third international conference on safe production and use...
TRANSCRIPT
Third International Conference on
Safe Production
and Use of Nanomaterials
Some of many challenges in nanotoxicology for Assessing Environmental and Human Safety (EHS):
• dose related concepts: in vivo; in vitro
• toxicity vs. adaptation
• nano – bio interactions: corona formation
• risk extrapolation: acute to subacute to chroniclab animals to humans
• case study: MWCNT
Outline
Respiratory Tract as Portal of Entry:Some Basics: Dose Concepts and Caveats
N Lar
0.0001 0.001 0.01 0.1 1 10 100 0.0
0.2
0.4
0.6
0.8
1.0
Diameter (µm)
Dep
osit
ion
Fra
ctio
n D
epos
ition
M
echa
nism
s Diffusion
Sedimentation
Impaction
Total deposition
Fractional Deposition of Inhaled Particles in the Human Respiratory Tract(ICRP Model, 1994; Nose-breathing)
Thoracic RespirableInhalable< 100um < 30um < 10um
Lower Respiratory Tract:
Tracheobronchial Alveolar
From: The CIBA Collection of Medical
Illustrations, Vol. 7, Respiratory System
Surface Areas of Respiratory Tract Regions at FRC
Rat Human
cm2 % of total cm2 % of total
Nasal 18.5 0.75 210 0.03
Trach-bronch 24 1.00 4149 0.65
Alveolar 2422 98.25 634620 99.32
Keyhani et al., 1997; Kimbell et al., 1997; Miller et al., 2011
Deposition per Unit Surface Area (cm2) over 8 Hour Exposure at 1 mg/m3 (nasal breathing, resting conditions)
Ext-Thor. Tr-Bronch. Alveolar0.1
1
10
100
1000
10000 Human Rat
Nanoparticle Size: CMD =20 nm; GSD = 1.0 Density:ρρρρ = 1 g/cm3
5724
1389
269391
1.75
7.2
ng/c
m2
JUSTIFYING — OR PUTTING INTO PERSPECTIVE? — EXPERIMENTAL DOSES/EXPOSURES:SHOULD THE DOSE RATE BE CONSIDERED?
EXAMPLES FOR NANOPARTICLES FROM THE CURRENT LITERATURE:
• “… amounts for a full working lifetime lie within the range of the highest in vitroassay concentrations … for nanoparticles on human, rat, and mouse cell lines.” (2011)
• “… an equivalent pulmonary burden in human to 10 µg in the rat [inhaled over 4 hrs.]would be achieved in roughly 5 years.” (2012)
• “… pulmonary deposition of 10 µg [4 hr. inhalation] is equivalent to the workers exposed at 0.1 mg/m3 for 27 workdays …” (2012)
• “The dose deposited in a person being exposed to the PEL for 20 eight-hr. workdayswould be equivalent to aspiration of a 20 µg SWCNT dose in the mouse.” (2007)
• “A steady-state exposure level of 200 µg/m3 over a number of years … would resultin an allometrically equivalent retained dose in human lungs to the 50 µg aspirateddose used in the mice ….” (2012)
From: Slikker Jr., et al. 2004
Conceptual Depiction of Factors for Considering Dose-dependent Transitions in Determinants of Toxicity
High
more material; d– as dry powder aerosol; liquid aerosolization methods likely require addition of dispersant
From: Morello et al., 2009
Intratracheal Instillation of Particle Suspension, Rat
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
Saline-Control
~ 99 % of lung lavage cells in control rats are Alveolar Macrophages
Rat Lung Lavage 24 hrs post Instillation of200 µg TiO2 Nanoparticles (25 nm) in Saline
Rat Lung Lavage 24 hrs post 4 hour Inhalationof TiO2 Nanoparticles (25 nm), Lung Dose ~200 µg
Rat Lung Lavage 24 hrs post 4 day Inhalationof TiO2 Nanoparticles (25 nm), Lung Dose ~200
µg
Dose-Rate matters as Determinant of Hazard
Toxicity and Adaptation:Identifying a Hazard
0
20
40
60
80Protein
0
2
4
6PMN's
Lung Lavage Parameters of Rats 4 hrs after 15 min. of Exposure to Fresh and Aged PTFE Fumes
(50-70 µg/m3: n=4/group, mean +/- St.Dev.)
% P
MN
Protein
(mg/m
l)
Sham Fresh Aged* p<0.05(ANOVA)
* *
0 0
STUDY DESIGN:ADAPTATION TO PTFE ULTRAFINE PARTICLE INHALATION
— RATS —
Day 1 Day 2 Day 3 Day 4
Group • • • 5 m i n. e x p o s u r e • • • 15-min. exposure
Adapted X X X X
Non-adapted sham sham sham X
Control sham sham sham sham
X = Ultrafine particle exposure: 5 x 105 part./cm3 = 50 µg/m3
0
20
40
60
80
100
PMNProtein
0
2
4
6
8* *
Lavage PMN and Protein Response in PTFE-fumeAdapted and Non-adapted F-344 Rats
Adapt 3 days for 5 min. each day - Day 4 exposure 15 min. @ 5x105 part/cm3
% P
MN P
rotein,mg/m
l
Sham Adapted Non-adapted
* P<0.05(ANOVA)n=5 n=6 (all died within 3 hours)
n=6
In Vitro Toxicity Testing:Challenges for In Vivo Extrapolation
Particle Transport/Deposition Processes for In Vitro Systems
From: Hinderliter et al., 2010
In Vitro Sedimentation, Diffusion and Dosimetry (ISDD) Model (Teeguarden et al.)
Tran
spor
t R
ate
In Vitro Transport Rates for TiO2 Particles from 10-500 nmMedia height = 3.1 mm
In Vitro Sedimentation, Diffusion and Dosimetry (ISDD) Model (Teeguarden et al.)
From: Hinderliter et al., 2010
Ce
ll v
iab
ilit
y (
%)
From: Cohen et al., 2012
0 1 2 3 4 50
25
50
75
100
Mass ( g)
Toxicity as a function of delivered and administered doses:10Ag/SiO2 (dBET = 5.3 nm) in RPMI, t = 24 hours
Administered Mass (MA)
Delivered Mass (MD)
µ
Concept and Impact of NP Coating
kDa
Albumin
Adsorption of Proteins (FBS) Is Inhibited by Surfactant Coating (Pluronic F127) (SDS PAGE)
(Dutta et al., 2007)
Casein Proteins Hemoglobulins Lactoglobulin
SWCNTBET surface 274 m2/g
Silica NP (10 nm)BET surface 212 m2/g
From: 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
Cytotoxicity of 10 nm SiO2 in RAW264.7 Cells (18 hr. incubation) Is Inhibited by Surfactant Treatment (Pluronic F127)
+
Safer by design?
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
Nano – Bio Interactions (Protein Corona Formation)Schematic drawing of the structure of NP-protein complexes in plasma:
NP physicochemical properties: Body compartment media:
protein/lipid adsorption and desorption patterns
biodispersion across barriers and in target tissues/cells
determine
determine
Modified from Müller and Heinemann, 1989
+
“Concept of Differential Adsorption”
— NP corona is the defining property —Cell interacts not with NP surface itself, but with a nano-object
specified by size, shape and structure of its protein corona
Walczyk et al., JACS 2010:
Respiratory TractEpithelial Lining Fluid
Lipids/Proteins
Secondary OrgansBiodistribution/Uptake
BloodPlasma Proteins
2 Time-points:1 and 24 hours
Analysis:NAA or ICP-MS
50 µg Au 15 µg AuLUNG MICROSPRAY –or– IV INJECTION
2 Portals of Entry
3 Coatings:CitrateSerum albuminPEG 5kD, 20kD
3 Sizes:5, 50, 200 nm
Biokinetics of Nanogold to Rats
Risk Assessment of Inhaled Nanoparticles:Concepts of Toxicity Testing
Adverse NP Effect:at portal of entry
and remote organs
ExperimentalAnimals
Humans
BiologicalMonitoring
(markers of exposure)
Occupational/Environmental
Monitoring
Public health/social/ economical/political
consequences
RegulationsExpos. Standards
Prevention/Intervention Measures
Biomed./Engineering
Exposure-Dose-Response Data
In VivoStudies(acute; chronic)
In Vitro Studies(non-cellular)
(animal/human cells)(subcellular distribution)
RiskCalculation
SusceptibilityExtrapolation Models
(high low)(animal human)
MechanisticData
Risk Assessment and Risk Management ParadigmFor Engineered Nanoparticles (NPs)
InhalationIngestion, Dermal
Biokinetics!
Dose-Metric!
Physico-chemicalParameters!
Modified from Oberdörster et al., 2005
Ris
k
Exposure
H a z a r d0 2 0 4 0 6 0 8 0 1 0 0
Risk:ExtremeVery highHighModerateLowVery lowMinimal
Risk = f (hazard; exposure)
in vivoHumansWorkplaceLaboratoryConsumer
in vitroBolus; ALI
Target cells,Tissues
Dose-Response
in vivoAnimals
Biokinetics(translocation;
corona formation)Dose-Response
Phys-chem. Properties Target OrgansRespirability
NOAELs; OELs; HECs
Phys-chem. Properties Endpoints; Ref. Material Hi-Lo Dose; Relevancy
MechanismsReproducibility
Exposure (assessment) Hazard (characterization)
Risk Assessment
Considering Exposure and Hazard for Risk Assessment
Concepts of Nanomaterial Toxicity Testing:
Inhal/Bolus( )
Long-term
In silicomodels
Dose - ResponseExposure – Dose - Response
ENM Risk Assessment Based on 3-Month Rat Inhalation Study
ENM to be evaluated“Positive” and “Negative” reference material (well characterized: phys/chem; biol/tox)
3-month rat multi-conc. inhalation; 3-6 months post-exposuresensitive endpoint: quantitative (BAL); functional (AM clearance); semiquantitative(histopath.); secondary target organ? Biokinetics: lung burden data (do rat data fit PSP kinetics?)
Hazard CharacterizationDose-response relationships by different
dosemetrics: mass; surface; volume; number
Steepest Slope Analysis:Ranking against reference material based on response per unit dose
Hazard greater or lower than reference?
Risk CharacterizationExposure-dose-response relationships
retained mass as metric
BMD analysis to derive subchronic rat BMCL (using established rat T½ )
BMD extrapolation to 2 yrs to derive chronic rat BMCL (using established rat T½ )
Dosimetric extrapolation to derive human BMCL (HEC) (using rat and human MPPD model )
Compare ENM BMCL to reference material BMCL
Comments:Consider modification of approach if:– 3 month study hasNOAEL vs. LOAEL– ENM is soluble– ENM low dose does not fit rat PSP kinetics
Hazard and Risk Chracterization:Case Study: MWCNT
Two Subchronic MWCNT Inhalation Sudies in Rats
Comparing MWCNT results with 5 other subchronic rat inhalation studies:
ultrafine carbon black
nano TiO2
micro TiO 2
cristalline silica
nickel subsulfide
negative
Reference materials
positive
90 - Day Inhalation, Rats: MWCNT, CB, SiO 2, Ni3S2, TiO2
Percent Increase of Lung Weight Above Controls
10 20 30 40 500
50
100
MWCNT (Pauluhn 2010)MWCNT (MaHock et al 2009)Carbon Black (Elder, et al, 2005)Ni3S2
SiO2 (Crist)nano TiO 2
micro TiO 2
(Oberdörster, et al, 1994)
(Oberdörster, unpub.data)
Exposure Concentration, mg/m 3
Lung
wei
ght,
% in
crea
se
As Function of Exposure Concentration
10 20 30 40 500
50
100
Exposure Concentration, mg/m 3
Lung
wei
ght,
% in
crea
se
As Function of Retained Lung Burden (Mass)
0
50
100
1 2 3 4 5 6 7Retained Lung Burden, mg
Lung
wei
ght,
% in
crea
se
As Function of Retained Particle Surface Area
5000 10000 150000
50
100
Retained Particle Surface Area, cm 2
Lung
wei
ght,
% in
crea
se
As Function of Retained Lung Burden (Volume )(based on bulk density)
5,000 10,0000
50
100
0
nano TiO 2 16%
@ 40,000 nl →→→→
Carbon Black 77% @ 32,440 nl →→→→
Retained Particle Volume, nl
Lung
wei
ght,
% in
crea
se
Hazard Ranking of Different (Nano)-Materials Based on Different Metrics and Steepest Slope of Exposure-Dose-Response Relationships
from Subchronic Rat Inhalation Studies (endpoint: lungweight increase)
Three Hazard Groupings:
Low: CB; TiO2 < 0.3 % lungwt. incr./cm2
Medium: MWCNT 0.3 – 1 % lungwt. incr./cm2
High: SiO2; Ni3S2 >1 % lungwt. incr./cm2
From: Oberdörster, 2002
Diffusion
Sedimentation
Impaction
HUMANS
RATS
Multiple Path Particle Dosimetry
model for human and rat
respiratory tract
Courtesy: B. Asgharian
Dosimetric Extrapolation of Inhaled Particles from Rats to Humans
Rat Human
Exposure [mg(m3)-1] Exposure (HEC) [mg(m3)-1]
Inhaled Dose [mg(kg)-1] Inhaled Dose [mg(kg)-1]
Deposited Dose µg(cm2)-1;µg(g)-1
Deposited Dose µg(cm2)-1;µg(g)-1
Retained (Accumulated) Dose[µg(g)-1; µg(cm2)-1]
EffectsAssumption: If retained dose is the same as in rats and humans, then effects will be the same
Breathing
Minute Volume
Tidal Volume, Resp. RateResp. Pause
Particle characteristicsAnatomy
ClearanceRetention
Regional Uptake(Metabolism, T½)