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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:
15 October 2014 - 11 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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:
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
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)
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).
- 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.
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.
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.
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.
Duration of test:
Final treatment was administered on test day 57.
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).

Examinations

Maternal 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: Dams 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.

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.

SACRIFICE
- 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, uterus (including cervix and oviducts), vagina) and all organs showing macroscopic lesions of all adult animals were preserved.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Ovaries were examined histopathologically; uterine content not examined as dams only sacrificed post-partum.
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early/late resorptions: No data; pre- and post-implantation loss were calculated from the number of corpora lutea, implantations and living foetuses.
Fetal examinations:
- External examinations: Yes: [all per litter]
- 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).

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:
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:
See Hansen (2015b)

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No embryotoxicity/teratogenicity effects seen at the 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 (F0) rats (12/sex/group) were administered diammonium hexachloroplatinate by gavage at 0 (corn oil), 10, 30 or 100 mg/kg bw/day. No adverse effects on reproductive parameters of parent males (F0 animals), or on developmental of offspring (FI generation), were observed at any dose, resulting in a NOAEL of 100 mg/kg bw/day. A single female with all dead implantations and two others with high post-implantation losses caused a slight increase in overall post-implantation loss and concurrent decrease in birth index, in dams at the highest tested dose.
Executive summary:

The potential ofdiammonium hexachloroplatinateto adversely affect the development of rats was investigated in a guideline reproductive and developmental screening study conducted according to OECD Test Guideline 421 and to GLP. The test material (in corn oil)was administered to rats 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. Pups were carefully examined for gross abnormalities at necropsy (on post-partum day 4).

 

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. Salivation and piloerection were also observed in low- and mid-dose animals. 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.

 

There was no developmental toxicity effect (survival index, body weight, gross abnormalities)at any dose level. Consequently, the NOAEL for developmental toxicity was 100 mg/kg bw/day, the highest dose tested.