Nanofibrillar cellulose in vitro study of cytotoxic and genotoxic properties

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1 Nanofibrillar cellulose in vitro study of cytotoxic and genotoxic properties Pitkänen, M. 1, Honkalampi, U. 2, von Wright, A. 2, Sneck, A. 1, Hentze, H-P. 1, Sievänen, J. 1, Hiltunen, J. 1, Hellén, E.K.O. 1 1 VTT Technical Research Center of Finland, P.O.Box 1000, FI VTT, Finland 2 BioSafe Special Laboratory Services Ltd, P.O.Box 1199, FI Kuopio, Finland Keywords: Nanofibrillar cellulose, cytotoxicity, genotoxicity, safety, characterization, nanocellulose, in vitro tests, NFC Nanomaterials will improve the performance of many products in future but at the same time they exhibit novel properties and may expose humans and environment to new risks. One of the most abundant and sustainable nanomaterials are nanocelluloses. They have a wide industrial application potential, which include e.g. composites and construction materials, porous materials, paper and board, coatings, functional surfaces, and functional additives. Actually, the term nanocellulose is widely used to describe a range of quite different cellulose-based nanomaterials. These differences are not only due to a wide variety of available raw materials sources, e.g. wood, crop residues, and bacteria, but also to different top-down and bottom-up production methods, and optional physical and chemical modifications. Wood pulp and powdered celluloses are generally recognized as safe and can be used for example as a raw material for food contact materials or even as food additives [1-3]. However, as with other nanomaterials, the biological effects of nanocelluloses cannot be predicted solely from the chemical nature of cellulose. The size, shape, aggregation properties, degree of branching and specific surface properties, among others, still poorly understood factors, may affect the interactions of cellulose nanofibers with cells and living organisms. This far, only a few studies on the safety of nanocelluloses have been published [4-7] In this study the cyto- and genotoxic properties of two wood-based nanocelluloses were addressed: 1P Masuko nano fibrillar cellulose (NFC) made from birch pulp by grinding it once with a laboratory-scale supermasscolloider equipment MKCA 6-2 from Masuko Sangyo Ltd. Arbocel MF40, which is a commercial nanofiber cellulose produced by Rettenmaier & Söhne GmbH (JRS). The sample was a dry powder with consistency of 99.5%. Methods to test the cyto- and genotoxigological properties of nanocelluloses were introduced. The biological effects were assessed using in vitro bioassays. Immediate cytotoxicity was studied in several human or animal cell lines and sublethal effects were tested on cultured human cells. The ability to damage DNA was assessed using a bacteriological assay. The samples were characterized using a combination of analytical methods, including scanning electron microscopy (SEM), viscosity and transmittance measurements, and nanoparticletracking-analysis (NTA, NanoSight Ltd.). The 1P Masuko NFC consists of long and curly fibrils with a high aspect ratio (Fig. 1A). The size range of fibrils is wide: Thickness varies from 20 to 1000 nm and fibril length up to several micrometers. The diameter of the smallest fibrils is around nm, but there are a lot of fibrils also in the size range of nm. Since the 1P Masuko NFC has been grinded only once, there are also some unfibrillated fibers.

2 The commercial Arbocel MF40 consists of whisker-like fibrils. Both fibril size and morphology differ clearly from the 1P Masuko NFC. The fibrils are shorter and needle like with a low aspect ratio (Figs. 1B and 1C). Most of the fibrils have a diameter in the range of nm. There are a lot of small fibrils but also some fiber fragments, whose length is in the micrometer range. The thickness of the thinnest fibrils is about 10 nm with a length up to nm. A B C Figure 1. SEM micrographs of A) 1 P Masuko NFC and B,C) Arbocel MF40. The scale bars are 20 µm (A), 30 µm (B) and 1 µm (C). The toxicological tests did not indicate any cyto- or genotoxic properties. The cytotoxicity was analyzed with Highest Tolerated Dose (HTD) and Total Protein Concentration (TPC) assays in mouse hepatoma (Hepa-1c7c), human keratinocyte (HaCat) and human cervix carcinoma (HeLa229) cell cultures. Neither were the nanocelluloses genotoxic, since no indication of damage in DNA, based on the Ames test, was observed. The results show that the applied tests suit well to screen toxicological aspects of nanocelluloses. However, method development is still needed e.g. to obtain effective and inert sterilization methods. In addition, further studies will be needed to address the toxicological properties of different nanocellulose qualities, especially those with smaller fibril dimensions. 1. FDA CFR Pulp. Available in Internet ( ): 2. Federal Institute for Risk Assessment. BfR Recommandations on Food Contact Materials. XXXVI Paper and board for food contact ( ). Available in Internet ( ): 3. Elintarviketurvallisuusvirasto Evira. Elintarvikkeiden lisäaineiden E-koodiavain (in Finnish) Available in Internet ( ): 4. O Connor B. Ensuring the safety of manufactured nanocrystalline cellulose. Presentation in OECD Conference July , Paris. 5. Rojas OJ, Laine J and Österberg M. Nanocellulose Materials, Functions and Environmental aspects. Presentation in OECD Conference on Potential Environmental Benefits of Nanotechnology, Paris France, July Vartiainen J, Sirola K, Pylkkänen L, Alenius H, Hokkinen J, Tapper U, Lahtinen P, Kapanen A, Putkisto K, Hiekkataipale P, Eronen P, Ruokolainen J, and Laukkanen A. Health and environmental safety aspects of friction grinding and spray drying of microfibrillated cellulose. Submitted to Cellulose Moreira S, Silva NB, Almeida-Lima J, Rocha HAO, Medeiros SRB, Alves J, Gama FM. In vitro study of genotoxicity and cell proliferation. Toxicology letters 189:

3 Nanofibrillar cellulose Assessment of cytotoxic and genotoxic properties in vitro Marja Pitkänen, Asko Sneck, Hans- Peter Hentze, Jenni Sievänen, Jaakko Hiltunen, Erkki Hellén Ulla Honkalampi, Atte von Wright Marja Pitkänen

4 Emergence of new materials sets new demands for product safety Nanomaterials will improve the performance of many products in future However, properties specific to nanomaterials might expose humans and environment to new risks The biological effects of nanomaterials, including nanocelluloses, cannot be predicted from the chemical composition Size, shape, aggregation properties, degree of branching and surface properties need also to be considered Nanomaterial/nanocellulose properties related to safety issues are not fully understood and need to be defined Marja Pitkänen

5 What is known about nanocelluloses safety? Nanocrystalline cellulose (NCC) did not indicate ecotoxicological concern B O Connor. Ensuring the Safety of Manufactured Nanocrystalline Celulose.OECD Conference Paris, July Bacterial cellulose (BC) nanofibers no genotoxicity in comet and Salmonella revision assays S Moreiraa et al. BC nanofibres: In vitro study of genotoxicity and cell proliferation. Toxicology Letters 189 (2009) Health and environmental safety aspects of microfibrillated cellulose has been recently assessed J Vartiainen et al. Health and environmental safety aspects of friction grinding and spray drying of microfibrillated cellulose. Submitted in Cellulose. Date Marja Pitkänen

6 Materials studied Nanofibrillar cellulose (NFC) Made from birch pulp by grinding for once using a laboratoryscale super mass colloider equipment MKCA 6-2 from Masuko Sangyo Ltd Arbocel MF40 Commercial ultrafine cellulose (UFC) produced by Rettenmaier & Söhne GmbH Dry powder with consistency of 99.5% Marja Pitkänen

7 Two different types of cellulose Fibrillar NFC Whisker-type UFC 20 µm 30 µm Long and curly fibrils with a high aspect ratio and wide size range Diameter: typically nm, with large fraction at nm Fibril length: up to several µm, also some fiber fragments Whisker-like short fibrils Diameter: Smallest fibrils nm, but also fiber fragments (up to µm range) Fibril length: Smallest fibrils nm Marja Pitkänen

8 Both materials contain variable size fragments Fibrillar NFC Whisker-type UFC 1 mm Light microscopy image of fibrillar NFC shows the presence of fiber fragments Whisker-type UFC micrograph shows short fiber fragments Marja Pitkänen

9 Clear difference in rheological properties between the materials Large particles Small particles Marja Pitkänen

10 A battery of in vitro methods are applied to assess cytotoxicity and genotoxicity Short-term toxicity (24-72 h exposures; change in total protein content), including estimates of highest tolerated dose (HTD) was studied using three well-characterized cell lines human keratinocyte line (HaCaT) human cervix carcinoma cell line (HeLa229) mouse hepatoma cell line (Hepa1) Sublethality was tested on cultured human cells human cervix carcinoma cells (HeLa229); inhibition of RNA synthesis Induction of mutation (genotoxicity) was assessed using a bacteriological assay (Ames test) Tester strain Salmonella typhimurium TA102 Marja Pitkänen

11 Sample preparation for toxicological testing Samples were initially dispersed into water Samples were subjected to UV radiation and subsequent dispersion in ethanol for microbiological cleansing Dry sample Contaminated with molds and bacteria UV treatment 254 nm, 16 h Sample (10% w/w) dispersed into EtOH 94% Diluted into culture medium Microbiologically clean sample Marja Pitkänen

12 Effect of nanocellulose on cellular morphology, protein content and rate of RNA synthesis Sample Cytotoxicity Sublethal effects HTD Hepa-1 HTD HaCaT HTD HeLa229 RNA synthesis inhibition assay Whisker-type UFC no changes in cells morphology no changes in cells morphology Not tested Not tested Fibrillar NFC no changes in cells morphology no changes in cells morphology no changes in cells morphology Did not reduce mrna synthesis in HeLa229 cells Highest doses Fibrillar NFC: 0.24 mg/ml Whisker-type UFC: 2mg/ml Marja Pitkänen

13 Effect of nanocellulose on cellular growth - Total Protein Content (TPC) assay Fibrillar NFC TPC HaCat 72h toxic Whisker-type UFC TPC HaCat 72h toxic Fibrillar NFC TPC Hepa-1 72h Whisker-type UFC TPC Hepa-1 72h nontoxic nontoxic nontoxic nontoxic toxic toxic Source: Date Marja Pitkänen

14 Assessment of mutagenicicity in strain TA102 in the Ames test (72h exposure) Fibrillar NFC Whisker-type UFC toxic toxic nontoxic nontoxic Marja Pitkänen

15 Summary The cytotoxic and genotoxic properties of two nanomaterials with different characteristics were studied in well-characterized in vitro model systems commonly applied in toxicity testing protocols The overall analysis indicated absence of cytotoxic and genotoxic properties of the nanomaterials, whereas consistently, effects were seen with the positive control agents Marja Pitkänen

16 Acknowlegments This work has been done in the Re-engineering Paper project belonging to the Intelligent and Resource Efficient Production Technologies (EffTech) program of the Forestcluster Ltd. Forestcluster Ltd Links: and portal.forestcluster.fi/ Marja Pitkänen