Nanotoxicology

A safety evaluation of nanomaterials

Rawiwan Maniratanachote

The 2nd National Conference in Toxicology

Miracle Grand Convention Hotel, Bangkok

December 17, 2009

Exposure to nanoparticles
Non-engineered particles

Engineered particles
- Free or in aerosol - Biopersistent - Catalytically active

Life Cycle Perspective

Human exposure

Human exposure

Ecological exposure

The nanotechnology consumer product inventory

More than 1000 nanoproducts already on the market
(As of August, 2009)

R = 0.9949

2005

2006

2007

2008

2009

2010

2012

http://www.nanotechproject.org/inventories/consumer/analysis_draft/

The closer the R is to 1, the better the model and the closer one can approximate a future outcome.

Nanomaterials Used in Commercial Products and Researches

Number of Nanotechnology products associated with specific materials

2009

Consumer products
Examples:

2006

Nanosilver cutting board Nanosilver baby mug Antibacterial kitchen ware Antibacterial textiles Nanosilver water storage tank
etc.
Silver Carbon zinc Silica Titanium Gold http://www.nanotechproject.org/inventories/consumer/analysis_draft/

Silver is among the most widely used NMs

Nano-sized silver particles have increased antibacterial properties

Nanotoxicology
An emerging discipline evolving from studies of nanomaterials An interdisciplinary field approach: Toxicology, materials science, medicine, molecular biology etc. 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

Oberdorster et al. 2005, Environ Health Perspect 113: 823-839.

Potential routes of nanomaterial exposure

micro
Blood cells

nano
DNA

12-15 m Hair

Actin

Local / Systemic adverse effects

Lung and Inhalation

Alveoli

Pulmonary Deposition as a Function of Particle Size

Potential Pathway for Nanoparticles in the Lung

Interstitialization pathway Clearance

Secretions Alveolar macrophage

Particle-laden macrophage

Bronchoalveolar space

Epithelium
Capillary Fibroblast Secretions Interstitial macrophage Lymph

Interstitium

Secretions

Modified from Donaldson et al. 1998, J Aerosol Sci, 29: 553-560.

Role of fiber length and biopersistence in determining adverse effects

Exposure Deposition Short fibers (<10 m) Macrophage clearance Long fibers (>20 m)

Non-biopersistent fibers

Biopersistent fibers Fibrosis / Cancer

Breakage

Dissolve

Ingestion

Gastrointestinal Tract and Site of Absorption

The Skin
In 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

Penetration through skin barrier

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 Species difference in hair follicle density
Species

Area Abdomen Back Back Back Back

Number of hair follicles/cm2 11 1 11 1 289 21 658 38 75 6

Human Pig Rat Mouse Hairless mouse

Bronaugh et al. 1982, Toxicol Appl Pharmacol 62: 481-488.

The skin from furry rodents results in overestimation of human skin penetration Pig has different lipid structures from human

NM effects as the basis of pathophysiology and toxicity

Experimental effects
ROS generation Mitochondrial perturbation Inflammation

Possible pathophysiological effects

Protein, DNA and membrane injury, oxidative stress Inflammation Energy failure, apoptosis, aponecrosis, cytotoxicity Tissue infiltration with inflammatory cells, fibrosis, granulomas, atherogenesis, acute phase protein expression Chronic inflammation, fibrosis, granulomas, interference in clearance of infectious agents Autoimmunity Mutagenesis, carcinogenesis
Adapt from: Nel et al., Science (2006) 311: 622-627.

Perturbation of phagocytic function, particle overload, mediator release Generation of neoantigens, breakdown in immune tolerance DNA damage

Particles characterization
The first step towards nanotoxicology studies 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

Material composition Electronic structure Bonded surface species Surface coating Solubility Contribution of surface species Environmental factors

Nel et al., Science (2006) 311: 622-627

Characterization of the particle

Analysis Morphology and compositions Size, size distribution Surface charge Specific surface area Metal contaminants Instrument SEM-EDX, TEM-EDX DLS (Nanosizer) Zeta potential analyzer BET surface area analyzer ICP, AA Transmission Electron Microscope (TEM)

Scanning Electron Microscope (SEM)

BET surface area analyzer

Nanosizer

Engineered Nanomaterials
Silver Carbon nanotubes Titanium
Silica Gold Zinc etc.

Silver
The most prevalent nanomaterials used in consumer products
Consumer products
Food packaging Odor resistant textiles Wound dressings
etc.

Potential adverse affects Potent bactericide

1. 2. Development of antibiotic resistant bacteria Harmful to beneficial bacteria which form symbiotic relationship to plants, animals and humans Disrupt ecosystem function

Most people are exposed daily to very low level of silver mainly in food and drinking water, and less in air.

Health Aspect
Argyria

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 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. New Eng J Med. May 20, 1999

A 56-year-old woman has had discolored skin since the age of 14

Silver containing products in Thailand

Phyto-Silver Balancing Day Cream Silver Citrate

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 washing

Geranio et al (2009), Environ. Sci. Technol. 43: 8113-8.

Human skin penetration of silver nanoparticles through intact and damaged skin
Larese et al. (2009), Toxicology 255: 33-37.

Human abdominal full thickness skins Silver nanoparticles (257.1 nm)

Franz diffusion cell method

Silver skin penetration at 24 h

100 nm

TEM micrograph of Ag nanoparticles-treated skin sample Ag nanoparticles are presented in deep stratum corneum

500 nm

Cytotoxicity of Silver nanoparticles from various studies

Cell type BRL 3A BRL 3A NIH 3T3 (Mouse fibroblast) A10 (Rat vascular smooth muscle) HCT 116 (Human colon cancer) A431 HT1080 (Human fibrosarcoma) mES MEF (Mouse embryonic fibroblasts) Macrophages Macrophages Macrophages Primary mouse fibroblast Primary mouse liver cells Size (nm) 15 100 1-100 1-100 1-100 7-20 7-20 25 25 15 30 55 7-20 7-20 Time
(h)

Assay MTT MTT MTT MTT MTT XTT XTT MTT MTT MTT MTT MTT XTT XTT

(g/ml)

IC50 24 19 <50 50 >50 12 11 >50 >50 28 33 >75 61 499

Reference
Hussain et al. 2005 Hussain et al. 2005 Hsin et al. 2008 Hsin et al. 2008 Hsin et al. 2008 Arora et al. 2008 Arora et al. 2008 Ahamed et al. 2008 Ahamed et al. 2008 Carlson et al. 2008 Carlson et al. 2008 Carlson et al. 2008 Arora et al. 2009 Arora et al. 2009

24 24 24 24 24 24 24 24 24 24 24 24 24 24

Carbon Nanotubes
Aditive for polymer composites Electronic field emitters Batteries Fuel cells Biological applications

SWCNTs: diameter of 1-2 nm, up to 100 m long MWCNTs: several layer of carbon cylinders diameter of 10-30 nm

1 MWCNT interactions with human epidermal keratinocytes

TEM

Monteiro-Riviere et al. (2005), Toxicol. Lett. 155: 377-384.

Dose-dependent cytotoxicity

Dose- and time-dependent increase in IL-8

Intracytoplasmic localization of MWCNTs

2 Pulmonary toxicity of SWCNTs in mice

Lam et al. (2004), Toxicol. Sci. 77: 126-134.

Control

Carbon black

Carbon nanotubes

Granulomas contained black particles Particles were scattered in alveoli

Lungs from mice instilled with 0.5 mg of a test material per mouse and euthanized 90 days after the single treatment

Pulmonary and Systemic Immune Response to Inhaled 3 MWCNTs

Mitchell et al. (2007), Toxicol. Sci. 100: 203-214.

Male mice whole-body inhalation to control air, 0.3, 1, 5 mg/m3 MWCNTs 7 or 14 days (6 h/day)
Control MWCNTs Control MWCNTs

Representative images from BALF collected from animals exposed for 14 days to 5 mg/m3

Many particle-laden and some enlarged macrophages

Inhalation of MWCNTs up to 5 mg/m3 did not cause significant lung inflammation or tissue damage They altered immune response functions

4 Exposure to carbon nanotube material

Maynard et al. (2004), J.Toxicol. Env. Health 67: 87-104.

Laboratory-based study: Aerosal release and dermal exposure during handling of unrefined SWCNT material 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. Estimated airborne concentration generated during handling were lower than 53 g/m3 Glove deposits of SWCNT during handling were between 0.2 - 6 mg/hand

Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma Chinese cases
Seven female workers (aged 1847 yrs), exposed to nanoparticles for 513 months Shortness of breath and pleural effusions admitted to hospital 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 nm in diameter Nonspecific pulmonary inflammation, pulmonary fibrosis and foreign-body granulomas of pleura The symptoms seen in the patients are "similar" to those seen in animals exposed to nanoparticles

Song et al (2009), Eur Respir J , 34:559-567

Titanium
TiO2
Anatase
Photocatalytic air purification Self cleansing surface Solar cell Paint Cancer therapy
Anatase

Rutile
Cosmetics Sunscreen products Food additives
Rutile

Schematic illustration of photo-activated TiO2

H2O2

eO2 + 2H+

Conduction band

3.2 eV

Valence band

H2O

hv

h+
OH + H+

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

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 2
Rahman et al. (2002), Environ. Health Perspect. 110: 797-800.

SHE cells treated with 10 g/cm2 UF-TiO2

Bisbenzimide (Hoechst 33258)staining

Cis

TiO2

M 24h NT 48h 24h

24h 24h
Microuclei formation Apoptotic bodies

DNA fragmentation

In vivo studies Comparative pulmonary toxicity inhalation and instillation studies with different TiO2 particle formulation
Warheit et al. (2005), Toxicol. Sci. 88: 514-524.

Experiment
Male SD rats, 8 weeks old (240-255 g)

SEM

Al = alumina = Al2O3 AMO = amorphous silica = SiO2

300 nm

BAL = bronchoalveolar lavage

Lung tissue section of a rat 1 year after 4-week exposure to 1130 mg/m3 Base TiO2

Proliferation of Base TiO2 particle-exposed alveolar epithelial cells

Lung tissue section of a rat 1 year after 4-week exposure to 1300 mg/m3 TiO2 formation III

Proliferation of fibroblast

Free particulates in alveolar spaces

Thickness of alveolar walls Particle containing macrophage Hyperplasia of alveolar epithelial cells

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)

The National Institute of Occupational Safety and Health

NIOSH recommended 10 critical research areas that will be used to address knowledge gap on health and occupational safety: 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

Safe handling of nanomaterials Worker

Avoid free air flow particles Maintain process containment Use personal protection equipments
Filtering facepiece respirators recommended for laboratory levels:

N95 and P100, P100 FFP2 and FFP3

(NIOSH-approved) (EN certified CE-Marked)

Rengasamy et al. (2009), Ann.Occup.Hyg. 53: 117-128.

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

Thank you for your attention

rawiwan@nanotec.or.th