perspectives. □General media: Newspapers. ▫ Le Monde (FR). ▫ The Financial
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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
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Nanotechnology Risks during production & use … ?
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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
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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
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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
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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
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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
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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
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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
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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)
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Results particles i.t.
Light Intensity (A.U.)
1000000
p