Registration Dossier

Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
with stated exceptions which do not impact the study integrity. See any other information on materials and methods for more information on GLP exceptions.
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane
EC Number:
219-863-1
EC Name:
2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane
Cas Number:
2554-06-5
Molecular formula:
C12H24O4Si4
IUPAC Name:
2,4,6,8-tetramethyl-2,4,6,8-tetravinyl-1,3,5,7,2,4,6,8-tetroxatetrasilocane

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Crl:CD1(ICR)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: one per cage in stainless steel cages
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form, ad libitum
- Water (e.g. ad libitum): municipal water, ad libitum
- Acclimation period: one week prior to the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test substance 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was determined to be soluble in corn oil at a concentration of 200 mg/mL.
- Concentration of test material in vehicle: 50 mg/mL, 100 mg/mL and 200 mg/mL
All stock dosing solutions utilized for dosing on the initial day of the MNT were evaluated. The concentrations of the test material in the dosing solutions used for the first day of dosing were verified using gas chromatography with mass spectrometry (GC/MS) detection and external standard quantitation. The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 89.8 to 93.8% of the targeted values + 1.0 to 7.0 % RSD, but the values are not given.

- Amount of vehicle (if gavage or dermal): Dosing solutions were administered in aliquots of 10 ml/kg bodyweight (bw)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions of the test material were prepared and used fresh on each of the two consecutive days of administration.

Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Test substance: The animals were dosed on two consecutive days (March 3, 2020 and March 4, 2020)
Positive control: Administered once
Post exposure period:
48 hours after the last dosing
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 male mice per group
Additional satellite animals were utilized to assess blood levels for the presence of test material to confirm systemic exposure. Individual animals were administered the test material (2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane) via oral gavage on two consecutive days at a targeted concentration of 2000 mg/kg body weight. Subsequent to the second gavage administration, blood samples from each animal were collected at terminal sacrifice at the following time points (one animal/time point): 15 minutes, 30 minutes, 60 minutes, 180 minutes, and ~48 hours, in accordance with the study design. Blood was collected via the orbital sinus in a manner similar to the collection of typical samples for the micronucleus analysis.
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): CP when administered by oral gavage has been shown to induce micronuclei in polychromatic erythrocytes of Crl:CD1(ICR) mice (De Boeck et al., 2005; Gollapudi et al., 1986).
- Route of administration: oral gavage
- Doses / concentrations: Dosing solutions were administered in aliquots of 10 ml/kg bodyweight (bw). CP was administered once only at dose of 40 mg/kg bw/day.

Examinations

Tissues and cell types examined:
Approximately 5,000 RETs were analysed per blood sample.
The number of normochromatic erythrocytes (NCE) and micronucleated NCE (MN-NCE), reticulocytes (RET) and micronucleated RET (MN-RET) was recorded for each sample and the frequency of MN-RET calculated to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoietic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RETs and MN-RETs observed.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels for the MNT were selected based upon the range finding test. Relatively non-toxic compounds were tested up to 2 g/kg body weight/day. The middle- and low-doses were approximately 1/2 and 1/4 of the maximum dose, respectively. The dose level for the positive control chemical, CP (40 mg/kg) was based upon our internal historical control database, which indicated a pronounced micronuclei induction in CD-1 mice at this dose level.

A dose range-finding test was conducted to aid the selection of dose levels for the MNT. The number of animals included in each dose group was based upon the use of the dose selection criteria. Male and female rats were dosed on September 3-4, 2019 (one/sex/dose) with 1000 or 2000 mg/kg bw/day of the test material on two consecutive days and observed for any signs of toxicity for at least 72 hours after the initial dosing. Based upon the results of the initial dosing, an additional two mice/sex/dose were dosed with 1000 or 2000 mg/kg bw/day on September 10-11, 2019. These animals were also observed for at least 72 hours after the initial dose for any signs of toxicity at the end of the study period. All animals were anesthetized with isoflurane, followed by euthanasia via CO2 and cervical dislocation.

Based upon the results of the range-finding study where no apparent sex differences were noted, only males were evaluated in the main study. The highest concentration tested was at the limit dose of 2000 mg/kg bw/day. The middle- and low-doses were 1000 mg/kg bw/day and 500 mg/kg bw/day, respectively.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): It takes approximately 48 hours for the treated erythroblasts to undergo one post-treatment division and mature into reticulocytes and reach a peak incidence in the peripheral blood (CSGMT, 1992). However, if the treatment induces cell cycle delay, or if there is a delayed absorption and metabolism of the test material, a longer sampling time (such as 72 hours) may be needed to recover the damage induced in the erythroblasts. The sampling time used in this study provided an opportunity to recover any delayed effect (i.e., effect induced by the first treatment) as well as a more immediate effect (i.e., effect induced by the second treatment) in the same treated animals.

DETAILS OF SLIDE PREPARATION:
Duplicate cell smears were prepared and stored to serve as a backup to flow cytometric analysis if required. Blood was collected into a microtainer tube coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears were prepared, fixed in methanol, and stored at room temperature. Slides were coded and manually scored with a fluorescent microscope for MN evaluation. Briefly, 2000 erythrocytes (normochromatic erythrocytes (NCE) and RET) were counted per animal and the percentage of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoietic activity indicative of cell toxicity. A minimum of 4000 RETs were scored per animal and the frequency of MN-RETs was determined to provide an indication of genotoxic potential. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RETs and MN-RETs observed.

METHOD OF ANALYSIS: Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry (MACSQuant Analyzer 10; Miltenyi Biotec) with traditional blood smears prepared as a backup.

OTHER: The mice were observed for positive signs of toxicity at least once on the day of dosing and subsequent days. The mice were weighed prior to dosing and on the day of their scheduled sacrifice in order to assess the extent of stress experienced by the animals following treatment.
Evaluation criteria:
A test material was considered positive in this assay if the following criteria were met:
• An increase in MN-RETs was statistically significant when compared to the negative control.
• An increase in MN-RET frequency was dose-related when evaluated with an appropriate trend test.
• A statistically significant increase in MN-RET frequency was out of historical negative control range.

A test material was considered negative in this assay if the following criteria were met:
• An increase in MN-RETs was not statistically significant when compared to the negative control.
• An increase in MN-RET frequency was not dose-related when evaluated with an appropriate trend test.
• An increased MN-RET frequency was within the historical negative control range.

Statistics:
MN-RETs and percent RETs were tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). If the results from Bartlett's test were significant, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations examined were the common log, the inverse, and the square root in that order. The data was reviewed, and an appropriate form of the data was selected and subjected to the following analysis.
Based on the similarity of the response between sexes in the RFT, the MN-RET data and the data on percent RETs were analysed by a one-way analysis of variance (only males were used) (Winer, 1971). Linear dose-related trend tests were performed for the pairwise comparisons yielding significant differences. The alpha level at which tests were conducted was 0.05. The MN-NCE, collected via flow cytometry, was not analyzed statistically and was only used as an adjunct end point to evaluate the biological significance of the MN-RET results. MN-NCE were not collected by manual scoring. The final interpretation of significance of the responses was based on both statistical outcome and biological relevance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: dose levels of 1000 and 2000 mg/kg bw/day 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane
- Solubility: not specified
- Clinical signs of toxicity in test animals: There were no appreciable changes in the body weights of mice. There were no observations of toxicity or mortality among the treated animals during the in-life portion of the range-finding test. There were also no remarkable changes in the body temperature of mice following treatment with the test material (Tables 6 and 7), although a slight (< 2°C), transient decrease was generally observed in both sexes 5 hours post-treatment. Based upon the results of the range-finding study where no apparent sex differences were noted, only males were evaluated in the main study.
- Evidence of cytotoxicity in tissue analyzed: not specified
- Rationale for exposure: The dose levels of 2000 and 1000 mg/kg bw/day are selected based on availability of acute oral toxicity data for the mouse
- Harvest times: not specified


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane did not induce a significant increase in the frequency of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. There were no significant differences in MN-RET frequencies between the groups treated with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of MN-RETs in the positive control group.
- % RET: The percent RET values observed in the test material-treated animals were decreased but not significantly different from the negative control values indicating that the test substance was not overtly toxic to the target tissue. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals. Analytical results of presence of the test material in the peripheral blood clearly indicated a significant quantity of 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyc lotetrasiloxane in all time course samples. These data confirmed the presence (> 5 µg/g at a minimum) of the parent material in the peripheral blood after oral (gavage) administration and demonstrates exposure of the target tissue (i.e., bone marrow).
- Appropriateness of dose levels and route: The dose levels were based on the dose range-finding study which was performed prior to the definitive study. The recommended route in OECD test guideline (474) is oral gavage.
- Statistical evaluation: yes

CONCENTRATION ANALYSIS: The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 89.8 to 93.8% of the targeted values + 1.0 to 7.0 % RSD.

Any other information on results incl. tables

See attachment for result tables.

Applicant's summary and conclusion

Conclusions:
2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane has been tested for the ability to induce micronuclei in vivo in a study, conducted according to OECD Test Guideline 474 and in compliance with GLP. In the study, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was administered at dose levels of 0 (negative control), 500, 1000 and 2000 (limit dose) as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. No treatment-related toxicity was observed. Exposure of the test material to the target tissue (i.e., bone marrow) was confirmed by the presence of the test material in the peripheral blood following and oral (gavage) administration. Based on the results of the study, it was concluded that the test material, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, did not induce a statistically significant increase in the frequency of micronucleated reticulocytes in the peripheral blood. Positive and vehicle controls were included and gave the expected results.