Diamminedichloropalladium

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: 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: Not applicable
- 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 2914 To: 06 June 2014 (last necropsy)

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 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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred or two weeks elapsed.
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually
- Any other deviations from standard protocol: The mating period for the high dose group was terminated after 14 days, as no evidence of oestrus cycling was observed in the females and only 2 out of 12 showed positive indication of mating.

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 given treated diet for 28 days (14 days pre-mating and 14 days mating/post-mating period), and were then 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.

Details on study schedule:

One generation study. Pups sacrificed on PND4.
- Age at mating of the mated animals in the study: At least 12 weeks

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
At the high dose, the achieved dose level was lower than the target dose of 1000 mg/kg bw/day probably due to the poor palatability of the diet

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. The palatability of the ssniff rodent diet containing DDP was investigated at 2200 and 4400 ppm (mg/kg diet) for 14 consecutive days (CiToxLAB study code 13/147-100PE).

The dietary levels of 1500, 4500 and 12,000 ppm were selected with the aim of achieving doses of at least 100 and 300 mg/kg bw/day in the Low and Mid dose groups and approximately 1000 mg/kg bw/day in the High dose group.

Animal food consumption per cage was measured twice a week and the mean daily feed intake per rat was calculated. Based on food consumption data, the mean dose level of each group was calculated for reporting purposes on the basis of mg/kg body weight/day.

The oral route was selected as it is one of the possible routes of human exposure.

- Rationale for animal assignment (if not random): Not applicable

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily.

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 AND COMPOUND INTAKE (if feeding study):
Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g twice a week (on the days of body weight measurements).

Oestrous cyclicity (parental animals):

Prior to necropsy, the stage of the oestrus cycle of all main females was determined by taking a vaginal smear, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Sperm parameters (parental animals):

Gross necropsy was performed on each animal, terminally one day after the last treatment. Special attention was paid to the organs of the reproductive system. Detailed histological examination was performed on the reproductive organs of all animals in the control and high dose groups. Special attention was paid to evaluation of the stages os spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Parameters examined in male parental generation: testis weight, epididymis weight, prostate weight, seminal vesicles with coagulating glands weight, sperm count in epididymides

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No data

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. In addition, pups were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (PND0 or PND1) and on PND4, with an accuracy of 0.01g. All the litters were checked daily for the number of viable and dead pups.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and pups euthanized at post-natal day (PND) 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals were sacrificed after a dosing period of 28 days.
- High dose female animals were sacrificed after a dosing period of 28 days due to deteriorating condition and evident changes in the oestrus cycle.
- Dams with offspring were sacrificed on post-partum day (PPD) 5.
- Additional females, assigned to satellite groups for toxicology endpoints were sacrificed after a dosing period of 28 days.

GROSS NECROPSY
- Adult animals were examined macroscopically at sacrifice for any abnormalities or pathological changes, with particular attention given to the reproductive organs.
- The number of implantation sites and corpora lutea were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The weighed organs and all organs showing macroscopic lesions from all adult animals were preserved. In addition, for all main males and all toxicology satellite females the organs and tissues listed in Table 1, or representative samples, were preserved.

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), except of minor order 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 and high dose group

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

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

- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries

From all Main males and from all Toxicology satellite females, the following organs were weighed in addition to the ones previously mentioned:

- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, 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.

Postmortem examinations (offspring):

SACRIFICE
Dead pups and pups killed on PPD4 were carefully examined externally for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS: Not conducted

Statistics:

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

Reproductive indices:

See Table 2: Formulas for Calculation of Mating and Fertility Indices

Offspring viability indices:

See Table 3: Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

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):

Marked body weight loss in the highest tested dose group

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to mild severity

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: (Food consumption decreased in the highest tested dose group.)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Disturbed as a consequence of malnutrition.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (Parenteral animals)
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 FOOD CONSUMPTION (Parenteral animals)
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.

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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The high dose group of females was terminated after 14 days of mating due to body weight loss and visible disturbance of the estrous cycle with positive signs of mating observed in only 2/12 females. Therefore, the evaluation of the reproductive parameters was not possible in this group.

There was no effect of treatment on the estrous cycle or on reproductive parameters at the mid or low dose level.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No reported effects.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no differences between the control and low and mid dose groups with regard to reproductive ability, mating and gestation indices. The mating and fertility indices were 100% in all groups. The gestation index was 92% in control and mid groups and 83% in the low dose group.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation). Mean pre-coital interval was 2.7 days in control and mid groups and 2.6 in the low dose group.

The mean duration of pregnancy was similar in the control and test item treated groups and was 22.1 days in control group and 22.0 days in the low and mid dose groups. All the parturitions were normal.

Compared to control, the number of corpora lutea and number of implantation sites were comparable to the control mean in the low and mid dose groups.

No significant differences were noted in pre/post-implantation loss, intrauterine, post-natal or total mortality values (%) in the low and mid dose groups.

The number of pups born did not differ significantly from the control mean in the low and mid dose groups.

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).

In males, weights of prostate, epididymis and seminal vesicles were decreased by approximately 50, 20 and 60% of control, respectively. In females, weights of ovaries and uterus were decreased by approximately 30 and 60%. All values attained statistical significance (p<0.01 and p<0.05 for ovaries). No differences were noted in absolute weights of testis.

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.

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 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.

Epididymis, prostate, seminal vesicle, mammary gland: the changes seen in these organs were minimal to mild reduction of epididymal sperm content (caput/body) in 6/12 and mild ductal atrophy in 1/12 males; minimal to moderate acinar atrophy of prostatic dorsolateral/ventral lobes in 8/12 males; minimal to moderate unilateral/bilateral atrophy of seminal vesicle in 10/12 males, were microscopically detected. Affected epididymides and seminal vesicles appeared small at necropsy but there were no macroscopic correlates for the changes in the mammary and prostate glands.

Uterus, ovary: the changes seen in these organs were mild to moderate atrophic myometrium and low glandular and luminal epithelia in 12/12 females in the uterus; minimal to moderate unilateral/bilateral decreased number of primary/secondary/tertiary follicles and corpora lutea in 11/12 females were observed in the ovary. With respect to these atrophic changes in the reproductive organs, only one main female (number 4505) achieved pregnancy.

Changes in the adrenal glands were characterized 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 adhered 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.

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.

Effect levels (P0)

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:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

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: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The number of viable pups on PND4 and the pup survival indices on PND0 and PND4 in the low and mid dose groups were comparable to control values.

The number of male and female pups was similar in the control and in the low and mid dose groups.

CLINICAL SIGNS (OFFSPRING)
A few pups were cannibalised and the incidence was similar to the control.

Lack of suckling was observed in a few pups with a similar incidence in the control.

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

SEXUAL MATURATION (OFFSPRING)
Not examined.

ORGAN WEIGHTS (OFFSPRING)
Not examined.

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

HISTOPATHOLOGY (OFFSPRING)
Not examined.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

373 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Overall reproductive toxicity

Reproductive 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 weights 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 a guideline (OECD TG 422) combined repeated dose and reproductive/developmental toxicity study, 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. Disruption to the estrous cycle was apparent in females at the highest tested dose, as a result of a marked bodyweight loss. No evidence of reproductive toxicity was observed at lower doses. The NOAEL of diamminedichloropalladium for reproductive toxicity was 373 mg/kg bw/day.

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 dietary concentrations 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 dosed for 14 days pre-mating, throughout the mating period and gestation, 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.

Special attention was paid to the reproductive organs. The numbers of implantation sites and corpora lutea were recorded. Histopathological examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure. A number of reproductive indices were calculated from the collected data (including mating, fertility and gestation indices).

No general toxicity was observed in the low- and mid-dose groups. 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. This weight loss caused disruption to the oestrous cycle of females, thus impacting on the mating performance and conception ability of the hgih dose rats. These animals were terminated on day 28. No test item-related microscopic changes were observed in the reproductive organs in the low- and mid-dose, nor effects on reproductive parameters (including fertility) or indications of maternal/foetal toxicity. Hence, the NOAEL of diamminedichloropalladium for reproductive toxicity was considered to be 373 mg/kg bw/day.