Safty Nanotoxicology

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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

5

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

Th k f tt ti


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