Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): Negative with and without 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

Referenceopen allclose all

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:
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.
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.
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:
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

Referenceopen allclose all

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:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes (incl. certificate)
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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.
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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.