Post on 06-Feb-2018
A common European approach to the regulatory testing of nanomaterials
Demonstration of a probe-sonicator calibration protocol for harmonizaton of batch dispersions used for
toxicological testing
A common European approachto the regulatory testing of nanomaterials
Keld Alstrup Jensen (NRCWE)
Y. Kembouche (NFA), M. Correia (DTU), A. Booth (SINTEF), Støen Lisbeth (SINTEF), C.R. Castro (UDL), D. Gonzales (LEITAT) J. Mejia (UNAMUR), S. Chevillard (CEA-LCE), K. Bastide (CEA ) M. Iafisco (ISTEC CNR)
P. Jalili (ANSES), B.S. Krause (BfR), K. Park (DWU), S. Sabelle (IIT), D. Cavallo (ISS INAIL), G. Lacroix (INERIS), A. Ribeiro (INMETRO), H. Louro (INSA), F. Barone (ISS),S. Mukherjee (KI), J Kim (KRISS), D Gonzales (LEITAT), K.
Robinson Kenny (NPL); S. Zienolddiny (STAMI), C. Cerrillo (TEKNIKER), C. Moneyrac (UCO),A. Sauter (UIB), J. Choi (UOS), M.van der Zande (WUR), and K. Löschner (DTU)
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A common European approach to the regulatory testing of nanomaterials
Variability is observed in (eco-)toxicological test results Material variability?
Exposure characteristics (agglomerates vs dispersed)?
Biological variability?
Variability in medium characteristics?
Variability in the assay – scales ?
Variability in the analytical methods?
Differences in preparation methods
Testing the test: Can we do it better?
The problem
Bilbao, June 9, 2016NANoREG general pressentation2
A common European approach to the regulatory testing of nanomaterials
Examples of difference due to test item preparation procedure
Bilbao, June 9, 2016NANoREG general pressentation3
Zincite
NM-110 (powder) ZnO in PBSZnO in Nanopure water
A)
A common European approach to the regulatory testing of nanomaterials
Dispersion protocol for in vitro studies
The NANOGENOTOX generic probe-sonicator dispersion protocol
Pre-wetting in 50 µL Ethanol
Dispersion medium 0.05% (v/v) BSA-water (standard 6 mL at 2.56 mg/mL)
Probe-sonicate with 13 mm probe for 16 min at 7.35 Watt (in cooled ice-water bath)
Characterize dispersion by DLS (and microscopy)
NANoREG general pressentation4
How can we reach comparability in exposure and test results in many different lab’s (20+)?
A common European approach to the regulatory testing of nanomaterials
The conceptual approach
Bilbao, June 9, 2016NANoREG general pressentation5
Probe-sonicator calibration protocol
In vivo In vitro ecotoxicology
Batch dispersion protocols
Harmonize de-agglemeration energies/efficiencies
Harmonize Initial Exposure Characteristics (per protocol)
Exposure characterization methods and protocols
Harmonized reporting to enable comparative analysis
Interpretation, interpolation, extrapolation, read-across ….
DLS
A common European approach to the regulatory testing of nanomaterials
Calibration of probe-sonicators
NANoREG general pressentation6
Requirements for implementationEasy-to-use and not very time-consuming
Reliable in the sense of giving a trustworthy result and being repeatableApply tools that are available in “all” nanotoxicology laboratories
Jensen et al. Final protocol for producing suitable MN exposure media. (June 2011). www.nanogenotox.eu
Taurozzi et al. Preparation of nanoparticle dispersions from powdered material using ultrasonic disruption. National Institute of
Standards and Technology. 2012.
Performance-calibration using benchmark material Calorimetric calibration of acoustic delivered energy
A common European approach to the regulatory testing of nanomaterials
The calibration protocol
1) Identifying the amplitude/duration to reach the acoustic delivered target energy
2) Verification of dispersion state using the DLS Zeta-average size of NM-200 (SAS) as benchmark material (NANOGENOTOX SOP)
Zave,mean = 210 to 270 nm and PDImean < 0.46 (n = 10 x 3)
3) Adjustment (if needed) of amplitude and/or duration to reach the target range of NM-200
NANoREG general pressentation7
min1605.035.7 pac MCt
TWattP
A common European approach to the regulatory testing of nanomaterials
Determination of the acoustic delivered energy
NANoREG general pressentation8
Bransonic 10% = 0.0036x + 22.248
R2 = 0.9941
Bransonic 15% = 0.0051x + 22.441
R2 = 0.9994Bransonic 20% = 0.0073x + 22.533
R2 = 0.9984
Sartorius; Ampl 30µm = 0.0041x + 21.086
R2 = 0.9892
Sartorius; Ampl 60µm = 0.006x + 20.948
R2 = 0.9966
Sartorius; Ampl 15µm = 0.0024x + 21.711
R2 = 0.9601
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0 100 200 300 400 500 600
Sonication Time [sec]
Tem
pe
ratu
re o
f so
no
cate
d m
ed
ium
[d
egr
ee
C]
Branson % Amplitude = 1.3026x + 0.4295
R2 = 1
Sartorius µm Ampl = 5.692x - 14.183
R2 = 0.9864
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14 16
Delivered Acoustic Power (W)
Am
plit
ud
e (
% o
r µ
m)
4.3°C above room temperature 6.3°C below room temperature
pac MCt
TWattP
)3(
n
t
T
0.5 L MilliQ-filtered water600 mL beaker
Thermometer ± 0.1CScale to determine (M)
Use the regression curve for Amplitude vs. Pac to
determine the amplitude setting to reach 7.35Watt
Adjust time of sonication if Pac cannot be reached
A common European approach to the regulatory testing of nanomaterials
Reaching the DLS performance criteria
NANoREG general pressentation9
NM200
0
2
4
6
8
10
12
1 10 100 1000 10000
dH [nm]
Inte
nsit
y [
%]
n=100 Z(ave) PDI
NM200 238 0.40
sigma 14 0.06
Use identified sonicator amplitude and duration to
prepare NM-200 batch dispersions according to
the NANOGENOTOX dispersion protocol (n=3)
Measure the hydrodynamic diameter of
the NM-200 batch dispersion (n=10 x 3)
If not within range (210 –270 nm; PDI 0.46), adjust duration (or amplitude) to
reach target values
Repeat preparation of batch dispersions
The “mother data” from NRCWE
A common European approach to the regulatory testing of nanomaterials
Interlaboratory performance testing
Bilbao, June 9, 20161010
NRCWE
SINTEF
CNR
UdL
LEITAT
IK4-Tekniker
NMBU
IIT
INAIL
INERIS
NPL
STAMI
UIB
INSA
ISS
INMETRO
KI
WUR
UNITO
KRISS
DWE Korea
UAB
ANSES
UNamur
CEA
UIB
UNITO
DTU-Food* (NANODEFINE)
A common European approach to the regulatory testing of nanomaterials
Many different sonicators
NANoREG general pressentation11
Partner number Probe sonicator Watt KHz probe diameter1a 400 Watt Branson Sonifier S-450D 400 20 131b 400 Watt Branson Sonifier S-450D 400 20 132 400 Watt Branson Sonifier S-450D 400 20 13
3 Bandelin, SONOPULS HD 2200 200 20 9
4 Heat Systems Sonicator Ultrasonic Processor XL (Pulsar or Continuous) 100 20 65b P1 Microson XL 2000, Qsonica, LLC 100 22.5 3.25c P2 Microson XL 2000, Qsonica, LLC 100 22.5 4.85a P4 Microson XL 2000, Qsonica, LLC 100 22.5 6.4
6 Sonics VC750 750 20 137 Heat System Misomix XL2020 550 20 138 QSONICA Q700 (with probe) 100 20 12.7
9 400 Watt Branson Sonifier S-450D 400 20 1310 Vibracell ultrasonifier Sonics and Materials Inc, USA 750 20 13
11 MSE Soniprep 150 (UK) 150 50 9.512 SONICS Vibra Cell VCX750 750 20 1313 Misonix Sonicator 3000 400 20 13
14 400 Watt Branson Sonifier S-450D 400 20 1315 SONICS Vibra Cell VCX750 750 20 1316 BransonS-450D 200 10 1317 Branson 250S 200 20 13
18 VibraCell VXC130 130 20 619 SCIENTZ-IID 950 20 NA20 Qsonica - Q700 700 20 3.221 Bandelin Sonopuls HD 3100 400 20 3
22 BransonS-450D 400 20 1323 Branson SLPe 150 20 6.424 Qsonica - Q700 700 20 1325 Vibra-Cell (Model VC 505,Sonics and materials,Ct,USA ) 500 20 1326 UP 400S Sonicator (Hielscher) 400 24 727 Branson Sonifier S-450D 400 20 13
A common European approach to the regulatory testing of nanomaterials
Examples of acoustic energy curves
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0
5
10
15
20
25
30
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0% 20% 40% 60% 80% 100% 120%Amplitude [%]
P(a
c) [
W]
arbitrary scale from µm amplitude
pac MCt
TWattP
A common European approach to the regulatory testing of nanomaterials
The DLS hydrodynamic size-distributions at calibrated settings
NANoREG general pressentation13
0
5
10
15
20
25
1 10 100 1000 10000 100000
Inte
nsi
ty [
%]
Hydrodynamic Size, dH [nm]
NM 200_NRCWE
NM 200_UDL
NM 200_SINTEF
NM-200_LEITAT
NM 200_INAIL
NM 200_INERIS
NM 200_NPL
NM 200_TEKNIKER
NM200_INSA
NM 200_CNR_ISTEC
NM 200_ISS
NM 200_DTU Food
NM-200_UIB
NM 200_UAB
NM 200_UNITO
NM 200_KRISS
NM 200_INMETRO
NM 200_DWE Korea
NM-200 KI
NM 200-IIT
NM 200-WUR
NM 200-STAMI
NM 200-CEA LCE
A common European approach to the regulatory testing of nanomaterials
Partner Zave PDI n1 250.6 19.7 0.419 0.070 11x102 218.1 2.7 0.329 0.029 3x103 254.0 13.9 0.402 0.042 3x104 250.2 10.3 0.413 0.040 3x105 251.1 16.5 0.418 0.058 6x106 264.9 41.1 0.337 0.095 3x107 248.9 16.8 0.282 0.052 3x108 266.6 18.4 0.414 0.056 4x109 243.5 10.4 0.384 0.033 3x10
10 264.7 31.1 0.395 0.042 3x1011 214.0 5.3 0.328 0.004 3x112 246.4 18.0 0.377 0.023 3x1013 #271.8 15.7 0.422 0.049 3x1014 *280.1 13.2 0.411 0.027 3x1015 231.4 8.3 0.348 0.028 3x1016 222.0 7.6 0.353 0.039 3x1017 216.5 3.9 0.341 0.019 2x1018 266.0 16.0 0.371 0.027 3x1019 253.4 13.0 0.459 0.042 3x1020 254.5 6.1 0.505 0.024 3x1021 247.7 3.1 0.167 0.013 3x1022 #272.4 11.4 0.380 0.020 2x323 #272.3 10.0 0.430 0.042 1x1024 CPS disc centrifuge25 *303.6 17.4 0.526 0.043 3x10
Average 252.7 13.7 0.384 0.038
24.9 0.079
Calibration possible!
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A common European approach to the regulatory testing of nanomaterials
A common European approach to the regulatory testing of nanomaterials
Performance testing and benchmarking
Type of MNMMNM Identification codes used by NANoREG*
Titanium Dioxide NM-100, NM-101, NM-103
Silicon Dioxide NM-200, NM-203
Zinc Oxide NM-110, NM-111
Cerium Dioxide NM-212
Barium Sulphate NM-220
Silver NM-300K, NM-302
Nanotubes (single and multi-walled) NM-400, NM-401, NM-411
Nanofibrillar celluloseNFC Fine, NFC Medium-coarse, UPM Biofibrils AS, UPM Biofibrils NS, UPM Bleached Birch Pulp
NANoREG general pressentation16
A common European approach to the regulatory testing of nanomaterials
NM-203NM-200
Performance on granular MNM
NM-302
200 µm
NM-103
A common European approach to the regulatory testing of nanomaterials
Performance on CNT and nanocellulose
50 µm