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Diss Factsheets

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 metabolic activation in Salmonella typhimurium strains TA98, TA100, TA 102, TA1535 and TA1537 (OECD Test Guideline 471) (BSL Bioservice, 2012).
Cytogenicity in mammalian cells: positive in Chinese hamster V79 cells with metabolic activation in repeat assay only (OECD Test Guideline 473) (BSL Bioservice, 2013).
Mutagenicity in mammalian cells: not required as a positive result was obtained from the in vitro cytogenicity assay.

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:
2012-01-20 to 2012-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidin operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
Expt I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate; Expt II: 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Remarks:
A. dest., BSL Lot No. 111219, 120105
Negative solvent / vehicle controls:
yes
Remarks:
EtOH, Applichem Lot No. 1R002540
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used. The experiment was repeated.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

- ACTIVATION
phenobarbiotol and beta-naphthoflavone induced rat liver S9. S9 mix included 5% S9 and glucose-6-phosphate and NADP as cofactors. 0.5 ml of S9 mix were added to 2.7 ml overlay agar including test substance or control and bacterial suspension, giving a final concentration of S9 of approximately 1%.
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium TA 102
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:
Precipitation was observed in all tester strains used in Experiment I and II (with and without metabolic activation). In Experiment I, precipitation of the test item was noted at dose levels of 2500 µg/plate and higher (without metabolic activation) and 5000 µg/plate (with metabolic activation). In Experiment II, precipitation of the test item was noted at 1580 µg/plate and higher (without metabolic activation) and 5000 µg/plate (with metabolic activation).

Mean number of revertant colonies - Experiment 1

Treatment µg/plate

TA98

TA100

TA1535

TA1537

TA102

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Negative control

22

31

104

120

7

11

5

6

225

311

Solvent control

22

31

87

112

6

12

4

4

244

315

31.6

27

35

102

136

8

11

7

5

236

292

100.0

17

33

106

126

8

15

8

5

227

342

316.0

24

35

92

121

4

15

4

3

248

317

1000

24

35

102

121

4

14

6

5

254

329

2500

32

39

109

114

2

9

3

4

256

295

5000

29

34

97

113

3

14

6

8

250

306

Positive control

372

2860

893

2060

726

123

105

246

1527

763

Mean number of revertant colonies - Experiment 2

Treatment µg/plate

TA98

TA100

TA1535

TA1537

TA102

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Negative control

20

25

132

127

11

7

7

8

230

300

Solvent control

25

30

110

115

13

7

6

7

201

279

15.8

28

35

129

133

12

15

7

10

246

340

50.0

27

37

114

137

9

14

4

7

223

342

158.0

26

30

117

151

6

15

4

8

249

346

500.0

25

30

118

136

9

12

2

4

243

308

1580.0

27

32

130

140

9

13

9

9

300

336

5000.0

30

24

122

138

12

14

5

7

257

350

Positive control

851

2524

855

2380

1212

152

149

188

2054

593

Conclusions:
2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested in a reliable bacterial mutagenicity study conducted according to OECD TG 471 and in compliance with GLP. No test-substance related increase in the number of revertants was observed when Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed with and without metabolic activation up to limit concentrations. Appropriate positive and solvent 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.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of S. typhimurium were exposed to 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (Experiment I) and 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate (Experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (Experiment I and II).

2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February 2012 to 28 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
MEM medium supplemented with:
10 % foetal bovine serum (FBS)
100 U/100 µg/mL Penicillin/Streptomycin solution
2 mM L-glutamine
0.25 mg/mL Amphotericin
25 µM HEPES

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: 2.5, 5.0 and 10.0 mM (-MA); 0.25, 2.5, 5.0 and 10.0 mM (+MA). Experiment II: 0.025, 0.035 and 0.040 mM (-MA); 0.4, 8.0 and 10.0 mM (+MA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol;

- Justification for choice of solvent/vehicle: The test item was dissolved in ethanol and diluted with cell culture medium (MEM) followed by ultrasonication for
around 5 min (final concentration of ethanol 1.0% v/v). After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity at a concentration of 1.0% (v/v) ethanol in the culture medium
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
600 µg/ml, without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
0.83 µg/ml, with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital (80 mg/kg bw) and β naphthoflavone (100 mg/kg bw) induced rat liver S9 was included in the S9 mix to a final protein concentration of 0.75 mg/ml. Cofactors were added to the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 5 mM NADP.

Test substance: a solution in ethanol was prepared (344.7 mg/ml; m. wt. 344.66 g/mol). This solution was diluted in MEM, then treated with ultrasonication for around 5 min, to improve the homogeneity of the resulting suspension.

METHOD OF APPLICATION: in medium;

DURATION

- Exposure duration: 4 hours (Experiment I, +/- MA, Experiment II +MA); 20 hours (Experiment II -MA).

- Expression time (cells in growth medium): 20 hours (Experiment I +/- MA and Experiment II +MA).

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Stock cultures in exponential growth were seeded into Quadriperm dishes containing at four slides. Two days after seeding the culture medium was replaced with test item suspension (with S9 mix as appropriate). The slides were divided into sets of two and at least one slide from each set was counted. Duplicate cultures were not used. The experiment was repeated.

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases (100 per set), containing 22+/- 1 centromeres, per concentration and validity controls were scored for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative cell density

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: the draft report states that "it is important to report polyploidy and/or endoreduplication when this is seen", but no further mention of endoreplication is made.

OTHER: Pre-Experiment for Toxicity I: with and without metabolic activation (exposure time 4 h): 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50, 1.0, 2.5, 5.0 and 10.0 mM
Pre-Experiment II: without S9 metabolic activation (exposure time 20 h):
0.0005, 0.0010, 0.0025, 0.005, 0.010, 0.025, 0.050, 0.075 and 0.100 mM
Evaluation criteria:
A positive result is determined by: a clear and dose-related increase in the number of cells with aberrations; a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells)
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
genotoxicity was observed only in the second experiment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic index reduced to 68% at 10 mM in first experiment; mitotic index reduced to 63% at 0.035 mM -MA, experiment II.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
M. I. 55% at 10 mM in experiment I; 49% at 0.04 mM in experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: not reported
- Evaporation from medium: no information
- Water solubility: not soluble in water
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:



ADDITIONAL INFORMATION ON CYTOTOXICITY:

Full tables of results are attached to this EPSR

Results Experiments I and II

Concentration mM

MI %

Cells counted

mean aberrant cells

inc gaps

exc gaps

Without metabolic activation, 4 h exposure, Experiment I

0 (medium)

100

200

3.5

3.0

0 (ethanol)

100

200

1.5

0.5

2.5

80

200

4.0

3.0

5

89

200

2.0

1.0

10

55

200

1.0

0.5

Positive control

100

200

10.0

8.0

With metabolic activation, 4 h exposure, Experiment I

0 (medium)

96

200

3.0

2.0

0 (ethanol)

100

200

1.5

1.0

0.25

82

200

1.5

1.0

2.5

90

200

2.0

1.0

5

102

300

3.7

2.3

10

68

200

4.0

3.0

Positive control

79

200

10.5

10.0

Without metabolic activation, 20 h exposure, Experiment II

0 (medium)

106

200

1.5

0.0

0 (ethanol)

100

200

1.5

2.0

0.025

105

200

2.0

2.0

0.035

63

200

3.0

2.5

0.040

12.5

200

1.5

0.0

Positive control

94

200

10.5

9.5

With metabolic activation, 4 h exposure, Experiment II

0 (medium)

 96

200

5.5

1.0

0 (ethanol)

 100

200

2.5

1.0

0.4

 99

400

5.3

3.3

8

 101

400

5.3

3.8

10

 88

200

8.0

6.5

Positive control

 

200

12.5

10

Conclusions:
2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested according to OECD 474 and in compliance with GLP, using Chinese hamster V79 cells. No evidence for induction of numerical chromosome aberrations was observed in either the initial or the repeat assay. Without metabolic activation, no biologically relevant increase in the proportion of cells with structural aberrations was observed in either of the two experiments. With metabolic activation, the negative result obtained in the first experiment was not reproduced in the second experiment. Replicate slides were counted in each experiment, but only single cultures were exposed to the controls and to each concentration the test item. It is noted that there is considerable variation between the replicate slides (for example, high dose metabolic activation group replicates providing results of 0 and 8 in experiment I; mid-dose replicates in experiment II results 4 and 1 without MA, 5 and 10 with MA). It is assumed that this lack of reproducibility was the reason that extra cells were counted. Appropriate positive, solvent and medium controls were included and gave results within the range of the historical controls. It was concluded by the authors of the draft report that the test substance is positive for the induction of structural chromosome aberrations under the conditions of the test. The reviewer notes that the differences between replicate slides; the lack of dose response in experiment 1; the unexplained differences between the solvent and medium controls; the different results in experiments 1 and 2 all call into question the biological relevance of the results. In addition, the differences between replicates mean that the absence of duplicate cultures may have had an impact on the results.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus assay: negative in male mice (OECD Test Guideline 474)

(Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 2020).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
with stated exceptions which do not impact the study integrity. See any other information on materials and methods for more information on GLP exceptions.
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Crl:CD1(ICR)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: one per cage in stainless steel cages
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form, ad libitum
- Water (e.g. ad libitum): municipal water, ad libitum
- Acclimation period: one week prior to the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test substance 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was determined to be soluble in corn oil at a concentration of 200 mg/mL.
- Concentration of test material in vehicle: 50 mg/mL, 100 mg/mL and 200 mg/mL
All stock dosing solutions utilized for dosing on the initial day of the MNT were evaluated. The concentrations of the test material in the dosing solutions used for the first day of dosing were verified using gas chromatography with mass spectrometry (GC/MS) detection and external standard quantitation. The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 89.8 to 93.8% of the targeted values + 1.0 to 7.0 % RSD, but the values are not given.

- Amount of vehicle (if gavage or dermal): Dosing solutions were administered in aliquots of 10 ml/kg bodyweight (bw)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions of the test material were prepared and used fresh on each of the two consecutive days of administration.

Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Test substance: The animals were dosed on two consecutive days (March 3, 2020 and March 4, 2020)
Positive control: Administered once
Post exposure period:
48 hours after the last dosing
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 male mice per group
Additional satellite animals were utilized to assess blood levels for the presence of test material to confirm systemic exposure. Individual animals were administered the test material (2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane) via oral gavage on two consecutive days at a targeted concentration of 2000 mg/kg body weight. Subsequent to the second gavage administration, blood samples from each animal were collected at terminal sacrifice at the following time points (one animal/time point): 15 minutes, 30 minutes, 60 minutes, 180 minutes, and ~48 hours, in accordance with the study design. Blood was collected via the orbital sinus in a manner similar to the collection of typical samples for the micronucleus analysis.
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): CP when administered by oral gavage has been shown to induce micronuclei in polychromatic erythrocytes of Crl:CD1(ICR) mice (De Boeck et al., 2005; Gollapudi et al., 1986).
- Route of administration: oral gavage
- Doses / concentrations: Dosing solutions were administered in aliquots of 10 ml/kg bodyweight (bw). CP was administered once only at dose of 40 mg/kg bw/day.
Tissues and cell types examined:
Approximately 5,000 RETs were analysed per blood sample.
The number of normochromatic erythrocytes (NCE) and micronucleated NCE (MN-NCE), reticulocytes (RET) and micronucleated RET (MN-RET) was recorded for each sample and the frequency of MN-RET calculated to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoietic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RETs and MN-RETs observed.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels for the MNT were selected based upon the range finding test. Relatively non-toxic compounds were tested up to 2 g/kg body weight/day. The middle- and low-doses were approximately 1/2 and 1/4 of the maximum dose, respectively. The dose level for the positive control chemical, CP (40 mg/kg) was based upon our internal historical control database, which indicated a pronounced micronuclei induction in CD-1 mice at this dose level.

A dose range-finding test was conducted to aid the selection of dose levels for the MNT. The number of animals included in each dose group was based upon the use of the dose selection criteria. Male and female rats were dosed on September 3-4, 2019 (one/sex/dose) with 1000 or 2000 mg/kg bw/day of the test material on two consecutive days and observed for any signs of toxicity for at least 72 hours after the initial dosing. Based upon the results of the initial dosing, an additional two mice/sex/dose were dosed with 1000 or 2000 mg/kg bw/day on September 10-11, 2019. These animals were also observed for at least 72 hours after the initial dose for any signs of toxicity at the end of the study period. All animals were anesthetized with isoflurane, followed by euthanasia via CO2 and cervical dislocation.

Based upon the results of the range-finding study where no apparent sex differences were noted, only males were evaluated in the main study. The highest concentration tested was at the limit dose of 2000 mg/kg bw/day. The middle- and low-doses were 1000 mg/kg bw/day and 500 mg/kg bw/day, respectively.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): It takes approximately 48 hours for the treated erythroblasts to undergo one post-treatment division and mature into reticulocytes and reach a peak incidence in the peripheral blood (CSGMT, 1992). However, if the treatment induces cell cycle delay, or if there is a delayed absorption and metabolism of the test material, a longer sampling time (such as 72 hours) may be needed to recover the damage induced in the erythroblasts. The sampling time used in this study provided an opportunity to recover any delayed effect (i.e., effect induced by the first treatment) as well as a more immediate effect (i.e., effect induced by the second treatment) in the same treated animals.

DETAILS OF SLIDE PREPARATION:
Duplicate cell smears were prepared and stored to serve as a backup to flow cytometric analysis if required. Blood was collected into a microtainer tube coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears were prepared, fixed in methanol, and stored at room temperature. Slides were coded and manually scored with a fluorescent microscope for MN evaluation. Briefly, 2000 erythrocytes (normochromatic erythrocytes (NCE) and RET) were counted per animal and the percentage of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoietic activity indicative of cell toxicity. A minimum of 4000 RETs were scored per animal and the frequency of MN-RETs was determined to provide an indication of genotoxic potential. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RETs and MN-RETs observed.

METHOD OF ANALYSIS: Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry (MACSQuant Analyzer 10; Miltenyi Biotec) with traditional blood smears prepared as a backup.

OTHER: The mice were observed for positive signs of toxicity at least once on the day of dosing and subsequent days. The mice were weighed prior to dosing and on the day of their scheduled sacrifice in order to assess the extent of stress experienced by the animals following treatment.
Evaluation criteria:
A test material was considered positive in this assay if the following criteria were met:
• An increase in MN-RETs was statistically significant when compared to the negative control.
• An increase in MN-RET frequency was dose-related when evaluated with an appropriate trend test.
• A statistically significant increase in MN-RET frequency was out of historical negative control range.

A test material was considered negative in this assay if the following criteria were met:
• An increase in MN-RETs was not statistically significant when compared to the negative control.
• An increase in MN-RET frequency was not dose-related when evaluated with an appropriate trend test.
• An increased MN-RET frequency was within the historical negative control range.

Statistics:
MN-RETs and percent RETs were tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). If the results from Bartlett's test were significant, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations examined were the common log, the inverse, and the square root in that order. The data was reviewed, and an appropriate form of the data was selected and subjected to the following analysis.
Based on the similarity of the response between sexes in the RFT, the MN-RET data and the data on percent RETs were analysed by a one-way analysis of variance (only males were used) (Winer, 1971). Linear dose-related trend tests were performed for the pairwise comparisons yielding significant differences. The alpha level at which tests were conducted was 0.05. The MN-NCE, collected via flow cytometry, was not analyzed statistically and was only used as an adjunct end point to evaluate the biological significance of the MN-RET results. MN-NCE were not collected by manual scoring. The final interpretation of significance of the responses was based on both statistical outcome and biological relevance.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: dose levels of 1000 and 2000 mg/kg bw/day 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane
- Solubility: not specified
- Clinical signs of toxicity in test animals: There were no appreciable changes in the body weights of mice. There were no observations of toxicity or mortality among the treated animals during the in-life portion of the range-finding test. There were also no remarkable changes in the body temperature of mice following treatment with the test material (Tables 6 and 7), although a slight (< 2°C), transient decrease was generally observed in both sexes 5 hours post-treatment. Based upon the results of the range-finding study where no apparent sex differences were noted, only males were evaluated in the main study.
- Evidence of cytotoxicity in tissue analyzed: not specified
- Rationale for exposure: The dose levels of 2000 and 1000 mg/kg bw/day are selected based on availability of acute oral toxicity data for the mouse
- Harvest times: not specified


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane did not induce a significant increase in the frequency of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. There were no significant differences in MN-RET frequencies between the groups treated with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of MN-RETs in the positive control group.
- % RET: The percent RET values observed in the test material-treated animals were decreased but not significantly different from the negative control values indicating that the test substance was not overtly toxic to the target tissue. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals. Analytical results of presence of the test material in the peripheral blood clearly indicated a significant quantity of 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyc lotetrasiloxane in all time course samples. These data confirmed the presence (> 5 µg/g at a minimum) of the parent material in the peripheral blood after oral (gavage) administration and demonstrates exposure of the target tissue (i.e., bone marrow).
- Appropriateness of dose levels and route: The dose levels were based on the dose range-finding study which was performed prior to the definitive study. The recommended route in OECD test guideline (474) is oral gavage.
- Statistical evaluation: yes

CONCENTRATION ANALYSIS: The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 89.8 to 93.8% of the targeted values + 1.0 to 7.0 % RSD.

See attachment for result tables.

Conclusions:
2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested for the ability to induce micronuclei in vivo in a study, conducted according to OECD Test Guideline 474 and in compliance with GLP. In the study, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was administered at dose levels of 0 (negative control), 500, 1000 and 2000 (limit dose) as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. No treatment-related toxicity was observed. Exposure of the test material to the target tissue (i.e., bone marrow) was confirmed by the presence of the test material in the peripheral blood following and oral (gavage) administration. Based on the results of the study, it was concluded that the test material, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, did not induce a statistically significant increase in the frequency of micronucleated reticulocytes in the peripheral blood. Positive and vehicle controls were included and gave the expected results.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data are available from reliable in vitro studies on 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane for mutagenicity to bacteria and for cytogenicity to mammalian cells and an in vivo mammalian micronucleus test.

2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested in a reliable bacterial mutagenicity study conducted according to OECD Test Guideline 471 and in compliance with GLP (BSL Bioservice, 2012). No test-substance related increase in the number of revertants was observed when Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed with and without metabolic activation up to limit concentrations. Appropriate positive and solvent 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.

2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested according to OECD Test Guideline 473 and in compliance with GLP, using Chinese hamster V79 cells (BSL Bioservice, 2013). No evidence for induction of numerical chromosome aberrations was observed in either the initial or the repeat assay. Without metabolic activation, no biologically relevant increase in the proportion of cells with structural aberrations was observed in either of the two experiments. With metabolic activation, the negative result obtained in the first experiment was not reproduced in the second experiment. Replicate slides were counted in each experiment, but only single cultures were exposed to the controls and to each concentration the test item. It is noted that there is considerable variation between the replicate slides (for example, high dose metabolic activation group replicates providing results of 0 and 8 in experiment I; mid-dose replicates in experiment II results 4 and 1 without MA, 5 and 10 with MA). It is assumed that this lack of reproducibility was the reason that extra cells were counted. Appropriate positive, solvent and medium controls were included and gave results within the range of the historical controls. It was concluded by the authors of the draft report that the test substance is positive for the induction of structural chromosome aberrations under the conditions of the test. The reviewer notes that the differences between replicate slides; the lack of dose response in experiment 1; the unexplained differences between the solvent and medium controls; the different results in experiments 1 and 2 all call into question the biological relevance of the results. In addition, the differences between replicates mean that the absence of duplicate cultures may have had an impact on the results.

Mutagenicity in mammalian cells: testing is not required as a positive result was obtained from the in vitro cytogenicity assay.

2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested for the ability to induce micronuclei in vivo in a study conducted according to OECD Test Guideline 474 and in compliance with GLP. In the study, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was administered at dose levels of 0 (negative control), 500, 1000 and 2000 (limit dose) as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. Doses were selected based on a dose range-finding study and only males were used for the MNT due to the results of the RFT, which showed no apparent difference in the toxicity between the sexes. No treatment-related toxicity was observed. Exposure of the test material to the target tissue (i.e., bone marrow) was confirmed by the presence of the test material in the peripheral blood following oral (gavage) administration. Based on the results of the study, it was concluded that the test material, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, did not induce a significant increase in the frequency of micronucleated reticulocytes in the peripheral blood. Positive and vehicle controls were included and gave the expected results (Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 2020).


Justification for classification or non-classification

Based on the available information, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008.