産総研:安全科学研究部門サイト > Development of Innovative Methodology for Safety Assessment of Industrial Nanomaterials > Research Activities and Results (June 2015) > Research and development item: ①(b) Sample preparation methods and characterization for equivalence evaluation

Research and development item: ①(b) Sample preparation methods and characterization for equivalence evaluation

Implemented by the National Institute of Advanced Industrial Science and Technology

Final objective: We provide nanomaterial suspensions for hazard testing and characterize the samples. We compile and release a technical instruction manual about methods and points of sample preparation and characterization for intratracheal administration testing.

Main result:
As a sample to be used for animal testing in order to establish sample preparation techniques for equivalence evaluation , we selected TiO2 from the following viewpoint and established preparation procedure for a standard suspension, which is the base for preparing other nanomaterial for testing:

  • TiO2 is a widely used all-purpose material.
  • TiO2 nanoparticles of various shapes and sizes are readily available as commercial products.

Since we observed that TiO2 particles easily agglomerate when the pH of their suspension is approximately 7 (neutral), instead of pure water, we used a solvent containing disodium phosphate (DSP), a dispersing agent that has been considered to be less hazardous to a living body. In addition, it was possible to prepare test materials other than TiO2 using pure water without DSP. We prepared a TiO2 suspension for hazard testing, using materials of various sizes, shapes and surface conditions, such as particles of 6–1,000 nm diameters, particle shapes ranging from spherical (aspect ratio 1) to needle-shaped (aspect ratio 13), and particle surfaces modified and unmodified with Al(OH)3.

As a more hazardous material relative to TiO2, which is a relatively low-hazard material, we selected NiO. We provide NiO suspensions prepared by using spherical particles with diameters ranging from 18 to 300 nm and fibrous particles with an aspect ratio of 1,000.

In the case of SiO2, whose hazard was expected to vary drastically depending on its crystallinity, we prepared samples of amorphous and crystalline materials (a quartz). For the amorphous SiO2, samples with different diameters (10–70 nm) and those with their surfaces modified with Al(OH)3 or a carboxylic acid were purchased as commercial items. In the case of crystalline materials, however, we prepared them by ball milling and centrifugation because the particle size of the commercially available items was limited. When crystalline SiO2 is milled, part of its crystalline structure becomes amorphous due to the mechanical damage; therefore, we decreased the amount of the amorphous component using an alkali solution and performed a quantitative evaluation of the amorphous component by X-ray diffraction analysis.

We also prepared suspension of CeO2 whose hazard is reported in several publications, while it is considered to be a substance of low solubility within a body of test animals.

Table ①(b) shows the materials used for preparation of the samples provided for animal testing.

We characterize physicochemical properties of nanoparticles in consideration of the standard characterization requirements recommended by the ISO, OECD, and others. Thus, the characterization includes evaluation of the shape and primary particle size of nanoparticles using a transmission and a scanning electron microscopes (TEM, SEM), measurement of the specific surface area of nanoparticles and calculation of their particle size from the results, evaluation of the zeta potential and isoelectric point of nanoparticles, their crystal structure by X-ray diffraction and measurement of the particle size (secondary particle size) in suspension using the dynamic light scattering method. We move forward with the characterizations of the whole provided samples.

Table①(b) List of materials used for preparation of samples provided for animal testing

Material Crystal Structure Particle Shape Diameter (catalog value, nm)
titanium
dioxide
anatase sphere 6
rutile / anatase sphere 21
rutile spindle long-D. 29, short-D. 8
rutile spindle long-D. 50-100, short-D. 10-20
rutile spindle
(Al(OH)3coat)
long-D. 50-100, short-D. 10-20
rutile sphere 1,000
rutile needle long-D. 1,700, short-D. 130
nickel
oxide
NaCl box 18
NaCl irregular <50
NaCl box 300
NaCl wire long-D. 20,000, short-D. 20
silicon
dioxide
amorphous sphere 10
amorphous sphere 70
amorphous sphere
(Al(OH)3coat)
70
amorphous sphere
(COOH coat)
70
α-quartz / amorphous irregular 80, milled + NaOH dissolution
α-quartz / amorphous irregular 200, milled
α-quartz irregular 300, (classification from 800 nm)
α-quartz irregular 300, (classification from 1,400 nm)
α-quartz irregular 1,400
cerium
dioxide
fluorite box 10