Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-18 to 2009-02-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2A (1996) and ICH S2B (1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick MD
- Age at study initiation: 6-8 weeks
- Weight at study initiation: range finding: males 30.1-33.5 g, females 24.8-26.7 g); definitive micronucleus study: males 28.6-33.4 g, females 23.8-26.3 g.
- Assigned to test groups randomly: under following basis: according to a computer-generated program, which is based on distribution according to body weight.
- Fasting period before study: no
- Housing: up to five per rodent Micro-Barrier cage
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark


IN-LIFE DATES: no information

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: based on good solubility/workability of the test article in the vehicle and compatibility with the test system and route of administration.
- Concentration of test material in vehicle: Stock solution 100 mg/ml diluted to appropriate doses: 25, 50, 100 mg/ml for definitive assay
- Lot/batch no. (if required): 3783J
Frequency of treatment:
Single treatment
Post exposure period:
24 and 78 hours
Doses / concentrationsopen allclose all
Dose / conc.:
25 other: mg/ml (nominal)
Remarks:
analysis of dosing solutions showed 85-115% of target solution
Dose / conc.:
50 other: mg/ml (nominal)
Remarks:
analysis of dosing solutions showed 85-115% of target solution
Dose / conc.:
100 other: mg/ml (nominal)
Remarks:
analysis of dosing solutions showed 85-115% of target solution
No. of animals per sex per dose:
5 (low and mid dose, and positive control); 10 (vehicle control); 15 (high dose)
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Justification for choice of positive control(s): none given in report - standard positive control substance
- Route of administration: intraperitoneal
- Doses / concentrations: concentration 2.5 mg/ml, dose 50 mg/kg bw

Examinations

Tissues and cell types examined:
Femoral bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on preliminary toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no additional information

DETAILS OF SLIDE PREPARATION: (slides) were prepared and stained with May-Gruenwald-Giemsa stain

METHOD OF ANALYSIS: Using a light microscope and a medium magnification (400X), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000X), the following cell populations and cellular components were evaluated and enumerated:
Polychromatic erythrocytes (PCEs)
PCEs stain bluish. PCEs are young erythrocytes (early stage of erythropoiesis) and are the target cells for evaluation of the test article clastogenicity. Two-thousand PCEs per mouse were scored for the presence of micronuclei resulting in evaluation of a total of 10,000 PCEs per each treatment group.
Normochromatic erythrocytes (NCEs)
NCEs stain pink (reddish). NCEs are mature erythrocytes (red blood cells) and are the final cell population formed during erythropoiesis. The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) was determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs). The incidence of MNCEs per 2000 PCEs was enumerated for each animal, but the results were not presented in the report
Micronuclei (M)
Micronuclei are round, darkly-staining nuclear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs).
The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (PCEs/ECs ratio).

Evaluation criteria:
A dose-dependent increase in the incidence of micronucleated polychromatic erythrocytes and one or more doses statistically elevated relative to the vehicle control would be considered a positive result.
Statistics:
Kastenbaum-Bowman Tables (binomial distribution, p ≤ 0.05)

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection (1000 and 2000 mg/kg), lethargy (2000 mg/kg); reductions in PCE/EC ratio (up to 11%) were observed in some test article groups relative to the vehicle control group.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000 and 2000 mg/kg bw
- Solubility: no information
- Clinical signs of toxicity in test animals: lethargy and piloerection at top and top two doses respectively
- Evidence of cytotoxicity in tissue analyzed: none
- Rationale for exposure: up to limit dose
- Harvest times: 3 days

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/CE (for Micronucleus assay): reductions of up to 22% observed in some test article groups. Reduction of this magnitude without a dose response suggests that the test article did not inhibit erythropoiesis.
- Appropriateness of dose levels and route: appropriate dose levels and route
- Statistical evaluation: appropriate statistical evaluation of results
- The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range.

Any other information on results incl. tables

Table 1: Summary of Bone Marrow Micronucleus Analysis Following a Single Intraperitoneal Administration of Dodecamethylcyclohexasiloxane (D6) in ICR Mice

Treatment

(20 ml/kg)

Sex

Time

(hr)

Number

of

Animals

PCE/Total

Erythrocytes

(Mean +/- SD)

Change from

Control (%)

Number of

MPCE/1000 PCE

(Mean +/- SD)

Number of

MPCE/PCE Scored

Corn Oil

 

M

24

5

0.426 ±0.05

-

0.1 ± 0.22

1/10000

F

24

5

0.396 ± 0.08

-

0.1 ± 0.22

1/10000

Dodecamethylcyclohexasiloxane (D6)

 

500 mg/kg bw

M

24

5

0.387 ± 0.04

-9

0.2 ± 0.27

2/10000

F

24

5

0.370 ± 0.09

-7

0.2 ± 0.27

2/10000

1000 mg/kg bw

M

24

5

0.380 ± 0.03

-11

0.01 ± 0.22

1/10000

F

24

5

0.393 ± 0.07

-1

0.01 ± 0.22

1/10000

2000 mg/kg bw

M

24

5

0.462 ± 0.02

8

0.04 ± 0.22

4/10000

F

24

5

0.449 ± 0.07

13

0.02 ± 0.27

2/10000

M

48

5

0.484 ± 0.03

-9

0.2 ± 0.27

2/10000

F

48

5

0.489 ± 0.15

-11

0.2 ± 0.27

2/10000

Cyclophosphamide

50 mg/kg

M

24

5

0.336 ± 0.04

-21

11.9 ± 1.43

*119/10000

F

24

5

0.348 ± 0.04

-12

10.9 ± 1.29

*109/10000

 

M

24

5

0.530 ± 0.03

-

0.2 ± 0.27

2/10000

Corn Oil

F

24

5

0.550 ± 0.03

-

0.2 ± 0.27

2/10000

*Statistically significant increase, p </= to 0.05 (Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:
Dodecamethylcyclohexasiloxane (D6) has been tested in a reliable in vivo micronucleus assay in ICR mice conducted according to OECD TG 474 and in compliance with GLP. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to control was observed in any test substance group up to limit concentrations at 24 and 48 hours after intraperitoneal administration of the test substance. Reductions in PCE/EC ratio (up to 11%) were observed in some test article groups, indicating that the test article had reached the target tissue. Administration of cyclophosphamide (the positive control) induced marked increases in micronucleated polychromatic erythrocytes. It is concluded that the test substance is negative for the induction of micronuclei in vivo under the conditions of the test.