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Toxicological information

Repeated dose toxicity: other routes

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Administrative data

Endpoint:
short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientifically principles, acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Glomerular calcification induced by bolus injection with dibasic sodium phosphate solution in Sprague-Dawley rats
Author:
Tsuchiya N, Matsushima S, Takasu N, Kyokawa Y, Torii M
Year:
2004
Bibliographic source:
Toxicol Pathol. 32(4):408-12; PMID: 15204963

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male Sprague-Dawley rats were treated with disodium hydrogenorthophosphate (0 - 408 mg/kg bw/day) by injection into the tail vain for 14 days. Urinalysis was carried out on days 7 and 13 of dosing, and rats were sacrificed for blood chemistry and histopathological examination of the kidneys.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dibasic sodium phosphate, Na2HPO4.
- Supplier: Wako Pure Chemical Industries Ltd., Osaka, Japan

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Clea Japan Inc. (Shiga, Japan)
- Age at study initiation: 5 weeks (35 days)
- Housing: 2 animals per cage in plastic cages in an animal room under controlled conditions
- Diet (e.g. ad libitum): pelleted diet (CA-1, Clea Japan Inc., Tokyo)
- Water (e.g. ad libitum): ad libitum by automatic watering system after filtration (30- and 3-µm pore filter) followed by UV-irradiation
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±3°C
- Humidity (%): 50±20%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intravenous
Vehicle:
physiological saline
Remarks:
pH adjusted to 5.5-6.5 with 1N sulfuric acid
Details on exposure:
The rats were administered disodium hydrogenorthophosphate at 4 mL/kg bw/min once daily via the tail vain through a polypethylene catheter connected to a plastic syringe mounted on an infusion pump (STC-525, Terumo Corp., Tokyo).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Animals were treated for 14 days.
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
360 mM disodium hydrogenorthophosphate at 8 mL/kg bw, equivalent to a dose of 408 mg/kg bw/day.
No. of animals per sex per dose:
Test group: 6 animals per time point
control group: 6 animals per time point
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: on days7 and 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
URINALYSIS
All rats were placed in individual metabolic cages and deprived of food and water, and urine was collected 4 hours after dosing on days 7 and 13. Analysed parameters are listed in table 1.

HISTOPATHOLOGY
Rats were euthanized under pentobarbital sodium anesthesia by exsanguination and necropsied. The kidneys were removed and weighed together (Mettler PM400). Organs listed in Table 2 were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin and examined microscopically. Paraffin sections were prepared and stained with hematoxylin and eosin (H&E). The kidney sections were also stained with von Kossa stain and periodic acid Schiff (PAS) reagent.

BLOOD CHEMISTRY
Immediately prior to necropsy, blood was collected from the posterior vena cava under sodium pentobarbital anesthesia in a vacuum blood-collecting tube containing sodium heparin. Parameters determined are listed in table 1.
Other examinations:
For electron microscopy, kidney tissues that were excised from 2 animals per group were fixed in 3% glutaraldehyde and 2% osmic acid and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy (JEM-1010, JEOL Ltd., Tokyo). Additionally, granular deposits in the glomerulus were analyzed using X-ray microanalysis (LINKISIS, Oxford).
Statistics:
Statistical significance was analyzed by Student’s t-test. All data are expressed as means ±SD.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormal clinical signs were observed in any group.

BODY WEIGHT AND WEIGHT GAIN
No body weight changes were observed in any group.

URINALYSIS
Treatment-related proteinuria was observed at day 7 and day 13 in the 284 and 408 mg/kg bw/day groups, with no significant difference in the degree of proteinuria between day 7 and 13.

CLINICAL CHEMISTRY
No abnormal changes were observed in any parameters in any treated groups when compared with the control animals.

ORGAN WEIGHTS
There were no statistically significant differences in the kidney weights of the phosphate-treated rats in comparison with the control group. No data were provided by the authors to corroborate this result.

GROSS NECROPSY
Kidneys from the 408 mg/kg group were pale and no remarkable gross lesions were observed in the other treated groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Glomerular lesions were observed in the kidneys of the phosphate-treated rats. In the 284 mg/kg bw/day group, there was minimal focal deposition of fine basophilic granules in the glomerular and parietal epithelium. In these rats there were no remarkable lesions in the capillary and glomerular basement membranes. Panglomerular deposition of basophilic granules was observed in the 408 mg/kg bw/day group animals with mild hypertrophy and vacuolization of the parietal epithelium. The capillary space tended to be widened at the vascular pole of the segmental tuft in some glomeruli. Also, in some glomeruli, there were PAS (carbohydrate)-positive rounded protuberances on the glomerular basement membranes, with capillary dilatation and glomerular collapse glomeruli secondary to pressure from pooled eosinophilic serous material within Bowman’s space. Basophilic granules and glomerular basement membranes with rounded protuberances were confirmed to have phosphate deposition by von Kossa staining. Minimal calcification was observed in the basement membrane of some proximal tubules of the 284 and 408 mg/kg bw/day group animals.
No remarkable changes were seen in the kidneys of the 1 and 28 mg/kg bw/day group rats. There were no lesions observed in any other organs including the bone and parathyroid.

ELECTRON MICROSCOPY
There were low-density lamellar structures at various sites in the glomeruli in the 284 and 408 mg/kg bw/day group rats. In the 284 mg/kg bw/day group rats, lamellar structures were observed within the Bowman’s space, glomerular epithelium, glomerular basement membrane, subepithelial spaces, mesangial matrix and parietal epithelium. These changes were more severe in the 408 mg/kg bw/day group rats. Higher magnification revealed high-density particles mixed with clusters of lamellar structures.
Aggregated vacuoles of various sizes were observed within the glomerular epithelium, glomerular membrane, mesangial matrix and parietal epithelium. Basement membranes were multifocally expanded and split by vacuoles protruding into the capillary lumens. A continuum between lamellar structures and vacuoles was observed within the parietal epithelial cells. Also, depending on the severity of the glomerular lesions, fusion of podocytes, increase in the number of microvilli, and large amounts of debris filling the Bowman’s space were observed. Detached capillary endothelium with accompanying vacuoles was observed associated with the expanded basement membranes; however, lesions inside the capillaries were minimal. There were no remarkable lesions in the lower dose groups.

X-ray microanalysis revealed peaks of calcium and phosphorus in the particles. Furthermore, a high content of lead and other heavy metals (including copper, osmium, uranium) were detected in the X-ray microanalysis, which was not explained by the authors.

Effect levels

Dose descriptor:
NOAEL
Effect level:
28 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3: Results of proteinuria detected by urinalysis. n=5 for each group.

Day 7

Treatment

Control

1 mg/kg

28 mg/kg

284 mg/kg

408 mg/kg

Minimala)

3

0

1

0

0

Mild

2

5

2

0

1

Moderate

0

0

2

4

0

Marked

0

0

0

1

4

Day 13

Treatment

Control

1 mg/kg

28 mg/kg

284 mg/kg

408 mg/kg

Minimala)

1

0

0

0

0

Mild

3

5

5

1

0

Moderate

1

0

0

2

0

Marked

0

0

0

2

5

a)Minimal: <15 mg/dL; Mild: 30 mg/dL; Moderate: 100 mg/dL; Marked: > 300 mg/dL;

 

Applicant's summary and conclusion

Conclusions:
There were no remarkable changes in blood chemistry, clinical signs, or body weight after disodium hydrogenorthophosphate administration. The kidneys of the highest-dose group were pale. Histopathology revealed panglomerular deposition of basophilic dense granules in which calcium and phosphorus were detected. In the high-dose groups, dose-dependent degeneration of the glomerular epithelium and parietal epithelium was detected. Electron microscopic examination revealed lamellar structures in the glomerular epithelium, basement membrane, mesangial matrix, parietal epithelium and Bowman’s space.
The results suggest that administration of high-dose (disodium hydrogenortho)phosphate transiently overloads the glomerular epithelium during filtration through glomerular capillaries and produces insoluble calcium salt and glomerular lesions, resulting in proteinuria.
Executive summary:

To investigate the early changes involved in glomerular calcification, 0-408 mg/kg bw/day disodium hydrogenorthophosphate was administered to rats via the tail vein for 14 days. Urinalysis was carried out on days 7 and 13 of the treatment and rats were sacrificed for clinical chemistry, histopathological and electron microscopic examination of the kidneys.

There were no remarkable changes in blood chemistry, clinical signs, or body weight after disodium hydrogenorthophosphate administration. The kidneys of the highest-dose group were pale. Histopathology revealed panglomerular deposition of basophilic dense granules in which calcium and phosphorus were detected. In the high-dose groups, dose-dependent degeneration of the glomerular epithelium and parietal epithelium was detected. Electron microscopic examination revealed lamellar structures in the glomerular epithelium, basement membrane, mesangial matrix, parietal epithelium and Bowman’s space.

The results suggest that administration of high-dose (disodium hydrogenortho)phosphate transiently overloads the glomerular epithelium during filtration through glomerular capillaries and produces insoluble calcium salt and glomerular lesions, resulting in proteinuria.