Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No evidence of mutagenic activity was detected in S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537, or in Escherichia coli strain WP2 uvrA, tested with Pt-divinyltetramethyldisiloxane at up to 5000 ug/plate in the presence or absence of a rat liver (S9) metabolic activation system (Scarcella, 2004).

In an in vitro mammalian cell micronucleus test, conducted according to OECD Test Guideline 487 and to GLP, 'Karstedt Concentrate’ was tested for 3 hours in the absence and presence of S9, and for 24 hours in the absence of S9 up to the limits of cytotoxicity (Lloyd, 2016). No evidence of micronuclei induction was evident with the 3-hour treatments, but statistically significant increases in mean micronuclei frequency were observed at the 24-hour timepoint.  

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 May to 8 September 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Minor deviations (plate not incubated in closed system), not expected to affected study validity
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The plates were incubated for approximately 72 hours at 37ºC. Due to an oversight the plates were not incubated in closed containers. The study authors felt that this deviation was not considered to have affected the integrity of the study.
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 and betanaphthoflavone-induced Sprague-Dawley rat liver S9.
Test concentrations with justification for top dose:
See attachment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Positive controls:
yes
Positive control substance:
9-aminoacridine
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation (Test 1) and in agar (plate incorporation) (Test 2)

DURATION
- Exposure duration: incubated for 72 hrs

NUMBER OF REPLICATIONS:
Duplicate (in Test 1) and triplicate (in Test 2)

DETERMINATION OF CYTOTOXICITY
Other: Thinning of background lawn

Evaluation criteria:
A two-fold or more increase in mean revertant numbers must be observed in two consecutive dose-levels or at the highest practicable dose level. In addition, there must be evidence of a dose-response relationship.
Statistics:
Regression analysis fits a regression line to the data by the least squares method, after root transformation of the plate counts.
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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitation of the test material was noted at 2500 and 5000 ug/plate in both tests, and in the first test also at 1600 ug/plate

RANGE-FINDING/SCREENING STUDIES: In the toxicity test, performed using the pre-incubation method, thinning of the background lawn was observed in all strains (with and without S9) at the higher dose-levels, except TA 98.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (with and without S9)

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
No evidence of mutagenic activity was detected in S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537, or in Escherichia coli strain WP2 uvrA, tested with Pt-divinyltetramethyldisiloxane at up to 5000 ug/plate in the presence or absence of a rat liver (S9) metabolic activation system.
Executive summary:

Pt-divinyltetramethyldisiloxane was examined for mutagenic activity in four histidine-dependant auxotrophs of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in Escherichia coli strain WP2 uvrA, in the absence or presence of S9, using both the pre-incubation method (Test 1) and plate incorporation method (Test 2). The test was conducted according to OECD Test Guideline 471 and to GLP.

 

The test material did not induce two-fold increases in the number of revertant colonies in the pre-incubation or plate incorporation assay, at any dose-level, in any tester strain, in the absence or presence of S9. In both tests, precipitation was noted at 2500 ug/plate and above, and also at 1600 ug/plate in test 1. Toxicity, as indicated by thinning of the background lawn, was observed at higher dose levels in all tester strains (with and without S9), except TA98.

 

In conclusion, no evidence of mutagenic activity was detected in S. typhimurium strains TA98, TA100, TA1535, and TA1537, or in Escherichia coli strain WP2 uvrA, tested with Pt-divinyltetramethyldisiloxane at up to 5000 ug/plate in the presence or absence of S9.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-Mar-2016 to 11-Nov-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
26-Sep-2014
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch number 9000503741
- Expiration date of the lot/batch: 22-Aug-2016
- Purity test date: 22-Feb-2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25 °C, protected from light and under nitrogen.
- Stability under test conditions: not assessed
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMF at 49.86 mg/mL; stability not assessed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not assessed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: Test article stock solutions were prepared by formulating Karstedt Concentrate [a liquid] under
subdued lighting in DMF, with the aid of warming, vortex mixing and ultrasonication (where required), to give the maximum required concentration. Subsequent dilutions were made using DMF. The test article solutions were protected from light and used within approximately 2 hours of initial formulation.
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material): not applicable

OTHER SPECIFICS:
Purity was 19.45% based on Platinum content (assumed 100% for testing)
Target gene:
not applicable
Species / strain / cell type:
lymphocytes:
Remarks:
human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood from healthy, non-smoking human donors
- Suitability of cells: as per guideline
- Cell cycle length, doubling time or proliferation index: assumed 24 hours
- Sex, age and number of blood donors if applicable: two females, aged 32 and 35 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: not applicable
- Modal number of chromosomes: not applicable
- Normal (negative control) cell cycle time: assumed 24 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HEPES-buffered RPMI medium containing 10% (v/v) heat-inactivated foetal calf serum and 0.52% penicillin / streptomycin [CO2 concentration not specified]
- Properly maintained: [yes/no] yes
- Periodically checked for Mycoplasma contamination: [yes/no] not applicable
- Periodically checked for karyotype stability: [yes/no] not applicable
- Periodically 'cleansed' against high spontaneous background: [yes/no] not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
3 hours treatment + 21 hours (without S9): 0.01-4 micrograms/mL
3 hours treatment + 21 hours (with S9): 2.5-50 micrograms/mL
24 hours treatment + 0 hours (without S9): 0.01-4 micrograms/mL
Concentrations based on results of cytotoxicity range-finding study
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: none
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Noscapine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 or 24 hours without S9; 3 hours with S9
- Expression time (cells in growth medium): 21 hours for 3-hour treatment; 0 hours for 24-hour treatment
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

STAIN (for cytogenetic assays): Acridine Orange

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8 °C prior to slide preparation for a minimum
of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 x g, for 2-3 minutes) and resuspended in a minimal amount
of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approximately 10 minutes. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED: 500/culture for replication index (RI); 1000/culture (2000/test concentration) for micronuclei

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: replication index (RI)
- Any supplementary information relevant to cytotoxicity:

RI, which indicates the relative number of nuclei compared to vehicle controls, was determined using the formula as follows:
RI = [number binucleate cells + 2 (number multinucleate cells)] / total number of cells in treated cultures

Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = [RI of treated cultures / RI of vehicle controls] x100

Cytotoxicity (%) is expressed as (100 – Relative RI).

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Methods, such as kinetochore antibody binding, to characterise whether micronuclei contain whole or fragmented chromosomes (if applicable): none
Rationale for test conditions:
As per guideline
Evaluation criteria:
Acceptance Criteria
Assay considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the 95th percentile of the observed current historical vehicle control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control
concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at
the time of harvest
5. The maximum concentration analysed under each treatment condition met the criteria specified.

Evaluation Criteria
For valid data, the test article was considered to have induced clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis.
Statistics:
Treatment of Data
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0, 32 or 55% at 0.3, 0.5 or 0.7 micrograms/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5, 24 or 46% at 0.5, 0.9 or 1.2 micrograms/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5, 38, 45 or 68% at 22.5, 32.5, 35.0 or 37.5 micrograms/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality: no effect
- Evaporation from medium: no data
- Water solubility: no data (the solubility limit in culture medium was less than 62.33 μg/mL)
- Precipitation: precipitate at 62.33 μg/mL (and higher) concentrations approximately 21 hours after test article addition to culture medium
- Definition of acceptable cells for analysis: Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
- Other confounding effects: none specified

RANGE-FINDING/SCREENING STUDIES: with and without S9; vehicle and untreated controls included; positive controls not included; 3 hours treatment + 21 hours (with and without S9), 24 hours treatment + 24 hours (in error) (without S9); concentrations tested were 1.814-500 micrograms/mL

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
Micronucleus study
3 hours + 21 hours (without S9)
vehicle control: 242 mono, 1620 bi, 138 multi, total 2000, RI 0.95
0.5 micrograms/mL: 145 mono, 809 bi, 46 multi, total 1000, RI 0.90
0.9 micrograms/mL: 291 mono, 696 bi, 13 multi, total 1000, RI 0.72
1.2 micrograms/mL: 497 mono, 498 bi, 5 multi, total 1000, RI 0.51
3 hours + 21 hours (with S9)
vehicle control: 221 mono, 1645 bi, 134 multi, total 2000, RI 0.96
22.5 micrograms/mL: 138 mono, 817 bi, 45 multi, total 1000, RI 0.91
32.5 micrograms/mL: 415 mono, 581 bi, 4 multi, total 1000, RI 0.59
35.0 micrograms/mL: 479 mono, 517 bi, 4 multi, total 1000, RI 0.53
37.5 micrograms/mL: 692 mono, 306 bi, 2 multi, total 1000, RI 0.31
24 hours (without S9)
vehicle control: 569 mono, 1354 bi, 77 multi, total 2000, RI 0.75
0.3 micrograms/mL: 278 mono, 687 bi, 35 multi, total 1000, RI 0.76
0.5 micrograms/mL: 492 mono, 503 bi, 5 multi, total 1000, RI 0.51
0.7 micrograms/mL: 657 mono, 343 bi, 0 multi, total 1000, RI 0.34

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
3 hours + 21 hours (without S9)
vehicle control: 0.68%
0.5 micrograms/mL: 1.03% p<=0.05
0.9 micrograms/mL: 0.68% ns
1.2 micrograms/mL: 0.65% ns
3 hours + 21 hours (with S9)
vehicle control: 0.80%
22.5 micrograms/mL: 0.90% ns
32.5 micrograms/mL:0.35% ns
35.0 micrograms/mL: 0.20% ns
37.5 micrograms/mL: 0.18% ns
24 hours (without S9)
vehicle control: 0.65%
0.3 micrograms/mL: 0.75% ns
0.5 micrograms/mL: 1.35% p<=0.05
0.7 micrograms/mL: 1.30% p<=0.05
- Indication whether binucleate or mononucleate where appropriate: binucleate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data:
3 hours + 21 hours (without S9)
19 experiments, 44 cultures
MNBN frequency: mean 0.54%, standard deviation 0.25, range 0.1-1.2%, 95% reference range 0.2-1.0%
3 hours + 21 hours (with S9)
23 experiments, 52 cultures
MNBN frequency: mean 0.51%, standard deviation 0.26, range 0.0-1.1%, 95% reference range 0.2-1.07%
24 hours (without S9)
7 experiments, 16 cultures
MNBN frequency: mean 0.64%, standard deviation 0.29, range 0.1-1.3%, 95% reference range 0.21-1.19%

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used] RI
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells] not applicable
Remarks on result:
other: 24-hour treatment + 0 hours
Conclusions:
In an in vitro mammalian cell micronucleus test, conducted according to OECD Test Guideline 487 and to GLP, 'Karstedt Concentrate’ was tested for 3 hours in the absence and presence of S9, and for 24 hours in the absence of S9 up to the limits of cytotoxicity (Lloyd, 2016). No evidence of micronuclei induction was evident with the 3-hour treatments, but statistically significant increases in mean micronuclei frequency were observed at the 24-hour timepoint.
Executive summary:

An in vitro mammalian cell micronucleus test was conducted according to OECD Test Guideline 487 and to GLP using human lymphocytes. Platinum(0)divinyltetramethylsiloxane complexes ('Karstedt Concentrate’) was tested at up to 4 ug/mL for 3 hours (+21 hours) with and without S9 and at up to 50 ug/mL for 24 hours (+0 hours) without S9. Untreated and vehicle controls were included and also positive clastogenic (with and without S9) and aneugenic controls (without S9). A cytokinesis block protocol was used.

 

At least three concentrations were selected for scoring from each treatment regimen based on cytotoxicity. A concentration-related increase in the frequency of micronuclei was seen in response to the 24-hour (+0 hours) treatment without S9. No such increase was seen for the 3 hour treatment (+21 hours) with or without S9. The vehicle and positive controls were valid.

 

It is concluded that, under the conditions of this in vitro study, 'Karstedt Concentrate’ induced micronuclei.

 

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A mammalian erythrocyte micronucleus test was conducted (according to OECD Test Guideline 474 and to GLP) using animals from a combined repeated-dose toxicity study with reproduction/ developmental toxicity screening test (conducted according to OECD Test Guideline 422 and to GLP). In the OECD 422 study, 'Karstedt Concentrate’ was administered daily (in corn oil) by oral gavage to a satellite group of rats (5/sex/group) for 28 days at 500 mg/kg bw/day. No treatment-related increase in micronuclei were reported.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Part of a combined repeated dose study (OECD 422) with reproductive and developmental toxicity screening.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2016-March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
The micronucleus analysis was performed at the end of the OECD 422 study (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test), following the principles of the OECD 474 guideline.
Deviations:
not applicable
Principles of method if other than guideline:
- Principle of test:
Measurement of micronuclei in peripheral blood obtained from rats tested in the OECD 422 study using Karstedt Concentrate.

- Short description of test conditions:
5 male and 5 female rats were included in each of six groups; the first received the vehicle (corn oil) control, the second (reported as Group 4) received the high-dose of Karstedt Concentrate (500 mg/kg bw/day) for 28 consecutive days. Groups 5-8 received 2 i.p. injections of either Mitomycin C (1 or 0.75 mg/kg bw) or vincristin sulphate (0.05 or 0.04 mg/kg bw).

24 hours after the final test item administration, blood samples were taken, prepared according to the MicroFlow instructions, and sent to Litron Laboratories for Flow Cytometry analysis.

- Parameters analysed / observed:
Numbers of normochromatic erythrocytes (NCE), micronucleated NCE, reticulocytes and micronucleated reticulocytes.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Mammalian somatic cell cytogenicity/aneugenicity assay
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories Germany, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant:
Yes.
- Age at study initiation:
Males and females were aged 81 days at first test material administration.
- Weight at study initiation:
Males: 407.0 - 474.3 g at first test material administration.
Females: 222.7 - 304.2 g at first test material administration.
- Fasting period before study:
Not specified.
- Housing:
Males and females kept in individual cages, except during mating period (see reproductive toxicity
section for further details).
- Diet (e.g. ad libitum):
Standard commercial feed ad libitum.
- Water (e.g. ad libitum):
Tap water ad libitum.
- Acclimation period:
6 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22°C (+/- 3°C)
- Humidity (%):
55% (+/- 15%)
- Air changes (per hr):
Not specified.
- Photoperiod (hrs dark / hrs light):
12 hrs light (150 lux)/12 hrs dark.
IN-LIFE DATES:
Males: end of the in-life period: 22 September 2016
Females: end of the in-life period: 17 October 2016.
Route of administration:
oral: gavage
Vehicle:
Corn oil.

- Justification for use and choice of vehicle (if other than water):
No justification specified (standard vehicle).
- Concentration in vehicle:
Not specified.
- Amount of vehicle (if gavage):
Constant dose volume of 5 mL/kg bw/day/animal.
- Lot/batch no. (if required):
Caesar and Loretz GmbH, Germany. Batch numbers 15296404 and 15296406.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were continuously stirred until the last animal of each group had been dosed.
Duration of treatment / exposure:
CONTROL AND TEST ITEM-TREATED ANIMALS
Males and females: 28 days [blood samples taken 24 hours after final dose, i.e. on study day 29]

POSITIVE CONTROL ANIMALS
Intraperitoneal injection
Once daily for 2 consecutive days [blood samples taken 24 hours after final dose]
Frequency of treatment:
Daily
Post exposure period:
None.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Maxiumum tolerated dose [see Repeated dose toxicity section for further details]
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C; vincristin sulphate
- Justification for choice of positive control(s): mitomycin C as per guideline; vincristin (vincristine) sulphate use not separately justified in report but known to be an acceptable positive control substance in the test. [Reference: Witt KL, Livanos E, Kissling GE, Torous DK, Caspary W, Tice RR, Recio L (2008) Comparison of flow cytometry- and microscopy-based methods for measuring micronucleated reticulocyte frequencies in rodents treated with nongenotoxic and genotoxic chemicals. Mutat Res. 649: 101-113].
- Route of administration: intraperitoneal injection.
- Doses / concentrations: mitomycin C 0.75 and 1.0 mg/kg bw/day; vincristin sulphate 0.04 and 0.05 mg/kg bw/day
Tissues and cell types examined:
Peripheral blood erythocytes and reticulocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: maximum tolerated dose in range-finder; top dose in repeated-dose/reproductive toxicity study (see Repeated dose toxicity section for details).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): animals were treated daily for 28 days and blood samples taken 24 hours after the final dose.

DETAILS OF SLIDE PREPARATION:
Litron Laboratories received 120 fixed rat blood samples (2/animal) from Study 33652. All fixed blood samples were stored in a freezer (–90 °C to –80 °C) until analysis.

Samples to be analysed (80) were washed by pipetting 12 ± 1 ml of cold Hank’s Balanced Salt Solution (HBSS) into each and cells were isolated by centrifugation. The cell pellets were stored on ice until staining and then refrigerated at 2 °C to 10 °C.

An aliquot (20 μL) of each washed blood sample was added to 80 μL of a solution containing RNase (to degrade RNA, 1 mg/mL), a fluorescently labeled (fluorescein isothiocyanate; FITC) antibody to the transferrin receptor to stain reticulocytes (RETs) (anti-CD71-FITC, 10 μL/mL), and a fluorescently labeled antibody (phycoerythrin; PE) to label platelets (anti-CD61-PE, 5 μL/mL) in a base of (Hanks' Balanced Salt Solution (HBSS). The samples were incubated in the staining solution for 30 ± 10 minutes at 2 °C to 10 °C and 30 ± 10 minutes at room temperature. After incubation, the cells were kept at 2 °C to 10 °C until analysis. A propidium iodide (PI) solution (2.0 mL ± 0.5 mL) was added to each sample immediately before flow cytometric analysis to stain all DNA, including MN in the cells.

Methanol-fixed blood from rats infected with Plasmodium berghei was used to configure the flow cytometer before analysis. Whereas MN are relatively rare and exhibit a heterogeneous DNA content, parasitized cells are prevalent and have a homogenous DNA content. These characteristics make them ideal for calibrating the flow cytometer for the MN scoring application.

METHOD OF ANALYSIS:
Each blood sample was analyzed by high-speed flow cytometry using CellQuest software, version 5.2 (Becton Dickinson, San Jose, CA). The stained cells were moved at a high velocity past an argon laser set to provide 488 nm excitation. Photomultiplier tubes collected the fluorescence emitted by each cell. Using the previously described staining procedure, the PI-stained DNA of the MN emitted a red fluorescence, the anti-CD71-FITC antibody emitted a high green fluorescent signal, and platelets were excluded based on their anti-CD61-PE fluorescence. All samples exhibited some degree of cellular aggregation, but they were analyzable and data was obtained. Upon successful analysis of the stained samples, each was discarded.
Evaluation criteria:
For test facility samples, up to 20,000 RETs (high CD71-positive) were evaluated for the presence of MN except sample 116 (mitomycin C) where 20,002 RETs were evaluated. Some samples exhibited cellular aggregation and fewer than 20,000 RETs were evaluated due to the aggregation.

The number of normochromatic erythrocytes (NCEs), MN-NCEs, RETs and MN-RETs are provided for each sample. The frequency of MN-RETs was calculated as an indication of genotoxic potential for samples where sufficient RETs were evaluated, and the % RET was determined to provide an indication of bone marrow toxicity.
Statistics:
One sample per male and female animal from Groups 1 (vehicle control), 4 (500 mg/kg bw/day), 6 (mitomycin C 0.75 mg/kg bw/day) and 8 (vincristin sulphate, 0.04 mg/kg bw/day) were analysed. Statistical methods were not used to evaluate the effect of treatment on the % MN-RET of the high dose group, as the % MN-RET of the male and female high dose groups were not higher than the % MN-RET of the corresponding negative controls. The means and standard deviations for both endpoints are presented for the four groups analysed (males and females).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No effect on %RET (i.e. no indication of bone marrow toxicity). However, animals in the main study treated with the same high dose (500 mg/kg bw/day) did exhibit statistically significantly reduced % RET, indicative of bone marrow toxicity.
Vehicle controls validity:
not specified
Remarks:
No historical control data are available for this study in this laboratory. The investigators recommend the use of published data instead.
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
This study was conducted on a satellite group of animals (5/sex/group) from an OECD 422 combined repeated-dose toxicity study with reproduction/ developmental toxicity screening test, in which the animals were treated with vehicle controls or the "high-dose" (500 mg/kg bw/day) group. There was no evidence of micronucleation and no effect on reticulocytes (suggestive of a lack of bone marrow toxicity). However, animals in the main study treated with the same high dose (500 mg/kg bw/day) did exhibit statistically significantly reduced % RET, indicative of bone marrow toxicity.

 

 

Conclusions:
A mammalian erythrocyte micronucleus test was conducted (according to OECD Test Guideline 474 and to GLP) using animals (satellite group) from a combined repeated-dose toxicity study with reproduction/ developmental toxicity screening test (conducted according to OECD Test Guideline 422 and to GLP). In the OECD 422 study, 'Karstedt Concentrate’ was administered daily (in corn oil) by oral gavage to a satellite group of rats (5/sex/group) for 28 days at 500 mg/kg bw/day. No treatment-related increase in micronuclei were reported.
Executive summary:

A mammalian erythrocyte micronucleus test was conducted according to OECD Test Guideline 474 and to GLP using animals from a combined repeated-dose toxicity study with reproduction/ developmental toxicity screening test, conducted according to OECD Test Guideline 422 and to GLP. In the OECD 422 study, platinum(0) 1,3-divinyl-1,1,3,3-tetramethyldisiloxane complexes ('Karstedt Concentrate’) was administered daily (in corn oil) by oral gavage to a satellite group of rats (5/sex/group) for 28 days at 500 mg/kg bw/day. Control animals received vehicle only. Positive control animals received mitomycin C at 0.75 or 1.0 mg/kg bw/day or vincristin sulphate at 0.04 or 0.05 mg/kg bw/day by intraperitoneal injection daily for 2 consecutive days.

 

Two blood samples per animal were fixed and made available for analysis. Evaluation of toxicity to the bone marrow and micronuclei was undertaken using one blood sample per animal from each of four groups (both sexes): vehicle control, high-dose test item, low-dose mitomycin C, low-dose vincristin sulphate. There was no evidence of toxicity to the bone marrow and no induction of micronuclei in animals treated with the test item. Statistically significant increases in the frequency of induced micronuclei were evident for both positive controls (in male and female animals).

 

No bone marrow toxicity was evident in test-item treated animals (in the satellite group). However, animals in the main study treated with the same high dose (500 mg/kg bw/day) did exhibit statistically significantly reduced % RET, indicative of bone marrow toxicity. It is also noted that systemic toxicity was reported in animals treated at the highest tested dose in the main OECD 422 study [See Repeated Dose toxicity section for details. An

analysis of the blood samples for platinum on test day 28/29 identified low levels of platinum in the plasma. As such, some bone marrow exposure occured.

It is concluded that, under the experimental conditions, 'Karstedt concentrate' failed to induce clastogenicity or aneugenicity.

 

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

Additional information

'Pt-divinyltetramethyldisiloxane' was examined for mutagenic activity in four histidine-dependant auxotrophs of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and in Escherichia coli strain WP2 uvrA, in the absence or presence of S9, using both the pre-incubation method (Test 1) and plate incorporation method (Test 2). The test was conducted according to OECD Test Guideline 471 and to GLP.The test material did not induce two-fold increases in the number of revertant colonies in the pre-incubation or plate incorporation assay, at any dose-level, in any tester strain, in the absence or presence of S9. In both tests, precipitation was noted at 2500 ug/plate and above, and also at 1600 ug/plate in test 1. Toxicity, as indicated by thinning of the background lawn, was observed at higher dose levels in all tester strains (with and without S9), except TA98.In conclusion, no evidence of mutagenic activity was detected in S. typhimurium strains TA98, TA100, TA1535, and TA1537, or in E. coli strain WP2 uvrA, tested with Pt-divinyltetramethyldisiloxane at up to 5000 ug/plate in the presence or absence of S9 (Scarcella, 2004).

An in vitro mammalian cell micronucleus test was conducted according to OECD Test Guideline 487 and to GLP using human lymphocytes. Platinum(0)divinyltetramethylsiloxane complexes ('Karstedt Concentrate’) was tested at up to 4 ug/mL for 3 hours (+21 hours) with and without S9 and at up to 50 ug/mL for 24 hours (+0 hours) without S9. Untreated and vehicle controls were included and also positive clastogenic (with and without S9) and aneugenic controls (without S9). A cytokinesis block protocol was used. At least three concentrations were selected for scoring from each treatment regimen based on cytotoxicity. A concentration-related increase in the frequency of micronuclei was seen in response to the 24-hour (+0 hours) treatment without S9. No such increase was seen for the 3 hour treatment (+21 hours) with or without S9. The vehicle and positive controls were valid. It is concluded that, under the conditions of this in vitro study, 'Karstedt Concentrate’ induced micronuclei (Lloyd, 2016)

A mammalian erythrocyte micronucleus test was conducted according to OECD Test Guideline 474 and to GLP using animals from a combined repeated-dose toxicity study with reproduction/ developmental toxicity screening test, conducted according to OECD Test Guideline 422 and to GLP. In the OECD 422 study, platinum(0) 1,3-divinyl-1,1,3,3-tetramethyldisiloxane complexes ('Karstedt Concentrate’) was administered daily (in corn oil) by oral gavage to a satellite group of rats (5/sex/group) for 28 days at 500 mg/kg bw/day. Control animals received vehicle only. Positive control animals received mitomycin C at 0.75 or 1.0 mg/kg bw/day or vincristin sulphate at 0.04 or 0.05 mg/kg bw/day by intraperitoneal injection daily for 2 consecutive days. Two blood samples per animal were fixed and made available for analysis. Evaluation of toxicity to the bone marrow and micronuclei was undertaken using one blood sample per animal from each of four groups (both sexes): vehicle control, high-dose test item, low-dose mitomycin C, low-dose vincristin sulphate. There was no evidence of toxicity to the bone marrow or induction of micronuclei in animals treated with the test item. Both positive controls had a statistically significant increase in micronucleus frequency. Since no bone marrow toxicity was evident in test-item treated animals (in the satellite group), an analysis of the blood samples for platinum is awaited to confirm exposure. It is noted that systemic toxicity was reported in animals treated at the highest tested dose in the OECD 422 study [see Repeated dose toxicity section for details]. It is concluded that, under the experimental conditions, 'Karstedt concentrate' failed to induce clastogenicity or aneugenicity.

 

Justification for classification or non-classification

No mutagenic activity was seen in a reliable in vitro gene mutation assay in bacteria. Although an increase in induced micronuclei was seen in an in vitro study in mammalian cells, no treatment-related effect on micronuclei formation was evident in an in vivo study where Karstedt concentrate was administered at a maximally tolerated dose. As such, ‘Karstedt concentrate’ does not meet the criteria for classification as a germ cell mutagen according to EU CLP criteria (EC 1272/2008).