Diammonium hexachloropalladate

Endpoint summary

Description of key information

In an OECD Test Guideline 407 study, to GLP, rats were administered diammonium hexachloropalladate by gavage for 28 days. The systemic NOAEL was the highest tested dose (100 mg/kg bw/day). Although there were some treatment-related effects at this dose (histological inflammation of the glandular stomach mucosa of both sexes and elevated mean white blood cell counts in males), these were considered to reflect a local irritant effect of the test substance rather than systemic toxicity (Matting, 2015). The critical oral NOAEL for diammonium hexachloropalladate (100 mg/kg bw/day) equates to an NOAEL of 30 mg/kg bw/day for palladium (based on MWt ratio).


  


In an GLP OECD Test Guideline 421 reproductive/developmental toxicity screening study (Török-Bathó, 2015), high dose (100 mg/kg bw/day) parental animals displayed only local effects in the glandular stomach mucosa, similar to those seen in the 28-day toxicity study. 




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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 May 2014 to 25 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study to GLP, slight deviations not anticpated to affect study validity or reliability.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

The actual temperature range (20.6-25.8 °C) slightly exceeded the targeted range (22±3°C). Also, the draft report was issued later than planned. These deviations are considered to have no effect on the validity or integrity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld
- Age at study initiation: Young adult rats, at least 8-9 weeks old at start. The age range within the study was kept to the minimum practicable.
- Weight at study initiation: males: 327 g – 377 g ; females: 201 g – 225 g
- Housing: Type II and/or III polypropylene/polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-25.8 °C, (target range 22±3°C)
- Humidity (%): 32 - 69 %, (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: From: To: 28 May 2014 to 25 June 2014

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations at concentrations of 1 mg/mL and 200 mg/mL were stable for at least 7 days refrigerated (2-8°C), including time (four hours) at room temperature for preparation. Based on these results, formulations were prepared weekly. Time elapsing between the end of formulation preparation and completion of the animal treatment did not exceed 7 days.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the formulation and preliminary analytical trials and in agreement with the Sponsor taking into account OECD test guideline recommendations.
- Concentration in vehicle: 2, 6 and 20 mg/ml for low, mid and high doses
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): MKBP7039V
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulations for concentration and checking of homogeneity was performed at the Test Site (Seibersdorf, Austria). Duplicate samples from the top, middle and bottom of each formulation were taken; one set for analysis and one set as back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for analysis to confirm the absence of the test item.

The formulations were homogenous. The actual concentrations were between 89.4% and 100.7% of nominal for the mid (6 mg/mL) and high dose level (20 mg/mL) and were therefore considered acceptable (±15%). For the lowest concentration (2 mg/mL), the mean measured concentration was 85% of nominal and therefore acceptable although, the actual concentration of two of the four preparations was more than 15% below nominal (weeks 1, 2, 3 and 4 were 84.1%, 80.3%, 86.8% and 88.9% respectively). This discrepancy is considered to have no impact on the outcome of the study.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

formulations were administered once daily by oral gavage

Remarks:

Doses / Concentrations:
0
Basis:
other: nominal: gavage

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
other: nominal: gavage

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
other: nominal: gavage

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
other: nominal: gavage

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a preliminary dose range finding study conducted at CiToxLAB Hungary Ltd. with the test item [CiToxLAB study code 13/194-100PE].
- Rationale for animal assignment (if not random): radomisation based on body weight
- Rationale for selecting satellite groups: none were selected
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): no data

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (for signs of morbidity and mortality); daily for general clinical observations
- Cage side observations: Changes in skin, fur, eyes, mucous membranes, occurence 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, stereotypes (e.g. excessive grooming, repetitive circling), '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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first treatment and weekly thereafter (in the morning).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded with a precision of 1g at randomization, on Day 0 and at least weekly, and prior to necropsy (fasted).

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: n/a

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): n/a
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the necropsy
- Anaesthetic used for blood collection: Yes (Pentobarbital)
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the necropsy
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last week of treatment (Day 22/23), each animal was subjected to the functional observation battery, including quantitative assessment of grip strength (manual and instrumental), manual assessment of sensory activity (auditory, visual and proprioceptive), and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessment of motor activity was measured.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / landing foot splay

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal euthanised under pentobarbital anaesthesia by exsanguinations on Day 28. After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY: Yes (see table 3)

Other examinations:

The following organs were weighed in surviving animal:
With precision of at least 0.01g
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus including cervix
Liver
Prostate complex (Prostate with seminal vesicle coagulating gland was measured as a complex)

With precision of at least 0.001g:
Adrenals
Ovaries
Thyroids with parathyroids

Statistics:

The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normally distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparison was performed using the Mann-Whitney U-test. The frequency of clinical observations and necropsy and histopathology findings was calculated as applicable.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality seen. Heightened startle response was noted in one control male from Day 16 until the end of the experiment. A transient noisy respiration was observed in one male in the mid dose group (30 mg/kg bw/day) on Day 21. These observations are unrelated to treatment with the test item.

BODY WEIGHT AND WEIGHT GAIN
There was no adverse effect of treatment on the body weight or body weight gain of the animals. On Day 14, statistically significantly higher (+7% and +8%) than control body weight values were observed in low and high dose group males (10 and 100 mg/kg bw/day respectively) but with no dose-response. A slight, but statistically significant increase in the body weight gain mean values for test group males for the first week of the study was attributed to low body weight gain by the control males.

FOOD CONSUMPTION
Administration of the test item did not cause any adverse effect on the food consumption of the animals.

FOOD EFFICIENCY
Not examined

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

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
An elevation in white blood cell count (WBC) group mean values was noted for high dose males, 100 mg/kg bw/day (p<0.05). This change is considered likely to be a secondary effect of the moderate inflammatory response observed in the gastric mucosa for a number of male animals in that group. In the low and mid dose groups there was no statistical difference in WBC. Female animals were not affected. [See Table under Any other information on results incl. tables, below].

Other minor differences observed in only one sex and/or without dose response were considered to be incidental, the outcome of normal biological variance and without toxicological relevance.

CLINICAL CHEMISTRY
No test item related effects were observed in clinical chemistry parameters. Minor statistical differences observed in only one sex and/or without dose response were considered to be incidental, the outcome of normal biological variance and without toxicological relevance.

URINALYSIS
Not examined

NEUROBEHAVIOUR
The functional observation battery did not reveal any test item related effects. For the control male with heightened startle response, the reaction to the finger approach and finger withdrawal tests, the touch response and position struggle tests was also heightened.

There was no test item-related effect observed from the landing foot splay measurements of the animals.

No test item-related effect was observed on the grip strength of the animals. A statistically significantly lower than control fore limb grip strength group mean value in the female mid dose group (30 mg/kg bw/day) was considered incidental to treatment.

No test item related effect was observed at the automated assessment of locomotor activity over a 60-minute period of observation. Sporadic statistically significant elevations in the total distance travelled mean values were observed for 20 to 25 minutes, 25 to 30 minutes and 50 to 55 minutes for the high dose males (100 mg/kg bw/day), and for 5 to 10 minutes and 40 to 45 minutes for the mid dose females (30 mg/kg bw/day). These changes were considered to be incidental to treatment and of no toxicological relevance based on the lack of consistent response in males and lack of dose-related effect in females and taking into account the pattern of response and the historical control ranges.

ORGAN WEIGHTS
There were no treatment-related effects on the weights of any of the organs. Minor differences observed in thymus and prostate absolute and/or relative organ weight group mean values in the low and mid dose groups only were considered to be incidental due to the lack of dose-response.

GROSS PATHOLOGY
Multifocal dark red discolouration of the glandular stomach mucosa was noted in 4/5 males in the high dose group (100 mg/kg bw/day) and was considered to be a treatment related effect. Females were unaffected. Other observations were considered not to be treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related microscopic findings were observed in the glandular stomach of both male and female High dose group animals.

Diffuse, mixed cellular inflammation correlated with necropsy of the glandular mucosa, with lymphoid follicle formation in all five males. These alterations were visualized grossly as dark/red discoloration of glandular mucosa in four males. A single organized ulcer of the glandular mucosa in one male, reactive focal hyperplasia of the superficial foveolar cells in 2/5 males, was also observed.

Other changes such as unilateral/bilateral, focal/multifocal tubular basophilia in the kidneys (observed in 2/5 High dose, 1/5 Mid dose and 1/5 Low dose males, no basophilia seen in the females), pyelitis in 1/5 Control female, proteinaceous cast in 1/5 High dose female, multifocal, mononuclear cell infiltrate in the prostate of 1/5 Control and 1/5 High dose male and multifocal, mononuclear cell infiltrate in the urinary bladder of 1/5 Control female, were recorded. All these changes had no apparent dose response in incidence/severity and were regarded as incidental or a common background.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicity was observed. Treatment related effects in the high dose group were considered to reflect a local irritant effect of the test substance rather than systemic toxicity.

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Haematology

Dose group	Sex	WBC (K/µL)	Historical mean	Individual range	Historical control range
Control	males	4.882	4.048	2.96 – 6.34	1.1 – 9.41
Low (10 mg/kg bw/day)	males	6.454 (+32%)		3.46 – 11.24
Mid (30 mg/kg bw/day)	males	7.574 (+55%)		4.69 – 11.07
High (100 mg/kg bw/day)	males	9.014 (+85%^*)		6.64 – 10.69

*=p<=0.05

Conclusions:

In a OECD guidleine study, to GLP, rats (5/sex/group) were administered diammonium hexachloropalladate by gavage for 28 consecutive days at 0 (corn oil), 10, 30 or 100 mg/kg bw/day. No treatment-related adverse systemic effects were observed in any group, resulting in a NOAEL for systemic toxicity of 100 mg/kg bw/day (the highest dose tested). Multifocal dark red discolouration of the glandular stomach mucosa was seen in male animals of the high dose group, with associated histological inflammatory changes of the gastric mucosa and a statistically significant elevation in mean white blood cell count. Similar inflammatory changes were evident histopathologically at a lower incidence in some females in the high dose group. These treatment related effects were considered to reflect a local irritant effect of the test substance (local LOAEL of 100 mg/kg bw/day). Therefore, the NOAEL for local effects is 30 mg/kg bw/day.

Executive summary:

The repeated dose toxicity of Diammonium hexachloropalladate was assessed in a 28 -day oral study on Wistar rats, performed according to GLP and OECD Test Guideline 407.

Four groups of rats (containing five males and five females) were gavaged with Diammonium hexachloropalladate via stomach tube at 0 (vehicle control, given corn oil), 10, 30 or 100 mg/kg bw/day. Rats were observed for signs of toxicity and mortality, and changes in body weight. Neurobehaviour was assessed (using a functional observation battery). A On day 28, blood samples were collected for the analysis of haematological parameters and clinical chemistry. This was immediately followed by sacrifice and scheduled necropsy, in which a comprehensive range of organs and tissues were examined macroscopically and microscopically.

Male animals of the high dose (100 mg/kg bw/day) group had multifocal dark red discolouration of the glandular stomach mucosa, with associated histological inflammatory changes of the gastric mucosa and a statistically significant elevation in mean white blood cell count. Similar inflammatory changes were evident histopathologically at a lower incidence in some females in the high dose group. These treatment-related effects in the high dose group were considered to reflect a local irritant effect of the test substances rather than systemic toxicity.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 100 mg/kg bw/day. The NOAEL for local effects was 30 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are no data available in humans relating to repeated dose exposure of diammonium hexachloropalladate. However, a reliable 28-day oral gavage repeated dose toxicity and a reproduction/developmental screening toxicity study have been conducted with diammonium hexachloropalladate.

In the first of these, the repeated dose toxicity of diammonium hexachloropalladate was assessed in a 28-day oral study on Wistar rats, conducted according to GLP and OECD Test Guideline 407 (Matting, 2015). Four groups of rats (containing five males and five females) were gavaged with diammonium hexachloropalladate via stomach tube at 0 (vehicle control, given corn oil), 10, 30 or 100 mg/kg bw/day. Rats were observed for signs of toxicity and mortality, and changes in body weight. Neurobehaviour was assessed (using a functional observation battery). On day 28, blood samples were collected for the analysis of haematological parameters and clinical chemistry. This was immediately followed by sacrifice and scheduled necropsy, in which a comprehensive range of organs and tissues were examined macroscopically and microscopically. Male animals of the high dose (100 mg/kg bw/day) group had multifocal dark red discolouration of the glandular stomach mucosa, with associated histological inflammatory changes of the gastric mucosa and a statistically significant elevation in mean white blood cell count. Similar inflammatory changes were evident histopathologically at a lower incidence in some females in the high dose group. These treatment-related effects in the high dose group were considered to reflect a local irritant effect of the test substance rather than systemic toxicity. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 100 mg/kg bw/day (the highest tested dose). The NOAEL for local effects was 30 mg/kg bw/day. The critical oral NOAEL for diammonium hexachloropalladate (100 mg/kg bw/day) equates to an NOAEL of 30 mg/kg bw/day for palladium (based on MWt ratio).

In support, in an OECD Test Guideline 421 reproductive/developmental toxicity screening study to GLP (Török-Bathó, 2015), high dose (100 mg/kg bw/day) parental animals displayed similar local effects in the glandular stomach mucosa as those seen in the 28-day toxicity study. However, no systemic effects were seen in the parental animals (resulting in a NOAEL of 100 mg/kg bw/day for systemic effects).

According to REACH Annex VIII (EC 1907/2006), repeated dose toxicity studies only need to be conducted on one species taking into consideration the most appropriate route of administration regarding human exposure. The compound is not expected to reach the lungs in appreciable quantities (based on respiratory tract deposition modelling data). Thus, inhalation will not be a significant route of exposure. Similarly, skin contact during production and/or use is expected to be negligible. As the oral route of exposure is considered the most appropriate, repeated dose toxicity studies were not carried out for the dermal or inhalation routes.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable repeated dose toxicity GLP study, conducted according to OECD guidelines.

Justification for classification or non-classification

No adverse systemic effects were seen in reliable guideline studies (28-day oral repeated dose and a reproductive/developmental screening assay) with diammonium hexachloropalladate. The local irritant response is not considered relevant for classification as STOT-RE. As such, the results of these studies indicate that classification of diammonium hexachloropalladate as STOT-RE is not required, according to EU CLP criteria (EC 1272/2008).