Decamethyltetrasiloxane

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 all strains tested (OECD TG 471) (Wagner, V. O., Hines, R. M., (2005)).
Cytogenicity in mammalian cells: read-across from structural analogue octamethyltrisiloxane: negative in CHO cells (OECD TG 473) (Madraymootoo and Rao (2008)).
Mutagenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (OECD TG 476) (Flanders, 2010).

The selected studies for bacterial and mammalian mutagenicity were the only studies available. They were conducted according to appropriate OECD guidelines and in compliance with GLP. The study selected for cytogenicity was the only available study for the surrogate substance. It was conducted according to an appropriate OECD guideline and in compliance with GLP.

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:

2005-09-15 to 2005-10-05

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

Deviations:

no

GLP compliance:

yes

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:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All salmonella strains + WP2 uvrA (with activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 (without activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 (without activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 (without activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA (without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 - 72 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; observation of background lawn density.

METABOLIC ACTIVATION: Aroclor induce rat liver S9; S9 mix included glucose-6-phophate and NADP as co-factors. 0.5ml of 10% S9 was added to 2 ml of top agar, 0.1 ml of tester strain and 0.05 ml of vehicle or test article giving a final concentration of approximately 2%.

Evaluation criteria:

A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2 uvrA and 3-fold of the solvent control for TA 1535 and TA 1537. There must be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of 2 increasing concentrations of test article.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or at least a moderate reduction in
the background lawn (background code 3, 4 or 5).

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Table 2a:  Toxicity and mutagenicity assay - plate incorporation (mean of 2 plates)

	TA98			TA100			TA1535
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	26	22	No	161	145	No	25	25	No
1.5	23	24	No	130	134	No	26	20	No
5	17	22	No	126	152	No	22	17	No
15	15	25	No	107	125	No	16	23	No
50	14	27	No	108	128	No	17	29	No
150	17	24	No	124	153	No	24	18	No
500	23	23	No	133	148	No	21	21	No
1500	16	20	No	115	129	No	27	20	No
5000	21	20	No	135	128	No	25	18	No
Positive Control	173	488	No	557	565	No	342	78	No

*solvent control with ethanol

Table 2b:  Toxicity and mutagenicity assay - plate incorporation (mean of 2 plates)

	TA1537			WP2 uvrA
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	11	9	No	28	13	No
1.5	8	8	No	17	25	No
5	8	8	No	22	19	No
15	7	8	No	27	17	No
50	6	5	No	20	19	No
150	7	8	No	22	24	No
500	7	7	No	16	24	No
1500	9	6	No	18	17	No
5000	8	10	No	22	17	No
Positive Control	871	62	No	141	203	No

*solvent control with ethanol

Table 3: Experiment 2 - preincubation Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)	—  MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	18	24	No	84	84	No	16	21	No
50	15	19	No	80	72	No	17	20	No
150	15	26	No	51	97	No	15	19	No
500	17	21	No	78	81	No	19	24	No
1500	23	25	No	67	90	No	17	24	No
5000	15	24	No	80	103	No	20	21	No
Positive control	96	124	No	283	279	No	184	64	No

*solvent control with ethanol

Table 3: Experiment 2 - preincubation Number of revertants per plate (mean of 3 plates)

	TA1537			WP2 uvrA
Conc. µg/plate	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	9	9	No	25	20	No
50	7	7	No	18	26	No
150	5	9	No	23	21	No
500	8	5	No	20	16	No
1500	7	3	No	21	18	No
5000	8	7	No	20	17	No
Positive control	505	29	No	124	104	No

*solvent control with ethanol

Conclusions:

Decamethyltetrasiloxane has been testing in a reliable study conducted according to OECD 471 1998 and under GLP up to limit concentrations. No increase in the number of revertants was observed at any concentration with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA in either the initial plate incorporation assay or the independent repeat experiment which used the pre-incubation method. It is considered that the test substance was negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2009 to 14 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

12.5-200 µg/ml (4h) and 1.56-50 µg/ml (24h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: none given in study report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation: 400 µg/ml (4 h exposure), 150 µg/ml (24 h exposure)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation: 2 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without metabolic activation); 24 h (without metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days:

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: % viability

OTHER EXAMINATIONS:
- Other: number of small and large colonies

OTHER: a preliminary toxicity assay was conducted up to a concentration of 3107 µg/ml (10 mM)

Evaluation criteria:

For a test material to demonstrate a mutagenic response it must produce a reproducible and dose-dependent statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value, exceeding the Global Evaluation Factor (GEF) value of 126 E10-06.

Statistics:

UKEMS statistical package used.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Changed to RTG 16% at 37.5 µg/ml (24h)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked change in pH was observed
- Effects of osmolality: did not increase by more than 50 mOsm
- Precipitation: a non-interfering precipitate of the test material observed at and above 100 μg/ml in the absence of metabolic activation, and at and above 150 μg/ml in the presence of metabolic activation.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: No dose-related toxicity was observed following 4 h exposure; marked toxicity was observed in the 24 h exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA: Control results were within the range of historical controls

CYTOTOXICITY: The toxicity observed in the main experiment differed from that in the preliminary cytotoxicity test. No explanation of this is given in the report.

Table 1 Preliminary toxicity test

Concentration (μg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
12.14	74	96	72
24.27	90	96	26
48.55	83	84	0
97.09	100	68	0
194.19	95	82	1
388.38	94	102	1
776.75	108	98	1
1553.5	100	102	4
3107	97	82	39

%RSG= Relative Suspension Growth

 

Table 2 Summary of results from main experiment, 4 h exposure

Treatment (μg/ml)	4-Hours-S-9			Treatment (μg/ml)	4-Hours+S-9
	%RSG	RTG	MF		%RSG	RTG	MF
0	100	1.00	89.99	0	100	1.00	114.53
12.5	112	1.29	89.46	12.5	113	1.04	91.57
25	111	1.28	82.23	25	106	1.20	95.58
50	121	1.42	89.78	50	102	1.05	101.72
100 P	110	1.28	79.77	100	105	1.09	93.79
150 P	109	1.28	86.92	150 P	104	1.10	88.74
200 P	122	1.38	86.12	200 P	106	1.17	98.22
Linear trend NS				Linear trend NS
Positive control	94	0.81	710.52	2	65	0.29	935.61

P Precipitate observed at the end of the exposure period.

* Not plated for viability or 5-TFT resistance

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF = 5-TFT resistant mutants/106 viable cells 2 days after treatment

 

Table 3 Summary of results from main experiment, 24 h exposure

Treatment (μg/ml)	24-Hours-S-9
	%RSG	RTG	MF
0	100	1.00	96.68
1.56 *	109
3.13	102	0.95	108.38
6.25	101	1.29	76.85
12.5	104	1.21	113.47
18.75	89	1.02	93.93
25	59	0.69	89.82
37.5	16	0.14	141.03
50 *	9
Linear trend NS
Positive control	92	0.69	992.94

* Not plated for viability or 5-TFT resistance

%RSG = Relative Suspension Growth = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

P(0) = number of negative wells/total wells plated

2 day viability %V = (-lnP(0) x 100)/number of cells/well

RCE = Relative Cloning Efficiency = (%V/mean solvent control %V)x100%

RTG = Relative Total Growth = (RCE x RSG)/100%

MF = 5-TFT resistant mutants/106 viable cells 2 days after treatment

Conclusions:

Decamethyltetrasiloxane has been tested according to OECD TG 476 and under GLP conditions for mutagenicity to mouse lymphoma L5178Y cells up to cytotoxic concentrations. No increase in mutant frequency was detected at any concentration after 4h exposure with and without metabolic activation and 24 h exposure without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in L5178Y 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:

key study

Study period:

2008-10-07 - 2008-10-26

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)

Qualifier:

according to guideline

Guideline:

other: EPA (TSCA) GLP 40 CFR Part 792

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium inc 10% FBS, 100 units penicillin/ml, 100 µg streptomycin/ml, 2mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, cell stocks not used beyond passage 20 to ensure karyotype stability
- Periodically "cleansed" against high spontaneous background: no information

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

4 hours (-MA): 2.5, 5, 12.5 µg/ml; 20 hours (-MA): 5, 10, 15 µg/ml; 4 hours (+MA): 10, 25 and 75 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: 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:

4 hours without metabolic activation : 0.2 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

4 hours with metabolic activation 10 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

20 hours without metabolic activation : 0.1 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition

ACTIVATION:
Aroclor 1254-induced rat liver S9: S9 mix included glucose-6-phosphate and NADP as co-factors.

Evaluation criteria:

The test article was considered to induce a positive response when the percentage of cells with aberrations was increased in a dose responsive manner with one or more concentrations being statistically significant (p<0.05). Values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant.

Statistics:

Fisher's Exact test and the Cochran-Armitage test to measure dose responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Substantial cytotoxicity observed at dose levels > 23.4 µg/ml in all three treatment groups

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of highest concentration approximately 7.5
- Effects of osmolality: considered acceptable, 2340 µg/ml was 281 mmol/kg, and did not exceed the osmolality of the solvent by more than 20%
- Precipitation: Visible precipitate in the form of oily droplets observed in treatment medium at 2340 µg/ml, dose levels < 702 µg/ml were soluble in treatment medium.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: included in report

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Summary table of cytogenetic analysis of CHO cells in the absence and presence of metabolic activation 

Treatment µg/ml	S9 Activation	Treatment time	Mean mitotic index (500 cells)	Metaphase cells scored	Mean aberrations per cell	Cells with aberrations
						Numerical       (%)	Structural (%)
Solvent control	-	4	11.2	200	0.000	0.0	0.0
2.5	-	4	9.9	200	0.005	0.0	0.5
5	-	4	9.8	200	0.000	0.5	0.0
12.5	-	4	9.3	200	0.000	0.0	0.0
Positive control	-	4	5.2	200	0.370	0.0	20.0
Solvent control	+	4	10.9	200	0.005	0.5	0.5
10	+	4	10.5	200	0.000	0.5	0.0
20	+	4	10.4	200	0.000	0.0	0.0
75	+	4	10.4	200	0.000	0.5	0.0
Positive control	+	4	3.3	200	0.350	1.5	18.0
Solvent control	-	20	10.9	200	0.000	0.0	0.0
5	-	20	8.9	200	0.005	0.0	0.5
10	-	20	8.9	200	0.005	0.5	0.5
15	-	20	5.5	200	0.000	0.0	0.0
Positive control	-	20	7.0	200	0.300	1.0	18.0

Conclusions:

Octamethyltrisiloxane has been tested in a reliable in vitro cytogenetic assay according to OECD TG 473 and under GLP. The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency in CHO cells. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations (is not clastogenic) in vitro under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data are available for decamethyltetrasiloxane (L4) from reliable studies on mutagenicity to bacterial and to mammalian cells. No data are available for cytogenicity to mammalian cells for the registered substance, however, a reliable study is available for the structural analogue, octamethyltrisiloxane (L3, CAS 107 -51 -7). Decamethyltetrasiloxane and octamethyltrisiloxane both hydrolyse slowly (measured), producing dimethylsilanediol and trimethylsilanol. Neither siloxanes nor silanediols/silanetriols are likely to contribute to genetic toxicity, and both registered and analogue substances have long hydrocarbon side-chains. It is therefore considered appropriate to read-across the in vitro mammalian cytogenicity study from octamethyltrisiloxane to the registered substance. Additional information is given in a supporting report (PFA (2013aa)) attached in Section 13 of the IUCLID 6 dossier.

L3 was selected as read-across substance because it has the same hydrolysis products as L4, and both substances hydrolyse slowly (see Section 4.1.1.1). Neither substance has any functional groups that are associated with genetic toxicity. The genetic toxicity data available for other substances from the analogue group are summarised in the Table below.

CAS	Name	Bacterial Mutagenicity	In VitroMammalian Cytogenicity	In VitroMammalian Mutagenicity	In VivoGenotox
107-46-0	Hexamethyldisiloxane	Negative Shin-Etsu (1994)	Negative Shin-Etsu (1995)	Negative Litton Bionetics (1978a)	Negative in chromosome aberration assay Dow Corning Corporation (1982)
107-51-7	Octamethyltrisiloxane	Negative Wagner, V. O. (2008)	Negative Madraymootoo, W. and Rao, M. (2008)	No data	No data
141-62-8	Decamethyltetrasiloxane	Negative Wagner, V. O., Hines, R. M,. (2005)	No data	Negative Flanders L (2010)	No data
141-63-9	Dodecamethylpentasiloxane	No data	Awaiting results; 3rd experiment planned	Awaiting results or waiver	Proposal of in vivo micronucleus study may be required depending on result of in vitro tests

Decamethyltetrasiloxane has been testing in a reliable study conducted according to OECD 471 1997 and under GLP up to limit concentrations (Wagner, V. O., Hines, R. M., (2005)). No increase in the number of revertants was observed at any concentration with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA in either the initial plate incorporation assay or the independent repeat experiment which used the pre-incubation method. It is considered that the test substance was negative for mutagenicity to bacteria under the conditions of the test.

No information is available for the registered substance on the potential for clastogenicity to mammalian cells, but information is available for the structural analogue, octamethyltrisiloxane, from a reliable in vitro cytogenetic assay conducted according to OECD TG 473 and under GLP (Madraymootoo and Rao (2008)). The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency in CHO cells. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in vitro (is not clastogenic) under the conditions of the test.

Decamethyltetrasiloxane has been tested according to OECD TG 476 and under GLP conditions for mutagenicity to mouse lymphoma L5178Y cells up to cytotoxic concentrations (Flanders L (2010)). No increase in mutant frequency was detected at any concentration after 4h exposure with and without metabolic activation and 24 h exposure without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in L5178Y cells under the conditions of the test.

The results of all the in vitro studies are negative, so there is no justification for proposal of in vivo testing.

)

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data, decamethyltrisiloxane is not classified for mutagenicity according to Regulation (EC)1272/2008.