Calcium bis(dihydrogenorthophosphate)

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Follows basic scientific and laboratory principles, article has been peer-reviewed.

Data source

Reference

Reference Type:

publication

Title:

Dietary phosphate modulates Atherogenesis and Insulin Resistance in Apolipoprotein E Knockout mice.

Author:

Ellam T, Wilkie M, Chamerlain J, Crossman D, Eastell R, Francis S and Chico JA

Year:

2011

Bibliographic source:

Arteriosclerosis, Thrombosis and Vascular Biology. 31:00-00

Report date:

2011

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Apolipoprotein E knockout mice were fed an atherogenic diet with low (0.2%), standard (0.6%), or high (1.6%) phosphate content.

GLP compliance:

not specified

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: oral

Test material

Test material information

Constituent 1

Reference substance name:

Calcium bis(dihydrogenorthophosphate)

EC Number:

231-837-1

EC Name:

Calcium bis(dihydrogenorthophosphate)

Cas Number:

7758-23-8

Molecular formula:

CaH4O8P2

IUPAC Name:

calcium dihydrogen phosphate

Constituent 2

Reference substance name:

Calcium hydrogenorthophosphate

EC Number:

231-826-1

EC Name:

Calcium hydrogenorthophosphate

Cas Number:

7757-93-9

Molecular formula:

CaHPO4

IUPAC Name:

calcium hydrogen phosphate

Details on test material:

- Name of test material (as cited in study report): study states that calcium monophosphate and calcium diphosphate were used in the study (assumed to be calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate).

Test animals

Species:

mouse

Strain:

other: Apolipoprotein E knockout mice

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: JAX LABS (JAX 2052) - breeding colony maintained in the laboratory.
- Age at study initiation: 8 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 6 light/6 dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

DIET: atherogenic diets (21% fat, 0.2% cholesterol, 0.03% cholate)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Phosphate content of test diets was confirmed by independent laboratory assessment (N.P, Analytical Laboratories, USA). Data not presented in article.

Duration of treatment / exposure:

12 and 20 weeks

Frequency of treatment:

Daily

Post exposure period:

No post-exposure period

Doses / concentrations

Remarks:

Doses / Concentrations:
0.2, 0.6 and 1.6% phosphate
Basis:
nominal in diet

No. of animals per sex per dose:

5 to 8 per group

Examinations

Examinations:

Blood Pressure Measurement
Systolic and diastolic blood pressure measurements were performed twice weekly on 4 animals per dietary group (total N=12) using tail cuff measurements (Visitech 2000, Visitech Systems, NJ, USA). Mean arterial pressure was calculated from each pair of systolic/diastolic measurements.

Biochemical Analyses
Fasting glucose was measured on whole blood (Medisense, Optium Xceed). Commercial sandwich ELISA assays were used to quantify plasma insulin (Crystalchem, USA), adiponectin (R&D systems, UK), parathyroid hormone (Immutopics, USA) and FGF23 (KAINOS laboratories, Japan), according to the manufacturers’ instructions. All other plasma biochemistry measurements were performed by autoanalyzer (Beckman Coulter DxC). LDL cholesterol was calculated from other lipid fractions according to the Friedewald formula1. Insulin resistance was measured by standard homeostatic model assessment2 (HOMA-IR) = fasting glucose (mmol/l) × fasting insulin (mU/l)/22.5.

Tissue collection
Mice were sacrificed by intraperitoneal injection of an overdose of pentobarbitone (4mg) and blood aspirated by cardiac puncture. The vasculature was then flushed with phosphate-buffered saline and perfusion-fixed by ventricular injection of 10% formalin. Thoracic aortae were dissected free of connecting tissue from the heart to the level of the diaphragm and fixed in 4% paraformaldehyde. Following fixation in 10% formalin, hearts and livers were dehydrated and embedded in paraffin wax. Epididymal fat pads, kidneys, lungs, livers and spleens were dissected free of connective tissue before weighing.

Results and discussion

Details on results:

Effects of Dietary Phosphate Group on Urea, Weight and Blood Pressure
Dietary phosphate intake had no significant effect on urea (7.3±0.5mmol/L, 8.0±0.5mmol/L and 7.0±0.5mmol/L for low, standard and high dietary phosphate groups respectively). Lower dietary phosphate intake was associated with a trend towards greater chow consumption and BMI, with a
significantly greater final weight and body length. With the exception of liver and epididymal fat pads, other organ weights (heart, lung, kidneys, and spleen) did not differ between groups. Blood pressure also did not differ between groups.
Increased dietary phosphate intake had no effect on aortic sinus atheroma at 12 weeks but was associated with significantly more atheroma at 20 weeks.
Calcification was absent by von Kossa staining, and lesions did not differ in vascular smooth muscle cell or macrophage content.
Epididymal fat pad mass was significantly greater in the low versus high dietary phosphate. Insulin resistance measured by homeostatic model assessment was increased 4-fold on a low-phosphate diet in comparison with other dietary groups.This was not accounted for by adiponectin, which did not differ between low and standard dietary phosphate groups. Hepatic steatosis was induced in the low-phosphate group and was accompanied by greater liver weight and alanine transaminase.

Applicant's summary and conclusion

Conclusions:

The authors conclude that a high-phosphate diet accelerates atherogenesis in apolipoprotein E K/O mice, whereas low phosphate intake induces insulin resistance. These data indicate that controlling dietary phosphate intake may influence development of both atherosclerosis and the metabolic syndrome.
It is worth noting that the dose levels used in the study are not representative of a typical human diet; the standard P diet used contained 2X the amount of phosphate found in a typical human diet and no effects were noted at this dose level. Further investigation of the conclusions drawn from this investigation will be required to prove beyond doubt the link between phosphorus and heart disease.
It is therefore concluded that although these finding may be of use to the medical community they are not relevant for assessment of calcium phosphates under REACH. As with all essential elements it is likely that an increase or a decrease in phosphate levels may have a detrimental effect on the health of the individual. However the normal levels of intake for inorganic phosphates fall well within the safe range identified in this study and the maximum tolerable daily intake of 70 mg/kg bw of P/day, from all sources, is not considered to induce any of the effects noted in the study. The levels of occupational exposure to inorganic phosphates will not significantly contribute to the overall daily intake of phosphrous and it is therefore not considered appropriate to investigate this further for the purpose of REACH registration.