ISO/TR 19057:2017

TECHNICAL ISO/TR
REPORT 19057
First edition
2017-10
Nanotechnologies — Use and
application of acellular in vitro
tests and methodologies to assess
nanomaterial biodurability
Nanotechnologies — Utilisation et application des tests in vitro sur
cellules et méthodes pour évaluer la biodurabilité des nanomatériaux
Reference number
©
ISO 2017
© ISO 2017, Published in Switzerland
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ii © ISO 2017 – All rights reserved

Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 2
5 Background including need for assessing the biodurability of particles .4
6 Aims and objectives . 6
7 Approaches for assessment of micrometre mineral particle and fibre biodurability .6
7.1 General . 6
7.2 Dissolution of nanomaterials versus their dispersion and biodegradation . . 7
8 Need for the assessment of nanomaterial biodurability . 7
9 Influence of different types of ligands and coatings on nanomaterial biodurability .8
10 Review of methodologies to assess micrometre mineral particle and fibre biodurability .8
10.1 General . 8
10.2 In vitro acellular methods . 8
10.3 Description of different simulated physiological media . 9
10.3.1 General. 9
10.3.2 Simulated lung airway lining fluids . 9
10.3.3 Simulated lung macrophage phagolysosomal fluid .10
10.3.4 Digestive system (saliva, gastric and intestinal fluids) .10
10.3.5 Simulated sweat (SSW) .11
10.4 Description of different simulated environmental media .11
10.4.1 General.11
10.4.2 Simulated natural freshwaters .11
10.4.3 Simulated seawater .11
10.4.4 Simulated estuarine waters .11
10.5 Description of different test systems to assess dissolution of particles and fibres .11
10.5.1 General.11
10.5.2 Static dissolution system .12
10.5.3 Continuous flow system (CFS) .12
10.5.4 Batch and batch filter systems .12
10.5.5 Tangential flow filtration system .12
10.6 Assessment of dissolved mass concentration post dissolution experiment . .13
10.6.1 General.13
10.6.2 Techniques based on physical principles .13
10.6.3 Techniques based on mechanical concepts .14
10.6.4 Techniques based on chemical principles .15
10.6.5 Ultraviolet-visible (UV-Vis) spectroscopy .16
10.6.6 One-dimensional mathematical models .16
10.6.7 Single particle inductively coupled plasma-mass spectrometry (spICP-MS) .16
11 Calculation of micrometre mineral particle biodurability .17
11.1 General .17
11.2 Dissolution kinetics, dissolution rates, and dissolution rate constants .18
11.3 Dissolution kinetics and dissolution rate of larger particles and fibres .18
11.4 Dissolution kinetics and dissolution rate of nanoparticles .20
11.5 Assessment of halftime estimates of particles and fibres .20
11.6 Assessment of lifetime estimates for particles and fibres .21
11.6.1 General.21
11.6.2 Shrinking sphere theory .21
11.6.3 Shrinking fibre theory .22
11.7 Assessment of halftime and lifetime estimates .23
12 Examples of micrometer mineral particles and fibres where biodurability was
assessed using in vitro acellular systems .24
12.1 Glass and asbestos fibres .24
12.2 Silicon dioxide (SiO ) .24
12.3 Talc .25
12.4 Tungsten oxide .25
12.5 Beryllium .25
13 Examples of nanomaterials where biodurability was assessed using in vitro
acellular systems .25
13.1 SWCNTs and MWCNTs .25
13.2 Silver nanoparticles (AgNPs) .26
13.3 Titanium dioxide (TiO ) .26
13.4 Zinc oxide (ZnO) .26
14 Biodurability of ligands .27
14.1 General .27
14.2 Examples of ligands attached to particles where biodurability has been assessed .27
14.3 Methodologies to assess the biodurability of the attached ligands .27
14.3.1 General.27
14.3.2 Gel permeation chromatography (GPC) .27
14.3.3 Matrix-assisted laser desorption ionization mass spectrometer (MALDI-MS) .28
14.3.4 Attenuated total reflectance-Fourier transform infrared
spectroscopy (ATR-FTIR) .28
14.3.5 Liquid chromatography coupled with mass spectrometry (LC-MS/MS) .28
15 Relationship with relevant international documents .28
15.1 Simulated sweat .28
15.2 Simulated sebum .29
15.3 Simulated lung fluids .29
15.4 Simulated digestive system fluids .29
16 Assessing the validity of assay/test systems .29
17 Biological relevance of the dissolution assay .30
18 Use of biodurability tests in risk assessment and its limitations .31
Annex A (informative) Tables of relevant information .32
Bibliography .36
iv © ISO 2017 – All rights reserved

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
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World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
TECHNICAL REPORT ISO/TR 19057:2017(E)
Nanotechnologies — Use and application of acellular in
vitro tests and methodologies to assess nanomaterial
biodurability
1 Scope
This document reviews the use and application of acellular in vitro tests and methodologies implemented
in the assessment of the biodurability of nanomaterials and their ligands in simulated biological and
environmental media.
This document is intended to focus more on acellular in vitro methodologies implemented to assess
biodurability and, therefore, excludes the general review of relevant literature on in vitro cellular or
animal biodurability tests.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
bioaccumulation
process of accumulation of a substance in organisms or parts
[SOURCE: ISO/TR 13329:2012, 3.3]
3.2
biodegradation
degradation due to the biological environment
Note 1 to entry: Biodegradation might be modelled by in vitro tests.
[SOURCE: ISO/TR 13329:2012, 3.4]
3.3
biodurability
ability of a material to resist dissolution (3.6) and mechanical disintegration from chemical and physical
clearance mechanisms
[SOURCE: ISO/TR 13329:2012, 3.5, modified]
3.4
biopersistence
ability of a material to persist in a tissue in spite of the tissue’s physiological clearance mechanisms and
environmental conditions
[SOURCE: EN 18748:1999]
3.5
dispersion
microscopic multi-phase system in which discontinuities of any state (solid, liquid or gas: discontinuous
phase) are dispersed in a continuous phase of a different composition or state
[SOURCE: ISO 26824:2013, 16.5]
3.6
dissolution
process of obtaining a solution containing the analyte of interest
Note 1 to entry: Dissolution is the act of dissolving and the resulting species may be molecular or ionic.S
[SOURCE: ISO 17733:2015, 3.4.10, modified]
3.7
ligands
atoms or groups joined to the central atom
[SOURCE: IUPAC Recommendations 1994]
3.8
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
Note 2 to entry: See also engineered nanomaterial, manufactured nanomaterial and incidental nanomaterial.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.9
nanoparticle
nano-object with all three external dimensions in the nanoscale
Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically by
more than three times), the terms nanorod or nanoplate are intended to be used instead of the term nanoparticle.
[SOURCE: ISO/TS 27687:2008, 4.1]
3.10
specific surface area for powders
absolute surface area of the sample divided by sample mass and is therefore expressed in units of
m /g or kg
[SOURCE: ISO 9277:2010, 3.11, modified]
3.11
suspension
heterogeneous mixture of materials comprising a liquid and a finely dispersed solid material
[SOURCE: ISO/TS 80004-6:2013, 2.13]
4 Symbols and abbreviated terms
AAS Atomic absorption spectroscopy
AF4 Asymmetrical flow field flow fractionation
AgNPs Silver nanoparticles
2 © ISO 2017 – All rights reserved

ATR-FTIR Attenuated total reflectance-Fourier transform infrared spectroscopy
AuNPs Gold nanoparticles
CdSe /ZnS Cadmium-selenium (core) and zinc sulfide (shell)
core shell
CE Capillary electrophoresis
CFS Continuous flow system
CNTs Carbon nanotubes
CPE Cloud-point extraction
CuONPs Copper oxide nanoparticles
Da Dalton
DMEM Dulbecco's Modified Eagle's Medium
DOM Dissolved organic matter
EPA US. Environmental Protection Agency
FFFF Flow field flow fractionation
FTIR Fourier transform infrared spectroscopy
GC-MS Gas chromatography–mass spectrometry
GPC Gel permeation chromatography
HPCIC High-performance chelation ion chromatography
HRP Horseradish peroxidase
ICP-MS Inductively coupled plasma-mass spectrometry
ICP-OES Inductively coupled plasma-optical emission spectrometry
SNMS/TOF-SIMS Secondary neutral mass spectrometry/time-of-flight secondary ion mass
spectrometry
LC-MS Liquid chromatography-mass spectrometry
LC-MS/MS Liquid chromatography-mass spectroscopy-mass spectroscopy
MALDI-TOF-MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
m/z Mass-to-charge ratios
MWCNTs Multi-wall carbon nanotubes
NaCl Sodium chloride
NP Nanoparticle
NIOSH U. S. National Institute for Occupational Safety and Health
NSL Nanosphere lithography
PAP Pulmonary alveolar proteinosis
PBPK Physiologically-based pharmacokinetic
PSF Phagolysosomal simulant fluid
QDs Quantum dots
QQQ Triple quadrupole
QTOF Quadrupole time-of-flight
REACH Registration, evaluation, authorisation and restriction of chemicals
RPMI Roswell Park Memorial Institute
SDS Sodium dodecyl sulfate
SiO Silicon dioxide
SLF Simulated lung fluid
spICP-MS Single particle inductively coupled plasma-mass spectrometry
SPE Solid-phase extraction
SPF Simulated physiological fluids
SPR Surface Plasmon Resonance
SS Simulated saliva
SSW Simulated sweat
SUF Simulant ultrafiltrate fluid
SWCNTs Single-wall carbon nanotubes
TBOs Tungsten blue oxides
TiO NPs Titanium dioxide nanoparticles
UV-Vis spectroscopy Ultraviolet-visible spectroscopy
WO Tungsten trioxide
ZnONPs Zinc oxide nanoparticles
5 Background including need for assessing the biodurability of particles
The tendency of a given inhaled particle or fibre to cause chronic disease is strongly related to the
duration of residence time in the pulmonary environment. Biopersistent particles and fibres are defined
as materials that resist clearance from the body through physical and chemical means. Resistance
to physical clearance may result through their resistance to phagocytosis by alveolar macrophages.
Resistance to clearance by chemical and physical means, also known as biodurability, may result
through their resistance to chemical dissolution. Particles and fibres that are amenable to dissolution
may release components which may contribute to their adverse health effects. Similar resistance to
chemical dissolution and/or biodegradation of the attached ligands in environmental media may also
produce biodurable nanomaterials.
Historical experiences with micrometre-scale mineral particles and fibres provide a useful model for
understanding the relationship between exposure, dose, and effect of nanomaterials in the human lung.
It has been shown that the residence time of mineral particles and fibres in the lung depends on their
4 © ISO 2017 – All rights reserved

mechanical clearance and the dissolution rates. The dissolution rate is governed mainly by particle and
fibre chemistry and the properties of the biological fluid of the tissue/cell environment in which they
are found.
Exposure
Internal dose
Clearance
Biopersistence
Biodurability
Effect
[ ]
Figure 1 — Relationship between exposure, dose, and effect of nanomaterials 1
[1]
Figure 1 presents a general framework proposed by Oberdörster et al. to evaluate potential adverse
effects from exposure to particles and fibres. According to this framework, biopersistence of the
particles and fibres is considered to be central to the production of health effects.
The respiratory tract may be divided into different zones, including the ciliated nasal and
tracheobronchial regions and the non-ciliated alveolar regions. Particles that deposit in each zone
interact with different cell populations with substantially different retention times and/or different
[2]
clearance pathways . The behaviour of inhaled particles in the respiratory tract and their alternative
fates of either deposition or exhalation in these various functional zones may depend upon the chemical
composition and the physical behaviour of the aerosol particles.
The importance of airway ciliated cells and alveolar macrophages in the clearance of micrometer-sized
particles from the lung surface has long been known. Due to the limited capability of macrophages
to recognize nanomaterials, inadequacy of this key clearance mechanism in peripheral lungs
[3][4]
was demonstrated . Concurrently, due to endocytotic processes and trans-cellular transport
mechanisms by other cells, translocation of nanomaterials to extra pulmonary organs has become
prominent. Biopersistence of nanomaterials through their resistance to clearance by alveolar
macrophages and also to their biodurability through resistance to breakage or dissolution and leaching
may therefore lead to their bioaccumulation. With their ability to translocate nanomaterials may
accumulate and be retained in critical target organs with the subsequent production of adverse health
effects. Translocation (disposition), accumulation and retention are, therefore, important aspects that
need to be considered when investigating the long-term toxicity of nanomaterials.
It is now well accepted that the toxicity of nanomaterials may very much be related to their
physicochemical properties including size, surface area, and surface characteristics including surface
chemistry. Once inside the cell, these and other physicochemical characteristics (surface composition,
surface activity) will determine their interaction with biological surroundings. The latter, in turn,
will determine the stability and biodurability of the surface and core of nanomaterials. Given the
strong sensitivity of many nanomaterial properties to their local environment, it should be noted
that biologically relevant changes in the physicochemical properties of a nanomaterial between
administration and deposition may have a significant impact on observed responses. As a result,
biodurability of nanomaterials in their local biological environments may impact on their long-term
toxicity.
Although the biodurability of certain larger particles can also be confirmed in different environmental
media, the importance of such biodurability in their long-term effects might not be of relevance in aquatic
organisms. This is shown to be the case with crystalline silica (quartz and cristobalite) for the fact that
they do not bioaccumulate in these biorganisms due to their very limited potential for uptake through
[5][6]
the gill or gut of fish . This might not be true for nanomaterials as the bioaccumulation of multiple-
[7]
wall carbon nanotubes (MWCNTs) has been shown in Daphnia magna, an aquatic invertebrate , of
[8]
gold nanoparticles (AuNPs) in clams marine bivalve Scrobicularia plana (Pan et al., 2012) and zinc
[9]
oxide nanoparticles (ZnONPs) in S. plana and Nereis diversicolor, copper oxide nanoparticles (CuONPs)
[10][11] [12]
in freshwater snail , titanium dioxide nanoparticles (TiO NPs) in Daphnia magna , and silver
[13]
nanoparticles (AgNPs) in deposit feeder such as the annelid Platynereis dumerilii have been shown.
6 Aims and objectives
Different nanomaterials are reported to be biopersistent as they resist breakage or dissolution and
clearance which in turn can lead to their bioaccumulation and subsequently their translocation and
distribution. It is, therefore, of great relevance to assess this property of nanomaterials and determine
their degradation half-lives in addition to their other properties. It is also of great relevance to study
the impact of surface coating and functional groups on biodurability in biological and environmental
surroundings.
The aim and objectives are
— the identification of tests performed and the methodologies implemented in the literature, and
— the description of the identified methodologies in the assessment of the biodurability of different
nanomaterials in different biological and environmental media.
The ultimate goal is that, by applying the identified methodologies, it is possible to assess the
biodurability of nanomaterials and their surface ligands. This is relevant in the assessment of their
long-term effects.
7 Approaches for assessment of micrometre mineral particle and fibre
biodurability
7.1 General
The toxicity of inhaled fibres is believed to be related to dose, biodurability and to their dimensions, with
long, thin fibres being potentially more carcinogenic than short, thick ones and to their biopersistence,
[14]
as fibres which dissolve rapidly in the lung are unlikely to induce long-term pathological changes .
The concept of biopersistence – resistance to clearance and to chemical dissolution - has therefore been
proposed as a key concept in the toxicity of mineral or synthetic fibres.
[15]
The critical role of dissolution on the potential health effects of inhaled fibres is well established .
Over the last several decades, there have been numerous publications on the relation between various
physicochemical characteristics, including chemical composition and diameter, of a synthetic vitreous
[16][17][18]
fibre and its dissolution rate in physiological saline solution .
Numerous animal in vivo experiments are described in the literature to assess clearance and chemical
instability, through their solubility, of different mineral and man-made synthetic fibres. For example,
particle clearance has been studied as a measure of physical persistence - the actual amount of fibre
remaining in the tissue and their solubility has been assessed through the release of metal constituents
[19][20][21][22][23]
in experimental animals .
Also, in vitro cellular systems have been described to study the chemical stability of particles using
macrophages, epithelial, and mesothelial cells where differences could be observed in the efficiency
6 © ISO 2017 – All rights reserved

of these cell types to change the chemical structure of the engulfed fibres emphasizing the differences
[24][25][26][27]
between phagosomal conditions between these cells .
In vitro acellular systems mimicking physiological conditions have been developed to study the release
of chemical constituents from fibres. Using different biological media simulants, cell-free chemical
dissolution assays were thought to provide valuable information on the behaviour of respired dusts in
biological surroundings.
A combination of the in vitro dissolution tests, cell-based assays (mono- and co-culture systems), in
silico methods, and information from existing in vivo studies, can provide a measure of biopersistence of
mineral particles and fibres. This document is limited to in vitro acellular systems and their applications
to nanomaterials.
7.2 Dissolution of nanomaterials versus their dispersion and biodegradation
The terms dissolution and dispersion have been defined with a distinction made between these two
[28]
terms . In this document, dissolution “denotes to the process of obtaining a solution containing the
analyte of interest. Dissolution is the act of dissolving and the resulting species may be molecular or
ionic” and dispersion “refers to the microscopic multi-phase system in which discontinuities of any
state (solid, liquid or gas: discontinuous phase) are dispersed in a continuous phase of a different
composition or state”. As per this definition, dissolution of nanomaterials will entail release of ions
to the surrounding solvent where the rate of dissolution will be dependent on size, chemistry, solvent
composition, and surface coating or functionalization of nanomaterials.
Dissolution of nanomaterials is an important property and is often a critical step in determining their
[29]
safety . Dissolution of metal- and metal oxide-based nanomaterials follows thermodynamic and
kinetic rules where size, shape, surface coating, aggregation state, and solution chemistry such as
pH, ionic components, and dissolved organic matter (DOM) affect the rate of their dissolution. As the
toxicity of the dissolved form can have different toxicological effects than the particulate, monitoring
[30]
dissolution kinetics is important . Subsequently, the determination of dissolution and dissolution
kinetics are recommended as part of the minimum requirements for nanomaterials to be characterized.
Biodegradation of organic and carbon-based nanomaterials on the other hand, is achieved through
enzymatic catalysis with subsequent complete breakdown and loss of nanomaterials characteristics.
The amenability to biodegradation seemed to be dependent on the type of surface functional groups
[31]
that they may carry .
8 Need for the assessment of nanomaterial biodurability
Biopersistence is one of the characteristics which are seen to determine the toxicity/pathogenicity/
[32]
carcinogenicity of ultrafine particles and also of nanomaterials . These characteristics are also
seen to alter their fate and biological distribution. Due to their small size, nanomaterials are likely
to translocate beyond the epithelial barrier into the interstitium where they accumulate for a long
[33][34][35]
time due to their resistance to phagocytic uptake . Those which are biodurable may cause
pulmonary inflammation, fibrosis, and cancer. Some of the interstitialized nanomaterials could be
translocated into the systemic circulation and some may induce impairment in extrapulmonary organs.
Once translocated, evidence seems to suggest that nanomaterials preferentially deposit in liver and
[36]
spleen resulting in prolonged retention and in some instances producing significant hepatotoxicity.
Biodurable nanomaterials maintain their particulate state which might increase the potential for their
[37]
bioaccumulation . Release of ions from nanomaterials that are soluble have also been shown to be
strongly associated with their toxicity while acute toxicological responses arise from nanomaterials
[38]
with high dissolution through released ions , low dissolution and non biodegradable nanomaterials
[39]
might provoke a range of long-term effects including carcinogenicity . When the sparingly soluble
nanomaterials are made soluble through surface functionalization or surface coating, when in contact
with body fluids, they might also disintegrate/dissolve to eventually exposing the biodurable core.
9 Influence of different types of ligands and coatings on nanomaterial
biodurability
The importance of surface properties of micrometre mineral particles and fibres was recently
emphasized as an indirect but critical factor in the manifestation of pathogenic activity as well as
in their biodurability under conditions of in vitro dissolution in biological fluids. Subsequently, in a
publication by the US. National Institute of Occupational Safety and Health (NIOSH), surfaces of mineral
particles and fibres were thought to be a controlling factor in their biopersistence, which is seen to be a
critical aspect in the mechanisms of continuing irritation or inflammatory response in causing fibrosis
[40]
or neoplastic transformation . For example, it could be shown that surface composition and surface-
associated activities of asbestos fibres were the major contributors to their potential to induce disease.
Similar observations have been made for crystalline silica in which surface modification by chemical
[41][42][43]
means has also been shown to alter their cytotoxicity .
Manipulation of surface properties of nanomaterials have also been investigated through surface
functionalization with a range of molecular groups for a number of reasons including an increase in
water dispersability to an otherwise non-water dispersible nanomaterials and for the increase of their
intracellular uptake with their subsequent accumulation in cellular lysosomes. As noted previously,
surface chemistry is an important nanomaterial property that will influence interactions with
biological fluids. The surface chemistry of nanomaterials can be changed drastically upon immersion in
[44]
a biological fluid . It is now well recognized that modification of nanomaterial surface chemistry can
yield novel properties and behaviours. Such modifications can also be used to promote biodurability
and minimize health risks. For example, accumulation in cellular lysosomes could be shown for dextran-
coated magnetic iron oxide nanoparticles as well as for single-wall carbon nanotubes (SWCNTs)
where they could be enzymatically degraded by lysosomal R-glucosidase, horseradish peroxidase,
myeloperoxidase, and heme oxygenase-1. This type of enzymatic degradation, however, might not be
possible for inert nanoparticles such as gold and therefore once inside the cell, they can deposit for
[45]
longer periods . Similar studies with carbon nanotubes (CNTs) have indicated that carboxylated
[46]
CNTs are more susceptible to biodegradation compared to other CNTs .
Loss of ligands from the surface of semiconductor nanocrystals in gastrointestinal fluids at low pH was
[47]
also reported with the subsequent increase in their toxicity . Moreover, the effect of this biological
fluid on the PEGylated quantum dots (QDs) was also investigated and showed that the fate of PEGylated
Cadmium-selenium (core) and zinc sulfide (shell) (CdSe /ZnS ) QDs depended on pH, ligand chain
core shell
[48][49]
length and presence of proteins and subsequently affecting their toxicity .
10 Review of methodologies to assess micrometre mineral particle and fibre
biodurability
10.1 General
Adaptation of traditional dissolution rate studies to mimic the physical and chemical conditions
encountered in biological tissues and organs allows investigators to estimate biodurability through
calculation of residence times based on the chemical dissolution mechanism. Particle and fibre
dissolution and breakdown in the body are important determinants of their biopersistence. The
physical and chemical mechanisms whereby particles and fibres may release ions through dissolution
and/or degrade in biological tissues and organs have been studied extensively in vitro using “simulated
biological fluids” (see Table A.1 to Table A.5). Such in vitro tests provide information on the biodurability
[50]
which impacts the biological effects of particles and fibres . In vitro dissolution tests have also
been suggested for screening for the biodurability of nanomaterials in environmental media (natural
[51][52]
freshwaters, simulated seawater, and simulated estuarine waters) (see Table A.8) .
10.2 In vitro acellular methods
In vitro acellular methods are comprised of two components, the simulated biological fluid and the
system to hold and retain the study material. In this first section, various simulated biological fluids
are described and in the next section available test systems are reviewed.
8 © ISO 2017 – All rights reserved

Over the last several decades, numerous publications have used various simulated biological fluids to
assess dissolution as a measure of biodurability of particles and fibres. Several studies have tried to
establish a relation between physicochemical characteristics such as chemical composition, density,
[53][15]
and diameter of a synthetic vitreous fibre and their dissolution rate in physiological fluids .
Several publications have also assessed the impact of different properties of biological fluids on the
dissolution of particles and fibres. These properties include pH 7,3 of the extracellular lung fluid and pH
4,5 of macrophage phagolysosomal fluid, presence of surfactants (e.g lung lining fluid), and presence of
certain biological compounds, including organic chelators such as sodium citrate that selectively bind
to surface ions (see Tables A.1 to A.5).
10.3 Description of different simulated physiological media
10.3.1 General
In this section, simulated biological fluids are described that are intended to mimic the lung and oral
as well as dermal exposure pathways. For the lung, generally there are two main compartments to
consider – the extracellular airway lining fluid having near neutral pH and the phagolysosomal fluid
of macrophage cells having acidic pH. For the oral route, particles will briefly come into contact with
saliva followed by gastric and interstitial fluids. To describe adequately the dissolution behaviour of
materials in the body, it is imperative that the simulant reflects the biochemical composition of the
fluids in the organ or tissue being modelled. The most common simulants used were therefore based
[54]
on the ionic composition of lung extracellular fluid described by Gamble which is near neutral pH
(7,2 to 7,4). Gamble's simulant fluid was also called Ringer's solutions, simulant ultrafiltrate fluid (SUF),
[55][56]
or simulant lung fluid (SLF) . The majority of experiments have, therefore, been conducted at
physiological pH (7,3), either at 25 °C or 37 °C. With evidence that dissolution of insoluble particles
occurs predominantly in phagolysosomes, investigators began to adjust the pH of lung extracellular
fluid-simulants to match that of the phagolysosome, i.e. pH 4,5 to 5.
Therefore, the simulated physiological fluids (SPF) that are discussed below have included: (1) the
lung airway lining simulant, (2) the phagolysosomal stimulant fluid, (3) the simulated saliva, (4) the
gastrointestinal fluids, as well as (5) sweat the composition of all of which are presented in Annex A.
For more information on the composition of various biological fluids and their simulants, the reader is
[57]
referred to Marques et al. .
While these SPF are considered useful analogues of human biofluids, they all suffer from the same basic
limitations. Firstly, SPFs have defined compositions and lack the dynamic conditions present in vivo.
For example, none of these fluids contain enzymes or oxidative cascades which can be important in
dictating the properties (composition, pH) of a SPF or biodurability of nanomaterials such as carbon
[58]
nanotubes . Additionally, unless specifically noted, proteins are omitted from most SPF for pragmatic
reasons (see 10.5.3); however, in vivo, proteins might serve as important binding molecules for dissolved
ions and influence their concentration at nanomaterial surfaces.
10.3.2 Simulated lung airway lining fluids
Aerosol materials that deposit in the lung are quickly immersed in the extracellular fluid that is excreted
[54]
by the lung tissue. The original composition of physiological fluids described by Gamble in 1967 has
served as a basis for a number of experimental derivations for human extracellular fluid, including the
[59] [60]
experimental solvents used by Scholze and Conradt and Kanapilly et al. .
Gamble’s Solution is a type of simulated lung airway lining fluid that is intended to mimic the surfactant
fluids released by Type II alveolar cells. The fluid fills the space between a
...