Decamethylcyclopentasiloxane

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) (RCC Cytotest cell research, 2003).
Cytogenicity in mammalian cells: Negative in Chinese hamster V79 cells (OECD TG 473) (Dow Corning Corporation, 2003).
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

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

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:

Phenobarbital beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and relative non-toxicity to bacteria, and on request of the sponsor.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains (with activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 98, TA 1537 (without activation)

Untreated negative controls:

yes

Remarks:

untreated

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:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2uvrA (without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION

- Preincubation period: 60 minutes

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

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; background lawn

Evaluation criteria:

The test article is considered a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA-98, TA-100 and WP2 uvrA) or thrice (strains TA-1535, TA-1537) the colony count of the corresponding solvent control is observed. An increase exceeding the threshold at only one concentration is judged as biologically relevant if produced in an independent second experiment. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of the negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation is required. Responses (number of revertants) to the test article were compared to concurrent
negative and positive controls.

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:

valid

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:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertants colony numbers of any the five tester strains was observed following treatment with D5 at any dose level, either in the presence or absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The controls were within historical values with the following exceptions: Strain TA 1535 without activation: number of revertants greater than historical values; small deviations were observed in the negative controls of two strains in the second experiment. These deviations do not affect the validity of the experiment.

Table 1 Experiment 1 plate incorporation: Number of revertants per plate (mean of 3 plates)

	TA 1535			TA 1537			TA 98
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Untreated	16	17	no	6	21	no	27	32	no
0*	13	27	no	8	15	no	28	44	no
33	13	14	no	5	13	no	30	41	no
100	14	17	no	5	11	no	32	42	no
333	10	18	no	7	16	no	34	42	no
1000	15	14	no	9	15	no	30	40	no
2500	14	16	no	7	11	no	33	39	no
5000	16	13	no	8	13	no	36	33	no
Positive control	884	161	-	58	67	-	273	658	-

*solvent control with ethanol

 

 Table 2 Experiment 1 plate incorporation: Number of revertants per plate (mean of 3 plates)

	TA 100			E coli WP2 uvrA
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Untreated	162	178	no	35	42	no
0*	152	164	no	40	47	no
33	166	194	no	38	36	no
100	156	190	no	45	39	no
333	151	215	no	35	43	no
1000	148	207	no	35	47	no
2500	164	206	no	26	46	no
5000	144	190	no	31	47	no
Positive control	773	970	-	768	180	-

*solvent control with ethanol

 

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

	TA 1535			TA 1537			TA 98
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Untreated	18	16	no	8	15	no	33	40	no
0*	15	21	no	9	12	no	38	48	no
33	11	17	no	11	14	no	32	39	no
100	16	20	no	10	9	no	34	46	no
333	21	19	no	8	15	no	40	48	no
1000	18	17	no	11	13	no	41	47	no
2500	15	15	no	9	10	no	40	48	no
5000	16	14	no	8	11	no	43	55	no
Positive control	1319	147	-	72	64	no	229	507	-

*solvent control with ethanol

 

 Table 4 Experiment 2 preincubation: Number of revertants per plate (mean of 3 plates)

	TA 100			E coli WP2 uvrA
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Untreated	178	248	no	38	31	no
0*	206	287	no	44	37	no
33	191	265	no	32	49	no
100	187	251	no	38	51	no
333	213	244	no	41	46	no
1000	185	239	no	36	38	no
2500	217	269	no	39	35	no
5000	229	318	no	47	43	no
Positive control	581	624	-	284	166	-

*solvent control with ethanol

Conclusions:

Decamethylcyclopentasiloxane has been tested according to OECD 471 and in compliance with GLP using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvrA. No test-substance related increase in the number of revertants was detected in the presence or absence of metabolic activation in either the initial (RCC Cytotest cell research, 2003) plate incorporation assay or the repeat experiment using pre-incubation, up to limit concentrations. Appropriate positive, solvent and untreated controls were included and gave expected results. The test substance is therefore considered to be 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:

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Expt. I: up to 0.2 ul/ml (without S9 mix) and up to 5 ul/ml (with S9 mix)
Expt II: up to 0.025 ul/ml (without S9 mix) and up to 5 ul/ml (with S9 mix) continuous exposure

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

Growth medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without activation)

Untreated negative controls:

yes

Remarks:

Growth medium

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 medium

DURATION

- Exposure duration: (without activation) 4 hours exp 1; 18, 28 hours exp 2. (with activation) 4 hours exp 1 and 2

- Expression time (cells in growth medium): (without activation) 18,18, 28 hours exp 1; (with activation) 18, 28 hours exp 2

NUMBER OF REPLICATIONS: 2 plates for each test concentration

NUMBER OF CELLS EVALUATED: 100 metaphase cells scored per plate

DETERMINATION OF CYTOTOXICITY

- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:

- Determination of polyploidy: determined as % of control

Evaluation criteria:

Chromosomes analysed for aberrations and statistical significance calculated.

Statistics:

Statistical significance was determined by means of the Fischer exact test (p<0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4 hrs at 0.039 to 0.6 ul/ml and for 24 hrs at 0.039 ul/ml and above in the absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Other confounding effects: In experiment 1, in the absence of S9 mix, oily drops were observed on the surface of the culture medium 4 hours after the start of treatment at 0.1 and 0.2 ul/ml.

Summary of results: mean of duplicate cultures, 100 cells per culture counted

Concentration μl/ml	Polyploid cells (% of control)	Cell numbers (% of control)	Mitotic indices (% of control)	Cells with aberrations (%)
				inc gaps	exc gaps	with exchanges
Experiment 1 without metabolic activation; 4 h exposure
0*	2.4	n.t.	100	1.5	0.5	0.0
0**	2.9	100	100	0.5	0.5	0.0
Positive control	2.7	n.t.	132	18.0	15.5	7.5
0.003	2.6	102	136	3.0	0.5	0.0
0.013	2.9	54	81	2.5	1.5	1.0
0.025	2.5	40	93	1.0	0.5	0.0
Experiment 2 without metabolic activation; 18 h exposure
0*	1.3	n.t	100	2.0	1.5	0.0
0**	1.9	100	100	0.5	0.0	0.0
Positive control	1.4	n.t.	140	11.5	9.0	3.0
0.003	1.4	90	112	1.5	0.5	0.0
0.006	1.7	96	75	1.5	0.5	0.0
0.013	2.1	31	39	0.0	0.0	0.0
Experiment 2 without metabolic activation; 28 h exposure
0*	1.8	n.t	100	1.0	0.5	0.0
0**	2.1	100	100	2.5	2.0	0.0
Positive control	1.7	n.t.	86	30.0	28.0	13.0
0.003	1.8	85	116	1.0	0.5	0.0
0.006	1.9	36	57	0.0	0.0	0.0
Experiment 1 with metabolic activation; 4 h exposure
0*	2.2	n.t	100	2.0	1.5	0.0
0**	2.6	100	100	3.0	2.0	0.5
Positive control	1.9	n.t.	98	14.0	13.0	5.5
3.0	2.1	77	93	4.0	3.0	0.5
4.0	1.9	95	87	3.0	2.0	1.0
5.0	2.0	99	92	2.5	1.5	0.5
Experiment 2 with metabolic activation; 4 h exposure
0*	1.1	n.t	100	0.0	0.0	0.0
0**	0.9	100	100	1.0	0.5	0.0
Positive control	1.0	n.t.	93	11.0	10.0	0.5
3.0	0.7	95	122	0.5	0.5	0.0
4.0	1.2	116	103	1.0	0.0	0.0
5.0	1.5	87	96	0.0	0.0	0.0

* negative control (untreated)

** solvent control (ethanol)

Conclusions:

Decamethylcyclopentasiloxane has been tested according to OECD 473 and in compliance with GLP in Chinese hamster lung fibroblasts (V79 cells). No test-substance related increase in the number of cells with structural or numerical aberrations was detected in the presence or absence of metabolic activation in either the initial or the repeat exposure when tested up to cytotoxic concentrations. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations (i.e. is not clastogenic) 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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no duplicates

Principles of method if other than guideline:

The procedure used is a modification of that reported by Clive and Spector (Mutation Research, 31 17-29, 1975).

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:

0.8-12.5 µl/ml (-S9); 0.8-6.4 µl/ml (+S9)

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

(with activation)

Evaluation criteria:

A compound is considered mutagenic if the minimum increase is at least 2.5 times greater than the solvent control value.

Statistics:

A mutation index was derived by dividing the number of clones found in the BUdR-containing selection medium by the number found in the same medium without BUdR. The ratio was then compared to that obtained from other dose levels and from positive and negative controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Responses were within the excepted ranges.

Table 2: Results of Mammalian Mutagenicity assay with L5178Y/TK^+/-  Mouse Lymphoma cells

Concentration µl/ml	Mutant* Frequency	Mutant* Frequency	%Relative Growth.	%Relative Growth.	Cytotoxicity (yes/no)
	— MA	+ MA	— MA	+ MA	-
Solvent Control	11.5	74.7	100	100	No
Negative Control	-	-	78	221.2	No
Positive Control	400	247.9	8.1	5.0	No
0.8	24.2	13.1	83.6	112.1	No
1.6	16.6	6.5	99.5	242.3	No
3.2	14.3	17	82.4	183.1	No
6.4	14.7	140	103.6	14.9	yes (+MA)
12.5	21.5	-	79.7	-	no

*Per 10⁶surviving cells

Solvent control with Ethanol

Conclusions:

Decamethylcyclopentasiloxane has been tested in a study conducted according to a protocol that is similar to OECD TG 476. No significant mutagenic activity was noted for the test substance with or without metabolic activation when tested in L5178Y TK+/- mouse lymphoma cells up to cytotoxic concentrations. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay inhalation study in rat: Negative (OECD TG 474) (RCC Cytotest cell research, 2004).
Unscheduled DNA synthesis inhalation study in rat: Negative (OECD 486) (RCC Cytotest cell research, 2004).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Age at study initiation: Males 8 weeks / Females 9 - 12 weeks


TEST ANIMALS
- Source: Chales River
- Age at study initiation: Males 8 weeks / Females 9 - 12 weeks
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 2 same sex animals in stainless steel cages in sealed chambers
- Diet: ad libitum, except during exposure periods
- Water: ad libitum, except during exposure periods
- Acclimation period: 5days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

inhalation

Vehicle:

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

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: 2200 L, stainless steel chambers equipped with glass doors

- Source and rate of air: Compressed air (40l/min)

- System of generating particulates/aerosols: Vapours generated by heating round-bottom flask containing test article

- Air flow rate: 380 l/min

Duration of treatment / exposure:

6 hours/day for 7 days

Frequency of treatment:

daily for 7 consecutive treatments

Dose / conc.:

160 ppm (nominal)

Remarks:

highest that can be prepared consistently based on vapour pressure

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene

- Route of administration: Oral gavage

- Doses / concentrations: 100 mg/kg b.w.

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): All animals were observed for mortality/moribundity twice daily during the 7 day exposure period, prior to and following exposure. Body weights were taken on the first day of the acclimatization period and on exposure days 1, 4 and 7.

DETAILS OF SLIDE PREPARATION: Primary hepatocytes were obtained by liver perfusion and cultures were established and exposed for 4 hours to 3HTdR, which is incorporated if UDS occurs. Cells were fixed to coverslips, which were contained in the culture dishes, and prepared for silver grain counting (autoradiography)

METHOD OF ANALYSIS: At least two slides/animal and 50 cells/slide were evaluated. The nuclear and cytoplasmic grain counts, as well as, the net grain counts (nuclear minus cytoplasmic grains) were reported separately.

Evaluation criteria:

A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points. A group average between 0 and 5 net grains is considered a marginal response.

Statistics:

Student's t-test for body weights; Mann-Whitney test for analysis of micronucleated polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean nominal concentration for the groups exposed to D5 (Groups 2, 5, 8 and 11) was 164.1 ± 3.2 ppm. The mean
analytical concentration for the same groups was 156.4 ± 3.1 ppm, thus, 4.7% lower than the mean nominal concentration.

There were no unscheduled deaths or clinical signs observed during the course of the study. There were no statistically
significant differences in body weight between the treatment and the control groups.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test item at any
of the treatment periods. The interindividual variations obtained for the numbers and the viabilities of the isolated
hepatocytes were in the range of the laboratory's historical control.

The test item did not induce any UDS in the hepatocytes of the treated animals as compared to the current vehicle
controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment
of the animals with the test item. Therefore, the net grain values obtained after treatment with the test item were
consistently negative. In addition, no substantial shift to higher values was obtained in the percentage distributions
of the nuclear grain counts.

In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Table 1 Mean net grains per nucleus

Treatment	Net grains per nucleus (males)		Net grains per nucleus (females)
Period	5/6 hours	16 hours	5/6 hours	16 hours
Air control	-9.57	-14.80	-6.51	-12.85
160 ppm D5	-9.72	-13.16	-7.26	-11.83
Positive control	45.65	17.99	34.27	13.41

Conclusions:

Decamethylcyclopentasiloxane has been tested according to OECD 486 and in compliance with GLP in an inhalation study in rat. No test substance related induction of unscheduled DNA synthesis in the hepatocytes of treated rats relative to the vehicle was observed. Appropriate positive control substance was used and gave expected results. it is concluded that the test substance is negative for the induction of unscheduled DNA synthesis in rat hepatocytes under the conditions of the test.

Reference 2

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)

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Age at study initiation: Males 8 weeks / Females 9 - 12 weeks

Route of administration:

inhalation: vapour

Vehicle:

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

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: 2200 L stainless steel chambers equipped with glass doors

- Source and rate of air: Compressed air

- Method of conditioning air: Compressed air (40l/min) was used to carry vapours from the heated round-bottom flask (containing the test article) to the chamber inlet where it was mixed with filtered dilution air (380 l/min).

- Air flow rate: 380 l/min

Duration of treatment / exposure:

6 hours

Frequency of treatment:

Daily for 7 consecutive days

Post exposure period:

24 hours

Dose / conc.:

160 ppm (nominal)

No. of animals per sex per dose:

Groups 1 - 9 each consisted of twelve animals/group (six animals/sex) while groups 10 - 11 consisted of only six
animals/group (females only).

Control animals:

yes, concurrent vehicle

Positive control(s):

One positive control group treated the same as the air control groups except dosed by oral gavage with
cyclophosphamide (CPA at 40 mg/kg b.w.) after the final exposure (group 9).

Tissues and cell types examined:

For the analysis of micronucleated erythrocytes, animals were sacrificed 24 hours following the last exposure and the marrow from the femora was harvested.

Details of tissue and slide preparation:

The erythrocytes were separated from the nucleated cells and smears were air-dried, stained and coverslipped. At least one slide was made from each bone marrow sample. At least 2000 polychromatic erythrocytes (PCE) were analysed/animal for micronuclei (chromosomal fragments or whole chromosomes induced by clastogens).

Evaluation criteria:

The ratio between polychromatic and normochromatic erythrocytes (NCE) was determined to assess cytotoxicity. For an acceptable test, the polychromatic/normochromatic erythrocyte ratio (PCE/NCE) should be within 20% of the negative control.

Statistics:

Statistical analysis was performed to determine if the mean number of micronucleated PCEs or NCEs in the test group
was significantly increased (p<0.05) compared to the control group.

Student's t-test for body weights; Mann-Whitney test for analysis of micronucleated polychromatic erythrocytes (PCE)
and normochromatic erythrocytes (NCE).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean nominal concentration for the groups exposed to D5 (Groups 2, 5, 8 and 11) was 164.1 ± 3.2 ppm. The mean
analytical concentration for the same groups was 156.4 ± 3.1 ppm, thus, 4.7% lower than the mean nominal concentration.

There were no unscheduled deaths or clinical signs observed during the course of the study. There were no statistically
significant differences in body weight between the treatment and the control groups.

The mean number of PCEs was not decreased after treatment with the test item as compared to the mean values of the
vehicle control, indicating that decamethylcyclo-pentasiloxane (D5) had no cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant
enhancement in the frequency of the detected micronuclei in both NCEs and PCEs after administration of the test item.
The mean values of micronuclei observed after treatment with decamethylcyclopentasiloxane (D5) were below or near to the
value of the vehicle control group.

CPA administered orally was used as a positive control which showed a statistically significant increase of induced micronucleus
frequency.

Table 1 Micronucleus results: males

Test group	NCEs with micronuclei (%)	PCEs with micronuclei (%)	PCE/NCE
Air control	0.030	0.190	2000/2226
160 ppm D5	0.040	0.100	2000/2435
40 mg/kg bw CPA*	0.020	0.775	2000/3263

*CPA Cyclophosphamide (positive control)

 

Table 2 Micronucleus results: females

Test group	NCEs with micronuclei (%)	PCEs with micronuclei (%)	PCE/NCE
Air control	0.025	0.125	2000/2493
160 ppm D5	0.060	0.100	2000/2214
40 mg/kg bw CPA*	0.045	0.380	2000/3086

*CPA Cyclophosphamide (positive control)

Conclusions:

Decamethylcyclopentasiloxane has been tested according to OECD 474 and in compliance with GLP in an inhalation study in rat. No increase levels of
micronucleated cells relative to control was observed in the bone marrow cells of the treated rats. Appropriate vehicle and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of micronuclei 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. In addition to the standard assays, there are results available from an in vitro sister chromatid exchange assay in L5178Y cells, and DNA damage in bacterial and L5178Y cells. The results of all the studies were in agreement.

D5 has been tested according to OECD 471 and in compliance with GLP using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvrA (RCC Cytotest cell research, 2003). No test-substance related increase in the number of revertants was detected in the presence or absence of metabolic activation in either the initial plate incorporation assay or the repeat experiment using pre-incubation up to limit concentrations. Appropriate positive, solvent and untreated controls were included and gave expected results. The test substance is therefore considered to be negative for mutagenicity to bacteria under the conditions of the test.

D5 has been tested according to OECD 473 and in compliance with GLP in Chinese hamster lung fibroblasts (V79 cells) (Dow Corning Corporation, 2003). No test-substance related increase in the number of cells with structural or numerical aberrations was detected in the presence or absence of metabolic activation in either the initial or the repeat exposure when tested up to cytotoxic concentrations. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations (i.e. is not clastogenic) under the conditions of the test.

D5 has been tested in a study conducted according to a protocol that is similar to OECD TG 476 (Litton Bionetics, 1978). No significant mutagenic activity was noted for the test substance with or without metabolic activation when tested in L5178Y TK+/- mouse lymphoma cells up to cytotoxic concentrations. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

D5 has been tested according to OECD 474 and in compliance with GLP in an inhalation study in rat (RCC Cytotest cell research, 2004). No increase levels of micronucleated cells relative to control was observed in the bone marrow cells of the treated rats. Appropriate vehicle and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.

D5 has been tested according to OECD 486 and in compliance with GLP in an inhalation study in rat (RCC Cytotest cell research, 2004). No test substance related induction of unscheduled DNA synthesis in the hepatocytes of treated rats relative to the vehicle was observed. Appropriate positive control substance was used and gave expected results. it is concluded that the test substance is negative for the induction of unscheduled DNA synthesis in rat hepatocytes under the conditions of the test.

Overall, the mutagenicity of D5 was investigated for the above endpoints. D5 neither induced gene mutations in bacteria in vitro, nor caused an increase in cells with chromosome aberrations. These negative results were confirmed under in vivo conditions. Treatment with D5 also neither led to an increase in cells with micronuclei in the bone marrow cells of mice, nor caused unscheduled DNA synthesis in rats. Consequently, D5 can be considered to have no genotoxic potential and additional tests are not required.

Justification for classification or non-classification

Based on the available data decamethylcyclopentasiloxane does not require classification for mutagenicity according to Regulation (EC) No. 1272/2008.