Diamminedichloropalladium

Repeated dose toxicity: oral

Administrative data

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:

22 April - 6 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

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

Constituent 1

Reference substance name:

Diamminedichloropalladium

EC Number:

238-269-3

EC Name:

Diamminedichloropalladium

Cas Number:

14323-43-4

Molecular formula:

Cl2H6N2Pd

IUPAC Name:

Diamminedichloropalladium

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Diamminedichloropalladium (II)
- Physical state: solid
- Analytical purity: >99.5%
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: 50.35% w/w palladium
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: 10912
- Expiration date of the lot/batch: 28 August 2014
- Stability under test conditions: No data
- Storage condition of test material: 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, at least 10 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: Males: 364 g – 416 g, Females: 224 g - 266 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 "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest Germany) as pellets and with, or without DDP (treated or control groups, respectively).
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption was provided from 500 ml bottle ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 24.5°C (target range 22±3°C)
- Humidity (%): 38 - 70 % (target range 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 April 2014 To: 6 June 2014

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: A standard ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” was used on this study as a vehicle.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Not applicable

DIET PREPARATION: Diamminedichloropalladium was incorporated into ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, at concentrations of 1500, 4500 and 12 000ppm. Each diet concentration was prepared as one batch of approximately 40 kg/concentration. The production and sampling procedure took place on 01 April 2014.

Production of DDP containing diet was as follows. Premixtures were produced by mixing an aliquot of the basal diet with the respective amount of the Diamminedichloropalladium; the premixtures were of 800 g for low and mid concentration and 2000 g for high concentration. These premixtures were then mixed with the remaining amount of the basal diet. After the mixing procedure the diets were pelleted. Pellets were prepared by simple compression; no binding agents, steam, external heat, or any other process or substance were used that might affect the test item or the quality of the diets. Similar diet preparation procedures were used to generate control diet (0 mg DDP/kg diet).

The prepared diets were stored at room temperature under dry conditions, in sewn bags pending and during transport to CiToxLAB Hungary Ltd. At CiToxLAB Hungary Ltd., the prepared diets were stored in areas designated for diet storage at room temperature, under dry conditions, pending transfer to the animal room at approximately 22±3°C during or prior to animal feeding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During and after the pelleting process, samples were taken for the validation and homogeneity tests, and as back up for analysis if required: 3 samples of about 30 g each from the control diet for validation; triplicate samples after 1 kg (“start”), 10 kg, 20 kg, and 40 kg (“end”) of 30 g each from diets containing DDP for the homogeneity test, and additional 7 samples (triplicate) of 30 g from the low and high concentrations for the validation.

Analysis of the diets for homogeneity and/or concentration of diamminedichloropalladium was performed at the Test Site using a validated ICP/AES method to determine the palladium content. The analytical method was validated in the concentration range of 2200 and 4400ppm as part of the 14-Day palatability study in rats (CiToxLAB study code 13/147-100PE, Test Site Phase code: CTL36).

Before analysis of the DDP content in the study diets, method validation was extended to cover the actual low and high dose diet concentrations used, which was 1500ppm and 12000ppm, respectively. Analysis of palladium concentration was performed from three representative samples at each concentration level and one control sample.

The diet samples were delivered together with a verified sample list stating the concentration, sample mass and sampling date.

Stability of diamminedichloropalladium in rodent diet was confirmed prior to this study by Fourier-transformation infrared spectroscopy (FT-IR), with reassurance that the chemical structure of the test item was not affected by diet matrix incorporation. The test item has a characteristic FT-IR spectrum that can be used to identify any chemical changes. The investigation was performed using powdered rodent diet (Ssniff Spezialdiäten GmbH) at concentration of 10000ppm, the minimal dietary concentration considered to give adequate results by IR analysis. (CiToxLAB Study Code: 13/147-905ANA, Non-GLP investigation).

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.

Females were fed test diet for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females of the High dose group were terminated following a 28-day treatment period (i.e. pre-mating and mating periods) due to the severity of the effect evident as body weight loss and disturbance of the oestrus cycle. In addition, positive signs of mating were observed in 2/12 females only. The high dose males were terminated at the same time.

Satellite females (toxicology endpoints) were treated similarly to the Main males, from Days 0 to 27 and were subjected to necropsy on Day 28.

Frequency of treatment:

A constant concentration of DDP in diet was administered to treated groups. Animals in Groups 2 to 4 were fed DDP-treated diet; Group 1 animals (control) were fed the control diet and handled in an identical manner to those in the test groups.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
121 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
373 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
831 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Main groups: 12 animals/sex /group, 4 groups. Animals originated from different units, to avoid brother/sister mating.

Satellite toxicology groups: 5 females/group, 4 groups

Control animals:

yes, plain diet

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, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Feed concentrations of 1500, 4500 and 12000 ppm were selected for the three treatment groups, in order to achieve target dose levels of 100, 300 and 1000 mg/kg bw/day, respectively.
- Rationale for selecting satellite groups: Assessment of toxicology endpoints
- Post-exposure recovery period in satellite groups: Necropsied 1 day after cessation of treatment
- Section schedule rationale (if not random): Not applicable

Positive control:

None

Examinations

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.

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 weekly in the morning. 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).

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment and during week 4
- Dose groups that were examined: All males and satellite females before treatment, and in the control group and high dose group males and satellite females, during week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes (Pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: 5 males/group (subgroup B) and all satellite females
- Parameters in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected immediately prior to scheduled necropsy.
- Animals fasted: Yes, overnight
- How many animals: 5 males/group (subgroup B) and all satellite females
- Parameters in table [2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (16 hours)
- Parameters in table [3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last week of treatment
- Dose groups that were examined: 5 males/group (subgroup A) and all satellite females
- Battery of functions tested: Selected animals were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind limb grip strength, performed on Day 24 for males and Day 26 for females. Motor activity was measured during the last week of treatment.

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 of the hind paws was measured.

Fore/hind limb grip strength measurements were conducted 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 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support; results are tabulated with individual and mean data.

Sensory reactivity to different type of stimuli (auditory, visual and proprioceptive) was investigated, qualitative assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were 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.

For observations including piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, tonic convulsions, diarrhoea, diuresis, salivation, lachrymation and vocalisation, the scoring system was 0 if absent or 1 if present. For reflexes including pinch responses, the scoring system was 0 for normal or -1 if absent. For the grasping and righting reflexes the scoring system was 0 for normal with -1 and -2 for slight and absent responses, respectively.

The respiration rate, startle, spatial locomotion, finger approach, finger withdrawal, visual placing, grip strength, body tone, positional struggle, skin, mucous membrane colour, limb tone and abdominal tone were scored as follows: -1 (decreased), 0 (normal), +1(increased). For locomotor activity, transfer arousal and touch escape response, the following scoring system was used: -2 (low), -1 (somewhat low), 0 (normal), +1 (somewhat high), +2 (high).

Motor activity assessment was conducted using the Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour during which a 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. The data from the high dose and control groups were evaluated for distance travelled in 5 minute segments.

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.

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.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows: on the retained organs in the control and high dose groups (subgroup A males and toxicology satellite females); all macroscopic findings (abnormalities) from all animals (were investigated at mid dose level in main group animals as follows: stomach of 9 females and adrenals of one male); on the retained reproductive organs of all animals of the control, mid and high dose group; adrenal gland, stomach, spleen, thymus, kidney, liver, pancreas, male’s mammary gland, vagina and heart from mid dose (subgroup A males and toxicology satellite females); stomach from the low dose animals (subgroup A males and toxicology satellite females)

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Marked body weight loss in the highest tested dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption decreased in the highest tested dose group

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

On day 28, a marked decrease was observed in lymphocyte and haemoglobin counts in both sexes at the highest tested dose. The former caused significant changes in the percentage distribution of the cells (p<0.01 except absolute lymphocyte values).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Differences observed in the high dose group include a decrease in total protein concentration, increase in alanine amino transferase, decrease in creatine and an increase in urea and serum chloride concentrations.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

lower urine volume and higher specific gravity measured in the highest dose males. Urine had a lower pH and was often opaque and dark. Leucocytes and bilirubin were found.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Minor differences seen in some grip-strength measures. Attributable to the malnutrition of the animals. Decreased total travel distance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower weights for almost all organs were noted in animals in the High dose groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thick and multifocal pale discoloration of glandular mucosa in the stomach observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the highest tested dose group, mild degenerative/atrophic changes affected the adrenal, epididymides, prostate, seminal vesicles, male mammary gland, uterus, ovary, spleen, thymus, liver and pancreas.

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality during the study.

Clinical signs were limited to brownish/black faeces in all animals in the mid and high dose groups from day 4. Piloerection was recorded during the last detailed observation for high dose satellite females on day 28 only.

BODY WEIGHT AND WEIGHT GAIN
Marked body weight loss noted in both sexes at the highest tested dose during the entire treatment period. On day 27, the mean body weight loss was approximately 19% of the initial body weight for males and 12% and 17% in females (main and satellite groups, respectively).

Lower body weight gain values were recorded in males and satellite females of the mid dose (373 mg/kg bw/day) group at the beginning of the treatment period (0-3 days). The difference was statistically significant (p<0.01) for the males only. Thereafter the body weight gains were similar, with the exception of higher values recorded for satellite females between days 3-7 and 14-17 (p<0.05).

The overall body weight gain values (days 0-27 in males and satellite females) did not differ significantly from the control. The mean body weight and body weight gain values of the mid dose level (373 mg/kg bw/day) main group females did not differ significantly from the control, with the exception of lower body weight gain values recorded at the beginning of the treatment period (not statistically significant) and between GD17-20 (p<0.05).

Body weights and body weight gain in the low dose group (121 mg/kg bw/day) were comparable to the control throughout the study.

FOOD CONSUMPTION
Significantly lower mean food consumption was noted in both males and females of the high dose group (831 mg/kg bw/day) during the entire treatment period. Statistical significance was reached in both sexes in the main groups between days 0-14 (p<0.01) and for males from the end of mating to day 27 (p<0.01) and from day 14 to end of mating (p<0.05). No statistical analysis was possible for main females from day 14-27 because of lack of concurrent control.

There was no adverse effect of treatment on food consumption at lower doses.

Statistically higher than control food consumption in all treated groups was attributed to frequently observed food spillage in the cage.

OPHTHALMOSCOPIC EXAMINATION
No test item related changes compared to pre-treatment.

HAEMATOLOGY
At the highest tested dose (831 mg/kg bw/day), a marked decrease was observed in lymphocyte counts in both sexes, which caused significant changes in the percentage distribution of the cells (i.e. decreased lymphocyte counts with concomitant increase in neutrophil granulocyte count in both males and females). A higher neutrophil granulocyte count was also measured in males of the low and mid dose groups. A statistically significant decrease in haemoglobin concentration was observed in males at the highest tested dose. A slight decrease mean corpuscular (erythrocyte) haemoglobin concentration (MCHC) value was noted in all test item treated male groups, although there was no dose response relationship and the differences were within the range of historical control values.

There were no toxicologically significant effects in females at mid or low dose levels. Other differences observed between the control and treated groups, occasionally attaining statistical significance, were regarded as incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.

No effect of treatment on blood clotting parameters, Activated Partial Thromboplastin Time (APTT) or Prothrombin Time (PTT) were observed.

CLINICAL CHEMISTRY
In the highest tested dose group, a decrease in total protein concentration in both males and females was observed corresponding to a decrease in albumin concentration (in females) without changes in the albumin:globulin ratio. This finding was attributed to the poor clinical condition of the animals. An increase in alanine aminotransferase activity (both sexes, p<0.01 in females only), decrease in creatinine (both sexes), increase in urea (females), and increase in serum chloride concentration (both sexes) were also observed in the high dose group.

There were no toxicologically significant effects noted at the mid or low dose levels. Other differences observed between the control and treated groups, occasionally attaining statistical significance were regarded as incidental or individual findings, which were not related to treatment.

URINALYSIS
Urinalysis findings were limited to the high dose animals.

Lower urine volume and slightly higher specific gravity were measured in the high dose males (p<0.01). A slightly lower mean urine pH value was also measured in these animals (p<0.01).

The appearance of the urine of 1/5 male and 3/4 females was opaque dark/dark yellow. In samples from all of these animals, an increased number of leucocytes were found. In all females and in 3/5 males, bilirubin was found in the urine.

No treatment-related effects were noted at the mid or low dose level.

NEUROBEHAVIOUR (Functional Observation Battery)
There were no toxicologically significant changes in the animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or treated groups, performed on days 24 and 26, in males and females, respectively.

The landing foot splay values were comparable with the control mean in all treated groups in males and females.

Minor differences were seen in some grip-strength measures at the high dose level. A slightly lower mean value for the hind limbs of males and the high dose attained statistical significance (p<0.05). Slightly lower values for limb grip strength in females were recorded in the mid and high dose groups (p<0.05 and p<0.01, respectively). Given the low magnitude of the differences and the loss of body weight observed at the high dose level, these responses were considered attributable to the animals' clinical condition and not a specific neurotoxic effect. The slightly lower fore-limb grip strength in females at the mid dose level was attributed to 1 of 5 females. Also, the control value for hindlimb grip strength in females was relatively high.

During the evaluation of motor activity, the total travelled distance was slightly lower in both males and females at the high dose level. The mean values were lower than the controls by approximately 29 and 44%, respectively, when evaluated for the 60 minute duration. The differences attained statistical significance (p<0.05). For males, lower mean values were recorded toward the end of the test, with statistical difference in periods 25-30 and 40-45 minutes (p<0.05).

When evaluated as individual values, significantly lower total travelled distance was noted in 3/5 males.

The total number of rearing was also slightly lower than the control mean at the high dose level, and was attributable to the marked body weight loss and not a specific neurotoxic effect.

In females, the lower total travelled distance values were generally measured at the beginning of the test with statistically significant differences compared to the controls noted for periods 5-20 minutes (p<0.05). Although the patterns of activity were similar to those of the control, lower values were recorded in other monitored parameters (e.g. the total number of rearing, average velocity and total time of moving fast). It should be noted that the individual values recorded for one control female were well above the values of the group mates.

ORGAN WEIGHTS
Compared to controls, significantly lower weights for almost all measured organs were noted in animals in the high dose groups (831 mg/kg bw/day). The differences versus control for absolute values of adrenals, heart, kidneys, liver, spleen and thyroid were in the range of approximately 20 and 40% in females and 16 and 47% in males. Thymus weights were decreased by approximately 50% in both sexes (absolute value). The differences were statistically significant (all organs p<0.01 except for adrenal glands in females, p<0.05).

A significant decrease in relative organ weight was observed in thymus of both males and females (p<0.01 and p<0.05, respectively), uterus (p<0.01), seminal vesicle and prostate (p<0.01 and p<0.05, respectively), compared to the control mean. Relative liver weight was lower than control mean by 18% in males and by 6% in females.

The changes were associated with the marked loss of body weight with consequential microscopic changes.

There were no toxicologically significant effects on organ weights in the mid or low dose groups.

GROSS PATHOLOGY
Macroscopic findings considered related to the test item were observed in the adrenal gland, prostate, seminal vesicle and glandular stomach at the high dose level while only glandular stomach changes were present at the mid dose level. The stomach changes consisted of thick and multifocal pale discoloration of glandular mucosa and were noted in high dose males and satellite females on day 28 as well as in mid and high dose females of the main groups necropsied on PPD5.

Small prostate and seminal vesicle was noted in 8/12 high dose males.

Diffuse bilateral pale discoloration of the adrenals was observed in 3/12 high dose males, and bilateral enlargement of the adrenals was seen in 1/12 mid dose males.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were noted in the adrenal gland, epididymis, prostate, seminal vesicle, male mammary gland, uterus, ovary, spleen, thymus, liver, pancreas and glandular stomach at the high dose level (831 mg/kg bw/day). Microscopic changes were also present in the glandular stomach of rats with macroscopic findings dosed at 373 mg/kg bw/day.

No test item related microscopic findings were noted at the low dose level (121 mg/kg bw/day).

At the top dose, degenerative/atrophic changes affected the adrenal, epididymides, prostate, seminal vesicles, male's mammary gland, uterus, ovary, spleen, thymus, liver and pancreas.

Changes in the adrenal glands were characterised by minimal to mild unilateral/bilateral decreased volume of cortical cells, and reduction of zona fasciculata/reticularis in 3/6 males and 2/5 females. These microscopic changes were seen grossly as pale discoloration in affected male rats.

Lymphoid atrophy was observed in the spleen in 2/5 males (mild severity) and in 3/5 females (minimal to mild severity). Minimal or moderate lymphoid atrophy of the thymus was found in 3/5 males and 1/5 females. There were no macroscopic correlates for these findings. The atrophic changes were in accordance with reduced organ weight and haematology results (decreased lymphocyte counts).

Changes in the liver were mild (males) or minimal to mild (females), diffusely decreased volume of cytoplasm/nuclei of the hepatocytes (with/without hepatocellular degeneration/necrosis or sinusoidal cell infiltrates) and these changes were present in 5/5 males. These microscopic changes correlated with a decreased liver weight, and liver to body weight ratios.

Microscopic changes seen in the pancreas were a minimal decrease of zymogen granules, which was present in 2/5 males and 2/5 females. There was no macroscopic correlate for this observation.

Stomach changes consisted of foreign eosinophilic material adhere to the glandular mucosa and were detected in animals on day 28 (in 4/9 males, 4/5 main group females and 5/5 satellite females). These histopathological findings corresponded with thick/pale discoloured glandular mucosa in affected rats.

At 373 mg/kg bw/day, foreign eosinophilic material was adhered to the glandular mucosa of the stomach in 1/5 males as well as 9/9 females.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

373 mg/kg bw/day (actual dose received)

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

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

2

Dose descriptor:

LOAEL

Effect level:

831 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Marked body weight loss occurred (malnutrition as a result of a reduction in food consumption due to the palatability of the test item in the diet)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mean test item intakes

DDP concentration in diet (mg/kg)	Mean test item intake (mg/kg bw/day)
	Main males	Satellite females	Combined for males and females
1500	109.6	132.4	121
4500	338.5	407.5	373
12000	619.0	1043.2	831

Mean body weight and body weight gain

	Groups/Concentration (ppm)
	Control	Low dose 1500	Mid dose 4500	High dose 12000
Males
Body weight on Day 27 (g)	468.8	474.4	462.1	316.3**	DN
	differences%	1.2	-1.4	-33
Body weight gain between Days 0-27 (g)	79.8	85.7	72.8	73.0**	DN
	differences%	7.4	-8.7	-192
Satellite Females
Body weight on Day 27 (g)	275.4	303.0*	286.6*	210.2**	DN
	differences%	10	4.1	-24
Body weight gain between Days 0-27 (g)	23.4	45.6**	34.0	-43.0**	DN
	differences%	95	45	-284
Main Females
Body weight on Day 14 (g)	262.6	260.8	265.7	217.6**	DN
	differences%	-0.7	1.2	-17
Body weight gain between Days 0-14 (g)	17.4	21.8	16.3	-33.3**	U
	differences%	25	-6.2	-291
Body weight on Day PP4 (g)	344.5	352.9	337.8	-	NS
	differences%	2.4	-2.0
Body weight gain between Days 0-PP4 (g)	99.1	112.2	88.3	-	NS
	differences%	13	-11

* =p<0.05; ** = p<0.01  

DN = Duncan's Multiple Range Test; U = Mann-Whitney U – test; NS = not significant

differences% = % differences vs. control

Applicant's summary and conclusion

Conclusions:

In an OECD Test Guideline 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study, to GLP, diamminedichloropalladium was administered to rats at dietary concentrations of 1500, 4500 and 12000 ppm (equivalent to doses of around 121, 373 and 831 mg/kg bw/day) for at least 28 days. A NOAEL of 373 mg/kg bw/day for general systemic toxicity was determined, due to the marked bodyweight loss observed at the highest tested dose (likely a consequence of the local effects in the glandular stomach combined with the poor test item palatability in the diet, and concomitant reduced food consumption)

Executive summary:

In an OECD Test Guideline 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study, diamminedichloropalladium was administered to rats (12/sex/group) at dietaryconcentrations of 1500, 4500 and 12000 ppm, equivalent to doses of around 121, 373 and 831 mg/kg bw/day. Males and high dose females were treated for 28 days, while low and mid dose females were treated up to postnatal day 5 (i.e. around 50 days in total). Additional females (5/group), assigned to satellite groups for toxicology endpoints, were treated for 28 days.

At the highest tested dose, animals displayed a marked body weight loss as a consequence of reduced food consumption, probably due to the local effects in the glandular stomach combined with the poor test item palatability in the diet. No general toxicity was observed in the low and mid dose groups (121 and 373 mg/kg bw/day respectively). Although some evidence of local toxicity was apparent in the glandular mucosa of the stomach at 373 mg/kg bw/day, this was not associated with any microscopic changes. Hence, the systemic no-observed-adverse-effect level (NOAEL) for diamminedichloropalladium was considered to be 373 mg/kg bw/day, due to the marked bodyweight loss observed at the highest tested dose.