Hexamethyldisiloxane

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 activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (similar to OECD TG 471) (Shin Etsu, 1994).
Cytogenicity in mammalian cells: negative in Chinese hamster lung fibroblasts (similar to OECD TG 473) (Shin Etsu, 1995).
Mutagenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (similar to OECD TG 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:

1994-03-24 to 1994-04-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restrictions were that duplicate plates were used, not triplicate, and 2-amino anthracene was the only control with metabolic activation. An authorised translation was available to the reviewer.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only duplicate plates; 2-AA only control +MA

Principles of method if other than guideline:

E coli Reverse mutation assay

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon (Salmonella strains); tryptophan operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

50 to 10000 µg/plate (range-finding);

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (dehydrated)
- Justification for choice of solvent/vehicle: insoluble in water

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

AF-2: TA98, TA 100 and E. coli WP2 uvrA without metabolic activation: 0.1 µg/plate (TA 98); 0.01 µg/plate (TA 100 and E. coli)

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

NaN3: TA 1535 without metabolic activation: 0.5 µg/plate

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-methoxy-6-chloro-9(3-(2-chloroethyl)-aminopropylamino) acridine 1µg/plate

Remarks:

ICR-191: TA 1537 without metabolic activation

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: µg/plate: 0.5 (TA 98), 1 (TA 100), 2 (TA1535 and 1537) and 1 (E.coli)

Remarks:

2AA: all strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: duplicate (triplicate for solvent controls); range-finding and main experiments evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn; reduction in number of revertants

OTHER: ACTIVATION: S9 mix included glucose-6-phosphate, NADP and NADPH as cofactors, and 10% S9. 1.5 ml S9 mix was added to 0.3 ml of bacteria and 0.15 ml test substance -concentration of S9 was therefore approximately 7.5% during pre-incubation.

Evaluation criteria:

The test substance was judged positive if the number of revertants was more than twice the negative control, and the response was dose-dependent and reproducible.

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

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

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

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

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1a Dose range finding test revertants per plate (mean of 2 plates)

Concentration µg/plate	TA 100			TA 1535			E. coli WP2 uvrA
	-MA	+MA	Cytotoxic	-MA	+MA	Cytotoxic	-MA	+MA	Cytotoxic
0*	135	141	no	15	15	no	42	48	no
50	123	129	no	11	16	no	43	46	no
100	121	127	no	15	19	no	43	50	no
200	132	125	no	10	16	no	47	56	no
500	127	121	no	14	16	no	44	51	no
1000	118	117	no	13	19	no	47	53	no
2000	123	127	no	13	19	no	46	50	no
5000	129	108	no	9	19	no	39	49	no
10000	123	123	no	13	20	no	52	53	no
Positive control	539	1026	-	420	306	-	508	457	-

* Solvent control with acetone, mean of three plates

Table 1b Dose range finding test revertants per plate (mean of 2 plates)

Concentration µg/plate	TA 98			TA 1537
	-MA	+MA	Cytotoxic	-MA	+MA	Cytotoxic
0*	27	31	no	11	19	no
50	26	29	no	8	18	no
100	28	31	no	8	16	no
200	27	33	no	10	19	no
500	33	27	no	6	19	no
1000	29	31	no	10	17	no
2000	24	32	no	11	13	no
5000	31	33	no	11	20	no
10000	32	32	no	9	12	no
Positive control	621	301	-	1581	203	-

* Solvent control with acetone, mean of three plates

Table 2a Main test revertants per plate (mean of 2 plates)

Concentration µg/plate	TA 100			TA 1535			E. coli WP2 uvrA
	-MA	+MA	Cytotoxic	-MA	+MA	Cytotoxic	-MA	+MA	Cytotoxic
0*	128	133	no	16	17	no	46	52	no
313	122	128	no	14	11	no	50	46	no
625	129	129	no	13	12	no	43	48	no
1250	140	125	no	16	15	no	42	48	no
2500	124	121	no	11	13	no	38	49	no
5000	137	127	no	13	14	no	41	51	no
10000	144	133	no	13	16	no	40	50	no
Positive control	645	1042	-	445	273	-	547	492	-

* Solvent control with acetone, mean of three plates

Table 2b Main test revertants per plate (mean of 2 plates)

Concentration µg/plate	TA 98			TA 1537
	-MA	+MA	Cytotoxic	-MA	+MA	Cytotoxic
0*	25	35	no	11	17	no
313	24	33	no	10	13	no
625	24	32	no	10	15	no
1250	26	31	no	12	14	no
2500	30	34	no	10	15	no
5000	22	40	no	10	10	no
10000	29	40	no	13	14	no
Positive control	627	318	-	1303	233	-

* Solvent control with acetone, mean of three plates

Conclusions:

Hexamethyldisiloxane has been tested according to a Japanese guideline that is similar to OECD 471 and under GLP, using the pre-incubation method. No evidence of test-substance induced increase in the number of revertants was observed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 or E. coli WP2 uvrA, with or without metabolic activation, up a concentration exceeding current limit concentrations in either the dose finding test or the main assay. Appropriate solvent, positive and sterility 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 substance.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

A certified translation from the original Japanese was reviewed.

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)

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 of solvent: none given in report

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 (gap, break, exchange etc) were recorded. A gap was defined as a clear discontinuity, wider than one chromatid but 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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 aberrations was observed with or without activation. No treatment-related polyploidy was observed.

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 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

Conclusions:

Hexamethyldisiloxane has been tested under GLP in a valid Japanese guideline study according to a protocol that is similar to OECD TG 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 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 with acceptable restrictions

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.

Key result

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, equivalent to approximately 200 µg/ml

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 valid and reliable study according to a protocol that is similar to OECD TG 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 study available

Genetic toxicity in vivo

Description of key information

Mammalian Bone Marrow Chromosome Aberration Test in rat (ip study) (similar to OECD TG 475): Negative (Dow Corning Corporation, 1982).

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:

comparable to guideline study with acceptable restrictions

Remarks:

It was not compliant with GLP.

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

- 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.

Key result

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: test substance (total number of aberrations observed)

		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

Table 2 Results of chromosome analysis in rat bone marrow cells: control substances (total number of aberrations observed)

		Vehicle control			Positive control
Sampling time (h)		6	24	48	48
Number of cells evaluated		500 approx	450 approx	500 approx	250 approx
Chromosome aberrations	Gaps	6	19	15	ND
	Breaks	3	12	8	145
	Other aberrations	0	0	0	4 quad, 6 tri, 5 del
Mitotic index (% range)		2-7	1-4	3-4	2-4

 N.R = Not Reported

quad = quadriradial tri = triradial del = deletion

Conclusions:

Hexamethyldisiloxane has been tested for the induction of chromosome aberrations in rat bone marrow cells in a valid study conducted according to a protocol that is similar to OECD 475. There was no evidence of induction of chromosomal damage in the bone marrow cells of rats following intraperitoneal 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

Information is available from reliable studies for all the required in vitro endpoints. Where there was more than one result for an endpoint the most reliable study available was chosen as key study. Where there was more than one reliable study, the most recent study was selected. The results of all the studies were in agreement.

Bacterial mutagenicity

Hexamethyldisiloxane has been tested according to a Japanese guideline that is similar to OECD 471 and in compliance with GLP, using the pre-incubation method (Shin Etsu, 1994). The study is considered to be reliability 2 as duplicate not triplicate plates were used. No evidence of test-substance induced increase in the number of revertants was observed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 or E.coli WP2 uvrA, with or without metabolic activation, up to a concentration exceeding current limit concentrations, in either the dose finding test or the main assay. Appropriate solvent, positive and sterility 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 substance. The key study is supported by a number of older, less reliable studies all of which reported negative results.

In vitro cytogenicity

Hexamethyldisiloxane has been tested under GLP in a valid Japanese guideline study according to a protocol that is similar to OECD TG 473 (Shin Etsu, 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.

Mutagenicity to mammalian cells

Hexamethyldisiloxane has been tested for mutagenicity in L5178Y cells in reliability 2 study conducted according to a protocol that is similar to OECD TG 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.

In addition, the test substance did not induce sister chromatid exchanges in mammalian cells, and an in vitro unscheduled DNA synthesis assay on mouse lymphoma L5178Y cells and DNA damage and repair assays using Saccharomyces cerevisiae and Escherichia coli W3110/polA+ P3478/polA- cells also gave negative results (Litton Bionetics, 1978).

In vivo

The conclusion reached from the in vitro results is confirmed by the negative result obtained when the substance was tested in vivo in a rat bone marrow chromosome aberration assay conducted according to a protocol that is similar to OECD 475 using intraperitoneal administration. The test substance was considered to be non-clastogenic (negative for the induction of chromosome aberrations) in rat bone marrow cells under the conditions of the test (Dow Corning Corporation, 1982). In addition, hexamethyldisiloxane does not include structural alerts for genetic toxicity (Benigni et. al., 2008). It is concluded that the available data does not indicate a potential for germ cell mutagenicity.

Benigni et. al. (2008).The Benigni/Bossa rule base for mutagenicity and carcinogenicity JR Scientific report EUR 23241 EN

Justification for classification or non-classification

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