1,1,3,3-tetramethyldisiloxane

Endpoint summary

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 TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2uvrA (OECD Test Guideline 471) (Dow Corning Corporation, 2009).
Cytogenicity in mammalian cells: read-across from analogous substance hexamethyldisiloxane: negative with and without metabolic activation in cultured human lymphocytes (similar to OECD Test Guideline 473) (Hita Research Laboratories, 1995).
Cytogenicity in mammalian cells: read-across from analogous substance 2,4,6,8-tetramethylcyclotetrasiloxane: negative with and without metabolic activation in peripheral human lymphocytes (OECD Test Guideline 473) (Harlan Cytotest Cell Research, 2010).
Mutagenicity in mammalian cells: read-across from analogous substance hexamethyldisiloxane: negative in L5178Y mouse lymphoma cells (similar to OECD Test Guideline 476) (Litton Bionetics, 1978).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-09-09 - 2008-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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-Naphtholflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 - 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate Experiment 2 - 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: As agreed with the sponsor THF was chosen for its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 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-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

Remarks:

TA1535, TA1537, TA98, TA100 - 2.5 µg/plate. 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-phospate and NADP as co-factors. S9 was 10% v/v in the cultures.
DURATION
- Preincubation period: 60 minutes at 37ºC
- Exposure duration: 48 - 72 hours at 37ºC

SELECTION AGENT (mutation assays): histidine deficient agar


NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

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


OTHER:

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the mean number of revertants exceeding the threshold twice (strains TA98, TA100 and WP2 uvrA) or thrice (strains TA1535 and TA1537) the mean 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 evaluation of the data required.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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

Species / strain:

E. coli WP2 uvr A

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
- Precipitation: none observed


ADDITIONAL INFORMATION ON CYTOTOXICITY: none observed

Summary results – Experiment I

Dose µg/plate	+/- metabolic activation	Average number of colony counts
		TA1535	TA1537	TA98	TA100	WP2 uvrA
Solvent control	-	11	7	27	123	44
Untreated control	-	9	9	23	119	44
3	-	15	8	28	116	40
10	-	13	11	25	111	44
33	-	13	9	31	128	51
100	-	14	13	35	115	48
333	-	8	8	19	101	40
1000	-	10	9	23	101	47
2500	-	16	12	25	108	50
5000	-	11	9	26	118	52
Positive control	-	1791	97	343	2013	1158
Solvent control	+	22	13	33	130	60
Untreated control	+	16	15	29	111	61
3	+	14	15	33	113	48
10	+	16	15	34	144	58
33	+	17	7	39	139	67
100	+	17	15	40	149	61
333	+	15	15	39	120	61
1000	+	15	13	38	138	56
2500	+	21	13	33	145	67
5000	+	17	12	39	145	65
Positive control	+	242	213	1452	1629	243

 

Summary results of Experiment II

Dose µg/plate	+/- metabolic activation	Average number of colony counts
		TA1535	TA1537	TA98	TA100	WP2 uvrA
Solvent control	-	15	13	32	142	49
Untreated control	-	17	19	30	166	40
33	-	18	12	28	144	54
100	-	14	12	37	123	52
333	-	15	10	27	139	46
1000	-	14	10	28	141	48
2500	-	14	11	32	146	47
5000	-	14	13	28	141	47
Positive control	-	2076	120	523	2172	275
Solvent control	+	19	13	32	156	63
Untreated control	+	18	13	35	160	59
33	+	18	13	34	156	59
100	+	15	14	31	157	69
333	+	22	13	32	162	65
1000	+	17	13	36	159	67
2500	+	19	10	36	161	58
5000	+	21	10	32	153	48
Positive control	+	302	210	1986	2372	266

Conclusions:

1,1,3,3-Tetramethyldisiloxane has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD Test Guideline 471, in compliance with GLP. No evidence of a test substance related increase in the number of revertants was observed with or without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2uvrA in the initial or the repeat experiments. Appropriate solvent and positive 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

A certified translation from the original Japanese was reviewed. The translated report indicates that aberrations including gaps were evaluated, and does not include historical data.

Qualifier:

according to guideline

Guideline:

other: Japan notification on partial revision of testing methods relating to new chemical substances nos 700, 1039 and 1014

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

similar to guideline but deviations in exposure time and evaluation criteria. No historical data included in report

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: CHL cells clone 11

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital, 5,6 benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

31.25 to 125 µg/ml (without), 100 to 400 µg/ml (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.5% methylcellulose aqueous solution was used to prepare suspensions of the test substance. Suspensions were used within 2 hours of preparation.
- Justification for choice ofsolvent: none given in report. Report states that the substance is insoluble in water and DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension, added to medium

DURATION
- Exposure duration: 6 hours with activation, 24 and 28 hours without
- Expression time (cells in growth medium): 24 or 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid added 2 hours before end of incubation period
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: duplicate cultures for each dose

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: other: cell growth inhibition and cell division inhibition


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: chromatid and chromosome structural aberrations (gao, break, exchange etc) were recorded, A gap was defined as a clear discontinuity, wider than one chromatid byt narrower than two, accompanied by minimal misalignment of the chromatid.

Evaluation criteria:

A dose related, reproducible increase in the incidence of cells with any aberration including gaps was evaluated as positive if the increase was 10% or more. An increase of 5% or more was evaluated as suspect positive, and an incidence of lower than 5% was evaluated as negative.

Species / strain:

mammalian cell line, other: CHL cells clone 11

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 µl/ml (without activation); 300 µg/ml (with activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

No treatment related increase in the percentage of cells with abarrations was observed with or without activation. No treatment-related polyploidy was observed.

The symbols used in Tables 2 and 3 are - negative; ++ positive (20% to lower than 50%); +++ positive (50% or higher)

Table 2: Results of chromosome analysis - without activation (% from total count from 2 cultures, 200 cells counted)

Treatment	Exposure time (h)	Concentration (µg/ml)	% polyploid cells	% cells with aberrations inc gaps	% cells with aberrations not inc gaps	Judgement
Solvent*	24	0	0	1	0.5	-
	48	0	0	2	0.5	-
Test substance	24	31.25	0	0	0	-
		62.5	0	1	1	-
		125	1	1.5	1.5	-
	48	31.25	0	0	0	-
		62.5	0	1	1	-
		125	0	1.5	1.5	-
Positive control	24	0.05	0	51.5	50.0	+++
	48	0.05	0	67.0	65.0	+++

*0.5% methylcellulose

 

Table 3: Results of chromosome analysis - with and without activation, 6 h exposure (% from total count from 2 cultures, 200 cells counted)

Treatment	S9 mix	Concentration (µg/ml)	% polyploid cells	% cells with aberrations inc gaps	% cells with aberrations not inc gaps	Judgement
Solvent**	-	0	0.5	0	0	-
	+	0	0.5	1	0.5	-
Test substance	_	100	0.5	2	1	-
		200*	Toxic			-
		400*	Toxic			-
	+	100	1	0.5	0.5	-
		200*	1	1	1	-
		400*	0	2.5	1.5	-
Positive control	-	10	0.5	0.5	0.5	-
	+	10	0	28.5	28.5	++

* precipitate

**0.5% methylcellulose

Table 4: Toxicity data

Concentration  (µg/ml)	Without metabolic activation				With metabolic activation
	24 h treatment		48 h treatment
	Growth rate %*	Mitotic index	Growth rate %*	Mitotic index	Growth rate %*	Mitotic index
0	100	+++	100	+++	100	+++
10	104.9	+++	103.8	+++	NT	NT
50	109.2	+++	108.1	+++	96.1	+++
75	88.1	+++	79.6	+++	NT	NT
100	53.6	+++	52.0	+++	92.7	+++
125	23.6	+	15.8	+	NT	NT
150**	13.3	+/-	14.4	+/-	85.8	+++
200**	10.4	-	10.6	-	74.3	+++
250**	11.3	-	9.2	-	53.6	+++
300**	12.1	-	7.8	-	51.9	++
400**	NT	NT	NT	NT	27.8	+
500**	NT	NT	NT	NT	6.6	-

* Mean value of relative rate of cell growth for two flasks

** Precipitation was observed

The following symbols apply to Table 4; not all are clearly defined in the report: +++ sufficient; ++ moderate; +rate (sic); +/- extremely [low] rate; - none

Conclusions:

Hexamethyldisiloxane has been tested in compliance with GLP in a valid Japanese guideline study according to a protocol that is similar to OECD Test Guideline 473. The test substance did not induce any chromosome aberrations with or without metabolic activation. There were no marked differences between replicate flasks. Expected results were obtained from vehicle and positive controls. It is concluded that the test substance is non-clastogenic (does not induce chromosome aberrations) in Chinese hamster lung cells under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting 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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

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 control^1	100.0	0.5	0.0	0.0
				Positive control^2	71.2	9.0	8.5^S	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 control^1	100.0	2.0	1.0	0.0
				Positive control^3	23.1	12.5	11.0^S	1.5
				83.6	103.2	1.0	1.0	0.0
				146.2	105.8	1.0	1.0	0.0
				255.9^P	105.3	2.5	2.5	0.5
			Exposure period 4 hrs with S9 mix
I		22 hrs		Solvent control^1	100.0	2.5	2.5	0.0
				Positive control^4	61.8	15.0	14.5^S	1.0
				83.6	110.9	3.5	1.5	0.0
				146.2	101.4	0.0	0.0	0.0
				255.9^P	87.7	2.0	1.5	0.0
II		22 hrs		Solvent control^1	100.0	2.0	1.5	0.0
				Positive control^4	27.2	9.0	8.5^S	0.5
				83.6	104.9	2.5	1.5	0.0
				146.2	110.4	1.5	0.5	0.0
				255.9^P	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 Test Guideline 473 and in compliance with 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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The restrictions were that the test concentrations were not duplicated.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

no duplicates

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

mouse liver S9

Test concentrations with justification for top dose:

0.0125, 0.025, 0.05, 0,1, 0.2 µl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): BUdR

NUMBER OF REPLICATIONS: no replicates

DETERMINATION OF CYTOTOXICITY
- Method: other: loss of growth potential

Evaluation criteria:

A substance is considered mutagenic if there is a dose response relationship over 3 dose levels; minimum increase at high level of dose response is at least times greater than the solvent control value; solvent control data are within normal range of spontaneous background mutation rates.

Statistics:

No statistical analysis was carried out.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.2 µl/ml with metaboic activation, equiv to approx 200 µg/ml. Testing was conducted up to limit concentration in absence of metabolic activation (no toxicity observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary cytotoxicity testing indicated toxicity at ≥ 0.2 µl/ml
Several scattered increases were thought by the authors to be the result of spurious fluctuations and cytotoxicity.

Table 1 Summary of mouse lymphoma mutagenicity results

Test substance concentration µl/ml	Activation	Relative growth %	Mutant frequency x 10E-06
Solvent control	-MA	100	23.7
Negative control	-MA	87.7	17.2
Positive control	-MA	20.3	515.5
0.0125	-MA	86.8	18.5
0.025	-MA	73.1	15.8
0.05	-MA	92.3	22.3
0.1	-MA	62.0	9.5
0.2	-MA	29.5	28.1
Solvent control	+MA	100	29.9
Negative control	+MA	94.9	23.1
Positive control	+MA	25.1	196.0
0.0125	+MA	90.4	42.1
0.025	+MA	68.4	26.3
0.05	+MA	72.5	40.3
0.1	+MA	90.4	21.1
0.2	+MA	0.1	50.7

Conclusions:

Hexamethyldisiloxane has been tested for mutagenicity in L5178Y cells in a reliable study according to a protocol that is similar to OECD Test Guideline 476. The test substance did not cause a biologically significant increase in the mutation frequency; solvent and positive controls gave expected results. It is concluded that the test substance is not mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian Bone Marrow Chromosome Aberration Test in rat (ip study) (similar to OECD Test Guideline 475): read-across from analogous substance hexamethyldisiloxane: negative (Dow Corning Corporation, 1991).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-11-26 to 1981-09-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

only male animals used. Number of cells counted for determination of mitotic index not indicated - probably ca. 100, should be 1000

Principles of method if other than guideline:

The test was performed according to the Rodent Bone Marrow Cytogenetic Assay as recommended by the Ad Hoc Committee on Chromosome Methodologies in Mutagen Testing (Toxicology and Applied Pharm 22: 269-275, 1972) with modifications per the EPA Gene-Tox Program Cytogenetics Committee (12/3 to 12/5, 1980, Washington D.C.).

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Age at study initiation: 14 - 16 weeks

- Weight at study initiation: 250 - 280 g for range finding. 290 - 430 g for cytogenetic study

- Housing: 6 per cage

- Diet (e.g. ad libitum): Charles River Agway

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 68 ± 3 °F (20 ± 1.7 °C)

- Humidity (%): approx 50

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: paraffin oil

- Lot/batch no. (if required): A7M02

- Purity: Laboratory Grade

Duration of treatment / exposure:

single treatment

Frequency of treatment:

Single IP injection

Post exposure period:

6, 24 and 48 hours

Dose / conc.:

255 mg/kg bw/day (nominal)

Dose / conc.:

515 mg/kg bw/day (nominal)

Dose / conc.:

1 030 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide, positive control agent, was included in the 24-hour group.  

- Route of administration: IP Injection

- Doses / concentrations: 22 mg/kg bw

Tissues and cell types examined:

Bone marrow from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on two range finding studies conducted to determine the maximum dose the animals could tolerate.


Range finding studies: Range finding study 1: Animals Injected intraperitoneally with 1676, 504, 168 and 50 mg/kg and observed once a day for 7 days for signs of toxicity. Range finding study 2: 10 animals injected with 3911, 1825, 521, 183 and 52 mg/kg. In main study animals were sacrificed at 6, 24 and 48 hours


DETAILS OF SLIDE PREPARATION: Approximately 4 slides were prepared for each animal.  The chromosomes were prepared by standard methods and Giemsa stained.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
6h group: Stock solution of 321 mg/ml; volumes injected were 1.0, 0.5 and 0.25 ml, resulting in the following doses calculated from body weight: 1030 mg/kg +/- 3.2%; 515 mg/kg +/- 5.6%; 255 mg/kg +/- 1.2%

24 hour group: animals were injected with 1.0 or 0.5 ml of 321 mg/ml stock solution, or 1.0 ml of 91 mg/ml stock solution, resulting in the following doses calculated from body weight: 1030 mg/kg +/- 0.8%; 515 mg/kg +/- 5.6%; 255 mg/kg +/- 3.1%

48 hour group: animals were injected with 1.0 or 0.45 ml of 426 mg/ml stock solution, or 1 ml of 102 mg/ml stock solution. This resulted in the following doses calculated from body weight: 1030 mg/kg +/- 3.1%; 515 mg/kg +/- 9.3%; 255 mg/kg +/- 2.4%

METHOD OF ANALYSIS: metaphase cells analysed by projecting the negatives with a darkroom enlarger onto a white counter

OTHER:

Evaluation criteria:

In general, a minimum of 100 metaphase cells from each animal were scored for incidence of chromosomal aberrations.

Statistics:

Statistical methods: Chi2 test for comparison of expected and observed distribution of the number of breaks; Wilcoxon test was used as a nonparametric test.

Sex:

male

Genotoxicity:

negative

Remarks:

on mitotic index

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

- Dose range: Exp 1: 1676, 504, 168, 50 mg/kg. Exp 2: 3911, 1825, 521, 183, 52 mg/kg

- Clinical signs of toxicity in test animals: No deaths observed in initial study. In second study 3 of 10 animals dosed with 3911 mg/kg died, while all the other animals survived till terminal sacrifice.

- Harvest times: 7 days exp 1, 14 days exp 2.

- High dose with and without activation: 1676 exp 1, 3911 exp 2

RESULTS OF DEFINITIVE STUDY

See table 1

Negative controls: frequencies of breaks were 0.54%, 2.49% and 1.47% at sacrifice at 6, 24 and 48 hours respectively.

Table 1:Results of chromosome analysis in rat bone marrow cells

		Low dose (255 mg/kg bw)			Mid dose (515 mg/kg bw)			High dose (1030 mg/kg bw)
Sampling time (h)		6	24	48	6	24	48	6	24	48
Number of cells evaluated		500 approx	500 approx	500 approx	500 approx	500 approx	500 approx	500 approx	500 approx	500 approx
Toxicity,specify effects
Chromosome aberrations	Gaps	6	11	3	3	6	25	2	12	14
	Breaks	5	2	9	10	15	6	5	6	7
	Other aberrations	0	0	0	0	0	0	0	0	0
Mitotic index (% range)		2 – 5	2 - 5	1 - 4	2 - 4	2 - 5	4 - 7	1 - 6	3 - 5	2 - 5
Polyploidy		N.R	N.R	N.R	N.R	N.R	N.R	N.R	N.R	N.R
Endo reduplication		N.R	N.R	N.R	N.R	N.R	N.R	N.R	N.R	N.R

 N.R = Not Reported

Conclusions:

Hexamethyldisiloxane has been tested for the induction of chromosome aberrations in rat bone marrow cells in a valid study. It did not induce chromosomal damage in the bone marrow cells of rats following i.p. injection. The test substance is considered to be non-clastogenic (negative for the induction of chromosome aberrations) in rat bone marrow cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

1,1,3,3-Tetramethyldisiloxane (H2-L2) has been tested for mutagenicity to bacteria, in three studies. The key study was conducted according to OECD Test Guideline 471, in compliance with GLP (Dow Corning Corporation, 2009). No evidence of a test substance related increase in the number of revertants was observed with or without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2uvrA in the initial or the repeat experiments. Appropriate solvent and positive 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.  This result is supported by two further reliable studies in bacteria, both of which reported negative results in appropriate ranges of bacterial strains (LPT, 2002) and Genetic Laboratory, 2006).

The available bacterial mutagenicity data for hexamethyldisiloxane (HMDS) have been included to the dataset to support read across for genetic toxicity.

The structural analogue hexamethyldisiloxane (HMDS) has been tested for cytogenicity, according to a protocol that is similar to OECD Test Guideline 473 and in compliance with GLP (Hita Research Laboratories, 1995). The test substance did not induce any chromosome aberrations with or without metabolic activation. There were no marked differences between replicate flasks. Expected results were obtained from vehicle and positive controls. It is concluded that the test substance is non-clastogenic (does not induce chromosome aberrations) in Chinese hamster lung cells under the conditions of the test.

In addition, to demonstrate that the Si-H group does not cause in vitro genetic toxicity, supporting data are used from a cyclic siloxane which contains this group, 2,4,6,8-tetramethylcyclotetrasiloxane (H-D4). H-D4 has been tested according to OECD Test Guideline 473 and in compliance with GLP (Harlan Cytotest Cell Research, 2010). 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.

Information on the mutagenicity to mammalian cells is provided by the structural analogue hexamethyldisiloxane (HMDS) which has been tested for mutagenicity in L5178Y cells in a reliable study conducted according to a protocol that is similar to OECD Test Guideline 476 (Litton Bionetics, 1978). The test substance did not cause a biologically significant increase in the mutation frequency; solvent and positive controls gave expected results. It is concluded that the test substance is not mutagenic under the conditions of the test.

Further information on the cytogenicity is provided by the structural analogue hexamethyldisiloxane (HMDS) which has been tested for the induction of chromosome aberrations in rat bone marrow cells in a valid in vivo study (Dow Corning Corporation, 1991). No evidence of the induction of chromosomal damage in the bone marrow cells of rats following i.p. injection was observed. The test substance is considered to be non-clastogenic (negative for the induction of chromosome aberrations) in rat bone marrow cells under the conditions of the test.

Justification for classification or non-classification

Based on the available in vitro and in vivo information, tetramethyldisiloxane is not classified for mutagenicity according to Regulation (EC) No 1272/2008.