nanotechnology activities at the joint research centre€¦ · joint research centre safety...

Joint Research Centre

Nanotechnology activities at the Joint Research Centre

• François Rossi

www.jrc.ec.europa.eu

Serving society

Stimulating innovation

Supporting legislation


Today, many products that contain “Nanomaterials” are already on the market:

– Automotive

– Chemical Industry

– Construction

– Cosmetics

– Electronics

– Energy

– Environment

– Food

– Nanomedicine

– Photonics

– Textiles

Nanotechnology is not a vision of the future.

“Nanomaterials” are already in our lives and there is a large potential for new applications!

Nanomaterials and Nanotechnology Big science of tiny things

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4 14/03/2012

Examples of product areas:

• Cosmetics / personal care

• Paints & coatings

• Household products

• Textiles

• Food (and ingredients)

• Food packaging

• and more…

Nanotechnology consumer products on the market


Materials : what makes them different?

• Size (1 - >100 nm)

• Large specific surface

• Special properties (electronic, mechanical, optical …)

• Chemical reactivity very different compared to bulk material

• Matrix dependent properties

• Many forms: fullerenes, nanotubes, nanocarriers, nanoemulsions, nanoencapsulates, …

5nm 30nm 40nm 50nm 80nm


Safety assessment of nanomaterials

JRC Nanotechnology for Policy Support

Nanomaterials in consumer products

• Developing methods for detection in food and consumer products

• Validating of analytical methods for nanomaterials

• Developing standardised protocols for toxicity testing

• Nanobiotechnology research activities:

protein-nanomaterial interaction, nanosensors

Provision of tools • Certified Reference Materials (IRMM)

• Hosting a repository of Representative Nanomaterials

• Hosting the NanoHub database


Safety/Risk Assessment

RISK CHARACTERIZATION

EXPOSURE ASSESSMENT

HAZARD IDENTIFICATION

HAZARD CHARACTERIZATION

RISK CHARACTERIZATION

EXPOSURE ASSESSMENT



EXPOSURE ASSESSMENT






Detection and characterisation

24 nm

Separate, quantify, characterise

Body fluid Protein

Corona ??

Uptake and cell interaction

72h exposure

0

20

40

60

80

100

120

1 10 100 1000

[Co] (µM)

CF

E (

%)

Conano ECVAM

Conano HRI

Cell response

NANO-SAFETY: In vitro Nano-Toxicology

Synthesis of NM

(radio)

labelling


Little crystals

Nanoparticles have many different shapes and sizes

SiO2

Stéfane Mornet, CEA Different sizes

Aggregates, Agglomerates

Silver

Thierry Darmanin, JRC

Different shapes

Carbon nanotubes


Adaptation of selected in vitro methods for

chemicals already recognised scientifically valid from OECD and/or European Centre for the Validation of Alternative Methods (ECVAM)

- Cytotoxicity: Colony forming Efficiency on Balb/3T3 cells (pre-

validation study by ECVAM)

- Morphological Transformation: Cell transformation assay on Balb/3T3 cells (pre-validation study by EURL-ECVAM; recommended in March 2012 to partially replace the rodent 2-year assay OECD Test Guidline 451 and 453)

- Genotoxicity: - Micronucleus test (MN) (OECD Test Guideline 487 in preparation) - Comet assay (considered for retrospective validation exercise by

ECVAM)


Objective: Organize inter-laboratory study of a series of in vitro tests in

nanotoxicology, namely CFE, MN, CTA on selected NM

Organizer: JRC- IHCP (SG7 of the OECD WPMN)

Participants: Invited participants from OECD WPMN

Tests: CFE, MN, CTA…

Materials: Au, SiO2, Ag, ZnO, CNT…

Results: Aiming at a full collaborative trial , terms to be defined at the OECD

WPMN.

Outcome: Assess the transferability of the methods to laboratories for an

eventual application for regulatory purposes

Inter-laboratory study

In vitro tests in Nanotoxicology


No upper size considered

No routine methods of directly “counting” particles*

High risk of false results by conversion of mass based measurements

A technical consideration of the definition

"Nanomaterial" means a natural, incidental or manufactured

material containing particles, in an unbound state or as an

aggregate or as an agglomerate and where, for 50 % or more of

the particles in the number size distribution, one or more external

dimensions is in the size range 1 nm - 100 nm.

COMMISSION RECOMMENDATION on the definition of nanomaterial

Many proteins and natural

polymers are nanosized

Few methods can

detect 1nm

Need to know how to consider

aggregate/agglomerates

Refers to primary

(constituent) particles

*Possible by EM or Coulter


Definition of Nanomaterial: Implementation

• Constituent particles – agglomerates - aggregates

• Validated size measurement methods

• Sample preparation

• Measurement techniques

• Evaluation and interpretation of data (number size distribution)

• Guidance

Implementation Issues

Nanomaterials in complex matrices (food, cosmetics, …)

Related Problem


• requirements for particle size measurement

• generic measurement issues

• capabilities of currently available measurement methods

JRC Reference Report Linsinger T. et al., EUR 25404, 2012 doi:10.2787/63490

Definition of Nanomaterial: Implementation


Candidate methods

• Counting methods (such as EM)

– can provide number based size distribution

– high number of particle needs to be measured

• Ensemble methods (such as DLS, MALS, XRD)

– intensity weighted sizes

– Conversion to number-based size distributions necessary

• Fractionation methods (such as FFF)

– can deal with polydisperse samples

– can be coupled with counting methods

– can become crucial for the implementation

of the definition

http://www.fei.com/products/dualbeams/quanta3d.aspx


From: Linsinger T. et al., EUR 25404, 2012 doi:10.2787/63490

Can deal with particular types of nanomaterials?

Method name

Measurement range and medium

size distribution of raw data

poly-dispersity

non-spherical particles

low-density materials

aggregates

Standards available?

Electron microscopy

1 nm and higher; dry

number-based long flat yes

Dynamic light scattering

5 nm to 500 nm; suspension

(no distribution, or scattering intensity-based)

yes

Centrifugal liquid sedimentation

20 nm and higher; suspension

extinction intensity-based

yes

Small-angle x-ray scattering

5 nm and higher; suspension

scattering intensity-based

yes

Field-flow-fractionation

1 nm to 200 nm; suspension

(depends on detector)

no

Particle tracking analysis

25 nm and higher, suspension

number-based no

Atomic force microscopy

1 nm and higher; dry

number-based yes

X-ray diffraction 1 nm and higher; dry

(no distribution measured)

yes


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Nano-Labelling

Labelling obligation for ingredients present in the form of nanomaterials

Titanium Dioxide (nano)

No hazard labelling Consumer information

will allow consumers to make a

choice

Control???

• Regulation (EC) No 1223/2009 on cosmetic products

• Regulation (EC) No 1169/2011 on the Provision of Food

Information to Consumers


Detection of Nanomaterials in Products

• Matrix structure at the nanoscale

• Detection and quantification of known/unknown material

• Inorganic/organic nanomaterials

• Matrix-Nanomaterial interaction

• Distinction from background

• Detection levels

• Analytical artifacts

• Availability of standard materials

No Validated Routine Methods

Problems Background:

Regulatory Issues

Definition

Labeling requests

Safety testing

Consumer information

Consumer behaviour


Objective: Organize inter-laboratory comparison of nanoparticle size distribution

measurements with reference to the proposed definition of a

nanomaterial .

Organizer: JRC-IHCP Start Date: May 2012

Participants: National control laboratories

Possible Methods: SEM/TEM, Field Flow Fractionation (FFF), Centrifugal Particle

Sedimentation, Analytical Ultracentrifugation – other valid alternatives can be considered

Materials: Aqueous dispersions of stabilized silver nanoparticles: multimodal size

distribution in the range 1-200nm

Results: Determine particle number size distribution in the range 1-200nm

and classify sample status according to the recommended definition

The performance of individual laboratories will be retained strictly

confidential

Outcome: Assess the state of readiness of EU analytical laboratories for an eventual

application of the proposed definition within a legislative framework

Particle size distribution measurement

Inter-laboratory comparison


Expert consultation in

development of

Standard Operation

Procedures

Inter-laboratory testing and validation

Sample PreparationSample Preparation

Separation/FractionationSeparation/Fractionation

Particle AnalyzerParticle Analyzer

Imaging/MicroscopyImaging/Microscopy Elemental AnalysisElemental Analysis

Sample PreparationSample Preparation

Separation/FractionationSeparation/Fractionation

Particle AnalyzerParticle Analyzer

Imaging/MicroscopyImaging/Microscopy Elemental AnalysisElemental AnalysisImaging/MicroscopyImaging/Microscopy Elemental AnalysisElemental Analysis

FFF/Hydrodynamic

Chromatography

Aqueous dispersions of Ag

NPs provided by JRC

DLS/MALS

SEM/TEM ICP-MS,

or AAS, ICP-OES


Objective: Organize inter-laboratory study of a single particle ICP-MS method for silver

nanoparticles (developed by RIKILT)

Organizer: RIKILT, JRC (IHCP, IRMM), Nanolyse partners

Participants: Invited participants from EU, US, Canada, Australia, etc.

Methods: Single Particle ICP MS

Materials: Focus on food Matrix: Food contact migration simulants

Results: Aiming at a full collaborative trial (i.e. at least 8 valid results, = 10-15

participants), preferably with laboratories already experienced in sp ICP-MS (in

case of no sufficient number of such labs selection criteria defined for

additional labs). A training workshop for the method will be offered at RIKILT for

participating labs.

Outcome: Assess the transferability of the methods to analytical laboratories for an

eventual application for regulatory purposes

Inter-laboratory study

Determination of silver nanoparticles by sp ICP MS


Representative Nanomaterials (NM series) from the European Commission - JRC

TiO2 Carbon Nanotubes

ZnO Silver

SiO2 Nanoclays

CeO2

Repository: Nanomaterials for Testing


Projects

Nanomaterials

• OECD list of representative NMs

• OECD sponsorship programme

• European NM Repository

• OECD WPMN

• FP 7 Projects

• National projects

• Institutional projects

JRC NanoHUB Database

Projects hosted today


DG ENV REACH, Definition implementation

DG ENTR REACH

DG SANCO Cosmetics, Food, Consumer Products

DG RTD Research projects => Legislation

EFSA Scientific Support – Food Legislation

ECHA Scientific Support – REACH / Databases

OECD Test Methods and Guidelines

ISO/CEN Standardisation

NBS - Nanotechnology for Policy Support


Standardization Activities

• Support to DG Environment by JRC (co-chair US/EU)

Safety Testing of a Representative Set of Manufactured Nanomaterials

Manufactured Nanomaterials and Testguidelines

The Role of Alternative Methods in Nanotoxicology

OECD Working Party on Manufactured Nanomaterials

CEN TC 352 Nanotechnology

ISO TC 229 Nanotechnology

ISO/TC24/SC4 (particle characterisation)

ISO/REMCO (reference materials)

www.jrc.ec.europa.eu Contact: [email protected]

Serving society

Stimulating innovation

Supporting legislation

Joint Research Centre (JRC)

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