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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 June 2014 - 09 August 2014 (last necropsy)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, conducted to GLP, on closely-related surrogate
Cross-reference
Reason / purpose for cross-reference:
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
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetraamminepalladium(2+) dichloride
- Substance type:
- Physical state: solid
- Analytical purity: Pd content: 41.69%
- Impurities (identity and concentrations): 15 ppm Pt; <2 ppm Ru, <5 ppm Rh; <10 ppm Ir; 4 ppm Au; 7 ppm Ag; 3 ppm Al; 1 ppm Ca; <2 ppm Co; <1 ppm Cr; 13 ppm Cu; <2 ppm Fe; <2 pmm Mg; <5 ppm Mn; <1 ppm Ni; <2 ppm Pb; <10 ppm Sb; <10 ppm Si; <5 ppm Sn; 18 ppm Zn.
- Isomers composition: not applicable
- Purity test date: 16 January 2014
- Lot/batch No.: 10214
- Expiration date of the lot/batch: 13 January 2015
- Stability under test conditions: The test item solution in aqueous ammonium-chloride buffer was stable over 7 days at room temperature and for 14 days when refrigerated (2-8ºC).
- Storage condition of test material: Controlled room temperature (15-25 deg C, below 70 RH%), protected from humidity

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, at least 10 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: Males: 338 g – 399 g, Females: 219 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment. - Fasting period before study: no data
- Housing: Type II and III polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany offered ad libitum
- Water (e.g. ad libitum): tap water from municipal supply as for human consumption from 500 ml bottle ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.8 °C (target range 22±3°C)
- Humidity (%): 40 - 70 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
Start of experiment (start of treatment): 24 June 2014
End of treatment: 22 July 2014 (males); 08 August 2014 (females)
End of experiment: 09 August 2014 (last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was used as the dosing vehicle.

The buffer was prepared as follows:
2.68 g ammonium chloride (Batch/Lot No.: BCBM5575V, Supplier: Sigma-Aldrich, Expiry: March 2015) was dissolved in 1000 ml distilled water (Batch/Lot No.: 0271113, Supplier: TEVA Pharmaceutical Works PLtd., Expiry: November 2016).
The pH was adjusted to 9 by adding approximately 1650-1850 µL of 29.1% ammonium hydroxide (Batch/Lot No.: SZBD2260V, Supplier: Sigma-Aldrich, Expiry: August 2015).

The first preparation was used for 7 consecutive days and was kept at room temperature. The next preparations were used for periods of up to 14 days, following confirmation of the stability, and the stock solution was kept refrigerated (2-8ºC). Dose solutions for treatment were diluted from the stock solution with aqueous ammonium-chloride buffer to the concentrations of 4.0 and 0.8 mg/mL, daily just before the treatment. The stock solution was prepared four times during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of the preliminary formulation trial and pilot DRF study (CiToxLAB study code: 13/195-100PE), an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

- Concentration in vehicle: The test item was formulated in the aqueous ammonium-chloride buffer as a stock solution at the concentration of 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was assessed in the concentration range of 5 mg/mL-200 mg/mL by Fourier-transformation infrared spectroscopy (FT-IR). According to the results the test item solution in aqueous ammonium-chloride buffer was stable over 7 days at room temperature and for 14 days when refrigerated (2-8ºC). (CiToxLAB Study code: 13/195-929AN).

Analysis of test item formulations was performed at the Test Site using a validated ICP method (ICP-AES) to determine the palladium content. The concentration measurements were performed on all preparations of the stock solutions and at the same time, from samples of diluted dose solutions. The sampling occasions coincided with the preparation of the stock solution.

Samples were taken from the test item formulations (including control) for concentration measurements. One set of samples was collected for analysis and one set retained as a back-up, for possible confirmatory analysis. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of the test item. No confirmatory analysis was required.

The measured concentrations of palladium in the formulations varied between 95.0 % and 111.9 % of the nominal. These results were within the range of acceptable values (85% - 115%).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurred. All pairs mated within the first 7 days from the onset of pairing.
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (and as Day 0 of pregnancy).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating period). They were then euthanized and subjected to necropsy examination.

Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum.
Frequency of treatment:
Once daily
Duration of test:
At least 28 days (males); Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation (22-23 days) and up to and including the day before necropsy (at least 4 days post-partum dosing).
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 by the Sponsor in consultation with the Study Director, based on available data, including the results of a dose range finding study (CiToxLAB study code 13/195-100PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Adult (parental) animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. The changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were performed once prior to first treatment (to allow for within-subject comparisons), and once a week thereafter. The animals were examined outside the home cage in a standard arena and at similar times on each occasion. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and prior to necropsy.

Adult females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before necropsy).

The body weights of the females were also recorded on GD4, 10 and 17 in order to give accurate treatment volumes, however, these data were not evaluated statistically.
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]. Any pups showing abnormalities in structure or behaviour will be subjected to necropsy with macroscopic examination
- Soft tissue examinations: Yes: gross only
- Skeletal examinations: Yes: gross only
- Head examinations: Yes: gross only
Statistics:
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. When Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Chi2 test was performed when feasible.
Indices:
Reproductive indices

The following indices were calculated for each group:

Male mating index [%] = (Number of males with confirmed mating/Total number of males cohabited) x 100 [Measure of male's ability to mate]
Female mating index [%] = (Number of sperm-positive females/Total number of females cohabited) x 100 [Measure of female's ability to mate]

Male fertility index [%] = (Number of males impregnating a female/Total number of males cohabited) x 100 [Measure of male's ability to produce sperm that can fertilise eggs]
Female fertility index [%] = (Number of pregnant females/Number of sperm-positive females) x 100 [Measure of female's ability to become pregnant]

Gestation index [%] = (Number of females with live born pups/Number of pregnant females) x 100 [Measure of pregnancy that provides at least one live pup]

Offspring viability indices

Survival index [%] = (Number of live pups (at designated time)/Number of pups born) x 100
Pre-implantation mortality [%] = (Number of Corpora lutea - Number of Implantations/Number of Corpora lutea) x 100
Intrauterine mortality [%] = (Number of implantations - Number of liveborns/Number of implantations) x 100
Total mortality [%] = (Number of implantations - Number of viable pups (PND4)/Number of implantations) x 100
% Males = (Number of pups examined - Number of males/Number of pups examined) x 100
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
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:
VIABILITY (OFFSPRING)
The number of viable pups on PND 0 and 4 was comparable for all groups including the control.
The incidence of mortality during 4 postnatal days was negligible and was 4 of 149 (Low dose), 5 of 146 (Mid dose), and 2 of 167 (High dose). No mortality occurred in control group.

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
There was no effect of treatment on the offspring body weight or body weight gain.

SEXUAL MATURATION (OFFSPRING)
Not examined

ORGAN WEIGHTS (OFFSPRING)
Not examined

GROSS PATHOLOGY (OFFSPRING)
No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.

HISTOPATHOLOGY (OFFSPRING)
Not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxicity, teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
no

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 tetraamminepalladium dichloride by gavage at 0 (in aqueous ammonium-chloride), 4, 20 or 100 mg/kg bw/day. No adverse effects on maternal toxicity, reproductive parameters, or on development of offspring, were observed at any dose, resulting in a NOAEL of 100 mg/kg bw/day
Executive summary:

The potential of a solution of Tetraamminepalladium dichloride to adversely affect the development of 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 (in aqueous ammonium-chloride buffer) by oral gavage. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post mating). Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). Three dose groups (4, 20, 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. 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.

 

There were no signs of maternal toxicity or developmental toxicity effect at any dose level. In conclusion, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for developmental toxicity was considered to be 100 mg/kg bw/day (the highest tested dose).