethylcyclotetrasiloxane to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1,000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

No reduced background growth was observed in all strains in both experiments with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed in all strains with and without S9 mix in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2,4,6,8 -tetramethylcyclotetrasiloxane at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, 2,4,6,8 -tetramethylcyclotetrasiloxane is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.