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)