Palladium

Endpoint summary

Description of key information

No relevant repeated dose toxicity data were identified for palladium.

 

Palladium is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and palladium dihydroxide'.

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 NOAEL was the highest tested dose (1000 mg/kg bw/day). Although some treatment related microscopic findings (mucosal discoloration in the non-glandular stomach, ileum, cecum, colon and/or rectum) were noted at dose levels of 300 and 1000 mg/kg bw/day, these were considered to result from direct (local) contact with the test substance rather than systemic toxicity (Török-Bathó, 2015). The oral NOAEL for palladium dihydroxide (1000 mg/kg bw/day) equates to an NOAEL of 758 mg/kg bw/day for palladium (based on MWt ratio).

No repeated dose toxicity studies by the inhalation or dermal route were identified, or are required.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2014 to 23 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Reason / purpose for cross-reference:

reference to same study

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

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)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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
- Purity: no data

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 acceptable stability 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 (i.e. around 50 days in total).

Frequency of treatment:

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

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: Nominal concentration

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.

Observations and examinations performed and frequency:

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals:
- Parameters in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters in table [2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters in table [3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
5 males and 5 females/group, “subgroup A”: Assessment of any potential test item related neurotoxicity was performed in the morning and prior to dosing, during the last exposure week (males on Day 24; females on PPD 4). Selected animals were subjected to the functional observation battery, including quantitative assessment of grip strength (manual and instrumental), and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessments of motor activity were performed.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured.

Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed at least 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data.
Manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were conducted and the general physical condition and behaviour of animals was tested. A modified Irwin test was performed.

Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system, where score “0” is given when the behaviour or reaction of the animal is considered normal, and -1 or -2, or +1 and +2 is given if the response is less, or heightened than expected in an untreated animal..

Quantitative assessment of motor activity was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity were monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings are made to produce the appropriate parameters. Obtained data was evaluated for distance travelled in 5 minute segments.


OTHER:

Sacrifice and pathology:

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

At the time of termination, body weight and the weight of the following organs from all adult animals were determined:

- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids

Paired organs were weighed together except testes and epididymides which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised.

The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10% buffered formalin solution.

The tissues and organs in Table 4 were retained from all animals.

For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• all macroscopic findings (abnormalities), except of minor order from all animals
• 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 retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

As no test item related pathology findings were noted, no additional histopathology evaluation was considered required.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During the study, two adult females were found dead. One female dosed at 300 mg/kg bw/day was found dead on GD20. On the previous day, this female showed decreased activity, hunched back and piloerection. Loss of body weight (15g) from day 17 was noted. Suppurative meningitis, pericarditis and pyometra were the cause of death for this female possibly related to an undetected partial misdosing leading to congestion / haemorrhage of the lungs. The uterus contained only intra-uterine deaths (13 in total). The second female dosed at 1000 mg/kg bw/day died on GD23. The cause of death was attributed to marked focal necrosis of the glandular stomach. Although this death occurred at the highest dose tested, it was an isolated incidence. Furthermore, the dose level of 1000 mg/kg bw/day was not associated with changes in clinical condition or with mortality in the dose range finding study (13/193-100PE). It is therefore concluded that the death of this female was not clearly related to treatment with the test-item.

No test item-related adverse effects or systemic clinical signs were noted following daily administration of the test item by oral gavage.

Black faeces were observed in Mid and High dose animals from Day 6 or Day 2, respectively.

In Mid dose females, skin paleness (Days 37 and 38) and slightly decreased activity (Days 37-41) in one animal were considered to be incidental.

BODY WEIGHT AND WEIGHT GAIN
No test item related effects were noted on the mean body weight and body weight gain values following daily administration of Palladium dihydroxide at dose levels up to and including 1000 mg/kg bw/day.

Occasional statistically significant differences were regarded as incidental and without toxicological significance.

FOOD CONSUMPTION
There were no test item-related differences in the mean daily food consumption in any test-item treated group when compared to the Control Groups.

Occasional statistically significant differences were regarded as incidental and of no toxicological significance.

HAEMATOLOGY
When compared to the controls, there were no differences that were considered toxicologically significant in the treated animals.

Variations noted for a few haematology parameters in males and females which attained statistical significance and the higher than control activated partial thromboplastin time (APTT) in the Low, Mid and High dose (p<0.05, p<0.01 and p<0.05, respectively showed no dose response.

In female animals statistically higher than control Neutrophils and Lymphocytes were recorded.

The individual values were within the historical control range for all dose groups. Evaluation of the mean and individual results in comparison with the control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental and of no toxicological significance.

CLINICAL CHEMISTRY
There were no toxicologically significant changes or adverse effects on serum chemistry that could be ascribed to Palladium dihydroxide.

A few clinical chemistry parameters in females were occasionally statistically significant, but were considered not to be toxicologically significant or related to treatment.

URINALYSIS
There was no effect of treatment noted during urinalysis.

All values were within the normal range and showed no dose or gender-dependent relationship, and were considered not to be of toxicological importance.

NEUROBEHAVIOUR
There were no treatment related effects.

There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.

There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total travelled distance was comparable to control for both sexes.

ORGAN WEIGHTS
Compared to controls, the absolute and relative weights of liver were slightly increased up to 13% in Mid and High dose groups, in females (evaluated on PPD4). The differences at 1000 mg/kg/day group attained statistical significance for absolute and brain related mean values (p<0.05). The changes were not associated with any findings in clinical pathology or microscopic changes and were considered to be of no toxicological significance.

Compared to controls, slightly lower weights of seminal vesicles were recorded for all treated males. The difference was approximately 6-18% and attained statistical significance (p<0.05 in Low and High dose) for absolute values and/or body weight and brain weight related mean values. These changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation and incidental to treatment.

Other variations noted in the absolute and/or relative organ weights adjusted for the terminal body or brain weight when compared to Controls, on occasion attaining statistical significance were noted in males and females (i.e. in right epididymis or testis in males and heart or adrenal weight in females).

However, in absence of any dose or gender response, or of any clinical pathology, macroscopic or microscopic changes, these variations were considered not to be related to treatment.

GROSS PATHOLOGY
Test item-related macroscopic findings were observed in the digestive contents of the cecum, colon and/or rectum, and in the non-glandular stomach mucosa at dose levels of 300 and 1000 mg/kg bw/day.

Black discoloration (corresponding to test item) of the digestive contents (caecum, colon and/or rectum) was observed in 6/23 Mid Dose and 22/23 High Dose animals. Brown discoloration of the non-glandular stomach mucosa was seen in 2/23 and 15/23 rats from the Mid Dose and High Dose groups, respectively.

Other changes were considered to be incidental or a common background.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related microscopic findings were noted in the non-glandular stomach mucosa, ileum, cecum, colon and/or rectum at dose levels of 300 and 1000 mg/kg.

In the non-glandular stomach mucosa, black granular foreign material, adhering to the mucosa, was observed in 2/2 Mid Dose rats, examined due to the presence of gross lesions, and in 15/18 High Dose animals. The adhered/luminal black granular foreign material also appeared in the ileum (2/10), caecum (7/10), colon (2/10) and rectum (1/10) from the High Dose groups.

No test item-related microscopic changes were noted in the reproductive organs, at a dose level of 1000 mg/kg bw/day.

Other changes were incidental or considered to be a common background.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

not specified

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 NOAEL was the highest tested dose (1000 mg/kg bw/day). Although some treatment related microscopic findings (mucosal discoloration in the non-glandular stomach, ileum, cecum, colon and/or rectum) were noted at dose levels of 300 and 1000 mg/kg bw/day, these were considered to result from direct (local) contact with the test substance rather than systemic toxicity.

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 (i.e. around 50 days in total).

 

Daily administration of palladium dihydroxide by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment, or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of up to 1000 mg/kg bw/day during the treatment period. There were no adverse treatment-related changes observed in organ weights for the adult animals of either sex.

 

There were no changes observed at histopathology considered indicative of treatment-related systemic toxicity. Further, no test item-related microscopic changes were noted in the reproductive organs.

 

Test item-related black discoloration of the digestive contents or brown mucosal discoloration in the non-glandular stomach, ileum, caecum, colon and/or rectum at necropsy, corresponded with black granular foreign material noted by light microscopy. These changes were considered to result from direct contact with the test item (i.e. local effects) and not to systemic toxicity. 

 

In conclusion, in this guideline GLP study, the systemic no-observed-adverse-effect level (NOAEL) of palladium dihydroxide was 1000 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Study period:

10 June 2014 to 23 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Justification for type of information:

Substance considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'

Reason / purpose for cross-reference:

read-across source

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

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)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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
- Purity: no data

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 acceptable stability 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 (i.e. around 50 days in total).

Frequency of treatment:

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

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: Nominal concentration

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.

Observations and examinations performed and frequency:

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals:
- Parameters in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters in table [2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters in table [3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
5 males and 5 females/group, “subgroup A”: Assessment of any potential test item related neurotoxicity was performed in the morning and prior to dosing, during the last exposure week (males on Day 24; females on PPD 4). Selected animals were subjected to the functional observation battery, including quantitative assessment of grip strength (manual and instrumental), and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessments of motor activity were performed.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured.

Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed at least 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data.
Manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were conducted and the general physical condition and behaviour of animals was tested. A modified Irwin test was performed.

Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system, where score “0” is given when the behaviour or reaction of the animal is considered normal, and -1 or -2, or +1 and +2 is given if the response is less, or heightened than expected in an untreated animal..

Quantitative assessment of motor activity was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity were monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings are made to produce the appropriate parameters. Obtained data was evaluated for distance travelled in 5 minute segments.


OTHER:

Sacrifice and pathology:

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

At the time of termination, body weight and the weight of the following organs from all adult animals were determined:

- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids

Paired organs were weighed together except testes and epididymides which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised.

The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10% buffered formalin solution.

The tissues and organs in Table 4 were retained from all animals.

For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• all macroscopic findings (abnormalities), except of minor order from all animals
• 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 retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

As no test item related pathology findings were noted, no additional histopathology evaluation was considered required.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During the study, two adult females were found dead. One female dosed at 300 mg/kg bw/day was found dead on GD20. On the previous day, this female showed decreased activity, hunched back and piloerection. Loss of body weight (15g) from day 17 was noted. Suppurative meningitis, pericarditis and pyometra were the cause of death for this female possibly related to an undetected partial misdosing leading to congestion / haemorrhage of the lungs. The uterus contained only intra-uterine deaths (13 in total). The second female dosed at 1000 mg/kg bw/day died on GD23. The cause of death was attributed to marked focal necrosis of the glandular stomach. Although this death occurred at the highest dose tested, it was an isolated incidence. Furthermore, the dose level of 1000 mg/kg bw/day was not associated with changes in clinical condition or with mortality in the dose range finding study (13/193-100PE). It is therefore concluded that the death of this female was not clearly related to treatment with the test-item.

No test item-related adverse effects or systemic clinical signs were noted following daily administration of the test item by oral gavage.

Black faeces were observed in Mid and High dose animals from Day 6 or Day 2, respectively.

In Mid dose females, skin paleness (Days 37 and 38) and slightly decreased activity (Days 37-41) in one animal were considered to be incidental.

BODY WEIGHT AND WEIGHT GAIN
No test item related effects were noted on the mean body weight and body weight gain values following daily administration of Palladium dihydroxide at dose levels up to and including 1000 mg/kg bw/day.

Occasional statistically significant differences were regarded as incidental and without toxicological significance.

FOOD CONSUMPTION
There were no test item-related differences in the mean daily food consumption in any test-item treated group when compared to the Control Groups.

Occasional statistically significant differences were regarded as incidental and of no toxicological significance.

HAEMATOLOGY
When compared to the controls, there were no differences that were considered toxicologically significant in the treated animals.

Variations noted for a few haematology parameters in males and females which attained statistical significance and the higher than control activated partial thromboplastin time (APTT) in the Low, Mid and High dose (p<0.05, p<0.01 and p<0.05, respectively showed no dose response.

In female animals statistically higher than control Neutrophils and Lymphocytes were recorded.

The individual values were within the historical control range for all dose groups. Evaluation of the mean and individual results in comparison with the control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental and of no toxicological significance.

CLINICAL CHEMISTRY
There were no toxicologically significant changes or adverse effects on serum chemistry that could be ascribed to Palladium dihydroxide.

A few clinical chemistry parameters in females were occasionally statistically significant, but were considered not to be toxicologically significant or related to treatment.

URINALYSIS
There was no effect of treatment noted during urinalysis.

All values were within the normal range and showed no dose or gender-dependent relationship, and were considered not to be of toxicological importance.

NEUROBEHAVIOUR
There were no treatment related effects.

There were no toxicologically significant changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.

There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total travelled distance was comparable to control for both sexes.

ORGAN WEIGHTS
Compared to controls, the absolute and relative weights of liver were slightly increased up to 13% in Mid and High dose groups, in females (evaluated on PPD4). The differences at 1000 mg/kg/day group attained statistical significance for absolute and brain related mean values (p<0.05). The changes were not associated with any findings in clinical pathology or microscopic changes and were considered to be of no toxicological significance.

Compared to controls, slightly lower weights of seminal vesicles were recorded for all treated males. The difference was approximately 6-18% and attained statistical significance (p<0.05 in Low and High dose) for absolute values and/or body weight and brain weight related mean values. These changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation and incidental to treatment.

Other variations noted in the absolute and/or relative organ weights adjusted for the terminal body or brain weight when compared to Controls, on occasion attaining statistical significance were noted in males and females (i.e. in right epididymis or testis in males and heart or adrenal weight in females).

However, in absence of any dose or gender response, or of any clinical pathology, macroscopic or microscopic changes, these variations were considered not to be related to treatment.

GROSS PATHOLOGY
Test item-related macroscopic findings were observed in the digestive contents of the cecum, colon and/or rectum, and in the non-glandular stomach mucosa at dose levels of 300 and 1000 mg/kg bw/day.

Black discoloration (corresponding to test item) of the digestive contents (caecum, colon and/or rectum) was observed in 6/23 Mid Dose and 22/23 High Dose animals. Brown discoloration of the non-glandular stomach mucosa was seen in 2/23 and 15/23 rats from the Mid Dose and High Dose groups, respectively.

Other changes were considered to be incidental or a common background.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related microscopic findings were noted in the non-glandular stomach mucosa, ileum, cecum, colon and/or rectum at dose levels of 300 and 1000 mg/kg.

In the non-glandular stomach mucosa, black granular foreign material, adhering to the mucosa, was observed in 2/2 Mid Dose rats, examined due to the presence of gross lesions, and in 15/18 High Dose animals. The adhered/luminal black granular foreign material also appeared in the ileum (2/10), caecum (7/10), colon (2/10) and rectum (1/10) from the High Dose groups.

No test item-related microscopic changes were noted in the reproductive organs, at a dose level of 1000 mg/kg bw/day.

Other changes were incidental or considered to be a common background.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

not specified

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 NOAEL was the highest tested dose (1000 mg/kg bw/day). Although some treatment related microscopic findings (mucosal discoloration in the non-glandular stomach, ileum, cecum, colon and/or rectum) were noted at dose levels of 300 and 1000 mg/kg bw/day, these were considered to result from direct (local) contact with the test substance rather than systemic toxicity.

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 (i.e. around 50 days in total).

 

Daily administration of palladium dihydroxide by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment, or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of up to 1000 mg/kg bw/day during the treatment period. There were no adverse treatment-related changes observed in organ weights for the adult animals of either sex.

 

There were no changes observed at histopathology considered indicative of treatment-related systemic toxicity. Further, no test item-related microscopic changes were noted in the reproductive organs.

 

Test item-related black discoloration of the digestive contents or brown mucosal discoloration in the non-glandular stomach, ileum, caecum, colon and/or rectum at necropsy, corresponded with black granular foreign material noted by light microscopy. These changes were considered to result from direct contact with the test item (i.e. local effects) and not to systemic toxicity. 

 

In conclusion, in this guideline GLP study, the systemic no-observed-adverse-effect level (NOAEL) of palladium dihydroxide was 1000 mg/kg bw/day.

The substance is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Overall, good-quality database which meets REACH Standard Information Requirements.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

No relevant human or laboratory animal repeated dose toxicity data with palladium were identified. 

 

Palladium is considered to be non-bioavailable following oral and dermal exposure, as evidenced by transformation/dissolution (cf. Skaeff 2011, section 4.8) and bio-elution (cf. Rodrigues 2012 a,b,c,d,e, section 7.12) test data. Palladium is not expected to reach the lungs in appreciable quantities (based on respiratory tract deposition modelling data - cf. Parr & Selck, section 4.5). Thus, inhalation will not be a significant route of exposure. Since a chemical is required to be bioavailable in order to induce systemic toxicity following repeated exposure, palladium is not considered to pose a systemic toxicity hazard. Finally, for animal welfare reasons, and considering that palladium is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide' with reliable experimental test data available for palladium dihydroxide, conducting new in vivo toxicity tests with palladium is considered as a last resort. Consequently, no testing for repeated dose toxicity of palladium is considered justified.

 

Justification for classification or non-classification

No repeated dose toxicity data are available for palladium. However, such effects are not expected, based on a lack of bioavailability following exposure via the oral, dermal and inhalation routes. Also, palladium is considered to fall within the scope of the read-across category 'Palladium, Palladium monoxide and Palladium dihydroxide'. Based on the test data with palladium dihydroxide and the expected lack of bioavailability of palladium, there is no evidence to classify it as STOT-RE according to EU CLP criteria (EC 1272/2008).