tetraammine palladium (II) hydrogen carbonate

Endpoint summary

Description of key information

In a GLP guideline study (EU Method B.7) rats were gavaged with tetraamminepalladium(II) hydrogen carbonate for 28 days. A NOAEL of 15 mg/kg bw/day is considered appropriate, based on microscopic changes in the spleen in high-dose (150 mg/kg bw/day) animals (Wragg et al., 1997).

 

In an OECD Test Guideline 421 reproductive/developmental toxicity screening study, to GLP, in rats with tetraamminepalladium(II) dichloride, the general systemic toxicity NOAEL for males was the lowest tested dose (4 mg/kg bw/day), on the basis of reduced growth at 20 and 100 mg/kg bw/day (Török-Bathó, 2015).

 

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

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 1996 to 12 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males: 166 to 224 g; females: 144 to 189 g
- Fasting period before study: no data
- Housing: groups of 5 in polypropylene grid-floor cages
- Diet (e.g. ad libitum): ad libitum standard pellet diet
- Water (e.g. ad libitum): ad libitum mains water
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 24 September 1996 to 6 November 1996

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: prepared weekly

VEHICLE
- Concentration in vehicle: 0, 0.15, 1.5 or 15 mg/ml
- Amount of vehicle (if gavage): 10 ml
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Theoretical test material concentrations of 0.15, 1.5 or 15 mg/ml (in distilled water) were analysed via high performance liquid chromatography (HPLC). To determine homogeneity, the top, middle and bottom of the samples were assessed. Stability was evaluated by testing 10 days after formulating. Concentrations of weekly test material formulations were assessed on weeks 1-4, with concentrations found to range between 94 and 107% of the nominal value.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
1.5, 15 or 150 mg/kg bw/day
Basis:
other: nominal

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 14-day range-finding study on 3/sex/dose of the same rat strain, given 0, 15, 150 or 400 mg/kg bw/day

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for mortality and overt clinical signs of toxicity immediately before dosing and one and five hours after dosing during the working week, and observed immediately before dosing and one hour after dosing on weekends

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 (the day before the start of treatment), 7, 14, 21 and 28, and at terminal kill (day 29).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly
- 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: No

FOOD EFFICIENCY: Yes
- 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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily (overt changes only)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all test and control animals
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Animals fasted: No
- How many animals: all test and control animals
- Parameters checked in table No.1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

No other examinations performed

Statistics:

One-way analysis of variance (ANOVA) was used to compare absolute and relative organ weights, haematological and clinical chemistry data, incorporating the F-max test for homogeneity of variance. The Kruskal-Wallis and Mann Whitney non-parametric ANOVA tests were used on data showing heterogeneous variances.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During week 3, reduced growth was seen in males and females given 150 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During week 3, reduced food consumption was seen in males given 150 mg/kg bw/day

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

During week 3, reduced food efficiency was seen in males given 150 mg/kg bw/day

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Reduced haemoglobin, erythrocyte count, haematocrit and mean corpuscular volume in males given 150 mg/kg bw/day. Two of these males had unusually high reticulocyte counts. Slight but significant rise in eosinophil levels in females given 150 mg/kg bw/day

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and relative kidney weights in females given 150 mg/kg bw/day

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pallor of the liver, kidneys and glandular gastric epithelium seen in one male given 150 mg/kg bw/day; reddening of the glandular gastric epithelium seen in one female given 150 mg/kg bw/day

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic changes seen in the spleens of females given 15 mg/kg bw/day, and in the liver, spleen, kidneys and stomach of males and females given 150 mg/kg bw/day. Increased mucus-secreting Goblet cells in glandular gastric mucosa of a 15 mg/kg bw/d male

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
No unscheduled deaths or toxicologically-relevant clinical signs of toxicity seen during the study.
Animals given 150 mg/kg bw/day showed increased salivation before or immediately after dosing from day 14, and sporadic incidences of increased salivation one hour after dosing, and noisy respiration. These effects were considered to result from an unpalatable and/or locally irritating formulation, or the dosing procedure, rather than compound-related systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN: See table 3 for details.
Reduced growth seen in males and females given 150 mg/kg bw/day during study week 3, compared to controls. One of the males showed a slight body weight loss on day 21. Growth was similar to controls during the final week of the experiment.

FOOD CONSUMPTION AND COMPOUND INTAKE:
21% decrease in food consumption in males given 150 mg/kg bw/day during study week 3 (mean 164 g/rat), compared to controls (mean 207 g/rat). Food efficiency also reduced in these males (mean 6%, compared to 15% in controls).

WATER CONSUMPTION AND COMPOUND INTAKE:
No significant effects seen.

HAEMATOLOGY: See table 4 for details.
Reduced haemoglobin, erythrocyte count, haematocrit and mean corpuscular volume in males given 150 mg/kg bw/day. The changes were not statistically significant, but many individual values fell outside the normally-expected range for rats of this age and strain. Two of these males also had unusually high reticulocyte counts. High-dose females showed a slight, but statistically significant, increase in eosinophil count, with two individual values outside the normally expected range. Other effects were not considered to be toxicologically significant.
Males given 150 mg/kg bw/day exhibited a slight, but statistically significant, increase in mean corpuscular haemoglobin concentration, but all individual values were within the normally expected range for rats of this age and strain. A slight, but significant, increase in lymphocyte count was seen in males given 15 or 150 mg/kg bw/day, but the highest individual value was seen in the 15 mg/kg bw/day group, making a dose-response relationship negligible.
Females given 150 mg/kg bw/day exhibited a slight, but statistically significant, increase in platelet count, but, in the absence of other associated changes indicating an adverse effect, this was considered to have arisen by chance.

CLINICAL CHEMISTRY: See table 4 for details.
No toxicologically significant changes in blood chemical parameters measured.
Males given 150 mg/kg bw/day showed a significant reduction in total plasma protein, a slightly increased plasma albumin/globulin ratio, slightly elevated plasma aspartate aminotransferase, and increased plasma glucose concentration compared to controls. The reduced protein level was not supported by an adverse effect on plasma albumin so was not considered to be toxicologically significant. The increased albumin/globulin ratio was thought to be due to a low control mean value. The increased plasma aspartate aminotransferase could not be associated with an adverse effect on a target organ or pathway, and was thought to have arisen by chance. All individual values for plasma glucose concentration in high-dose males were within the normally expected range, so were not considered to be of toxicological importance.
Levels of plasma creatinine were slightly, but significantly, reduced in females given 150 mg/kg bw/day, and plasma inorganic phosphorus levels were slightly increased. As the plasma creatinine change was not indicative of a toxic effect, and no concomitant change in plasma calcium was seen for the phosphorus findings, neither effect was considered toxicologically significant.

ORGAN WEIGHTS: See table 5 for details.
Increased absolute and relative kidney weights seen in females given 150 mg/kg bw/day, compared to controls. Findings not considered to be of toxicological relevance were: slight decrease in absolute, but not relative, liver weight in males given 150 mg/kg bw/day; increased absolute brain and ovary weights in females given 1.5 or 15 mg/kg bw/day, but not at 150 mg/kg bw/day (i.e. could not be related to dose).

GROSS PATHOLOGY:
One male given 150 mg/kg bw/day showed pallor of the liver, kidneys and glandular gastric epithelium. One female showed reddening of the glandular gastric epithelium. Findings not considered to be of toxicological significance were: an enlarged right testis in one male given 150 mg/kg bw/day and a small right testis in one male given 1.5 mg/kg bw/day (in the absence of histopathological data suggesting the testes as target organs); hydronephrosis of the kidney seen in one control and one 15 mg/kg bw/day female (a common condition for laboratory rats of this age and strain).

HISTOPATHOLOGY: NON-NEOPLASTIC: See table 6 for details.
Hepatic, splenic, renal and gastric abnormalities seen. Reduced glycogen type hepatocyte vacuolisation, and reduced splenic Perl's positive haemosiderin pigment accumulation seen in males and females given 150 mg/kg bw/day. The latter effect was also "probably" seen in females given 15 mg/kg bw/day. Deposits of Perl's negative pigment found in the renal proximal tubular epithelium of males and females given 150 mg/kg bw/day. Numbers of mucus-secreting Goblet cells were increased in the glandular gastric mucosa of males and females given 150 mg/kg bw/day and in one male given 15 mg/kg bw/day, with associated mucosal and sub-mucosal inflammatory cell infiltrates and mucosa ulceration in some cases.

A draft commentary on the report (Harleman, 1997) describes the reduced pigment deposition in females given 15 mg/kg bw/day as "slight" and notes that the "degree of pigment deposition in the spleen varies marked in rats". The commentary goes onto say that the "absence of pigment deposition is indicative of a toxicologic effect", whereas "slight variations of pigment deposition are normal and do not have any physicological, pathological, or toxicological significance". The findings are not considered indicative of a systemic adverse effect in this group. Findings in the stomach were considered indicative of local irritation, and not systemic toxicity.

HISTORICAL CONTROL DATA (if applicable): Available.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathology (treatment-related hepatic, splenic, renal and gastric abnormalities)

Critical effects observed:

not specified

Table 3: Average body weights and body weight gains during 28 days of treatment (standard deviation in brackets)

Dose (mg/kg bw/day)	Body Weights (g)					Total Weight Gain
	Week 0	Week 1	Week 2	Week 3	Week 4	g	% of control
	Male
0	204  (13)	258 (15)	299 (17)	331 (19)	364 (21)	160	100
1.5	204 (4)	258 (7)	303 (17)	341 (25)	373 (31)	169	106
15	189 (15)	241 (14)	284 (19)	313 (21)	339 (23)	150	94
150	197 (13)	250 (16)	287 (21)	297 (23)	329 (31)	132	83
	Female
0	161 (6)	181 (7)	198 (6)	216 (9)	232 (7)	71	100
1.5	155 (6)	175 (11)	198 (13)	213 (13)	227 (13)	72	101
15	161 (7)	187 (9)	209 (13)	229 (17)	246 (17)	85	120
150	165 (14)	186 (17)	206 (20)	219 (22)	236 (22)	71	100

Data obtained from pages (46-7) in the study report.

Table 4: Group mean haematological and blood chemistry values (standard deviation in brackets)

Doses (mg/kg bw/day)	0	1.5	15	150	0	1.5	15	150
	male				female
Number of animals/group	5	5	5	5	5	5	5	5
Haematology(day 28)
- RBC (10^12/L)	7.38 (0.25)	7.40  (0.29)	7.26  (0.26)	6.16*  (1.35)	7.20 (0.25)	7.14 (0.23)	7.14 (0.40)	7.22 (0.13)
- MCV (FL)	57.2 (1.1)	57.4 (0.5)	58.6 (2.2)	54.4** (1.3)	56.5 (1.4)	56.9 (1.2)	57.2 (1.0)	55.9 (1.7)
- MCH (pg)	20.4 (0.3)	21* (0.2)	21* (0.7)	20 (0.2)	20.3 (0.4)	20.6 (0.4)	20.4 (0.5)	20.3 (0.6)
- MCHC (g/dL)	35.7 (0.7)	36.5 (0.2)	35.9 (0.9)	36.8* (0.8)	36.0 (1.1)	36.2 (0.3)	35.7 (0.6)	36.4 (1.2)
- HCT (%)	42.2 (1.2)	42.5  (1.9)	42.5 (0.5)	33.5 (7.7)	40.7 (1.6)	40.6 (1.5)	40.8 (1.8)	40.3 (1.5)
- HGB (g/dL)	15.1 (0.6)	15.5  (0.7)	15.2  (0.5)	12.3**  (2.7)	14.6 (0.3)	14.7 (0.5)	14.6 (0.7)	14.7 (0.4)
- WBC (10^9/L)	11.4 (2.5)	14.0 (2.4)	15.6 (5.5)	15.2 (2.1)	9.9 (2.2)	11.8 (5.0)	9.7 (2.2)	9.8 (1.6)
- Neut (10^9/L)	2.77 (1.20)	3.62 (1.75)	3.36 (1.53)	3.06 (1.67)	2.49 (0.73)	2.49 (1.36)	2.29 (0.88)	3.26 (1.69)
- Lymph (10^9/L)	8.41 (1.73)	10.30 (1.46)	11.82* (4.38)	11.90* (0.98)	7.25 (1.68)	9.09 (3.73)	7.18 (1.92)	6.23 (1.04)
- Mono (10^9/L)	0.00 (0.00)	0.03 (0.07)	0.07 (0.10)	0.00 (0.00)	0.02 (0.04)	0.00 (0.00)	0.02 (0.04)	0.06 (0.09)
- Eos (10^9/L)	0.24 (0.19)	0.10 (0.16)	0.33 (0.18)	0.26 (0.10)	0.14 (0.05)	0.22 (0.10)	0.17 (0.10)	0.29* (0.09)
- Bas (10^9/L)	0.00 (0.00)	0.00 (0.00)	0.00 (0.00)	0.00 (0.00)	0.00 (0.00)	0.00 (0.00)	0.00 (0.00)	0.00 (0.00)
- CT (secs)	27 (1)	26 (2)	28 (1)	27 (2)	27 (2)	27 (2)	26 (2)	26 (1)
- PLT (10^9/L)	1074 (118)	1102 (93)	1012 (113)	1294 (280)	993 (53)	1020 (111)	1076 (91)	1180* (183)
- Retic (%)	8 (2)	9 (1)	10 (2)	17 (13)	8 (3)	8 (2)	7 (3)	8 (2)
Blood chemistry(day 28)
- urea (mg/dL)	33 (2)	33 (4)	34 (4)	34 (1)	37 (4)	36 (5)	32 (6)	44 (7)
- glucose (mg/dL)	141 (8)	145 (7)	143 (9)	168*** (5)	153 (7)	153 (12)	140 (8)	174 (38)
- total protein (g/dL)	6.94 (0.32)	6.93 (0.41)	6.93 (0.53)	6.16** (0.30)	6.93 (0.41)	7.14 (0.40)	6.94 (0.30)	6.71 (0.24)
- albumin (g/dL)	3.44 (0.06)	3.42 (0.12)	3.40 (0.15)	3.31 (0.12)	3.77 (0.18)	3.80 (0.20)	3.76 (0.17)	3.70 (0.20)
- A/G ratio	0.99 (0.09)	0.99 (0.15)	0.98 (0.15)	1.17* (0.08)	1.19 (0.06)	1.14 (0.06)	1.18 (0.02)	1.23 (0.07)
- sodium (MMOL/L)	143 (2)	142 (1)	143 (1)	143 (1)	144 (2)	145 (1)	145 (2)	144 (2)
- potassium (MMOL/L)	5.02 (0.17)	5.18 (0.20)	4.94 (0.23)	4.77 (0.44)	4.76 (0.41)	4.70 (0.44)	4.81 (0.16)	5.06 (0.17)
- chloride (MMOL/L)	106 (2)	105 (1)	107 (2)	105 (2)	108 (2)	109 (2)	108 (1)	107 (2)
- calcium (MMOL/L)	2.69 (0.04)	2.66 (0.09)	2.69 (0.04)	2.64 (0.11)	2.68 (0.07)	2.70 (0.12)	2.71 (0.04)	2.66 (0.07)
- phosphorus (MMOL/L)	2.69 (0.15)	2.78 (0.22)	2.84 (0.13)	2.90 (0.27)	2.29 (0.30)	2.35 (0.11)	2.29 (0.17)	2.68* (0.27)
- ASAT (IU/L)	95 (15)	91 (9)	95 (11)	116* (13)	94 (10)	93 (8)	91 (7)	106 (12)
- ALAT (IU/L)	84 (13)	78 (7)	83 (13)	82 (11)	76 (18)	63 (13)	60 (9)	62 (4)
- AP (IU/L)	584 (66)	521 (124)	568 (90)	529 (114)	393 (67)	354 (59)	365 (46)	391 (104)
- creatinine (mg/dL)	0.53 (0.02)	0.55 (0.03)	0.54 (0.04)	0.54 (0.05)	0.61 (0.03)	0.60 (0.02)	0.58 (0.04)	0.56* (0.03)
- bilirubin (mg/dL)	0.11 (0.02)	0.11 (0.02)	0.11 (0.01)	0.12 (0.03)	0.10 (0.01)	0.09 (0.01)	0.10 (0.02)	0.10 (0.01)
- CHE (KU/L)	0.15 (0.03)	0.16 (0.04)	0.15 (0.02)	0.15 (0.03)	0.75 (0.18)	0.83 (0.31)	0.89 (0.14)	0.73 (0.18)

Statistics: * P < 0.05   ** P < 0.01  *** P < 0.001

Conclusions:

In a GLP guideline study (EU Method B.7), rats were gavaged with tetraamminepalladium(II) hydrogen carbonate for 28 days. A NOAEL of 15 mg/kg bw/day was obtained based on microscopic changes in the spleen in high-dose animals (150 mg/kg bw/day)

Executive summary:

In a GLP study, performed in accordance with EU Method B.7, male and female rats were treated orally with tetraamminepalladium(II) hydrogen carbonate for 28 days.

 

Groups of Sprague-Dawley rats (5/sex) were given daily gavage administrations of tetraamminepalladium(II) hydrogen carbonate at 0 (distilled water), 1.5, 15 or 150 mg/kg bw/day for 28 days. Throughout the study, animals were observed daily for mortality and overt clinical signs of toxicity. Body weight and food and water consumption were monitored. At the end of the study, blood samples were taken for haematological and clinical chemistry assessments, and animals were killed and subject to gross necropsy. The major organs of all treated and control animals were weighed, and the adrenals, heart, kidney, liver, spleen, stomach and testes of high-dose and control animals were examined microscopically. Having been identified as possible target organs, the liver, spleen, kidney and stomach were subsequently examined microscopically in all treated and control animals.

 

No unscheduled deaths or treatment-related overt clinical signs of toxicity were seen over the course of the study. During study week 3, growth was reduced in high-dose males and females, and high dose males demonstrated reduced food consumption and food efficiency – but these effects were not seen during week 4. High-dose males demonstrated reduced haemoglobin, erythrocyte count, haematocrit and mean corpuscular volume, with two rats having unusually high reticulocyte counts. A slight but significant rise in eosinophil levels was seen in high-dose females. At necropsy, absolute and relative kidney weights were increased in high-dose females. One high-dose male had a pale liver, kidney and glandular gastric epithelium, while one high-dose female had a reddened glandular gastic epithelium. Numbers of mucus-secreting Goblet cells were increased in the glandular gastric mucosa of one male given 15 mg/kg bw/day, indicating a local irritant effect of treatment. Treatment-related microscopic changes were seen in the spleens of females given 15 mg/kg bw/day (slightly reduced splenic haemosiderin deposition), and in the liver, spleen, kidneys and stomach of males and females given 150 mg/kg bw/day.

A draft commentary on the report notes that the "degree of pigment deposition in the spleen varies marked in rats" and that the "absence of pigment deposition is indicative of a toxicologic effect". However, "slight variations of pigment deposition are normal and do not have any physiological, pathological, or toxicological significance". Therefore, the overall no-observed-adverse-effect level (NOAEL) was considered to be 15 mg/kg bw/day, with the critical effect being microscopic changes in the spleen (no haemosiderin deposition in 5/5 males and 2/5 females, compared to 0 controls) in high-dose animals. Changes seen in the stomach have been considered indicative of local irritation.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

other: Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June - 09 August 2014 (last necropsy)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study, conducted to GLP. However, as a reproducitve and developmental screening assay, it has some limitations compared to a standard repeated dose assay assessing general systemic toxicity (e.g. no haematology, clinical chemistry or urinalysis was conducted, and histopathology was limited to the high dose group).

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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, from SPF colony
- Age at study initiation: Young adult rats, at least 10 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no data
- Housing: Type II and III polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany offered ad libitum
- Water (e.g. ad libitum): tap water from municipal supply as for human consumption from 500 ml bottle ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.8 °C (target range 22±3°C)
- Humidity (%): 40 - 70 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
Start of experiment (start of treatment): 24 June 2014
End of treatment: 22 July 2014 (males); 08 August 2014 (females)
End of experiment: 09 August 2014 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

other: an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was used as the dosing vehicle.

The buffer was prepared as follows:
2.68 g ammonium chloride (Batch/Lot No.: BCBM5575V, Supplier: Sigma-Aldrich, Expiry: March 2015) was dissolved in 1000 ml distilled water (Batch/Lot No.: 0271113, Supplier: TEVA Pharmaceutical Works PLtd., Expiry: November 2016).
The pH was adjusted to 9 by adding approximately 1650-1850 µL of 29.1% ammonium hydroxide (Batch/Lot No.: SZBD2260V, Supplier: Sigma-Aldrich, Expiry: August 2015).

The first preparation was used for 7 consecutive days and was kept at room temperature. The next preparations were used for periods of up to 14 days, following confirmation of the stability, and the stock solution was kept refrigerated (2-8ºC). Dose solutions for treatment were diluted from the stock solution with aqueous ammonium-chloride buffer to the concentrations of 4.0 and 0.8 mg/mL, daily just before the treatment. The stock solution was prepared four times during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of the preliminary formulation trial and pilot DRF study (CiToxLAB study code: 13/195-100PE), an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

- Concentration in vehicle: The test item was formulated in the aqueous ammonium-chloride buffer as a stock solution at the concentration of 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the vehicle was assessed in the concentration range of 5 mg/mL-200 mg/mL by Fourier-transformation infrared spectroscopy (FT-IR). According to the results the test item solution in aqueous ammonium-chloride buffer was stable over 7 days at room temperature and for 14 days when refrigerated (2-8ºC). (CiToxLAB Study code: 13/195-929AN).

Analysis of test item formulations was performed at the Test Site using a validated ICP method (ICP-AES) to determine the palladium content. The concentration measurements were performed on all preparations of the stock solutions and at the same time, from samples of diluted dose solutions. The sampling occasions coincided with the preparation of the stock solution.

Samples were taken from the test item formulations (including control) for concentration measurements. One set of samples was collected for analysis and one set retained as a back-up, for possible confirmatory analysis. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of the test item. No confirmatory analysis was required.

The measured concentrations of palladium in the formulations varied between 95.0 % and 111.9 % of the nominal. These results were within the range of acceptable values (85% - 115%).

Duration of treatment / exposure:

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

Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
4 mg/kg bw/day
Basis:
other: nominal dose

Remarks:

Doses / Concentrations:
20 mg/kg bw/day
Basis:
other: nominal dose

Remarks:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director, based on available data, including the results of a dose range finding study (CiToxLAB study code 13/195-100PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Adult (parental) animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. The changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were performed once prior to first treatment (to allow for within-subject comparisons), and once a week thereafter. The animals were examined outside the home cage in a standard arena and at similar times on each occasion. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and prior to necropsy.

Adult females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before necropsy).

The body weights of the females were also recorded on GD4, 10 and 17 in order to give accurate treatment volumes, however, these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 and then weekly thereafter.

FOOD EFFICIENCY: No data

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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

SACRIFICE
- Male animals: All animals were sacrificed on test day 29, after a dosing period of 28 days.
- Maternal animals: Dams were sacrificed following at least 4 days post-partum dosing.

GROSS NECROPSY
- Gross necropsy consisted of external appearance. The cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Special attention was paid to the organs of the reproductive system. The number of implantation sites and the number of corpora lutea were recorded. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on testes, epididymides and ovaries in the control and high dose groups. In addition, gross lesions (the stomachs of 9 males and 5 females from the High Dose) were also examined microscopically. The stomachs of two control males and females were also examined for comparison.

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

Statistics:

Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight growth reductions in the two highest dose groups (slight transient body weight loss in the highest dose group)

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Discolouration of the glandular stomach and epididymis were apparent in the highest tested dose group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Effects on the glandular stomach apparent in the highest tested dose group

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no clinical signs related to treatment. All animals were clinically normal.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights of males treated at 100 mg/kg/day were slightly lower than controls from Week 1 onwards. This was due in part to the higher mean body weight for the control group. The differences from control attained statistical significance (p<0.01 on Days 7, 14, 21). Compared to the controls, mean value was approximately 7% lower on Day 27, and the difference was statistically significant (p<0.05).

Compared to the control, significantly lower body weight gain was noted for High dose males during Weeks 1 (p<0.01) and 3 (not statistically significant) resulting in lower overall (Days 0-27) gain value by 25% (p<0.05). In 5 of 12 males at 100 mg/kg/day suppressed body weight gain or slight body weight loss was recorded at the beginning of the treatment period (Days 0-3). For one male no body weight gain or a slight body weight loss was noted during the last week of the treatment. This animal had the most severe changes in the stomach in the form of mild inflammation and ulceration in the glandular stomach.

Body weight of males at 20 mg/kg/day was lower than control mean throughout the entire treatment period by 6-7%, and the differences were statistically significant (p<0.01 on days 7, 14 and p<0.05 on days 21 and 27). The body weight gain values of these males were significantly lower, than control mean during Weeks 1 (p<0.01) and 2 (not statistically significant). The overall (Days 0-27) body weight gain value was lower than the control mean by 22% (p<0.05). The individual body weight gain values were within the control range, with the exception of one male which had consistently suppressed body weight gain or slight body weight loss throughout the entire treatment period.

The mean body weight of males at 4 mg/kg/day did not differ significantly from the control mean.

It should be noted, that the mean body weight of control males was slightly higher (by approximately 2%) on Day 0 and the body weight values were in the higher range. In addition, two control males had overall body weight gain value higher than group mean by 25-30%.

Females at 100, 20 and 4 mg/kg/day had mean body weights and body weight gains comparable to the controls throughout the entire treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The average food consumption of males in all test item treated groups was lower, than control during Week 2 by approximately 12-13% at 4 and 100 mg/kg/day, and by 18% at 20 mg/kg/day. The differences attained statistical significance: p<0.01 for Low and Mid dose groups and p<0.05 for High Dose group, but were not clearly treatment related due to the lack of dose-response.

Lower than control mean food consumption was also recorded for all test item treated groups of males from the end of mating period up to Day 21. The differences were statistically significant (p<0.05) and attributed to the higher control value.

There were two males in the control group with food consumption well above the control mean.

The average food consumption in females in all test item treated groups was comparable with the control mean throughout the entire treatment period.

The test item formulations were found to be in the range of 95.0 and 111.9 % of of nominal concentrations and were therefore considered acceptable.

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable
OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOUR: No data

ORGAN WEIGHTS: There were no test item related effects on organ weights. Occasional statistically significant differences in absolute testis and prostate weight were attributed to the lower body weights of the animals and therefore of no toxicologically significance.

GROSS PATHOLOGY: At necropsy, test item-related findings were observed at 100 mg/kg/day (High dose) in the form of focal/multifocal dark red discoloration of the glandular mucosa of the stomach in 9/12 males and 5/12 females.

Unilateral, focal, yellow/green discoloration of the epididymis was noted in 1/12 males in the High dose.

HISTOPATHOLOGY: NON-NEOPLASTIC: Test item-related microscopic findings were observed in the glandular stomach of animals at 100 mg/kg/day (High dose). In all males with macroscopic changes (dark red discoloration), congestion was noted in the glandular mucosa of the stomach (9/9). In addition, 7/9 of these males had minimal to mild, mixed cellular infiltration, and minimal to mild mixed cellular inflammation was observed in 2/9 males. In one animal organizing ulcer in the glandular mucosa was found additionally. In 5/5 all High dose females congestion of the glandular mucosa of the stomach with minimal, mixed cellular infiltration was detected. The observation was in agreement with necropsy finding.

No test item-related microscopic findings were noted in the reproductive system of the males and females from the High dose group. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally present.

The spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

The unilateral, focal, yellow/green discoloration of the epididymis in 1/12 males in the High dose was microscopically identified as moderate spermatocoele and was regarded as a background observation.

The other microscopic changes were regarded as incidental or background.

HISTOPATHOLOGY: NEOPLASTIC: Not applicable

HISTORICAL CONTROL DATA: Not applicable

OTHER FINDINGS: The spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decreased body weight gain at 20 and 100 mg/kg bw/day.

Critical effects observed:

not specified

Conclusions:

In an OECD Test Guideline 421 reproductive/developmental toxicity screening study, to GLP, in rats with tetraamminepalladium dichloride, the general systemic toxicity NOAEL for males was the lowest tested dose (4 mg/kg bw/day), on the basis of reduced growth at 20 and 100 mg/kg bw/day.

Executive summary:

In a OECD Test Guideline 421 reproductive/developmental toxicity study, conducted according to GLP, rats (12/sex/group) received a solution of tetraamminepalladium dichloride by gavage at doses of 0, 4, 20, or 100 mg/kg bw/day for at least 28 days (males were dosed for 28-days in total, while females received treatment for a longer period of time [incorporating the gestation period and proceeding up until postpartum day 4, i.e. around 7-8 weeks]). This reproductive and developmental screening assay, has some limitations compared to a standard repeated dose assay for assessing general systemic toxicity. Notably, no haematology, clinical chemistry or urinalysis. Histopathology was limited to the high dose group and focused on the reproductive organs and gross lesions.

Effects on the glandular stomach (including discolouration, inflammation, and congestion) were seen in the high-dose animals, and likely reflects a local effect of treatment. These effects in the glandular stomach may have contributed to the significantly reduced body weight gain seen in males at 20 and 100 mg/kg bw/day (growth of females was unaffected).The NOAEL for systemic toxicity was 4 mg/kg bw/day on the basis of reduced growth in males at 20 and 100 mg/kg bw/day.

Reference 3

Endpoint:

repeated dose toxicity: oral, other

Remarks:

other: Reproduction/Developmental Toxicity Screening Test

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

24 June - 09 August 2014 (last necropsy)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study, conducted to GLP. However, as a reproducitve and developmental screening assay, it has some limitations compared to a standard repeated dose assay assessing general systemic toxicity (e.g. no haematology, clinical chemistry or urinalysis was conducted, and histopathology was limited to the high dose group).

Justification for type of information:

Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the acetate, chloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide, dichloride and di(hydrogencarbonate) are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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, from SPF colony
- Age at study initiation: Young adult rats, at least 10 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no data
- Housing: Type II and III polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany offered ad libitum
- Water (e.g. ad libitum): tap water from municipal supply as for human consumption from 500 ml bottle ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.8 °C (target range 22±3°C)
- Humidity (%): 40 - 70 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
Start of experiment (start of treatment): 24 June 2014
End of treatment: 22 July 2014 (males); 08 August 2014 (females)
End of experiment: 09 August 2014 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

other: an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was used as the dosing vehicle.

The buffer was prepared as follows:
2.68 g ammonium chloride (Batch/Lot No.: BCBM5575V, Supplier: Sigma-Aldrich, Expiry: March 2015) was dissolved in 1000 ml distilled water (Batch/Lot No.: 0271113, Supplier: TEVA Pharmaceutical Works PLtd., Expiry: November 2016).
The pH was adjusted to 9 by adding approximately 1650-1850 µL of 29.1% ammonium hydroxide (Batch/Lot No.: SZBD2260V, Supplier: Sigma-Aldrich, Expiry: August 2015).

The first preparation was used for 7 consecutive days and was kept at room temperature. The next preparations were used for periods of up to 14 days, following confirmation of the stability, and the stock solution was kept refrigerated (2-8ºC). Dose solutions for treatment were diluted from the stock solution with aqueous ammonium-chloride buffer to the concentrations of 4.0 and 0.8 mg/mL, daily just before the treatment. The stock solution was prepared four times during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of the preliminary formulation trial and pilot DRF study (CiToxLAB study code: 13/195-100PE), an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

- Concentration in vehicle: The test item was formulated in the aqueous ammonium-chloride buffer as a stock solution at the concentration of 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the vehicle was assessed in the concentration range of 5 mg/mL-200 mg/mL by Fourier-transformation infrared spectroscopy (FT-IR). According to the results the test item solution in aqueous ammonium-chloride buffer was stable over 7 days at room temperature and for 14 days when refrigerated (2-8ºC). (CiToxLAB Study code: 13/195-929AN).

Analysis of test item formulations was performed at the Test Site using a validated ICP method (ICP-AES) to determine the palladium content. The concentration measurements were performed on all preparations of the stock solutions and at the same time, from samples of diluted dose solutions. The sampling occasions coincided with the preparation of the stock solution.

Samples were taken from the test item formulations (including control) for concentration measurements. One set of samples was collected for analysis and one set retained as a back-up, for possible confirmatory analysis. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of the test item. No confirmatory analysis was required.

The measured concentrations of palladium in the formulations varied between 95.0 % and 111.9 % of the nominal. These results were within the range of acceptable values (85% - 115%).

Duration of treatment / exposure:

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

Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
4 mg/kg bw/day
Basis:
other: nominal dose

Remarks:

Doses / Concentrations:
20 mg/kg bw/day
Basis:
other: nominal dose

Remarks:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director, based on available data, including the results of a dose range finding study (CiToxLAB study code 13/195-100PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Adult (parental) animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. The changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were performed once prior to first treatment (to allow for within-subject comparisons), and once a week thereafter. The animals were examined outside the home cage in a standard arena and at similar times on each occasion. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and prior to necropsy.

Adult females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before necropsy).

The body weights of the females were also recorded on GD4, 10 and 17 in order to give accurate treatment volumes, however, these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 and then weekly thereafter.

FOOD EFFICIENCY: No data

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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

SACRIFICE
- Male animals: All animals were sacrificed on test day 29, after a dosing period of 28 days.
- Maternal animals: Dams were sacrificed following at least 4 days post-partum dosing.

GROSS NECROPSY
- Gross necropsy consisted of external appearance. The cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Special attention was paid to the organs of the reproductive system. The number of implantation sites and the number of corpora lutea were recorded. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on testes, epididymides and ovaries in the control and high dose groups. In addition, gross lesions (the stomachs of 9 males and 5 females from the High Dose) were also examined microscopically. The stomachs of two control males and females were also examined for comparison.

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

Statistics:

Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight growth reductions in the two highest dose groups (slight transient body weight loss in the highest dose group)

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Discolouration of the glandular stomach and epididymis were apparent in the highest tested dose group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Effects on the glandular stomach apparent in the highest tested dose group

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no clinical signs related to treatment. All animals were clinically normal.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights of males treated at 100 mg/kg/day were slightly lower than controls from Week 1 onwards. This was due in part to the higher mean body weight for the control group. The differences from control attained statistical significance (p<0.01 on Days 7, 14, 21). Compared to the controls, mean value was approximately 7% lower on Day 27, and the difference was statistically significant (p<0.05).

Compared to the control, significantly lower body weight gain was noted for High dose males during Weeks 1 (p<0.01) and 3 (not statistically significant) resulting in lower overall (Days 0-27) gain value by 25% (p<0.05). In 5 of 12 males at 100 mg/kg/day suppressed body weight gain or slight body weight loss was recorded at the beginning of the treatment period (Days 0-3). For one male no body weight gain or a slight body weight loss was noted during the last week of the treatment. This animal had the most severe changes in the stomach in the form of mild inflammation and ulceration in the glandular stomach.

Body weight of males at 20 mg/kg/day was lower than control mean throughout the entire treatment period by 6-7%, and the differences were statistically significant (p<0.01 on days 7, 14 and p<0.05 on days 21 and 27). The body weight gain values of these males were significantly lower, than control mean during Weeks 1 (p<0.01) and 2 (not statistically significant). The overall (Days 0-27) body weight gain value was lower than the control mean by 22% (p<0.05). The individual body weight gain values were within the control range, with the exception of one male which had consistently suppressed body weight gain or slight body weight loss throughout the entire treatment period.

The mean body weight of males at 4 mg/kg/day did not differ significantly from the control mean.

It should be noted, that the mean body weight of control males was slightly higher (by approximately 2%) on Day 0 and the body weight values were in the higher range. In addition, two control males had overall body weight gain value higher than group mean by 25-30%.

Females at 100, 20 and 4 mg/kg/day had mean body weights and body weight gains comparable to the controls throughout the entire treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The average food consumption of males in all test item treated groups was lower, than control during Week 2 by approximately 12-13% at 4 and 100 mg/kg/day, and by 18% at 20 mg/kg/day. The differences attained statistical significance: p<0.01 for Low and Mid dose groups and p<0.05 for High Dose group, but were not clearly treatment related due to the lack of dose-response.

Lower than control mean food consumption was also recorded for all test item treated groups of males from the end of mating period up to Day 21. The differences were statistically significant (p<0.05) and attributed to the higher control value.

There were two males in the control group with food consumption well above the control mean.

The average food consumption in females in all test item treated groups was comparable with the control mean throughout the entire treatment period.

The test item formulations were found to be in the range of 95.0 and 111.9 % of of nominal concentrations and were therefore considered acceptable.

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable
OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOUR: No data

ORGAN WEIGHTS: There were no test item related effects on organ weights. Occasional statistically significant differences in absolute testis and prostate weight were attributed to the lower body weights of the animals and therefore of no toxicologically significance.

GROSS PATHOLOGY: At necropsy, test item-related findings were observed at 100 mg/kg/day (High dose) in the form of focal/multifocal dark red discoloration of the glandular mucosa of the stomach in 9/12 males and 5/12 females.

Unilateral, focal, yellow/green discoloration of the epididymis was noted in 1/12 males in the High dose.

HISTOPATHOLOGY: NON-NEOPLASTIC: Test item-related microscopic findings were observed in the glandular stomach of animals at 100 mg/kg/day (High dose). In all males with macroscopic changes (dark red discoloration), congestion was noted in the glandular mucosa of the stomach (9/9). In addition, 7/9 of these males had minimal to mild, mixed cellular infiltration, and minimal to mild mixed cellular inflammation was observed in 2/9 males. In one animal organizing ulcer in the glandular mucosa was found additionally. In 5/5 all High dose females congestion of the glandular mucosa of the stomach with minimal, mixed cellular infiltration was detected. The observation was in agreement with necropsy finding.

No test item-related microscopic findings were noted in the reproductive system of the males and females from the High dose group. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally present.

The spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

The unilateral, focal, yellow/green discoloration of the epididymis in 1/12 males in the High dose was microscopically identified as moderate spermatocoele and was regarded as a background observation.

The other microscopic changes were regarded as incidental or background.

HISTOPATHOLOGY: NEOPLASTIC: Not applicable

HISTORICAL CONTROL DATA: Not applicable

OTHER FINDINGS: The spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decreased body weight gain at 20 and 100 mg/kg bw/day.

Critical effects observed:

not specified

Conclusions:

In an OECD Test Guideline 421 reproductive/developmental toxicity screening study, to GLP, in rats with tetraamminepalladium dichloride, the general systemic toxicity NOAEL for males was the lowest tested dose (4 mg/kg bw/day), on the basis of reduced growth at 20 and 100 mg/kg bw/day.

Executive summary:

In a OECD Test Guideline 421 reproductive/developmental toxicity study, conducted according to GLP, rats (12/sex/group) received a solution of tetraamminepalladium dichloride by gavage at doses of 0, 4, 20, or 100 mg/kg bw/day for at least 28 days (males were dosed for 28-days in total, while females received treatment for a longer period of time [incorporating the gestation period and proceeding up until postpartum day 4, i.e. around 7-8 weeks]). This reproductive and developmental screening assay, has some limitations compared to a standard repeated dose assay for assessing general systemic toxicity. Notably, no haematology, clinical chemistry or urinalysis. Histopathology was limited to the high dose group and focused on the reproductive organs and gross lesions.

Effects on the glandular stomach (including discolouration, inflammation, and congestion) were seen in the high-dose animals, and likely reflects a local effect of treatment. These effects in the glandular stomach may have contributed to the significantly reduced body weight gain seen in males at 20 and 100 mg/kg bw/day (growth of females was unaffected).The NOAEL for systemic toxicity was 4 mg/kg bw/day on the basis of reduced growth in males at 20 and 100 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

4 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

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

No relevant human data were identified. However, a reliable 28-day oral gavage repeated dose toxicity and a reproduction/developmental screening toxicity study (both in rats) have been conducted with tetraamminepalladium(II) hydrogen carbonate and tetraamminepalladium(II) dichloride, respectively. Tetraamminepalladium(II) dichloride and hydrogen carbonate fall within the scope of the read-across category "tetraamminepalladium salts". See IUCLID section 13 for full read-across justification report.

 

The repeated dose toxicity of tetraamminepalladium(II) hydrogen carbonate was assessed in a 28-day oral GLP study conducted according to EU Method B.7. Groups of Sprague-Dawley rats (5/sex) were given a daily gavage administration of tetraamminepalladium(II) hydrogen carbonate at 0 (distilled water), 1.5, 15 or 150 mg/kg bw/day for 28 days. Throughout the study, animals were observed daily for mortality and other overt clinical signs of toxicity. Body weight and food and water consumption were monitored. At the end of the study, blood samples were taken for haematological and clinical chemistry assessments, and animals were killed and subject to gross necropsy. The major organs of all animals were weighed, and the adrenals, heart, kidney, liver, spleen, stomach and testes of high-dose and control animals were examined microscopically. Having been identified as possible target organs, the liver, spleen, kidneys and stomach were subsequently examined microscopically in all treated and control animals. No unscheduled deaths or treatment-related overt clinical signs of toxicity were seen over the course of the study. During study week 3, growth was reduced in high-dose males and females, and high-dose males demonstrated reduced food consumption and food efficiency – but these effects were not seen during week 4. High-dose males demonstrated reduced haemoglobin, erythrocyte count, haematocrit and mean corpuscular volume, with two rats having unusually high reticulocyte counts. A slight but significant rise in eosinophil levels was seen in high-dose females. At necropsy, absolute and relative kidney weights were increased in high-dose females. One high-dose male had a pale liver, kidney and glandular gastric epithelium, while one high-dose female had a reddened glandular gastric epithelium. A draft commentary on the report notes that the "degree of pigment deposition in the spleen varies marked in rats" and that the "absence of pigment deposition is indicative of a toxicologic effect". However, "slight variations of pigment deposition are normal and do not have any physiological, pathological or toxicological significance". Therefore, the overall NOAEL was considered to be 15 mg/kg bw/day, with the critical effect being microscopic changes in the spleen (no haemosiderin deposition in 5/5 males and 2/5 females, compared to 0 controls) in high-dose animals. Changes seen in the stomach have been considered indicative of local irritation (Wragg et al., 1997).

 

In a OECD Test Guideline 421 reproductive/developmental toxicity study, conducted according to GLP, rats (12/sex/group) received a solution of tetraamminepalladium(II) dichloride by gavage at doses of 0, 4, 20, or 100 mg/kg bw/day for at least 28 days (males were dosed for 28 days in total, while females received treatment for a longer period of time [incorporating the gestation period and proceeding up until postpartum day 4, i.e. around 7-8 weeks]). This reproductive and developmental screening assay has some limitations compared to a standard repeated dose assay for assessing general systemic toxicity (e.g. no haematology, clinical chemistry or urinalysis, and histopathology was limited to the high dose group). Effects on the glandular stomach (including discolouration, inflammation, and congestion) were seen in the high-dose animals, and likely reflect a local effect of treatment. These effects in the glandular stomach may have contributed to the significantly reduced body weight gain seen in males at 20 and 100 mg/kg bw/day (growth of females was unaffected). Although likely a consequence of local effects on the stomach, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 4 mg/kg bw/day on the basis of reduced growth in males at 20 and 100 mg/kg bw/day (Török-Bathó, 2015). [This NOAEL of 4 mg/kg bw/day is used as the health-precautionary critical point of departure for DNEL derivation purposes.] The critical oral NOAEL for tetraamminepalladium(II) dichloride (4 mg/kg bw/day) equates to an NOAEL of 1.76 mg/kg bw/day for palladium (based on MWt ratio).

Justification for classification or non-classification

Based on the histopathological effects on the spleen observed at the top dose (150 mg/kg bw/day) in the 28-day rat study, tetraamminepalladium(II) hydrogen carbonate should be classified as STOT-RE 2 according to EU CLP criteria (EC 1272/2008). This is in-line with the harmonised classification for this substance. The observed effects on the stomach are likely the result of local irritancy.