Nanomaterial toxicity through a Safer by Design approach on Mytilus edulis hemocytes

Objectives:

• Develop a high throughput screening approach (HTS) for testing in vitro toxicity thresholds for nanomaterials (NMs) using mussel Mytilus edulis hemocytes

• Test NMs from industrial partners for safe-by-design and life cycle analysis

Conclusion and Perspectives:

• Current Results suggest that SiΩC99 (40nm) has lower cytotoxic potential than SiΩC99 (75nm)

• Further toxicity assays are currently in progress (oxidative stress, apoptosis, immunity, cell metabolism) in order to evaluate the response of mussel hemocytes

towards SiΩC99 (40 and 75nm)

• In addition, pure Si nanomaterials will be provided for baseline comparison

• Results on in vitro assays will be validated through in vivo exposures

HTS & Nanomaterials• Rapidly developing technologies makes it

difficult to screen all NMs in a realistic timeframe

• Conclusion on NM toxicity lacks a

comprehensive toxicological library for

comparison between NMS1

• HTS takes advantage of advances in robotics

and computational techniques to improve the

throughput of in vitro techniques

• HTS can be used establish thresholds of NMs

toxicity for the development of a regulatory

framework4

• Implementation of safer-by-design approach

through life cycle analysis:

• Current research focused on nanomaterials

prepared at different sizes at production

• Selection of least toxic nanomaterial for

production of product

Nanomakers SiΩC99

• Silicon Carbide Nanomaterials

Coated in Carbon used to improve

battery life

• Compare toxicity for two sizes of

preparation of nanomaterial (40 &

75nm)

Dynamic Light Scattering (DLS)

Barrick A., Châtel A., Bruneau M., Mouneyrac C.

Mer Molécules Sante (MMS), Université Catholique de l’Ouest, 3 place André Leroy, BP10808, 49008 Angers Cedex 01

1. Jung S.K., Qu X., Aleman-Meza B., Want T., Riepe C., Liu Z., Li Q., Zhong W. Multi-endpoint high-throughput study of nanomaterial toxicity in Caenorhabditis elegans. Environmental Science & Technology. 49,2477-2485.

2. Arts J., Hado M., Irfan M.A., Keene A., Kreiling R., Lyon D., Maier M., Michel K., Petry T., Sauer U., Warheit D., Wiench K., Wohlleben W., Landsiedel R. A decision-making framework for the grouping and testing of nanomaterials (DF4nanogrouping). Regulatory toxiciology and pharmacology. S1-27, 71.

3. Cao A., Mercado L., Ramos-Martinez J.C., Barcia R. 2003. Primary cultures of hemocytes from Mytilus galloprovincialis lmk: expression of IK-2Rα subunit. Aquaculture. 108, 216.

4. Katsumit A., Arostegui I., Valsami-Jones E., Cajaraville M. Cytotoxicity of Au, Zno and SiO2 nps using in vitro assays with mussel hemocytes and gill cells: relevance of size, shape and additives. Nanotoxicology. 1-9.

5. Nel A., Xia T., Meng H., Wang X., Lin S., Ji Z. Zhang H. 2013. Nanomaterial toxicity testing in the 21st century: use of a predictive toxicological approach and high throughput screening. Acc Chem Res. 607-621 (3)46.

6. Oomen A., Bleeker E., Bos P., Broekhuizen F., Gottardo S., Groenwold M., Hristozov D., Hund-Rinke K., Irfan M.A., Marcomini A., Peijnenburg W., Rasmussen K., Jimenez A., Scott-Fordsmand J., Tongeren M., Wiench K., Wohlleben W., Landsidel R. 2015. Grouping and read-across approaches for risk assessment of

nanomaterials. International Journal of Environmental Research and Public Health. 12,13415-13434. 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."

Start of Experiment (0 Hours)

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)

SiΩC99 (40nm) in Culture Media 45.24±0.4425 0.389 82.49±22.64 (97) 2856±1636 (3) -

SiΩC99 (40nm) Stock Solution 40.03±1.363 0.549 160.7 (60) 21.82 (39) 1621 (1)

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)

SiΩC99 (75nm) Stock Solution 68.38±2.027 0.571 206.7±16.6 (75.2) 24.4±9.5 (24.4) 441.9±1395 (0.4)

End of Experiment (24 Hours)

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)

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 (40nm) Stock 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 (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)

SiΩC99 (75nm) Stock Solution 131.7±1.029 0.421 216.9±10.89 (91.5) 27.23±3.413 (8.3) 991.7±2091 (0.3)

NANoREG SOP for Dispersion

• 2.56 mg/mL solution prepared by pre-wetting powder with

ethanol and dispersed in 0.05% BSA solution

• Branson S-450 sonicator with 13mm probe

• 7056J of energy using 10% amplitude for 16 minutes

• DLS measured at 200µg/mL concentration

Developing an HTS Strategy for Nanomaterials on M. edulis Hemocytes

Establish and Maintain a Mixed

Primary Cell Culture on

Hemocytes for 24 Hours2,3

In vitro Toxicity Screening: Alamar

Blue and Reactive Oxygen Species

(ROS)4

Hemolymph Extraction from the

M. edulis Posterior Adductor

Mussel

24 Hour exposure to NMs

(0.001µg/mL – 200µg/mL)

Chemical

Composition

Particle Size/

Range

Surface

Characteristics

Solubility and

Hydrophobicity

Biological

Reactivity

Biological

Uptake

Screening of nanomaterials to

establish database characterizing life

cycle events6

Toxicity Profiles

Alamar Blue Reactive Oxygen Species

• Results suggests that SiΩC99 (75nm) significantly decreased cell

viability compared to SiΩC99 (40nm)

• LC(50) of SiΩC99 (75nm) is 100µg/mL whereas for SiΩC99

(40nm), it is >200µg/mL

• Mussels exposed to SiΩC99 (75nm) presented significantly higher

levels of ROS compared to SiΩC99 (40nm) for tested

concentrations ranging from 0.1 to 50 ug/ml

Cell Viability Oxidative Stress

Life

Cycle

Analysis

High Throughput Screening (HTS)

Raw Powder

Production

Production of

Product

Commercial Use

End of Life

Sample Preparation

Toxicity Screening & Data Collection

Data Analysis, Hit

Identification & Visualization5

Data Mining: Cluster Analysis & Structure-

Activity Relationships

QSARS

Grouping Strategy & Read

Across Approaches

Life Cycle Analysis on Chemical

Products2,5