Trimethyl[2-(phosphonooxy)ethyl]ammonium chloride, calcium salt (1:1)

Endpoint summary

Description of key information

The available data refer to the degradation products of calcium phosphorylcholine chloride (choline and inorganic phosphate):

- Key study. Method similar to OECD 452. The lethal dose 0 (LD0) of Choline chloride is 500 mg/kg/day (1%) in rats, after 72-week oral administration.

- Key study. Method similar to OECD 408 and according to ISO 10993. Beta-calcium pyrophosphate has a NOAEL higher than 30 mg/kg/day in rats, after 90-day oral administration.

- Key study. OECD 408. Tetrasodium pyrophosphate was found to have a NOAEL of  500 mg/kg/day and the target organ appears to be the kidney in rats, after 90-day oral administration.

- Supporting study: Phosphoric acid has a NOAEL>386 mg/kg/day in rats, after administration in diet for more than twelve months.

Based on this information, it can be concluded that the substance phosphoryl choline chloride has a

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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

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

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.

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

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

Key result

Critical effects observed:

no

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 10^3/mm^3)	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 10^6/mm^3)	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 10^3/mm^3)	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 ± 0.0003^a	0.0066 ± 0.0003^a	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.

 

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.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

only 30 males and 1 dose

GLP compliance:

no

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Fisher Chemical Company

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kigston, NY
- Age at study initiation: not specified
- Weight at study initiation: 50-60 g
- Fasting period before study: no
- Housing: three per cage in plactic shoebox cages
- Diet (e.g. ad libitum): ad libitum a natural ingredient chow (Wayne Laboratory Blox Allied Mills, Inc., Chicago, IL) alone or supplemented with 1% choline cloride
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS: not specified

IN-LIFE DATES: 103 weeks

Route of administration:

oral: feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): 1% choline (Fisher Chemical Company) in a natural ingredient ground chow (Wayne Laboratory Blox Allied Mills, Inc., Chicago, IL)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

72 weeks

Frequency of treatment:

Daily

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Recalculated dose of 1%

No. of animals per sex per dose:

30 males

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for 16 weeks and biweekly thereafter.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Two animals per group were sacrificed at 28 weeks for an interim assessment of the experiment's progress. Afterwards all the animals dying or sacrificed during the study were necropsied. liver weights and body weights were taken at necropsy. All internal organs were examined.

HISTOPATHOLOGY: Yes
The livers and all other organs bearing gross abnormalities were routinely removed and fixed in buffered formalin. They were then sectioned and stained with hematoxylin and eosin for histological examination.

Statistics:

The comparisons between the incidences of tumors in different groups were analysed by Fischer's exact test. The survival data were analysed by the computer program developed by Thomas et al. (1977). Kaplan-Meier survival curves were derived by using this procedure. Comparison among survival of different experimental groups were made by Cox's test and P value based on the chi-square test from Cox's analysis. The incidences of liver and lung tumors and of leukemias in each group were based upon the number of animals surviving at 1 year since the first instance of each of the tumors occurred between weeks 55 and 60.

Clinical signs:

not examined

Mortality:

no mortality observed

Description (incidence):

Choline did not seem to alter survival of rats in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No marked effect on growth was 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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences between control and treated groups.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No significant differences between control and treated groups.

Other effects:

not specified

Key result

Dose descriptor:

other: LD0

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: neoplastic

mortality

Key result

Critical effects observed:

no

Table 1. The histological distribution of liver tumors in male weaning F344 rats fed withsaline or 1% of choline.

Group	Survival at wk			Body wt at wk		Relative liver wt (%)	No. of animals bearing liver tumors
							Neoplastic nodules	Hepatocellular carcinomas	Cholangiomas + cholangiocarcinomas	Lung metastases
Control	28	28	23	253	408	3.60	2	1	0	0
Choline chloride 1%	28	28	24	258	406	3.40	2	0	0	0

Conclusions:

The lethal concentration 0 of Choline chloride in rats is 500 mg/kg/day (1%), after 72-week oral administration.

Executive summary:

A 72-week repeated dose toxicity study was performed on rats similar to OECD 452. Choline chloride was orally administered to 30 male Fischer 344 rats, at 1% (500 mg/kg/day). Body weights were taken at weekly intervals for 16 weeks and biweekly thereafter. Two animals per group were sacrificed at 28 weeks for an interim assessment of the experiment's progress. Afterwards, all the animals dying or sacrificed during the study were necropsied. Liver weights and body weights were taken at necropsy. The livers and all other organs bearing gross abnormalities were routinely removed and fixed in buffered formalin. They were then sectioned and stained with haematoxylin and eosin for histological examination. No marked effect on growth was observed and no mortality occurred throughout the study. The histological distribution of liver tumours showed no significant differences between the control and treated group. Under these conditions, Choline chloride has a lethal concentration 0 of 500 mg/kg/day in rats (1%), after 72-week oral administration.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1952

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: To evaluate the effect of a prolonged intake of phosphoric acid in rats
- Short description of test conditions: Rats were fed to appetite on a basal diet and phosphoric acid added at different concentrations. The groups were kept in the experiment after 6 months, and observations continued through 2 filial generations.
- Parameters analysed / observed: Effect on growth in 3 generations, reproduction and blood parameters of the first two generations.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Diet (e.g. ad libitum): basal diet ad libitum.
- Water (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Details on route of administration:

Basal diet containing casein 6%, dried whole milk 10%, whole wheat flour 34.5%, potato flour 33%, groundnut oil 6.5%, cod liver oil 0.5%, linseed oil 0.5%, brewer's yeast 5% and salt mixture 4%, phosphoric acid at 0.4 or 0.75%.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

More than 52 weeks

Frequency of treatment:

Daily

Dose / conc.:

0.4 other: %

Remarks:

21 mg/kg/day (extrapolation from level of compound in diet assuming 0.35-kg rat eats 18 g food/day)

Dose / conc.:

0.75 other: %

Remarks:

386 mg/kg/day (extrapolation from level of compound in diet assuming 0.35-kg rat eats 18 g food/day)

No. of animals per sex per dose:

No data

Control animals:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: No data

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: 18 g food/0.35 kg/day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: No data
- Parameters checked: No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: Not specified
- How many animals: No data
- Parameters checked: No data

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

There was no treatment-related mortality in the animals treated with the test substance during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no differences in weight between control and treated groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no differences in food consumption between control and treated groups.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There was no differences in haematological parameters between control and treated groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no differences in clinical biochemistry parameters between control and treated groups.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

386 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

Key result

Critical effects observed:

no

Conclusions:

Phosphoric acid has a NOAEL>386 mg/kg/day in rats, after administration in diet for more than twelve months.

Executive summary:

A long-term repeated dose toxicity study of phosphoric acid was performed on rats. Male and female rats were administered on a basal diet with the addition of phosphoric acid at 0.4% or 0.75%. The groups were kept in the experiment after six months, and observations continued through two filial generations (more than twelve months). Effect on growth in three generations, reproduction and blood parameters of the first two generations. No adverse effects were noted at up to 0.75%. The NOEL for this study was 0.75% or more of phosphoric acid in the diet, which was equivalent to 386 mg/kg/day or more, assuming a 0.35 kg rat consumes 18 g food/day. Therefore, the test item has a NOAEL>386 mg/kg/day in rats, after administration in diet for more than twelve months.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Remarks:

Not specified in the publication

Limit test:

no

Specific details on test material used for the study:

- Source: Sigma-aldrich (Korea)
- Batch number: 048K0049

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Co., Ldt. (Gyeonggi-do, Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: young adults
- Fasting period before study: not specified
- Housing: animals were housed in groups of two to three in stainless steel wire mesh cages
- Diet (e.g. ad libitum): comercial roden show (2.0 Mrad gamma-ray sterilized EP pellet, Cargill Agri purina Korea Ltd, korea) ad libitum
- Water (e.g. ad libitum): sterilized tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3ºC
- Humidity (%): 55 ± 5%
- Air changes (per hr): 10-18 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hour light (from 8:00 to 20:00)/ 12 hour dark

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

Five times a week

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 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 doses were selected based on the results of the acute toxicity study and a 14-day dose range study (No toxicity or mortality was observed in any of the five male and female rats treated with Tetrasodium pyrophosphate at the dose of 2000 mg/kg for 14 days).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:at the end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during the last week of administration
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: glucose, bilirubin, ketone body, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocyte levels

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see tables 3 and 4)

HISTOPATHOLOGY: Yes (see table 5)

Statistics:

The results are presented as the mean ± standard deviation (SD). The differences in parameters (BWs, organ wtights, and the results of the blood biochemistry and hematology) between groups in the acute and sub-chronic toxicity test were assessed by a standard two-way analysis of variance (ANOVA). If these showed statistical significance, Duncan's of Dunnett's multiple range test were used to compare groups (SPSS version 12.0 [SPSS Inc., Chicago, IL, USA]). P-vallues < 0.05 were considered statistically significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no clinical signs in rats administered with 2000 mg/kg of tetrasodium pyrophosphate.
Female rats treated with 500 and 1000 mg/kg of the test substance showed hair loss. No significant clinical signs were observed in any other group.

Mortality:

no mortality observed

Description (incidence):

There were no dead animals in the treatment group administered with 2000 mg/kg of tetrasodium pyrophosphate.
There was no treatment-related mortality in the animals treated with the test substance during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The terminal body weight of male rats treated with 1000 mg/kg of test substance was lower than that of the control group, while there were no differences in body weight between the 250, 500 mg/kg, and control group. There was no treatment-related change in body weight in female rats.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A significant decrease in food consumption was observed in the 1000 mg/kg group of males at week 8 and 9. Food consumption in females increased significantly in the 1000 mg/kg group at week 4. There were no significant changes in food consumption in the other groups (Table 1).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no significant changes in hematology in the 250 mg/kg group.
WBC and neutrophil counts were ignificantly increased in males and females of the 1000 mg/kg group, whereas the lymphocyte count was significantly lower in males and females of the 1000 mg/kg group, whereas the lymphocyte count was significantly lower in males and females of the 1000 mg/kg geoup. RBC, HB, HCT, PT, and APTT were signidicantly reduced in the males of the 1000 mg/kg group, and PT was significantly reduced in males of the 500 mg/kg group (Table 2).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no significant changes in the serum biochemistry of males and females in the 250 mg/kg group.
Total protein was significantly reduced in both sexes in the 500 and 1000 mg/kg group. Albumin was significantly reduced in males of the 500 and 1000 mg/kg group, and in females of the 1000 mg/kg group. The A/G ratio was significantly increased in females of the 500 and 1000 mg/kg group. T-BIL was significantly increased in femaels of the 1000 mg/kg group, and ALP was significantly reduced in females of the 1000 mg/kg group, but ALT was significantly reduced in females of the 1000 mg/kg group. AST was significantly increased in males of the 1000 mg/kg group. TG were significantly increased in both sexes in the 1000 mg/kg group, while Ca, IP, Na, K, and Cl were significantly reduced in both sexes in this group. IP was also reduced in males of the 500 mg/kg group (Table 3).

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no specific symptoms in urinalysis at the doses of 250, 500 and 1000 mg/kg.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no significant changes in organ weight in males and females of the 250 mg/kg group. The relative weight of the liver in the males of the 500 mg/kg and 1000 mg/kg groups showed a significant increase. The absolute and relative weight of the liver in females of the 500 mg/kg and 1000 mg/kg groups were also significantly incrased (Table 4,5).

Gross pathological findings:

no effects observed

Description (incidence and severity):

There was no grossly visible findings or lesions in any group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No exposure-related histopathological changes were observed in any of the organs examined in SD rats, with the exception of kidney lesions. Cortical tubular basophila of the renal tubule was more evident in males of the 1000 mg/kg group and mineralization of the kidney was evident in females of the 1000 mg/kg group. Other background lesions were observed in the 0, 250, 500 and 1000 mg/kg groups in both sexes (Table 6).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Food consumption of SD rats orally treated with tetrasodium pyrophosphate for 90 days.

	Male				Female
	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
1	27.1 ± 1.0	27.0 ± 1.6	26.7 ± 0.9	27.1 ± 1.8	19.1 ± 1.4	20.6 ± 1.7	19.9 ± 1.5	20.2 ± 1.0
2	30.4 ± 0.9	28.5 ± 6.4	30.7 ± 1.7	29.2 ± 1.4	21.1 ± 2.6	21.3 ± 3.9	21.4 ± 2.1	20.3 ± 2.1
3	33.8 ± 1.9	34.1 ± 2.2	32.9 ± 3.1	30.4 ± 3.6	22.6 ± 2.2	22.6 ± 2.7	21.8 ± 2.1	21.7 ± 1.0
4	30.9 ± 1.2	31.6 ± 2.5	31.8 ± 6.6	28.5 ± 3.2	19.7 ± 2.5	20.5 ± 1.4	21.3 ± 1.5	22.7 ± 1.3*
5	33.4 ± 2.8	30.8 ± 1.2	31.8 ± 2.7	30.5 ± 2.1	21.7 ± 0.6	20.3 ± 3.3	19.3 ± 2.5	22.9 ± 2.7
6	29.3 ± 3.9	27.9 ± 2.6	29.2 ± 1.9	27.9 ± 2.9	18.1 ± 2.0	17.1 ± 3.7	18.2 ± 1.8	19.2 ± 1.3
7	33.1 ± 2.7	33.3 ± 2.6	33.8 ± 1.2	32.9 ± 3.2	24.3 ± 2.0	21.9 ± 2.0	23.2 ± 2.8	23.9 ± 2.9
8	31.5 ± 2.1	31.3 ± 2.1	33.0 ± 1.6	28.3 ± 0.5^Ɨ	20.8 ± 2.0	20.3 ± 2.9	21.7 ± 2.8	21.0 ± 2.2
9	31.8 ± 2.1	31.3 ±1.7	30.5 ± 1.1	28.2 ± 2.5*	20.6 ± 3.8	18.8 ± 2.7	19.9 ± 1.4	19.8 ± 2.1
10	32.7 ± 2.0	31.6 ± 2.3	31.9 ± 1.1	28.8 ± 1.8	21.2 ± 1.3	18.9 ± 3.8	21.2 ± 2.4	19.4 ± 1.7
11	29.8 ± 2.7	30.3 ± 3.0	29.6 ± 2.0	27.8 ± 1.6	20.5 ± 3.5	19.5 ± 2.3	21.1 ± 3.7	21.0 ± 3.9
12	33.8 ± 0.5	33.5 ± 0.5	33.2 ± 0.6	33.4 ± 0.5	20.2 ± 1.8	19.7 ± 3.1	21.5 ± 2.0	20.3 ± 2.9
13	30.1 ± 2.2	29.4 ± 2.3	31.2 ± 1.8	28.5 ± 1.7	20.1 ± 1.5	18.6 ± 1.9	20.4 ± 3.6	21.1 ± 2.5

*p<0.05,^Ɨp<0.01

Table 2. Hematological values of SD rats orally treated with tetrasodium pyrophosphate for 90 days.

	Male				Female
	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
WBC (K)	7.20 ± 1.83	6.62 ± 0.71	7.76 ± 1.58	9.35 ± 1.31*	3.72 ± 1.86	6.84 ± 2.09	4.44 ± 0.66	6.39 ± 1.83^Ɨ
Neutrophil (%)	14.2 ± 4.6	18.6 ± 6.2	20.1 ± 6.6	24.8 ± 4.9	12.2 ± 3.7	17.7 ± 5.0	17.3 ± 7.3	20.9 ± 6.3*
Lymphocyte (%)	80.1 ± 4.5	74.8 ± 6.2	73.6 ± 6.9	68.7 ± 6.0	81.4 ± 5.0	74.4 ± 6.1	75.4 ± 7.4	73.2 ± 7.0*
Monocyte (%)	3.6 ± 1.1	3.9 ± 1.6	3.4 ± 1.1	4.1 ± 1.8	3.6 ± 1.5	3.7 ± 0.2	3.8 ± 1.7	3.3 ± 0.9
Eosinophil (%)	1.4 ± 0.5	1.9 ± 0.5	2.3 ± 0.7*	1.8 ± 0.6	2.1 ± 0.9	1.9 ± 0.3	2.6 ± 1.1	2.1 ± 0.6
Basophil (%)	0.2 ± 0.1	0.2 ± 0.1	0.2 ± 0.1	0.3 ± 0.1	0.2 ± 0.1	0.2 ± 0.0	0.2 ± 0.1	0.2 ± 0.1
RBC (M)	8.56 ± 0.19	8.43 ± 0.34	8.55 ± 0.42	8.19 ± 0.19^Ɨ	7.82 ± 0.28	8.16 ± 0.40	7.96 ± 0.23	7.82 ± 0.25
Hb (g/dl)	15.3 ± 0.5	14.9 ± 0.7	14.8 ± 0.8	14.4 ± 0.3^Ɨ	14.4 ± 0.5	14.5 ± 0.6	14.6 ± 0.4	14.1 ± 0.4
Hct (%)	45.5 ± 1.4	44.3 ± 1.8	44.4 ± 2.2	43.1 ± 0.8^Ɨ	42.3 ± 1.5	43.1 ± 2.0	42.8 ± 1.3	42.0 ± 1.2
MCV (fL)	53.1 ± 1.1	52.5 ± 1.3	52.0 ± 2.7	52.7 ± 0.9	54.1 ± 1.2	52.0 ± 2.4	53.8 ± 1.3	53.7 ± 0.9
MCH (pg)	17.9 ± 0.4	17.6 ± 0.5	17.3 ± 1.0	17.6 ± 0.4	18.5 ± 0.5	17.5 ± 0.9	18.3 ± 0.5	18.1 ± 0.2
MCHC (g/dL)	33.8 ± 0.5	33.5 ± 0.5	33.2 ± 0.6	33.4 ± 0.5	34.1 ± 0.4	33.0 ± 1.4	34.1 ± 0.2	33.7 ± 0.4
Reticulocyte (%)	2.04 ± 0.34	2.1 ± 0.3	2.38 ± 0.52	2.37 ± 0.38	1.79 ± 0.41	2.11 ± 0.26	2.02 ± 0.49	2.27 ± 0.42
PLT (K)	1148.2 ± 124.4	1071.6 ± 98.4	1213.9 ± 123.4	1264.5 ± 171.4	1153.7 ± 171.4	1153.2 ± 105.1	1169.5 ± 427.3	1441.4 ± 210.2
PT (sec)	17.7 ± 0.5	17.6 ± 0.6	16.7 ± 0.6^Ɨ	16.9 ± 0.6^Ɨ	16.9 ± 0.8	17.1 ± 0.6	17.1 ± 1.0	16.2 ± 0.9
APTT (sec)	20.3 ± 1.2	19.5 ± 1.9	19.4 ± 1.2	18.4 ± 1.1*	16.4 ± 0.8	18.8 ± 1.5	16.5 ± 0.9	16.7 ± 0.8

WBC: white blood cell, RBC: red blood cell, Hb haemoglobin, Hct: haematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular haemoglobin, MCHC: mean corpuscular hemoglobin concentration, PLT. Platelet, PT: prothrombin time, APTT: activated partial thromboplastin time.

*p<0.05,^Ɨp<0.01

 

Table 3. Biochemical values of SD rats orally treated with tetrasodium pyrophosphate for 90 days.

	Male				Female
	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
TP (g/dL)	6.2 ± 0.4	6.0 ± 0.2	5.4 ± 0.3^Ɨ	4.8 ± 0.3^Ɨ	6.7 ± 0.3	6.5 ± 0.3	5.8 ± 0.5^Ɨ	4.9 ± 0.3^Ɨ
Albumin (g/dL)	2.5 ± 0.2	2.4 ± 0.1	2.3 ± 0.1*	2.1 ± 0.1^Ɨ	3.0 ± 0.2	2.9 ± 0.2	2.7 ± 0.2	2.3 ± 0.2^Ɨ
A/G ratio	0.67 ± 0.05	0.68 ± 0.06	0.73 ± 0.02	0.74 ± 0.05*	0.78 ± 0.06	0.84 ± 0.05	0.88 ± 0.06*	0.87 ± 0.06*
T-BIL (mg/dL)	0.078 ± 0.010	0.071 ± 0.010	0.075 ± 0.015	0.076 ± 0.031	0.104 ± 0.010	0.096 ± 0.013	0.083 ± 0.017	0.075 ± 0.024^Ɨ
ALP (U/L)	270 ± 49.0	278 ± 36.0	277 ± 74.0	227 ± 55.0	146 ± 40.0	139 ± 35.0	118 ± 23.0	103 ± 26.0*
AST (U/L)	99 ± 21.0	117 ± 20.0	103 ± 16.0	138 ± 25.0^Ɨ	113 ± 32.0	128 ± 50.0	109 ± 25.0	110 ± 25.0
ALT (U/L)	35 ± 5.0	37 ± 10.0	31 ± 4.0	39 ± 13.0	28 ± 7.0	39 ± 28.0	24 ± 4.0	17 ± 1.0^Ɨ
CRN (mg/dL)	0.6 ± 0.0	0.6 ± 0.0	0.6 ± 0.0	0.6 ± 0.1	0.7 ± 0.1	0.7 ± 0.1	0.7 ± 0.0	0.7 ± 0.1
BUN (mg/dL)	11.5 ± 1.4	11.5 ± 2.2	12.2 ± 1.3	13.6 ± 1.7	12.8 ± 2.6	14.3 ± 1.7	12.8 ± 1.7	11.8 ± 1.8
CHOL (mg/dL)	71 ± 14.0	67 ± 8.0	78.0 ± 19.0	75 ± 11.0	78 ± 18.0	81 ± 16.0	79 ± 12.0	74 ± 8.0
TG (mg/dL)	118 ± 29.0	106 ± 38.0	178 ± 68.0	183 ± 57.0*	25 ± 17.0	39 ± 22.0	91 ± 68.0	141 ± 96.0*
GLU (mg/dL)	143 ± 21.0	138 ± 15.0	139 ± 24.0	132 ± 14.0	143 ± 26.0	137 ± 13.0	128 ± 8.0	135 ± 18.0
Ca (mg/dL)	10.0 ± 0.2	9.9 ± 0.3	9.7 ± 0.4	9.2 ± 0.3^Ɨ	10.1 ± 0.4	10.0 ± 0.3	9.7 ± 0.2	9.0 ± 0.2^Ɨ
IP (mg/dL)	7.3 ± 0.4	6.7 ± 0.5	6.4 ± 0.5^Ɨ	5.7 ± 0.4^Ɨ	6.1 ± 0.6	6.1 ± 0.8	5.6 ± 0.4	4.7 ± 0.4*
CK (IU/L)	354 ± 221.0	504 ± 233.0	449 ± 268.0	617 ± 181	533 ± 259.0	556 ± 203.0	569 ± 433	612 ± 318.0
Na (mmol/L)	142 ± 2.0	141 ± 2.0	140 ± 2.0	139 ± 1.0	142 ± 1.0	141 ± 1.0	140 ± 1.0^Ɨ	139 ± 2.0
K (mmol/L)	4.6 ± 0.3	4.6 ± 0.1	4.6 ± 0.2	4.2 ± 0.3*	4.3 ± 0.2	4.2 ± 0.2	4.3 0.2	4.0 ± 0.3
Cl (mmol/L)	107 ± 2.0	108 ± 1.0	106 ± 2.0	103 ± 3.0*	107 ± 1.0	106 ± 1.0	105 ± 1.0	105 ± 2.0

TP: total protein, T-BIL: total bilirubin, ALP: alkaline phosphatase, AST: aspartate aminotransferase, ALT: alanine aminotransferase, CRN: creatinine, BUN: blood urea nitrogen, CHOL: total cholesterol, TG: triglycerides, GLU: glucose, Ca: calcium, IP phosphorus, CK: cratitnine kinase, Na: sodium, K: potassium, Cl: chloride.

*p<0.05,^Ɨp<0.01

 

Table 4. Absolute organ weights of SD rats orally treated with tetrasodium pyrophosphate for 90 days.

Organs (g)	Male				Female
	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
Liver	14.54 ± 1.24	14.26 ± 2.17	16.28 ± 2.55	14.53 ± 1.59	7.33 ± 0.83	7.25 ± 0.87	8.09 ± 0.60	9.06 ± 1.09
Kidney left	1.77 ± 0.15	1.76 ± 0.23	1.82 ± 0.17	1.69 ± 0.19	0.99 ± 0.13	0.97 ± 0.07	1.02 ± 0.07	1.06 ± 0.10
Kidney right	1.70 ± 0.19	1.74 ± 0.20	1.82 ± 0.20	1.72 ± 0.18	1.00 ± 0.09	0.89 ± 0.32	1.05 ± 0.06	1.08 ± 0.12
Adrenal gland left	0.036 ± 0.008	0.029 ± 0.004	0.031 ± 0.005	0.035 ± 0.006	0.032 ± 0.007	0.034 ± 0.003	0.033 ± 0.007	0.036 ± 0.005
Adrenal gland right	0.035 ± 0.004	0.032 ± 0.004	0.030 ± 0.004	0.031 ± 0.004	0.033 ± 0.007	0.034 ± 0.005	0.034 ± 0.006	0.037 ± 0.009
Testis/ovary left	1.76 ± 0.13	1.76 ± 0.13	1.74 ± 0.15	1.70 ± 0.11	0.041 ± 0.011	0.041 ± 0.013	0.043 ± 0.009	0.046 ± 0.008
Testis/ovary right	1.76 ± 0.11	1.73 ± 0.13	1.73 ± 0.14	1.71 ± 0.11	0.043 ± 0.011	0.043 ± 0.008	0.044 ± 0.006	0.045 ± 0.008
Brain	2.14 ± 0.09	2.10 ± 0.12	2.10 ± 0.08	2.07 ± 0.11	1.96 ± 0.10	1.95 ± 0.13	1.95 ± 0.11	1.97 ± 0.11

*p<0.01

 

Table 5. Relative organ weights of SD rats orally treated with tetrasodium pyrophosphate for 90 days.

Organs (g)	Male				Female
	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
Liver	2.5 ± 0.16	2.53 ± 0.18	2.76 ± 0.17*	2.80 ± 0.17*	2.41 ± 0.10	2.42 ± 0.10	2.81 ± 0.18^Ɨ	2.94 ± 0.25
Kidney left	0.30 ± 0.03	0.31 ± 0.02	0.32 ± 0.03	0.33 ± 0.03	0.33 ± 0.04	0.33 ± 0.04	0.35 ± 0.03	0.34 ± 0.04
Kidney right	0.31 ± 0.03	0.31 ± 0.02	0.32 ± 0.01	0.33 ± 0.03	0.33 ± 0.02	0.30 ± 0.11	0.36 ± 0.02	0.35 ± 0.04
Adrenal gland left	0.006 ± 0.001	0.005 ± 0.001	0.005 ± 0.001	0.007 ± 0.001	0.010 ± 0.002	0.011 ± 0.002	0.011 ± 0.003	0.012 ± 0.001
Adrenal gland right	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.011 ± 0.002	0.012 ± 0.002	0.012 ± 0.002	0.012 ± 0.003
Testis/ovary left	0.30 ± 0.02	0.32 ± 0.04	0.30 ± 0.04	0.33 ± 0.04	0.014 ± 0.04	0.014 ± 0.004	0.014 ± 0.003	0.015 ± 0.003
Testis/ovary right	0.30 ± 0.01	0.31 ± 0.04	0.30 ± 0.05	0.33 ± 0.04	0.014 ± 0.03	0.015 ± 0.003	0.014 ± 0.001	0.015 ± 0.003
Brain	0.37 ± 0.03	0.38 ± 0.04	0.37 ± 0.04	0.40 ± 0.04	0.65 ± 0.08	0.66 ± 0.09	0.65 ± 0.05	0.64 ± 0.05

*p<0.05,^Ɨp<0.01

 

Table 6. Incidence of histopathological findings in SD rats orally treated with tetrasodium pyrophosphate for 90 days.

Findings	Male				Female
	Group 1	Group 2	Group 3	Group 4	Group 1	Group 2	Group 3	Group 4
Liver
Centrolobular vacuolation	0	0	1	0	0	0	0	0
Periportal vacuolation	0	0	0	0	0	0	0	2
Parenchymal degeneration	0	0	0	0	0	0	0	0
Kidney
Cortical tubular basophilia	3	3	3	7	0	1	0	6
Medullary tubular basophilia	0	0	0	0	0	0	1	0
Interstitial inflammatory cells	2	3	1	0	0	0	0	0
Cortical mineralization	0	0	0	0	0	0	0	3
Medullary mineralization	0	0	0	0	0	0	2	9
Medullary cyst	2	0	0	0	1	0	0	0
Papillary epithelial hyperplasia	0	1	0	0	0	0	0	0
Cortical scarring	0	0	2	0	1	0	0	0
Transitional epithelial hyperplasia	0	0	2	0	0	0	0	0
Pelvic transitional epithelial Inflammatory cells	0	0	0	1	0	0	0	0
Adrenal gland
Cortical vacoulation	4	7	5	2	0	0	0	1
Hypertrophy-zona glomerulosa	0	0	1	0	0	0	0	0
Cortical cyst	0	0	0	1	0	0	0	0
Heart
Myocardial inflammatory cells	1	8	6	5	0	0	2	0
Myocardial fibrosis	2	1	3	0	0	0	0	4
Spleen
Extramedullary hematopoiesis	1	1	1	0	0	0	0	0

*p<0.05,^Ɨp<0.01

Conclusions:

Tetrasodium pyrophosphate was found to have a NOAEL of 500 mg/kg/day and the target organ appears to be the kidney in rats.

Executive summary:

A 90 -day repeated dose toxicity study was performed on Sprague-Dawley rats according to OECD 408. Tetrasodium pyrophosphate was orally administered to 10 male and 10 female Sprague-Dawley rats, at 0, 250, 500 and 1000 mg/kg/day five times a week for 90 days. Body weights were recorded at baseline and weekly after. Mortality, feed intake, and body weight were monitored. There were no significant changes in body weight in the 1000 mg/kg treatment group, or food consumption, urinalysis, and haematology in any group. The levels of total protein, albumin, A/G ratio, triglyceride, calcium and inorganic phosphate were altered at doses 500 and 1000 mg/kg. However, no changes were observed at the dose of 250 mg/kg. No mortality occurred throughtout the study and no clinical signs were observed. Regarding histopathological findings, cortical tubular basophila of the kidney increased at the dose of 1000 mg/kg, but not at doses of 250 and 500 mg/kg. No significant changes were observed in other organs at doses of 250, 500 and 1000 mg/kg. Based on these results, the test item was found to have a NOEL and a NOAEL of 250 and 500 mg/kg/day respectively and the target organ appears to be the kidney in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 2

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Key study: A 72-week repeated dose toxicity study was performed on rats similar to OECD 452. Choline chloride was orally administered to 30 male Fischer 344 rats, at 1% (500 mg/kg/day). Body weights were taken at weekly intervals for 16 weeks and biweekly thereafter. Two animals per group were sacrificed at 28 weeks for an interim assessment of the experiment's progress. Afterwards, all the animals dying or sacrificed during the study were necropsied. Liver weights and body weights were taken at necropsy. The livers and all other organs bearing gross abnormalities were routinely removed and fixed in buffered formalin. They were then sectioned and stained with haematoxylin and eosin for histological examination. No marked effect on growth was observed and no mortality occurred throughout the study. The histological distribution of liver tumours showed no significant differences between the control and treated group. Under these conditions, Choline chloride has a lethal concentration 0 of 500 mg/kg/day in rats (1%), after 72-week oral administration.

- Key study: 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.

- Key study: A 90 -day repeated dose toxicity study was performed on Sprague-Dawley rats according to OECD 408. Tetrasodium pyrophosphate was orally administered to 10 male and 10 female Sprague-Dawley rats, at 0, 250, 500 and 1000 mg/kg/day five times a week for 90 days. Body weights were recorded at baseline and weekly after. Mortality, feed intake, and body weight were monitored. There were no significant changes in body weight in the 1000 mg/kg treatment group, or food consumption, urinalysis, and haematology in any group. The levels of total protein, albumin, A/G ratio, triglyceride, calcium and inorganic phosphate were altered at doses 500 and 1000 mg/kg. However, no changes were observed at the dose of 250 mg/kg. No mortality occurred throughout the study and no clinical signs were observed. Regarding histopathological findings, cortical tubular basophila of the kidney increased at the dose of 1000 mg/kg, but not at doses of 250 and 500 mg/kg. No significant changes were observed in other organs at doses of 250, 500 and 1000 mg/kg. Based on these results, the test item was found to have a NOEL and a NOAEL of 250 and 500 mg/kg/day respectively and the target organ appears to be the kidney in rats.

- Key study: A long-term repeated dose toxicity study of phosphoric acid was performed on rats. Male and female rats were administered on a basal diet with the addition of phosphoric acid at 0.4% or 0.75%. The groups were kept in the experiment after six months, and observations continued through two filial generations (more than twelve months). Effect on growth in three generations, reproduction and blood parameters of the first two generations. No adverse effects were noted at up to 0.75%. The NOEL for this study was 0.75% or more of phosphoric acid in the diet, which was equivalent to 386 mg/kg/day or more, assuming a 0.35 kg rat consumes 18 g food/day. Therefore, the test item has a NOAEL>386 mg/kg/day in rats, after administration in diet for more than twelve months.

Justification for classification or non-classification

Based on available data, the substance is not classified for repeated dose toxicity according to CLP Regulation (EC) No. 1272/2008.