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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Metabolisation experiment as part of a dissertation. Tests performed according to accepted scientific methods.

Data source

Reference
Reference Type:
other: Dissertation
Title:
the effect of a prolonged intake of phosphoric acid and citric acid in rats
Author:
Bonting SL
Year:
1952

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
Two metabolism experiments were carried out. Calcium and phosphorus balances, the urinary excretion of ammonia, urea, total base and volume, pH and titratable acidity of the urine were determined. Experiments with radioactive phosphorus were perfomed as well: endogenous fecal phosporus excretion and phosphorus retention of the bone were determined.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Orthophosphoric acid
EC Number:
231-633-2
EC Name:
Orthophosphoric acid
Cas Number:
7664-38-2
Molecular formula:
H3O4P
IUPAC Name:
phosphoric acid
Details on test material:
- Name of test material (as cited in study report): Phosphoric acid
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
other: Mus norvegicus
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar Institute (U.S.A.)
- Age at study initiation: 8.5 months
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: metabolism cages
- Diet (e.g. ad libitum): food was given ad libitum every other day
- Water (e.g. ad libitum): automatic drinking water supply system
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): constant temperature of 25°C
- Humidity (%): more or less constant
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The acids were added to the basal diet in the required amounts. The mixture was homogenized by rubbing it through a sieve. phosphoric acid 89%, sp.gr. 1.75, diluted to a solution containing 500 mg/mL H3PO4 before use, of pharmacopoeia quality, was used through the investigation

DIET PREPARATION
All animals received the same basal diet, which was designed to resemble the average Dutch diet as closely as possible, especially with respect to the acid-base balance. Linseed oil was added four months after the beginning of the experiments, when a slight dermatosis of the tail was observed in a considerable number of the rats grown on this diet, which was ascribed to linolenic acid deficiency. This diet is slightly acidogenic: urine was obtained with a mean pH of 5.87 (standard error 0.03) from the rats. The composition of the basal diet is specified in the field 'Any other information on results including tables'.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
Duration and frequency of treatment / exposure:
Experiment 1: 6 weeks of basal diet, 8 weeks basal diet + acid + 6 weeks basal diet
Experiment 2: without control period; all animals had received the diet during 8 months previous to this metabolism experiment, enabling us to compare the metabolism of animals who had received the diets already during a considerable part of their life time (5 weeks of comparison).
Doses / concentrations
Remarks:
Doses / Concentrations:
Experiment 1: 0.40%
Experiment 2: 0.40%
No. of animals per sex per dose / concentration:
8 females per dose; in preliminary experiments it was found that female rats adapted themselves better to the metabolism cages than male rats. Therefore in the two experiments, adult female rats were used. in the endogenous fecal phosphorus excretion experiment 10 female rats (aged 7 months, diet during 6 months) were used. In the phosphorus retention experiment of the tibia 33 male rats of the different diet groups were used. Of each group, 11 animals, aged 13 months, who received the diets during 12 months previously.
Control animals:
other: control period in the first experiment: 6 weeks on basal diet before and after the experiment for all the animals tested
Positive control reference chemical:
No data
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): not applicable
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: blood, urine, faeces, tissues, kidney, bone
- Time and frequency of sampling:
first experiment phosphorus balance: 480 balance figures of 24 animals during 20 weeks; no data on second experiment
urine: collection period of 7 days
- From how many animals: it was not possible to give all experimental data in detail, mostly averages were reported
- Method type(s) for identification:
Inorganic phosphorus (blood): colorimetric method of Sumner (1944)
Calcium content (blood): deproteinization with trichloroacetic acid -> calcium precipitated with ammonium oxalate. Calcium oxalate dissolved in sulfuric acid and is tirated with KMnO4
Total base content (serum, liver and muscle): ion exchange method; bicarbonate was determined by the Van Slyke method
Alkaline reserve/total CO2 content (serum): determined with manometric method of Van Slyke
Chloride (blood, liver and muscle): iodometric determination of Van Slyke
Alkaline phosphatase (blood): method of Bessey, Lowry and Brock (1946)
Total phosphorus (liver and muscle): wet-ashing method of Neumann (1902)
Potassium and sodium (liver and muscle): flame photometric determination
Water and ash content (kidneys): one kidney: alkaline phosphatase determination; one kidney: determination of water and ash content
Ash content, P content and Ca content from tibia (bone): Ca content determined by method of Wang (1935) without deproteinization
Urine: pH and titratable acidity: 1st determination: Beckman pH meter; titratable acidity: method of Henderson and Palmer (1914); ammonia, urea and total nitrogen: ammonium and urea determined with microdiffusion technique by Conway (1947); in addition phosphorus, calcium, total base and chloride content were determined according to methods mentioned above
Feces: phosphorus, calcium and total nitrogen; latter was determined by the Kjeldahl method
Radioactive phosphorus experiments: endogenous fecal phosphorus retention; phosphorus retention of the bone: activity measurements were carried out using a Geiger Muller counting tube and an electronic scaling circuit followed by a mechanical register.
- Limits of detection and quantification: no data


TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): Not applicable
Statistics:
test of Wilcoxon: phosphorus balance, tissue analysis

Results and discussion

Preliminary studies:
In the preliminary experiments it was found that female rats adapted themselves better to the metabolism cages than male rats.

Toxicokinetic / pharmacokinetic studies

Details on excretion:
Two metabolism experiments were performed where the phosphorus balance was determined, being the difference between the total phosphorus intake and the sum of urinary and fecal phosphorus excretion in mg/d of phosphorus. Strictly spoken only inorganic urinary phosphorus was determined, but as this compromises generally 95 to 99% of the total urinary phosphorus the difference is negligible. In both metabolism experiments a significantly increased phosphorus retention was found on administration of phosphoric acid. The ratio of urinary to fecal phosphorus excretion was increased in both experiments, but significantly only in the first experiment. The phosphoric acid increased the phosporus balance and the urinary phosphorus excretion markedly, while the fecal phosphorus excretion was not increased or onlyl very little. The results of the experiments with radioactive phosphorus are the following: In this experiment the percentage of endogenous phosphorus in the total fecal phosphorus excretion was determined. No significant differences were found for either of the acid diets in the percentage of endogenous fecal phosphorus, in the ratio of urinary to fecal activity excreted per day and in the total activity excreted per day. Only the ratio urinary to fecal phosphorus excretion per day for the group of 0.40% H3PO4 was increased significantly, a fact found already in the metabolism experiments described above. There is however a tendency towards a parallel increase in the ratio of the activities excreted daily in urine and feces. Results of phosphorus retention of the bone are the following: in this experiment no appreciable effect of the acid diets on the phosphorus metabolism of the bone in the animals was found.

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
No details provided in the dissertation

Any other information on results incl. tables

Treatment of results: in order to obtain values that could be compared irrespectivelly of differences in body size of the animals, results were divided by the 2/3 power of the body weights. The 2/3 power of the body weight should approximately be proportional to the body surface.

The most outstanding results of the chemical experiments were:

- The alkaline reserve of an acidotic condition in the animals tested, following from the alkanline reserve of the blood

- The absence of a decrease in the calcium balance

- The absence of an increase in the fixed base excretion

- The absence of a decrease in the nitrogen balance as well as of an increase in the urinary ammonia plus urea nitrogen excretion

- The complete similarity between the two metabolism experiments, proving that the effects of the phosphoric acid on the mineral metabolism was the same after prolonged intake as after a relatively short one.

These results prove that the animals could ingest phosphoric acid in the quantity given during a considerable period of life without disturbance of the fixed base and nitrogen metabolism. A noteworthy feature of the results for the nitrogen metabolism is the decrease of the urea output accompanying the increased ammonia excretion, the sum of urinary ammonia and urea nitrogen remaining constant. According to the results of the metabolization experiments the phosphoric acid activated the following neutralization mechanisms:

- the excretion of ammonia by the kidney

- the phosphoric shift, resulting in an increased titratable acidity and a decreased pH of the urine

- the excretion of acid calcium phosphates in the feces instead of Ca3(PO4)2. Neutralization by means of the blood bicarbonate was not found to have taken place.

The evidence obtained from the metabolism experiments and the tissue analyses proved that the prolonged intake of the acid diets did not produce an acidotic condition. No evidence of demineralizing effect of the acid diets was found in the metabolism experiments as well as in the tissue analyses.

From the experiments with radioactive phosphorus no evidence of changes in the endogenous fecal phosphorus excretion and in the phosphorus retention of the bone was found. Apart from an increased retention of phosphorus and an increased urinary phosphorus excretion no evidence of any fundamental changes in the phosphorus metabolism was found. Some further experiments concerning the effect on the mineral composition of the bone are needed.

Applicant's summary and conclusion

Conclusions:
As in man the same mechanisms are known to function in the neutralization and excretion of ingested acid as found in the rat, it is believed justified in expecting from the results of the experiments in this dissertation that no harm can result from a prolonged consumption of beverages acidulated with phosphoric acid.

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