Palladium (II) di(4-oxopent-2-en-2-oate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Remarks:

Read-across from study conducted on an analogue, palladium di(4-oxopent-2-en-2-oate)

Adequacy of study:

key study

Study period:

10 June 2014 to 23 July 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP, on closely-related surrogate

Justification for type of information:

Palladium di(4-oxopent-2-en-2-oate) is considered to be a suitable read-across analogue. See section 13 for full justification report.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Palladium dihydroxide

EC Number:

235-219-2

EC Name:

Palladium dihydroxide

Cas Number:

12135-22-7

Molecular formula:

H2O2Pd

IUPAC Name:

palladium(2+) dihydroxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Palladium dihydroxide
- Physical state: Brown powder
- Analytical purity: Pd content 71.9% w/w, which represents 94.9% w/w Pd(OH)2
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 9045/00-F3
- Expiration date of the lot/batch: 14 June 2015
- Stability under test conditions: acceptable stability for 14 days at room temperature
- Storage condition of test material: Controlled room temperature (15-25 deg C below 70 RH%)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

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, approximately 11 weeks old at starting and 13 weeks at mating
- Weight at study initiation: Males: 355 g – 413 g, Females: 222 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
- Fasting period before study:
- Housing: Type II and/or III polypropylene/polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- 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 ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 24.6 °C (target range 22±3°C)
- Humidity (%): 33 – 66 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily

IN-LIFE DATES: From: 10 June 2014 To: 26 July 2014 (last necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance has low solubility in water (0.000001 g/l)
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml (for control, low-, mid-, and high dose levels)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): MKBQ9948V / MKBP7039V

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days (all pairs mated within the first 5 days of the mating period) .
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared weekly or biweekly, based on the stability assessment results. Stability of the test item in the selected vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to 13/193-929AN).

Analysis of Palladium dihydroxide formulation samples of 1 – 200 mg/mL concentration range showed no decrease of concentration and was consideredacceptable stabilityle for 14 days at room temperature. The infrared spectra both in the liquid phase and the solid phase (in CsI disk) showed no presence of degradation products and support the stability of the test item in corn oil for 14 days (according to 13/193-929AN).

Analysis of test item formulations for concentration and homogeneity was performed in the Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken and analysed from test item formulations and all concentrations. Sample analysis was made on 2 occasions for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.

Formulations prepared on Day 0 (for use over 7 days), were sampled and sent to the Test Site for rapid formulation analysis to provide confirmation on formulation concentration and homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.

Description of the analytical method and the results of the formulation analysis are included in an analytical Phase report, provided by the Principal Investigator in Appendix 4 of the study report.

Duration of treatment / exposure:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4

Frequency of treatment:

Test item or negative control treated animals were administered the dosing formulations daily on a 7 days/week basis

Details on study schedule:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

12 animals/sex/group, 4 groups (48 male, 48 female rats)

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 and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

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

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. 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 were 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, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weight of the female animals was additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (on the days of body weight measurement).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

The following parameters parameters were also evaluated, as detailed in 7.5.1 Repeated dose toxicity: oral:
Haematology
Clinical chemistry
Urinalysis
Neurobehavioural examination

Oestrous cyclicity (parental animals):

Oestrous cycle assessed during the mating period, until a sperm positive vaginal smear or a vaginal plug was identified

Sperm parameters (parental animals):

Testis sperm content assessed. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure (see 'Postmortem exaimnations (Paranteral animals)' below for further details).

Litter observations:

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Data were recorded to provide information on:
- Mean pup body weight (per pup within the group and per litter) on PND 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4
- Survival Index of pups on postnatal Days 0 and 4
- Sex ratio % (on postnatal Days 0 and 4)

Postmortem examinations (parental animals):

Surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Special attention was paid to the organs of the reproductive system. Notably, a detailed histological examination of the reproductive organs of all animals of the control and high dose group and of all males (testes, epididymides, prostate gland, seminal vesicles with coagulation gland and preputial gland) that failed to sire and all females (uterus, cervix, ovary, oviduct and vagina for females) that failed to deliver healthy pups.

The number of implantation sites and of corpora lutea were recorded in the females as applicable.

The weight of a range of organs was determined, including those of the reproductive system (including the ovaries and uterus (including cervix) for the females, and the testis, epididymis, seminal vesicles and prostrate gland weights for the males).

Postmortem examinations (offspring):

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). 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. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Reproductive indices:

See Table below: Formulas for Calculation of Mating and Fertility Indices

Offspring viability indices:

See Table below: Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed. The numbers of implantation sites and corpora lutea were also unaffected by treatment. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Results: F1 generation

Details on results (F1)

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or on necropsy observations.

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:

viability

clinical signs

gross pathology

other: Developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide for at least 28 days, the systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day)

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4.

No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed. The numbers of implantation sites and corpora lutea were also unaffected by treatment. Special attention was paid to the organs of the reproductive system. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported.

In conclusion, under the conditions of this study, the systemic and reproductive toxicity no-observed-adverse-effect level (NOAEL) of palladium dihydroxide was 1000 mg/kg bw/day (the highest tested dose).