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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2014 - 07 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study, conducted 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 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): tetraammine platinum (II) dinitrate
- Substance type: organometallic
- Physical state: liquid
- Analytical purity: in the range 94.33 % to 100.88% (calculation of test item formulations based on the analysis of the platinum content)
- Impurities (identity and concentrations): chloride, < 0.1 % (w/w)
- Composition of test material, percentage of components: platinum, 3.030 % (w/w)
- Isomers composition: not applicable
- Purity test date: 20 August 2013
- Lot/batch No.: CPI-15448
- Expiration date of the lot/batch: August 2014
- Stability under test conditions: 12 months
- Storage condition of test material: at room temperature (10 - 25 deg C), in a tightly closed container

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 GmbH
Sandhofer Weg 7
97633 Sulzfeld
Germany
- Age at study initiation (on test day 1): 73 days
- Weight at study initiation: males: 352.4 - 398.3 g; females: 231.0 - 291.4 g
- Fasting period before study: no data
- Housing: except during mating, the dams were housed singly in cages. No data on housing of males. Granulated textured wood was used as bedding material, and the cages were cleaned and changed once per week
- Diet (e.g. ad libitum): Commercial ssniff(r) R/Z V1324 was offered ad libitum
- Water (e.g. ad libitum): drinking water was offered ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 deg C
- Humidity (%): 55 +/- 15 % relative humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light. About 150 lux at approximately 1.5 m room height

IN-LIFE DATES:
First administration (at age 73 days): 21 May 2014
End of in-life part (males): 18 June 2014
End of in-life part (females): 07 July 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item formulations were freshly prepared once weekly. The test item, supplied as a solution, was further diluted in tap water to the appropriate concentrations.

VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no. (if required): not applicable
- Purity: not applicable
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy had occurred or two weeks
- Proof of pregnancy: presence of sperm or a vaginal plug referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly in a standard cage
- Any other deviations from standard protocol: one female, which showed no evidence of copulation after 14 days of mating, was sacrificed 16 days later
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of approximately 5 mL were taken from each of the weekly prepared test item formulations and stored at -20 deg C or colder until shipment to the GLP analytical laboratory.
Duration of treatment / exposure:
The animals were treated for the following periods:
- males: 2 weeks prior to mating, during the mating period, and approximately 2 weeks post mating until 28 days' dosing was completed. From test day 1 up to and including test day 28.
- females: Throughout the study. Beginning 2 weeks prior to mating up to and including day 3 post-partum. From test day 1 and test day 40 (first sacrificed females) or test day 47 (last sacrificed females).
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a 14-day dose-range finding study (results not included here). A top dose of 1000 mg/kg/day was tested without apparent toxicity.
Positive control:
No.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Signs of illness or reaction to treatment were recorded immediately after administration. Otherwise, animals were observed daily for behaviour, external appearance and nature of the faeces. Animals were checked regularly throughout the working day (07:00 - 15:45). On Saturdays and Sundays, regular checks were made between 07:00 and 11:00, with a final check at approximately 15:30. Further checks were made early in the morning and again in the afternoon of each working day, and up to around midday on weekends, to look for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed 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 and on day 4 post-partum.

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Drinking water consumption was monitored by visual appraisal of the drinking water bottles throughout the study.
Oestrous cyclicity (parental animals):
No data.
Sperm parameters (parental animals):
No data. Histopathological examination of the testes and epididymides suggested no significant effect on spermatogenesis. This is supported by 100% pregnancies of mated females.
Litter observations:
STANDARDISATION OF LITTERS
Not applicable.

PARAMETERS EXAMINED
The following parameters were examined in offspring at birth and day 4 post-partum:
Number of pups (absolute); number of pups (per dam); number of still births (absolute and per dam); number of pups with malformations (absolute and per dam).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external gross abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals were sacrificed on test day 29, after a minimum total dosing period of 28 days.
- Maternal animals: Dams with offspring were sacrificed on day 4 post-partum. One female, which showed no evidence of copulation, was sacrificed 16 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal macroscopic examination for any abnormalities or pathological changes. Special attention was paid to the reproductive organs.
- The numbers of implantation sites and corpora lutea were recorded.
- The ovaries, testes, epididymides, accessory sex organs (coagulating gland, preputial gland, prostate, seminal vesicle, uterus (including cervix and oviducts), and vagina) and all organs showing macroscopic lesions were preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The testes (2) and epididymides (2) of the male animals were weighed.
- Histopathologic examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups.
- Detailed histopathologic examination was performed on one testicle and one epididymis of all males in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure.
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 by using the SHAPIRO-WILKS test and the BARTLETT test. Data not normally distributed or with heterogeneous variances between the groups were stepwise log- or rank-transformed.

One-way analysis of variance (ANOVA) was performed with non-transformed or log-transformed data. The KRUSKAL-WALLIS test was used for rank-transformed data.

In case of significant differences (found by ANOVA or KRUSKAL-WALLIS test), inter-group comparisons with the control group were made by parametric or non-parametric DUNNETT multiple comparison tests (p ≤ 0.05 and p ≤ 0.01).

Other parametrical values, such as number and weight of the neonates, were analysed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01). Prior to the DUNNETT test homogeneity of variances was tested using the BARTLETT test. In case of heterogeneity of variances, the STUDENT's t-test was carried out (p ≤ 0.05 and p ≤ 0.01).

Statistical analyses of non-parametrical data like the reproductive indices were performed using the following settings:
FISHER exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

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
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 1000 mg/kg bw/day, a statistically significant (p <= 0.01) reduced body weight (reduced by 8.5% compared to controls) was noted for female rats on lactation day 4 only, and similarly for the body weight at autopsy. No effect on the body weight of male rats, or food consumption of either sex.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
No effects

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility/reproductive parameters
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects seen at highest tested dose
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No general systemic effects seen at highest tested dose
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No general systemic effects seen at 250 mg/kg bw/day
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased body weight at day 4 post-partum only.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
No effects

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
No effects

ORGAN WEIGHTS (OFFSPRING)
No effects

GROSS PATHOLOGY (OFFSPRING)
No effects

HISTOPATHOLOGY (OFFSPRING)
No effects

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse 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 421 reproduction and developmental toxicity screening study, to GLP, parental rats (12/sex/group) were administered tetraammineplatinum(II) dinitrate by oral gavage at up to 1000 mg/kg bw/day for 14 days pre-mating, through mating, and (for females) throughout gestation and up to lactation day 3. No adverse effects on reproductive parameters, or on development of offspring, were observed at any dose, resulting in a reproductive toxicity NOAEL of 1000 mg/kg bw/day.
Executive summary:

The potential of a solution of tetraammineplatinum dinitrate(II) to adversely affect the fertility and reproductive parameters of CD rats was investigated in a reproductive and developmental screening study conducted according to OECD Test Guideline 421 and to GLP. The test material was administered by oral gavage for at least 28 days. Males were dosed for 14 days pre-mating and 14 days mating/post mating. Females were dosed for 14 days pre-mating, through gestation and up to post-partum day 3 (test day 40-47). Three dose groups (50, 250 and 1000 mg/kg bw/day) and a control group were used, each containing 12 animals of each sex.

 

Parental (F0) animals were observed for clinical signs of toxicity throughout the study, with body weights and food consumption monitored. At necropsy, animals were subjected to external and internal macroscopic examinations for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. The numbers of implantation sites and corpora lutea were recorded. Histopathological examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure. A number of reproductive indices were calculated from the collected data (including mating, fertility and gestation indices).

 

The only clinical sign of toxicity was a significantly reduced body weight in high-dose females at the end of the study on post-partum day 4. There was no impact on food consumption in males or females. Thus, the NOAEL for general toxicity was considered to be 250 mg/kg bw/day.

 

No test item-related microscopic changes were noted in the reproductive organs, and there was no impact on fertility or on the measured reproductive parameters at any dose level. Thus, the NOAEL for reproductive toxicity was 1000 mg/kg bw/day, the highest dose tested.