This document summarizes a study that evaluated the toxicity of three engineered nanomaterials (ENMs) - silicon (Si) nanoparticles, and carbon-coated silicon carbide (SiΩC99) nanoparticles of 40nm and 75nm size - using a high throughput screening approach on marine mussel (Mytilus edulis) hemocytes. The study found that the 75nm SiΩC99 nanoparticles were generally more toxic than the 40nm SiΩC99 or 40nm Si nanoparticles, as evidenced by their effects on various biomarkers like oxidative stress, DNA damage, membrane transport, immune response and detoxification. The results indicate that both size and carbon coating influence the toxicity profile of the ENMs, with

1. Project #646221, Funded by the Horizon 2020
Framework Programme of the European Union
Nanomaterial Toxicity through a High Throughput
Screening Approach on
Mytilus edulis hemocytes
Andrew Barrick, Mélanie Bruneau, Amélie Châtel, Catherine Mouneyrac
COST, Aveiro 2017

2. Regulatory Requirements
Key characteristics associated with release, exposure, behavior and
hazard
Standardization of methods
Nano-specific risk assessment strategies
Research Needs to Meet Regulatory Goals
Fill data gaps associated with implementing grouping strategies
and read across approaches
Development and validation of grouping strategies
Methods to rapidly screen ENMs (High Throughput Screening)
Current State of Regulation for ENMS
Novel Materials
Requiring Testing
Tested and
Approved
Products

3. NANoReg II Project Objectives
Development and Implementation of Grouping and Safe-by-Design
approaches within regulatory frameworks
Safer by
Design
Large range of nano products
Difficult to test all and create regulatory
policy
High Throughput Screening
approach for NM categorization
Development of grouping strategy
for read across approach's
Case studies on ENMs
from industrial partners

4. What is High Throughput Screening?
Use of advances in technology to increase the throughput of in vitro screening
Probes key biological events in pathways that can lead to toxicity
Term HTS has been used for measurements on isolated DNA, cell cultures and
endpoints on whole organism testing
HTS Platform of hatching success for Danio reiro
exposed to different concentrations of CuO
Nanoparticles (Lin et al., 2011)

5. No Clear Definition of HTS for Ecotoxicology
Development of HTS Assays Data Analysis
Hit Identification
Data Mining/Modeling
Selection of Endpoints
Establish Chemical Library
Judson et al., 2012 (EPA Tox21 Project)
• 96 well plate or higher
• Concentration response format
• Quantitative read-out
• Simultaneous cytotoxicity measurements

6. CASE STUDY: Implementation of SbD Strategy
Industrial Partner : scale up SiC production to 1 ton/year
• Triggers REACH protocols
• Determine which product is safer for the environment
• Limited information on environment risk of SiC
Pure
Silicon
(40nm)
Carbon Coated
Silicon Carbide
SiΩC99 (40nm)
Carbon Coated
Silicon Carbide
SiΩC99 (75nm)
Environmental Risk Assessment

7. STRATEGY
In vitro testing on marine mussel M. edulis hemocytes
24h hemocyte
maintenance in culture
Hemolymph extraction
SiΩC99(40nm) SiΩC99(75nm)
Raw Si (40nm)
24 Hour Cytotoxicity (HTS)
In vitro exposure at sublethal concentrations for
biomarker analysis
Oxidative stress, membrane transport, immune response,
apoptosis, lipid peroxidation, cytoskeleton, DNA damage
qPCR (HTS)
Comet assay
Biochemical analysis
In vivo Exposure
(Same assays)
LC(50): 0.001-200µg/mL (6 replicates + 2 blanks)

8. Dispersion of ENMs: NANoREG SOP
2.56mg/mL in 0.05%
Bovine Serum
Albumin (BSA)
Solution
Sonication: 10% Amplitude
for 16 Minutes
Dilution to 200µg/mL: BSA
Solution or Cell Culture
Media
Dynamic Light Scattering &
Zeta Potential: Start and End
of experiment

9. DLS
Condition Z-average (d-nm) PDI Peak 1: d-nm (%) Peak 2: d-nm (%) Peak 3: d-nm (%)
Culture Media 17±0.984 0.408 20.6±10.54 (73.7) 1238± 1804(25.4) 1816±2364 (0.9)
Si (40nm) in BSA Solution 46.71±0.4946 0.558 139.2±20.01 (75.8) 16.79±9.529 (24.1) 0.4599±1.454 (0.1)
Si (40nm) in Culture Media 87.27±1.083 0.339 140.3±12.1 (98.8) 1358±2059 (0.9) 1.933±5.8 (5.8)
SiΩC99 (40nm) BSA Solution 40.03±1.363 0.549 160.7 (60) 21.82 (39) 1621 (1)
SiΩC99 (40nm) in Culture Media 45.24±0.4425 0.389 82.49±22.64 (97) 2856±1636 (3) -
SiΩC99 (75nm) BSA Solution 68.38±2.027 0.571 206.7±16.6 (75.2) 24.4±9.5 (24.4) 441.9±1395 (0.4)
SiΩC99 (75nm) in Culture Media 134.4±2.629 0.53 384.8±78.8 (72.9) 55.98±27.49 (26.5) 927.7±1951 (0.6)
Condition Z-average (d-nm) PDI Peak 1: d-nm (%) Peak 2: d-nm (%) Peak 3: d-nm (%)
Culture Media 18±2.978 0.412 17.66±10.4 (68.2) 431.8±1133 (29.5) 2608±2298 (2.3)
Si (40nm) BSA Solution 94.78±1.351 0.317 148±5.712 (90.5) 22.76±2.475 (9.5) -
Si (40nm) Culture Media 175±3.82 0.253 233.8±24.16 (99.8) 431.6±1365 (0.2) -
SiΩC99 (40nm) BSA Solution 39.145±0.697 0.5364 138.492±59.07 (62.25) 65.69±99.2 (37.37) 397.8±1256.9 (0.37)
SiΩC99 (40nm) in Culture Media 81.2±1.6 0.4817 230.38±59.1 (75.4) 448.8±99.2 (23.9) 839.5±1256.9 (0.77)
SiΩC99 (75nm) BSA Solution 131.7±1.029 0.421 216.9±10.89 (91.5) 27.23±3.413 (8.3) 991.7±2091 (0.3)
SiΩC99 (75nm) in Culture Media 173.9±1.325 0.509 368.6±90.44 (83) 53.43±34.03 (16.4) 1443±2322 (0.7)
End of Experiment (24 Hours)
Start of Experiment (0 Hours)

10. Zeta Potential
Condition Zeta Potential (mV)
Culture Media -5.00±1.22
Si (40nm) in BSA Solution -22.1±0.32
Si (40nm) in Culture Media -6.48±1.34
SiΩC99 (40nm) BSA Solution -6.96±0.21
SiΩC99 (40nm) in Culture Media -6.48±1.31
SiΩC99 (75nm) BSA Solution -11.53±0.252
SiΩC99 (75nm) in Culture Media -9.65±3.02
Condition Zeta Potential (mV)
Culture Media -6.94±1.83
Si (40nm) BSA Solution -28.7±1.67
Si (40nm) Culture Media -5.66±1.31
SiΩC99 (40nm) BSA Solution -4.64±0.171
SiΩC99 (40nm) in Culture Media -5.66±1.31
SiΩC99 (75nm) BSA Solution -14.51±4.47
SiΩC99 (75nm) in Culture Media -6.08±0.10
Start of Experiment (0 Hours)
End of Experiment (24 Hours)

11. Cell Viability
0
50
100
150
200
250
300
0 0.001 0.01 0.1 1 10 25 50 100 200
Percent
Control
(%)
Concentration (µg/mL)
Increases in cell viability for Si and SiΩC99 (40nm) mainly higher
concentrations
Decrease in cell viability for SiΩC99 (75nm)
Si (40nm)
SiΩC99 (40nm)
SiΩC99 (75nm)
Alamar Blue

12. SOD
Oxidative stress
ROS
CAT
Induction of ROS, Catalase and SOD gene expression with SiΩC99
Inhibition of Catalase with Si (40nm)
Si (40nm)
SiΩC99 (40nm)
SiΩC99 (75nm)

13. 0
10
20
30
40
50
60
70
80
0 0.01 0.1 1 10 H2O2
%
Tail
DNA
Concentration (µg/mL)
Comet Assay
Comet Assay
Genotoxicity
All 3 ENMs cause DNA damage at higher concentrations
DNA damage linked to ROS or direct effect of ENM?
Si (40nm)
SiΩC99 (40nm)
SiΩC99 (75nm)

14. Cellular Metabolism
Actin: Induction of gene expression at 1µg/mL for SiΩC99, 10µg/mL for Si (40nm)
Cytoskeleton:
Formation of
Cellular
Extensions,
Endocytosis
Release of
Hydrolytic
Enzymes
Actin
Lysozyme
Lysozyme: Only SiΩC99 (40nm) induced expression, inhibition of expression
with Si (40nm) at 0.1µg/mL
Si (40nm)
SiΩC99 (40nm)
SiΩC99 (75nm)

15. SiΩC99 (75nm) inhibits membrane transport. Si (40nm) increases membrane
transport
Membrane Transport
Both SiΩC99 increased expression of PgP gene
Activity of membrane
transporters: P-
glycoproteins
PgP
Si (40nm)
SiΩC99 (40nm)
SiΩC99 (75nm)

16. Detoxification
Induction of Metallothionein gene expression with SiΩC99
GST
Metallothionein
Induction of GST with SiΩC99. Inhibition of GST gene expression at 0.1µg/mL
Si(40nm)
Si (40nm)
SiΩC99 (40nm)
SiΩC99 (75nm)

17. Conclusions on Case Study
Biological
Endpoint
Si (40nm) SiΩC99 (40nm) SiΩC99 (75nm)
Cell Viability
Oxidative
Stress
Genotoxicity
Membrane
Transport
Immune
Response
Detoxification
SiΩC99 cause more toxic effects and lower concentrations than Si (40nm)
No Effect Observed Effect Observed
SiΩ99 (75nm) is more toxic than SiΩC99 (40nm)

18. Conclusions and Perspectives
Case Study
SiΩC99 (75nm) appears to be more toxic than SiΩC99 (40nm)
and Si (40nm)
• Effect of size?
• Effect of carbon coating?
• Potential effect of sedimentation in assay?
• Differences in administered dose and effective dose?
Each ENM displays a different toxic signature
• Different pathways of toxicity?
• Continue in vitro analysis (additional gene expressions, biochemical assays)
Conduct in vivo analysis

19. Acknowledgments
Thank you for your attention
Acknowledgements: The research contained within this publication was funded by the European Union’s Horizon 2020
research and innovation program NANoREG2 under grant agreement 646221.
"The sole responsibility of this publication lies with the author. The European Union is not responsible for any use that may be
made of the information contained therein."

20. ENM Behavior in Culture Media
Spectroscopic Analysis: Sedimentation of ENMs after 24 hours
Concentration
(µg/mL)
0 0.001 0.01 0.1 1 10 25 50 100 200
Si (40nm) BSA 0 0 0 0 0.065 0.39 0.33 0.44 0.71 1.47
Si (40nm)
Culture Media
0 0 0 0 0.1 0.45 3.54 7.62 20.87 44.37
SiΩC99 (75nm)
BSA
0 0 0 0 0.14 0.69 1.36 1.54 2.46 3.75
SiΩC99 (75nm)
Culture Media
0 0 0 0 0.82 7.21 18.80 48.14 99.63 184.06
SiΩC99 (40nm)
BSA
0 0 0 0 0.15 0.90 0.90 0.97 1.64 1.64
SiΩC99 (40nm)
Culture Media
0 0 0 0 0.77 5.18 12.98 26.04 46.56 58.22
Strong sedimentation effect from 10 µg/mL for both sizes of SiΩC99
and from 25 µg/mL of Si in culture medium