Aluminium tri-sec-butanolate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

The influence of citrate, maltolate and fluoride on the gastrointestinal absorption of aluminium at a drinking water-relevant concentration: A 26-Al and 14C study.

Author:

Zhou et al.

Year:

2008

Bibliographic source:

J Inorg Biochem 2008; 102: 798-808

Materials and methods

Objective of study:

absorption

toxicokinetics

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

not specified

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium potassium sulphate

IUPAC Name:

Aluminium potassium sulphate

Constituent 2

Reference substance name:

Aluminium sodium bis(sulphate)

EC Number:

233-277-3

EC Name:

Aluminium sodium bis(sulphate)

Cas Number:

10102-71-3

Molecular formula:

Al.2H2O4S.Na

IUPAC Name:

aluminium sodium bis(sulphate)

Test material form:

solid: particulate/powder

Details on test material:

Source of aluminium ion for iv infusion:
AlK(SO4)2 (source and purity not specified)

Radiolabelled materials:
26-Al (0.5 Ci/mole, 26-Al:27-Al ratio=1:34) in 0.1 N HCl was obtained from Purdue Rare Isotope measurement Lab (PRIME Lab).

14-C-citric acid (109 Ci/mole) was obtained from Amersham Biosciences.

14-C maltol (50.9 Ci/mole) was custom synthesized by Perkin Elmer.

Other chemicals:
Sodium fluoride and other chemicals used in producing the oral dosing solutions were obtained from Sigma.

Radiolabelling:

yes

Remarks:

organic ligands (citrate and maltolate) were labeled with 14-C and the Al (as AlK(SO4)2) with 26-Al for the gastric administration and 27-Al for the iv infusion dose.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 270±18 g
- Diet: Food access was limited to a 10% protein diet (Harlan Teklad 95215) available to the animals from 8am until 6pm each day for the 7 days prior to dosing. This diet is designed to minimize the retention of food in the GI tract.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose solution formulation:
Solutions of aluminium and the different ligands for oral dosing were prepared on the day of administration from stock solutions. The solutions were kept at room temperature for 1 hour and then the pH adjusted to (X) by addition of NaOH.

The aluminium solution for iv infusion was prepared by dissolving AlK(SO4)2 in saline followed by sterilization by filtration through a 0.22 μm filter.

Duration and frequency of treatment / exposure:

Single oral dosing with radiotracer.

Doses / concentrations

Remarks:

Doses / Concentrations:
The 22 rats were randomly assigned to receive an oral dosing of 1 mL Milli-Q purified water or 1 mL of a solution of 65 μM Al (52 ng [1 nCi] 26-Al) and ca. 1700 ng 27-Al). The rats also received an iv infusion of 100 μg Al/kg/h (as AlK(SO4)2) from 14 hours prior to 24 hours after the administration of the oral dose of radio-labelled dosing solution to maintain a plasma Al concentration of ca. 500 ng/mL.

No. of animals per sex per dose / concentration:

4 treatment groups; 5 animals per group and an additional 2 animals to monitor 26-Al and 14-C contamination of samples.
One animal was assigned to receive iv infusion of saline to measure endogenous serum Al concentration to give a total of 23 male animals.

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Not applicable.

Details on study design:

Twenty-two rats were randomly assigned to oral dosing with:
(a) 1 mL Milli-Q water negative control (n=2);
(b) 1 mL 26-Al labeled solution with no additional ligands (“free Al3+“, pH adjusted to about 5; n=5);
(c) 1 mL 26-Al labeled solution with added 14-C labeled citrate (Al:citrate=1:1; citrate concentration=30 nmol (3270 nCi); pH adjusted to about 7; n=5);
(d) 1 mL 26-Al labeled solution with added 14-C labeled maltolate (Al:maltolate=1:3; maltolate concentration=30 nmol (1530 nCi); pH adjusted to about 7; n=5); or
(e) 1 mL 26-Al labeled solution with added fluoride (Al:F=1:4; pH adjusted to about 4; n=5).
The rats also received an iv infusion of 100μg Al/kg/h (as AlK(SO4)2) from 14 hours prior to 24 hours after the administration of the oral dose of 26-Al radiolabelled dosing solution in order to maintain a plasma Al concentration of ca. 500 ng/mL. Al rats were implanted with two femoral cannulae 1 day before oral dosing to allow iv administration of the 27-Al solution and blood withdrawal (upstream cannula).
“Oral Al absorption was determined in the un-anaethestized rat”.
Although not stated explicitly, the 22 rats were anaesthetized, although the method was not described. Only one rat was un-aneasthetised.
From 08:00 until 18:00h for the 7 days prior to gastric dosing, the rats were only given access to a 10% protein diet (Harlan Teklad 95215) that minimized gastric retention. A pilot study (Yokel et al., 2001) had shown that this led to no food in the stomach prior to dosing.
Blood samples were taken 1 hour before and at 0.25 (0.4 mL), 0.5 (0.4 mL), 0.75 (0.4 mL), 1 (0.4 mL), 1.25 (0.4 mL), 1.5 (0.4 mL), 2 (0.4 mL), 4 (0.4 mL), 8 (0.6 mL) and 24 hours (2.2 mL) after the single oral dose. The volume withdrawn was replaced by injection of saline. Blood samples were analysed for total Al, 26-Al and 14-C. Blood urea nitrogen (BUN) and creatinine were also determined in the sample taken at 24 hours to investigate renal function. If BUN or creatinine were above the normal range (30 and 1 mg/dL, respectively), the rat was replaced.

Details on dosing and sampling:

Dosing:
Oral doses were administered intra-gastrically. To determine whether a significant amount of Al was lost by adsorption to the syringe or gastric feeding tube, the oral delivery procedure was simulated by dispensing the solutions into a plastic tube. The concentrations of Al in the delivered solutions was quantified and compared with the concentrations in the original solutions. The results showed no significant adsorption.

Sampling:
See above.

Chemical Analyses/Calculations:
The Al speciation in the oral dosing solutions was calculated using the SPECIES software (Academic Software, Trimble, Otley, UK).

The presence of insoluble Al in dosing salutations was measured by Al analysis (without 26Al) before and after passage through a 0.22 μm filter. The unfiltered and filtered solutions were measured using ETAAS (electrothermal atomic absorption spectrometry).

Total Al
Total Al in blood was analysed by ETAAS (Perkin Elmer 4100 Zl Spectrometer) using serum samples diluted 10-fold with 0.2% HNO3 containing 2.5mM Mg as a matrix modifier. Standards were prepared in the same matrix. The authors report that the serum samples were analysed repeatedly until the total Al concentration relative standard deviation (coefficient of variation) was <10%.

26-Al and 14-C
The levels of radiotracer were determined by accelerator mass spectrometry (AMS). Two rats were used for quality control purposes. Serum samples were taken 4 hours after the oral administration of 52 ng 26-Al and 1700 ng 27-Al (65 nmoles = 65 μM). Quality control 14-C serum samples were collected 2 hours after administration of 30 nmol 14-C citrate with equimolar 27-Al.

4 mg of a 27-Al standard (ICP/DCP standard, Aldrich) were added to a 100 μL aliquot of each serum sample for determination of the 26-Al:27-Al ratio and quantification of the absolute level of 26-Al by comparison with the added 27-Al. After drying overnight (80ºC), digesting in 2mL of 70:30 mixture of nitric acid and hydrogen peroxide, the sample was heated to evaporation. The residue was then dissolved in 35% nitric acid, dried overnight in a porcelain crucible, ashed at 1000 ºC for 2 hours and analysed for 26-Al at the PRIME Lab (Purdue University).

The 14-C was also determined at the PRIME Lab. 50μL serum samples were frozen and micro-centrifuge tubes prior to sending. The carbon in the samples was converted to graphite by lyophilization, combustion and graphitization (no details included in the article (Record et al., cited).

One quality control sample was processed with each serum sample batch. Five 26-Al quality control samples had an RSD (relative standard deviation) of 3.8%. Five 14-C quality control serum samples had an RSD (relative standard deviation) of 4.9%. The authors report that samples with a percent error >10% or 20% for 26-Al and 14-C, respectively, were not included in the analysis.

Statistics:

Normality of the distributions was tested using the Kolmogorov-Smirnov test and equality of variances by Bartlett’s test for 26-Al (4 treatments) and F-test for 14-C (two treatments). One-way ANOVA was used to test for significant treatment effects on absolute Al bioavailability, Cmax and Tmax between the four Al treatments. 26-Al bioavailability results were transformed by taking the square root prior to one-way ANOVA. A significance level of p=0.05 was used in all tests.
Other quantitative assessments, modeling and software:
Pharmacokinetic analysis of the 26-Al serum results was done using WinNonlin. One and two compartment models were used to find best fit values for AUC, Cmax and Tmax. The mean total Al serum concentration was calculated as:
AUC for total Al/time period i.e. 1 to 24h.

Oral 26-Al bioavailability was calculated as:
(AUC for 26-Al x 27Al infusion rate)/(mean total Al serum concentration x 26-Al dose)

Results and discussion

Preliminary studies:

Aluminium speciation calculations
“free Al3+” dosing solution: pH=5, similar amounts of Al3+, Al(OH)2+, with a smaller concentration of Al(OH)2+ (see attached picture)
Al with citrate: pH=7, 80% trimer Al3(citrate)3(OH)4- and 20% monomer Al(citrate)-.
Al with maltolate: pH=7, 64% Al(mal)3, 25% Al(OH)3, ca. 5% Al(OH)4-, ca. 5% Al(mal)2+.
Al with fluoride: pH ca. 4, 55% AlF2+, 40% AlF3, 4% AlF2+, 1.7% AlF4-.
Overall, the calculated speciation results and visual observation of the solutions showed that any Al present in the dosing solutions as insoluble Al(OH)3 remained dispersed in a colloidal form.

Levels of aluminium in dosing solutions
Before and after 0.22 μm filtration:
In solutions of “free Al3+” with pH=7, the authors report analyzable amounts of 11%, 13%, 51% and 52% for concentrations of 20, 65, 200 and 2000 μM before filtering and 10%, 4%, 1.5% and 1.5% in the filtered solutions suggestive of Al(OH)3 precipitate removed by the 0.22μm pore size. The speciation calculations did not predict significant precipitation. In solutions containing the ligands and 65 μM Al, filtering did not significantly change the amount of Al.

Levels of blood urea nitrogen and serum creatinine:
Levels of BUN and serum creatinine were within the normal range (3.9 to 17.8 mg/dL and 0.2 to 0.5 mg/dL, respectively). No animals were excluded on the basis of these results.

Levels of Al in serum:
The total Al level in the serum of the rat that was not infused was ca. 50 ng/mL. The average in rats that did receive the 27-Al iv infusion was 639±168 ng/mL.

Levels of 26-Al in serum:
All samples were within the ≤ 10% analytical error. Results were normally distributed with the exception of Tmax for 26-Al. Variances were similar between groups.

Levels of 14-C in serum:
Seven samples (from 7 different rats) exceeded the >20% analytical error limit and were excluded. Results were normally distributed. Variances were equal with the exception of Cmax values for 14-C (Kolmogorov-Smirnov Test, p=0.028).


26-Al mean±s.d. (range) fg/mL 14-Cmean±s.d. (range) fg/mL
Prior to oral dosing 0.71±0.76 (0-3.27) Not reported
Non-treated rats 1.07±1.16 (0-4.64) Not reported

Levels of aluminium in feed and drinking water: Not reported.
Mortality and general toxicity: No deaths were mentioned. As animals were anaesthetized and femoral cannulae used, animal morbidity would be hard to assess.
Food consumption: Not reported.
Water consumption: Not reported.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The shapes of the serum 14-C and 26-Al time-concentration curves were similar but 14-C levels were ca. 100-times 26-Al levels, suggesting dissociation of the Al-citrate complex in the GI tract.

Details on distribution in tissues:

Total Al Serum Levels:
The total Al in the serum of the rat that did not receive the 27-Al infusion was “ca. 50 ng/mL”. In the rats that did receive the infusion, the concentration was 639±168 ng/mL.

Details on excretion:

Not assessed.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

Cmax: 659

Remarks:

for free ion

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

Tmax: 1.2

Remarks:

for free ion

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

Not assessed.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

The bioavailability of the free ion is 0.29%

Any other information on results incl. tables

Toxicokinetic parameters:

Results for Systemic Bioavailability

Parameter	Free ion	Citrate	Maltolate	Fluoride	ANOVA/ t-test
26-Al oral bioavailability (%)	0.29±0.11	0.61±0.31	0.50±0.25	0.35±0.10	P=0.11
- square root transform					P=0.14
26-Al Cmax (fg/mL)	659±195	1073±250	881±356	880±295	P=0.18
26-Al Tmax (h)	1.2±0.9	1.0±1.1	1.3±1.0	1.0±0.9	P=0.90

 

Based on the results in the table above, there was no statistically significant difference between the different Al-forms with respect to systemic 26-Al bioavailability when administered by the oral route under the conditions used in the study. 

The radiolabel concentrations in the serum returned to baseline values by 24 hours after the dose was administered.

The absence of food in the stomach was required to prevent interference from competing ligands “to test the hypothesis that Al maltol and Al citrate were absorbed intact”.

Applicant's summary and conclusion

Conclusions:

Tis study was designed to assess the bioavailability of the substances not the bioaccumulation potential.
This study observed no statistically significant difference in the systemic bioavailability of aluminium administered orally in the presence and absence of different ligands at equimolar concentrations to aluminium at drinking water relevant concentrations.

Executive summary:

Zhou et al. (2008) studied the absolute oral bioavailability of Al when introduced as the Al³⁺ion alone, or as a complex with citrate, maltolate, and fluoride at an Al concentration relevant to drinking water. The primary objective was to test the hypothesis that the absolute oral bioavailability of Al in rats is the same when dosed as the Al³⁺ion in the absence of added ligands as when in the presence of citrate, maltolate, or fluoride at a dose relevant to daily consumption of Al in drinking water by humans.

 

Male Fisher 344 rats (n=23, body weight 270 ± 18 gram) were used in the study. Bioavailability was studied by using²⁶Al as a tracer and accelerator mass spectrometric analysis. The rats had free food and drinking water access throughout the study except for the period from 14 h before to 4 h after oral dosing. To minimize food retention in the stomach before dosing, animals’ access to food was limited to a 10% protein diet (Harlan Teklad 95215). Two femoral venous cannulae were implanted in all rats 1 day prior to oral dosing to avoid Al contamination of withdrawn blood. Blood was withdrawn 1 h prior to, and 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, 8, 24 h after oral dosing. The quantification of²⁶Al,²⁷Al and¹⁴C was performed in serum. Levels of blood urea nitrogen (BUN) and creatinine in the 24 h sample were used to check renal function.

 

Animals (n=5/group) were randomly assigned to receive oral²⁶Al by gastric administration in the absence of ligands or presence of citrate, maltolate or fluoride. The total Al:ligand ratio was 1:1 for citrate, 1:3 for maltolate and 1:4 for fluoride. Each rat received a 1 mL dose of a solution containing ~50 ng (1 nCi)²⁶Al and 1700 ng²⁷Al; the Al concentration in the administered solution was ~ 65μM. The ligands, citrate and maltolate, were administered as [¹⁴C]-citrate and [¹⁴C]-maltolate. The pH of the administered solution was adjusted to about 5 for Al in the absence of ligands, about 7 for Al in the citrate or maltolate solutions, and to about 4 for Al with fluoride. The control rats (n=2) similarly received intragastric administration of water without²⁶Al.

 

Aluminium speciation in the administered solutions from pH 2 to 8 was predicted by computer modeling.Species calculations showed that, at pH 7, nearly all of the citrate and 70% of the maltolate should be bound to Al in the prepared dosing solutions.

 

The authors reported that, in the absence of ligands or in the presence of citrate, maltolate and fluoride, the reported C_max(fg/mL) values for²⁶Al were 659±195, 1073±250, 881±356, and 880±295, respectively; T_max(h) values for²⁶Al were 1.2±0.9, 1.0±1.1, 1.3±1.0, and 1.0±0.9 for the Al ion, and in the presence of citrate, maltolate, and fluoride, respectively. The serum¹⁴C concentration was much higher after Al citrate and Al maltolate administration than²⁶Al, suggesting considerable dissociation of Al from ligands in the gastrointestinal (GI) tract.

 

The absolute oral bioavailability of²⁶Al (%) was estimated to be 0.29±0.11, 0.61±0.31, 0.50±0.25, and 0.35±0.10 for Al ion, in the presence of citrate, maltolate, and fluoride, respectively. Although Al bioavailability was 2.1-, 1.7- and 1.2-fold greater after administration as the citrate, maltolate, and fluoride, respectively, compared with the ion, the differences were not statistically significant.