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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following key in vitro genetic toxicity tests are available for the registered substance hexamethylcyclotrisiloxane (D3; CAS 541-05-9; EC No. 208-765-4):

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (similar to OECD Test Guideline 471, but not in compliance with GLP) (Litton Bionetics, 1978a).

Cytogenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (similar to OECD Test Guideline 473 and in compliance with GLP) (Litton Bionetics, 1978b).

Mutagenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (similar to OECD Test Guideline 476 but not in compliance with GLP) (Litton Bionetics, 1978c).

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
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
plates not replicated, range of strains not compliant with current guideline
Principles of method if other than guideline:
Methods for detecting carcinogens and mutagens with Salmonella mammalian-microsome mutagenicity test. Mutation Res. 31, 347-364. 1975.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA-1535, TA-1537, TA-1538, TA-98, TA-100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.1, 1.0, 10 and 500 µg/plate
Vehicle / solvent:
ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1-nitrosoguanidine
Remarks:
TA1535 and TA100 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: quinacrine mustard
Remarks:
TA1537 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 and TA1538 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: anthraquinone
Remarks:
TA100 and TA1535 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: aminoquinoline
Remarks:
TA1537 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: acetyl aminofluorene
Remarks:
TA1538 and TA98 with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation);

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

NUMBER OF REPLICATIONS: no replicates

DETERMINATION OF CYTOTOXICITY
- Method: no information
Evaluation criteria:
A substance producing a positive dose response over three concentrations, with lowest increase equal to twice the solvent control (TA1535, 1537, 1538) or the highest increase at least twice (TA 100) or 2-3 times (TA 98) is considered positive
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
All positive controls produced significantly increased revertants, while the negative controls did not.
No testing strains increased the number of revertants relative to the negative controls when tested with and without metabolic activation.  The maximum number of revertants in the solvent control was 230 (TA 100) and in the treated cultures was 292. The positive control gave a maximum of >1000 revertants (TA 1535)
Conclusions:
Hexamethylcyclotrisiloxane has been tested for mutagenicity to bacteria in a valid in vitro study in S. typhimurium TA1535, TA1537, TA1538, TA98, TA100, conducted according to a protocol similar to OECD Test Guideline 471 but prior to GLP compliance. The test material did not increase the number of revertants in any of the strains tested in a valid, reliable and reproducible study when tested with or without metabolic activation up to a limit concentration. Positive and solvent controls were included and gave the expected results. The test substance is concluded to be negative for mutagenicity to bacteria under the conditions of this test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
number of cells scored for aberrations (50 cells per dose were scored; current guideline requires 200)
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0.0976, 0.1953, 0.3906, 0.7812, 1.5625, and 3.1250 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 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:
other: 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): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): BUdR (20-hour incubation)
STAIN (for cytogenetic assays): Giesma


NUMBER OF REPLICATIONS: Six doses evaluated, no replications

NUMBER OF CELLS EVALUATED: 50

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Non-activation assay: The procedure used is a modification of that reported by Clive and Spector (Mutation Research, 31:17-29, 1975). Prior to treatment, cells were cleaned of spontaneous TK-/- by growing them in a medium containing thymidine, hypoxanthine, methotrexate, and glycine (THMG). The test compound was added to the cleansed cells in growth medium at the predetermined doses for four hours. The treated cells were washed, fed, and divided into two equal aliquots. One aliquot was used in the gene mutation assay (see supporting studies). BUdR was added for approximately 20 hours or two-cell doublings in order to incorporate it into the DNA strands.

Activation assay: The activation assay differs from the non-activation assay in the following manner only. Two millilitres of the reaction mixture were added to 10 ml of growth medium. The desired number of cleansed cells was added to this mixture, and the flask was incubated on a rotary shaker for four hours. The incubation period was terminated by washing the cells twice with growth medium. The cells were assayed as described previously for chromosome aberrations.
Statistics:
Statistical methods: Type and frequency of aberrations per cell (%) and number of cells with two or more aberrations (%)of the test substance were compared to concurrent negative and positive controls.  Statistical significance was determined based on employing a t-statistic.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0.016 mg/ml concentration for non-activated study and 1 mg/ml for activated study
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: The positive and solvent control results were comparable to the historical data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Statistical methods: Type and frequency of aberrations per cell (%) and number of cells with two or more aberrations (%)of the test substance were compared to concurrent negative and positive controls.  Statistical significance was determined based employing a t-statistic.

The tables below summarize the results of the cytogenetic analysis.  Both concurrent and cumulative controls are reported.  

Table 1: Non-activation


 Compound & concentration

 Total number of cells ab/cell (%)

 Type & frequency of more ab.

 No cells w/2 or %

 Mitotic index

 Negative control (media)

 

 

 

 

 Concurrent

 50

 0 (0.0

 0 (0.0

 6.2

 Cumulative

 550

 15 (2.7)

 1 (2.0)

 7.6

 Solvent control (EtOH)

 

 

 

 

 Concurrent

 50

 1tb (2)

 0 (0.0)

 5.8

 Cumulative

 550

 

 0 (0.0)

 8.8

 Positive control (EMS)

 

 

 

 

 Concurrent

 50

 5tb, 1d, 6af

 1 (2.0)

 3.5

 Cumulative

 451

 34 (7.5)

 19 (4.2)

 5.8

 Test compound (mg/ml)

 

 

 

 

 0.0976

 50

 1tb (2.0)

 0 (0.0)

 7.2

 0.1953  50  1f, 2af, 1min (6.0)  1 (2.0)  8.6
 0.3906  50  0 (0.0) 0 (0.0)   4.3
 0.7812  50 0 (0.0)   0 (0.0)  4.9
 1.562  50  1af (2.0)  0 (0.0)  7.6
 3.1250  50  2af (4.0)  0 (0.0)  6.4


(%) = Percent cells/aberrations
Mitotic index based on 500 cells. 

Table 2: without activation

 Compound & concentration

 Total number of cells ab/cell (%)

 Type & frequency of more ab.

 No cells w/2 or %

 Mitotic index

 Negative control(media)

 

 

 

 

 Concurrent

 50

 1t (2.0)

 0 (0.0

 5.6

 Cumulative

 550

 19 (3.5)

 1 (0.2)

 7.9

 Solvent control (EtOH)

 

 

 

 

 Concurrent

 50

  0 (0.0)

 0 (0.0)

 5.6

 Cumulative

 550

 4 (0.8)

 1 (0.2)

 7.5

 Positive control (DMN)

 

 

 

 

 Concurrent

 24

 1af, 2t

 0 (0.0)

 2.9

 Cumulative

 481

 84 (17.5)

 21 (4.4)

 5.4

 Test compound (mg/ml)

 

 

 

 

 0.0976

 41

 1tb, 1af (4.9)

 0 (0.0)

 2.4

 0.1953  14

 0 (0.0)

  0 (0.0)

 1.8

 0.3906

 50

 1tb (2.0)

1 (2.0) 

 4.6

 0.7812

 22

0 (0.0) 

 0 (0.0)

 2.1

 1.562

 50

 1tb, 1af (4.0)

 0 (0.0)

 5.2

 3.1250

 50

 2tb, 2af (8.0)

 0 (0.0)

 5.1

  
(%) = Percent cells/aberrations
Mitotic index based on 500 cells. 

af = acentric fragment
f = fragment
min = minute chromosome
t = translocation
tb = chromatid break

The highest value for chromosome aberrations was at a value of > 3 mg/ml in the presence of metabolic activation.  This increase was based on two chromatid breaks and 2 acentric
fragments and could be accounted for by toxicity.

Conclusions:
Hexamethylcyclotrisiloxane has been tested for the ability to induce chromosome aberrations in mouse lymphoma L5178Y cells in a valid study, conducted according to a protocol similar to OECD Test Guideline 473 and in compliance with GLP. A low level of chromosome damage was observed at a concentration of about 1 mg/ml or higher. These results could be explained by the slight cytotoxicity seen at these levels. It is concluded that the test substance is negative for cytogenicity under the conditions of the test when tested with or without metabolic activation up to cytotoxic concentration. Positive and solvent controls were included and gave the expected results.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
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:
rat liver S9
Test concentrations with justification for top dose:
0.067, 0.130, 0.270, 0.530, and 1.06 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) ethanol
Untreated negative controls:
yes
Remarks:
growth medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
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
Remarks:
ethanol
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): growth medium

NUMBER OF REPLICATIONS: triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth
Statistics:
Colonies were counted on an electronic colony counter that resolves all colonies greater than 200 microns in diameter.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 0.016 mg/ml concentration for non-activated study and 1 mg/ml for the activated study
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was exposed at concentrations up to 1.06  mg/ml for four hours. No increases in the mutant frequencies were  observed even under test conditions exhibiting cell death.
Conclusions:
Hexamethylcyclotrisiloxane was tested for mutagenicity in mouse lymphoma L5178Y cells in a valid study, conducted according to a protocol similar to OECD Test Guideline 476 but prior to GLP. No increase in the number of revertants was observed when tested with and without metabolic activation up to cytotoxic concentrations. Positive, negative and solvent controls were included and gave the 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

The following key in vivo genetic toxicity tests are available for the registered substance hexamethylcyclotrisiloxane (D3; CAS 541-05-9; EC No. 208-765-4):

Mammalian Bone Marrow Chromosome Aberration Test: Negative in rat (OECD Test Guideline 475 and in compliance with GLP) (Bioassay Systems, 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:
1980-11-25 to 1981-03-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
number of cells scored for MI; presentation of results
Principles of method if other than guideline:
Method: other
GLP compliance:
yes
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
- Source: Charles River
- Age at study initiation: 50-60 days
- Weight at study initiation: 200-440 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: 6/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 50
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
intraperitoneal
Vehicle:
Vehicle: DMSO
Duration of treatment / exposure:
6, 24 or 48 hours
Frequency of treatment:
Single ip injection of test and positive and solvent controls
Dose / conc.:
125 mg/kg bw (total dose)
Dose / conc.:
225 mg/kg bw (total dose)
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
400 mg/kg bw (total dose)
Dose / conc.:
515 mg/kg bw (total dose)
Dose / conc.:
1 080 mg/kg bw (total dose)
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
-cyclophosphamide
- Justification for choice of positive control(s): none given in report
- Route of administration: ip injection
- Doses / concentrations: 21 mg/kg bw
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: initial cytotoxicity assay.
The doses used in the cytogenetic studies were based on the range-finding experiments. Animals were injected IP with hexamethylcyclotrisiloxane prepared in glass tubes at doses of 1081, 721, 360, 180, and 72 mg/kg. No deaths were observed. A second range-finding experiment was not performed. All subsequent cytogenetic studies were performed using polypropylene tubes to prepare samples.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were sacrificed at 6, 24, or 48 hours after injection. Colchicine was injected 2 to 2.5 hours before sacrifice at a final dose of approximately 1.5 mg/kg.

DETAILS OF SLIDE PREPARATION: Approximately four slides were prepared from each animal. Slides were fixed, stained with Giesma and permanently mounted.

METHOD OF ANALYSIS: Suitable cells (with properly condensed and well spread chromosomes) were photographed. Projected photographs were examined for chromosomal breaks or gaps, chromatid breaks or gaps, complex rearrangements, polyploidy and large translocation or deletions. Small translocation or deletions could not be detected.
Statistics:
If the compound did not cause chromosomal damage, then the distribution of breaks per animal for all the negative control and test animals is expected to follow the Poisson distribution. A comparison of the expected and observed distribution values was performed using the Chi2 test as a measure of "goodness to fit". The Wilcoxon test was used as a non-parametric test to compare the distribution of breaks per animal between the negative control animals and the highest test doses. Details of both statistical evaluations are given in the text, Statistical Methods, Snedecor, G.W. and Cochran, W.G., Iowa State University Press, Ames, Iowa, 1980.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
on mitotic index. Deaths occurred at highest dose.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
For the cytogenetic experiments, hexamthylcyclotrisiloxane prepared in polypropylene tubes produced mortalities at high dose levels. The dosage groups analyzed in the cytogenetic studies were based on the maximum tolerated dose for each sacrifice group. In the 48-hour sacrifice group, doses of 1080 mg/kg killed 5 of 6 animals and 515 mg/kg killed 1 of 6 animals. In the 24-hour group, the highest dose level (400 mg/kg) killed 3 of 6 animals, but all 6 animals injected with 300 mg/kg lived. In the 6-hour assay, all of the animals injected with the highest dose, 300 mg/kg, lived.

There was no evidence that hexamethylcyclotrisiloxane induced chromosomal damage in rats following IP injection. A summary of the total number of cells analyzed for each dose at each time of sacrifice is summarized as follows: For animals injected with 125 mg/kg the frequencies of breaks were 1.40%, 2.02%, and 1.99% for the three times of sacrifice (6, 24, and 48 hours, respectively); frequencies of 0.71%, 2.17%, and 2.35% were seen for animals injected with 225 mg/kg at the three times of sacrifice. For animals dosed with 300 mg/kg, the frequencies of breaks were 2.07% and 1.01% at 6 and 24 hours; 0.90% breaks were seen with animals dosed with 400 mg/kg at 24 hours; and 1.94% breaks were seen in animals injected with 515 mg/kg at 48 hours. No complex rearrangements such as quadriradials, triradials, or ring chromosomes were detected in animals injected with the test substance. For the negative control animals, the frequencies of breaks were 0.85%, 0.58%, and 2.23% for sacrifice times of 6, 24, and 48 hours, respectively. The animals injected with cyclophosphamide had both a large number of breaks and complex rearrangements. A comparison of the frequencies of breaks for doses of hexamethylcyclotrisiloxane with the negative control at the three time points shows no significant differences. These frequencies are similar to those recorded for control animals in previous testing. The frequency of breaks in previous negative control animals ranged from 0 to 2.58%, a range compatible with all the results obtained in this project. The conclusion based on comparison to historical data is confirmed by a statistical analysis using the Wilcoxon test that demonstrated that there was no difference between the number of breaks per animal for the negative control groups and the test groups. The use of the Chi2 test as a measure of "goodness to fit" indicated that the distribution of breaks per animal did not closely follow the Poisson distribution. However, the Wilcoxon test demonstrated that there was no difference between the number of breaks per animal for the negative control groups and the test groups.

Data for the following dose groups is summarized below:
a. 48-hour sacrifice time: 515, 225, and 125 mg/kg

b. 24-hour sacrifice time: 400, 300, 225, and 125 mg/kg

c. 6-hour sacrifice time: 300, 225, and 125 mg/kg


Summary of Chromosome Aberration Data at three respective
intervals
                                        
Test   Dose   No.                
Chem. (mg/kg) cells   G*  B* O*  MI*(%)
DMSO:
6 hrs  --   142     1     1       0      4.6
              101     0     2       0      5.4
              116     5     1       0      5.8
              120     1     1       0      7.3
              107     0     0       0      4.7

24 hrs* --    120     0     0       0      7.3
              101     2     0       0      3.7
              103     1     2       0      3.0
              95      0     1       0      2.7
              94      0     0       0      6.8

48 hrs  --  117     0     0       0      4.8
              108     1     3       0      5.7
              92      0     5       0      3.5
              113     2     4       0      7.4
              107     0     0       0      5.2

Test material:
6 hrs  300  112     0     3       0      3.7
              120     1     1       0      4.8
              113     0     3       0      4.3
              115     0     0       0      3.4
              120     0     5       0      3.5
       225  114     3     0       0      6.9
              108     0     1       0      4.9
              120     0     0       0      3.3
              109     1     3       0      2.9
              110     2     3       0      4.6
       125  98      1     1       0      6.6
              107     2     1       0      7.9
              106     2     1       0      4.9
              118     2     3       0      3.6
24 hrs 400    117     0     1       0      4.9
              106     3     1       0      5.9
       300  121     0     2       0      3.0
              115     0     0       0      9.9
              136     3     0       0      5.1
              108     3     1       0      7.9
              114     4     3       0      5.0
       225  104     0     1       0      10.0
              119     0     0       0      8.3 
              115     0     4       0      5.8
              138     0     3       0      7.8
              122     1     5       0      8.9
       125  113     0     0       0      7.7
              121     0     2       0      7.8
              110     0     0       0      4.4
              131     0     6       0      5.1
              119     0     4       0      6.0
48 hrs 515  106     3     4       0      9.9   
              109     0     0       0      6.7
              146     2     3       0      12.4
       225  102     1     3       0      6.4
              115     0     3       0      6.1
              109     0     3       0      9.3
              109     0     0       0      6.8
              103     0     1       0      6.4
              100     0     5       0      5.4
       125  112     0     3       0      9.5
              119     1     4       0      6.9
              43      0     0       0      5.0
              118     0     2       0      5.1
              111     0     1       0      3.1
            
Cyclophosphamide
24 hrs   21 109     ND    >64     7Quad  4.2
                                    3Tri
              120     ND    >67     2Quad  5.4
                                    1Tri
              107     ND    >48     2Quad  2.5
                                    2Tri
---
*: G=Gaps; B=Breaks; O=Other; MI=Mitotic Index
ND = not determined
Quad = quadriradial
Tri = Triradial

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Hexamethylcyclotrisiloxane has been tested for mutagenicity to bacteria in a valid in vitro study in S. typhimurium TA1535, TA1537, TA1538, TA98, TA100, conducted according to a protocol similar to OECD Test Guideline 471 but prior to GLP compliance. The test material did not increase the number of revertants in any of the strains tested in a valid, reliable and reproducible study when tested with or without metabolic activation up to a limit concentration. Positive and solvent controls were included and gave the expected results. The test substance is concluded to be negative for mutagenicity to bacteria under the conditions of this test (Litton Bionetics, 1978a).

Hexamethylcyclotrisiloxane has been tested for the ability to induce chromosome aberrations in mouse lymphoma L5178Y cells in a valid study, conducted according to a protocol similar to OECD Test Guideline 473 and in compliance with GLP. A low level of chromosome damage was observed at a concentration of about 1 mg/ml or higher. These results could be explained by the slight cytotoxicity seen at these levels. It is concluded that the test substance is negative for cytogenicity under the conditions of the test when tested with or without metabolic activation up to cytotoxic concentration. Positive and solvent controls were included and gave the expected results (Litton Bionetics, 1978b).

Hexamethylcyclotrisiloxane was tested for mutagenicity in mouse lymphoma L5178Y cells in a valid study, conducted according to a protocol similar to OECD Test Guideline 476 but prior to GLP. No increase in the number of revertants was observed when tested with and without metabolic activation up to cytotoxic concentrations. Positive, negative and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test (Litton Bionetics, 1978c).

Hexamethylcyclotrisiloxane has been tested for mutagenicity to bacteria in a valid in vitro study in S. typhimurium TA1535, TA1537, TA1538, TA98, TA100, conducted according to a protocol similar to OECD Test Guideline 471 but prior to GLP compliance. The test material did not increase the number of revertants in any of the strains when tested with or without metabolic activation. The test substance is concluded to be negative for mutagenicity to bacteria under the conditions of this test (Dow Corning Corporation, 1979).

Hexamethylcyclotrisiloxane has been tested in E coli cells which are deficient in DNA polymerase, and in non-mutant cells for its ability to cause DNA damage. No indication of modification of DNA by the test substance was observed. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for DNA damage under the conditions of this test (Litton Bionetics, 1978d).

Hexamethylcyclotrisiloxane has been tested for DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro. No evidence of primary DNA damage was obtained. An increase in the change in elution was observed at the high dose when tested without metabolic activation, but it appeared to be the result of toxicity. It is concluded that the test substance is negative for the induction of damage to DNA under the conditions of the test (Litton Bionetics, 1978e).

Hexamethylcyclotrisiloxane has been tested for DNA damage and repair in an in vitro sister chromatid exchange assay, conducted according to a protocol similar to EU Method B.19 (Sister Chromatid Exchange Assay In Vitro) but prior to GLP compliance. The substance was slightly cytotoxic at 0.016 and 1 mg/ml when tested in the absence and presence of metabolic activation, respectively. Slight elevations in the sister chromatid exchange per cell were observed at  concentration near 1 mg/ml or higher with no associated increase in chromosomal aberrations which reduced the implications of the SCE's. Positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for the induction of damage to DNA under the conditions of the test (Litton Bionetics, 1978f).

Hexamethylcyclotrisiloxane has been tested for DNA damage and repair in an in vitro yeast cytogenic assay, not conducted according to OECD test guidelines and pre-dating GLP compliance. Positive and negative controls were included and gave the expected results. Hexamethylcyclotrisiloxane did not demonstrate genetic activity in this assay and was considered not mutagenic under these test conditions (Litton Bionetics, 1978g).

Hexamethylcyclotrisiloxane has been tested for the ability to induce chromosome aberrations in vivo in a study, conducted according to a protocol similar to OECD Test Guideline 475 and in compliance with GLP. The test substance did not cause an increase in the frequency of chromosomal breaks or aberrations in bone marrow cells of rats.  It is concluded that hexamethylcyclotrisiloxane is negative for clastogenicity (causing chromosomal damage) under the conditions of the test (Bioassay Systems, 1982).

Justification for classification or non-classification

Based on the available in vitro and in vivo genetic toxicity data, hexamethylcyclotrisiloxane does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.