1. Testing strategy for nanomaterials
Maria Dusinska
maria.dusinska@nilu.no
No
Norwegian Institute for Air Research
Kjeller, Norway
http:www. nilu.no
National coordinator for NanoSafety
Health Effects Laboratory, GLP certified
Barcelona, November 29, 2013

2. Nanotechnology is an technology that deals with structures
ranging from approximately 1-100nm in at least one dimension
Nanoparticles have larger
surface/volume ratio and are
potentially more reactive.
Barcelona, November 29, 2013

3. Oxidised lipids,
thiols, ROS
Respiratory
chain
mitochondria
Surface
markers
Cytokines
Proteins RNA
Cathepsins
lysosome

4. Barcelona, November 29, 2013

5. Nanomaterials regulation
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

6. 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
Primary properties
Secondary properties
Aglomeration/aggregation
Protein corona
Size distribution
Stability of dispersion
Barcelona, November 29, 2013
Chemical composition
Crystalline structure
Size, Shape
Surface composition
Surface charge
Surface area
Purity/inpurities
Porosity

7. NP characterisation. Primary properties
U-Fe3O4,
no coating
8±3nm
Fluorescent
140nm
OCFe3O4,
coating
8±3nm
Powder
Water
dispersion
Water
dispersion
Anatase/rutile
Unknown
Ti, O
TiO2
21nm
Chem compos
Nano SiO2
SiO2,
25 nm
Fluorescen
t SiO2,
50 nm
Water dispersion
Water
dispersion
Water
dispersion
Powder
Spinel
(octahedral)
Spinel
(octahedral)
Amorphous
Amorphous
Amorphous
C, H, O
Fe, O
Fe, O
Si, O
Si, O
Si, O
Not applicable
0.33
26
2.8
-2
-2
Not
applicable
Irregular
Not applicable
Oblong
Oblong
Round/
Round/
oblong
Phase
Crystal
structure
PLGA
oblong
TEM/EDX
Particle
concent (%) 1
Shape-TEM
Crystallite size
distributionTEM (nm)
Surface areaBET (m2/g)
Pore volumeBET (mL/g)
Surface
chemistry
Zeta potential
milliQ pH7 (mV)
Irregular,
rectangular
15-60
Not applicable
5-12
5-13
15-30
25-50
5-30
61
Not applicable
Not applicable
Not applicable
Not applicable
Not applicable
Not applicable
Uncoated
Uncoated
Oleate micelle
coating
Uncoated
Not
applicable
Not
applicable
Uncoated
226
0.13
Not
applicable
Not
applicable
Uncoated
-30.2
-43.4
-31.9
-2.8
- 20
- 22
0.7
Uncoated
Free oleate (960 ppm), Na (26.000 ppm), Ca (1.300 ppm), K (730 ppm)

8. Biological test system
In vitro vs in vivo
2D culture representing solid tissue
Blood model:
cells in suspension
Barcelona, November 29, 2013

9. In vitro
Blood: human blood cellsleucocytes, granulocytes,
monocytes, etc. TK6
Outcome
Blood
Automation and validation
Central nervous system
Barrier transport
Digestive system
Genotoxicity
Lung
Oxidative stress
Liver
Inflammation/Immunotoxicity
Vascular system
Placenta
Central nervous
system :
HCEC, EC219, Murine
N11 microglial cells
Mechanism studied
ex vivo
Liver: hepatocytes, kupffer
cells and liver sinusoidal
endothelial cells (LSEC),
HepG2
Vascular: HCEC,
EC219, ECp23, HL1
System
Kidney: monkey
kidney Cos-1 cells,
human HEC
in vivo
Placenta: Placenta
perfusion, BeWo cells
Kidney
Digestive system :
Caco 2, CacoGonlet,
CacoReady TM,
Colon HT29
Lung: bronchial epithelial
cell lines (16HBE, NCIH and
Calu-3, and human alveolar
type 2 cells A549, HBEC)

10. 1 min
30 min
NP characterization
Size distribution and stability of oleic acid coated iron oxide in various culture media
Conc.: 0.25 mg/ml)
Medium
(Conc.: 0.25 mg/ml)
Hydrodynamic diameter (nm)
DMEM
Very large agglomerates,
DMEM +10% FBS
Trimodal distribution,
DMEM-HG
Very large agglomerates,
DMEM-HG +10% FBS
1
> 900
Size stability
with time 1
< 10 min
~ 2 days
> 2000
< 5 min
Bimodal distribution,
36 and 153
~ 3 days
RPMI
Trimodal distribution,
18, 73 and 232
~ 2 days
RPMI +10% FBS
1
18, 86 and 237
Bimodal distribution,
39 and 165
~ 3 days
DMEM- F12-HAM
by DLS
DMEM-F12-HAM +10% FBS
Bimodal distribution,
31 and 132
~ 3 days
Bimodal distribution,
36 and 153
~ 3 days

11. 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
Barcelona, November 29, 2013

12. Biological effect depends on dispersion
DP1
DP2
DP 1 with serum
DP2: without serum
The state of aggregation
of NPs is important.
25
*
TiO2 DP1
Tail intensity (%)
DNA damage (comet assay)
after 24h exposure of EUE cells
to TiO2 NPs; 2 dispersions.
30
20
TiO2 DP2
*
15
10
5
1
0
by DLS
0
Magdolenova et al, JEM, 2012
0,12
0,6
3
TiO2 NPs (μg/cm2)
15
75

13. absorbance at 540 nm
1.2
MTT assay: without cells
A
1
B
0.8
PLGA
TiO2
0.6
0.4
0.2
Interference
Nanomaterial interference
with read out systems has
been observed for
colorimetric/fluorescens
spectrophotometric assays
0
0
0.5
5
50
NP concentration (µg/ml)
200
(A) Uncoated and (B) oleic acid coated
Fe3O4 NPs, PLGA-PEO NPs and TiO2 NPs.
Interference observed: WST-1, MTT, lactate dehydrogenase, neutral red,
propidium iodide, 3H-Tymidine incorporation, automated cell counting, proinflammatory response evaluation (ELISA for GM-CSF, IL-6 and IL-8), and
oxidative stress detection (monoBromoBimane, dichlorofluorescein, NO assays).
Barcelona, November 29, 2013

14. NP properties and interferences with assays
NP properties:
Light
adsorption
Light
scattering
Aggregation/
Agglomeration
Surface
reactivity
Dissolution
Adsorption/Reaction with
Assay
reagents
Problematic
techniques:
Problematic
assays:
Spectrophotometry
Spectrofluorometry
Flow cytometry
Cell counting
WST-1, MTT, NR, 3H-T,
Cell proliferation,
mBBr, DCF, Griess reagent…
PI uptake…
Biomolecules
Chemistry
Biochemistry
Immunochemistry
MTT, 3H-T…
ELISA, LDH…
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

15. Adoptation of Protocol for Cytokinesis
Blocked Micronucleus assay OECD 487
Interference with cytochalasin B
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.
Cos-1 cells exposed to TiO2 NP (UP7 dispersion
a. Cyt B simultaneously with NPs;
b. Cyt B added 2 hr after NPs;
c. Cyt B added 24 hr after NPs
% MN in binucleated cells
protocol) in concentration 75µg /cm2
9
8
7
6
5
4
3
2
1
0
pos. ctrl
Magdolenova et al., 2012
neg. ctrl
a
b
c

16. Adoptation of Protocol for Cytokinesis Blocked
Micronucleus assay OECD 487
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

17. The comet assay results of all NanoTEST NPs
Only TiO2 and coated
Fe3O4 showed
genotoxic effect
35
OC-Fe3O4
U-Fe3O4
30
PLGA-PEO
Fl-25 SiO2
Strand Breaks
25
Fl-50 SiO2
TiVedisp
TiUPdisp
20
15
10
5
0
0
20
40
60
80
16HBE140
BeWo b30
Cos-1
HEK293
HCEC
Human Lymphocytes
TK6
Rat Hepatocytes
Kupffer
Dose (g/cm2)
Cell-line/ nanomaterial
evaluation of the effect

18. Final Remarks
•
•
•
•
•
•
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

19. 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
NO
Bacterial Reverse Mutation Test
OECD 471
NO
Sacharomyces cerevisiae, Gene Mutation Assay Mitotic OECD 480, 481
NO
Recombination Assay
DNA Damage and Repair, Unscheduled DNA Synthesis in OECD 482
NO
Mammalian Cells in vitro
In vitro Mammalian Cell Micronucleus Test
OECD 487
In vitro Comet assay (Single-Cell Gel Electrophoresis)
JaCVAM/ECVAM
Cell Transformation assay
Draft Guideline

20. And NanoTEST
consortium

21. @qualitynano
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, info@qualitynano.eu
Website: http://www.qualitynano.eu
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

22. QualityNano overview
Transnational
Access (TA)
Networking
Activities (NA)
Funded access to
Training modules and
15 European
Best practice for nanosafety
Nano-characterisation sites
assessment
Joint Research
Activities (JRA)
Development of tools and
Protocols for nanosafety
assessment

23. Transnational Access Sites

24. 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

25. Overview of the Calls
http://www.qualitynano.eu/access/all-equipment.html
4th call over 80 applications
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%
5th TA Call Deadline: 20st December 2013
~ 4 monthly calls

26. Contact Details
+353 1 716 2459
TA@qualitynano.eu
info@qualitynano.eu
www.qualitynano.eu
@qualitynano