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Repeated dose toxicity: oral

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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

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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: ISO 10993 Biological Evaluation of Medical Devices Part II (1995): test for systemic/6.7 subchronic oral application
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

Constituent 1
Chemical structure
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 pyrophosphate
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.