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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
effects on body weight may have contributed to the results of this study, limited data available

Data source

Reference
Reference Type:
publication
Title:
Morphologic and functional alterations of erythroid cells induced by long-term ingestion of aluminium.
Author:
Vittori et al.
Year:
1999
Bibliographic source:
J Inorg Biochem. 1999 Aug 30; 76(2):113-20.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Aluminium citrate was administered to ten female Sprague Dawley rats with drinking water at a concentration of 80 mmol/L for 8 months. Eight female rats that received drinking water without added aluminium citrate were used as controls. Blood was collected at the end of the treatment. After blood collection, the animals were sacrificed, femoral bone marrow was removed and smears were prepared. The remaining bone marrow cells were conditioned for in vitro clonal assays of haematopoietic progenitors (late colony-forming unit-erythroid, CFU-E) and in vitro cellular iron uptake. Livers, kidneys, spleens, brains and the remaining femora were removed and prepared for subsequent analysis for Al content. Haemoglobin, hematocrit, reticulocyte counts, circulating haptoglobin, plasma iron and aluminium, free haemoglobin in plasma and total iron-binding capacity were determined. Morphology of circulating erythrocytes and erythropoietic cells in peripheral blood films and bone marrow smears were studied under light and electronic microscopes.
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Aluminium citrate
EC Number:
250-484-4
EC Name:
Aluminium citrate
Cas Number:
31142-56-0
IUPAC Name:
aluminum citrate
Details on test material:
- Name of test material (as cited in study report): Aluminium citrate
- Analytical purity: No data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: No information.
- Age at study initiation: 21 days
- Weight at study initiation: 101±8 g
- Housing: No information.
- Diet: “Standard diet” was given to all rats. Water to the control rats was provided ad libitum.
- Water: No information about access to water for the treated animals.


ENVIRONMENTAL CONDITIONS
“Controlled temperature, humidity and lighting were maintained throughout the experimental period.” No details provided. “Controlled temperature, humidity and lighting were maintained throughout the experimental period.” No details provided.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
“Al citrate solution (1:1) was freshly prepared by mixing equimolar solutions of aluminium chloride and sodium citrate.”
Al content in the diet was 10 µmol/g food; in the drinking water - 5 µmol/L. Each rat received 6-11 µmol Al/g b.w. daily with food and drinking water.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data.
Duration of treatment / exposure:
8 months
Frequency of treatment:
Daily, 7 days per week.
Doses / concentrations
Dose / conc.:
302 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
80 mmol/L
Basis:
nominal in water
Based on daily intake of 0.037 L water/day and an average body weight of 267 g --> the daily dose is 302 mg/kg bw
No. of animals per sex per dose:
10 animals in the treated group and 8 animals in the control group; all female
Control animals:
other: Control animals received drinking water without added aluminium citrate
Details on study design:
Random assignment to experimental groups.
Positive control:
No data.

Examinations

Observations and examinations performed and frequency:
Observations and clinical examination:
- Observation for morbidity and mortality: no information
- Food consumption: no information
- Body weights: only final body weights reported
- Water consumption: no information

Haematology:
Haematology parameters were assessed at the end of treatment: hematocrit, blood haemoglobin (the cyanmethaemoglobin method), reticulocyte count, morphology of circulating erythrocytes (under light and electronic microscopes) and of erythropoietic cells in peripheral blood films and bone marrow smears (under light microscope).

Femoral bone marrow cells were conditioned for in vitro clonal assays of haematopoietic progenitors (late colony-forming unit-erythroid, CFU-E) and in vitro cellular iron uptake.

CFU-E assay (developmental ability of haematopoietic progenitors)
Bone marrow cells were flushed into minimal essential medium Eagle, α-modification (α-MEM) containing 2% heat inactivated bovine serum. Single cell suspensions were obtained. Triplicate cell cultures stimulated with 0.25 U/ml recombinant human erythropoietin were carried out in α-MEM-methylcellulose medium. Erythroid colonies of 8 or more hemoglobinised cells derived from the most differentiated erythropoietic progenitors were considered as CFU-E

In vitro cellular iron uptake
Bone marrow cells from 6 treated and 4 control animals were suspended in liquid medium, supplemented with 5% foetal bovine serum, 0.4 µM apotransferrin and stimulated with recombinant human erythropoietin (0.75 U/ml). Triplicate cell cultures were incubated for 24 hours at 100% humidity and 37°C in an atmosphere containing 5% CO2 in the presence of 59Fe citrate. After the incubation, two aliquots were taken and washed twice with phosphate-buffered saline. In one aliquote, 59Fe radioactivity (characteristic gamma-radiation) was measured to determine cell 59Fe uptake. The cell pellet in the second aliquot was mixed with 1 ml cyanmethaemoglobin, 0.2 ml 0.1 M HCl and vortexed with 1.5 ml cyclohexanone. The emulsion was centrifuged, and the organic layer containing 59Fe-haem as cyanmethematin was counted.

Blood chemistry:
The following parameters were determined in plasma:
- haptoglobin (radial immunodiffusion technique)
- free haemoglobin (a colored product produced by Hb reaction with benzidine in acid solution and hydrogen peroxide was measured spectrophotometrically)
- iron concentration
- total iron-binding capacity (TIBC)

Sacrifice and pathology:
The animals were sacrificed after the treatment had been terminated and blood drawn. Histopathological examination was not performed.
Other examinations:
Toxicokinetics/Biodistribution:
Aluminium in plasma, liver, kidney, spleen, brain and femur samples was determined using an atomic absorption spectrometer coupled with a graphite furnace atomiser.
Aluminium in circulating erythrocytes was determined by scanning electron microscopy in combination with energy dispersive X-ray analysis (EDAX)
Measures to prevent contamination with Al
Double deoinized water was used to prepare solutions and culture media. Laboratory glassware and plastic ware were treated with 30% HCl and then carefully rinsed with double deoinized water. Al-free vials used for acid solutions from digested tissues were prepared by immersion in 15% HNO3 for 48 hours.

Statistics:
The results were expressed as mean±standard deviation or as median and range. The non-parametric Mann-Whitney U-test was used for comparisons between the experimental groups.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final body weight was significantly lower in the Al treated group than in the control group (272±25 g and 361±38 g, respectively; p<0.001).
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
No data
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality:
No information

Body weight:
Final body weight was significantly lower in the Al treated group than in the control group (272±25 g and 361±38 g, respectively; p<0.001).

Haematology:
Significantly lower haematocrit (38.8±4.29% vs. 43.1± 3.58%, p<0.05) and blood Hb concentration (137±10.1 g/L vs. 148±8.5 g/L) in the Al treated rats than in the control rats.
Mature erythrocyte morphology
Anisocytosis (abnormal cell size), anisochromia (non-uniformity of cell staining) and poikilocytosis (abnormal cell shape) were observed in the blood films from the Al-treated rats. Scanning electron microscopy showed red cell fragments (schistocytes), unusually thin cells (leptocytes), crenated and target cells and other abnormal cells not seen in control samples.
Reticulocyte count
Significantly higher in the Al treated group than in the control group (1.8% vs. 1.2%; p<0.05).
Bone marrow
A significant inhibition of CFU-E growth (≈65% relative to the control group; p<0.005) was seen in the Al treated group.
A significant (p<0.05) reduction of 59Fe uptake in the bone marrow cells of Al exposed rats compared to the control rats.

Clinical Chemistry (blood):
Plasma iron concentration and total iron-binding capacity: no significant group differences (indicates that the Al treated animals were not depleted of iron).

Plasma Hb concentrations: no significant group differences.

Plasma haptoglobin concentration: significantly lower in the Al treated animals than in the control animals (p<0.05). This decrease and the presence of erythrocytes with abnormal shape are indicative of intravascular haemolysis.

Blood urea concentration: no significant group differences (indicates that kidney function was not altered by Al administration).


Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Tissue Metal Levels

Al concentrations in the bone, spleen, liver, kidney and plasma were significantly higher in the Al treated group than in the control group. No significant group differences in brain Al concentrations. There was no correlation between plasma Al concentrations and Al levels in the organs or any other biochemical data.

Scanning electron microscopy combined with EDAX detected Al inside circulating erythrocytes with abnormal shape from animals in the Al treated group.

Applicant's summary and conclusion

Conclusions:
The results of this study suggest that Al affects erythropoiesis in rats with normal renal function.
Executive summary:

Aluminium citrate was administered to ten female Sprague Dawley rats with drinking water at a concentration of 80 mmol/L for 8 months. Eight female rats that received drinking water without added aluminium citrate were used as controls. Blood was collected at the end of the treatment. After blood collection, the animals were sacrificed, femoral bone marrow was removed and smears were prepared. The remaining bone marrow cells were conditioned for in vitro clonal assays of haematopoietic progenitors (late colony-forming unit-erythroid, CFU-E) and in vitro cellular iron uptake. Livers, kidneys, spleens, brains and the remaining femora were removed and prepared for subsequent analysis for Al content. Haemoglobin, hematocrit, reticulocyte counts, circulating haptoglobin, plasma iron and aluminium, free haemoglobin in plasma and total iron-binding capacity were determined. Morphology of erythrocytes and erythropoietic cells in peripheral blood films were studied under light and electronic microscopes. Plasma iron concentration and total iron-binding capacity were not different in the control and the Al treated rats, indicating that the Al treated animals were not depleted of iron. There were no significant group differences in blood urea concentration, which suggests that kidney function was not altered by Al administration. Significantly lower haematocrit and blood Hb concentration were observed in the Al treated rats than in the control rats. Significantly higher reticulocyte count, abnormal erythrocyte morphology, a significant inhibition of CFU-E growth and a significant reduction of59Fe uptake in the bone marrow were reported in the Al treated rats. Plasma haptoglobin concentration was significantly lower in the Al treated animals than in the control animals. This and the presence of abnormal erythrocytes in the Al treated rats are indicative of intravascular haemolysis. Scanning electron microscopy combined with EDAX detected Al inside circulating erythrocytes with abnormal shape from animals in the Al treated group. Al concentrations in the bone, spleen, liver, kidney and plasma were significantly higher in the Al treated group than in the control group. No significant group difference in brain Al concentrations was seen. There was no correlation between plasma Al concentrations and Al levels in the organs or any other biochemical data. The results of this study suggest that Al affects erythropoiesis in rats with normal renal function.