Günter Oberdorster_How to assess the risks of nanotechnology?

The Responsible Development of Nanotechnology:
Challenges and Perspectives
Ne3LS Network International Conference 2012
November 1-2, 2012, Montréal, Canada
Plenary Session 1:
How to assess the risks of nanotechnology?
Analysis and discussion of the various risk facets

Günter Oberdörster
Department of Environmental Medicine
University of Rochester
November 1, 2012

OUTLINE

Introductory remarks

Nanoparticle characteristics

Dosing the respiratory tract

Nanoparticle Biokinetics

Nanoparticle – protein interactions

Hazard/Risk characterization

Nanoscale Materials
Water Glucose Antibody Virus Bacteria Cancer Cell A Period Tennis Ball

10-1 1 10 102 103 104 105 106 107 108

Nanometers

Bucky-ball Carbon Gold ZnO Nanorods Nano-onion
Nanotube Nanoshell

NANOTECHNOLOGY

THE BRIGHT! and THE DARK?
Multiple Applications/Benefits Consumer Fears/Perceived Risks
• Structural Engineering • Safety: Potential adverse effects
• Electronics, Optics • Environmental Contamination
• Food and Feed Industry • Inadvertent Exposure
(inhalation, dermal, ingestion)
• Consumer Products
• Susceptible Subpopulation
• Alternative Energy
• Societal Implications
• Soil/Water Remediation
• Nanomedicine:
– therapeutic • Nanotoxicology:
– diagnostic
– drug delivery Safety Assessment of
– cancer engineered Nanomaterials and of
– nanosensors Nanotechnology enabled Applications
– nanorobotics

Some Headlines in the Popular Press:
Nanoparticles (NPs)
kill workers
cause cancer
damage DNA
soften your brain
cause lung damage
cause genetic damage
in sunscreen cause Alzheimer’s?
are carbon nanotubes the next asbestos?

Nanotoxicology - Hype Cycle
All NPs are “toxic”

Realistic Assessment of Hazard and Risk
Hazard

Increasing Insight of
Nanotox Concepts

Appreciation of Reality

Effects and Biokinetics Time
of UFP (1990s)

Conceptual Depiction of Factors for Considering
Dose-dependent Transitions in Determinants of Toxicity

From: Slikker Jr., et al. 2004

Nano TiO2 repeated bolus instillation into mouse:
7.5 mg into mouse = 17.5 grams into human nose!

…

DPPC/Alb-Dispersed Mitsui Multiwalled Carbon
Nanotubes (MWCNTs)

Induction of mesothelioma in p53+/- mouse by intraperitoneal
application of multi-wall carbon nanotube
Takagi et al., J. Toxicol. Sci. 33 (No. 1): 105-116, 2008

Carbon nanotubes introduced into the
abdominal cavity of mice show asbestos-like
pathogenicity in a pilot study
CRAIG A. POLAND, RODGER DUFFIN, IAN KINLOCH, ANDREW MAYNARD,
WILLIAM A. H. WALLACE, ANTHONY SEATON, VICKI STONE, SIMON BROWN
WILLIAM MACNEE, AND KEN DONALDSON

Nature Nanotechnology/Vol. 3/July 2008

Induction of mesothelioma by a single intrascrotal administration
of multi-wall carbon nanotube in intact male Fischer 344 rats
Sakamoto et al, J.Tox. Sci., 34, 65-76, 2009

MWCNT in Subpleural Tissues, Visceral Pleura and
Pleural Space of Mice Following Oro-Pharyngeal
Aspiration (80 µg) From: Mercer et al., 2010

VP VP
Mac

Day 28 Day 28

Day 56
Day 56
VP PS

Ly VP
Mo

VP: visceral pleura; Mac: alveolar macrophage; Mo: monocyte; Ly: lymph vessel; PS: pleural space

Many Challenges in Nanotoxicology

Which testing strategy to assess environmental and
human safety (EHS) concerns?

• in vitro – in vivo design/methodologies
• acute – chronic toxicities
• dose, dosimetry, dosemetric related issues
• correlations between phys.chem. properties and toxicity
• nano-bio interactions: corona formation
• risk extrapolation: laboratory animals to humans
• human exposure assessment

Safety Assessment of Engineered Nanomaterials
ENM Synthesis
Sources
Characterization – Properties

Life Cycle Releases
Exposure From Cradle to Grave
Pathways

Dispersion, Transformation

Air, Water, Soil, Sediment
Receptor
Interactions

Exposure Assessment Hazard Characterization
Environment; Human Eco/Human Toxicity

Risk Assessment
Risk Characterization

Predictive Bioinformatics
Models
Predicting nanostructure- induced responses

What is different: Nanoparticles vs Larger Particles (respiratory tract as portal-of-entry)
Nanoparticles (<100 nm) Larger Particles (>500 nm)
General characteristics:
Ratio: number/surface area/volume high low
Agglomeration in air, liquids likely (dependent on medium; surface) less likely
Deposition in respiratory tract diffusion; throughout resp. tract sedimentation, impaction, inter-
ception; throughout resp. tract
Protein/lipid adsorption in vitro very effective and important for bio- less effective
kinetics and effects
Translocation to secondary target organs: yes generally not (to liver under “overload”)
Clearance
— mucociliary probably yes efficient
— alv. macrophages poor efficient
— epithelial cells yes mainly under overload
— lymphatic yes under overload
— blood circulation yes under overload
— sensory neurons (uptake + transport) yes not likely
Proteinl/lipid adsorption in vivo yes some
Cell entry/uptake yes (caveolae; clathrin; lip. rafts; diffusion) yes (primarily phagocytic cells)
— mitochondria yes no
— nucleus yes (<40 nm) no
Effects (caveat: dose!) :
at secondary target organs yes (no)
at portal of entry (resp. tract) yes yes
— inflammation yes yes
— oxidative stress yes yes
— activation of signaling pathways yes yes
— genotoxicity, carcinogenicity probably yes some

Physico-chemical NP Properties of Relevance for Toxicology

Size (aerodynamic, hydrodynamic)
Size distribution
Shape Properties can change
Agglomeration/aggregation
-with: method of production
Density (material, bulk)
preparation process
Surface properties: storage
- area (porosity)
- charge -when introduced into
- reactivity physiol. media, organism
- chemistry (coatings, contaminants)
- defects
Solubility/Sol-Rate (lipid, aqueous, in vivo)
Crystallinity
Biol. contaminants (e.g. endotoxin)

Key parameter: Dose!

Particle Number and Particle Surface Area per 10 pg/cm3 Airborne Particles
(Unit density particles)

Particle Diameter Particle Number Particle Surface Area
nm N/cm3 µm2/cm3

5 153,000,000 12,000
20 2,400,00 3,016
250 1,200 240
5,000 0.15 12

Small size, high number per mass, and surface chemistry
confer both desirable and undesirable properties.

Detailed Physico-chemical characterization of NP is essential.

Surface Molecules as Function of
Particle Size
60%

50%
% Surface Molecules

40%
Ns/N Ns/N
30%

20%

10%

0%
1 10 100 1000 10000
Dp [nm]
Diameter (nm)

From Fissan, 2003

Which Dose-Metric?
Percent of Neutrophils in Lung Lavage 24 hrs after
Intratrachael Dosing of Ultrafine and Fine TiO2 in Rats
▲ Fine TiO2 (200nm)
■ Ultrafine TiO2 (25nm)
● Saline
50 UltrafineTiO 2 (~20nm)
ultrafine TiO 2 (20nm) 40
pigment grade TiO 2 (~200nm) Fine TiO 2 (~250nm)
saline
40
30
% Neutrophils

% Neutrophils
30

20
20

10 10

0 0
0 500 1000 1500 2000
108 10 9 1010 10 11 10 12 10 13
Particle Mass, ug Particle Number

Particle Mass vs Particle Number

Which Dose-Metric?
Percent of Neutrophils in BAL 24 hrs after Instillation of TiO 2 in Rats
Correlation with Particle Surface Area
50

40 ultrafine TiO 2 (~25nm)
fine TiO 2 (~200nm)
saline
% Neutrophils

30

20

10

0
0 50 100 150 200 250
2
Particle Surface Area, cm

Dosing the Respiratory Tract
Impact of Dose-Rate

Fractional Deposition of Inhaled Particles in the Human Respiratory Tract
(ICRP Model, 1994; Nose-breathing)

1.0
Total deposition

Deposition Fraction
0.8

0.6

0.4

0.2

0.0
0.0001 0.001 0.01 0.1 1 10 100
Diameter (µm)
Mechanisms
Deposition
Impaction
Diffusion

Sedimentation
Inhalable Thoracic Respirable
< 100um < 30um < 10um

High

more material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant

Intratracheal Instillation of Particle Suspension, Rat

From: Morello et al., 2009

Comparing Responses when the same Lung Dose of TiO2 Nanoparticles

is administered by Inhalation or intratracheal Instillation

4 Groups of Rats:

1. Controls (instillation of saline)
2. 200 µg TiO2 (25 nm) instillation into lungs
3. Four hour inhalation to deposit 200µg TiO2 into lungs
4. Four days of 4 hour daily inhalation to deposit 200µg TiO2 into lungs

24 hours after dosing lungs were lavaged and
inflammatory cellular and biochemical parameters determined

Dose-Rate matters as Determinant of Hazard

Biokinetics andTranslocation
of Inhaled Nanoparticles

Exposure and Biokinetics of Nanoparticles
Confirmed routes
Potential routes

Exposure Media Air, water, clothes Drug Delivery Air Food, water

Deposition Injection Inhalation Ingestion

Skin Respiratory Tract
Uptake Pathways trach- GI-tract
nasal bronchial alveolar

Lymph
CNS
Blood
PNS (platelets, monocytes, Liver
endothelial cells)
Translocation
and Distribution
Lymph

Bone Marrow Other sites
(e.g. muscle, placenta) Kidney Spleen Heart

Excretory Sweat/exfoliation Urine Breast Milk Feces
Pathways
(Modified from Oberdörster et al., 2005)

Confirmed routes
Potential routes




Lymph
CNS
Blood
endothelial cells)
Translocation
and Distribution
Lymph


Pathways
Translocation rates are very low!

Confirmed routes
Potential routes
Translocation and Elimination Pathways from Respiratory Tract



?
Lymph
CNS
Blood
endothelial cells)
Translocation
and Distribution
Lymph

<~6 nm

Pathways
GI-tract and kidney as major excretory organs

FROM RESPIRATORY TRACT TO BRAIN:
POTENTIAL TRANSLOCATION PATHWAYS OF NANOPARTICLES

CSF: Cerebrospinal Fluid
BBB: Blood-Brain Barrier Nose
CSBB: Cerebrospinal Brain Barrier Olfactory Mucosa
Nasopharyngeal Mucosa

Inhaled Nanoparticles
Brain
Olfactory Bulb
Trigem. Ganglion BBB
Blood

Lymph
CSF

Lower Respirat.
Tract

Rat, Right Nostril Occlusion Model:
Accumulation of Mn in Right and left Olfactory Bulb 24 Hours after Exposure
to Ultrafine (~30nm) Mn Oxide Particles (n=3-5, mean+/-SD)
left olfactory bulb right olfactory bulb

1.0
ng Mn/mg wet weight

0.8

0.6

0.4

0.2

0.0
Unexposed Exposed
right nostril occluded
Elder et al, 2006

Mn concentration in lung and brain regions of rats following
12 days ultrafine Mn-oxide exposure (mean +/- SD)
(465 µg/m3; 17x106 part./cm3; CMD: 31 nm; GSD: 1.77)
2.0
*
Control
12 days

1.5
ng Mn/mg wet wt

1.0

*
* *
0.5 *

0.0
Lung Olfactory Trigeminal Striatum Midbrain Frontal Cortex Cerebellum
Cortex
Elder et al, 2006

Inflammation in the Brain Regions where
Mn Signal was Found

mRNA Levels TNF- Protein

8 TNF-a 40
MIP-2
GFAP
Neurogenin
7
Relative Intensity, fold increase

MnSOD
NCAM

Relative Intensity, fold increase
Na-K-Cl co-trans. 30
6
ATP-rel. K-channel

5

4 20

3

2 10

Controls 1

0 0
Olfactory Frontal Midbrain Striatum Cerebellum Olfactory Frontal Midbrain Striatum Cerebellum
Bulb Cortex Bulb Cortex

Elder et al, 2006

Protein Adsorption on Nanoparticles

“Concept of Differential Adsorption”
Modified from Müller and Heinemann, 1989

NP physicochemical properties: Body compartment media:
•Size •Respiratory tract lining fluid
•Surface size and chemistry •Gastrointestinal milieu
•Charge + •Plasma proteins
•Surface hydrophobicity •Other mucosal membranes
•Redox activity •Intra/extracellular fluid
•Others

determine
protein/lipid adsorption and desorption patterns

determine

biodispersion across barriers and in target tissues/cells

Altered plasma composition in disease state is likely to change adsorption pattern

Nano – Bio Interactions
Schematic drawing of the structure of NP-protein complexes in plasma:

Two Layers of Proteins of the “Core” Nanoparticle :
An outer “weak” layer of protein corona And an inner “hard”layer of stable,
rapidly exchanging with free proteins slowly exchanging corona of proteins
(red arrows) From: Walczyk et al., 2010

Adsorption of Proteins (FBS) Is Inhibited by Surfactant Coating (Pluronic F127)
(SDS PAGE)

kDa

Albumin

SWCNT Casein Proteins Silica NP (10 nm)
BET surface 274 m2/g Hemoglobulins BET surface 212 m2/g
Lactoglobulin

(Dutta et al., 2007)

Cytotoxicity of 10 nm SiO2 in RAW264.7 Cells (18 hr. incubation)
Is Inhibited by Surfactant Treatment (Pluronic F127)

+

From: Dutta et al., 2007

Hazard and Risk Characterization

Risk Assessment and Risk Management Paradigm
For Engineered Nanoparticles (NPs)

Physico-chemical
Parameters! Inhalation
Ingestion, Dermal Public health/social/
Adverse NP Effect: economical/political
at portal of entry consequences
Biological
and remote organs Monitoring
(markers of exposure) Regulations
Expos. Standards
Experimental Humans Prevention/Intervention
Animals Occupational/ Measures
Environmental Biomed./Engineering
Monitoring
Biokinetics!

Exposure-Dose- Risk
Response Data Calculation

In Vivo Studies Susceptibility
(acute; chronic) Extrapolation Models
(high low)
(animal human)
In Vitro Studies
(non-cellular)
(animal/human cells)
(subcellular distribution) Mechanistic
Data
Dose-Metric!

Modified from Oberdörster et al., 2005

Concepts of Nanomaterial Toxicity Testing:
Considering Exposure and Hazard for Risk Assessment
Exposure – Dose - Response Dose - Response

Phys-chem. Properties in vivo Phys-chem. Properties
in vivo Target Organs Inhal/
in vitro
Humans Respirability
Animals ( Bolus
) Endpoints; Ref. Material
Bolus; ALI
Hi-Lo Dose; Relevancy
Workplace Biokinetics Target cells,
NOAELs; OELs; (translocation; Mechanisms
Laboratory corona formation) Tissues
HECs Reproducibility
Consumer Dose-Response Dose-Response

Long-term

In silico
models

Exposure (assessment) Hazard (characterization)

Risk Assessment

Dosimetric Extrapolation of Inhaled Particles from Rats to Humans
Rat Human

Exposure [mg(m3)-1] Exposure (HEC) [mg(m3)-1]
Breathing
Minute
Volume
Inhaled Dose [mg(kg)-1] Inhaled Dose [mg(kg)-1]
Tidal Volume, Resp. Rate
Resp. Pause
Particle characteristics
Anatomy
Deposited Dose µg(cm2)-1; Deposited Dose µg(cm2)-1;
µg(g)-1 µg(g)-1
Clearance
Retention
Regional Uptake
(Metabolism, T½)

Retained (Accumulated) Dose
[µg(g)-1; µg(cm2)-1]

Effects
Assumption: If retained dose is the same as in rats and humans, then effects will be the same

Two Subchronic MWCNT Inhalation Sudies in Rats

Günter Oberdorster_How to assess the risks of nanotechnology?

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