Disodium tetrachloropalladate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20/12/2020 - 9/7/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

13820-53-6

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): sodium chloropalladite
- Substance type: brown powder
- Physical state: solid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Purity test date: no data
- Lot/batch No.: 041118/A
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: supplied in closed glass jar; no further details

Specific details on test material used for the study:

Pd content: 36.11%
Brown-red hygroscopic crystals
Expiry dates: 24 August 2021 / 24 August 2021 (as per CoA on the 2 batches)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl:WI (Wistar) rats (Rattus norvegicus)

Details on species / strain selection:

The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

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, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 11/12 weeks old (females/males) at start of the experiment and 14/15 weeks old (females/males) at mating.
- Weight at study initiation: Males: 389-484 g, females: 242-286 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: not applicable
- Housing:
*Cage type:Type II and/or III polycarbonate
*Bedding / nesting: SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201022 / 03027201024 / 03027201208, Expiry date: 22 October 2023 / 24 October 2023 / 08 December 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200405 /05072200824, Expiry date: 05 April 2023 / 24 August 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Details of the bedding and nesting materials were included in the raw data binder and archived.
*Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Males were individually housed after their mating finished until the end of the study.
*Animal Enrichment: Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities. Nesting material allowed normal nesting behaviour. Certified cardboard hiding tubes (GLP Mini Fun Tunnels, LBS (Serving Biotechnology) Ltd, Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals, details of the quality were documented in the raw data and archived

- Diet (e.g. ad libitum):During acclimatisation, animals were provided with ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. For two weeks before the treatment start, animals received the study control diet to determine the baseline level for food consumption. The supplier provided analytical certificates for the batches used, which were documented in the raw data and archived. The diet was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

- Water (e.g. ad libitum): Animals received tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Quality control analysis of the water was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, Address: H-8200 Veszprém, József Attila u. 36., Hungary). The quality control results are retained in the Archives of the Test Facility, one certified copy is kept and archived in the raw data binder.

- Acclimation period: Environmental acclimation period for the study was 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-24.2℃ (target range: 19-25℃)
- Relative humidity (%): 31-63% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours light daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: 29 December 2020 (first vaginal smear sampling) - 24 March 2021 (last necropsy)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared diets were stored for no more than 13 weeks; diets were considered to be stable for this period. Test item stability was demonstrated by an IR analytical method.
- Mixing appropriate amounts with (Type of food): The test item was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” to generate the test concentrations required for the study (1000, 3000 and 10000 ppm were prepared). First, the control diet was produced from the standard rodent diet. The different test item enriched diets were produced as follows. Test item (as solid crystals) was added to the standard diet and mixed for up to approximately 12 minutes (approximately 6 minutes for premix preparation, and after addition of water (5%) for an additional 6 minutes for preparation of the complete diets). Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, 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 disodium tetrachloropalladate /kg diet added).
The pelleting process was considered not to induce an “unmixing” of the diets or particle segregation and prevents the potential settling out of the test item particles that could occur in handling/transport of powdered diets.
- Storage temperature of food:
The prepared diets were stored at room temperature under dry conditions in sewed bags.

Details on mating procedure:

Mating began after the animals had attained full sexual maturity, 3 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred for up to 5 days, except in one case where the mating lasted for 15 days. A vaginal smear was prepared daily during the mating period and stained with 1% (w/v) aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Remarks:

Analysis of test item concentration and/or homogeneity in the diets was performed using extraction from diet and a validated method at the Test Site.

Details on analytical verification of doses or concentrations:

Sampling for the pre-treatment measurements (before the start of use):
Representative test item-containing diet samples (5 x 25 grams) for each concentration and representative blank-diet samples of first batch were sampled at the Test Facility and one sample from the middle of the diet container (1 x 25 grams) was collected for the control diet. The first analysis for each dose group (including the control diet) was performed prior to the start of the treatment to confirm the dietary concentrations.
Representative test item-containing diet samples (5 x 10 grams) were collected at the Test Facility from five different places of the diet container (in duplicates) from each dose group of the second batch, additionally one sample from the middle of the diet container (in duplicates) was collected for the control diet.

Sampling after the treatment phase:
For additional analysis, representative diet samples (5 x 10 grams) were collected at the Test Facility from five different places of the diet container (in duplicates) from each dose group, additionally one sample from the middle of the diet container (in duplicates) was collected for the control diet.
Acceptance criteria of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 20%.
Acceptance criteria of the homogeneity was that the CV of replicates (from 5 different locations) should be less than 10%.

The mean concentrations of test item in the diet were in the range of 90.0 to 109.9% of nominal concentrations (within the ±20% limit), thus they were considered acceptable.
Diets were homogeneous.

Duration of treatment / exposure:

Males were dosed for 35 days (21 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 21 days pre-mating, for up to 4 days (except one Mid dose female where it was 15 days) mating period, through gestation (22-25 days) and up to and including the day before necropsy (at least 13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. All selected F1 offspring was terminated on Day 13 post-partum (F1 offspring selected for blood sampling was terminated on PND4 due to technical reasons and litters were culled on PND4). In order to allow for overnight fasting of dam prior necropsy on PND14, offspring was euthanized on PPD/PND13, and the dams on PPD/PND14.

Frequency of treatment:

A constant concentration of disodium tetrachloropalladate in diet were administered to all animals within a defined dosing group throughout the in-life period. Animal food consumption per cage was measured and the mean daily food intake per rat was calculated.

Details on study schedule:

Control diet administration for both sexes began after 12 days acclimatisation. Dosing of both sexes began after the acclimatisation (12 days) and pre-exposure period (14 days), and it was performed 3 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.

The first day of dosing of each animal was regarded as Day 0.

Males were dosed for 35 days (21 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.

Females were dosed for 21 days pre-mating, for up to 4 days (except one Mid dose female where it was 15 days) mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.

All selected F1 offspring was terminated on Day 13 post-partum (F1 offspring selected for blood sampling was terminated on PND4 due to technical reasons and litters were culled on PND4). In order to allow for overnight fasting of dam prior necropsy on PND14, offspring was euthanized on PPD/PND13, and the dams on PPD/PND14.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm

Remarks:

target dosing level: 100 mg/kg bw/d

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

target dosing level: 300 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

10 000 ppm

Remarks:

target dosing level: 1000 mg/kg bw/d

No. of animals per sex per dose:

12 animals/sex/group

Control animals:

yes

Details on study design:

- Dose selection rationale:
The concentrations of the test item in diet was selected based on the results of a 14-day dietary toxicity study in rats. In the 14-day study groups of 5 rats/sex were given diets containing 0 ppm (Control), 1000 ppm, 3000 ppm or 10000 ppm Disodium tetrachloropalladate. There was no mortality and no treatment-related clinical signs. Reductions were noted for the body weight, body weight gain, body weight at autopsy and the food consumption of the male rats dosed with 10000 ppm Disodium tetrachloropalladate.
-All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
- Fasting period before blood sampling for clinical biochemistry: All animals including the randomly selected animals for blood sampling were fasted (overnight period of food deprivation, in case of females this happened after the litter had been necropsied).
- Rationale for selecting satellite groups: no satellite animals included
- Post-exposure recovery period in satellite groups: no satellite animals included
- Section schedule rationale (if not random): random
- Dose range finding studies: 14-DAY DOSE-RANGE-FINDING STUDY IN RATS OF DISODIUM TETRACHLOROPALLADATE BY ORAL ADMINISTRATION VIA THE DIET, Hansen, 2020 (LPT, Hamburg, Germany, LPT Report No. 36051)

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
°Animals were inspected for signs of morbidity and mortality twice per day (at the beginning and end of each working day). The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration.
°Any clinical sign noted during dosing or at any other occasions were recorded at the time seen.
°General (routine) clinical observations were made once a day, during the pre-treatment and treatment period in the afternoon (pm). No general clinical observations were made on the day of necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
°Detailed clinical observations were made daily until PND14 or termination (males). These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). 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) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
°Pertinent behavioural changes, signs of difficult or prolonged parturition were recorded including onset, degree and duration of signs as applicable.
°On Gestation Day 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
°Mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.


BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g on Day 0 (randomisation and the first day of exposure) and weekly thereafter and at termination. Extra body weight measurements were performed daily from Day 8 to Day 15 for High dose animals because of body weight decrease during the first week. Parent females were weighed on Gestation Days (GD) 0, 4, 7, 11, 14, 17 and 20, and on PPD (Post-partum Day) 0 (within 24 hours after parturition), 4, 7 and 13.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g weekly (on a body weight measurements day). Food consumption was measured on Gestation Days (GD) 0, 4, 7, 11, 14, 17 and 20, and on PPD (Post-partum Day) 0, 4, 7 and 13.
Main daily food consumption was calculated for each interval.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Animal food consumption per cage was measured once weekly and the mean weekly food consumption and daily feed intake per rat were calculated. Based on food consumption data, the mean test item intake of each group was calculated for reporting purposes on the basis of mg/kg body weight/day, in addition food conversion efficiency (g/g) was calculated as [weekly body weight gain (g)/weekly food consumption (g)]. Individual data was calculated, the food consumption data was also reported on a cage basis (two animals per cage).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Oestrus cycles was monitored by vaginal smears daily during the pre-exposure period before the treatment started. Any females that failed to show a 4-5 day cycle were not included in the study. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating (during the pre-mating and mating periods).
Additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
°organ weight: testis and epididymis (individual weight)
°testes and epididymides were retained in modified Davidson’s fixative
°For the adult animals, detailed histological examination was performed with special attention to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On PND 4, litters were culled to yield, as nearly as possible, 5 males and 5 females per litter. Pups to be culled within each litter was selected at random. In litters of insufficient size where the number of male or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
°Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
°Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4, PND 7 and PND 13, with accuracy of 0.01 g.
°All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) was also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.
°On PND 4, litters were culled. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
°The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0).
°Number of nipples/areolae in male pups was recorded on PND 13.
°One male and one female pup per litter were previously selected for culling for blood sampling on PND 13.
°All surviving pups were necropsied on PND 13. Pups were examined for gross lesions.

GROSS EXAMINATION OF DEAD PUPS:
yes; the pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
not included

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
not included

Postmortem examinations (parental animals):

There was not mortality of parental animals in this study.
SACRIFICE
- Male animals: males were euthanized after 36 days of dose administration, gross necropsy was performed on each adult animal, animals were sacrificed under anaesthesia by exsanguination
- Maternal animals: females were euthanized on PND14, gross necropsy was performed on each adult animal, animals were sacrificed under anaesthesia by exsanguination

GROSS NECROPSY
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened and the appearance of the tissues and organs were 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.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. The number of implantation sites and of corpora lutea was recorded in the females.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all adult animals was determined:
• With a precision of 0.01 g: testes, epididymides, prostate, seminal vesicles with coagulating glands measured from all males; brain, heart, kidneys, liver, spleen and thymus measured in case of 5 males/group and 5 females/group. Uterus (including cervix) was measured for all females.
• With a precision of 0.001 g: adrenals (in case of 5 males/group), thyroids with parathyroids (from all animals, after fixation)
Paired organs were weighed together except of testes and epididymides which were weighed individually (paired organ weights as applicable are summarised). Individual and/or paired absolute organ weight are reported for each animal and relative organ weight (to body and brain weight) was calculated and reported.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
For all adult males, testes, epididymides, prostate, seminal vesicle with coagulating gland, mammary gland area, thyroid and gross lesions were retained. In addition, on completion of the macroscopic examination the following tissues and organs were retained only for 5 males/ group.
For all the females, ovaries, uterus (with cervix), mammary gland area and gross lesions were retained. In addition, on completion of the macroscopic examination the following tissues and organs were retained only for 5 females/ Control, Low, Mid dose group and all females from High dose.
In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology (below) 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 selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group)
• all macroscopic findings (abnormalities), except of minor order from all animals
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND13
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
°pups were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 pups was also recorded.
°thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.

Statistics:

At the Test Facility, the statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS 9.2 (when using Provantis) to be documented in the raw data and reported.

Reproductive indices:

Male mating index
Female mating index
Male fertility index
Female fertility index
Gestation index

Offspring viability indices:

survival index (Survival index on PND13 will be calculated from number of pups after culling on PND4 instead of number of pups born)
Pre-implantation mortality
Intrauterine mortality
Total morality
Sex ratio (female)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: No clinical signs observed

Females:
°Piloerection was observed in 1/12 Mid dose female on Day 58, 6/12 High dose females from Day 48 to Day 61. The longevity of the symptom was 7 days.
°Hunched posture was observed in 4/12 High dose female animals from Day 48 to Day 61. The symptom lasted for 7 days.
°Alopecia on both forelimbs was observed in 1/12 High dose female from Day 43 until Day 57.
°Red discharge around nose was observed 1/12 High dose female on Day 49.
°Thin fur (neck ventral area) was observed in 1/12 Low dose female on the day of death.
°Nodule (neck ventral area) was observed in 1/12 Control and 1/12 Low dose female on the day of death.
Alopecia, red discharge, thin fur and nodule was considered incidental. The limited High dose female observations of hunched posture and piloerection were considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
Low & mid dose: no test item related effect
High dose: Statistically significant body weight loss (p<0.01) during the first 7 days, thereafter the growth rate was similar to control (fully adapted to the diet, suggesting a palatability effect), although weights remained below control (statistically significant at most time points).

Females:
Low & mid dose: unaffected by treatment until the last week of lactation when the Mid dose females lost ~23g body weight, corresponding with the increased food intake (because of lactation) and hence a greater intake of test item on a mg/kg/day basis.
High dose: in the first week of treatment a lower body weight gain was observed (not statistically significant, probably a palatability effect) but by week 3 High dose females gained more weight than the controls, indicating a good acceptance of the diet. However, from the start of pregnancy to the study termination the High dose females consistently gained less weight than controls (generally not enough to be statistically significant) Statistically significantly decreased body weight and body weight gain was observed from GD17 to GD20 and during lactation (p<0.05 and p<0.01). Overall gestation weight gain was 25.3% below the control value. The final body weight of High dose females (PPD13) was 22% below controls, which is considered to indicate that this dose level exceeded the MTD.

cfr. Tables ‘Selected body weight parameters of parental animals (male)’ and ‘Selected body weight parameters of parental animals (female)’ in Section ‘Any other information on results incl. tables’

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related reduced food consumption was seen in the High dose group males and females.

Statistically significantly lower (p<0.01) food consumption values were recorded in the High dose male group; the most marked difference was in the first 7 days (19.9%). A similar trend was seen in High dose females during the pre-mating (not statistically significant) and gestation and lactation periods. A statistically significant decrease in the food consumption of High dose females was noted during the lactation period when compared to control (by 43.1%, p<0.01), correlating with the body weight effects. This finding was considered to be test item related; no effect was seen in lower dose groups.

cfr. Table ‘Selected daily food consumption parameters of parental animals’ in Section ‘Any other information on results incl. tables’

The test item intake values (mg/kg bw/day) were calculated from the weekly individual body weight, weekly mean food intake and nominal diet concentration. The mean achieved dose levels (combined for males and females) were 94.6, 271.7 and 786.1 mg/kg bw/day in the Low (1000 ppm), Mid (3000 ppm) and High (10000 ppm) dose groups, respectively.
For the males in all dose groups, the actual achieved test item intake values were lower than the target dose levels. In case of females, the mean test item intake was above the achieved dose levels for the lactation, but this was related to the higher food consumption during lactation (due to the physiological reasons).
During the pre-mating period the weekly mean test item intake was 708 to 746 mg/kg bw/day, when growth appeared to be acceptable, but as the food intake increased during pregnancy the daily intake was more than 800 mg/kg bw/day and the body weight gain declined; during lactation the mean intake was above 1000 mg/kg bw/day, when the growth rate was most affected (~70% lower than control). The Mid dose females had a normal body weight gain until the last week of lactation, when the test item intake increased to 483 mg/kg bw/day, before this week with lower intake there were no apparent effects of treatment.

cfr. Table ‘Mean test item intake of parental animals’ in Section ‘Any other information on results incl. tables’

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

There was an effect on food conversion efficiency in the High dose male group in the first 7 days (when they lost body weight) and in the last few days of the Gestation period for the High dose female group.

cfr. Table ’Food Conversion Efficiency’ in Section ’Any other information on results incl. tables’

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related effects were observed in the High dose male and female groups.
Male:
Low & Mid dose: no effects observed
High dose:
°Statistically significantly decreased monocyte relative % and the data were outside of the historical control range (considered treatment related)
°decreased (but not statistically significant) haemoglobin concentration, haematocrit %, mean cell haemoglobin, mean platelet volume and mean cell volume indicating slight anaemia

Female:
Low dose: no effect observed
Mid dose: Statistically significantly decreased reticulocyte relative % which was out of the historical control range but was considered to be unrelated to treatment in the absence of a similar change in the High dose group.
High dose: The haematological differences seen were large enough to be considered as adverse.

Several other values in the Mid and Low dose groups were outside of the historical control range, although the concurrent controls were at the low end of the normal range and the red blood cell counts were normal.

cfr. Table ‘Summary of selected haematology parameters’ in section ‘Any other information on results incl. tables’

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male:
Low & mid dose: no effects
High dose: Statistically significantly increased (p<0.05) chloride concentration and the value was outside of the historical control range. It is considered that the small increase in plasma chloride was not adverse and may be related to the chlorine in the test item.

Female:
Low dose: no effects
Mid dose: slightly increased sodium concentration (p<0.05). It is considered that the small increase in plasma chloride was not adverse and may be related to the chlorine in the test item)
High dose: Statistically significantly increased sodium concentration (p<0.01), potassium (p<0.05), chloride concentration (p<0.01) and ALT/GPT (p<0.05) was detected in the High dose female group. The values of the last two parameters were outside of the historical control range and were considered as potentially test item related effects. It is considered that the small increase in plasma chloride was not adverse and may be related to the chlorine in the test item. Changes in ion levels can be associated with the physiological effects of metal test item ions; none of the differences were large enough to be considered as adverse, in the absence of any histopathological changes.

cfr Table ‘Summary of selected clinical chemistry parameters’ in section ‘Any other information on results incl. tables’

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly decreased T4 hormone concentration level was recorded in the High dose group of parental males and females. The data for males were outside of the historical control range therefore it was considered as test item related effect. The T4 data for females were within the normal range, it was not different from control (No thyroid hormone concentration level differences were observed for the pups, all values were normal (despite the pups being much smaller than controls)).
No relevant changes were noted in the absolute or relative (to body / brain) thyroid weights of the parental males (although the High dose was slightly lower than control, the values are close to, or even above the historic mean value, indicating there was no real weight reduction). No statistically significant or biological relevant changes were observed on the thyroid weight in the female dosed groups. No histopathology (microscopic) findings were detected in any adult High dose males or females.
In summary, there were effects on the thyroid hormone levels in parental males and females that were ascribed to the test item. The observed effects in the High dose animals on thyroid hormone levels is considered as secondary effect because of the systemic toxicological effect or stress on these animals where the High dose was considered to have exceeded the MTD.

cfr. Table ‘Selected parameters related to thyroid hormone levels’ in Section ‘Any other information on results incl. tables’

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no 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 in the Irwin test or during the assessment of grip strength and landing foot splay.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with a steady reduction in activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related changes were observed.
The decreased cellularity (lymphocytes) in the thymus, correlated with necropsy findings and organ weight changes were considered as test item-related non-adverse change.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, or a common background.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No test item-related changes were observed.
The decreased cellularity (lymphocytes) in the thymus, correlated with necropsy findings and organ weight changes were considered as test item-related non-adverse change.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, or a common background.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Pre-exposure period:
Each female selected for the study showed regular cycles (mean cycle length of 4.00 days was observed in all groups) before starting the treatment period.
Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.00, 4.02, 4.02 and 4.00 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in one Mid dose female but as there was no dose related trend and the mating or pregnancy rate was not affected, this finding was not considered as a test item related effect.
Prolonged dioestrus was noted for one Control female but this occurrence did not affect to the mating or pregnancy.
Pseudo-pregnancy was noted for one Low, for two Mid dose and one High dose female, but this frequency was in line with the normal, expected range and the animal successfully mated and delivered later.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating and fertility index were 100% in all groups (males and females). The gestation index was 100% in all dose groups.
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) occurred mostly within 5 days of pairing (cohabitation), except one Mid dose female (15 days). The mean duration of mating was 2.42, 2.17, 3.75 and 2.83 days in the Control, Low, Mid and High dose groups, respectively.

Organ weights:
°In the Low and Mid dose male groups, there were no treatment related organ weight differences.
°The High dose male had a terminal body weight of ~7% below control (so most organs could be expected to be similarly below the control organ weight) hence the weight adjusted for body weight is the more appropriate statistical comparison for most organs. Although there were some statistically lower absolute mean organ weights (eg seminal vesicle), and weights adjusted for brain weight, all the data were within the historical control range. Taking into account the lower terminal body weights and the historical control ranges (shown in the table below) it is considered that there were no male organ weights adversely affected by treatment.

Macroscopic Findings: No test item-related changes were observed at necropsy.

Microscopic Findings: No test item-related changes were observed.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating and fertility index were 100% in all groups (males and females). The gestation index was 100% in all dose groups.
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) occurred mostly within 5 days of pairing (cohabitation), except one Mid dose female (15 days). The mean duration of mating was 2.42, 2.17, 3.75 and 2.83 days in the Control, Low, Mid and High dose groups, respectively.
There was no effect of treatment noted during the gestation period, parturition and post-partum period in any of dose groups.
The mean duration of pregnancy was comparable in the Control and test item treated groups .
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
There were no statistically significant differences in the number of implantation sites. The number of implantation sites of treated groups was within the HCD and there was no indication of a dose response or an effect of treatment.
The number of live born pups in the High dose group was statistically significantly lower than the control. This was mainly influenced by the results for one female with a gestation length of 24 days and no live born pups. The mean was within the historical mean range hence the numerical difference is considered not to reflect an adverse effect.
There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.

cfr. tables in section 'Any other information on results incl. tables'

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: The NOAEL for reproductive effects of the parental generation was considered to be 10000 ppm (corresponding to 786 mg/kg bw/day) since no test item related adverse reproductive effects were observed.

Target system / organ toxicity (P0)

Critical effects observed:

no

System:

other: none

Organ:

other: none

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or abnormalities were recorded for any pups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND 0, PND 4 and PND 13 as well as pup survival indices on PND 0, PND 4 and PND 13 were comparable to control values in the Low and Mid dose groups but the number of viable pups was statistically significantly lower in the High dose group.
There were 4 litters where the prenatal mortality was 14% or higher. These pups/litters also had marked reductions in body weight and food consumption.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Low dose group pups: no effect observed
Mid dose group: pup weights were not different to control to PND4, after which they were statistically below control, but the values for weight and growth were fully in line with historic control data (above the mean). On PPD13 data were within the historical control range hence the Mid dose group was considered to be unaffected by treatment.
High dose group: PND0 pup weights were slightly below control (p<0.05) and the growth of these pups remained significantly below control (the overall weight gain to PND13 was less than 50% of the control value). The body weight effect on High dose females was considered to be test item related and resulted in effects on pup body weight.

cfr. table in section 'Any other information on results incl. tables'

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance.
No relevant statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.

cfr table in section 'Any other information on results incl. tables'

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention. No nipples/areolae were present in any of the male pups on PND13.

cfr table in section 'Any other information on results incl. tables'

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were observed up to 10000 ppm.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis
High dose group pups: No thyroid hormone concentration level differences were observed for the pups, all values were normal (despite the pups being much smaller than controls).
The PND13 High dose pup body weights were about 50% the size of the control pups, the thyroid gland weights were ~43% below control, which is fully in line with the body weight effect, hence it is considered that there was no test item weight effect directly on the thyroids.

cfr. Table in section 'Any other information on results incl. tables'

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: /

Target system / organ toxicity (F1)

Critical effects observed:

no

System:

other: none

Organ:

other: none

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Summary of reproductive parameters (males)

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Number of treated animals	12	12	12	12
Number of males used for mating	12	12	12	12
Number of pre-terminal death	0	0	0	0
Number of successful mating	12	12	12	11
Number of infertile animals	0	0	0	0
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	100	100	100

 

Summary of reproductive parameters (females)

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Number of treated animals	12	12	12	12
Number of females used for mating	12	12	12	12
Number of sperm positive females	12	12	12	12
Number of females with no implantation sites	0	0	0	0
Number of pregnant females	12	12	12	12
Number of found dead pregnant	0	0	0	0
Number of pregnant females with live born(s)	12	12	12	11
Number of pregnant females with stillborn pups	0	0	0	1
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	100	100	100
Female gestation index (%)	100	100	100	92

 

Summary of the pregnancy evaluation

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Number of evaluated females	12	12	12	12
Number of pregnant females	12	12	12	12
Duration of pregnancy (days)	22.67	22.67	22.67	22.83	NS
Number of implantations, mean	17.33	16.42	17.17	15.50	NS
Number of pups born, mean	15.83	14.75	15.50	13.67	NS
Number of live born pups, mean	15.58	14.75	15.25	12.50*	DN
Pre-natal mortality, mean	1.75	1.67	1.92	3.00	NS
Pre-natal mortality (%), mean	9.91	10.27	11.12	18.11	NS
Post-natal mortality, mean	0.33	0.00	0.33	0.36	NS
Post-natal mortality (%), mean	1.96	0.00	2.04	2.55	NS
Total mortality, mean	2.08	1.67	2.25	3.33	NS
Total mortality (%), mean	11.82	10.27	12.93	20.16	NS

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Duncan’s multiple range test, NS: Statistically not significant when compared to the control.

 

Summary of survival (offspring)

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Number of evaluated litters	12	12	12	12
Number of pups born, mean	15.83	14.75	15.50	13.67	NS
Number of live born pups, mean	15.58	14.75	15.25	12.50*	DN
Number of living pups on PND13, mean	10.00	10.00	9.83	8.25	NS
Pre-natal mortality, mean	1.75	1.67	1.92	3.00	NS
Pre-natal mortality (%), mean	9.91	10.27	11.12	18.11	NS
Post-natal mortality on PND0-4, mean	0.33	0.00	0.33	0.36	NS
Post-natal mortality on PND0-4 (%), mean	1.96	0.00	2.04	2.55	NS
Total mortality on PND4, mean	2.08	1.67	2.25	3.33	NS
Total mortality on PND4 (%), mean	11.82	10.27	12.93	20.16	NS
Post-natal mortality on PND0-13, mean	0.33	0.00	0.33	0.36	NS
Post-natal mortality on PND0-13 (%), mean	1.96	0.00	2.04	2.55	NS
Total mortality on PND13, mean	2.08	1.67	2.25	3.33	NS
Total mortality on PND13 (%), mean	11.82	10.27	12.93	20.16	NS
Survival index on PND0	98.21	100.00	98.75	88.96	NS
Sex ratio % (Female) on PND0	48.51	45.29	48.50	57.50	NS
Survival index on PND4	98.04	100.00	97.96	97.45	NS
Sex ratio % (Female) on PND4	48.05	45.29	49.46	57.44	NS
Survival index on PND13	100.00	100.00	100.00	90.91	NS
Sex ratio % (Female) on PND13	49.17	50.00	52.92	51.44	NS

Statistical significance compared to control: * = p<0.05

DN: Duncan’s multiple range test, NS: Statistically not significant when compared to the control.

 

Summary of the offspring mortality

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Number of evaluated litters	12	12	12	12$
Number of pups born	190 / 12	177 / 12	186 / 12	164 / 12	NS
Number of cannibalized pups	2 / 2	0 / 0	3 / 2	/ 3
Number of autolyzed pups	5 / 3	0 / 0	4 / 3	21 / 4
Number of stillborn pups	0 / 0	0 / 0	0 / 0	0 / 0
Number of live born pups	187 / 12	177 / 12	183 / 12	150** / 11	CH
Number of found dead pups (born alive)	3 / 1	0 / 0	3 / 2	14 / 3
Number of living pups on PND0	187 / 12	177 / 12	183 / 12	150**/11	CH
Number of cannibalized pups (PND0-13)	2 / 2	0 / 0	3 / 2	7 / 3
Number of autolyzed pups (PND0-13)	5 / 3	0 / 0	4 / 3	21 / 4
Number of found dead, intact pups (PND0-13)	0 / 0	0 / 0	0 / 0	0 / 0
Total number of pups died (born alive)	25 / 12	20 / 12	27 / 12	50** / 11	CH
Culled for blood sampling on PND4	22 / 12	22 / 12	22 / 11	19 / 10
Culled for litter standardization on PND4	41 / 10	35 / 10	39 / 10	18 / 6
Number of viable pups on PND13	120 / 12	120 / 12	118 / 12	99 / 10	NS

Notes: Statistical significance compared to control: * = p<0.05, p<0.01; CH: Chi square test

NS: Statistically not significant when compared to the control

$: In case of a High dose litter (#4512), all the pups died by PND7.

 

Selected body weight data (offspring)

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Number of evaluated litters	12	12	12	11^§
Mean pup body weight (PND0), g	6.6	6.7	6.5	5.8*	DU
Mean pup body weight (PND4), g	11.3	11.9	11.4	8.5**	DN
Mean pup body weight gain (PND0-4), g	4.7	5.3	4.9	2.7**	DN
Mean pup body weight (PND13), g	34.2	35.3	30.7**	18.6**	DN
Mean pup body weight gain (PND4-13), g	22.8	23.3	19.4*	9.8**	DU
Mean pup body weight gain (PND0-13), g	27.6	28.5	24.2**	12.7**	DN

Data were rounded to one decimal place.
§: In case of a High dose litter (#4512), all the pups died by PND7.

 

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

Anogenital distance

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Male pups
Number of evaluated litters	12	12	12	11
Anogenital distance, litter mean of males (mm)	3.49	3.47	3.47	3.50	NS
Minimum / Maximum value, litter mean (mm)	3.2 / 3.8	3.2 / 3.9	3.2 / 3.9	3.1 / 3.9
Female pups
Number of evaluated litters	12	12	12	11
Anogenital distance, litter mean of females (mm)	1.90	1.86	2.01	1.70	NS
Minimum / Maximum value, litter mean (mm)	1.7 / 2.5	1.4 / 2.5	1.5 / 2.6	1.3 / 2.4

Group mean values were rounded to two decimal places.

NS: Statistically not significant when compared to the control.

 

Selected parameters related to thyroid hormone levels

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)

PND 13 pups
Number of evaluated litters	12	12	12	10
T4 concentration (ng/mL)	42.67	42.00	38.33	42.00
HC range: 32-52	difference (%)	-1.6	-10.2	-1.6
Thyroid gland weights (g)	0.0051	0.0054	0.0054	0.0029**
HC range: 0.0042-0.0064	difference (%)	5.7	4.9	-43.4
Thyroid gland / body weight (%) (x10^-4)	1.5057	1.5283	1.7660*	1.5801
HC range: 1.448-2.092 (x10^-4)	difference (%)	1.5	17.3	4.9

Data (group mean values) were rounded to two or four or five decimal places. Thyroid and parathyroid weights were measured together. Thyroid gland weight for one male and one female pup per litter were determined. Pups blood (serum) was pooled for T4 (thyroxin) determination.

HC: Historical control. NS: Statistically not significant when compared to the control

Statistical significance compared to control: * = p<0.05, ** = p<0.01. Bold letter: data out of the historical control range. DN: Dunnett’s Multiple Range test, U: Mann-Whitney U-test

Applicant's summary and conclusion

Conclusions:

A GLP-compliant study according to OECD N°422 was performed to obtain information on the possible effects on toxicity and reproductive performance of disodium tetrachloropalladate following repeated (daily) administration at a constant concentration in pelleted diet to Wistar (Crl:WI) rats at 3 dose levels (1000, 3000 and 10000 ppm test item in diet). A control group received non-treated control diet.

Under the conditions of this study, the dietary administration of disodium tetrachloropalladate to Wistar rats did not result in test item related mortality.
The NOAEL for systemic toxicity of the parental generation was considered to be 3000 ppm (corresponding to 272 mg/kg bw/day) based on effects on body weight, body weight gain and food consumption at 10000 ppm.
The NOAEL for reproductive effects of the parental generation was considered to be 10000 ppm (corresponding to 786 mg/kg bw/day) based on no test item related adverse reproductive effects.
The NOAEL for pup development and survival was considered to be 3000 ppm (corresponding to 272 mg/kg bw/day). Effects on the mean High dose pup body weight and body weight gain were considered as secondary to maternal toxicity, as High dose females showed body weight loss during gestation and the effect on final body weights exceeded the MTD. Dam intake was >1000 mg/kg bw/day during lactation for High dose females.

Executive summary:

A GLP-compliant study according to OECD N°422 was performed to obtain information on the possible toxic effects of disodium tetrachloropalladate following repeated (daily) administration at a constant concentration in pelleted diet to Wistar (Crl:WI) rats at 3 dose levels (1000, 3000 and 10000 ppm test item in diet). A control group received non-treated control diet.

Under the conditions of this study, the dietary administration of disodium tetrachloropalladate to Wistar rats did not result in test item related mortality.

Piloerection and hunched posture were observed in some High dose female during the lactation period only.

High dose males lost weight (~12 g) in the first 7 days but recovered and showed normal growth rate for the remaining 4 weeks of the treatment (suggesting a palatability effect). High dose females also had lower body weight gain in the first 7 days of the study (not-statistically significant). The total body weight gain during gestation was lower than control (particularly in the last few days) and during lactation growth was ~66% lower than controls in the High dose females. Their final body weight (PPD13) was 22% below controls (which is considered to exceed the MTD). There was no effect on body weight in Mid dose females until the last 7 days of gestation where there was a weight loss of ~23 g (when the mg/kg bw/day intake of test item was maximal). Mid and Low dose males and Low dose females were unaffected. Lower food intake values tended to reflect the body weight effects.

Food conversion efficiency in the High dose male group was reduced in the first 7 days (when they lost body weight) but was normal thereafter; food conversion in the High dose female group was less efficient in the last few days of the Gestation period. At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the test groups compared to control group.

Decreases in haemoglobin and related parameters were seen in High dose females suggesting slight anaemia. Minor test item-related findings were seen in the remaining haematology and clinical pathology parameters but were considered not adverse.

No test item effect on oestrus cycle of parental females was noted.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding was recorded for F1 pups at necropsy. The number of live born pups in the High dose group was statistically significantly lower than control. Mean pup body weight and subsequent pup body weight gain was decreased in the High dose group.

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

The levels of T4 and thyroid gland weights were decreased in parental males and females at the High dose only. The observed effects in the High dose animals on thyroid hormone levels are considered as secondary to the systemic toxicological effect or stress in these animals where the High dose was considered to have exceeded the MTD.

Under the experimental conditions of this study no test item related effects were observed on nipple retention, anogenital distance external reproductive organs, appearance, histopathology and reproductive performance.

The NOAEL for systemic toxicity of the parental generation was considered to be 3000 ppm (corresponding to 272 mg/kg bw/day) based on effects on body weight, body weight gain and food consumption at 10000 ppm.

The NOAEL for reproductive effects of the parental generation was considered to be 10000 ppm (corresponding to 786 mg/kg bw/day) based on no test item related adverse reproductive effects.

The NOAEL for pup development and survival was considered to be 3000 ppm (corresponding to 272 mg/kg bw/day). Effects on the number of High dose live born pups, mean High dose pup body weight and body weight gain were considered as secondary to maternal toxicity, as High dose females showed body weight loss during gestation and the effect on final body weights exceeded the MTD. Dam intake was >1000 mg/kg bw/day during lactation for High dose females.