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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
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:
other: 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)
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
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.
Details on mating procedure:
- Impregnation procedure: cohoused
If cohoused:
- 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
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.
Duration of test:
40-47 days (for females)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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.

Examinations

Maternal 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.

POST-MORTEM EXAMINATIONS: Yes
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), 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Dead pups and pups killed on day 4 post-partum were carefully examined externally for gross abnormalities.

- External examinations: Yes: [all]
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
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)
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

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
Historical control data:
No data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No embryotoxicity / teratogenicity seen at highest tested dose

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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 gavage at 0, 50, 250 or 1000 mg/kg bw/day. No adverse effects on reproductive parameters or development of offspring were observed at any dose, resulting in a developmental NOAEL of 1000 mg/kg bw/day.
Executive summary:

The potential of a solution of tetraammineplatinum dinitrate to adversely affect the development of CD rat pups 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. Histopathologic 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. On postnatal day 4, pups were carefully examined for gross abnormalities at necropsy.

 

The only sign of toxicity was a significantly reduced body weight in high-dose females at the end of the study on post-partum day 4. Thus, the NOAEL for general toxicity was deemed to be 250 mg/kg bw/day.

 

There was no developmental toxicity effect at any dose level. Thus, the NOAEL for developmental toxicity was 1000 mg/kg bw/day, the highest dose tested.