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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with limitations which do not impair the overall conclusion from the data

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Objective of study:
other: Bioaccessibility
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Solubility of test item in simulated human fluids. Principle of test is similar to Transformation/Dissolution testing according to OECD Series 29 (2001)
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
sodium hexahydroxoantimonate
EC Number:
251-735-0
Cas Number:
33908-66-6
Molecular formula:
NaSb(OH)6
IUPAC Name:
sodium hexahydroxoantimonate
Constituent 2
Reference substance name:
sodiumhexahydroxoantimonate
IUPAC Name:
sodiumhexahydroxoantimonate
Details on test material:
- Name of test material (as cited in study report): Sodium hexahydroxoantimonate (NaSb(OH)6)
- Physical state: White powder
- Analytical purity: > 99.5 %
- Purity test date: 2009-01-21
- Batch No.: 61049-070901J100000
- Expiration date of the batch: 2010-09
- Storage condition of test material: tightly closed, well ventilated place

Test animals

Species:
other: in vitro (simulated human body fluids)
Details on test animals or test system and environmental conditions:
The test item was exposed to five different test media at a pH range from 1.6 to 7.4. The following synthetic biological fluids were used:

• Phosphate‐buffered saline (PBS, pH 7.4), a standard physiological solution that mimics the ionic strength of human blood serum. It is widely used in the research (e.g. Norlin et al, 2002) and medical health care community (e.g. Hanawa et al, 2004, Okazaki and Gotoh, 2005) as a reference test solution for comparison of data under simulated physiological conditions.
• Gamble’s solution (GMB, pH 7.4) mimics the interstitial fluid within the deep lung under normal health conditions (Stopford et al, 2004).
• Artificial sweat (ASW, pH 6.5) simulates the hypoosmolar fluid, which is excreted from the body upon sweating. The fluid is recommended in the available standard for testing of nickel release from nickel containing products (EN 1811, 1998).
• Artificial lysosomal fluid (ALF, pH 4.5) simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions (Stopford et al, 2004).
•Artificial gastric fluid (GST, pH 1.6) mimics the very harsh digestion milieu of high acidity in the stomach (Hamel et al, 1998; ASTM, 2003).

The test media were selected in order to simulate human exposure as far as possible, e.g. skin contact. Ingestion to the gastro-intestinal tract can either be direct, or previously inhaled particles can be translocated from the respiratory tract to the gastro-intestinal tract by mucociliary clearance. It should be stressed though, that the different test media only simulate physiological conditions to a limited extent, as the complexity and function of the real body fluids are difficult to simulate. However, in vitro results in such synthetic biological media can, in a simple way, provide information that could be relevant for a real situation.

The test solutions were prepared using ultra‐pure water and chemicals of analytical grades.

The pH of ALF and GMB was adjusted using 50% NaOH and 25% HCl, respectively. The pH of ASW and PBS was adjusted with 1% ammonia solution and 50% NaOH, respectively.

Artificial gastric fluid was prepared according to the ASTM standard using 4 g of 25% HCl solution diluted with ultra‐pure water to 1 L (ASTM D5517, 2003).

References:
ASTM D5517-03 (2003), ”Standard Test Method for Determining Extractability of Metals from Art Materials”

Administration / exposure

Route of administration:
other: in vitro (simulated human body fluids)
Details on study design:
Experimental Procedure:
Triplicate samples were prepared for exposure in different test media, each for two different time periods. In addition, one blank sample (without addition of any test item) containing only the test solution was incubated together with triplicate samples for each time period. An amount of 5 ± 0.5 mg of the test item were weighed using a Mettler AT20 balance (readability: 2 μg), and placed in a PMP Nalgene® jar. A volume of 50 mL of the test solution (no adjustment of solution volume to powder mass was made) was added to the Nalgene® jar containing the test item, before incubated in a Platform‐Rocker incubator SI 80 regulated at 37 ± 2°C. The solution was gently shaken (bi‐linearly) with an intensity of 25 cycles per minute for 2 and 24 hours, respectively.
Details on dosing and sampling:
The standard loading of 0.1 g/L was selected. It allows a comparison of the generated data with results from the OECD Transformation/Dissolution test (OECD, 2001) and similar bioaccessibility tests conducted with other materials under the same conditions.

The time periods for exposure of the test item were selected to have some relevance to the inhalation/ingestion scenario and to enable comparison with other reported metal release/dissolution data generated for similar time periods. The approximate time for the gastric phase of digestion is about 2 hours, and therefore this exposure time period was considered relevant for testing in artificial gastric fluid (Hamel et al, 1998). The 24 hour exposure was selected as a standard time duration that is relatively easy to compare with existing metal release/dissolution data as well as toxicity data for further evaluation of the bioaccessibility of released metals. Moreover, it can be assumed that human exposure to particles last no longer than 24 hours at ambient conditions.

After exposure, the samples were allowed to cool to ambient room temperature before the final pH of the test solution was measured. The test medium was then separated from the powder particles by centrifugation at 3000 rpm for 10 minutes, resulting in a visually clear supernatant with remaining particles in the bottom of the centrifuging tube. The supernatant solution was decanted into a polypropylene storage flask and acidified to a pH less than 2 (not needed in the case of artificial gastric fluid) with 65% pure HNO3 prior to solution analysis.

Results and discussion

Any other information on results incl. tables

Particle characterisation

Surface area, particle size distribution and morphology

BET:

The specific surface area, measured by BET-analysis is 0.25 m²/g. It should be underlined that this specific surface area is measured by nitrogen absorption and includes also the surface of surface pores.

SEM:

Morphology observation of the test item was performed by using scanning electron microscopy, SEM. A large size distribution was observed from the SEM images. The particles are cuboid-formed and of larger size with a wide size distribution. Energy dispersive spectroscopy (EDS), which analyses the elemental composition for elements with an atomic number larger than, or equal to sodium, and which has an information depth of about 1 µm, showed that Aluminium was locally detected in particles of NaSb(OH)6 (between 1 and 2 wt%, slightly above the limit of detection of approximately 1 wt% for this instrument).

PBS:

This measurement illustrates how the test item behaves in the biological fluids investigated. Measurements of the particle size distribution in solution are conducted when dispersed particles are continuously pumped through the laser diffraction system. The results reflect the stability of possible clusters formed due to agglomeration/aggregation. The measured particle size distribution is: Median (d0.5) = 30.2 µm), 10% (d0.1) = 8.7 µm, and 90% (d0.9) = 79.4 µm) [percentile values as the percentage in volume (mass); results are reported as the average value of six replicate measurements].

There are no indications for agglomeration during the exposure to the different synthetic biological fluids investigated. The particle size distribution measurements and SEM images indicate a relatively large particle size distribution without any agglomerates at higher particle sizes. There is a large number of small particles and there are relatively large particles as well.

X-ray photoelectron spectroscopy

The composition of the outermost surface oxide was investigated at five different areas (locations) of particles of the test item by means of X-ray photoelectron spectroscopy. A small peak corresponding to carbon contamination was observed. A surface contamination layer of carbon (285 eV) is always observed to different extent due to the surface history, and its source is usually atmospheric.

The Sb 3d, O 1s, Na 1s, and Cl 2p peaks were considered. No peak corresponding to metallic Sb was observed. Antimony was present in its oxidized state in the outermost surface layer. The state of oxidation could not be determined due to peak overlapping for different antimony oxides and the oxygen peak. Sodium and a small amount of chloride were also detected on the surface.

The relative surface composition of antimony, oxygen, sodium and chloride in the surface layer of particles of NaSb(OH)6 is: 49 % Sb, 35 % O, 16% Na and 0.4 % Cl.

Solubilty in simulated human fluids

The dissolution of the NaSb(OH)6particles indicates a pH, composition and time dependence. In GST (pH 1.6), the most acidic test media, the NaSb(OH)6particles dissolved to the highest extent and were nearly completely dissolved after 24 hours of exposure (73% (2h), 94% (24h)). In ALF (pH 4.5), the particles dissolved also to a relatively high extent (20% (2h), 71% (24h)). In ASW (pH 6.5), 15% of the particles dissolved after 2 hours of exposure and 61% after 24 hours of exposure. In weakly alkaline media, PBS and GMB (pH 7.4), the NaSb(OH)6particles dissolved at a lower percentage (PBS: 9% (2h), 29% (24h) and GMB: 3% (2h) and 33% (24h)).

Table: Antimony transformed [mass%], equivalent to percentage of the amount particles dissolved

 

GMB

PBS

ASW

ALF

GST

2h

3.1±3.3

8.7±0.3

15.1±0.3

19.8±3.3

72.5±8.8

24h

32.5±4.5

29.2±2.5

60.8±1.1

71.4±7.7

94.3±8.7

 

The release rate of antimony from particles of NaSb(OH)6decreased with time in all media with the exception of GMB. As the particles were nearly completely dissolved in GST (pH 1.6), the release rate of antimony (since no or little material is left to be dissolved) can hardly be assessed. In ALF (pH 4.5) and ASW (pH 6.5), the release rate of antimony decreased slightly with time, indicating a sparse formation of a protective surface layer with low barrier properties to hinder or reduce the extent of chemical dissolution with time. In PBS (pH 7.4), the release rate of antimony decreased more, indicating the formation of some surface barrier/ protection with time hindering further dissolution. In GMB (pH 7.4), the release rate of antimony did not decrease with time, in fact the release rate increased slightly when calculated based on the last known surface area approach (the surface area at 24 hours of exposure for the release rate at 24 hours of exposure). These calculations indicate that a protective surface layer was not formed to hinder/reduce further dissolution and/or an additional dissolution processes caused for instance by chemical salts in the complex solution of GMB, after a certain exposure time period. The release rate of antimony decreased to the lowest extent (or even slightly increased) in GMB (pH7.4) and in ALF (pH 4.5), the most complex test media, both simulating lung fluids. Solution composition and pH seem to affect the dissolution behaviour of this compound.

Table: Release rate of antimony [mg/cm²/h] and the standard deviation of triplicate samples in the different media.

 

GMB

PBS

ASW

ALF

GST

2h

0.0031±0.0033

0.0086±0.0003

0.015±0.0003

0.0196±0.0033

0.0717±0.0087

24h

0.0027±0.0004

0.0025±0.0002

0.0051±0.0001

0.0059±0.0007

0.008±0.001

 

Table: Release rate of antimony [mg/cm²/h] after recalculation of the surface after dissolution (the start time values have been used, i.e. the original surface for the 2 hour exposure metal release rate and the surface after 2 hours of exposure for the 24 hour exposure metal release rates) and the standard deviation of triplicate samples in the different media.

 

GMB

PBS

ASW

ALF

GST

2h

0.003±0.003

0.009±0.001

0.015±0.001

0.02±0.004

0.072±0.009

24h

0.003±0.001

0.003±0.001

0.006±0.001

0.007±0.001

0.019±0.002

 

Table: Release rate of antimony [mg/cm²/h] after recalculation of the surface after dissolution (the end time values have been used, i.e. the surface after 2 hours of exposure for the 2 hour exposure metal release rate and the surface after 24 hours of exposure for the 24 hour exposure metal release rates) and the standard deviation of triplicate samples in the different media.

 

GMB

PBS

ASW

ALF

GST

2h

0.003±0.004

0.01±0.001

0.017±0.001

0.023±0.004

0.176±0.016

24h

0.004±0.001

0.004±0.001

0.01±0.001

0.014±0.002

0.052±0.003

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: refer to the executive summary
Particles of sodium hexahydroxoantimonate dissolved at least partially in all tested media with the degree if dossolution depending on the composition of the solution.
Executive summary:

Sodium hexahydroxoantimonate (NaSb(OH)6) was exposed to five different synthetic body fluids for two different time periods, 2 and 24 hours. In parallel to the generation of bioaccessibility data, particle and surface characterisations were conducted.

- Particles of NaSb(OH)6 revealed a large number of smaller sized particles (about 1µm) and large size distributions (up to > 100µm).

- A pH and solution composition dependence was observed for the dissolution.

- Particles of NaSb(OH)6 dissolved better in acidic media: GST-pH 1.6: 94%, ALF-pH 4.5: 71%, ASW- pH 6.5: 61%, PBS-pH 7.4: 29 %, and GMb-pH 7.4: 32.5% after 24 hours of exposure.

- Rates of antimony release from NaSb(OH)6 particles decreased with time in all media except in GMB. In GMBantimony releaseremained constant indicating that a protective surface layer against further dissolution may have been formed. The release rate differed in the media as follows: (release rates in mg /cm2/h corrected for measured surface area after 2 and 24 h): GST (0.176 to 0.052) > ALF (0.023 to 0.014) > ASW (0.017 to 0.01) > PBS (0.01 to 0.004) > GMB (0.003 to 0.004).

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