Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: 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:
other: The study was conducted according to a test protocol that is similar to the appropriate OECD test guideline, with acceptable restrictions. The restrictions were that the results are not clearly presented. It was compliant with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals 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


Administration / exposure

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
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 225, 300, 400, 515 and 1,080 mg/kg
Basis:

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

Examinations

Tissues and cell types examined:
Bone marrrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: initial cytotoxicity assay

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 chromosmal breaks of gaps, chromatid breaks of 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.

Results and discussion

Test results
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

Any other information on results incl. tables

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.  

For the cytogenetic experiments, hexamethylcyclotrisiloxane
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.

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
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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance, hexamethylcyclotrisiloxane (CAS No.541-05-9), 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.