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NanotoxicologyNanotoxicologyA safety evaluation of nanomaterialsA safety evaluation of nanomaterials
Rawiwan Maniratanachote
December 17, 2009
The 2nd National Conference in Toxicology
Miracle Grand Convention Hotel, Bangkok
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Exposure to nanoparticlesExposure to nanoparticles
Non-engineered particles
Engineered particles
- Free or in aerosol
- Biopersistent
- Catalytically active
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Life Cycle PerspectiveLife Cycle Perspective
Human exposure
Human exposure Ecological exposure
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The nanotechnology consumer product inventory
http://www.nanotechproject.org/inventories/consumer/analysis_draft/
2005 2006 2007 2008 2009 2010 2012
The closer the R is to 1, the better the model and the closer one canapproximate a future outcome.
R = 0.9949
More thanMore than 10001000
nanonanoproducts alreadyproducts already onon
the marketthe market(As of August, 2009)(As of August, 2009)
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http://www.nanotechproject.org/inventories/consumer/analysis_draft/
Silver Carbon zinc Silica Titanium Gold
Number of Nanotechnology products associatedwith specific materials
Nanomaterials Used in Commercial Products and Researches
Consumer productsExamples:
Nanosilver cutting boardNanosilver baby mugAntibacterial kitchen wareAntibacterial textilesNanosilver water storage tanketc.
Nano-sized silver particles haveincreased antibacterial propertiesSilveris among the most widely used NMs
2009
2006
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Nanotoxicology
The small size facilitates uptake into cells andtranslocation to reach sensitive target sites
The greater surface area per mass makes NMs more
biologically active
An interdisciplinary field approach: Toxicology,materials science, medicine, molecular biology etc.
An emerging discipline evolving from studies of nanomaterials
Oberdorster et al. 2005, Environ Health Perspect113: 823-839.
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Potential routes of nanomaterial exposure
Local / Systemic adverse effectsLocal / Systemic adverse effects
Hair
Blood cells
micro nano
DNA
Actin
12-15 m
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Lung and InhalationLung and Inhalation
Pulmonary Deposition as a Function of Particle Size
Alveoli
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Potential Pathway for Nanoparticles in the LungInterstitialization
pathwayClearance
Alveolarmacrophage
Secretions
Particle-laden
macrophage
Capillary
Secretions Interstitial
macrophage Secretions
Fibroblast
Lymph
Epithelium
Interstitium
Broncho-alveolar
space
Modified from Donaldson et al. 1998, J Aerosol Sci, 29: 553-560.
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Role of fiber length and biopersistence in determiningRole of fiber length and biopersistence in determining
adverse effectsadverse effects
Exposure
Deposition
Long fibers
(>20 m)
Short fibers
(
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Gastrointestinal Tract and Site of Absorption
IngestionIngestion
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The SkinThe Skin
In healthy skin, theepidermis providesexcellent protectionagainst particlespread to the dermis
Damaged skin allows
micrometer-sizeparticles access to thedermis and regionallymph nodes Effectson the immune system
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The skin from furry rodents results in overestimation of human
skin penetration
The stratum corneum is an excellent skin barrierFactors influence in penetration test for nanomaterials
Hair foll icle densitySize of hair follicle opening
Lipid structures and contents
Penetration through skin barrier
Species difference in hair follicle density
Species Area Number of hair follicles/cm2
Human Abdomen 11 1Pig Back 11 1Rat Back 289 21
Mouse Back 658 38Hairless mouse Back 75 6Bronaughet al. 1982, Toxicol Appl Pharmacol 62: 481-488.
Pig has different lipid structures from human
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NM effects as the basis of pathophysiology and toxicity
Adapt from: Nel et al., Science (2006) 311: 622-627.
ROS generation Protein, DNA and membrane injury,
oxidative stress Inflammation
Mitochondrial perturbation Energy failure, apoptosis, apo-
necrosis, cytotoxicity
Inflammation Tissue infiltration with inflammatorycells, fibrosis, granulomas,atherogenesis, acute phase protein
expression
Perturbation of phagocytic function , Chronic inflammation, fibrosis,particle overload , mediator release granulomas, interference in
clearance of infectious agents
Generation of neoantigens , breakdown Autoimmunity
in immune tolerance
DNA damage Mutagenesis, carcinogenesis
Experimental effects Possible pathophysiological effects
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The first step towards nanotoxicology studiesParticles characterization
To ensure that the results are reproducible
To provide basis for understanding the
properties of nanoparticles that determinetheir biological effects
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Physicochemical characteristic of nanoparticels
Nel et al., Science (2006) 311: 622-627
Material composition
Electronic structure
Bonded surface species
Surface coating
Solubility
Contribution of surfacespecies
Environmental factors
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Characterization of the particle
Analysis Instrument
Morphology and compositions SEM-EDX, TEM-EDX
Size, size distribution DLS (Nanosizer)
Surface charge Zeta potential analyzer
Specific surface area BET surface area analyzer
Metal contaminants ICP, AA
Scanning Electron Microscope (SEM)
Transmission ElectronMicroscope (TEM)
NanosizerBETsurface area analyzer
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Engineered NanomaterialsEngineered Nanomaterials
Silver Carbon nanotubes Titanium Silica Gold Zinc
etc.
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Potential adverse affects Potent bactericide
1. Development of antibiotic resistant bacteria
2. Harmful to beneficial bacteria which form symbiotic relationship
to plants, animals and humans Disrupt ecosystem function
Consumer products Food packaging
Odor resistant textiles
Wound dressingsetc.
The most prevalent nanomaterials used in consumer products
Silver
Most people are exposed daily to very low level of silver mainly in
food and drinking water, and less in air.
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At the age of 11 the patient was given nose drops of
unknown composition for allergies, and three years
later her skin turned gray. She was thought to have
argyria, and a skin biopsy at the age of 15 confirmed
the presence ofsilver deposition.
The facial pigmentation was diffuse until the age of
36, but it became patchy after dermabrasion. The
patient has had no other related problems.
Colloidal silver products sold in the early 1900s had
silver concentrations as high as 30 percent.
Suspensions of silver, available now in some health
food stores and pharmacies, are touted for the
treatment of many disorders, including the acquired
immunodeficiency syndrome, cancer, sore throats,
meningitis, parasites, chronic fatigue, andacne,
without substantiation.
BRUCE A. BOUTS, M.D.
Argyria
A 56-year-old woman has had discolored skinsince the age of 14
New Eng J Med. May 20, 1999
Health Aspect
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Phyto-Silver BalancingDay Cream
Silver Citrate
Silver containing products in Thailand
And more
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Washing studies
Nanoparticle silver released into water from commercially
available sock fabricsBenn and Westerhoff (2008), Environ. Sci. Technol. 42: 4133-9.
The behavior of silver nanotextiles during washing
Geranio et al (2009), Environ. Sci. Technol. 43: 8113-8.
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Franz diffusion cell method
Human skin penetration of si lver nanoparticles through intact and
damaged skin
Larese et al. (2009), Toxicology 255: 33-37.
TEM micrograph of Agnanoparticles-treated skin sample
500 nm
100 nm
Ag nanoparticles are presented indeep stratum corneum
Silver skin penetration at 24 h
Human abdominal full thickness skins
Silver nanoparticles (257.1 nm)
C toto ici t of Sil er nanoparticles from ario s st dies
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Cell type Size (nm)Time
(h)Assay IC50
(g/ml)Reference
BRL 3A 15 24 MTT 24 Hussainet al. 2005
Primary mouse fibroblast 7-20 24 XTT 61 Arora et al. 2009
Primary mouse liver cells 7-20 24 XTT 499 Arora et al. 2009
BRL 3A 100 24 MTT 19 Hussainet al. 2005
1-100
1-1001-100
7-20
7-20
25
25
Macrophages 15 24 MTT 28 Carlson et al. 2008
Macrophages 30 24 MTT 33 Carlson et al. 2008
Macrophages 55 24 MTT >75 Carlson et al. 2008
NIH 3T3 (Mouse fibroblast) 24 MTT 50 Hsin et al. 2008
A431 24 XTT 12 Arora et al. 2008
HT1080 (Human fibrosarcoma) 24 XTT 11 Arora et al. 2008
mES 24 MTT >50 Ahamed et al. 2008
MEF (Mouse embryonic fibroblasts) 24 MTT >50 Ahamed et al. 2008
Cytotoxici ty of Silver nanoparticles from various studies
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Carbon Nanotubes
SWCNTsSWCNTs:: diameter of 1-2 nm, up to 100 m long
MWCNTs:MWCNTs: several layer of carbon cyl inders diameter of 10-30 nm
Aditive for polymer composites Electronic field emitters Batteries Fuel cells
Biological applications
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MWCNT interactions with human epidermal keratinocytes
Monteiro-Riviere et al. (2005), Toxicol. Lett. 155: 377-384.
Intracytoplasmic localizationof MWCNTs
Dose-dependent cytotoxicity
Dose- and time-dependent
increase in IL-8
TEM
1
P l t i i t f SWCNT i i
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Lungs from mice insti lled with 0.5 mg of a test material per mouse and euthanized
90 days after the single treatment
Control Carbon black Carbon nanotubes
Granulomas contained black particles
Particles were scattered in alveoli
Pulmonary toxicity of SWCNTs in mice
Lam et al. (2004), Toxicol. Sci. 77: 126-134.2
Pulmonary and Systemic Immune Response to Inhaled
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Pulmonary and Systemic Immune Response to Inhaled
MWCNTsMitchell et al. (2007), Toxicol. Sci. 100: 203-214.
3
Male mice whole-body inhalation to control air, 0.3, 1, 5 mg/m3 MWCNTs
7 or 14 days (6 h/day)
Many particle-laden and
some enlarged macrophages
Representative images from BALF collected fromanimals exposed for 14 days to 5 mg/m3
ControlMWCNTsControl MWCNTs
Inhalation of MWCNTs up to 5mg/m3 did not cause signif icant
lung inflammation or tissue damage
They altered immune response
functions
Exposure to carbon nanotube material
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Exposure to carbon nanotube material
Maynard et al. (2004), J.Toxicol. Env. Health 67: 87-104.4
Laboratory-based study:Aerosal release and dermal exposureduring handling of unrefined SWCNT material
Estimated airborne concentration generated during handlingwere lower than 53 g/m3
Glove deposits of SWCNT during handling were between 0.2 - 6mg/hand
With sufficient agitation, SWCNT can release fine particles
into the air
The aerosol concentrations generated while handling unrefined
material in the field at the work loads and rates observed werevery low .
Exposure to nanoparticles is related to pleural effusion
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Exposure to nanoparticles is related to pleural effusion,
pulmonary fibrosis and granulomaSong et al (2009), Eur Respir J, 34:559-567
Seven female workers (aged 1847 yrs), exposed to nanoparticles
for 513 months
Two of them died after working for months without proper protectionin a paint factory using nanoparticles,
Their lung tissues and fluids contained nanoparticles about30 nmin diameter
The symptoms seen in the patients are "similar" to those seen in
animals exposed to nanoparticles
Chinese cases
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Shortness of breath and pleural effusions admitted to hospital
Nonspecific pulmonary inflammation, pulmonary fibrosis andforeign-body granulomas of pleura
Tit i
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TiOTiO22
AnataseAnatase Photocatalytic air purification Self cleansing surface Solar cell Paint Cancer therapy
RutileRutile Cosmetics Sunscreen products Food additives
Anatase
Rutile
Titanium
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hv
e
-
h+
O2
+ 2H+
H2O
OH + H+
3.2 eV
Valence band
Conduction band
H2O
2
Schematic illustration of photo-activated TiO2
Anatase
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Quantitative determination of OH radical generation
and its cytotoxicity induced by TiO2-UVA treatmentUchino et al. (2002), Toxicol. in Vitro 164: 629-635.
Electron spin resonance (ESR)/ spin-traping with DMPO
1. Formation of OH-DMPO adducts is dependent on
concentration ofAnatase and intensity ofUVA
1
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2. Effect of crystal form of TiO2 on DMPO-OH radical
production
Anatase produces more OH radical than rutile
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3. Relationship between OH radical production and
viability of CHO cells
Cytotoxicity is dependent on OH radical generation
E id th t lt fi tit i di id i d d
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Evidence that ultrafine titanium dioxide induced
micronuclei and apoptosis in SHE fibroblastsRahman et al. (2002), Environ. Health Perspect. 110: 797-800.
SHE cells treated with 10 g/cm2 UF-TiO2 CisNT
TiO2
48h 24hM
DNA fragmentation
Apoptot ic
bodies
Bisbenzimide (Hoechst 33258)staining
Microuclei
formation
24h24h
24h
2
In vivo studies
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Comparative pulmonary toxicity inhalation and insti llation studies
with different TiO2 particle formulationWarheit et al. (2005), Toxicol. Sci. 88: 514-524.
Experiment
Male SD rats, 8 weeks old (240-255 g)
Al = alumina = Al2O3AMO = amorphous silica = SiO2
SEM
300 nm
3
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BAL = bronchoalveolar lavage
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Proliferation of Base
TiO2 particle-exposed
alveolar epithelial cells
Lung tissue section of a rat 1 year after 4-week
exposure to 1130 mg/m3 Base TiO2
Lung tissue section of a rat 1 year after 4-week
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g y
exposure to 1300 mg/m3 TiO2 formation III
Proliferation of
fibroblast
Thickness of alveolar walls Particle containingmacrophage
Hyperplasia of alveolarepithelial cells
Free particulates in
alveolar spaces
Surface treatment can influence toxicity of TiO2 particles in the lung
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Percent neutrophils recovered from BAL fluids of
saline and TiO2-instilled rats (2 and 10 mg/kg)
The National Institute of Occupational Safety and Health
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Exposure assessment
Toxicity and internal dose
Epidemiology and surveillance
Risk assessment
Measurement methods
Engineering controls and personal protective equipment
Fire and explosion safety
Recommendations and guidance
Communication and information
Applications
NIOSH recommended 10 critical research areas that will be used to
address knowledge gap on health and occupational safety:
Safe handling of nanomaterialsSafe handling of nanomaterials
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Worker
Avoid free air flow particlesMaintain process containmentUse personal protection equipments
Filtering facepiece respirators recommended for laboratory levels:
N95N95 and P100P100, FFP2FFP2 and FFP3FFP3Rengasamy et al. (2009),Ann.Occup.Hyg. 53: 117-128.
(NIOSH-approved) (EN certified CE-Marked)
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Operating area Local exhaust system equipped with a
particular filter eg. HEPA H14 Glove box
Cleaning
Vacuum cleaning (to avoid dust explosion) Nanoparticles are trapped in liquid-filled drum
Waste disposal Collect in specific drums Treat as hazardous waste
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Thank you for your attention