Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2014 - 30 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, conducted according to GLP.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): dihydrogen hexahydroxyplatinate.
- Substance type: powder.
- Physical state: solid.
- Analytical purity: no data.
- Impurities (identity and concentrations): no data.
- Composition of test material, percentage of components: 64.83% platinum w/w.
- Isomers composition: not applicable.
- Purity test date: 28 May 2013.
- Lot/batch No.: CZ0185.
- Expiration date of the lot/batch: June 2018.
- Stability under test conditions: 5 years.
- Storage condition of test material: At +10 to +25 deg C, in a tightly closed container, protected from 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, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Age at study initiation: 67 days (at first test item administration).
- Weight at study initiation: males: 326.4 - 402.7 g; females: 189.4 - 246.0 g (at first test item administration).
- Fasting period before study: no data.
- Housing: animals were housed singly, except during the mating period.
- Diet (e.g. ad libitum): certified commercial diet, offered ad libitum.
- Water (e.g. ad libitum): tap water, offered ad libitum.
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22degC +/- 3degC (maximum range).
- Humidity (%): relative humidity of 55% +/- 15% (maximum range).
- Air changes (per hr): no data.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light, about 150 lux.

IN-LIFE DATES:
From: July 2014.
To: 23 October 2014 (males); 14 November 2014 (females).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: test item formulations were prepared once weekly and stored in a tightly closed container at room temperature (+10 to +25 degC) until use.

DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable.
- Mixing appropriate amounts with (Type of food): not applicable.
- Storage temperature of food: not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was found to be unstable in water and other aqueous vehicles. Stability for 7 days in corn oil was demonstrated in an identity and stability investigation, and thus corn oil was taken forward as the vehicle for this study.
- Concentration in vehicle: 20, 60 or 200 mg test item/mL vehicle.
- Amount of vehicle (if gavage): 5 mL/kg bw/day.
- Lot/batch no. (if required): Batch nos. 13249003 and 13249006, Caesar & Loretz GmbH, 40721 Hilden, Germany.
- Purity: no data.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy occurred, or two weeks.
- Proof of pregnancy: presence of sperm or a vaginal plug referred to as the day of conception (day 0 of pregnancy).
- After successful mating each pregnant female was caged (how): except during mating, animals were housed singly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of approximately 10 mL were collected once weekly, at the time of preparation of the formulation, and analysed for homogeneity and concentration.
Duration of treatment / exposure:
Males were dosed from test days 1-35 (inclusive), including 2 weeks prior to mating, the mating period and approximately 2 weeks post-mating. Females were dosed from test day 1 (2 weeks prior to mating), throughout mating and gestation, until day 3 post-partum or the day before sacrifice (from test day 41 for the first sacrificed females to test day 58 for the last sacrificed female).
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based a 14-day dose range finding study conducted at the same testing laboratory. In the dose range finding study, male and female rats were treated with 1000 mg test item/kg bw/day. No animal died prematurely, there were no test item-related adverse effects, and no test item-related changes noted at necropsy.
- Rationale for animal assignment (if not random): computer-generated randomisation programme.
- Rationale for selecting satellite groups: not applicable.
- Post-exposure recovery period in satellite groups: not applicable.
- Section schedule rationale (if not random): not applicable.
Positive control:
No.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after dosing and, additionally, regularly throughout the working day (7:00 to 15:45 on Monday to Friday; 7:00 to 11:00 with a final check at 15:00 on Saturday and Sunday).
- Cage side observations included any behavioural changes, signs of illness or reaction to treatment. Skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns were recorded, together with the onset, intensity and duration of any signs observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly, once before the first administration and once a week thereafter.
- Signs recorded included: changes in skin, fur, eyes, mucous membranes; occurrence of secretions, excretions and autonomic activity; changes in gait, posture and response to handling; behavioural changes.

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During gestations, females were weighed on days 0, 7, 14 and 20, within 24 hours of parturition (day 1 post-partum), and day 4 post-partum.

FOOD AND WATER CONSUMPTION:
- Food consumption for each animal determined: Yes.
- Food intake per animal was determined using the total amount of food given to and left by each animal.
- Drinking water consumption for each animal determined: Yes, by daily visual appraisal throughout the study.
Oestrous cyclicity (parental animals):
No data.
Sperm parameters (parental animals):
No data.
Litter observations:
STANDARDISATION OF LITTERS
Not applicable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, alive and dead, as an absolute number and number per dam; stillbirths (absolute and per dam); postnatal mortality (pup numbers were recorded at parturition and post-partum day 4); presence of gross anomalies (absolute/per dam).

GROSS EXAMINATION OF DEAD PUPS:
Yes. dead pups were carefully examined externally for gross abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed on test day 36.
- Maternal animals: All surviving dams with offspring were sacrificed on day 4 post-partum.
- Females showing no signs of littering were sacrificed 24 days after the last day of the mating period.

GROSS NECROPSY
- Adult animals were examined macroscopically at sacrifice for any abnormalities or pathological changes, with particular attention given to the reproductive organs.
- The number of implantation sites and corpora lutea were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The epididymides, mammary glands (females only), ovaries, prostate, seminal vesicle, testes, uterus (including cervix and oviducts) and vagina of all adult animals were preserved and examined histopathologically. The epididymides, testes, ovaries and uterus (including cervix and oviducts) of all animals were weighed individually before fixation.
Postmortem examinations (offspring):
Dead pups and pups killed on day 4 post-partum were carefully examined externally for gross abnormalities.
Statistics:
Analysis of normal distribution and homogeneity of variances was performed using the Shapiro-Wilks test and the Bartlett test.
One-way analysis of variance was performed using Anova or the Kruskal-Wallis test.
Intergroup comparisons, in the case of significant differences, were made using the Dunnett test.
Statistical analyses of non-parametrical data were performed using Fisher's exact test or the Chi-squared test.
Reproductive indices:
The following indices were calculated for each group:
Male fertility Index [%] = (No. of males with confirmed female insemination/Number of rats used) x 100
Female fertility Index [%] = (Number of pregnant rats/Number of rats used) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including those, that delivered at term, aborted or had fully resorbed litters.
Gestation Index [%] = (Number of dams with live pups/Number of pregnant rats) x 100
Offspring viability indices:
For each litter and group the following indices were determined:
Birth Index [%] = (Total number of pups born (alive + dead)/Number of implantation scars) x 100
Live Birth Index [%] = (Number of pups alive on day 0/1 of lactation/Total number of pups (alive + dead)) x 100
Survival Index [%] = (Number of pups alive on day 4/Number of pups alive on day 0/1) x 100
Pre-implantation loss [%] = ((corpora lutea – implantations)/corpora lutea) x 100
Post-implantation loss [%] = ((implantations - living neonates)/implantations) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test item-related premature deaths were reported. Two female rats (one from the low-dose group and one from the mid-dose group) died during laboratory examination on test day 15, but their deaths were considered as not test item-related.

There were no changes in behaviour, external appearance, faeces or other clinical signs in any treated group, or the control, at any point during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no test item-related changes in body weight or body weight gain in male rats, in the pre-mating, mating, and post-mating periods, or in females during the pre-mating period, gestation and lactation.FOOD AND WATER CONSUMPTION

No test item-related changes in food consumption were noted between the control groups and any treated rats. There was a statistically significant decrease in food consumption in mid-dose females during the second week of the test; this showed no dose-response relationship and was considered not to be related to treatment.

The consumption of drinking water, assessed by daily visual appraisal, was not altered by treatment.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not examined (gavage study).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no test item-related difference in fertility index in any control or treated rats. Insemination was confirmed in 100% of female rats paired with males; pregnancy was confirmed in 100% of high-dose females, and 91 or 92% of control, low- and mid-dose animals (1 female/group did not conceive). No statistically significant difference in pre-coital time or gestational length was reported.

The mean number of corpora lutea, implantation sites, and relative and absolute pup numbers were not affected by treatment with the test item. In total, six stillbirths were noted, with at least one in each group (including the control). These were considered to be spontaneous. There was no test item-related differences reported in birth index, live birth index, or pre- or post-implantation losses.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related change in organ weights was reported in any treatment group. A statistically significant decrease in absolute liver weight was noted in low-dose females. This was within the range of the background data for the testing laboratory.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test item-related changes were observed in macroscopic examination of the internal organs and tissues of all test animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No microscopic changes in the examined organs or tissues were seen in any treated animals.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No general systemic or reproductive effects seen at highest tested dose

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No test item-related differences in survival of pups from treated dams or the controls were noted. Survival index in all groups (treated and control) was >98%.

CLINICAL SIGNS (OFFSPRING)
Not examined.

BODY WEIGHT (OFFSPRING)
There was no difference in mean pup body weight or total litter weight between control and treated dams.

SEXUAL MATURATION (OFFSPRING)
Not examined.

ORGAN WEIGHTS (OFFSPRING)
Not examined.

GROSS PATHOLOGY (OFFSPRING)
No gross abnormalities were observed on any pups.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No general systemic effects seen in the F1 generation at highest tested dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of dihydrogen hexahydroxyplatinate for at least 35 days (including 2-weeks pre-mating and throughout mating), no effects on any measured reproductive or fertility parameters were observed. The systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day).
Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, CD rats (12/sex/group) were orally administered dihydrogen hexahydroxyplatinate by stomach tube (gavage) at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were dosed for 35 days (2 weeks pre-mating, during the mating period and for approximately 2 weeks post-mating). Females were dosed for 14 days pre-mating, through mating, gestation and up to post-partum day 3 (test day 41-58).

 

There were no reported changes to reproductive parameters (including number of corpora lutea, implantation sites, number of pups, fertility and gestation indices, birth index and pre- and post-implantation losses). The systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day).