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No
Testing strategy for nanomaterials
Maria Dusinska
Norwegian Institute for Air Research Kjeller, Norway http:\\www. nilu.no
National coordinator for NanoSafety Health Effects Laboratory, GLP certified
Barcelona, November 29, 2013
Nanotechnology is an technology that deals with structures
ranging from approximately 1-100nm in at least one dimension
Barcelona, November 29, 2013
Nanoparticles have larger surface/volume ratio and are potentially more reactive.
mitochondria lysosome
Oxidised lipids, thiols, ROS
Surface markers
Cytokines
Respiratory chain
Cathepsins
Proteins RNA
Barcelona, November 29, 2013
To ensure sustainable development of nanotechnologies, urgent needs for regulation of nanomaterials is needed. EU already framed some regulation on nano-containing products:
Nano-cosmetics: Article 16 of the Cosmetic Regulation (EC) 1223/2009 from 11.1. 2013, Industry must notify the Commission of cosmetic products containing nanomaterials.
The Scientific Committee on Consumer Safety SCCS is performing risk assessments on cosmetics containing nanomaterials.
French Registry for Engineered Nanomaterials - From 1. 1 2013 France requires mandatory registration of “substances with nanoparticle status” that manufacturers produce, import, distribute, or formulate (Articles L. 523-1 to L. 523-5 of the French Environmental Code).
Until April 2013, 457 companies have made 1991 declarations
Nanomaterials regulation
Barcelona, November 29, 2013
Physico-chemical properties The novel size-dependent properties of nanomaterials make them desirable in technical and commercial uses. Biological interactions depend on the physico-chemical properties
Chemical composition
Crystalline structure
Size, Shape
Surface composition
Surface charge
Surface area
Purity/inpurities
Porosity
Primary properties Secondary properties
Aglomeration/aggregation Protein corona Size distribution Stability of dispersion
TiO2
21nm
PLGA
140nm
OC-
Fe3O4,
coating
8±3nm
U-Fe3O4,
no coating
8±3nm
Fluorescent
SiO2,
25 nm
Fluorescen
t SiO2,
50 nm
Nano SiO2
Phase
Powder Water
dispersion
Water
dispersion
Water dispersion Water
dispersion
Water
dispersion
Powder
Crystal
structure
Anatase/rutile Unknown Spinel
(octahedral)
Spinel
(octahedral)
Amorphous Amorphous Amorphous
Chem compos
TEM/EDX
Ti, O C, H, O Fe, O Fe, O Si, O Si, O Si, O
Particle
concent (%) 1
Not applicable 0.33 26 2.8 - 2 - 2 Not
applicable
Shape-TEM Irregular Not applicable Oblong Oblong Round/
oblong
Round/
oblong
Irregular,
rectangular
Crystallite size
distribution-
TEM (nm)
15-60 Not applicable 5-12 5-13 15-30 25-50 5-30
Surface area-
BET (m2/g) 61 Not applicable Not applicable Not applicable Not
applicable
Not
applicable 226
Pore volume-
BET (mL/g) 0.13 Not applicable Not applicable Not applicable Not
applicable
Not
applicable 0.7
Surface
chemistry
Uncoated Uncoated Oleate micelle
coating
Uncoated Uncoated Uncoated Uncoated
NP characterisation. Primary properties
Zeta potential
milliQ pH7 (mV) -30.2 -43.4 -31.9 -2.8 - 20 - 22
Free oleate (960 ppm), Na (26.000 ppm), Ca (1.300 ppm), K (730 ppm)
Barcelona, November 29, 2013
Blood model: cells in suspension
In vitro vs in vivo
2D culture representing solid tissue
Biological test system
Blood
SystemA
uto
matio
n a
nd
va
lidatio
nMechanism studied Outcome
Vascular system
Liver
Lung
Central nervous system
Digestive system
Placenta
Kidney
Ba
rrier
tran
sp
ort
Infla
mm
atio
n/Im
mu
no
toxic
ity
Ge
no
tox
icity
Oxid
ativ
e s
tres
s
Vascular: HCEC, EC219, ECp23, HL1
Blood: human blood cells- leucocytes, granulocytes, monocytes, etc. TK6
Liver: hepatocytes, kupffer cells and liver sinusoidal endothelial cells (LSEC), HepG2
Lung: bronchial epithelial cell lines (16HBE, NCIH and Calu-3, and human alveolar type 2 cells A549, HBEC)
Placenta: Placenta perfusion, BeWo cells
Digestive system : Caco 2, CacoGonlet, CacoReady TM, Colon HT29
Central nervous system : HCEC, EC219, Murine N11 microglial cells
Kidney: monkey kidney Cos-1 cells, human HEC
In vitro ex vivo in vivo
NP characterization
Size distribution and stability of oleic acid coated iron oxide in various culture media Conc.: 0.25 mg/ml)
1 by DLS
Medium
(Conc.: 0.25 mg/ml)
Hydrodynamic diameter (nm) 1
Size stability
with time 1
DMEM Very large agglomerates, > 900 < 10 min
DMEM +10% FBS Trimodal distribution, 18, 86 and 237 ~ 2 days
DMEM-HG Very large agglomerates, > 2000 < 5 min
DMEM-HG +10% FBS Bimodal distribution, 36 and 153 ~ 3 days
RPMI Trimodal distribution, 18, 73 and 232 ~ 2 days
RPMI +10% FBS Bimodal distribution, 39 and 165 ~ 3 days
DMEM- F12-HAM Bimodal distribution, 31 and 132 ~ 3 days
DMEM-F12-HAM +10% FBS Bimodal distribution, 36 and 153 ~ 3 days
1 min 30 min
Barcelona, November 29, 2013
Interaction with assay components, presence of serum and problems with exposure conditions.
Our as well as result from literature show that serum content in exposure media can influence NM uptake and toxic response. Forming of protein corona. Coating Coating change the behaviour and cellular uptake of the NPs.
Uncoated iron oxide - uptake
Coated iron oxide poor uptake
Magdolenova et al, 2013 Nanotoxicology
DP1 DP2
0
5
10
15
20
25
30
0 0,12 0,6 3 15 75
Tail
inte
nsi
ty (
%)
TiO2 NPs (μg/cm2)
TiO2 DP1
TiO2 DP2
*
*
DNA damage (comet assay) after 24h exposure of EUE cells to TiO2 NPs; 2 dispersions.
Biological effect depends on dispersion
1 by DLS
DP 1 with serum DP2: without serum
The state of aggregation of NPs is important.
Magdolenova et al, JEM, 2012
Barcelona, November 29, 2013
MTT assay: without cells
0
0.2
0.4
0.6
0.8
1
1.2
0 0.5 5 50 200
NP concentration (µg/ml)
ab
so
rb
an
ce a
t 540 n
m
A
B
PLGA
TiO2
Nanomaterial interference with read out systems has been observed for colorimetric/fluorescens spectrophotometric assays
(A) Uncoated and (B) oleic acid coated Fe3O4 NPs, PLGA-PEO NPs and TiO2 NPs.
Interference
Interference observed: WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, 3H-Tymidine incorporation, automated cell counting, pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6 and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, NO assays).
Aggregation/ Agglomeration
Surface reactivity
NP properties and interferences with assays
Light scattering
Light adsorption
Assay reagents
Spectrophotometry Spectrofluorometry
Flow cytometry Cell counting
WST-1, MTT, NR, 3H-T, mBBr, DCF, Griess reagent…
Biomolecules
ELISA, LDH… Cell proliferation, PI uptake…
MTT, 3H-T…
NP properties:
Problematic techniques:
Problematic assays:
Adsorption/Reaction with
Chemistry Biochemistry
Immunochemistry
Dissolution
Chemistry Biochemistry
MTT, LDH…
Guadagnini et al: Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests, Nanotoxicology 2013, accepted
Cytokinesis block assay detects mutagenic / clastogenic and aneugenic effects and provides a measure of both chromosome breakage and chromosome loss. MN frequencies, multinucleated cells, apoptosis/necrosis and in vitro proliferation rates scored on the slides.
0
1
2
3
4
5
6
7
8
9
pos. ctrl neg. ctrl a b c
% M
N in
bin
ucl
eat
ed
ce
lls
Cos-1 cells exposed to TiO2 NP (UP7 dispersion protocol) in concentration 75µg /cm2
Magdolenova et al., 2012
a. Cyt B simultaneously with NPs; b. Cyt B added 2 hr after NPs; c. Cyt B added 24 hr after NPs
Adoptation of Protocol for Cytokinesis Blocked Micronucleus assay OECD 487
Interference with cytochalasin B
Exposure conditions and time of the treatment • At least 24h exposure with NPs is important to complete cell cycle Cyt B must be added 24h and allowing endocytosis • Cytochalasin B must be added separately from NPs treatment allowing NPs to enter cells Suggested treatment: 24h exposure to NPs then add Cyt B for another 24h
Adoptation of Protocol for Cytokinesis Blocked Micronucleus assay OECD 487
Dose (g/cm2)
0 20 40 60 80
Str
an
d B
rea
ks
0
5
10
15
20
25
30
35
OC-Fe3O4
U-Fe3O4
PLGA-PEO
Fl-25 SiO2
Fl-50 SiO2
TiVedisp
TiUPdisp
The comet assay results of all NanoTEST NPs
Cell-line/ nanomaterial evaluation of the effect
Only TiO2 and coated Fe3O4 showed genotoxic effect
16HBE140
BeWo b30
Cos-1
HEK293
HCEC
Human Lymphocytes
TK6
Rat Hepatocytes
Kupffer
• Both primary and secondary characterizations are crucial • Level of serum influence behavior and uptake of NPs • Dispersion protocol is critical • Exposure conditions should represent in vivo situation • Coating influence the behaviour and cellular uptake of the NPs • Relevant positive and negative control should be always included to NPs testing • Concentration used and cytotoxicity data • The experimental outcome can be affected by the cell type used, by the toxicity read out system, by the exposure time, by the dispersion method and the dispersion stability
Final Remarks
REACH regulation for genotoxicity
In vitro Mammalian Chromosome Aberration Test OECD 473
In vitro Mammalian Cell Gene Mutation Test OECD 476
In vitro Sister Chromatid Exchange in Mammalian Cells OECD 479
Bacterial Reverse Mutation Test OECD 471
Sacharomyces cerevisiae, Gene Mutation Assay Mitotic
Recombination Assay
OECD 480, 481
DNA Damage and Repair, Unscheduled DNA Synthesis in
Mammalian Cells in vitro
OECD 482
In vitro Mammalian Cell Micronucleus Test OECD 487
In vitro Comet assay (Single-Cell Gel Electrophoresis) JaCVAM/ECVAM
NO
NO
NO
Cell Transformation assay Draft Guideline
NO
And NanoTEST consortium
NUID UCD | NHM | IOM | JRC | BFR | KIT | FUNDP | IST | UNIVLEEDS | NILU | HMGU | LMU | CIC | UU | ICN |
DLO/RIKILT | WU | DGUV | TAU | VITO | SMU | TCD | FIOH | UOE | CNRS | INERIS | UoB | HWU | RIVM
QualityNano: A pan-European Infrastructure for Quality in NMs Safety Testing
Grant agreement n°: SP4-Capacities-2010-262163
Start and end dates: 1st February 2011 – 31st January 2015
Coordinator: University College Dublin, Ireland
Prof. Kenneth A. Dawson, [email protected]
Website: http://www.qualitynano.eu
@qualitynano
QualityNano overview
Networking Activities (NA)
Joint Research Activities (JRA)
Transnational Access (TA)
Funded access to 15 European
Nano-characterisation sites
Training modules and Best practice for nanosafety
assessment
Development of tools and Protocols for nanosafety
assessment
Transnational Access process
4 TA Technological categories:
A) Nanomaterial synthesis
B) Nanomaterial labelling & pre-processing
C) Nanomaterial characterization in situ & ex situ
D) Nanomaterial exposure assessment
www.QualityNano.eu/access
Number of Applications granted in 1-3 Calls
Call 1 Call 2 Call 3
Submitted 37 40 41
Eligible 36 40 38
Sent to Review 35 38 38
Granted 21 19 33
Success rate 60,0 % 50,0 % 80%
Overview of the Calls
http://www.qualitynano.eu/access/all-equipment.html
5th TA Call Deadline: 20st December 2013 ~ 4 monthly calls
4th call over 80 applications
Contact Details
+353 1 716 2459
www.qualitynano.eu
@qualitynano