Disodium tetrachloropalladate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

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.

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

Details on route of administration:

The oral route was selected as it is one of the possible routes of human exposure.
The way of diet administration was in compliance with the relevant OECD No. 422 guideline

Vehicle:

unchanged (no vehicle)

Details on oral 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.

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 (#3508) 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.

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

Observations and examinations performed and frequency:

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). 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:
°Any clinical sign noted during dosing or at any other occasions were recorded at the time seen.
°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).
Furthermore, 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:
- 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).

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

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 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). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes
pentobarbital anaesthesia (Euthanimal 40% (400 mg/mL sodium pentobarbital solution) supplied by Alfasan Nederland BV, The Netherlands was used by intraperitoneal injection.
- Animals fasted: Yes
- How many animals: 5 males and 5 females/group (randomly selected) and 12 High dose females
- Parameters checked:

PARAMETERS (UNITS) // METHODS
RBC Red Blood Cell (erythrocyte) count, (1012/L) M/µL // Automatic laser cell count
WBC White Blood Cell (leukocyte) count, (109/L) K/µL // Automatic laser cell count
Hgb Haemoglobin concentration, (g/dL) //Determination of cyan-methemoglobin absorbance
Hct Haematocrit (relative volume of erythrocytes) (%) //Calculated value
MCV Mean Corpuscular (erythrocyte) Volume (fL) //Laser cell volume determination
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg) //Calculated value
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL) //Calculated value
RDW Red Cell (erythrocyte) width (%) // Distribution Width Laser detection
Plt Platelet (thrombocyte) count, (109/L) K/µL // Automatic laser cell count
MPV Mean Platelet Thrombocyte volume (fL) // Cell volume determination by laser
RETIC % Reticulocyte count (%) // Comparative value based on laser light detection
NE % Neutrophil (%) // Cell differentiation based on myeloperoxidase activity
LY % Lymphocyte (%) // Cell differentiation based on myeloperoxidase activity
MO % Monocyte (%) // Cell differentiation based on myeloperoxidase activity
BA % Basophil (%) // Cell differentiation based on myeloperoxidase activity
EO % Eosinophil (%) // Cell differentiation based on myeloperoxidase activity
LUC % Large Unstained Cells (%) // Cell differentiation based on myeloperoxidase activity
Coagulations
APTT Activated Partial Thromboplastin Time (sec) // Factors of intrinsic coagulation system are activated by incubation the plasma with the optimal amount of phospholipids, a surface activator and calcium ions.
PT Prothrombin Time (sec) // The coagulation cascade is activated by incubating plasma with the optimal amount of thromboplastin and calcium.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 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). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Animals fasted: Yes
- How many animals: 5 males and 5 females/group (randomly selected) and 12 High dose females
- Parameters checked:
PARAMETERS (UNITS) // METHODS
Glucose Blood sugar concentration (mmol/L) // Endpoint (340/410 nm)
T-BIL Total Bilirubin concentration (μmol/L) // Diazo-sulfanilic acid method (Endpoint, 555/600 nm)
Urea Urea concentration (mmol/L) // Urease with GLDH (Kinetic (RRA), 340/410 nm)
Chol. Cholesterol concentration (mmol/L) // Enzymatic (Endpoint (EPA), 505/694 nm)
Creat. Creatinine concentration (μmol/L) // Enzymatic, colorimetric (540-570 nm)
Phos. Phosphorus concentration (mmol/L) // Phosphomolibdate/UV method
(Endpoint, 340/658 nm)
Na+ Sodium concentration (mmol/L) // Potentiometric (Ion-Selective Electrode)
K+ Potassium concentration (mmol/L) // Potentiometric (Ion-Selective Electrode)
Ca++Calcium concentration (mmol/L) // Potentiometric (Ion-Selective Electrode)
Cl- Chloride concentration (mmol/L) // CPC (Endpoint (EPA), 545/658 nm)
Tot. Prot. Total Protein concentration (g/L) // Biuret (Endpoint (EPA), 545 nm)
Alb. Albumin concentration (g/L) // BCG staining (Endpoint (EPA), 596/694 nm)
A/G Alb/glob ration // Calculated value
AST/GOT Aspartate Aminotransferase activity (U/L) // Modified IFCC (Kinetic (RRA), 340/410 nm)
ALT/GPT Alanine Aminotransferase activity (U/L) // Modified IFCC (Kinetic (RRA), 340/410 nm)
GGT Gamma-Glutamyl transferase activity (U/L) // Modified IFCC (Kinetic, 410/478 nm)
ALKP Alkaline Phosphatase activity (U/L) // Modified IFCC (Kinetic (increasing), 405 nm)


PLASMA/SERUM HORMONES/LIPIDS: Yes
For thyroid hormone analysis, blood samples were taken over a period of no more than 2 hours per day, by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
• from all litters culled on PND 4 (1 pooled sample per litter, is held for possible future evaluation) and from 1 pup/sex/litter on PND13 (total T4)
• from all dams on PND 14 (females), for evaluation of total T4,
• from all adult males at termination (total T4).
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. Timing are documented in the raw data.

URINALYSIS: Yes
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours. The evaluation of the urine samples was performed by using of Medi-Test URYXXON® Stick 10 Urinalysis-strips:
PARAMETERS (UNITS) // METHODS
LEU / Leukocyte // Reflection photometry
NIT / Nitrite // Reflection photometry
pH Reflection // photometry
PRO / Protein // Reflection photometry
GLU / Glucose // Reflection photometry
UBG / Urobilinogen // Reflection photometry


NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observational Battery and locomotor activity measurement was performed on five rats per sex per group on one occasion. Male rats were evaluated shortly (on Day 31) before scheduled euthanasia, prior to blood sample collection; female rats were evaluated during the lactation period, (on PPD3-8) before scheduled euthanasia.
- Time schedule for examinations: Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 31 am, females on PPD3-8 am). In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
- Dose groups that were examined: Five males and five females/group were randomly selected
- Battery of functions tested: Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross findings
Lungs with bronchi 8
Skeletal muscle (quadriceps)
Adrenals
Lymph node 9
Small intestine 10
Animal identification 1
Ovary
Spinal cord 11
Aorta 2
Oviduct
Spleen
Brain 3
Pancreas
Sternum with marrow
Epididymis
Pituitary
Stomach
Eye with the optic nerve 4
Prostate
Testis
Oesophagus
Salivary gland (including mandibular, sublingual and parotid glands)
Thymus
Femur with marrow
Thyroid with parathyroid gland 4
Heart 5
Tongue
Kidney
Sciatic nerve
Trachea
Large intestine 6
Seminal vesicle with coagulating gland
Urinary bladder
Extraorbital lachrymal gland
Uterus 12
Harderian gland
Skin, subcutis with mammary gland (inguinal)
Vagina
Liver7
Legend:
1. Fixation and preservation only
2. Aorta thoracic and abdominal
3. 7 section according to the NTP recommendations
4. If applicable, parathyroids and optic nerves were examined histologically only if present in routine sections
5. Section including both ventricles and atria, septum with papillary muscle
6. Caecum, colon and rectum
7. Liver, 3 lobes, left lateral, right medial, caudate
8. Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal
9. Mandibular and mesenteric
10. Duodenum, ileum and jejunum with Peyer’s patches
11. Transverse sections, 3 levels: cervical, thoracic and lumbar.
12. Horns, body and cervix

HISTOPATHOLOGY: Yes
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.

ORGAN WEIGHT:
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.

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.

Results and discussion

Results of examinations

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.

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'

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

Description (incidence and severity):

Males:
Low & mid dose: no treatment related organ weight differences. Sporadic statistical differences were without a dose response.
High dose: 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, 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.

Females:
Low dose: no differences in organ weights that were considered to be with a treatment relationship.
Mid dose: heart weight was statistically lower than control, but the absence of any histological change in this organ indicates a lack of an adverse test item effect.
High dose:
°heart weight was statistically lower than control, but the absence of any histological change in this organ indicates a lack of an adverse test item effect.
°The High dose females were ~20% below the control terminal body weight (so most organs could be expected to be similarly below the control organ weight). The absolute organ weight of the thymus in the High dose group decreased by 42.0%, the relative to body weight decreased by 27.0% and the relative to brain weight decreased by 39.7% compared to Controls (data within the historical control range), correlated with necropsy and microscopic findings and was considered as stress induced. None of the organs when adjusted for body weight were statistically below control, other than thyroids, which were a similar absolute weight to control, but statistically greater when adjusted for body weight. As the terminal body weight was reduced by ~20%, which is considered as in excess of the MTD (the OECD guideline suggests 10% difference is suitable for a High dose effect), this statistical difference is not considered to indicate a specific thyroid effect (also the histopathology was normal).

cfr. table 'Selected organ weight data' in section 'Any other information on results incl. tables'

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed at necropsy. The small thymus in the High dose female group correlated with microscopic findings and organ weight changes were considered as test item-related non-adverse change.
All other changes were considered as incidental or a common background.

Neuropathological findings:

not examined

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table: Selected body weight parameters of parental animals (male)

Parameters	Dose groups (dietary concentration)
	Control	Low dose	Mid dose	High dose
	(0 ppm)	(1000 ppm)	(3000 ppm)	(10000 ppm)
Males, Body weight on Day 7 (g)	452.0	452.8	452.3	422.3*	DN
	difference (%)	0.2	0.1	-6.6
Males, Body weight gain Day 0-7 (g)	16.3	18.6	17.4	-11.8**	DN
	difference (%)	14.4	7.2	-172.8
Males, Body weight on Day 34 (g)	515.9	522.5	511.0	478.6*	DN
	difference (%)	1.3	-1.0	-7.2
Males, Body weight gain Day 0-34 (g)	80.2	88.3	76.2	44.4**	DN
	difference (%)	10.1	-5.0	-44.6
Males, Body weight gain Day 7-34 (g)	63.9	69.7	58.8	56.3	NS
	difference (%)	9.0	-8.1	-12.0

Data (group mean values; n=12) were rounded to one decimal place.

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

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

 

Table: Selected body weight parameters of parental animals (female)

Parameters	Dose groups (dietary concentration)
	Control	Low dose	Mid dose	High dose
	(0 ppm)	(1000 ppm)	(3000 ppm)	(10000 ppm)
Females, Body weight on Day 21	285.8	288.8	293.4	278.8	NS
	difference (%)	1.0	2.7	-2.4
Females, Body weight gain Day 0-21 (g)	22.4	24.8	29.0	14.3	NS
	difference (%)	10.4	29.4	-36.4
Females, Body weight on GD20 (g)	457.7	455.2	470.6	410.9**	DN
	difference (%)	-0.5	2.8	-10.2
Females, Body weight gain GD 0-20 (g)	168.1	162.8	169.5	125.6**	DN
	difference (%)	-3.1	0.8	-25.3
Females, Body weight gain PND 0-7 (g)	19.0	26.8	29.5	5.6
	difference (%)	41.2	55.3	-70.6
Females, Body weight on PPD13 (g)	391.3	401.1	368.6*	305.3**	DN
	difference (%)	2.5	-5.8	-22.0
Females, Body weight gain PPD 0-13 (g)	37.8	40.4	6.2**	13.0**	DN
	difference (%)	6.8	-83.7	-65.6
Females, Body weight gain Day 0-PPD13 (g)	127.8	137.0	104.8**	40.8**	D
	difference (%)	7.2	-18.1	-68.1

Data (group mean values; n=12) were rounded to one decimal place.

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

DN: Dunnett’s test; D: Duncan’s test; NS: Statistically not significant when compared to the control.

 

Table: Selected daily food consumption parameters of parental animals

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Males, Food consumption Day 0-21 (g/animal/day)	27.90	29.36	28.77	24.15**	DN
	difference (%)	5.2	3.1	-13.4
Males, Food consumption Day 0-34 (g/animal/day)	28.33	29.72	28.87	25.10**	DN
	difference (%)	4.9	1.9	-11.4
Females, Food consumption Day 0-21 (g/animal/day)	20.32	21.82	22.25	19.64	NS
	difference (%)	7.4	9.5	-3.3
Females, Food consumption GD 0-20 (g/animal/day)	28.01	29.25	29.48	25.35	NS
	difference (%)	4.4	5.2	-9.5
Females, Food consumption Day 0-7 (g/animal/day)	47.76	50.63	48.76	28.51**	DU
	difference (%)	6.0	2.1	-40.3
Females, Food consumption Day 7-13 (g/animal/day)	70.92	74.99	61.19**	38.81**	DN
	difference (%)	5.7	-13.7	-45.3
Females, Food consumption PPD 0-13 (g/animal/day)	58.45	61.87	54.50	33.26**	DU
	difference (%)	5.9	-6.8	-43.1

Data were rounded to two decimal places.

GD: Gestation Day, PPD: Post-partum Day.

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

 

Table: Mean test item intake of parental animals

Test Item Intake (mg/kg bw/day)
ppm in diet	Males (Day 0 - termination)	Females (Day 0 – end of mating)	Females (Gestation)	Females (Lactation)	Mean
1000	62.65	78.03	82.49	155.14	94.58
3000	184.49	235.91	242.83	423.39	271.66
10000	571.67	729.99	762.68	1080.18	786.13

 

Table: Food conversion efficiency

Parameters	Dose groups
	Control	1000 ppm	3000 ppm	10000 ppm
Male, Food conversion efficiency (Day 0-34)	0.08290	0.08683	0.07754	0.05158**	DN
	difference (%)	4.7	-6.5	-37.8
Female,   Food conversion efficiency (Day 0-21)	0.05194	0.05362	0.06139	0.03413	NS
	difference (%)	3.2	18.2	-34.3
Female,   Food conversion efficiency (GD 0-20)	0.30029	0.27803	0.28806	0.24782**	DN
	difference (%)	-7.4	-4.1	-17.5
Female,   Food conversion efficiency (PPD 0-13)	0.05074	0.04996	0.00877**	0.03021	DN
	difference (%)	-1.5	-82.7	-40.5

GD: Gestation Day, PPD: Post-partum Day.

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

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

 

Table: Summary of selected Haematology parameters

Parameters	Dose groups (dietary concentration)
	Control	Low dose	Mid dose	High dose
	(0 ppm)	(1000 ppm)	(3000 ppm)	(10000 ppm)
Males
Haemoglobin concentration (g/dL)	13.98	13.98	14.26	13.30	NS
HC range: 13.74-15.70	difference (%)	0.0	2.0	-4.9
Haematocrit (%)	42.06	42.26	42.76	40.28	NS
HC range: 41.07-48.60	difference (%)	0.5	1.7	-4.2
Mean Cell Volume (fL)	50.80	51.24	51.26	49.66	NS
HC range: 51.66-57.10	difference (%)	0.9	0.9	-2.2
Mean Cell Haemoglobin (pg)	16.90	16.98	17.06	16.42	NS
HC range: 16.60-19-08	difference (%)	0.5	0.9	-2.8
Mean Platelet Volume (fL)	6.50	6.62	7.00	6.58	NS
HC range: 6.72-9.72	difference (%)	1.8	7.7	1.2
Monocytes relative (%)	3.32	3.16	3.42	1.98*	DN
HC range: 2.26-4.56	difference (%)	-4.8	3.0	-40.4
Females
Haemoglobin concentration (g/dL)	13.96	13.88	14.62	12.28**	DN
HC range: 12.7-16.0	difference (%)	-0.6	4.7	-12.1
Haematocrit (%)	41.16	40.60	42.90	36.46**	DN
HC range: 39.0-46.5	difference (%)	-1.4	4.2	-11.4
Mean Cell Volume (fL)	57.74	56.16	55.52	53.06**	DN
HC range: 54.4-61.9	difference (%)	-2.7	-3.8	-8.1
Mean Cell Haemoglobin (pg)	19.58	19.18	18.92	17.86**	DU
HC range: 18.2-21.4	difference (%)	-2.0	-3.4	-8.8
Mean Platelet Volume (fL)	7.62	7.56	6.82	6.05**	DN
HC range: 6.9-8.9	difference (%)	-0.8	-10.5	-20.6
Monocytes relative (%)	2.52	2.42	3.00	2.39	NS
HC range: 1.5-4.7	difference (%)	-4.0	19.0	-5.1

Data (group mean values, n=5 or 12) were rounded to two decimal places

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

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control. Bold letters: data out of the historical control range.

 

Table: Summary of selected clinical chemistry parameters

Parameters	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Males
Sodium concentration (mmol/L)	144.0	144.7	144.5	144.5	NS
HC range: 137.8-151.4	difference (%)	0.5	0.3	0.4
Potassium concentration (mmol/L)	5.52	5.30	5.34	5.62	NS
HC range: 4.82-6.60	difference (%)	-4.0	-3.3	1.8
Chloride concentration (mmol/L)	100.90	102.26	100.46	104.50*	DN
HC range: 95.5-104.1	difference (%)	1.3	-0.4	3.6
Alanine Aminotransferase activity	62.2	46.4	47.2	58.8	NS
HC range: 33.6-141.6	difference (%)	-25.4	-24.1	-5.5
Females
Sodium concentration (mmol/L)	137.4	140.7	141.1	142.3**	DU
HC range: 133.6-150.1	difference (%)	2.4	2.7	3.6
Potassium concentration (mmol/L)	4.96	5.00	5.64	5.78*	DN
HC range: 4.9-6.7	difference (%)	0.8	13.7	16.4
Chloride concentration (mmol/L)	95.32	97.18	102.66*	104.98**	DN
HC range: 92.1-103.0	difference (%)	2.0	7.7	10.1
Alanine Aminotransferase activity	58.6	63.0	98.4	167.8*	DU
HC range: 49.8-98.0	difference (%)	7.5	67.9	186.3

Data (group mean values, n=5 or 12) were rounded to one or two decimal places

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

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

 

Table: 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)
Males
Number of evaluated animals	12	12	12	12
T4 concentration (ng/mL)	48.67	47.58	46.08	36.42**	DN
HC range: 37-91	difference (%)	-2.2	-5.3	-25.2
Thyroid gland weights (g)	0.0302	0.0290	0.0275	0.0243**	DN
HC range: 0.019-0.029	difference (%)	-3.9	-8.8	-19.6
Thyroid gland / body weight (%)	0.00617	0.00587	0.00566	0.00531	NS
HC range: 0.004-0.006	difference (%)	-4.8	-8.3	-13.9
Females
Number of evaluated animals	12	11	12	12
T4 concentration (ng/mL) HC: 18-103	32.83	32.27	28.50	24.08**	DN
	difference (%)	-1.7	-13.2	-26.6
Thyroid gland weights (g) HC range: 0.018-0.026	0.0210	0.0221	0.0202	0.0206	NS
	difference (%)	5.2	-3.9	-2.0
Thyroid gland / body weight (%) HC range: 0.0-0.01	0.00594	0.00601	0.00578	0.00744**	DN
	difference (%)	1.2	-2.8	25.2
PND 13 pups
Number of evaluated litters	12	12	12	10
T4 concentration (ng/mL)	42.67	42.00	38.33	42.00	NS
HC range: 32-52	difference (%)	-1.6	-10.2	-1.6
Thyroid gland weights (g)	0.0051	0.0054	0.0054	0.0029**	U
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	U
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

 

Table: Selected organ weight data

Organ weight	Dose groups (dietary concentration)
	Control (0 ppm)	Low dose (1000 ppm)	Mid dose (3000 ppm)	High dose (10000 ppm)
Males
Terminal body weight, g	489.7	495.3	484.9	457.4	NS
	difference (%)	1.2	-1.0	-6.6
Adrenal glands, relative to brain (%) HC: 2.359-4.505	3.185	3.233	3.373	2.696*	DN
	difference (%)	1.5	5.9	-15.4
Thyroid/Parathyroid glands, absolute (g) HC: 0.009-0-059	0.0302	0.0290	0.0275	0.0243**	DN
	difference (%)	-3.9	-8.8	-19.6
Thyroid/Parathyroid glands, relative to body (%) HC: 0.004-0.006	0.00617	0.00587	0.00566	0.00531	NS
	difference (%)	-4.8	-8.3	-13.9
Thyroid/Parathyroid glands, relative to brain (%) HC: 0.421-2.892	1.375	1.321	1.236	1.107**	DN
	difference (%)	-3.9	-10.1	-19.4
Seminal vesicle, absolute (g)	2.943	2.575*	2.676	2.547*	DU
HC: 1.52-3.31	difference (%)	-12.5	-9.1	-13.5
Seminal vesicle, relative to brain (%) HC: 69.7-152.6	134.1	117.7	121.3	116.7*	DU
	difference (%)	-12.3	-9.6	-13.0

 

Females
Terminal body weight, g	352.8	367.8	350.6	283.7**	DU
	difference (%)	4.3	-0.6	-19.6
Adrenal glands, relative to brain (%) HC: 3.8-5.1	4.159	4.095	3.940	3.017**	DN
	difference (%)	-1.5	-5.3	-27.5
Thyroid/Parathyroid glands, absolute (g) HC: 0.018-0.026	0.0210	0.0221	0.0202	0.0206	NS
	difference (%)	5.2	-3.9	-2.0
Thyroid/Parathyroid glands, relative to body (%) HC: 0.0-0.01	0.00594	0.00601	0.00578	0.00744**	DN
	difference (%)	1.2	-2.8	25.2
Thyroid/Parathyroid glands, relative to brain (%)	1.005	1.068	0.989	1.030	NS
HC: 0.9-1.3	difference (%)	6.3	-1.6	2.5
Ovaries, relative to brain (%)	6.26	6.21	6.08	5.32*	DU
HC: 5.4-7.7	difference (%)	-0.8	-3.0	-15.0
Liver, relative to brain (%)	636.75	672.44	626.83	556.24**	DN
HC: 566.2-749.1	difference (%)	5.6	-1.6	-12.6
Brain, relative to body (%)	0.594	0.563	0.596	0.719**	DU
HC: 0.5-0.64	difference (%)	-5.1	0.4	21.1
Heart, relative to brain (%)	57.46	57.10	52.44**	44.28**	DN
HC: 51.8-62.3	difference (%)	-0.6	-8.7	-22.9

Data were rounded to one to three decimal places.

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

DN: Dunnett’s Multiple Range test, DU: Dunn test, NS: Statistically not significant when compared to the control. Bold letters: data out of the historical control range.

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

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. 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.

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 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.

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.