tetraammine palladium (II) hydrogen carbonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 1996 to 12 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Reference 2

Reference Type:

other: Commentary

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tetrammine Palladium Hydrogen Carbonate

IUPAC Name:

Tetrammine Palladium Hydrogen Carbonate

Constituent 2

Reference substance name:

134620-00-1

Cas Number:

134620-00-1

IUPAC Name:

134620-00-1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): tetrammine palladium hydrogen carbonate
- Substance type: no data
- Physical state: pale yellow solid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: DF0445
- Expiration date of the lot/batch: no data
- Stability under test conditions: formulations prepared weekly were stable for at least 10 days
- Storage condition of test material: compound stored at room temperature, shielded from light; formulations stored at approximately 4 °C, in the dark

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.