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

Life Cycle PerspectiveLife Cycle Perspective

Human exposure

Human exposure Ecological exposure

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

Gastrointestinal Tract and Site of Absorption

IngestionIngestion

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

BAL = bronchoalveolar lavage

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

Percent neutrophils recovered from BAL fluids of saline and TiO2-instilled rats (2 and 10 mg/kg)

• 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

rawiwan@nanotec.or.th

Thank you for your attention