Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 October 2014 - 11 December 2014
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 421 (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): diammonium hexachloroplatinate.
- Substance type: organometallic.
- Physical state: solid.
- Analytical purity: salt assumed 100% pure.
- Impurities (identity and concentrations): total metallic impurities (excluding Na) < 0.1%; sodium 0.2%.
- Composition of test material, percentage of components: 37.99% w/w platinum.
- Isomers composition: not applicable.
- Purity test date: 01 July 2013.
- Lot/batch No.: UZ0129-1.
- Expiration date of the lot/batch: 5 years from the date of manufacture [01 July 2018] if packaging is intact and material is stored under described conditions.
- Stability under test conditions: as above.
- Storage condition of test material: At +10degC to +25degC, 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 of Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Age at study initiation: (P) 66 days ; (F1) n/a.
- Weight at study initiation: (P) Males: 313.7-369-1 g; Females: 192.6-248.0 g; (F1) n/a.
- Fasting period before study: no.
- Housing: animals were housed singly (except during mating).
- Use of restrainers for preventing ingestion (if dermal): not applicable.
- Diet (e.g. ad libitum): certified commercial diet, offered ad libitum.
- Water (e.g. ad libitum): drinking water, offered ad libitum.
- Acclimation period: 7 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: August 2014.
To: 11 December 2014.

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 (10-25degC) 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: 2, 6 or 20 mg test item/mL vehicle.
- Amount of vehicle (if gavage): 5 mL/kg bw/day.
- Lot/batch no. (if required): Batch no. 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 57 for the last sacrificed female).
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
low dose
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
intermediate dose
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
high dose
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 considering the results of a 14-day dose-range finding study (results not included) and a 28-day repeated-dose oral toxicity study (Hansen, 2015a).
Positive control:
No positive control used.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes. Animals were observed daily for behaviour, external appearance and nature of the faeces.
- Time schedule: Animals were checked for any signs of illness or reaction, immediately after administration of the test item. In addition, animals were checked regularly throughout the working day (07:00 - 15:45 Monday to Friday; 07:00 - 11:00 Saturday and Sunday, 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 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. Pups were weighed within 24 hours of parturition and on day 4 post-partum.

FOOD AND WATER CONSUMPTION:
- Food consumption for each animal was recorded on a weekly basis during the pre-mating and gestation period, and on day 4 post-partum. 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 was determined by visual appraisal throughout the study.
Oestrous cyclicity (parental animals):
No data.
Sperm parameters (parental animals):
Not examined.
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 were sacrificed after a dosing period of 35 days, on test day 36.
- Dams with offspring were sacrificed on day 4 post-partum.

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 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 of all adult animals were preserved.
- The testes and epididymides of the male animals were weighed.
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).

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 animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2/12 female animals in the high-dose group died prematurely. Clinical signs in high-dose group were salivation, piloerection, pale faeces and increased water consumption. Other clinical signs reported in mid- and low-dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight and reduced body weight gain in high-dose group. Reduced food consumption in high-dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight and reduced body weight gain in high-dose group. Reduced food consumption in high-dose group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related differences were noted for the organ weights of the epididymides and the testes
Gross pathological findings:
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:
effects observed, treatment-related
Description (incidence and severity):
Increased post-implantation loss and decreased birth index in high-dose females.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Two high-dose females died prematurely (8 or 23 days after the start of treatment). Both animals had displayed piloerection and reduced motility ante mortem, among other clinical signs of toxicity. At necropsy, one of the animals showed reddening in the cardiac stomach and caecum and greyish discolouration of the thymus. The intestines were filled with liquid and the spleen was reduced in size. The only reported necropsy finding in the second female that died prematurely was a partial detachment of the stomach mucosa.

In surviving high-dose animals, slight to extreme salivation, piloerection, and/or pale faeces were reported. Increased water consumption was observed in high-dose males. Salivation and piloerection were also observed in low- and mid-dose animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no reported test item-related differences in body weight or body weight gain in low- or mid-dose males or females at any point during the study.

In males, body weights in the high-dose group were significantly reduced at the end of the study compared to the control group (mean body weight of high-dose animals was 16% below the controls). This was accompanied by a significant reduction in body weight gain; across the whole study, body weight gain of high-dose males was only approximately half as large as the control animals (18.6% vs 41.5%).

During the pre-mating period, the weight of high-dose females was reduced by 3.3% compared to controls (not statistically significant); body weight gain was reduced accordingly. Body weight reductions of high-dose females during gestation (up to 11.8% below controls) and lactation (12.9% below controls) were also reported.

Food consumption at the end of test week 1 was statistically significantly reduced, by 33.8% in high-dose males and 21% in females compared to controls. Food intake improved during the course of the study, and at the end of test week 2 the reduction in males was 8.2%. Food consumption in high-dose females was within the range of the control group. No test item-related differences in food consumption were seen in females during the gestation or lactation periods.

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

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined. However, no test item-related microscopic changes were reported in histopathological examinations of epididymides or testes of the high-dose males. Detailed examination revealed no test item-related effects on the qualitative stages of spermatogenesis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The male fertility index was 100%; all male rats were confirmed to have successfully inseminated their female.

No test item-related differences in the fertility index or gestation index of the female rats was noted for any treated groups. In the high-dose group, there was a statistically significant increase in the number of resorptions and consequently a statistically significant slight increase in post-implantation loss (18% vs 4.8% in controls). This also led to a statistically significant reduction in birth index (82.7% vs 95.2% in controls). There was no difference in the live birth index.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related differences were noted for the epididymides or testes in any treated males.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In high-dose males, 4/12 animals showed test item-related kidney changes (increased volume, external appearance and consistency) and 3/12 showed stomach changes (haemorrhagic foci, reddened mucosa, ulcer and white/thickened forestomach).

One high-dose female showed kidney lesions (dilated renal pelvis in both kidneys), and 2/12 animals had stomach changes (detached mucosa and white/thickened forestomach; reddened cardiac part with thin/transparent fundus part).

HISTOPATHOLOGY (PARENTAL ANIMALS)
However, no test item-related microscopic changes were reported in histopathological examinations of epididymides or testes of the high-dose males or the ovaries of high-dose females.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Slightly increased post-implantation loss and decreased birth index were noted in the high-dose group. This was attributed to a single female with all dead implantations and 2 other females with post-implantations of 23.1% and 18.2%.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
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 index were reported between controls and pups from dams in any treatment group. The survival index was almost 100% for each group.

CLINICAL SIGNS (OFFSPRING)
No data on clinical signs of toxicity observed in pups.

BODY WEIGHT (OFFSPRING)
No test-item related difference between body weights of pups from control dams and pups from treated dams when weighed on lactation (post-partum) days 1 and 4.

SEXUAL MATURATION (OFFSPRING)
Not investigated.

ORGAN WEIGHTS (OFFSPRING)
Not investigated.

GROSS PATHOLOGY (OFFSPRING)
No test item-related gross abnormalities were reported in pups from any treated dam after sacrifice on post-partum day 4.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 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 the 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 (F0) rats (12/sex/group) were administered diammonium hexachloroplatinate by oral gavage at up to 100 mg/kg bw/day for 14 days pre-mating, through mating, and (for females) throughout gestation and up to lactation day 3. Clinical signs of toxicity (including premature death, reduced body weight and growth) were observed in the high-dose groups, resulting in morphological changes to the kidneys and stomach. There was a significant increase in post-implantation loss and an accompanying decrease in birth index (although live birth index was unaffected). The study NOAEL was established to be 30 mg/kg bw/day for both systemic and reproductive toxicity.
Executive summary:

The potential of diammonium hexachloroplatinateto 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(in corn oil) was administered by oral gavage. Males were dosed for 35 days (14 days pre-mating, during the mating period and for approximately 14 days post mating). Females were dosed for 14 days pre-mating, through mating, gestation and up to post-partum day 3 (test day 41-57). Three dose groups (10, 30 and 100 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.

 

Two females in the high-dose group died prematurely. Surviving high-dose animals displayed slight to extreme salivation, piloerection, and/or pale faeces were reported. Increased water consumption was observed in high-dose males. Salivation and piloerection were also observed in low- and mid-dose animals. In the high-dose group, body weights were significantly reduced compared to controls, and cellular changes to the stomach and kidneys were also reported. A significantly increased percentage post-implantation loss was reported in high-dose females, leading to a significant reduction in birth index (although live birth index was unaffected). No test item-related microscopic changes were noted in the reproductive organs at any dose level.

 

The study NOAEL was established to be 30 mg/kg bw/day for both systemic and reproductive toxicity.