Calcium bis(metaphosphate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Data source

Reference

Reference Type:

publication

Title:

A 90-day subchronic toxicity study of beta-calcium pyrophosphate in rat

Author:

Lee JH, Chang B-S, Ryu H-S & Lee C-K

Year:

2009

Bibliographic source:

Drug and Chemical Toxicology, 32(3): 277-282

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ISO 10993 Biological Evaluation of Medical Devices Part II (1995): test for systemic/6.7 subchronic oral application

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

yes

Remarks:

only one dose tested at 30 mg/kg bw/day, no immulogoical or neurological effects examined

Principles of method if other than guideline:

Evaluation of toxicity from subchronic oral administration of β-calcium pyrophosphaste to male and female Sprague-Dawley rats.

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dicalcium pyrophosphate

EC Number:

232-221-5

EC Name:

Dicalcium pyrophosphate

Cas Number:

7790-76-3

Molecular formula:

Ca2O7P2

IUPAC Name:

dicalcium diphosphate

Constituent 2

Reference substance name:

Beta calcium pyrophosphate

IUPAC Name:

Beta calcium pyrophosphate

Test material form:

other: aqueous extract of beta calcium pyrophosphate

Details on test material:

β-calcium pyrophosphate (β-CPP) was prepared by reacting high-purity Ca2P2O7 (Sigma-Aldrich Co, St Louis, Missouri, USA; 99.99 %;) with CaCO3 (High Purity Chemicals, Sakado, Japan; 99.99 %) in the solid state. Porous β-CPP was prepared by using polyurethane foams with randomly interconnected pores (60 ppi). Polyurethane foams were coated with β-CPP slurry and then burned and sintered at 1,100 to 1,300°C for 2 hours. The resulting porous β-CPP contained interconnected pores (pore size 300-500 μm) and a porosity of 80%, which is similar to that of natural spongy bone.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dae Han Biolink Inc, ChoongChung-BukDo, Korea
- Age at study initiation: 5 weeks
- Weight at study initiation: 182 ± 8 g (treated males); 163 ±7 g (treated females); 183 ± 8 g (control males); 163 ± 6 g (control females)
- Fasting period before study: No
- Housing: metal cages
- Diet (e.g. ad libitum): γ-irradiated (25-40 kGy; Greenpai, Yugookun, Korea) commercial feed (Purina feed for rats; Nestle Purina Pet Care Co, St Louis, USA)
- Water (e.g. ad libitum): autoclaved (121 °C for 15 min)
- Acclimation period: 7 days in individual cages

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 45-50%
- Air changes (per hr): room air turnaround was 12-18 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (07:00 to 19:00) and 12 hours dark

IN-LIFE DATES: From: Day minus 7 To: Day 90

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: saline

Details on oral exposure:

Aqueous extracts for the administration experiments were prepared by dissolving 0.1026 g of β-CPP in 34.2 mL of saline at 70 ± 2°C for 24 hours.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily between 10:00 am and 12:00 am

Doses / concentrations

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: maximum required dose for repeated dose toxicity studies regarding biological evaluation of medical devices

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily during the treatment period to detect mortality, morbidity and signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: at baseline and then weekly until scheduled sacrifice 90 days later

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at baseline and towards the end of the treatment period.
- Dose groups that were examined: all rats

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 90
- Anaesthetic used for blood collection: not specified
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: red blood cell count (RBC, white blood cell count (WBC), haemaglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular haemaglobin (MCH), mean corpuscular haemaglobin concentration (MCHC) and platelet count, prothrombin times (PTs) and activated partial thromboplastin time (aPTT), differential blood cell counts (i.e of lymphocytes, neutrophils, basophils, eosinophils and monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 90
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: total protein, albumin, glucose, total cholesterol, triglyceride (TG), total bilirubin, blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), Cl, Ca, K and P

URINALYSIS: Yes
- Time schedule for collection of urine: no data
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: urine color, Specific gravity (SG), pH, leukocyte esterase, nitrate, protein, glucose, ketone bodies, urobilinogen and bilirubin, and occult blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

- On day 90, all animals were anaethetised with ether, euthanised by exsanguination, weighed, and blood samples were collected from abdominal aorta for haematology and serum biochemistry.
- Each animal was subjected to complete necropsy including external body macroscopic examinations.
- The weights of heart, liver, lung, kidney (both), spleen, adrenal glands (both), brain, thymus, pituitary gland, testes (both) and ovaries (both) were determined.
- Organ-to-body-weight and organ-to-brain-weight ratios were calculated.
- At sacrifice, samples of the tissues and organs listed above, and all gross lesions, were fixed and preserved in 10% neutral buffered formalin.
- Tissue samples from all animals were further processed for histopathology. The following tissues and organs were sectioned at 2 μm and H&E (haematoxylin and eosin) stained: digestive system (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, liver, gall bladder, salivary gland and pancreas); urinary system (kidney and urinary bladder); respiratory system (lung and trachea); cardiovascular system (heart and aorta); haematopoietic system (spleen, thymus, lymph nodes and bone marrow); endocrine system (adrenal, pituitary, thyroid and parathyroid glands); nervous system (brain and spinal cord); muscular skeletal system (skeletal muscle, femur and sternum); male reproductive system (testes, epididymides, prostate and seminal vessel); female reproductive system (ovaries, uterus, mammary glands and vagina), skin, tongue and eyes.

Statistics:

Mean and statistical deviations were calculated for all quantitative data. If warranted, and based on group size constraints, the test and control groups were compared by using one-way analysis of variance, followed by the Dunnett's multiple comparison test. Homogeneity of variances was tested using Bartlett's test and, when differences were significant (P<0.05), the Kruskal-Wallis test was performed. When those results were significant, the Wilcoxon-Mann-Whitney rank-sum test and the Nemenye-Kruskal-Wallis multiple comparisons were performed. The Chi-square test was used to determine the significances of histopathological changes.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

β-CPP and control group animals gained weight equally over the treatment period (see Table 1, attached)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

no treatment-related abnormalities in treated or control animals during the test period

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Activated partial thromboplastin time (aPTT) showed significantly decreased values in female rats. But these values were all in the range of the reference values and are therefore considered as normal and not treatment-related.
For detailed information see Table 2_1 in "any other information on results" and Table 2 (attached).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cholesterol and AST (GOT) showed significantly decreased values in female rats and Cloride ions were significantly increased in males But these values were all in the range of the reference values and are therefore considered as normal and not treatment-related.
For detailed information see Table 2_1 in "any other information on results" and Table 2 (attached).

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

- No mortalities occurred during the study, and no clinical signs or behavioural or motor activity changes were observed that could have been attributed to treatment.
- No diet-related effects on feed intake were observed.
- No statistically significant differences in mean body weight gain (see Table 1, attached) or mean feed efficiency were observed between the β-CPP and control groups, or between male and female rats in the two groups.
- No significant differences were observed between β-CPP and control males or females in terms of urinalysis response variables by random sampling.
- Mean serum chloride level in β-CPP-treated males (104 ± 1) was significantly higher (P = 0.026) than in controls (102 ± 2).
- Mean aPTT, cholesterol and AST(GOT) levels in β-CPP females (25.5 ± 3; 102 ± 18 and 133 ± 34 respectively) were significantly lower (P = 0.021, P = 0.049 and P = 0.026 respectively) than in control females (28.1 ± 2.0; 112 ± 11 and 170 ± 21). However, the four parameters were within the normal range in both female subgroups (see Table 2, attached).
- No differences were observed between β-CPP and control groups with respect to other haematologic response and response variables.
- No differences were found between the two study groups or between males and females in these groups with respect to absolute or relative organ weights (see Table 3, attached)
- Microscopic histopathology revealved no evidence of changes that could be attributed to β-CPP treatment. All observed findings were typical for the Sporague-Dawley strain.

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 30 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 2_1: Significant differences in hematology and biochemical values of male and female rats
	RV	Control	Treated	p-value
Female rats
aPTT (seconds)	18.4-45	28.1±2.0	25.5±3.0	0.0210*
Cholesterol (mg/dL)	40-130	112±11	102±18	0.0491*
AST (GOT) (IU/L)	47-155	170±21	133±34	0.0256*
Male rats
Cloride (mmol/L)	98-110	102±2	104±1	0.0262*
RV = reference value * groups significantly different (p<0.05) by the one-way analysis of variance test or the Wilcoxon rank-sum test.

Applicant's summary and conclusion

Conclusions:

No adverse effects were detected in male or female rats when β-calcium pyrophosphate was fed orally at 30 mg/kg bw/day.