Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): Negative with and without activation in all strains tested (OECD 471) (Harlan Cytotest Cell Research, 2010a)
Cytogenicity in mammalian cells: Negative with and without activation in cultured human lymphocytes (OECD 473) (Harlan Cytotest Cell Research, 2010b)
Mutagenicity in mammalian cells: Negative with and without activation in L5178Y mouse lymphoma cells (OECD TG 476) (Harlan Laboratories, 2010)

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
Histidine for Salmonella.
Tryptophan for E. coli
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
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.
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.

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 17 2010 to July 15 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
lymphocytes: peripheral human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos' modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/betanaphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: 19.5 - 3000 µg/ml. Experiment 2: 19.5 - 3000 µg/ml -S9; 19.5-319.9 µg/ml +S9
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation: 7.5 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation: 825.0 µg/ml (Exp. I) and 770.0 µg/ml (Exp. II)
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In
Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours
after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium
DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: about 1.5 cell cycles
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON
microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded
as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well
spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the
mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).
Species / strain:
lymphocytes: peripheral human
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:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item 2,4,6,8-tetramethylcyclotetrasiloxane, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations
in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment
II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II)
after the start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal
aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2400.0 μg/ml (approx. 10 mM) was chosen with respect to the OECD Guideline for in
vitro mammalian cytogenetic tests considering the molecular weight of the test item.
In Experiment I in the presence of S9 mix and in Experiment II in the absence and presence of S9 mix, visible precipitation of the test
item in the culture medium was observed at 255.9 μg/ml and above at the end of treatment. No relevant influence in the osmolarity or
pH value was observed (Exp. I: solvent control: 396 mOsm, pH 7.2 versus 364 mOsm and pH 7.2 at 2400.0 μg/ml; Exp. II: solvent
control: 398 mOsm, pH 7.5 versus 364 mOsm and pH 7.5 at 2400.0 μg/ml). Phase separation was observed in Experiment I at 447.8
μg/ml and above in the absence and presence of S9 mix.
In this study, at both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity
indicated by clearly reduced mitotic indices could be observed.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural
chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.5 % aberrant cells,
excluding gaps) were similar to the range of the solvent control values (0.0 - 2.5 % aberrant cells, excluding gaps) and within the range of
the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770.0 or 825.0 μg/ml) or CPA (7.5 μg/ml) were used as positive controls and showed distinct increases
in cells with structural chromosome aberrations.

Summary of results of the chromosomal aberration study with 2,4,6,8-tetramethylcyclotetrasiloxane

Exp.

Preparation

Test item

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

 

 

 

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

 

 

Exposure period 4 hrs without S9 mix

 

I

22 hrs

Solvent control1

100.0

0.5

0.0

0.0

 

 

 

Positive control2

71.2

9.0

8.5S

0.5

 

 

 

783.7

104.9

0.5

0.5

0.0

 

 

 

1371.4

110.1

0.5

0.5

0.0

 

 

 

2400.0

102.6

3.0

1.5

0.0

 

 

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

2.0

1.0

0.0

 

 

 

Positive control3

23.1

12.5

11.0S

1.5

 

 

 

83.6

103.2

1.0

1.0

0.0

 

 

 

146.2

105.8

1.0

1.0

0.0

 

 

 

255.9P

105.3

2.5

2.5

0.5

 

 

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

2.5

2.5

0.0

 

 

 

Positive control4

61.8

15.0

14.5S

1.0

 

 

 

83.6

110.9

3.5

1.5

0.0

 

 

 

146.2

101.4

0.0

0.0

0.0

 

 

 

255.9P

87.7

2.0

1.5

0.0

 

II

22 hrs

Solvent control1

100.0

2.0

1.5

0.0

 

 

 

Positive control4

27.2

9.0

8.5S

0.5

 

 

 

83.6

104.9

2.5

1.5

0.0

 

 

 

146.2

110.4

1.5

0.5

0.0

 

 

 

255.9P

109.2

1.0

1.0

0.0

 

*  Including cells carrying exchanges

P  Precipitation occurred at the end of treatment

S  Aberration frequency statistically significant higher than corresponding control values

1   Ethanol 0.5 % (v/v)

2     EMS 825.0 µg/mL

3     EMS 770.0 µg/mL

4   CPA      7.5 µg/mL

Conclusions:
2,4,6,8-Tetramethylcyclotetrasiloxane has been tested according to OECD 473 and under GLP. No statistically significant nor biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed with or without activation in either the initial or repeat experiments. It is concluded that the test substance is negative for the induction of chromosome aberrations in mammalian cells under the conditions of the study.
Executive summary:

The test item 2,4,6,8-tetramethylcyclotetrasiloxane, dissolved in ethanol, was assessed for its potential to induce structural chromosomal

aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were scored for structural chromosomal

aberrations.

The highest applied concentration in this study (2400.0 μg/ml of the test item, approx. 10 mM) was chosen with regard to the molecular

weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in

accordance with OECD Guideline 473.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying

structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural

chromosome aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 9th 2010 - September 20th 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
autosomal thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
75-2400 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol.
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to cell cultures
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix: 13.0 - 19.5 µg/ml = 0.12 - 0.18 mM
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix: 3.0 – 6.0 µg/ml = 10.7 – 21.4 µM
Details on test system and experimental conditions:
Experimental Performance
In the mutation experiment 1E+07 (3+06 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 ml RPMI medium with 3 % horse serum (15 % during 24 h exposure) will be exposed to various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls are performed in parallel. After 4 h (24 h in the second experiment without metabolic activation) the test item is removed by centrifugation (425 g, 10 min) and the cells are washed twice with "saline G". Subsequently the cells are resuspended in 30 ml complete culture medium and incubated for an expression and growth period of totally 48 h.
The cell density is determined each day and adjusted to 3x10⁵ cells/ml, if necessary. The relative suspension growth (RSG) of the treated cell cultures is calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector (11).
After the expression period the cultures are seeded into microtitre plates. Cells from each experimental group will be seeded into 2 microtitre plates so that each well contains approximately 4x10³ cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency) will be determined by seeding about 2 cells per well into microtitre plates (same medium without TFT). The plates are incubated at 37°+/- 1.5 °C in 4.5 % CO2/95.5 % humidified air for 10 - 15 days. Then the plates will be evaluated.
Complete Culture Medium
RPMI 1640 medium (GIBCO, invitrogen) supplemented with 15 % horse serum (HS, GIBCO, invitrogen) (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/ml Penicillin/Streptomycin, 220 µg/ml Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal (11).
Selective Medium
RPMI 1640 (complete culture medium) by addition of 5 µg/ml TFT.
Saline G Solution
The "saline G" solution is composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose 1100 mg
Na2HPO4x7H2O 290 mg
KH2PO4 150 mg
pH: 7.2
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1000000 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent con­trols of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
Statistics:
A linear regression (least squares) will be performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Summary of results of Experiments I and II

Dose level µg/ml

S9 mix

Relative total growth

Mutant colonies/108cells

Relative total growth

Mutant colonies/108cells

Experiment I – 4 hr treatment

Culture I

Culture II

Solvent control

-

100.0

151

100.0

63

75.0

-

Culture was not continued

Culture was not continued

150.0

-

125.0

107

64.0

58

300.0 (p)

-

165.5

80

84.3

104

600.0(p)

-

153.3

89

102.0

59

1200.0(p)

-

108.3

114

130.0

39

2400.0(p)

-

147.6

83

78.4

66

Positive control

-

37.9

421

23.2

246

Experiment I – 4 hr treatment

Culture I

Culture II

Solvent control

+

100.0

124

100.0

144

75.0

+

Culture was not continued#

Culture was not continued#

150.0

+

86.0

136

233.1

33

300.0

+

77.6

122

72.1

134

600.0

+

86.5

119

71.5

215

1200(p)

+

66.3

153

68.5

124

2400(p)

+

89.5

111

109.3

109

Positive control A

+

51.2

215

60.8

203

Positive control B

+

46.6

303

25.4

447

Experiment II – 24 hr treatment

Culture I

Culture II

Solvent control

-

100.0

128

100.0

87

75.0

-

Culture was not continued#

Culture was not continued#

150.0

-

80.7

118

100.5

79

300.0

-

65.2

95

170.2

79

600.0

-

96.0

92

78.1

103

1200(p)

-

70.7

111

84.6

106

2400(p)

-

78.1

82

105.9

74

Positive control

-

45.4

592

30.4

747

Experiment II – 4 hr treatment

 

 

 

 

Solvent control

+

100.0

106

100.0

102

75.0

+

Culture was not continued#

Culture was not continued#

150.0

+

132.2

88

214.2

85

300.0

+

140.5

78

95.4

129

600.0

+

125.5

81

146.3

107

1200(p)

+

134.4

98

239.8

56

2400(p)

+

99.7

122

168.6

74

Positive control A

+

75.6

191

41.1

222

Positive control B

+

34.1

414

25.8

441

# culture was not continued since a minimum of four concentrations is required by the guidelines

(p) phase separation visible to the unaided eye

Conclusions:
2,4,6,8-Tetramethylcyclotetrasiloxane has been tested according to OECD 476 and under GLP. No increase in the number of mutant colonies was observed with or without activation in either the initial or repeat experiments. It is concluded that the test substance is negative for the induction of mutations in mammalian cells under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Valid information is available for in vitro gene mutation in bacteria as well as for in vitro cytogenicity and mutagenicity in mammalian cells.

 

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 (Harlan Cytotest Cell Research, 2010a).

2,4,6,8-Tetramethylcyclotetrasiloxane has been tested according to OECD 473 and under GLP. No statistically significant nor biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed with or without activation in either the initial or repeat experiments. It is concluded that the test substance is negative for the induction of chromosome aberrations in mammalian cells under the conditions of the study (Harlan Cytotest Cell Research, 2010b).

2,4,6,8-Tetramethylcyclotetrasiloxane has been tested according to OECD 476 and under GLP. No increase in the number of mutant colonies was observed with or without activation in either the initial or repeat experiments. It is concluded that the test substance is negative for the induction of mutations in mammalian cells under the conditions of the study (Harlan Laboratories, 2010).

Justification for classification or non-classification

Based on the available data, 2,4,6,8 -tetramethylcyclotetrasiloxane does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.