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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24

25

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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

35

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