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NanotoxicologyNanotoxicologyA safety evaluation of nanomaterialsA safety evaluation of nanomaterials
Rawiwan Maniratanachote
December 17, 2009
The 2nd National Conference in ToxicologyMiracle Grand Convention Hotel, Bangkok
Exposure to nanoparticlesExposure to nanoparticles
• Non-engineered particles
• Engineered particles
- Free or in aerosol
- Biopersistent
- Catalytically active
The nanotechnology consumer product inventory
http://www.nanotechproject.org/inventories/consumer/analysis_draft/
2005 2006 2007 2008 2009 2010 2012
The closer the R² is to 1, the better the model and the closer one can approximate a future outcome.
R² = 0.9949
More than More than 10001000nanonanoproducts already products already on on
the marketthe market(As of August, 2009)(As of August, 2009)
http://www.nanotechproject.org/inventories/consumer/analysis_draft/
Silver Carbon zinc Silica Titanium Gold
Number of Nanotechnology products associated with specific materials
Nanomaterials Used in Commercial Products and Researches
Consumer productsExamples:
• Nanosilver cutting board• Nanosilver baby mug• Antibacterial kitchen ware• Antibacterial textiles• Nanosilver water storage tank• etc.
“Nano-sized silver particles have increased antibacterial properties”Silver is among the most widely used NMs
2009
2006
Nanotoxicology
• The small size facilitates uptake into cells and translocation 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 Perspect 113: 823-839.
Potential routes of nanomaterial exposure
Local / Systemic adverse effectsLocal / Systemic adverse effects
Hair
Blood cells
“micro” “nano”DNA
Actin
12-15 µm
Lung and InhalationLung and Inhalation
Pulmonary Deposition as a Function of Particle Size
Alveoli
Potential Pathway for Nanoparticles in the LungInterstitialization pathway
Clearance
Alveolar macrophage
SecretionsParticle-laden macrophage
Capillary
Secretions Interstitial macrophage Secretions
Fibroblast
Lymph
Epithelium
Interstitium
Broncho-alveolarspace
Modified from Donaldson et al. 1998, J Aerosol Sci, 29: 553-560.
Role of fiber length and biopersistence in determining Role of fiber length and biopersistence in determining adverse effectsadverse effects
Exposure
Deposition
Long fibers (>20 µm)
Short fibers (<10 µm)
Macrophage clearance Non-biopersistent
fibersBiopersistent
fibers
DissolveFibrosis / Cancer
Breakage
The SkinThe SkinIn healthy skin, the epidermis provides excellent protection against particle spread to the dermis
Damaged skin allows micrometer-size particles access to the dermis and regional lymph nodes Effects on the immune system
• The skin from furry rodents results in overestimation of human skin penetration
• The stratum corneum is an excellent skin barrier• Factors influence in penetration test for nanomaterials
• Hair follicle density• Size of hair follicle opening• Lipid structures and contents
Penetration through skin barrier
Species difference in hair follicle densitySpecies Area Number of hair follicles/cm2
Human Abdomen 11 ± 1Pig Back 11 ± 1Rat Back 289 ± 21Mouse Back 658 ± 38Hairless mouse Back 75 ± 6
Bronaugh et al. 1982, Toxicol Appl Pharmacol 62: 481-488.
• Pig has different lipid structures from human
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 inflammatory cells, fibrosis, granulomas,atherogenesis, acute phase protein expression
Perturbation of phagocytic function, Chronic inflammation, fibrosis,‘‘particle overload,’’ mediator release granulomas, interference in
clearance of infectious agentsGeneration of neoantigens, breakdown Autoimmunityin immune toleranceDNA damage Mutagenesis, carcinogenesis
Experimental effects Possible pathophysiological effects
The first step towards nanotoxicology studiesParticles characterization
• To ensure that the results are reproducible• To provide basis for understanding the
properties of nanoparticles that determine their biological effects
Physicochemical characteristic of nanoparticels
Nel et al., Science (2006) 311: 622-627
• Material composition
• Electronic structure
• Bonded surface species
• Surface coating
• Solubility
• Contribution of surface species
• Environmental factors
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 Electron Microscope (TEM)
NanosizerBET surface area analyzer
Engineered NanomaterialsEngineered Nanomaterials• Silver• Carbon nanotubes• Titanium• Silica• Gold• Zinc• etc.
Potential adverse affects“Potent bactericide”
1. Development of antibiotic resistant bacteria2. Harmful to beneficial bacteria which form symbiotic relationship
to plants, animals and humans Disrupt ecosystem function
Consumer products• Food packaging• Odor resistant textiles• Wound dressings
etc.
“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.
At the age of 11 the patient was given nose drops of unknown composition for “allergies,” and three yearslater her skin turned gray. She was thought to have argyria, and a skin biopsy at the age of 15 confirmed the presence of silver 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 skin since the age of 14
New Eng J Med. May 20, 1999
Health Aspect
Phyto-Silver Balancing Day Cream
การเคลือบสารซิลเวอรนาโนบริเวณคียบอรดและที่วางมือ ผงซักฟอกที่มีสวนผสมของซิลเวอรนาโน
ผลิตภัณฑระงับกลิ่นกายที่มีสวนผสมของ Silver Citrate
Silver containing products in Thailand
And more…
Washing studies
• Nanoparticle silver released into water from commercially available sock fabrics
Benn and Westerhoff (2008), Environ. Sci. Technol. 42: 4133-9.
• The behavior of silver nanotextiles during washingGeranio et al (2009), Environ. Sci. Technol. 43: 8113-8.
Franz diffusion cell method
Human skin penetration of silver nanoparticles through intact and damaged skin
Larese et al. (2009), Toxicology 255: 33-37.
TEM micrograph of Ag nanoparticles-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 (25±7.1 nm)
Cell type Size (nm) Time (h) Assay IC50
(µg/ml) Reference
BRL 3A 15 24 MTT 24 Hussain et 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 Hussain et al. 2005
1-100
1-100
1-100
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
A10 (Rat vascular smooth muscle) 24 MTT 50 Hsin et al. 2008
HCT 116 (Human colon cancer) 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
Cytotoxicity of Silver nanoparticles from various studies
Carbon Nanotubes
SWCNTsSWCNTs: : diameter of 1-2 nm, up to 100 µm longMWCNTs: MWCNTs: several layer of carbon cylinders diameter of 10-30 nm
• Aditive for polymer composites• Electronic field emitters• Batteries• Fuel cells• Biological applications
MWCNT interactions with human epidermal keratinocytesMonteiro-Riviere et al. (2005), Toxicol. Lett. 155: 377-384.
Intracytoplasmic localization of MWCNTs
Dose-dependent cytotoxicity
Dose- and time-dependent increase in IL-8
TEM
1
Lungs from mice instilled 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 particlesParticles were scattered in alveoli
Pulmonary toxicity of SWCNTs in miceLam et al. (2004), Toxicol. Sci. 77: 126-134.
2
Pulmonary and Systemic Immune Response to Inhaled MWCNTs
Mitchell 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 from animals exposed for 14 days to 5 mg/m3
ControlMWCNTsControl MWCNTs
• Inhalation of MWCNTs up to 5 mg/m3 did not cause significant lung inflammation or tissue damage
• They altered immune response functions
Exposure to carbon nanotube materialMaynard 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 handling were lower than 53 µg/m3
• Glove deposits of SWCNT during handling were between 0.2 - 6 mg/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 were very low.
Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma
Song et al (2009), Eur Respir J , 34:559-567
• Seven female workers (aged 18–47 yrs), exposed to nanoparticles for 5–13 months
• Two of them died after working for months without proper protection in a paint factory using nanoparticles,
• Their lung tissues and fluids contained nanoparticles about 30 nmin diameter
The symptoms seen in the patients are "similar" to those seen inanimals exposed to nanoparticles
Chinese cases
5
• Shortness of breath and pleural effusions admitted to hospital
• Nonspecific pulmonary inflammation, pulmonary fibrosis and foreign-body granulomas of pleura
TiOTiO22
AnataseAnatase• Photocatalytic air purification • Self cleansing surface• Solar cell• Paint • Cancer therapyRutileRutile• Cosmetics• Sunscreen products• Food additives
Anatase
Rutile
Titanium
hv
e-
h+
O2 + 2H+
H2O
OH• + H+
3.2 eV
Valence band
Conduction band
H2O2
Schematic illustration of photo-activated TiO2
Anatase
Quantitative determination of OH radical generation and its cytotoxicity induced by TiO2-UVA treatment
Uchino 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 of Anatase and intensity of UVA
1
2. Effect of crystal form of TiO2 on DMPO-OH radical production
Anatase produces more OH radical than rutile
3. Relationship between OH radical production and viability of CHO cells
Cytotoxicity is dependent on OH radical generation
Evidence that ultrafine titanium dioxide induced micronuclei and apoptosis in SHE fibroblasts
Rahman et al. (2002), Environ. Health Perspect. 110: 797-800.
SHE cells treated with 10 µg/cm2 UF-TiO2 CisNT
TiO2
48h 24hM
DNA fragmentation
Apoptotic bodies
Bisbenzimide (Hoechst 33258)staining
Microucleiformation
24h24h
24h
2
Comparative pulmonary toxicity inhalation and instillation studies with different TiO2 particle formulation
Warheit et al. (2005), Toxicol. Sci. 88: 514-524.
In vivo studies
Experiment• Male SD rats, 8 weeks old (240-255 g)
Al = alumina = Al2O3AMO = amorphous silica = SiO2
SEM
300 nm
3
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 exposure to 1300 mg/m3 TiO2 formation III
Proliferation of fibroblast
• Thickness of alveolar walls• Particle containing
macrophage• Hyperplasia of alveolar
epithelial cellsFree particulates in
alveolar spaces
Surface treatment can influence toxicity of TiO2 particles in the lung
• 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:
The National Institute of Occupational Safety and Health
Worker• Avoid free air flow particles• Maintain process containment• Use personal protection equipments
Filtering facepiece respirators recommended for laboratory levels:N95N95 and P100P100, FFP2FFP2 and FFP3FFP3
Rengasamy et al. (2009), Ann.Occup.Hyg. 53: 117-128.(NIOSH-approved) (EN certified CE-Marked)
Safe handling of nanomaterialsSafe handling of nanomaterials
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