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Jürgen Pauluhn Germany
A weight-of-evidence approach for setting OELs for poorly soluble, low-toxicity nano-particles
Days
0 20 40 60 80 100 120 140 160 180
Lu
ng
Do
se
0
50
100
150
200
250
non-overload
overload
exposure post-exposure
Washington, DC – Sept. 10, 2012
Outline
2
Principal Mode of Action of low-toxicity PSPs
Pharmacokinetic model to interrelate the cumulative
lung dose with associated chronic changes
Verification of kinetic modelling by retrospective
analysis of inhalation studies with fine and ultrafine
PSPs
The kinetic cornerstones of low-toxicity PSPs must be
observed for designing long-term inhalation studies
Derivation of Occupational Exposure Levels (OELs/
DNELs) based on a unifying rat-human overload-
threshold concept
Summary and conclusions
Outline
Chronic Exposure Paradigms require focus on the Chronic Mechanisms of inhaled low-toxicity PSPs
The metric of dose depends on the chronic Mode of Action:
– Instant effect at the initial site of deposition.
– Transient surfactant dysfunction, coating and opsonation of PSPs.
– Phagocytosis of PSPs by alveolar macrophages (AM).
– Chemotactic stimuli for AMs and PMNs to migrate into the alveoli.
– Any increased pool of AMs increases the lung burden and residence time of retained PSPs.
– OELs are designed for preventing overload-like effects to occur. Study Day
0 20 40 60 80 100 120
Lung B
urd
en [
l P
Mre
sp/m
³]
0
10
20
30
40
50
60
Lung E
ffects
[A
.U]
MoAI + MoAII
MoAI
lung burden
Epithelial lining fluid
Type I pneumocyte
MoAII
500nm
MoAI Nociception
Blood capillary
3
Interstitial clearance (0-order) may contribute to total lung burden but necessarily not alveolar burden.
Study Day
0 10 20 30 40 50 60
Lu
ng B
urd
en
[m
g S
ub
sta
nce
resp/k
gra
t]
0
20
40
60
80
100
Putative Mechanisms of inhaled low-toxicity Nanoparticles
Type II Cell activation
Dissolution/ bioavailability
Substance- specific
Retention-related indirect mode of action (cumulative, chronic)
• Generic & particle-specific System variables: • Size of AM • Total number of AM • Retained volume dose per AM
Direct mode of action (acute)
time
MoAI
4
Surfactant dysfunction
MoAII
Cx t [mg/m³ x days]
0 500 1000 1500 2000 2500 3000 3500
TC
C [
#10
6/L
ung]
0
20
40
60
80
100
Particle exposure groups
Air control groups
5
The Principal chronic Mode of Action and Lung-Overload are interrelated
• Low-toxicity PSPs do not show any (subcompartmental) solubility.
• Increasing cumulative lung burdens cause particle-displacement volume-dependent increase in total lung cells (Vd).
• Prevailing experimental evidence suggests that the particle volume is a lead metric to understand and model the AM-mediated kinetics of PSPs.
• Kinetic hallmarks can differentiate adaptive from overload effects as well as particle-specific effects.
CL
Vt d )2ln(2/1
Repeated Exposure
Vd
Vd
or
Particle Translocation to the LALNs occurs secondary to Overload-dependent Inflammation
Study Day
0 50 100 150 200
LA
LN
Part
icle
Burd
en [
g F
e3O
4/O
rgan]
0
200
400
600
800
1000
1200
control
10 mg/m³
20 mg/m³
50 mg/m³
100 mg/m³
* *
*
**
**
**
**
PMN (% based)
Study Day
0 50 100 150 200
Re
lative
to
Co
ntr
ol [%
]
0
2000
4000
6000
8000
10000
12000
control
10 mg/m³
20 mg/m³
50 mg/m³
100 mg/m³
PMN (absolute)
Study Day
0 50 100 150 200 250
Re
lative
to
Co
ntr
ol [%
]
0
10000
20000
30000
40000
50000
control
10 mg/m³
20 mg/m³
50 mg/m³
100 mg/m³
**
*
**
*
Baytubes (r: 0.2 g/cm³)
13-wks+26 wks postexposure
Magnetite (r: 4.6 g/cm³) 4-wks+26 wks postexposure
As long as the overload-threshold is not exceeded, the kinetics of PSPs appears to be predominated by the AM-mediated clearance.
Lung Associated Lymph Node Burden
Study Day
0 50 100 150 200 250 300
LA
LN
Bu
rde
n [
ng C
o/O
rga
n]
0
20
40
60
80
100
120
140
0.1 mg/m³
0.4 mg/m³
1.5 mg/m³
6 mg/m³
Study Week
8 13 17 26 39
PM
N C
ou
nt
(x 1
04 c
ells
/lu
ng)
0
1000
2000
3000
4000
5000
**
**
**
**
**
******
**
****
*
*
*
6
Volumetric Capacity of Alveolar Macrophages and Overload-Threshold (MoAII)
dosepulmonarycumulativekgPMl
mAMthresholdOverload
ratresp
v ratkg
/2.4
1042.0100
6107 31010
%6
• Total AMvol
• Respirability • Inhaled volume • Time to attain overload threshold
Human-specific
Rat-specific
Parameterization of Vd:
*
*: empirically confirmed in the current 13-week rat inhalation study
*
Generic NOAELhuman
Average AM Volume ( m³)
Average AM Counts in Lung
AM Volume per Lung ( m³/kg bw)
Rat (kg - based) 1166 6 x 10 7 7 x 10 10
Human (70 kg) 4990 9 5 x 10 11
7.0 x 10
Volume-load of AM-pool 6%: no change in pool-size (= kinetic NOAEL)
Generic NOAELrat
7
³54.0
³2
3
19.0
069.0
³19.0
3
229.0
/2.4
m
PMl
kgm
kgPMldoseDaily
kg
m
kgPMl
resp
dayrat
dayratpulm
lung
dayrat
ratpulm
The kinetic threshold of PM-volume-dependent lung overload is the theoretical volumetric NOAEL for any poorly soluble, granular low-toxicity particulate
Volumetric Capacity of Alveolar Macrophages and Overload-Threshold (MoAII)
Cumulative alveolar threshold volume dose:
Alveolar ventilation:
Chronic inhalation exposure concentration (6 h/day, 5-times/wk):
Constrains: AM = f(C x t); ØAM = constant
8
Cumulative Threshold Dose: The Transition from Homeostasis to Overload (increased Vd)
³29.0
1
³29.05.4
2.4
]/³[]/[][5.4
2.42.42.4
mf
l
mf
l
daymVlunggdayflungg
l
lung
l
ratkg
l
cum
PM
cum
PM
Lratkgcumratkg
PM
ratkg
PMPM
]³
[100
³29.0
1
m
mg
Deposl
mg
mf
lNOAEL
pulmonaryPMcum
PMpred
r
Daily exposure dose to attain the overload threshold:
9
Starting point for dose selection for repeated inhalation exposure studies
PM: respirable fraction of particulate matter likely to be deposited in the pulmonary region
Estimation of generic No-observed-adverse Effect Levels (NOAELs) for any PSP and Study Type
Study Day
0 100 200 300 400 500
Lu
ng
Bu
rde
n [
l P
Mre
sp/k
gra
t]
0
1
2
3
4
5
0.86 l/day - 5 days
0.24 l/day - 4 week
0.105 l/day - 13-weeks
0.069 l/day - 2 yr
Lung overload threshold
5.1724.0
2.4
40105.0
2.4
61069.0
2.4
4
13
104
wkscum
wekscum
wkscum
f
f
f
Not necessarily applicable to 1-wk studies since MoAI
-related acute effects may confound retention-related
chronic effects.
Study Day
0 200 400 600 800
Lu
ng
Bu
rde
n [
l P
Mre
sp/k
gra
t]
0
1
2
3
4
5
0.069 l/m³ - 2 yr (100% steady-state)
0.069 l/m³ - 13 weeks (67% steady-state)
0.069 l/m³ - 4 weeks (29% steady-state)
0.069 l/m³ - 5 days (8% steady-state)
Lung overload threshold
Daily dose to attain threshold Dose-Timeadj
1.5
2.3
3.4
X3.4
X3.4
10
Cumulative Volume Concentration [ l PMresp
/m³]
1 10 100 1000
PM
N C
ou
nt
[x 1
04 c
ells
/lu
ng]
0
1000
2000
3000
4000
5000
Volume Dose [ l/lung]
1 10 100
AlOOH-40 nm (4 wks)
AlOOH-10 nm (4 wks)
Fe2O3 (4-wks)
MWCNT (13 wks)
Fe2O3 (13 wks)
Prediction based on overload threshold
Cumulative Volume Concentration [ l PMresp
/m³]
0.1 1 10 100 1000
Re
ten
tio
n H
alf-t
ime
[d
ays]
0
200
400
600
800
1000
Volume Dose [ l PM/lung]
0.1 1 10 100
Fe3O4-13 wks
Fe3O4-2 wks
Carbon Black-13 wks
uTiO2-13 wks
AlOOH-40nm-4wks
AlOOH-10nm-4wks
MWCNT-13 wks
pTiO2-13 wks
Overload-threshold
y=64.2+797.6*(1-exp(-0.008*x))
1. Kinetic threshold (homeostasis) precedes the effect threshold (inflammation, PMN)
2. Any elimination t½ > 1 year due to kinetic overload does deliver relevant information for hazard identification
Cumulative Volume Dose & Elimination t1/2
Cumulative Volume Dose & BAL-Neutrophils
The kinetics of adaptive changes in the Vd and increased PMNs are interrelated
BAL-PMN [%] - Predicted
0 5 10 15 20 25 30
BA
L-P
MN
[%
] -
Observ
ed
0
10
20
30
40
BAL-PMN 4-week study
BAL-PMN 13-week study
PMN 3.7% at 5 l PM/m³ x days (overload threshold)
11
BAL-TCC
Cumulative Retained Particle Volume [l PMresp
/m³ x days]
0 10 20 30 40 50 60 70
BA
L-C
ell
Co
un
t [x
10
6]
0
20
40
60
80
100
4-week (males)
13-week (males and females combined)
BMCL (95%): 10.1 l PMresp
/m³ x days = 11.6 mg/m³
12
Fe3O4
The cumulative Lung Dose, not duration, deter-mines the terminal t1/2 and the Study Outcome
C x t [mg/m³ x days]
0 1000 2000 3000 4000 5000 6000
Ha
lf-t
ime
t1/2 [
days]
50
100
150
200
250
300
350
400
1-week (simulated)
4-weeks (simulated)
4-weeks (empirical)
13-weeks (simulated)
BAL-PMN
Cumulative Retained Particle Volume [l PMresp
/m³ x days]
0 10 20 30 40 50 60 70
Cyto
dife
ren
tia
ls [
%]
0
10
20
30
40
50
4-week (males rats)
13-week (males and females combined)
PMN 3.7% at 5 l PM/m³ x days (overload threshold)
BMCL (95%): 4.5 l PMresp
/m³ x days = 5.2 mg/m³
1. C x t determines the cumulative lung burden, t1/2, and the associated intensity of effect.
2. Time-related aggravations of effect did not occur.
(Fe3O4)
(Fe3O4)
(Fe3O4)
13 weeks
Concentration [mg/m³]
0 0.1 0.4 1.5 6
Cyto
diffe
ren
tia
ls [
x1
04 c
ells/lu
ng
]
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
Macrophages
non classifiable
PMN
Lymphocytes
foamy cells
39 weeks
Concentration [mg/m³]
0 0.1 0.4 1.5 6
Cyto
diffe
ren
tials
[x10
4 c
ells
/lun
g]
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
Macrophages
non classifiable
PMN
Lymphocytes
foamy cells
MWCNT
Study Day
0 50 100 150 200 250 300
Lu
ng B
urd
en [
l P
Mre
sp/m
³]
0
50
100
150
200
250
300
350
0.1 mg MWCNT/m³ - terminal t1/2
: 87 days
0.4 mg MWCNT/m³ - terminal t:1/2
: 175 days
1.5 mg MWCNT/m³ - terminal t:1/2
: 428 days
6 mg MWCNT/m³ - terminal t:1/2
: 788 days
Overload-Threshold
postexposure period
Proof-of-Principle for low Density PSPs 13-Week Rat Baytubes Inhalation Study (Pauluhn, 2010)
³2.0
8
100
40
2.0
³29.0
1)(
m
mg
m
lELANO predicted
NOAELB-BAL-PMN%: 0.16 mg/m³ (BMDL)
13
Proof-of-Principle for high Density PSPs 13-Week Rat Magnetite Inhalation Study (Pauluhn, 2012)
Concentration [mg/m³]
0 5 15 50
Cyto
diffe
ren
tia
ls [
%]
0
20
40
60
80
100
120
Macrophages
non classifiable
PMN
Lymphocytes
foamy cells
Concentration [mg/m³]
0 5 15 50
Cyto
diffe
ren
tia
ls [
x1
04 c
ells
/lu
ng]
0
1000
2000
3000
4000
5000
6000
7000
Macrophages
non classifiable
PMN
Lymphocytes
foamy cells
Study Day
0 50 100 150 200 250 300
Lu
ng
Bu
rde
n [
l F
e3O
4-R
esp
/m³]
0
20
40
60
80
5 mg/m³ - t1/2 = 94 days
15 mg/m³ - t1/2 = 177 days
50 mg/m³ - t1/2 = 392 days
Overload threshold
³5
8
100
40
6.4
³29.0
1)(
m
mg
m
lELANO predicted
BAL-Total Cell Count measurements most sensitive to detect changes in Vd (t1/2)
15
Proof-of-Principle for unit Density PSPs 2 Year Toner Rat Inhalation Study (Muhle, 1991)
Toner
Study Day
0 200 400 600 800 1000
Lung B
urd
en [
l P
Mre
sp/m
³]
0
50
100
150
200
250
300
1 mg/m³ - terminal t1/2
: 82 days
4 mg/m³ - terminal t1/2
: 209 days
16 mg/m³ - terminal t1/2
: 680 days
Overload-Threshold where: r = 1.2 g/cm³ (toner) fvi = 61 PMresp = 5.3%
³3.1
3.5
100
61
2.1
³29.0
1)(
m
mg
m
lELANO
ipredicted
³0.1)(
m
mgELANO
iobserved Based on BAL_PMN
Hypothesis verified
Empirical t1/2 at 1, 4 und 16 mg/m³: 81 (75-88), 173 (135-241) and 568 (306-1000 days)
]³
[100
³29.0
1
m
mg
Deposl
mg
mf
lNOAEL
pulmonaryPMcum
PM
r
15
16
Repeated Exposure Inhalation Studies: Compar-ison of Predicted and empirical NOAELs
MW
CNT
Toner C
B
Fe3O4
Fe3O4
Concentr
ation [
mg/m
³]
0
2
4
6
8
10
12
NO(A)ELobserved
NO(A)ELpredicted
13 wks
13 wks
13 wks
4 wks
2 yrr=0.2
r=1.2 r=1.8
r=4.6
]³
[100
³29.0
1
m
mg
Deposl
mg
mf
lNOAEL
pulmonaryPMcum
PM
r
r=4.6
17
Study Day
0 100 200 300 400
Lung B
urd
en [
l P
SP
-Resp/m
³]
0
20
40
60
80
0.11 l/m³ - t1/2 =96 days
0.33 l/m³ - t1/2 = 167 days
1.1 l/m³ - t1/2 = 384 days
Lung overload threshold
Study Day
0 200 400 600 800 1000
Lung
Burd
en [
l P
SP
-Re
sp
/m³]
0
50
100
150
200
250
300
0.07 l/m³ - t1/2 = 113 days
0.21 l/m³ - t1/2 = 324 days
0.7 l/m³ - t1/2 = 754 days
Lung overload threshold
Study Day
0 200 400 600 800 1000
Lung
Burd
en [
l P
SP
-Re
sp
/m³]
0
20
40
60
80
100
0.07 l/m³ - t1/2 = 113 days
0.14 l/m³ - t1/2 = 210 days
0.28 l/m³ - t1/2 = 428 days
Lung overload threshold
Study Day
0 50 100 150 200 250 300
Lung
Burd
en [
l P
SP
-Re
sp
/m³]
0
10
20
30
40
50
60
0.26 l/m³ - t1/2 = 94 days
0.78 l/m³ - t1/2 = 152 days
2.6 l/m³ - t1/2 = 327 days
Lung overload threshold
Long-term Inhalation Studies with PSPs should not exceed the MTD (= t1/2 >1 year)
Kinetic Modeling-based Study Design for Inhalation Testing of low-toxicity (Nano)Particles
4-wk Study 13-wk Study
2-yr Study (3x) 2-yr Study (2x)
Targeted spacing: x 10 Actual spacing: x 50
Targeted spacing: x 2 Actual spacing: x 15
Concentration [ul PSPrespirable
/m³]
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Elim
ina
tio
n H
alf-t
ime
[d
ays]
0
100
200
300
400
500
600
700
800
4-week - 3x
13-wk - 3x
2-yr - 3x
2yr - 2x
Concentration [ul PSPrespirable
/m³]
0.0 0.5 1.0 1.5 2.0 2.5 3.0
PM
N in B
ALF
[%
]
0
10
20
30
40
50
4-week - 3x
13-wk - 3x
2-yr - 3x
2yr - 2x
Kinetic Modeling-based Study Design for Inhalation Testing of low-toxicity (Nano)Particles
There is an apparent dependence of BAL-PMN on TCC (Vd) and the cumulative volumetric particle lung burden. In the absence of any increased TCC, BAL-PMN% <4% appear to be toxicologically insignificant.
MTD
Basis: Vd-dependence of BAL-PMNs
18
Volumetric Capacity of Alveolar Macrophages and Overload-Threshold (MoAII) in Human Workers
dosepulmonarycumulativekgPMl
mAMthresholdOverload
humanpulm
v humankg
/30
103100
6105 31011
%6
• Respirability • Inhaled volume
Human-specific
Parameterization of Vd:
Generic NOAELhuman
*
*: empirically confirmed in the current 13-week rat inhalation study
*
Average AM Volume ( m³)
Average AM Counts in Lung
AM Volume per Lung ( m³/kg bw)
Rat (kg - based) 1166 6 x 10 7 7 x 10 10
Human (70 kg) 4990 9 5 x 10 11
7.0 x 10
Volume-load of AM-pool 6%: no change in pool-size (= kinetic NOAEL)
19
CL
Vt d )2ln(2/1
• t1/2 should be 7-times higher due to 7-times higher Vd which means t1/2 = 420 days
Volumetric Capacity of Alveolar Macrophages and Overload-Threshold (MoAII) in Workers
When accounting for the species-specific differences in the kinetic hallmarks of PSP (AM-mediated clearance only), the cumulatively retained pulmonary dose for attaining the overload-threshold is similar in rats and humans. Hence, following adjustment for ventilation and respirability, the paradigm devised for rats are also applicable to humans.
20
Estimation of Human Equivalent Lung Dose – Rat- or Human-based (MWCNT)
92.0492.0
452.0
3.0
70
164.0
075.0
10
086.0/
A
H
HaHE
AaAEHAlungburden
BW
BW
FV
FVAF
94.01063
1059
3.0
70
60
400
105.3
101.2
2ln
2ln13
13
13
10
2/1
2/1/
A
H
AHd
HAdHAclearance
BW
BW
tV
tVAF
Ventilation x Deposition:
Clearance:
Parameterization of Vd:
per rat = 2.1 x 1010 (300 g)
per human = 3.5 x 1013
Ventilation rat (0.8 L/kg-min) = 240 ml/min (300 g rat) x 360 min = 0.086 m³/day Ventilation human: 10 m³/day
21
*
*: empirically confirmed in the current 13-week rat inhalation study
*
Average AM Volume ( m³)
Average AM Counts in Lung
AM Volume per Lung ( m³/kg bw)
Rat (kg - based) 1166 6 x 10 7 7 x 10 10
Human (70 kg) 4990 9 5 x 10 11
7.0 x 10
Study Day
0 100 200 300 400 500
Lu
ng
Bu
rde
n [
l P
Mre
sp/k
gra
t]
0
1
2
3
4
5
0.86 l/day - 5 days
0.24 l/day - 4 week
0.105 l/day - 13-weeks
0.069 l/day - 2 yr
Lung overload threshold
Derivation of OEL (MWCNT): Empirical Approach
ionStudyDuratHAclearance
HAburdenlung
AAFAF
AFELANOOEL
1)(
/
/
)(³
1.05.194.0
92.0
³16.0 Baytubes
m
mg
m
mgOEL BMDL
yearsdaysDurationStudyAF 2905.1069.0
105.0
(chronic exposure)
22
³1.02.0
94.0
92.0
³5.0
m
mg
l
mg
m
lOELgeneric
Which means the theoretical mechanism-based approach verifies the empirical approach and vice versa. The NOAEL depends on kinetic factors (dosimetry) and not yet unknown Mode of Actions.
Generic threshold rat
6h/day-5-times/week
Rat-to-human differences
in lung dosimetry-kinetics-
exposure duration/day.
Assumption: aerosol size in
inhalation chamber = workplace
Density of agglome-
rates at workplace
(chronic exposure)
OEL Calculation for MWCNT (Baytubes):
Theoretical Approach
eagglomerat
HAclearance
HAburdenlungrespirable
genericAF
AF
m
PMlOEL
1
³
5.0
/
/
23
Study Day
0 100 200 300 400 500
Lu
ng
Bu
rde
n [
l P
Mre
sp/k
gra
t]
0
1
2
3
4
5
0.86 l/day - 5 days
0.24 l/day - 4 week
0.105 l/day - 13-weeks
0.069 l/day - 2 yr
Lung overload threshold
24
ionStudyDuratHAclearance
HAburdenlung
AAFAF
AFELANOOEL
1)(
/
/
)(³
4
069.0
105.094.0
92.0
³6.5 43OFe
m
mg
m
mgOEL BMDL
(chronic exposure based
on 13 wk study & prediction)
OEL Calculation for Fe3O4 :
Empirical Approach
)(³
4
069.0
24.094.0
92.0
³1.15 43OFe
m
mg
m
mgOEL BMDL
(chronic exposure based
on 4 wk study & prediction)
Summary: Computational Design of Repeated Exposure Inhalation Studies
MWCNT
Study Day
0 50 100 150 200 250 300
Lung B
urd
en [
l P
Mre
sp/m
³]
0
50
100
150
200
250
300
350
0.1 mg MWCNT/m³ - terminal t1/2
: 87 days
0.4 mg MWCNT/m³ - terminal t:1/2
: 175 days
1.5 mg MWCNT/m³ - terminal t:1/2
: 428 days
6 mg MWCNT/m³ - terminal t:1/2
: 788 days
Overload-Threshold
postexposure period
• Anticipation of threshold Cxt for overload (= no-observed-adverse effect concentration, NOAEL).
• NOAEL, LOAEL and MTD can be rationalized based on the dynamic overload- dependent increased Vd and associated t1/2.
• Appropriate postexposure period can be selected to verify simulated clearance and reversibility for particles being ‘inactivated’ (MoAI 0).
• Principal hypothesis: the effect follows the cumulative dose and ‘acute-on-chronic effects’ should be negligible.
]³
[100
³29.0
1
m
mg
Deposl
mg
mf
lNOAEL
pulmonaryPMcum
PM
r
25
• Inhalation studies should be designed to meet the kinetic cornerstones of generic PSPs and observing the kinetic MTD (t1/2 > 1 year).
• The metric of dose must be defined by the mechanism of target organ injury which is the volumetric overload of alveolar macrophages.
• Therefore, the most important metrics for the OEL-derivation of Poorly Soluble Particle-like structures is the agglomerate/aggregate volume.
• Adequately designed and executed 4-week inhalation studies can reliably predict the chronic OELs of low-toxicity PSPs in the absence of overriding lung-overload-related confounders.
eagglomerat
HAclearance
HAburdenlungrespirable
genericAF
AF
m
PMlOEL
1
³
5.0
/
/
]³
[100
³29.0
1
m
mg
Deposl
mg
mf
lNOAEL
pulmonaryPMcum
PM
r
Conclusion The Effect-Threshold is determined by the threshold of increased Vd. This
threshold appears to apply to all low-toxicity PSPs for both rats and humans.
26
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