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perspectives. □General media: Newspapers. ▫ Le Monde (FR). ▫ The Financial Times (UK). ▫ De Standaard (BE). □Fiction. ▫PREY by Michael Crichton ...
Nanotech and toxicity studies

Peter Hoet K.U.Leuven Pneumology, Lung Toxicology [email protected]

Overview  Introduction: Setting the scene  Nanoparticles – ultrafine particles  Do we need to worry?

 Nanotoxicology?     

General view Physico-chemical properties Can nanoparticles enter the human body Persistent material Other…

 Conclusions  Requirements for risk assessment Brussels 20051217 ECOPA workshop

100 nm

Nanomaterials Brussels 20051217 ECOPA workshop from Brook et al. Circulation

2004, 109, 2655-71

Definition?

 What to test?

 European Academy at Bad Neuenahr: “Nanotechnology (therefore man-made) is dealing with functional systems based on the use of subunits with specific size dependent properties of the individual sub-units or of a system of those.”  very general, does no exact size  100 nm  excludes non purpose made materials such as soot and dust.  life cycle analysis Brussels 20051217 ECOPA workshop

PM10 and mortality (short term) Estimated % change in daily mortality per 10 g/m3 change

Pope, Ch.31 in Holgate et al. 1999 Brussels 20051217 ECOPA workshop

Do we need to worry  Sakai et al. Effect of relocating to areas of reduced atmospheric particulate matter levels on the human circulating leukocyte count. J Appl Physiol 2004, 97, 1774-80  Japanese Antarctic Research Expedition 1999-2001  39 men, 24-57 y, 16 smokers  1999-2001: 516 days, 336 days in Antarctica  PM number densities:  to < 1% of levels in Japan  Blood: total WBC , PMN , monocytes  Brussels 20051217 ECOPA workshop

Sakai et al. J Appl Physiol 2004, 97, 1774-80

Brussels 20051217 ECOPA workshop

Nanotechnology Risks during production & use … ?

Brussels 20051217 ECOPA workshop

Introduction: conclusion  The field to study, how to define the “nano”  Inhalation of nanomaterials has an effect on human health:  Short term & long term effects  E.g. studies of pope et al.

 Dose dependent

Brussels 20051217 ECOPA workshop

Question 1: Nanotoxicology?  Does nanotech pose a specific problem? Is it just symptomatic and exemplary for any new technology; in term of risk, health and environmental impact?

Brussels 20051217 ECOPA workshop

General view Exposure: dose x time  response Patterns of exposure

Patterns of response

acute or high dose

(clinically) manifest

chronic low dose

subtle and/or long-term  cancer  reproductive effects (endocrine disrupters)  neurodegenerative disease  immunologic susceptibility  …

Epidemiology Long-term studies (Time) No specific nano-problem

Brussels 20051217 ECOPA workshop

Defining the material

no universal “nanoparticle” Origin • natural • unintentionally released • manufactured („old“, „new“)

Nanocapsules

Chem. composition • metals/ metal oxides • polymers, carbon • semiconductors • biomolecules • compounds ...

Ultrafine Aerosols

Nanoparticulate Materials Quantum Nanodots particles Nanotubes

Aggregation state • single particles • aggregates • agglomerates

Dispersion in • gases (aerosols) • liquids (e.g. gels, ferrofluids) • solids (e.g. matrix materials)

Shape/Structure • spheres • needles • platelets • tubes

Surface modification • untreated (as obtained in production process) • coated (e.g. conjugates, polymeric films) • core/shell particles (e.g. spheres, capsules)

Brussels 20051217 ECOPA workshop

BrusselsHealth 20051217 ECOPA workshop Oberdörster et al Environmental Perspectives , 113, 2005

Gurr et al, Toxicology, 2005 (absence of photoactivation) Brussels 20051217 ECOPA workshop

Question 1: Nanotoxicology?  General view: No specific nano-problem  Defining material:  There is no universal “nanoparticle” to fit all the cases  Physico-chemical characteristics  Crystal structure, Size, aggregation, dissolution, … Correct characterization + Correct test conditions Specific attention is needed! + how to report dose? + detection in biological samples

Brussels 20051217 ECOPA workshop

Nanoparticles enter the body?  Enter the systemic circulation via skin exposure?

 Penetration of the skin barrier is size dependent, nano-sized particles are more likely to enter more deep into the skin than larger ones.  Contact place  Motion  Materials, which can dissolve or leach from a particle (e.g. metals)

 skin sensitisation?

 The observation that particles in the skin can be phagocytised by langerhans cells or other cells is a possible road towards skin sensitisation. (Tinkle et al.) Brussels 20051217 ECOPA workshop

Systemic effects of inhaled nanoparticles? Uptake in CNS via olfactory bulb

BrusselsHealth 20051217 ECOPA workshop Oberdörster et al Environmental Perspectives , 113, 2005

Systemic effects of inhaled nanoparticles? Local inflammation – systemic effects

Inhaled particles (all sizes)  pulmonary inflammation + systemic release of mediators

Inflammatory mediators

Brussels 20051217 ECOPA workshop

Systemic effects of inhaled nanoparticles? Uptake in blood via alveoli inhaled ultrafine particles (Ø < 0.1 m) pass into the circulation “direct” effects on cardiovascular endpoints

Brussels 20051217 ECOPA workshop

Systemic uptake of nanomaterials after inhalation in humans Inhalation of 99mTc-carbon particles (“Technegas”) b

r 200000 g 150000 / 100000 M P 50000 C 0

largely particle-bound

n=5

1 5 10 Nemmar et al. Circulation 2002,105, 411

20

30

45

60

Time (min) Brussels 20051217 ECOPA workshop

60 min

Question 1: Nanotoxicology?  General view: No specific nano-problem  Defining material:  There is no universal “nanoparticle” to fit all the cases  Physico-chemical characteristics  Crystal structure, Size, dissolution, … Correct characterization + Correct test conditions Specific attention is needed!

Nanoparticles can enter the human body (cells):  Penetration via the skin? No, but…  Via the inhalation (and the intestines.) Yes, important Translocation over biological membranes:  Mechanisms involved?  Toxico-kinetics  target organs Brussels 20051217 ECOPA workshop

Persistent material  Dose: the quantity of dust inhaled over time  Dimension: aspect ratio (1/3)  Respirable? (Deposition)

 Durability: or biopersistence.  Defense: Macrophages - Mucociliary clearance Decades, years

Months, weeks

Brussels 20051217 ECOPA workshop

Peto J, Lancet 1995 (UK) – Br J Cancer 1999 (EU) Brussels 20051217 ECOPA workshop

2 mg/rat Carbon nanotube Grounded carbon nanotube Masson trichrome  Fibrotic response

Hematoxylin

Muller et al Toxicology and Applied Pharmacology 207 (2005) 221– 231

Question 1: Nanotoxicology?  General view: No specific nano-problem  Defining material:  There is no universal “nanoparticle” to fit all the cases  Physico-chemical characteristics  Crystal structure, Size, dissolution, … Correct characterization + Correct test conditions Specific attention is needed!

 Nanoparticles can enter the human body (cells):  Penetration via the skin? No, but…  Via the inhalation (and the intestines.) Yes, important Translocation over biological membranes

 Inhaled fibrous material  under investigation – long term?  Biopersistence?  Other target organ-tissue or cells: blood, brain, liver ….  Secretion of nanoparticles … (Kidney) (under investigation)  Unexpected results …. a long way to go Brussels 20051217 ECOPA workshop

Final Conclusions Risk characterization Hazard Evaluation (toxicity) • • • •

Dose response relation

Exposure evaluation

No or little data + no long term studies • Technically difficult Immunotox – Genotox – Teratotox? ToxicoKinetics - Target organs? Risk assessment NEED FOR STRATEGY

Cost-benefit analysis

Risk perception

Risk management Brussels 20051217 ECOPA workshop

Requirements for risk assessment Need for strategy – Two roads  Fundamental - mechanistic research

 Chemical and physical properties must be known  Toxicokinetics! (targets)  How do nanomaterials cross biological barriers?  Long term studies  Allergenicity – carcinogenicity – teratogenicity - …

 During development of new materials

 Precaution  Preliminary screening: High throughput  In vitro & ex vivo testing   

Testing cytotoxicity – release of inflammatory cytokines Ex vivo: potential to generate radicals (interaction with cellular membranes)

 Type & level exposure  

Occupational & consumer Used in food - drugs (chronic exposure)

 Post-marketing monitoring (Epidemiology) Brussels 20051217 ECOPA workshop

Nanotech and toxicity studies

Contains Nanomaterials Thank You For Your Attention

Scheme for a Preliminary Risk Assessment of Candidate Nanoparticles Aerosol release during Production Handling Processing

YES

rapidly soluble?

NO

aspect ratio > 100:1 ? YES

Direct exposure to Customers Workers Environment

Toxicological Screening Lung toxicity Systemic effects Oxidative stressor Endocrine disruptor Sensitiser/Adjuvant

NO

length < 3μm ?

YES

YES

Ecotoxicological Screening Persistence Long range transport Biomagnification

diameter < 100 nm

NO

NO

NO NO

Low priority

Intermediate priority

Brussels 20051217 ECOPA workshop

YES or UNKNOWN

High priority

Requirements for risk assessment Need for strategy  Fundamental Case per case approach  “The” nanoparticle does not exist  Chemical and physical properties must be known

 Type & level exposure  Occupational & consumer  IF used in food - drugs (chronic exposure): other rules!

 Toxicology  Testing as novel chemicals?  Scientifically valid and agreed Strategy 

High throughput (in vitro test in TIER 1)

 Fundamental research needs:  Toxicokinetics! (targets)  (Allergenicity – carcinogenicity – teratogenicity - …)

 Post-marketing monitoring (Epidemiology) Brussels 20051217 ECOPA workshop

Olfactory transport?  Rodents (50 %) vs humans (5 %)  Probably important neuro-toxic compounds e.g. metals  Parkinson disease, aging

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Warheit et al ToxSci 2004

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Bernstein J ALLERGY CLIN IMMUNOL VOLUME 114, NUMBER 5, 2004

Hierarchical oxidative stress model

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Personal worry: optimism: Low exposure levels & response Patterns of exposure

Patterns of response

acute or high dose

clinically manifest

chronic low dose

subtle and/or long-term  cancer  reproductive effects (endocrine disrupters)  neurodegenerative disease  immunologic susceptibility  …

STE century 21Epidemiology (Time)can manage! Human

Brussels 20051217 ECOPA workshop

Do we need to worry (Why do we worry?) Epidemiology Short-term & long-term effects

PM10 & mortality/morbidity (short term) Stylized summary: % change per 10 g/m3 change in PM10

Pope, Ch.31 in Holgate et al. 1999 Brussels 20051217 ECOPA workshop

PM10 and mortality (long term) Pope et al. Lung cancer, cardio-pulmonary mortality, and long-term exposure to fine particulate air pollution. JAMA 2002, 287, 1132-41  ACS; 1982-98; 500,000 adults; 51 U.S. metropolitan areas  average adjusted relative risk per 10 g/m3 PM2.5  all-cause: 1.06 (1.02-1.11)  cardiopulmonary: 1.09 (1.03-1.16)  lung cancer: 1.14 (1.04-1.23)  all other causes: 1.01 (0.95-1.06) controlling for age, sex, race, smoking, education, marital status, BMI, alcohol, occupation, diet RR for smoking 2.58, 2.89, 14.80 Brussels 20051217 ECOPA workshop

Quantum dots  < 50 nm  Carboxyl and amine modified organic layer

5-10 nm ZnS

20-30 nm CdSe Brussels 20051217 ECOPA workshop

Mouse lung Dosing: Quantum dots 100 g i.v. Sampling 1 h. after dosing

Brussels 20051217 ECOPA workshop

Known Risks & Knowledge gaps Toxicology / Human Health  There is no universal “nanoparticle” to fit all the cases  Physico-chemical characteristics  Crystal structure, Size, dissolution, …

 Nanoparticles can enter the human body:  Penetration via the skin? No, but…  Via the inhalation and the intestines. Yes, important  Health risks of inhaled fibrous material

under investigation

? Other target organ-tissue or cells Brain, blood, liver (Kupffer cells)…. Not much is know concerning the secretion of nanoparticles … (Kidney) under investigation

Unexpected results a long way to go Brussels 20051217 ECOPA workshop

ECOPA workshop Muller et al Toxicology and Brussels Applied20051217 Pharmacology 207 (2005) 221– 231

Macrophage + asbestos fiber

BAL + Asbestos bodies Brussels

20051217 ECOPA workshop

 The first health studies on carbon nanotubes (CNTs) proclaimed that working with CNTs) was unlikely to be associated with health risks while most recent results demand a closer look and further research.  It is quite difficult to compare different studies because there are several factors that are very important:  type of CNTs (SWTNs are found to be more toxic than MWNTs)  the way the CNTs are produced (diameter and the presence of different catalytic particles Co, Ni, Y, Fe, …)  the way the CNTs were instilled to the testing animals (concentration mgof CNTs per volume of liquid or mg CNTs per weight of testing animals)

Brussels 20051217 ECOPA workshop

Results particles i.t.

Light Intensity (A.U.)

1000000

p