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
2
VTT Technical Research Center of Finland, P.O.Box 1000, FI-02044 VTT, Finland
BioSafe Special Laboratory Services Ltd, P.O.Box 1199, FI-70211 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 20-60 nm, but there are a lot of fibrils
also in the size range of 100-350 nm. Since the 1P Masuko NFC has been grinded only once,
there are also some unfibrillated fibers.
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 20-100 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 400-500 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 21 §§ 186.1673 Pulp. Available in Internet (5.7.2010):
http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?c=ecfr&tpl=/index.tpl
2. Federal Institute for Risk Assessment. BfR Recommandations on Food Contact Materials.
XXXVI Paper and board for food contact (1.6.2009). Available in Internet (5.7.2010):
http://bfr.zadi.de/kse/faces/resources/pdf/360-english.pdf
3. Elintarviketurvallisuusvirasto Evira. Elintarvikkeiden lisäaineiden E-koodiavain (in Finnish)
Available in Internet (5.7.2010):
http://www.evira.fi/uploads/WebShopFiles/1197635026443.pdf
4. O’Connor B. Ensuring the safety of manufactured nanocrystalline cellulose. Presentation in
OECD Conference July 15-17 2009, 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, 15-17 July 2009.
6. 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 5.7.2010.
7. 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 189:235-241.
Nanofibrillar cellulose – Assessment of
cytotoxic and genotoxic properties in
vitro
Marja Pitkänen, Asko Sneck, HansPeter Hentze, Jenni Sievänen, Jaakko
Hiltunen, Erkki Hellén
Ulla Honkalampi, Atte von Wright
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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Emergence of new materials sets new demands for
product safety
•
Nanomaterials will improve the performance of many products in
future
•
•
The biological effects of nanomaterials, including nanocelluloses,
cannot be predicted from the chemical composition
•
•
However, properties specific to nanomaterials might expose humans and
environment to new risks
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
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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2
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 15-17 2009
•
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) 235–241.
•
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
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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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%
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
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Two different types of cellulose
Fibrillar NFC
Whisker-type UFC
20 µm
Long and curly fibrils with a high
aspect ratio and wide size range
•
•
Diameter: typically 20-60 nm, with
large fraction at 100-350 nm
30 µm
Whisker-like short fibrils
•
Diameter: Smallest fibrils 20-100 nm,
but also fiber fragments (up to µm range)
•
Fibril length: Smallest fibrils 400-500 nm
Fibril length: up to several µm, also
some fiber fragments
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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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
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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Clear difference in rheological properties between
the materials
Large particles
Small particles
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
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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
•
•
•
•
Sublethality was tested on cultured human cells
•
•
human keratinocyte line (HaCaT)
human cervix carcinoma cell line (HeLa229)
mouse hepatoma cell line (Hepa1)
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
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
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Sample preparation for toxicological testing
Dry sample
Contaminated with
molds and bacteria
•
•
Samples were initially dispersed
into water
Samples were subjected to UV
radiation and subsequent
dispersion in ethanol for
microbiological cleansing
UV treatment
254 nm, 16 h
Sample (10% w/w)
dispersed into
EtOH 94%
Diluted into culture
medium
Microbiologically clean
sample
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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Effect of nanocellulose on cellular morphology,
protein content and rate of RNA synthesis
Sample
Cytotoxicity
HTD
Hepa-1
Sublethal effects
HTD
HaCaT
HTD
HeLa229
RNA synthesis
inhibition assay
no changes in
Whisker-type UFC
cells’
morphology
no changes in
cells’
morphology
Not tested
Not tested
no changes in
cells’
morphology
no changes in
cells’
morphology
no changes in
cells’
morphology
Did not reduce
mRNA synthesis in
HeLa229 cells
Fibrillar NFC
Highest doses
• Fibrillar NFC: 0.24 mg/ml
• Whisker-type UFC: 2mg/ml
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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Effect of nanocellulose on cellular growth - Total Protein
Content (TPC) assay
Fibrillar NFC – TPC HaCat 72h
Whisker-type UFC – TPC HaCat 72h
nontoxic
nontoxic
toxic
toxic
Fibrillar NFC – TPC Hepa-1 72h
Whisker-type UFC – TPC Hepa-1 72h
nontoxic
nontoxic
toxic
toxic
Source:
Date
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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Assessment of mutagenicicity in strain TA102 in the
Ames test (72h exposure)
Whisker-type UFC
Fibrillar NFC
toxic
toxic
nontoxic
nontoxic
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Marja Pitkänen
27.9.2010
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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
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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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: www.forestcluster.fi/ and portal.forestcluster.fi/
2010 Intl Conf on Nanotechnology for the Forest Products Ind
Marja Pitkänen
27.9.2010
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