Registration Dossier

Administrative data

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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
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:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were continuously stirred until the last animal of each group had been dosed.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn oil. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each dose solution were analysed by ICP-OES for platinum content, at dates throughout the study period.
Duration of treatment / exposure:
Males and females were dosed from 2 weeks prior to mating and during the mating period.
Males were further dosed after the mating period for a total treatment duration of at least 28 days.
Females were dosed throughout gestation and at least up to and including day 13 post-partum (total of 49-62 days).
Frequency of treatment:
Daily
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses selected on the basis of a 14-day range-finding study (see Supporting study (Hansen, 2016) in this endpoint).
- Rationale for selecting satellite groups:
Satellite groups (5/sex/group) were administered vehicle only or the high dose (500 mg/kg bw/day) for 28 days. Blood samples were taken prior to the final dose, and at 3, 6, 12 and 24-hours post administration. Plasma was analysed for elemental platinum as an analytical marker for the test compound (analysis carried out by Allessa, and included as an appendix to the study report). Blood taken from these animals was also assessed for micronucleus formation (see in vivo Micronucleus ESR for full details).
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least once daily.
- Cage side observations included: skin/fur, eyes, mucuous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least once daily.

BODY WEIGHT: Yes.
- Time schedule for examinations: On the first day of dosing, weekly thereafter, and at termination. During gestation, females were weighed on days 0, 7, 14 and 20, and within 24 hours of parturition.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified.

OPHTHALMOSCOPIC EXAMINATION: Not specified.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: At the end of the pre-mating period.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (overnight).
- How many animals: 5/sex, randomly selected from each group.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: At the end of the pre-mating period.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (overnight).
- How many animals: 5/sex, randomly selected from each group.
- Parameters checked in Table 2 were examined.

URINALYSIS: Not specified.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Males: test day 43 or 50 (shortly before scheduled sacrifice); females: test day 65-71 (shortly before scheduled sacrifice). Screening was carried out two hours after dosing, and before any blood sampling.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity (auditory, visual, proprioceptive stimuli); grip strength; motor activity.

IMMUNOLOGY: Not specified.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
All tissues and organs were examined macroscopically.

The weight of the following organs was recorded before fixation (where applicable):
Adrenals, kidney (2), epididymis (2), liver, uterus (incl. cervix), ovary (2), spleen, testicle (2), thyroid, thymus, combined weight of prostate+seminal vesicles+coagulating glands. Paired organs were weighed individually and identified as left or right.

The brain and heart of 5 animals/sex/group were also weighed.

Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY: Yes.
The following organs/parts were fixed for microscopic examination: adrenal gland (2), bone, bone marrow (os femoris), brain, epididymis (2), eye with optic nerve (2), all gross lesions observed, heart, intestine, kidney and ureter (2), liver, lungs with bronchi and bronchioles, lymph node (1 cervical and 1 mesenteric), mammary gland, skeletal muscle, sciatic nerve, oesophagus, ovary and oviduct, pituitary, prostate/seminal vesicles/coagulating gland, spinal cord (3 sections), spleen, stomach, testicle (2), thyroid, thymus, tissue masses or tumours, tongue, trachea, urinary bladder, uterus (incl. cervix), vagina.
Statistics:
Homogeneity of variances and normality of distribution were tested using the Bartlett's and Shapiro-Wilks test. In case of heterogeneity and/or non-normality of distribution, Anova and Dunnet's test were used. Non-parametrical values were evaluated using the Fisher or Chi-squared tests.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All males of the high-dose group displayed increased salivation, considered to be treatment-related and adverse.

4/10 high-dose females also displayed increased salivation, also considered to be treatment-related and adverse.
Mortality:
mortality observed, treatment-related
Description (incidence):
Males:
500 mg/kg bw/day: no deaths.

Females:
500 mg/kg bw/day: One premature death (during lactation).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
500 mg/kg bw/day: significantly lower body weight than controls during the whole study period (e.g. 6.5%, 11.5% and 14.4% below controls on test days 22, 29 and 52, respectively).

Females:
500 mg/kg bw/day: no effects on body weights pre-mating or during lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
Males:
500 mg/kg bw/day: lower food consumption week 1 of study, approximately half control value.
125 mg/kg bw/day: lower food consumption week 1 of study, approximately 15% below control value.
Food consumption normalised between test days 22 and 28, so these observations were not considered to be adverse.

COMPOUND INTAKE (from feed):
Not applicable.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Group mean white blood cell (WBC) counts (absolute values) were higher than the control mean for all treated groups, but the effect only achieved statistical significance at 500 mg/kg bw/day. These increases were particularly associated with dose-related elevations in mean neutrophil and lymphocyte counts (absolute values), which were themselves statistically significant at the high-dose level. For the high-dose group, statistically significant increases in monocytes and large unclassified cell means were also noted. A dose-related decrease in the mean reticulocyte (%) counts was evident for all treated groups when compared to the respective control mean. A statistically significant difference was present only at 125 and 500 mg/kg bw/day, although the effect in the mid-dose fell within historical control ranges for this effect (and, as such, was not considered by the study authors to be related to treatment).

Females:
Mean white blood cell (WBC) counts (absolute values) showed minor increases at the mid- and high-dose (although neither value achieved statistical significance), and which again appeared to be primarily related to limited increases in neutrophil and lymphocyte group mean values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males:Increases in group mean alanine aminotransferase (ALAT) values were noted for all treated groups, which achieved statistical significance at 125 and 500 mg/kg bw/day. However, the magnitude of this effect was limited in degree (only the high-dose value approached the upper boundary of the historical control dataset). Clear treatment-related differences in other measured serum enzyme levels were not evident.
Several other statistically significant effects were detected in group mean values at 500 mg/kg bw/day: concentrations of albumin (decrease), total cholesterol (increase), protein (decrease) and calcium (decrease).

Females:
No statistically significant changes were seen in any of the clinical chemistry parameters of females.
There was a dose-related decrease in aP, but statistical significance was not achieved at any dose level.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
One mid-dose animal showed slight signs of positive geotropism; another had slight diarrhoea. One low-dose and one high-dose animal showed "slight influences" during the wire manoeuvre test, and males in these two groups showed a decreased reaction to a tail pinch. All observations were considered to be spontaneous and not related to treatment.

Females:
One mid-dose female was noted with salivation and a slightly reduced urination. This was considered to be spontaneous and not related to treatment.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males:
Statistically significant increases in group mean liver and kidney weights relative to body weight were evident at 500 mg/kg bw/day. However, there were no differences between treated and control groups in respect of absolute liver or kidney weights, which had values residing within historical control ranges. [Note that there were changes in clinical chemistry at this dose level.]
The group mean absolute weights of the spleen, thymus and prostate/seminal vesicle were statistically significantly decreased at 500 mg/kg bw/day (although when evaluated relative to body weight, the decreases were not statistically significant).
The study investigators did not consider the above observations in males to be evidence of treatment-related adverse effects.

Females:
Dose-related increases in group mean relative and absolute adrenal weights were recorded at 125 and 500 mg/kg bw/day; the effect achieving statistical significance at the high-dose, and with the majority of the individual organ weights being outside of the historical control range. Concomitant stress hormone measurements were not conducted during this study, but the possibility exists that the basis of these changes was stress-related rather than due to organ toxicity.
Slightly elevated mean kidney weights were noted for the high-dose group (only the relative right kidney weight value being statistically significant).
A marginal, and non-statistically-significant, increase in relative and absolute mean liver weight was apparent at 500 mg/kg bw/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
500 mg/kg bw/day: one animal had lungs with multiple red foci, correlating with test-item microscopic findings, and considered to be treatment related.

Females
All doses: Haemorrhagic foci in the stomach were noted in 1/10 control females and 0/10, 3/10 and 1/10 females in the low, intermediate and high-dose groups, respectively. These findings considered to be not related to treatment, as these lesions were not seen in males, and there was no evident dose dependency.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females:
500 mg/kg bw/day: Lung - increased pigment deposition (4/5 males, 2/6 females); haemorrhages (3/5 males, 4/6 females); and granulomatous inflammation (4/5 males, 4/6 females). Incidence of pigment deposition and inflammation was statistically significant in males.
The one female that died during the study period was found to have marked granulomatous inflammation of the lungs. The investigators concluded that the pulmonary effects noted for the male and female animals were considered to be a foreign body response to test item in the lung rather a toxicological effect.

Females:
500 mg/kg bw/day: Statistically significantly increased incidence of hypertrophy of the adrenal cortex in 3/6 females. [Note that there were changes in adrenal organ weight at this dose level.] However, evidence of similar hypertrophic change was not found when tissues from mid-dose group females were examined.
No test item-related microscopic changes in the reproductive organs of high-dose males or females were detected. Qualitative evaluation of the stages of spermatogenesis in males of this treatment group revealed no abnormalities.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A limited decrease in Thyroid Hormone T4 concentration (17.5% below the mean value of the control group, p ≤ 0.05) was noted in male animals of the high-dose group. T4 measurements for females of all treated groups were unaffected, as were those for males receiving 30 or 125 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
In a combined repeated-dose toxicity study with reproduction/developmental toxicity screening test, conducted according to OECD Test Guideline 422 and to GLP, 'Karstedt Concentrate’ was administered daily (in corn oil) by gavage to groups of rats (10/sex/dose) for at least 28 days (males) or 49-62 days (females) at 30, 125 or 500 mg/kg bw/day. Treatment-related adverse effects were observed in the high dose animals (including reduced food consumption and body weight, haematological and clinical chemistry changes, and lesions to the lungs). The NOAEL for systemic toxicity was established as 125 mg/kg bw/day.
Executive summary:

In a combined repeated-dose toxicity study with reproduction/developmental toxicity screening test, conducted according to OECD Test Guideline 422 and to GLP, platinum(0) 1,3-divinyl-1,1,3,3-tetramethyldisiloxane complexes ('Karstedt Concentrate’) was administered daily (in corn oil) by gavage to groups of rats (10/sex/dose) for at least 28 days (males) or 49-62 days (females) at 30, 125 or 500 mg/kg bw/day. Control animals received vehicle only.

In the high-dose group, one female died, and reduced body weights were seen in males. On commencement of treatment, food consumption was transiently lower in males in the mid- and high-dose groups, but with a very marginal degree of effect evident for the former. Haematological examination revealed effects on white blood cell parameters mainly in males in the high-dose group. A statistically significant reduction in group mean reticulocyte count was also evident at the high- and mid-dose (though the value for the latter was within the historical control range for this parameter). Statistically significant effects were noted for several clinical chemistry parameters in males receiving 500 mg/kg bw/day, although effect severity was limited. A dose-related increase in absolute and relative adrenal gland weight was seen in females at 125 and 500 mg/kg bw/day (only reaching statistical significance at the higher dose, where histopathology indicated the existence of an accompanying adrenal cortical hypertrophy). Males showed a limited decrease in thyroid hormone T4 concentration at the highest tested dose.

 

On the basis of the observed treatment-related adverse effects seen in the high-dose animals, the NOAEL was considered to be 125 mg/kg bw/day.

 

Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
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:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were continuously stirred until the last animal of each group had been dosed.
VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn oil. 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 mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy, or 2 weeks.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each dose solution were analysed by ICP-OES for platinum content, at dates throughout the study period.
Duration of treatment / exposure:
Males and females were dosed from 2 weeks prior to mating and during the mating period.
Males were further dosed after the mating period for a total treatment duration of at least 28 days.
Females were dosed throughout gestation and at least up to and including day 13 post-partum (total of 49-62 days).
Frequency of treatment:
Daily
Details on study schedule:
Not applicable
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses selected on the basis of a 14-day range-finding study (see Supporting study in this endpoint).
- Rationale for selecting satellite groups:
Satellite groups (5/sex/group) were administered vehicle only or the high dose (500 mg/kg bw/day) for 28 days. Blood samples were taken prior to the final dose, and at 3, 6, 12 and 24-hours post administration. Plasma was analysed for elemental platinum as an analytical marker for the test compound (analysis carried out by Allessa, and included as an appendix to the study report). Blood taken from these animals was also assessed for micronucleus formation (see in vivo Micronucleus ESR for full details).
Positive control:
None.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least once daily.
- Cage side observations included: skin/fur, eyes, mucuous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.
DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least once daily.
BODY WEIGHT: Yes.
- Time schedule for examinations: On the first day of dosing, weekly thereafter, and at termination.
During gestation, females were weighed on days 0, 7, 14 and 20, and within 24 hours of parturition.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified.
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Monitored daily by visual appraisal.

OTHER: see Repeated dose toxicity for details of other examinations.
Oestrous cyclicity (parental animals):
Yes; determined by evaluating vaginal smears and confirmed by microscopic examination at necropsy.
Sperm parameters (parental animals):
Parameters examined in male parental (F0) animals:
Weights of testes, epididymes, and combined weight of prostate/seminal vesicles/coagulating glands, and histopathological examination of the same. A detailed examination of the qualitative stages of spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, following a randomisation scheme.
- 8 top 10 pups/litter; excess pups (at least 2, preferably female) were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, thyroid hormone levels (T4).

GROSS EXAMINATION OF DEAD PUPS:
Yes. Dead pups and pups sacrified at day 13 post-partum were carefully examined externally for gross abnormalities. External reproductive genitals were examined for signs of altered development. The thyroid from 1 male and 1 female pup/litter was weighed and fixed for histopathological examination.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed on test day 52 (approximately 2 weeks post-mating, for a total of 37 treatment days).
- Maternal animals: All surviving animals were sacrificed on day 13 post-partum (between test days 66 and 77, for a total of 51-62 treatment days).

GROSS NECROPSY
All tissues and organs were examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weight of the following organs was recorded before fixation (where applicable):
Adrenals, kidney (2), epididymis (2), liver, uterus (incl. cervix), ovary (2), spleen, testicle (2), thyroid, thymus, combined weight of prostate+seminal vesicles+coagulating glands. Paired organs were weighed individually and identified as left or right.

The brain and heart of 5 animals/sex/group were also weighed.

The following organs/parts were fixed for microscopic examination: adrenal gland (2), bone, bone marrow (os femoris), brain, epididymis (2), eye with optic nerve (2), all gross lesions observed, heart, intestine, kidney and ureter (2), liver, lungs with bronchi and bronchioles, lymph node (1 cervical and 1 mesenteric), mammary gland, skeletal muscle, sciatic nerve, oesophagus, ovary and oviduct, pituitary, prostate/seminal vesicles/coagulating gland, spinal cord (3 sections), spleen, stomach, testicle (2), thyroid, thymus, tissue masses or tumours, tongue, trachea, urinary bladder, uterus (incl. cervix), vagina.
Postmortem examinations (offspring):
SACRIFICE
- All F1 animals were sacrificed on day 13 post-partum.

GROSS EXAMINATION OF DEAD PUPS:
Yes. Dead pups and pups sacrified at day 13 post-partum were carefully examined externally for gross abnormalities. External reproductive genitals were examined for signs of altered development. The thyroid from 1 male and 1 female pup/litter was weighed and fixed for examination.
Statistics:
Homogeneity of variances and normality of distribution were tested using the Bartlett's and Shapiro-Wilks test. In case of heterogeneity and/or non-normality of distribution, Anova and Dunnet's test were used. Non-parametrical values were evaluated using the Fisher or Chi-squared tests.
Reproductive indices:
Male fertility index (%): 100 x (Males with confirmed female insemination/number of rats used).
Female fertility index (%): 100 x (Pregnant rats/total rats).
Gestation index (%): 100 x (Dams with live pups/pregnant rats).
Offspring viability indices:
Birth index (%): 100 x (Total pups born {alive or dead}/number of implantation scars).
Live birth index (%): 100 x (Number of pups alive on day 0/1 of lactation/total number of pups {alive or dead}).
Viability index (%): 100 x (Pups alive on day 4/Pups alive on day 0/1).
Pre-implantation loss (%): 100 x ((Corpora lutea - implantations)/corpora lutea).
Post-implantation loss (%): 100 x ((Implantations - living foetuses)/implantations).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All males of the high-dose group displayed increased salivation, considered to be treatment-related and adverse.
4/10 high-dose females also displayed increased salivation, also considered to be treatment-related and adverse.
Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
Males:
500 mg/kg bw/day: no deaths.
Females:
500 mg/kg bw/day: One premature death (during lactation period).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
500 mg/kg bw/day: significantly lower body weight than control during the whole study period (e.g. 6.5%, 11.5% and 14.4% below controls on test days 22, 29 and 52, respectively).
Females:
500 mg/kg bw/day: no effects on body weights pre-mating or during lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
Males
500 mg/kg bw/day: lower food consumption week 1 of study, approximately half control value.
125 mg/kg bw/day: lower food consumption week 1 of study, approximately 15% below control value.
Food consumption normalised between test days 22 and 28, so these observations were not considered to be adverse.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Group mean white blood cell (WBC) counts (absolute values) were higher than the control mean for all treated groups, but the effect only achieved statistical significance at 500 mg/kg bw/day. These increases were particularly associated with dose-related elevations in mean neutrophil and lymphocyte counts (absolute values), which were themselves statistically significant at the high-dose level. For the high-dose group, statistically significant increases in monocytes and large unclassified cell means were also noted. A dose-related decrease in the mean reticulocyte (%) counts was evident for all treated groups when compared to the respective control mean. A statistically significant difference was present only at 125 and 500 mg/kg bw/day, although the effect in the mid-dose fell within historical control ranges for this effect (and, as such, was not considered by the study authors to be related to treatment)

Females:
Mean white blood cell (WBC) counts (absolute values) showed minor increases at the mid- and high-dose (although neither value achieved statistical significance), and which again appeared to be primarily related to limited increases in neutrophil and lymphocyte group mean values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Increases in group mean alanine aminotransferase (ALAT) values were noted for all treated groups, which achieved statistical significance at 125 and 500 mg/kg bw/day. However, the magnitude of this effect was limited in degree (only the high-dose value approached the upper boundary of the historical control dataset). Clear treatment-related differences in other measured serum enzyme levels were not evident.
Several other statistically significant effects were detected in group mean values at 500 mg/kg bw/day: concentrations of albumin (decrease), total cholesterol (increase), protein (decrease) and calcium (decrease).

Females:
No statistically significant changes were seen in any of the clinical chemistry parameters of females.
There was a dose-related decrease in aP, but statistical significance was not achieved at any dose level.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
One mid-dose animal showed slight signs of positive geotropism; another had slight diarrhoea. One low-dose and one high-dose animal showed "slight influences" during the wire manoeuvre test, and males in these two groups showed a decreased reaction to a tail pinch. All observations were cons idered to be spontaneous and not related to treatment.

Females:
One mid-dose female was noted with salivation and a slightly reduced urination. This was considered to be spontaneous and not related to treatment.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females:
500 mg/kg bw/day: Lung - Increased pigment deposition (4/5 males, 2/6 females), haemorrhages (3/5 males, 4/6 females) and granulomatous inflammation (4/5 males, 4/6 females). Incidence of pigment deposition and inflammation was statistically significant in males.
The one female that died during the study period was found to have marked granulomatous inflammation of the lungs. The investigators concluded that the pulmonary effects noted for the male and female animals were considered to be a foreign body response to test item in the lung rather a toxicological effect.

Females:
500 mg/kg bw/day: Statistically significantly increased incidence of hypertrophy of the adrenal cortex in 3/6 females. [Note that there were changes in adrenal organ weight at this dose level.] However, evidence of similar hypertrophic change was not found when tissues from mid-dose group females were examined.
No test item-related microscopic changes in the reproductive organs of high-dose males or females were detected. Qualitative evaluation of the stages of spermatogenesis in males of this treatment group revealed no abnormalities.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A limited decrease in Thyroid Hormone T4 concentration (17.5% below the mean value of the control group, p ≤ 0.05) was noted in male animals of the high-dose group. T4 measurements for females of all treated groups were unaffected, as were those for males receiving 30 or 125 mg/kg bw/day.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test item-related influences on oestrous cycle in any treatment group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the qualitative stages of spermatogenesis. The histopathological examination performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) also did not reveal any test item-related effects.
Reproductive performance:
no effects observed
Description (incidence and severity):
No adverse effect on the fertility index, the gestation index, the precoital time or the gestation length in the P0 animals.
[Adverse effects on post-implantation loss and number of stillborn pups discussed below; F1 section.]

Key result
Dose descriptor:
NOAEL
Remarks:
Fertility and reproduction parameters
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Critical effects observed:
not specified
Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
not specified
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
No differences considered to be related to treatment were noted for the mean number of: corpora lutea; implantation sites; total pups born (alive and dead); live born pups; and the number of stillbirths when comparing the control group to the low and intermediate dose groups (30 or 125 mg/kg bw/day). Related reproductive indices were unaffected. An increased post-implantation loss (dam/group) was observed for all treatment groups. When calculated as a group index [%], the differences achieved statistical significance. In the high-dose group (500 mg/kg bw/day), the post-implantation loss index was clearly above the range of the background historical control data, and was considered to be related to treatment. The respective index for the low-dose group was noted to be within the historical control dataset for this parameter, whilst that for the mid-dose group just exceeded the historical control upper bound. In the case of the latter group the data were skewed due to the influence of a high post-implantation loss for a single animal (no 56). Therefore, it was concluded by the investigators that the post-implantation loss differences observed for the low- and mid-dose groups may have been spontaneous in nature rather than treatment-related. For the high-dose (500 mg/kg bw/day), a statistically significantly decreased live birth index was noted (due to a total of 7 stillbirths from 3 different dams), which was considered to be related to treatment. This was accompanied by lower pup weights [see 'Body weight and weight changes' section (F1)]. A possible effect in relation to decreased mean pup survival was also evident for the high-dose group, but this was due only to the influence of 2 litters (one was lost due to the death of dam, whilst the other was a total litter loss). Survival in the remaining 7 litters at this treatment level was good and similar to that of the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day, a statistically significant depression in mean pup bodyweight was evident at all timepoints (viz. approximately 15%, 18%, and 32% below control values at d1, d4, and d13, respectively). [data combined for sexes; similar extent of difference in both sexes]
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No difference was noted for the weights of the left and right thyroid glands between the pups of the control group and the pups of the treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The external macroscopic examination after sacrifice revealed no test item-related gross abnormalities.
Histopathological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were noted in the thyroid glands of either sex.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
ENDOCRINE PARAMETERS
No effects on offspring endocrine parameters [nipple retention in male pups and anogenital distance in male and female pups].

No notable differences were apparent between control and treated groups in respect of thyroid hormone T4 levels at lactation day (LD) 4. However, by LD 13, a modest reduction in group mean serum T4 level was evident for the pups of dams which received 500 mg/kg bw/day, but not for the pups of the other treated groups. Since the reductions were observed in both male and female pups from the former group, and were statistically significant, this finding was considered to be treatment-related. These changes were not associated with thyroid organ weight effects or adverse histopathological findings.
Description (incidence and severity):
not applicable
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
Pre-natal development (conception to birth)
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased live birth index and increased post-implantation loss
Key result
Dose descriptor:
NOAEL
Remarks:
Post-natal development of pups
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Critical effects observed:
not specified
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
not specified
Conclusions:
In a combined repeated-dose toxicity study with reproduction/developmental toxicity screening test, conducted according to OECD Test Guideline 422 and to GLP, 'Karstedt Concentrate’ was administered daily (in corn oil) by gavage to groups of rats (10/sex/dose) for at least 28 days in males (during pre-mating, mating and post-mating) or 49-62 days in females (during pre-mating, mating, gestation and lactation) at 30, 125 or 500 mg/kg bw/day. Parental (P0) effects were observed (including reduced food consumption and body weight, and haematological and clinical chemistry changes). The NOAEL for adverse effects on fertility and reproductive performance of the P0 generation was established at 500 mg/kg bw/day, the highest dose tested. In the F0 generation, increased post-implantation loss and increased number of stillborn pups (decreased live birth index), and reduced pup body weights and reduced viability index (increased mortality during lactation), were reported at the highest tested dose, leading to an NOAEL of 125 mg/kg bw/day for adverse effects on pre- and post-natal development.
Executive summary:

In a combined repeated-dose toxicity study with reproduction/developmental toxicity screening test, conducted according to OECD Test Guideline 422 and to GLP, platinum(0) 1,3-divinyl-1,1,3,3-tetramethyldisiloxane complexes ('Karstedt Concentrate’) was administered daily (in corn oil) by oral gavage to groups of rats (10/sex/dose) for at least 28 days in males (during pre-mating, mating and post-mating) or 49-62 days in females (during pre-mating, mating, gestation and lactation) at 30, 125 or 500 mg/kg bw/day. Control animals received vehicle only.

In the high-dose group, one female died, and reduced body weights were seen in males. On commencement of treatment, food consumption was transiently lower in males in the mid- and high-dose groups, but with a very marginal degree of effect evident for the former. Haematological examination revealed effects on white blood cell parameters mainly in males in the high-dose group. A statistically significant reduction in group mean reticulocyte count was also evident at the high- and mid-dose (though the value for the latter was within the historical control range for this parameter). Statistically significant effects were noted for several clinical chemistry parameters in males receiving 500 mg/kg bw/day, although effect severity was limited. A dose-related increase in absolute and relative adrenal gland weight was seen in females at 125 and 500 mg/kg bw/day (only reaching statistical significance at the higher dose, where histopathology indicated the existence of an accompanying adrenal cortical hypertrophy). Males at the highest tested dose showed a decrease in thyroid hormone T4 concentration, whereas females were unaffected.

 

No adverse effect on the fertility index, the gestation index, the precoital time or the gestation length was reported, resulting in a NOAEL of 500 mg/kg bw/day for fertility and reproductive performance of the P0 generation.

In the F1 generation, increased post-implantation loss and increased number of stillborn pups (decreased live birth index), and reduced pup body weights and reduced viability index (increased mortality during lactation), were reported at the highest tested dose (500 mg/kg bw/day), leading to an NOAEL of 125 mg/kg bw/day for adverse effects on pre- and post-natal development. By lactation day 13, there was a modest reduction in group mean serum thyroid hormone (T4) level evident for the pups of dams which received 500 mg/kg bw/day, but not for the pups of the other treated groups; this finding was considered to be related to treatment, but was not associated with thyroid organ weight effects or adverse histopathological findings.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Yellow to brown, aromatic liquid
- Storage: At +10°C to +25°C under inert gas, kept in a tightly closed container and stored in a dry place, protected from heat and direct sunlight.

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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

Examinations

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).

Results and discussion

Test results
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.

Any other information on results incl. tables

 

 

Applicant's summary and conclusion

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.