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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Dose: 30 mg/kg bw/day instead of 1000 mg/kg bw/day
Qualifier:
according to guideline
Guideline:
other: ISO 10993 Biological evaluation of Medical Devices
Version / remarks:
Part 11 (1995): tst for systemic toxicity/6.7 subchronic oral application
Deviations:
no
GLP compliance:
not specified
Remarks:
Not specified in the publication
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Tricalcium bis(orthophosphate)
EC Number:
231-840-8
EC Name:
Tricalcium bis(orthophosphate)
Cas Number:
7758-87-4
Molecular formula:
Ca.2/3H3O4P
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: beta-calcium pyrophosphate (beta-CPP) was prepared by reacting high-purity Ca2P2O7 (99.99% Sigma-aldrich Co., St. Louis, Missouri, USA) with CaCO3 (99.99% High Purity Chemicals, Sakado, Japan) in the solid state. Porous beta-CPP was prepared by using polyurethane foams with randomly interconnected pores (60 ppi). Polyurethane foams were coated with beta-CPP slurry and then burned and sintered at 1.100-1300ºC for 2 hours. The resulting porous beta-CPP contained interconnected pores (pore size, 300-500 µm) and a porosity of 80%, which is similar to that of natural spongy bone.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: to produce aqueous extracts for the administration experiments, 0.1026 g of beta-CPP was dissolved in 34.2 mL of saline at 70 ± 2ºC for 24 hours.

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 Biolin, Inc., ChoongChung-BukDo, Korea
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 5 week-old
- Weight at study initiation: males: 183 ± 8 g, females: 163 ± 6 g
- Fasting period before study: no
- Housing: not specified
- Diet (e.g. ad libitum): commercial feed (Purina feed for rats, Nestle Purina Pet Care Co., St. Louis, USA) ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: food irradiated (25-40kGy, Greenpia, Yugookun, korea), water autoclaved (121ºC, 15 minuts)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 45-50%
- Air changes (per hr): 12-18 times/ hour
- Photoperiod (hrs dark / hrs light): 12 hours (from 7:00 to 7:00 pm)

IN-LIFE DATES: 90 days

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
physiological saline
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): physiological saline is used for beta-calcium pyrophosphate extraction.

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrations
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the maximum dose required for repeated-dose toxicity studies was chosen

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice-daily

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

FOOD EFFICIENCY:
Average daily consumptions of water and food were determined weekly by weighing, and overall mean weekly consumptions during the 90-day treatment period were calculated.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 90
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Not specified
- How many animals: All animals
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 90
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during the 90-day treatment period
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: urine volume and urine color, specific gravity (SG), pH, leukocyte esterase, nitrite, protein, glucose, ketone bodies, urobilinogen, bilirrubin and occult blood.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes
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 (hematoxylin and eosin) stained: digestive system (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, liver, gall bladder, salivary gland, and pancreas), urinary system (kidney and urinary bladder), respiratory system lung and trachea), cardiovascular system (heart and aorta), hematopoietic system (spleen, thymus, limph nodes, and bone marrow), endocrine system (adrenal, pituitary, thyroid, and parathyroid glands), nervious system (skeletal muscle, femur, and sternum), male reproductive system (testes, epididymides, prostate, and seminal vesicle), female reproductive system (ovaries, uterus, mammary glands, and vagina), skin, tongue, and eyes.
Statistics:
Mean and standard deviations were calculate for all quantitative data. If warrented 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 by using Barlett's test, and when differences were significant (P<0.05), the Kruskal-Wallis test was performed. When these results were significant, the Wilcoxon-Mann-Whitney rank-sum tests, and 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
Description (incidence and severity):
No clinical signs or motor activity changes were observed that could be attributed to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortalities ocurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Control and treated groups gained weight equally over the treatment period. No statistically significant differences in mean body weight gain were observed.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Description (incidence and severity):
No diet-related effects on feed intake were observed. No statistically significant differences in mean body weight gain or meanfeed efficiency were observed.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related abnormalities in treated or control animals during the test period were observed.
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean serum chloride level in beta-CPP-treated males was signifiicantly higher than in controls. Mean aPTT (activated partial thromboplastin time), cholesterol, and AST(GOT) levels in beta-CPP females were significantly lower than in control females. Nevertheless, these four parameters were within the normal range in both female subgroups (see table 2).
No differences were observed between the beta-CPP and control groups with respect to other hematologic response and response variables.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant differences were observed between beta-CPP and control males or famales in terms of urinalysis response variables by randon sampling.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No behavioral changes were observed that could be attributed to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No differences were found between the two study groups with respect to other hematologic response and response variables.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology revealed no evidence of changes that could be attributed to beta-CPP treatment. All observed findings were typical for the Sprague-Dawley strain.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1. Body-weight changes in male and female rats given control and test substance (mean ± standard deviation, g)

 

Control

Treat

P-value

 

Male

Female

Male

Female

Male

Female

Body weight week 0

183 ± 8

163 ± 6

182 ± 8

163 ± 7

0.7771

0.8614

Body weight week 1

231 ± 10

187 ± 7

231 ± 8

189 ± 12

0.8414

0.7871

Body weight week 2

283 ± 11

211 ± 12

287 ± 9

211 ± 16

0.3943

0.9580

Body weight week 3

311 ± 13

228 ± 16

321 ± 11

225 ± 17

0.0693

0.6310

Body weight week 4

335 ± 17

240 ± 15

346 ± 14

235 ± 15

0.1533

0.5384

Body weight week 5

355 ± 20

250 ± 15

367 ± 19

248 ± 20

0.2108

0.8170

Body weight week 6

375 ± 20

262 ± 15

388 ± 22

261 ± 17

0.1935

0.8990

Body weight week 7

381 ± 21

265 ± 17

395 ± 23

260 ± 14

0.1703

0.4692

Body weight week 8

397 ± 22

272 ± 19

408 ± 25

272 ± 20

0.2900

0.9364

Body weight week 9

407 ± 22

276 ± 15

421 ± 26

274 ± 18

0.2051

0.8067

Body weight week 10

421 ± 23

279 ± 18

432 ± 26

282 ± 19

0.3290

0.6678

Body weight week 11

431 ± 23

282 ± 16

445 ± 26

283 ± 19

0.1938

0.9063

Body weight week 12

439 ± 25

287± 16

456 ± 27

285 ± 17

0.1491

0.7587

 

Table 2. Hematology and biochemical values of male and female rats (mean ± standard deviation)

 

RV

Control

Treat

P-value

 

 

Male

Female

Male

Female

Male

Female

WBC (x 103/mm3)

3.0 -15.0

5.5 ± 1.2

5.1 ± 1.1

5.5 ± 0.6

4.3 ± 1.2

0.8496

0.2720

RBC (x 106/mm3)

5.0 -12.0

7.12 ± 0.52

6.64 ± 0.39

7.34 ± 0.4

6.67 ± 0.43

0.2413

0.6500

Hb (g/dL)

11.1 – 18.0

14.6 ± 0.6

13.9 ± 0.6

14.7 ± 0.7

14.3 ± 0.6

0.5960

0.3219

Hct (%)

36.0 -52.0

40.6 ± 2.6

38.0 ± 1.8

41.1 ± 2.8

39.0 ± 2.1

0.6498

0.4488

Platelet (x 103/mm3)

500-1300

691.7 ± 59.1

717.1 ± 27.6

671.6 ± 54.5

701.7 ± 58.7

0.5706

0.3074

MCV (fL)

44-69

57.0 ± 2.3

57.3 ± 1.6

56.0 ± 2.0

58.5 ± 1.6

0.3029

0.1383

MCH (pg)

12.0-24.5

20.6 ± 1.2

21.0 ± 1.1

20.1 ± 0.8

21.4 ± 1.0

0.3066

0.2890

MCHC (g/dL)

21.6-42.0

36.0 ± 1.2

36.7 ± 1.1

35.9 ± 1.3

36.6 ± 1.2

0.4718

0.4263

Monocyte (%)

0-5

1.4 ± 1.4

2.1 ± 1.7

1.0 ± 0.7

1.5 ± 1.5

0.6641

0.4176

Basophil (%)

0-1.5

0.1 ± 0.3

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.3306

1.0000

Eosinophil (%)

0-6

0.0 ± 0.0

0.1 ± 0.3

0.0 ± 0.0

0.0 ± 0.0

1.0000

0.3306

Neutrophil (%)

9-34

20.8 ± 11.4

18.7 ± 5.4

19.0 ± 2.9

17.4 ± 7.5

0.9396

0.5699

Lymphocyte (%)

65-85

77.7 ± 11.5

79.1 ± 5.6

80.0 ± 3.1

81.1 ± 7.5

0.9394

0.5948

PT seconds (seconds)

12.5-18.7

15.4 ± 0.4

15.4 ± 0.7

15.5 ± 0.6

15.4 ± 0.6

0.3047

0.8201

aPTT (seconds)

18.4-45

28.3 ± 3.1

28.1 ± 2.0

28.8 ± 2.2

25.5 ± 3.0

0.9397

0.0210*

Calcium (mg/dL)

5.3-13.0

9.8 ± 0.3

9.8 ± 0.2

9.8 ± 0.4

9.9 ± 0.3

0.8188

0.3761

Phosphorus (mg/dL)

5.6-9.6

7.3 ± 0.6

6.8 ± 0.6

7.1 ± 0.4

7.3 ± 1.1

0.3422

0.3825

Glucose (mg/dL)

50-135

112 ± 35

73 ± 16

120.0 ± 24

89 ± 22

0.6232

0.1618

BUN (mg/dL)

15-21

25 ± 2

25 ± 3

23 ± 3

24 ± 4

0.1008

0.4700

Choresterol (mg/dL)

40-130

104 ± 16

112 ± 11

99 ± 15

102 ± 18

0.5960

0.0491*

T.Protein (g/dL)

5.6-7.6

6.1 ± 0.2

6.3 ± 0.2

6.2 ± 0.2

6.3 ± 0.2

0.1075

1.0000

Albumin (g/dL)

3.8-4.8

6.6 ± 10.0

3.8 ± 0.1

3.5 ± 0.1

3.8 ± 0.1

0.9687

0.4097

T.Bil. (mg/dL)

0.20-0.55

0.1 ± 0.0

0.2 ± 0.1

0.1 ± 0.0

0.1 ± 0.1

1.0000

0.6934

Alkaline phosphatase (IU/L)

60-300

59 ± 12

39 ± 8

58 ± 6

41 ± 5

0.9698

0.5681

AST (GOT) (IU/L)

47-155

135 ± 46

170 ± 21

123 ± 31

133 ± 34

0.6500

0.0256*

ALT (GPT) (IU/L)

17-56

36 ± 7

29 ± 5

32 ± 5

26 ± 3

0.4256

0.0792

Creatinine (mg/dL)

0.2-0.8

0.6 ± 0.1

0.7 ± 0.0

0.6 ± 0.1

0.7 ± 0.1

0.1197

0.6770

Na (mmol/L)

135-145

146 ± 2

146 ± 1

147 ± 2

145 ± 2

0.8777

0.6696

K (mmol/L)

3.5-5.5

4.7 ± 0.3

4.0 ± 0.2

4.7 ± 0.2

4.1 ± 0.3

0.6193

0.3381

Cl (mmol/L)

98-110

102 ± 2

104 ± 1

104 ± 1

104 ± 1

0.0262*

0.0738

TG (mg/dL)

0-200

55 ± 16

48 ± 13

43 ± 11

46 ± 15

0.1400

0.7616

A/G

-

1.3 ± 0.1

1.5 ± 0.2

1.3 ± 0.1

1.5 ± 0.1

0.3384

0.4594

RV, reference values

* Groups significantly different (P<0.05) by the one-way analysis of variance test or the Wilcoxon rank-sum test.

RBC: red blood count; WBC: white blood count; MCV: mean corpuscular volume (MCV); MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; PTs: prothrombin times; aPTT: activated partial thromboplastin time; TG: triglyceride; BUN: blood urea nitrogen; ALT: alanine transaminase; AST: aspartate transaminase; ALP: alkaline phosphatase.

 Table 3. Summary of male and female rats absolute and relative organ weights (mean ± standard deviation, g).

 

Control

Treat

P-value

 

Male

Female

Male

Female

Male

Female

Body weight

417 ± 25

271 ± 17

428 ± 28

265 ± 20

0.3595

0.5097

Brain

1.960 ± 0.083

1.789 ± 0.159

2.005 ± 0.074

1.840 ± 0.055

0.2206

0.5706

Thymus

0.296 ± 0.041

0.249 ± 0.024

0.357 ± 0.080

0.240 ± 0.071

0.1041

0.4961

Lung

1.566 ± 0.169

1.364 ± 0.089

1.697 ± 0.265

1.321 ± 0.095

0.4274

0.3053

Spleen

0.711 ± 0.063

0.556 ± 0.051

0.699 ± 0.082

0.543 ± 0.054

0.7159

0.5813

Pituitary

0.012 ± 0.003

0.014 ± 0.003

0.012 ± 0.003

0.013 ± 0.003

0.9404

0.7743

Heart

1.354 ± 0.135

0.874 ± 0.043

1.355 ± 0.137

0.881 ± 0.070

0.9754

0.8089

Prostate

0.688 ± 0.167

 

0.683 ± 0.086

 

0.5450

 

Liver

10.550 ± 1.200

6.050 ± 0.339

10.485 ± 0.979

5.790 ± 0.589

0.8965

0.2420

Adrenal

0.025 ± 0.002

0.032 ± 0.005

0.023 ± 0.003

0.027 ± 0.005

0.1394

0.0665

Kidney

1.261 ± 0.113

0.797 ± 0.049

1.265 ± 0.080

0.764 ± 0.046

0.9371

0.1365

Testis

1.794 ± 0.139

 

1.844 ± 0.102

 

0.3748

 

Ovary

 

0.064 ± 0.009

 

0.067 ± 0.014

 

0.5067

Relative brain

0.0047 ± 0.0003a

0.0066 ± 0.0003a

0.0047 ± 0.0003

0.0070 ± 0.0005

0.8871

0.1016

Relative thymus

0.0007 ± 0.0001

0.0009 ± 0.0001

0.0008 ± 0.0001

0.0009 ± 0.0002

0.0731

0.8117

Relative lung

0.0038 ± 0.0003

0.0050 ± 0.0003

0.0040 ± 0.0006

0.0050 ± 0.0004

0.5674

0.6598

Relative spleen

0.0017 ± 0.0002

0.0021 ± 0.0001

0.0016 ± 0.0002

0.0020 ± 0.0002

0.4365

0.7751

Relative pituitary

0.00003 ± 0.00001

0.00005 ± 0.00001

0.00003 ± 0.00000

0.00005 ± 0.00001

1.0000

0.7007

Relative heart

0.0032 ± 0.0002

0.0032 ± 0.0002

0.0032 ± 0.0002

0.0033 ± 0.0002

0.3104

0.3480

Relative prostate

0.0017 ± 0.0004

 

0.0016 ± 0.0002

 

0.9694

 

Relative liver

0.0253 ± 0.0016

0.0224 ± 0.0017

0.0245 ± 0.0011

0.0218 ± 0.0018

0.2118

0.4048

Relative adrenal

0.00006 ± 0.00001

0.00012 ± 0.00002

0.00005 ± 0.00001

0.00010 ± 0.00002

0.1519

0.1602

Relative kidney

0.0030 ± 0.0002

0.0030 ± 0.0003

0.0030 ± 0.0002

0.0029 ± 0.0001

0.5535

0.2790

Relative testis

0.0043 ± 0.0003

 

0.0043 ± 0.0003

 

0.9388

 

Relative ovary

 

0.0002 ± 0.0000

 

0.0003 ± 0.0000

 

0.6506

aExpressed as a percentage of body weight.

 

Applicant's summary and conclusion

Conclusions:
Beta-calcium pyrophosphate has a NOAEL higher than 30 mg/kg/day in rats, after 90-day oral administration.
Executive summary:

A 90 -day repeated dose toxicity study was performed on Sprague-Dawley rats similar to OECD 408. Beta-calcium pyrophosphate was orally administered to 10 male and 10 female Sprague-Dawley rats, at 30 mg/kg/day. Body weights were recorded at baseline and weekly after. Mortality, feed intake, and body weight were monitored. Urinalysis was assessed during the 90-day treatment period. On day 90, all animals were anesthetized, euthanized, and weighed, and blood samples were collected for haematology and serum biochemistry. In males, mean serum chloride level in beta-CPP-treated was significantly higher than in controls. In females, mean aPTT (activated partial thromboplastin time), cholesterol, and AST(GOT) levels in the treated group were significantly lower than in the control group. No mortality occurred throughout the study and no clinical signs were observed. Under these conditions, the test item was found to have a NOAEL higher than 30 mg/kg/day in rats.