1. Overview: Health, Safety, Issues for
Nanotechnology

2. Potential bio-accumulation of nanoscale particles.
• Accumulation of a substance within a species can occur due to lack of
degradation or excretion.
• Many nanoparticles are not biodegradable.
• If nanoparticles enter organisms low in the food web, they may be
expected to accumulate in organisms higher in the food web.
Very little is understood about possible health
effects of nanoparticle exposure

3. Potential human hazards for nanoscale particulates.
Inhalation: Inhaled particles induce
inflammation in respiratory tract,
causing tissue damage. Example:
Inhalation of silica particles in
industrial workers causes “silicosis”.
Dermal exposure: Particles may enter
body through the skin. Potential
hazards are unknown at present.
Ingestion: nanoparticles may cause liver
damage. Ingested nanoparticles (i.e. for
oral drug delivery) have been found to
accumulate in the liver. Excessive
immune/inflammatory responses cause
permanent liver damage.
Other: ocular, ….

4. Potential bio-uptake of nanoscale particulates.
• Nanoparticles may enter living cells via:
– Endocytosis
• Receptor activation for initiation
– Membrane penetration
• Generally occurs with very hydrophobic
particles
– Transmembrane channels
• May be seen with very small nanoparticles
(< 5 nm?)

5. Nanoparticle Toxicity
Nanoparticles affect biological behaviour at
cellular, subcellular, protein, and gene levels
5

6. Examples of specific Nanomaterials Tested
Study Focus effects investigated
Cytotoxicity Affinity to cell aluminum oxide (Al2O3), cerium oxide (CeO2), cupric
membranes, oxidative oxide (CuO) dendrimers, iron oxide (Fe2O3), nickel oxide
damage, structure- (NiO), silicon dioxide (SiO2), titanium dioxide (TiO2),
function relationships, zinc oxide (ZnO)
mechanisms
Dermal toxicity Dermal absorption, cadmium celenide (CdSe), fullerenes, iron (Fe)
cutaneous toxicity,
General toxicity Human blood aluminum oxide (Al2O3), cadmium celenide (CdSe),
coagulation, induction cadmium telluride (CdTe) dendrimers, fullerenes, gallium
of inflammatory gene nitride (GaN)Geranium, lead selenide (PbSe), nanofibers,
expression, nanowires, quantum dots, silicon dioxide (SiO2), quantum
genotoxicity dots, titanium dioxide (TiO2), zinc sulfide (ZnS)
Pulmonary toxicity Oxidative stress, aluminum oxide (Al2O3), cerium oxide (CeO2), cupric
inflammation, surface oxide (CuO) dendrimers, gold (Au), iron oxide (Fe2O3),
coating effects, multiwalled nanotubes (MWNT), nickel oxide (NiO),
nano/non-nano effects, silicon dioxide (SiO2), single walled nanotubes (SWNT),
new/aged agglomerated silver (Ag), titanium dioxide (TiO2), zinc oxide (ZnO)
effects, clearance
mechanisms
Translocation/Disposition Translocation to sites aluminum oxide (Al2O3), iron oxide (Fe2O3), titanium
distant from original dioxide (TiO2), silicon dioxide (SiO2), zinc oxide (ZnO)
exposure, persistence in
vivo.

7. Possible Health Effects
Inhalation
Pulmonary inflammatory reaction
– Persistent inflammation is likely to lead to diseases such as
fibrosis and cancer. Thus it is important to control
inflammation. This can be done if we can:
- (i) determine the critical dose of particles that initiates
inflammation and
- (ii) set exposure limits, according to the relevant metric, so that
such a dose cannot be reached within a lifetime exposure
scenario.

8. Wahrheit (Dupont), January 2004.
Optical micrograph of lung tissue from a rat exposed to single-
wall carbon nanotubes (1 mg/kg) 1 week post exposure. Note
the early development of lesions surrounding the instilled
SWCNT (arrows) and the nonuniform, diffuse pattern of
single-wall carbon nanotube particulate deposition in the lung
(X 100).
Low-magnification micrograph of lung tissue from a rat
exposed to single-wall carbon nanotubes (1 mg/kg) at 1 month
postinstillation. Note the diffuse pattern of granulomatous
lesions (arrows). It was interesting to note that few lesions
existed in some lobes while other lobes contain several
granulomatous lesions—and this was likely due to the
nonuniform deposition pattern following carbon nanotube
instillation. Magnification X 20.
Higher magnification optical micrograph of lung tissue from
a rat exposed to single-wall carbon nanotubes (1 mg/kg) at 1
month postinstillation exposure. Note the discrete, multifocal
mononuclear granuloma centered around the carbon
nanotube material (arrows). Magnification X 400.
D. B. Wahrheit et. al. Toxilogical Sciences 77, 117-125 (2004)

10. Concerns about granulomas and fibers.
Granulomas (miscropic nodules), consisting of particles, live and
dead cells, and debris and could impair cellular and physiological
(gas exchange) lung functions and give rise to fibrosis, more
defined nodules, and other lesions.
Fibers are generally of more health hazard than other forms of
particulates. It is well established that the pathogenicity of a
fiber in the lungs directly correlates with its
biopersistency(Oberdorster 2000).
NTs are totally insoluble and probably one of the most
biologically nondegradable man-made materials.Determining how
the NT-induced granulomas progress would require a longer-
duration study with this biopersistent material.

11. Observations and tentative conclusions.
• Granulomas were observed in lungs 7 d or 90 d after an instillation
of 0.5 mg NT per mouse (also in some with 0.1 mg);
• NT, regardless synthetic methods, types and amounts of residual
catalytic metals, produced granulomas;
• Lung lesions in the 90-d NT groups, in most cases, more pronounced
than those in the 7-d groups.
• Our study shows that, on an equal-weight basis, if carbon nanotubes
reach the lungs, they are much more toxic than carbon black and can
be more toxic than quartz, which is considered a serious occupational
health hazard in chronic inhalation exposures.
• If fine NT dusts are present in a work environment, exposure
protection strategies should be implemented to minimize human
exposures.

12. Control of Nanoparticles
Exposure by inhalation
- Filtering respirators or air supplied
respirators may be used as a last
option to control exposure to
nanoparticles.
- Probably the efficiency will be high for
all but the smallest nanoparticles (less
than 2 nanometers).
- The respirator must fit properly to
prevent leakage. The white powder around the
nostrils shows that this mask
did not have a tight fit.

13. Possible Health Effects
Ingestion
Nanoparticles can be swallowed and therefore available for transfer to other
body organs via the gastro-intestinal compartment.
There is also some evidence that smaller particles can be transferred more
readily than their larger counterparts across the intestinal wall (Behrens et al;
2002).
Little is currently known about the health effects of nanoparticles on the liver
and kidneys as well as the correct metric for describing the nanoparticle dose
in these organs.
Another area which merits further research is the transfer of nanoparticles
across the placenta barrier. Exposure to nanoparticles during the critical
window of fetal development may lead to developmental damage in the
offspring.

14. Control of Nanoparticles
Ingestion exposure
- Occurs from hand-to-mouth contact
- Control by using gloves when handling
nanoparticle products
- Hand washing before eating, drinking
or smoking is also important

15. Possible Health Effects
Dermal exposure
• Harmful effects arising from skin exposure may either occur locally within
the skin or alternatively the substance may be absorbed through the skin
and disseminated via the bloodstream, possibly causing systemic effects.
• Dermal absorption of ultrafine particles (nanoparticles) has not been well
investigated and suggested that ultrafine particles may penetrate into hair
follicles where constituents of the particles could dissolve in the aqueous
conditions and enter the skin. Direct penetration of the skin has been
reported by Tinkle et al (2003) for particles with a diameter of 1000
nm, much larger than nanoparticles.
• It is reasonable to postulate that nanoparticles are more likely to
penetrate, but this has not yet been demonstrated. Several
pharmaceutical companies are believed to be working on dermal
penetration of nanoparticles as a drug delivery route.

16. Control of Nanoparticles
Skin Exposure
• Skin penetration may occur
mainly in the later stages of
the process, recovery or
surface contamination.
• Some evidence shows that
nanoparticles penetrate into
the inner layers of the skin and
possibly beyond, into the
blood circulation.

17. Environmental Fate/Transport and Environmental Toxicity
Examples of specific effects Nanomaterials Tested
Study focus investigated
Aquatic fate Impact on water migration through alumina, magnetite, nanofibers, silicon
soil, chemical behavior in carbide, silicon dioxide (SiO2), single walled
estuarine systems, fate in potable nanotubes (SWNT), titanium dioxide (TiO2),
water, uptake by aquatic organisms zinc oxide (ZnO)
Environmental Microbial biomass, organic carbon cadmium celenide (CdSe), cupric oxide
toxicity assimilation rates, deposit (CuO), iron oxide (Fe2O3), molybdenum
feeding, uptake, estuarine disulfide (MoS2), nanofibers, quantum dots,
invertebrates, toxicity in drinking silicon dioxide (SiO2), single walled
water, fish, frogs, bacteria, fungi, nanotubes (SWNT), titanium dioxide (TiO2),
daphnia, algae zinc oxide (ZnO)
Fate in air Emission minimization, sampling fullerenes, silicon dioxide (SiO2), single
and analysis, nucleation rate walled nanotubes (SWNT) sulphuric acid
(H2SO4)
Fate in Desorption and release from aluminum oxide (Al2O3), cadmium celenide
soils/sediment nanoparticle surfaces, disposition (CdSe), hyroxylated fullerenes, magnetite
of contaminants,
Cross media Effects of oxygen, chlorine, UV carbon nanofibers, fullerenes, titanium
fate/Transport light dioxide (TiO2), zinc oxide (ZnO)

18. Health Effects: Many questions, not many answers.
• In what ways might employees be
exposed to nanomaterials in
manufacture and use?
• In what ways might nanomaterials
enter the body during those
exposures?
• Once in the body, where would the
nanomaterials travel, and how would
they interact physiologically and
chemically with the body’s systems?
• Will those interactions be harmless, or
could they cause acute or chronic
adverse effects?
• What are appropriate methods for
measuring and controlling exposures to
nanometer-diameter particles and
nanomaterials in the workplace?

19. Health Risk Studies
These six federal agencies are conducting studies of potential health risks
of nanomaterials:
- The National Institute of Environmental Health Sciences (including the
National Toxicology Program);
- The National Institute for Occupational Safety and Health (NIOSH);
- The Environmental Protection Agency (EPA);
- The Department of Defense;
- The Department of Energy (DOE);
- The National Science Foundation (NSF)
INAIL

20. Problem areas for regulation of particulates.

21. PhysicochemicalCharacterization
Size measurement of Nanoparticles Using DLS
Size Measurement of Nanoparticles Using Atomic Force
Microscopy
Measuring the Size of Nanoparticles Using Transmission
Electron Microscopy
Determination of polymeric NP in Rat Blood with Mass
Spectrometry
Quantification of Free and Chelated Gadolinium Species in
Nanoemulsion-Based Magnetic Resonance Imaging
Zeta Potential

22. In Vitro Characterization
Detection of Endotoxin Contamination Detection
of Microbial Contamination
Detection of Mycoplasma Contamination
Targeting
Cell Binding/Internalization
Analysis of Hemolytic Properties of Nanoparticles Analysis
of Platelet Aggregation
Analysis of Nanoparticle Interaction with Plasma Proteins by
2D PAGE
Coagulation Assay
Detection of Nitric Oxide Production by Macrophage

23. TOXICITY
Oxidative Stress
Hep G2 Hepatocyte Glutathione Assay
Hep G2 Hepatocyte Lipid Peroxidation Assay (MDA)
Cytotoxicity (necrosis) assay (MTT and LDH Release
Cytotoxicity (apoptosis) assay (Caspase 3 Activation)
Autophagy Assay: Analysis of MAP LC3I to LC3-II Conversion
by Western Blot

24. In Vivo Characterization
Efficacy
Therapeutic
Imaging
Tissue Distribution
Clearance
Half-life
Systemic exposure (plasma AUC)
Single and Repeat-Dose Toxicity