Dipotassium hydrogenorthophosphate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Study on analogous substance submitted as supporting data only.

Data source

Reference

Reference Type:

publication

Title:

Early Events Involving Glomerular Calcification Induced by Dibasic Sodium Phosphate Solution in Rats

Author:

Tsuchiya N, Torii M, Narama I, Matsui T

Year:

2008

Bibliographic source:

J Toxicol Pathol 21:229-37; DOI: 10.1293/tox.21.229

Materials and methods

Objective of study:

distribution

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male Sprague-Dawley rats were treated with disodium hydrogenorthophosphate (409.6 mg/kg bw/day) by injection into the tail vain. Urinalysis was carried out on days 1, 3, 5 and 8 of dosing, and rats were sacrificed on days 2, 4 and 9 for histopathological and electron microscopic examination of the kidneys.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium hydrogenorthophosphate

EC Number:

231-448-7

EC Name:

Disodium hydrogenorthophosphate

Cas Number:

7558-79-4

Molecular formula:

H3O4P.2Na

IUPAC Name:

disodium hydrogen phosphate

Details on test material:

- Name of test material (as cited in study report): dibasic sodium phosphate, Na2HPO4g.
- Analytical purity: >=99% (supplier information)
- Supplier: Kanto Chemical Co. Inc. Tokyo, Japan

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Clea Japan Inc. (Shiga, Japan)
- Age at study initiation: 5 weeks (35 days)
- Housing: individually 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

Details on exposure:

The rats were administered 360 mM disodium hydrogenorthophosphate at 4 mL/kg bw/min for 2 minutes 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).

Duration and frequency of treatment / exposure:

once daily for up to 8 days

Doses / concentrations

Remarks:

Doses / Concentrations:
360 mmol/L, equivalent to 409.6 mg/kg bw/day

No. of animals per sex per dose / concentration:

5

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no

Details on study design:

HISTOPATHOLOGY:
Rats were euthanized by exsanguination by cutting both the abdominal aorta and vena cava under pentobarbital sodium anesthesia. And were then necropsied. The kidneys were removed and weighed together (Mettler PM400). Organs listed in Table 2 were microscopically examined. These organs were fixed in 10% neutral buddered formalin, processed routinely and embedded in paraffin. Paraffin sections were prepared and stained with hematoxylin and eosin (H&E). The kidney sections were also stained with von Kossa and periodic acid Schiff (PAS) reagents and periodic acid methenamine-silver (PAM) stain.

IMMUNOHISTOCHEMISTRY:
Kidney sections were subjected to indirect immunoperoxidase staining using the following primary antibodies:
- PCNA (proliferating cell nuclear antigen)
- Podoplanin (specific for podocytes and parietal cells)
- Desmin

For electron microscopic observation, kidneys that were excised from 2 animals per group at each time point 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 under an electron microscope (JEM-1010, JEOL Ltd., Tokyo).

Statistics:

Statistical significance was analyzed by Student’s t-test for body and kidney weights and the Mann-Whitney test for immunohistochemistry. All data are expressed as means ±SD.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:

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
Increased urinary protein was first detected on day 3 in 2 out of 10 treated rats (100 mg/dL/4 h). Almost all treated animals had increased urinary protein excretion on day 5 with values ranging from 100 – 1000 mg/dL/4 h. By day 8, all treated rats showed moderate to severe proteinuria. In the control group, although several rats had minimal to mild proteinuria, no time course increase was observed.
Data are summarized in table 1.

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.

HISTOPATHOLOGY: NON-NEOPLASTIC
No significant changes were observed in the saline control group. The first renal lesions were observed on day 4 of treatment as minimal focal deposition of calcium salts within the parietal epithelial cells. After 8-day repeated dosing, focal calcification of the glomerular basement membrane, mesangium, and parietal epithelial cells were evident. Hypertrophy and increased mitotic figures were also visible.
There were no lesions observed in the other organs including the bone and parathyroid.
The results are summarized in table 2.

IMMUNOHISTOCHEMISTRY
There were no differences in podoplanin expression or the number of PCNA-positive cells between the treated and untreated rats on days 2 and 4.
On day 9, the number of podoplanin-positive podocytes and parietal epithelial cells were significantly lower and desmin expression was significantly higher compared to control values.
The results are summarized in table 2.

ELECTRON MICROSCOPY
After single administration of disodium hydrogenorthophosphate, a number of small vacuoles were observed within the Bowman’s space. High-density particles (calcium phosphates?) adhered to the membranes of the vacuoles and clusters of debris were found within the Bowman’s space.
On day 4, low-density lamellar structures were observed within the Bowman’s space, the glomerular basement membrane and the mesangial matrix. Some of these structures protruded into the vascular cavity and the podocytes lining the glomerular basement membrane exhibited hypertrophy. Bowman’s space was filled with large amounts of debris.
On day 9, degeneration of the podocytes was more severe and lamellar structures accumulated partially within parietal epithelia, podocytes and glomerular basement membrane.
The results are summarized in table 2.

Metabolite characterisation studies

Metabolites identified:

not measured

Any other information on results incl. tables

Table 1: Detection of proteinuria by urinanalysis.

Treatment	Day 1		Day 3		Day 5		Day 8
	Control	Treated	Control	Treated	Control	Treated	Control	Treated
Nr. of animals	9	15	6	10	3	5	3	5
Negative	0	0	0	0	0	0	0	0
Minimal	4	11	6	4	2	0	3	0
Mild	5	4	0	4	1	1	0	0
Moderate	0	0	0	2	0	3	0	1
Marked	0	0	0	0	0	1	0	4

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

 

Table 2: Findings for disodium hydrogenorthophosphate treated rats

Findings		Days of administration
		Single	3 days	8 days
Urinalysis	Proteinuria	-	P	P
Microscopy	Calcification:parietal epithelial cells	-	P	P
	Calcification: Glomerular basement membrane / podocytes	-	-	P
	Calcification: Mesangium	-	-	P
	Hypertrophy of parietal epithelial cells	-	-	P
	Proliferation and/or activation of parietal epithelial cells	P	P	P
	Decrease of podoplanin expression in parietal epithelial cells	-	-	P
	Decrease of podoplanin expression in podocytes	-	P	P
	Desmin expression in glomeruli	-	-	P
Electron microscopy	Small vacuoles in Bowman’s space and podocytes	P	-	-
	Lamellar body deposition	-	P	P
	Effacement / degeneration of podocytes	-	P	P
	Increase of microvilli	-	P	P
	Thickening of glomerular basement membrane	-	-	P
	Electron-dense capillary endothelium	P	P	P

P: present; - not present

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The detected glomerular changes are consistent with the onset of proteinuria, which were considered due to protein leakage secondary to glomerular injury characterized by degneratoin of podocytes. On day 9 of treatment, calcification was observed in all parts of glomeruli.

Executive summary:

To investigate the early changes involved in glomerular calcification, 409 mg/kg bw/day disodium hydrogenorthophosphate was administered to rats via the tail vein for up to 8 days. Urinalysis was carried out on days 1, 3, 5, and 8 of the treatment and rats were sacrificed on days 2, 4, and 9 for histopathological and electron microscopic examination of the kidneys.

Following single dosing, there were no gross or pathological findings but electron microscopy revealed a number of vacuoles within the Bowman’s space. On day 4, minimal focal mineralization was observed within the parietal epithelial cells. Lamellar structures with effacement of podocytes, an increased number of microvilli and large amounts of debris filling the Bowman’s space were the main electron microscopic changes on days 4 and 9. Increased urinary protein expression correlated well with the glomerular changes. These results suggest that the onset of glomerular calcification is preceded by primary podocytes damage.