subchronicinhalation toxicity evaluation of silver nanoparticles · 2014-11-11 ·...
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Subchronic Inhalation Toxicity Evaluation of silver
nanoparticles
IL JE YU, PhD, DABT, CIH, CSP, RQAP-GLP, UK-EU Registered Toxicologist
KEMTI
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Backgrounds
• Wide use in silver nano products due to its anti-microbial activity in Korea
• Increasing exposed population
• No definite data on silver nano aerosol inhalation
• Applicability of current occupational exposure levels of silver particles to silver nanoparticles
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Silver nano applications in Korea
• Bedding
• Washer
• Water purification
• Tooth paste
• Shampoo
• Rinse
• Nipple &Nursing bottle
• Fabrics
• Socks
• Deodorant
• Filter
• Kitchen utensils
• Toy
• Mattress
• Humidifier
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Maynard, 2006. Nanotechnology: The Next Big Thing, or Much Ado about Nothing?
Numbers of products associated with specific materials
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90 day inhalation toxicity study
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Silver nanoparticle generation method
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Silver nanoparticle generation
□ Silver nanoparticle generator (ISO 10801)
□ Based on OECD Guideline for Testing of Chemicals No. 413 'Subchronic Inhalation Toxicity: 90-day Study‘
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□ Silver nanoparticle exposure condition
Silver nanoparticlesTest substance
10 male SD rat / dose group10 female SD rat / dose group
Animal
6 hr/day, 5 day/week, 90 daysDuration
High : 3.0±0.2 ×106 particles/cm3
Middle : 1.5±0.2 ×106 particle/cm3
Low : 0.7±0.2 ×106 particle/cm3Dose
InhalationRoute
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Test substance characterization
□ Dose monitoring : Nanoparicle monitoring method (ISO 10808)
□ Nanoparticle distribution (ISO 10808) : diameter, number, surface area, volume : DMAS. CPC
□ Shape, CMD, Purity: TEM-EDX (ISO 10808)
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Evaluation items
□ Body weight□ Food consumption : 1 / week
□ Eye : Opthalmoscope □ Urin : Urisacn strip 10 test, Urine analyzer
□ Necropsy :
□ Organ weight : Brain, thymus, lung, heart, liver, spleen, kidney, adrenal, testis, ovary, olfactory bulb
□ Blood biochemistry
□ Hematology
□ Coagulation test
□ Histopathology
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Lung Function Test
□ Pulmonary toxicity Biomarkers
BAL (bronchoalveolar lavage) cell distribution 평가
: total cell count, macrophage, PMN, lymphocyte 측정
: total protein, albumin 및 LDH
Lung function test : tidal volume, minute volume, frequency, inspiratory time, expiratory time, peak inspiratory flow, peak expiratory flow
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□ Silver nanoparticle distribution
514.78±3.746.61±0.032.85±0.0118.93(0.05)Total
535.06±4.356.78±0.042.85±0.0119.13(0.06)Down
494.50±5.976.43±0.052.86±0.0118.72(0.07)Up
High
133.13±1.052.37±0.011.43±0.0018.33(0.03)Total
132.51±1.492.36±0.021.42±0.0118.35(0.05)Down
133.75±1.482.39±0.021.44±0.0118.30(0.05)Up
Middle
48.94±0.471.08±0.020.66±0.0018.12(0.04)Total
48.53±0.661.08±0.040.66±0.0018.13(0.06)Downb
49.36±0.671.08±0.030.67±0.0018.12(0.05)Upa
Low
0000Control
Mass(μg/m3)
Surface(× 109 nm2/cm3)
Number(×106 particles/cm3)
Diameter†
(nm)SiteGroup
Mean ± S.E
Distribution of silver nano particle
† : GM(GSD)
※ HEPA filter 를 통과한 공기 중 분진 수 : channel 1 – 1.62±0.07 개/min
channel 2 – 0.39±0.03 개/min
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Maintenance of concentrations during 90 day exposure period
Distribution of silver nanoparticles concentrations during90-days
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Particle shape and purity by TEM-EDX
400 nm
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Particle distribution by TEM
Log normal distribution of silver nanoparticles
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Body weight changes
Day
-7 0 7 14 21 28 35 42 49 56 63 70 77 84 91
Wei
ght (
g)
200
300
400
500UnexposedLowMiddleHigh
A
Initiation of exposure
Termination of exposure
Necropsy
Day
-7 0 7 14 21 28 35 42 49 56 63 70 77 84 91
Wei
ght (
g)
150
200
250
300UnexposedLowMiddleHigh
B
Initiation of exposure
Termination of exposure
Necropsy
ab
bb
b bc c b c c
c
Body weight of rats exposed to silver nanoparticles. A : male, B : female(a : p < 0.05, middle vs. other groups, b : p < 0.05, middle vs. low and high groups, c : p < 0.05, middle vs. high group)
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Lung weight change
R
R
0.77 ±0.030.67 ±0.040.70 ±0.010.68 ±0.03
1.10±0.060.94 ±0.040.95 ±0.050.92 ±0.04
0.38 ±0.01
0.49 ±0.02
Control
0.41 ±0.01
0.57 ±0.03
High
0.37 ±0.02
0.50 ±0.02
Low
L
L 0.51 ±0.02Male
0.35 ±0.01Female
Middle
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Histopathology of Liver
□ Liver
Control x100 Bile duct hyperplasia x100 Vacuolization x100
Hepatocytes swelling x200 Siderotic near central vein x200
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Histopathology of Lung
□ Lung
Control x100 Perivasculitis x100 Alveolitis x200
Granulomatous lesions x100 Histiocytosis x400 Inflammation x100
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Bronchoalveolar lavage
Total cell Macrophage PMN Lympocyte
Cel
l No.
(*10
6 )
0.00
0.02
0.40
0.60
0.80
1.00 UnexposedLowMiddleHigh
A B
Total cell Macrophage PMN Lympocyte
Cel
l No.
(*10
6 )
0.00
0.01
0.40
0.60
0.80
1.00 UnexposedLowMiddleHigh
Cell no. of total cell, macrophage, PMN, and lymphocyte of rats exposed to silver nanoparticles, A : male, B : female.
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Bronchoalveolar lavage
A
Albumin (ug/ml) LDH (IU/L) Total protein (ug/ml)0
20
60
80
100 UnexposedLowMiddleHigh
a
B
Albumin (ug/ml) LDH (IU/L) Total protein (ug/ml)0
20
40
60
80
100
120
140 UnexposedLowMiddleHigh
b
c
Values of albumin, LDH and total protein of rats exposed to silver nanoparticles, A : male, B : female. a : Significant different from high vs. low and middle groups, p < 0.05, b : Significant different from high vs. other groups, p < 0.01,c : Significant different from high vs. other groups, p < 0.05
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Lung function test (male)
Minute volume Tidal volume
Day
-7 0 7 14 21 28 35 42 49 56 63 70 77 84 91
Tida
l vol
ume
(mL)
0.15
0.20
0.25
0.30
0.35UnexposedLowMiddle High
a b
c c
e
de f
g
h
h
i
c c
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Lung Function Test (Female)
Minute volume Tidal volume
Day
-7 0 7 14 21 28 35 42 49 56 63 70 77 84 91
Tida
l vol
ume
(mL)
0.12
0.16
0.20
0.24
UnexposedLowMiddle High
a
b
c
d d
f g
e
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Tissue distribution of silver nanoparticles (Male)
0.372630.241.242.150.8622.87S.E.
4.31a14645.42a18.63a30.48c9.49a132.97aMEAN
High:
0.20904.170.951.610.412.88S.E.
1.82b5450.29b7.89b17.103.58b13.75MEAN
Middle :
0.0866.030.730.770.330.98S.E.
0.68613.57†3.456.441.633.52MEAN
Low :
0.020.250.340.380.200.20S.E.
0.090.771.120.510.850.70MEAN
Control :
BloodLungBrainOlfactory bulbKidneyLiverTESTS:
a : p<0.01, high vs. other groups, b : p<0.01, middle vs. control and low group, c : p<0.01, high vs. other groups (dose-dependant),
† : p<0.05, male vs. female in low group. Unit ng/g of wet tissue
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S.E.
MEAN
High
S.E.
MEAN
Middle
S.E.
MEAN
Low
S.E.
MEAN
Control
TESTS:
0.601880.312.412.747.0424.50
6.86a,‡20585.63a19.97a32.84c37.66a,†71.08a
0.22641.101.191.324.272.50
2.10b4241.17b10.22b13.7511.8112.07
0.1475.500.460.750.571.40
0.85295.924.097.432.614.55
0.010.100.260.740.180.31
0.051.010.662.260.940.90
BloodLungBrainOlfactory bulbKidneyLiver
Female
a : p<0.01, high vs. other groups, b : p<0.01, middle vs. control and low group, c : p<0.01, high vs. other groups (dose-dependant),
† : p<0.05, female vs. male in high group, ‡ : p<0.01, female vs. male in high group. Unit ng/g of wet tissue
Tissue distribution of silver nanoparticles (Female)
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Applicability of existing OECD Test guidelines to Nanoparticle toxicity testing
OK19-25°C, 30-70%Temperature, Humidity
Particle number, Surface area, Mass, Composition, CMD etc.
Gravimetric and chemical analysis
Characterization of test atmosphere
OKWithin ±20% of the mean concentration
Concentration
need to be improvedParticle size distribution(1 time/concentration level/week)
Monitoring of exposure condition
NO Mass median aerodynamic diameter (1-4μm)Geometric standard deviation (1.5-3.0)
Particle size
OK1 control + 3doseGroup
OK6hrs/day/5-7day/weekExposure time
OKLess than 5% of test chamberAnimal volume
OK12-15hrs, 19-24%Air change/hr, Oxygen
OKHead/nose-only or whole bodyMode of exposure
Applicability to nanoparticleOECD TG 412/413Items
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Sex difference in tissue distribution of silver nanoparticle (kidney) (ng/g of wet tissue)
F
M
F
M
69.32±22.3416.77±2.812.82±0.850.02 ±0.0
24.32±10.635.98±2.361.3±0.590.02 ±0.01Oral
11.81 ±4.27
3.58 ± 0.41
Middle
37.66 ±7.04
9.49 ± 0.86
High
0.94 ±0.18
0.85± 0.20
Control
1.63± 0.33
Inh2.61 ±0.57
LowSex
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Particle deposition in the lungs
0
0.2
0.4
0.6
0.8
1
0.01 0.1 1 10
Dep
ositi
on F
ract
ion
Particle diameter / µm
Alveolar DepositionTracheobronchial Deposition
Head depositionTotal
Source: Multiple Pathway Deposition Model (MDEP), CIIT
Modeled lung deposition. Mouth and nose breathing, person at rest.
Permission from Maynard
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LFT decrease
Day
-7 0 7 14 21 28 35 42 49 56 63 70 77 84 91
Tida
l vol
ume
(mL)
0.15
0.20
0.25
0.30
0.35UnexposedLowMiddle High
a b
c c
e
de f
g
h
h
i
cc
Welding fume Silver nanoparticle
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Deposit dose
36 m312 m3Inh. Vol/d
117 mg (L), 311 mg (M), 1205 mg (H)35100 mg (L),
58230 mg (H)
Total dose
80%30%Deposition fraction
~20%
10530 mg (L)
17469 mg (H)
540 m3 (60 d),
65 mg/m3, 108 mg/m3
WF (100 nm)
~20%LFT decrease
94 mg (L), 275 mg (M), 1141 mg (H)Deposit dose
2340 m3 (90 d), Total inh volume
0.049 (L), 0.133 (M), 0.515 mg/m3 (H)Exp. dose
Ag nano (20 nm)
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Target organs for silver nanoparticles and NOAEL
133Mass (μg/m3)
2.37×109Surface area (nm2/cm3)
1.43×106Number (particle/cm3)
rat
Target organs: Liver and Lung
NOAEL
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Acceptable concentration
85163Mass (μg/m3)
1.5×1092.9×109Surface area (nm2/cm3)
9.1×1051.75×106Number (particle/cm3)
General populationWorkers
※ ACGIH or MOL Silver TLV : 100 ㎍/m3 (Argyria),
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Nano specific inhalation toxicity study
BAL, Coagulation, etcEvaluation
Mass, number, surface area, Dosimetry
Nano specific pathology (TEM, Raman, Confocal),
Pathology
Target organ determination, toxicokinetics, durability, clearance
Tissue distribution
Shape, diameter, purity, dispersion, surface chemistry, hygroscopicity, aggregation/agglomerization, purity
Particle characterization
Obstructive disease, restrictive diseaseLung function test
Nano specific particle generation
Dispersed vs aggregated, monodispersed vspolydispersed
Particle generation
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Conclusions
• Collaboration between toxicologist and engineer is strongly recommended to study the health and environmental effects of nanoparticle exposure.
• New dosimetry is required for inhalation toxicity testing.
• Nano-specific parameters are required for nanotoxicity evaluation.