Iso TR 10993-22-2017

TECHNICAL ISO/TR
REPORT 10993-22
First edition
2017-07
Biological evaluation of medical

devices —
Part 22:
Guidance on nanomaterials
Évaluation biologique des dispositifs médicaux —
Partie 22: Lignes directrices sur les nanomatériaux
Reference number
ISO/TR 10993-22:2017(E)
© ISO 2017
ISO/TR 10993-22:2017(E)
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ii © ISO 2017 – All rights reserved

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Contents Page
Foreword .................................................. ................................................... ................................................... ................................................... ............................... v
Introduction .................................................. ................................................... ................................................... ................................................... ..................... vi
1 Scope .................................................. ................................................... ................................................... ................................................... ...................... 1
2 Normative references .................................................. ................................................... ................................................... .............................. 2
3 Terms and definitions .................................................. ................................................... ................................................... ............................. 2

4 General principles .................................................. ................................................... ................................................... ....................................... 5
4.1 General considerations .................................................. ................................................... ................................................... ............ 5
4.2 Biological evaluation of nanomaterials ................................................... ................................................... ....................... 6
4.3 Categorization of nanomaterials .................................................. ................................................... ........................................ 6
4.4 Nanomaterial equivalence ................................................... ................................................... ................................................... ... 8
5 Characterization of nanomaterials ................................................... ................................................... ............................................. 8
5.1 General considerations .................................................. ................................................... ................................................... ............ 8
5.2 Characterization parameters and methods ................................................... ................................................... ........... 10
5.3 Use of reference materials ................................................... ................................................... ................................................... 14
6 Sample preparation .................................................. ................................................... ................................................... ................................ 1 5
6.1 General considerations .................................................. ................................................... ................................................... ......... 15
6.2 Special considerations for nanostructured materials .................................................. ...................................... 15
6.3 Special considerations for nano-objects .................................................. ................................................... ................... 15
6.4 I dentity, s to rage and s tab ility o f s to ck nano materials .................................................. ..................................... 17
6.5 D es crip tio n o f chemical co mp o s itio n o f s to ck and do s ing dis p ers io ns .............................................. 17
6.6 C haracterizatio n o f s to ck dis p ers io ns .................................................. ................................................... ......................... 18
6.7 C haracterizatio n o f do s ing s o lutio ns p rep ared f ro m s to ck dis p ers io ns ............................................ 18
6.8 Dose metrics .................................................. ................................................... ................................................... ................................... 19
6.9 Additional considerations .................................................. ................................................... ................................................... .. 19
6.9.1 Endotoxin .................................................. ................................................... ................................................... .................... 19
6.9.2 Sterilization .................................................. ................................................... ................................................... ............... 20
7 Release of nano-obj ects from medical devices ................................................... ................................................... ............ 2 1
7.2 Degradation products .................................................. ................................................... ................................................... ............ 22
7.3 Releas e of nano - o b j ects by wear .................................................. ................................................... ..................................... 22
7.4 In situ processing ................................................... ................................................... ................................................... ...................... 22
8 Toxicokinetics .................................................. ................................................... ................................................... ............................................... 2 2
8.2 Facto rs influencing the toxico kinetics .................................................. ................................................... ........................ 23
8.2 .1 Phys ico chemical p ro p erties .................................................. ................................................... ........................... 23
8.2.2 Biomolecular adsorption .................................................. ................................................... .................................. 24
8.2.3 Exposure route .................................................. ................................................... ................................................... ....... 25
8.2.4 Dose .................................................. ................................................... ................................................... .................................. 25
8.2.5 Species and gender .................................................. ................................................... ................................................ 25
8.2.6 Measurement techniques .................................................. ................................................... ................................. 26
9 Toxicological evaluation .................................................. ................................................... ................................................... .................... 2 6
9.2 In vitro cyto toxicity tes ting .................................................. ................................................... ................................................... 27
9.2.1 General considerations .................................................. ................................................... ....................................... 27
9.2 .2 C o ns ideratio n o f f
nano material inter erence with the as s ays ................................................ 28
9.2.3 Consideration of relevant dose and dose metrics .................................................. .......................... 28
9.2 .4 C o ns ideratio n o f nano - o b j ect kinetics ................................................... ................................................... . 28
9.3 Geno toxicity, carcino genicity and rep ro ductive toxicity .................................................. ................................ 29
9.3.2 In vitro geno toxicity tes ts .................................................. ................................................... ................................. 30
9.3.3 In vivo geno toxicity tes ts .................................................. ................................................... ................................... 31
© ISO 2017 – All rights reserved iii
ISO/TR 10993-22:2017(E)
9.3 .4 C arcino genicity .................................................. ................................................... ................................................... ....... 32

9.3 .5 Rep ro ductive toxicity .................................................. ................................................... ........................................... 32
9.4 I mmuno toxicity, irritatio n and s ens itizatio n .................................................. ................................................... ........ 33
9 . 4. 2 I mmuno toxicity .................................................. ................................................... ................................................... ...... 33
9.4.3 Sensitization .................................................. ................................................... ................................................... ............. 34
9.4.4 Irritation .................................................. ................................................... ................................................... ....................... 35
9.5 H aemo co mp atib ility .................................................. ................................................... ................................................... ................ 36
9.5 .2 C o mp lement sys tem activatio n .................................................. ................................................... ................... 37
9.5 .3 S p ecific co ns ideratio ns f o r haemo co mp atib ility tes ting .................................................. .......... 37
9.6 Sys temic toxicity .................................................. ................................................... ................................................... ......................... 37
9.7 Pyro genicity.................................................. ................................................... ................................................... .................................... 38
9.8 Implantation ................................................... ................................................... ................................................... .................................. 39
10 Presentation of characterization and test results .................................................. ................................................... ..... 39
11 Risk assessment .................................................. ................................................... ................................................... ......................................... 40
11.2 Exposure assessment .................................................. ................................................... ................................................... ............. 42
1 1 .3 B io lo gical hazard identificatio n ................................................... ................................................... ....................................... 43
1 1 .4 Ris k es timatio n .................................................. ................................................... ................................................... ............................ 44
1 1 .5 Ris k evaluatio n .................................................. ................................................... ................................................... ............................. 44
12 Biological evaluation report .................................................. ................................................... ................................................... .......... 44
Bibliography .................................................. ................................................... ................................................... ................................................... .................. 46
iv © ISO 2017 – All rights reserved

ISO/TR 10993-22:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work o f 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 o f
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
di fferent types o f ISO documents should be noted. This document was dra fted 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 o f the elements o f this document may be the subject o f
patent rights. ISO shall not be held responsible for identi fying any or all such patent rights. Details o f
any patent rights identified during the development o f 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 in formation given for the convenience o f users and does not
constitute an endorsement.
For an explanation on the voluntary nature o f standards, the meaning o f ISO specific terms and
expressions related to con formity assessment, as well as in formation about ISO’s adherence to the
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 194, Biological and clinical evaluation of
medical devices .
A list of all parts in the ISO 10993 series can be found on the ISO website.
© ISO 2017 – All rights reserved v

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Introduction
This document is intended as guidance for the biological evaluation of medical devices that contain,
generate or are composed o f nanomaterials. Multiple definitions have been developed for the term
nanomaterial. For the purposes o f this document, the ISO definition will be used: A material is considered
a nanomaterial when it has a size at the nanoscale including external and internal dimensions, i.e. when
it has a size or is composed o f structures with a length o f approximately between 1 nm and 100 nm
(ISO/TS 80004-1:2015). For regulatory purposes, it is advisable to check i f specific national or regional
regulatory definitions are applicable. It should be realized that other characteristics (e.g. nanospecific
properties) might also be included in such definitions.
Morphological structures created on the surface of a medical device can also have sizes in the nanoscale.
Therefore, possible effects of such structures on the biological response to the device also need to be
considered.
Nano-objects having a length range from 1 nm to 100 nm can be generated during the li fe cycle o f a
medical device, so the evaluation of possible adverse effects due to the generation of nano-objects either
from preparation, use, wear or degradation of medical devices needs to be addressed. This applies to
medical devices manufactured using nanomaterials and medical devices that are manufactured not
using nanomaterials but having the potential to generate nanoscale wear and/or degradation particles.
For the biological evaluation o f medical devices, knowledge on the potential generation and/or release
of nano-objects from such materials is essential.
The procedures as described in the ISO 10993 series for the biological evaluation of medical devices
can be used for the biological evaluation of those medical devices that contain nano-objects that are
not released from such a device as they are an integrated part o f the device. However, when release
o f the nano-objects is possible, a sa fety evaluation should also be per formed on the released nano-
objects. In addition to evaluating a medical device, nanomaterial components or constituents can also
be separately evaluated.
This document provides trained professionals, in the context of medical device evaluation, a general
approach to biological evaluation of nanomaterials and addresses how the other parts of the ISO 10993
series can be used when dealing with the evaluation o f nanomaterials. It is likely that the various assays
as described in the ISO 10993 series are not always appropriate as such in the testing o f nanomaterials.
Nanomaterials by themselves can be present as powders or colloid dispersions, but also can be present
in medical devices while incorporated in a matrix, as nanostructured material or as surface structures
on materials and/or medical devices. In general, nanomaterials themselves need to be evaluated
instead o f extracts as usually used when testing biomaterials or medical devices. Nanomaterials pose
specific challenges when applying test systems commonly used for medical device evaluation and when
interpreting test results.
The field o f nanotechnology, development o f nanomaterials and the evaluation o f potential toxicity
o f such materials are emerging fields and this document represents only the knowledge at the time
of writing. Although appropriate tools and methods for evaluation of nanomaterials are still under
development, data on the characteristics and biological effects of nanomaterials should be provided in
order to address sa fety issues in their application in the medical device field, taking into consideration
a risk/benefit analysis.
This document provides guidance on how to perform a biological evaluation for those medical devices
that contain, generate, or are composed o f nanomaterials within a risk management process as
described in ISO 10993.
vi © ISO 2017 – All rights reserved

TECHNICAL REPORT ISO/TR 10993-22:2017(E)
Biological evaluation of medical devices —

Part 22:
Guidance on nanomaterials
1 Scope
This document describes considerations for the biological evaluation of medical devices that are
composed of or contain nanomaterials. In addition, this guidance can also be used for the evaluation
of nano-objects generated as products of degradation, wear, or from mechanical treatment processes
(e.g. in situ grinding, polishing of medical devices) from (components of) medical devices that are
manufactured not using nanomaterials.
This document includes considerations on the:
— characterization of nanomaterials;
— sample preparation for testing of nanomaterials;
— release of nano-objects from medical devices;
— toxicokinetics o f nano-objects;
— biological evaluation of nanomaterials;
— presentation of results;
— risk assessment o f nanomaterials in the context o f medical device evaluation;
— biological evaluation report;
— nanostructures on the sur face o f a medical device, intentionally generated during the engineering,
manufacturing or processing of a medical device.
The following are excluded from this document:
— natural and biological nanomaterials, as long as they have not been engineered, manu factured or
processed for use in a medical device;
— intrinsic nanostructures in a bulk material;
— nanostructures on the sur face o f a medical device, generated as an unintentional by-product during
the engineering, manufacturing or processing of a medical device.
NOTE Examples of unintentional nanostructures on the surface of a medical device are extrusion draw lines
and machining/tool marks.
This document is intended to provide a general framework and highlights important aspects which
need to be considered when assessing the sa fety o f medical devices composed o f, containing and/or
generating nano-objects. Additionally, the document identifies several common pit falls and obstacles
which have been identified when testing nanomaterials compared to bulk materials or small molecule
chemical species. As a technical report (TR), this document represents the current technical knowledge
related to nanomaterials. No detailed testing protocols are outlined or provided. This document can
serve as a basis for future documents containing detailed protocols with a focus on nanomaterial
testing.
© ISO 2017 – All rights reserved 1

ISO/TR 1 0993 -2 2 : 2 01 7(E)
2 Normative references
The following documents are re ferred to in the text in such a way that some or all o f their content
constitutes requirements o f this document. For dated re ferences, only the edition cited applies. For
undated re ferences, the latest edition o f the re ferenced document (including any amendments) applies.
ISO 10993 (all parts), Biological evaluation of medical devices
ISO/TR 13014, Nanotechnologies — Guidance on physico-chemical characterization of engineered
nanoscale materials for toxicologic assessment
ISO 14971, Medical devices — Application of risk management to medical devices

3 Terms and definitions
For the purposes o f this document, the terms and definitions given in ISO 10993 (all parts), ISO/TR 13014
and ISO 14971 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http://www.iso .org/obp
— IEC Electropedia: available at http://www.electropedia .org/
3 .1
aggregate
particle comprising strongly bonded or fused particles where the resulting external sur face area is
significantly smaller than the sum o f calculated sur face areas o f the individual components
Note 1 to entry: The Get
forces holding
more FREEan aggregate
standardstogether are strongSharing
from Standard forces, for example,
Group andcovalent bonds, or those
our chats
resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.5, modified — definition and Note 1 to entry changed]
3.2
agglomerate
collection o f weakly bound particles or aggregates (3.1) or mixtures of the two where the resulting
external surface area is similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces, or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4]
3.3
engineered nanomaterial
nanomaterial (3.7 ) designed for a specific purpose or function
[SOURCE: ISO/TS 80004-1:2015, 2.8]
3 .4
incidental nanomaterial
nanomaterial (3.7 ) generated as an unintentional by-product o f a process
Note 1 to entry: The process includes manu facturing, bio-technological or other processes.
2 © ISO 2017 – All rights reserved

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Note 2 to entry: See “ultrafine particle” in ISO/TR 27628:2007, 2.21.

[SOURCE: ISO/TS 80004-1:2015, 2.10]
3.5
manufactured nanomaterial
nanomaterial (3.7 ) intentionally produced to have selected properties or composition
[SOURCE: ISO/TS 80004-1:2015, 2.9]
3 .6
nanofibre
nano-object (3.8) with two similar external dimensions in the nanoscale (3.12) and the third dimension
significantly larger
Note 1 to entry: A nanofibre can be flexible or rigid.
Note 2 to entry: The two similar external dimensions are considered to di ffer in size by less than three times and
the significantly larger external dimension is considered to di ffer from the other two by more than three times.
Note 3 to entry: The largest external dimension is not necessarily in the nanoscale.
[SOURCE: ISO/TS 80004-6:2013, 2.6]
3 .7
nanomaterial
material with any external dimension in the nanoscale (3.12) or having internal structure or surface
structure in the nanoscale
Note 1 to entry: This generic term is inclusive o f nano-object (3.8) and nanostructured material (3.17).
Note 2 to entry: See also 3.3 to 3.5.
Note 3 to entry: For regulatory purposes, it is advisable to check i f specific national or regional regulatory
definitions are applicable. It should be realized that di fferent size ranges or other properties might be included in
such definitions.
[SOURCE: ISO/TS 80004-1:2015, 2.4, modified — Note 2 to entry modified and Note 3 to entry added]
3.8
nano - obj ect
discrete piece of material with one, two or three external dimensions in the nanoscale (3.12)
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3 .9
nanoparticle
nano-object (3.8) with all external dimensions at the nanoscale (3.12) where the length of the longest
and the shortest axes o f the nano-object do not di ffer significantly
Note 1 to entry: I f the dimensions di ffer significantly (typically by more than 3 times), terms such as nanofibre or
nanoplate may be pre ferred to the term nanoparticle.
[SOURCE: ISO/TS 80004-2:2015, 4.4]

3 .10
nanoplate
nano-object (3.8) with one external dimension in the nanoscale (3.12) and the two other external
dimensions significantly larger
Note 1 to entry: The smallest external dimension is the thickness o f the nanoplate.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Note 2 to entry: The two significantly larger dimensions are considered to di ffer from the nanoscale dimension
by more than three times.
Note 3 to entry: The larger external dimensions are not necessarily in the nanoscale.
[SOURCE: ISO/TS 80004-6:2013, 2.4]
3 .11
nanorod
solid nanofibre (3.6)
[SOURCE: ISO/TS 80004-2:2015, 4.7]
3 .1 2
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
Note 2 to entry: Properties impacting biocompatibility can also occur at larger sizes, e.g. between 100 nm and 1 um.
[SOURCE: ISO/TS 80004-1:2015, 2.1, modified — Note 2 to entry was added]
3 .1 3
nanoscale phenomenon
effect attributable to the presence of nano-objects (3.8) or nanoscale (3.12) regions
[SOURCE: ISO/TS 80004-1:2015, 2.13]
3 .14
nanoscale property
characteristic of a nano-object (3.8) or nanoscale (3.12) region
[SOURCE: ISO/TS 80004-1:2015, 2.14]
3 .15
nanoscience
study, discovery and understanding o f matter, where size- and structure-dependent properties and
phenomena mani fest, predominantly in the nanoscale (3.12), distinct from those associated with
individual atoms or molecules, or extrapolation from larger sizes of the same material
[SOURCE: ISO/TS 80004-1:2015, 2.2]
3 .16
nanos tructure
composition of inter-related constituent parts in which one or more of those parts is a nanoscale
(3.12) region
Note 1 to entry: A region is defined by a boundary representing a discontinuity in properties.
[SOURCE: ISO/TS 80004-1:2015, 2.6]
3 .17
nanostructured material
material having internal nanostructure (3.16) or surface nanostructure
Note 1 to entry: This definition does not exclude the possibility for a nano-object (3.8) to have internal structure
or surface structure. If external dimension(s) are in the nanoscale (3.12), the term nano-object is recommended.
[SOURCE: ISO/TS 80004-1:2015, 2.7]

ISO/TR 1 0993 -2 2 : 2 01 7(E)
3 .18
nanotechnology
application o f scientific knowledge to manipulate and control matter predominantly in the nanoscale
(3.12 ) to make use o f size- and structure-dependent properties and phenomena distinct from those
associated with individual atoms or molecules, or extrapolation from larger sizes of the same material
Note 1 to entry: Manipulation and control includes material synthesis.
[SOURCE: ISO/TS 80004-1:2015, 2.3]
3 .19
nanotube
hollow nanofibre (3.6)
[SOURCE: ISO/TS 80004-2:2015, 4.8]
3.20
representative tes t material
RTM
material, which is su fficiently homogenous and stable with respect to one or more specified properties,
and is implicitly assumed to be fit for its intended use in the development o f measurement and test
methods that target properties other than those for which homogeneity and stability have been
demonstrated
[SOURCE: ISO/TS 16195:2013; 3.1 , modified — Notes 1 and 2 to entry deleted]
4 General principles
4.1 General considerations
Nanomaterials are manu factured and used because o f the specific properties that can be associated
with the decrease in size accompanied by an increase o f sur face area. Also, materials with dimensions
in the size range >100 nm or <1 micron can elicit properties different from those in the macroscale
(>1 micron). For these types o f particulate materials, it might be considered to per form an assessment
similar to nanomaterials in the size range between 1 nm and 100 nm.
The biological evaluation o f any material or medical device intended for use in humans should form
part o f a structured biological evaluation program within a risk management process in accordance
with ISO 14971 and ISO 10993-1. The risk management process is applicable to devices that contain
or are composed o f nanomaterials. The risk management process is also applicable to devices that
generate nano-objects as products of degradation, wear, or from mechanical treatment processes (e.g.
in situgrinding, polishing o f medical devices). Similarly, i f there is release o f nano-objects, there are
specific challenges in the sa fety evaluation o f such products. The sa fety evaluation and risk assessment
of nanomaterials requires a special focus as various nanomaterials consisting of the same chemical
substance can have a di fferent toxicological risk profile depending on a number o f variables, including
size, sur face chemistry, physicochemical properties and intended application. For medical devices
that are composed o f or that contain nanomaterials, the sa fety evaluation program should specifically
address issues related to the sa fety evaluation o f nanomaterials. The ISO 10993 series, ISO/TR 13014,
ISO 14971, and References [5 ],[14],[15 ],[16 ],[21],[23 ],[24],[28 ],[46 ],[47 ] and [49 ] deal with biological
evaluation of medical devices and various aspects of nanomaterials.
Nanomaterials have sizes similar to structures at subcellular levels including DNA, and thus
(theoretically) can reach and interact with such structures. Also, medical devices utilizing materials
with nanoscale internal structures or with surface nanoscale features associated with coatings,
functionalization, or with other topographical features on the nanoscale, that are intended as part of
the functionality o f the device, can have specific and unique properties that might need to be addressed
in the biological evaluation. For example, it has been shown that nanoscale sur face topography can
influence cell alignment, cell morphology, cell signalling, gene expression and extracellular matrix
[52 ] [53 ] [54] .

ISO/TR 10993-22:2017(E)
The release of nano-objects and the use of free nanomaterials are considered to pose the highest
potential for risk in view o f the potential internal exposure that can occur.
4.2 Biological evaluation of nanomaterials

ISO 10993-1:2009, Annex A, provides a framework for the development o f an assessment program o f
the biological risks that should be considered depending upon a device’s type and duration o f body
contact. This framework is also generally applicable to devices that contain, generate or are composed
o f nanomaterials. Such testing should be based on each device’s merits. Special considerations apply to
the ISO 10993 series of tests due to the presence of nanomaterials, as outlined in this document.
ISO 10993-1 provides guidance on the risk management process, which includes hazard identification,
exposure assessment and risk estimation. This process is generally su fficiently robust and flexible
to provide a basis for evaluation o f nanomaterials, even though they can have properties that can
be di fferent from conventional ones. This process, including biological evaluation strategy, program
content and acceptance criteria o f the risk related to the nanomaterials as required by ISO 10993-1,
should be planned, carried out and documented by knowledgeable and experienced pro fessionals.
The initial step in the biological evaluation of nanomaterials is to gather existing information on that
particular nanomaterial according to the general approach as described in ISO 10993-1. Literature
review of clinical and non-clinical data should be carried out according to ISO 10993-1:2009, Annex C
to provide a rigorous and objective summary o f available in formation about the nanomaterial and
its intended application. Reference [55 ] has summarized several places where in formation about
nanomaterials can be found. Following the logic o f ISO 14971 and ISO 10993-1, i f the biological sa fety
assessment concludes from existing data that the identified risks are acceptable, no further testing is
needed. Otherwise, additional information should be obtained. In order to use existing data for the
biological evaluation, demonstration o f nanomaterial equivalence is necessary (see 4.4).
4.3 Categorization of nanomaterials

The exposure assessment and hazard identification should be based on the characteristics o f the
finished medical device and the intended use. Hazard identification should consider the physicochemical
and toxicological properties of the nanomaterial, including additives and processing aids. Exposure
assessment should consider the concentration of nanomaterial used in the medical device, intended use
and exposure route, and the rate and pattern of release and estimated patient exposure. The manner
in which the nanomaterial is incorporated into the finished medical device can significantly alter the
exposure characteristics [56 ] . General considerations for different categories of nanomaterials and
medical devices are presented in Table 1 . Certain devices might fall into more than one category, in
which case evaluation appropriate to each category should be considered. The evaluation o f any device
that does not fall into one of the categories described should follow the general principles contained in
ISO 10993-1, along with any special considerations outlined within this document.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Table 1 — Considerations for biological evaluation of medical devices

that contain, generate, or are composed of nanomaterials
C ategory a Type of nanomaterial Considerations in addition to the biological

in the medical device evaluation according to I SO 10 9 93 -1
— Consider potential cellular or tissue effects due to direct

interaction with sur face nanostructures (beneficial or
adverse).
— Consider potential o f structures to be released (break
1 Surface nanostructures off) from the surface.
— Consider potential o f nano-objects to be generated by
degradation, wear or mechanical treatment processes.
— Consider characterization of nanostructures (see
Clause 5).
— Consider potential cellular or tissue effects due to direct
interaction with surface-bound nano-objects/nano-materi-
als (beneficial or adverse).
Nano-objects bound to or incor- — Consider potential of nano-objects to be released from
2 porated within a medical device; the device.
without intention to be released — Consider potential o f nano-objects to be generated by
— Consider characterization o f physicochemical properties
of the nano-objects (see Clause 5).
— Consider release kinetics (rate and quantity) o f the na -
no-objects and contact duration of the medical device.
— Consider potential cellular or tissue effects due to
direct interaction with nano-objects/nanomaterials (benefi -
cial or adverse).
Nano-objects/nanostructures on the — Consider characterization o f physicochemical properties
3 surface of or within a medical device; of the released nano-objects (see Clause 5).
with intentional/expected release
from the device — Consider toxicokinetics and tissue distribution o f the
nano-objects (see Clause 8).
— Consider biological evaluation of the nano-objects (see
Clause 9).
— Consider potential o f nano-objects to be generated by
4 Nano-object medical device — Consider toxicokinetics and tissue distribution o f the
nano-objects (see Clause 8).
— Consider biological evaluation of the nano-objects (see
Clause 9).
a A device can contain nanomaterials in more than one category.
b Nano-objects can be generated from a medical device that does not contain nano-objects.
c Degradation, wear or treatment of a medical device containing nano-objects can generate new or
unintended nano-objects.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Table 1 (continued)
C ategor y a Type of nanomaterial Cons iderations in addition to the biolo gical

in the medical device evaluation according to I SO 10 9 93 -1

— Clause 7 describes additional considerations for
nano-objects
Nano-objects b released from a medi- cessing. released by wear or generated by in situ pro -
cal device as product of degradation,
5 wear, or from mechanical treatment c — Consider toxicokinetics and tissue distribution o f the
processes (e.g. in situ grinding or nano-objects (see Clause 8).
polishing) — Consider biological evaluation of the generated nano-ob-
jects (see Clause 9).
— Consider contact duration and release kinetics (rate and
quantity).
a A device can contain nanomaterials in more than one category.
b Nano-objects can be generated from a medical device that does not contain nano-objects.
c Degradation, wear or treatment of a medical device containing nano-objects can generate new or
unintended nano-objects.
4.4 Nanomaterial equivalence
Proper identification and characterization o f the nanomaterial is essential. For nanomaterials,

equivalence is dependent on multiple factors. Chemical composition alone is not su fficient to
demonstrate equivalence as nanomaterial-specific properties can also be influenced by a number
of other factors such as size, shape and surface properties of the nanomaterial and/or the source
(manufacturer) ofGet
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can only be claimed i f properly demonstrated and justified by accompanying data.
In general, extrapolation o f results by using existing data from other products using/containing similar
nanomaterials, or from the corresponding parent compound of the same substance is not applicable,
although such products can give an indication o f possible sa fety concerns. I f testing is considered
necessary, it should be per formed on the actual product and/or any nanomaterials which can come into
contact with patients.
5 Characterization of nanomaterials
5 .1 General considerations
Knowledge o f the physicochemical properties o f nanomaterials is essential to understanding their

behaviour in biological systems. The physicochemica l characterization is necessary for the identification
o f a specific nanomaterial. Characterization o f the physicochemical properties o f nanomaterials/nano-
objects incorporated in a device and/or created by degradation, wear or mechanical treatment processes
o f the device is thus an important step in completing its biological evaluation. Physicochemical
characterization can also be use ful in the screening o f potential new nanomaterials for suitability
in a medical device for a proposed application. In addition, a proper characterization is necessary to
establish or confirm the specifications o f a nanomaterial in a defined medium and conditions.
Physicochemical characterization addresses three fundamental questions about a nanomaterial used
in or released from a medical device.
— C hemical composition:What is it made of?
— What does it look like?
Physical description:
— How does it interact with the surrounding environment?

E xtrinsic properties:

ISO/TR 1 0993 -2 2 : 2 01 7(E)
T he phys ico chem ic a l prop er tie s a s s o ci ate d with the s e que s tion s encomp a s s a wide range o f na no - obj e c t
cha rac teri s tic s . I n ke epi ng with gu idance for eva luation o f conventiona l materi a l s used i n me d ic a l
device s , the phys ico chem ic a l charac teri z ation o f nano - s c a le d materi a l s i s targe te d to prop er tie s that
are relevant to the biological evaluation and the intended use of the device (clinical application and
f
du ration o f
us e) . G enera l pri nciple s or chem ic a l, phys ico chem ic a l, morpholo gic a l and top o graph ic a l
characterization of materials used in medical devices are covered in ISO 10993-18 and ISO/TS 10993-
19. T he identi fic ation a nd quanti fic ation o f de gradation pro duc ts i n me d ic a l device s are add re s s e d
i n I S O 10 9 9 3 -9, I S O 10 9 9 3 -1 3 , I S O 10 9 9 3 -14 a nd I S O 10 9 9 3 -1 5 . D e tai le d guidance s p e c i fic a l ly for the
phys ico chem ic a l ch arac teri z ation o f nanomateria l s i s emergi ng. Re cently publ i she d guidance i nclude s
I S O/ T R 1 3 014 and I S O/ T R 141 8 7 a nd gu idance s publ i she d b y the E urop e an C om m i s s ion for fo o d a nd
feed, cosmetic ingredients and medical devices [57 58 59 . ] [ ] [ ]
ISO/TR 13014 lists the following as properties of engineered nanomaterials to be characterized in the
context of toxicological testing:
— chemical composition;
— pu rity;
— object size and size distribution;

— aggregation and agglomeration state;
— shape;
— surface area;
— s u r face chem i s tr y;
— surface charge;
— s olubi l ity;
— d i s p ers ibi l ity.
These properties should be viewed as a starting point for the evaluation of nanomaterials used in a
device; characterization of additional properties might be indicated depending on the design, intended
u s e and we a r charac teri s tics o f the device . E xample s o f o ther phys ico chem ic a l prop er tie s th at m ight b e
con s idere d on a c a s e -b y- c a s e b a s i s i nclude:
— cr ys ta l l i n ity;
— p oro s ity;
— redox potential;
— ( pho to) c ata lys i s;
— radical formation potential;

— o c ta nol/water p a r tition co e fficient (m ight no t b e applic able for s ol id materia l s) .
In order to ob ta i n the re qu i re d data as i nd ic ate d ab ove, mu ltid i s cip l i na r y col lab oration s a mong
toxicolo gi s ts , phys ic a l chem i s ts , engi ne ers and o ther s ubj ec t- are a e xp er ts are ne ce s s a r y i n developi ng a
releva nt a nd rel i able ch arac teri z ation pro gra m for a s p e c i fic nanomateri a l contai n i ng device .
Sp e c i fic guidance for cha rac teri z ation i s ava i l able for cer tai n nanomateri a l s , e . g. s i ngle and mu ltiwa l l
carbon nanotubes (SWCNT, MWCNT), nanoscale calcium carbonate powder, nanoscale titanium dioxide
1) .
p owder, gold and s i lver nanop ar ticle s , and nano clays
1) For details, see http://www.iso .org/iso/home/store/catalogue_tc/cata logue_tc_browse.htm?commid= 381983 .

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Devices incorporating nanostructured surfaces can require characterization of morphological features

in addition to characterization o f the physicochemical properties listed above. Modification o f medical
device sur face structure at the nanoscale continues to be explored with the objective o f modi fying
cell and microbial interactions with devices. The measurement parameters required for the adequate
characterization o f sur face architecture depend on the specific application [60 ] [61 ] . For example, Webb
et al. [42 ] propose the use of a minimum of three statistical parameters as a standard for describing the
vertical and horizontal nanoarchitecture of surfaces in the context of bacterial adhesion.
— Average surface roughness (Ra): the average deviation of surface height values from the mean plane.
— Surface area difference (Rsa): the increase in sur face area caused by roughness, i.e. the percentage
difference between the actual surface area and the projected surface area. This parameter can be
used i f it is possible to precisely measure the sur face area.
— Peak count (R pc): the number o f peaks in the measured profile.
In addition, peak height and peak-to-peak distance can be use ful spatial parameters.
Nanoporous materials are being developed for use in device applications including drug delivery and
tissue scaffolding. Useful information for characterization of porous materials includes:
— size and structure of pores or voids;
— density o f pores or voids;
— distribution of the pores or voids.
Nanomaterials should be characterized in the form they will be delivered to the end-user, i.e. as the
final device. Representative samples from the final device or materials processed in the same manner
as the final device can also be characterized, in order to directly evaluate a nanomaterial component. In
addition, characterization
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toxicological studies and biocompatibility testing. For biological sa fety evaluation, the above re ferred
physicochemical characteristics o f the nanomaterials should be determined also in the biological
medium used in the test system. The interaction between these media and the nanomaterials can have
a pro found influence on the behaviour o f the nanomaterials in the test system. This factor should be
taken into account during the test procedures and during the evaluation o f the test results.
The contact with body fluids can alter the sur face characteristics o f nanomaterials and thus their
biological behaviour which can have an impact on the hazard caused by a nanomaterial (see 8.2.2).
The isolation and characterization o f nano-objects generated by wear o f medical devices remains a
challenge. In vitro methods for simulating the in situ generation of nanomaterials/nano-objects should
be representative of the clinical use.
5 .2 Characterization parameters and methods
Table 2 summarizes fundamental parameters that are useful as a starting point for characterization of
nanomaterials used in medical devices. Detailed information on the relevance of these parameters to
biological evaluation is provided in ISO/TR 13014.
Table 2 also provides examples of methods that can be used to provide quantitative and/or qualitative
data for each of these parameters, based on information provided in ISO/TR 13014. This list should be
considered a dynamic compilation, as development o f best practices for nanomaterial characterization
is an area of ongoing research and discussion. The listed methods include some developed for
conventional particle analysis, as well as others developed specifically for nanoscaled materials.
Multiple methods are o ften available for characterization o f a specific physicochemical parameter. A
single characterization method might not provide an accurate assessment of the parameter (e.g. size
distribution, sur face composition). In such cases, a complementary method, i f available, might be
necessary to provide an adequate assessment o f the property to be characterized; two independent
methods for characterization might be needed. It is important to note that the results obtained
ISO/TR 1 0993 -2 2 : 2 01 7(E)
for a particular property using di fferent methods might not be directly comparable and that there
are currently few harmonized methods for physicochemical assessment o f nanomaterials to aid
development o f a robust test plan. The method(s) selected for characterization should be justified on
the basis o f the nanomaterial type and form, and proposed use o f the device. For example, for nano-
object size determination, at least one microscopy technique [e.g. transmission electron microscopy
(TEM), con focal laser scanning microscopy (CLSM)] should be used as indicated in several guidance
documents [57 ] [58 ] [59 ] . A report was published by the EU-JRC describing possibilities and pitfalls on
nano-object size measurements [62 ] .
Because characterization o f nanomaterials is o ften scientifically and technically challenging, a quality
assurance programme and laboratory best practices should be considered. The selection o f techniques,
analysis o f nanomaterials and interpretation o f nanomaterial properties should be per formed by
pro fessionals appropriately qualified by training and experience. In per forming the analyses, care ful
consideration should be given to sample preparation procedures to ensure that the data obtained are
representative of the material used in the device. All aspects of the characterization process should be
care fully documented to ensure transparency and reliability o f results. Methodology used should be
demonstrated to be appropriate for the nanomaterials investigated.
Table 2 — Key physicochemical characteris tics and examples of measurement methods
Relevant
C haracteris tic Meas urand E xample meas urement methods I SO guidance on
methodolo gy
X-ray fluorescence
X-ray photoelectron spectroscopy
Auger electron spectroscopy
Fourier X-ray di ffraction (XRD trans form
The number and identity in frared spectroscopy) ISO 22309
of elements alone or in (FTIR), Raman and other molecular ISO 22489
Chemical molecules (can be
composition expressed as a chemical spectroscopies ISO 24173
and purity formula Thermogravimetric analysis ISO 13084
Level or concentration of UV/visible spectrometry ISO 18144
unintended constituents Scanning electron microscopy + XRD or
(impurities) energy dispersive x-ray spectroscopy (EDS)
Nuclear magnetic resonance
(Single particle) Inductively coupled
plasma- mass spectrometry (spICP-MS)

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Table 2 (continued)
Relevant
C haracteris tic Meas urand E xample measurement methods I SO guidance on
methodology
:
Particle s ize Dynamic light scattering
Equivalent spherical Small angle X-ray scattering ISO 9276 series
diameter for particles Size exclusion chromatography ISO 9277
displaying a regular
geometry Analysis o f images o f scanning electron ISO 13318 series
Length of one or several microscopy (SEM), transmission electron ISO 13320
specific aspects o f the microscopy (TEM), or scanning probe
particle geometry microscopy (SPM) ISO 22412
Particle size Di fferential mobility analysis ISO 13322 series
and particle size Centrifugal liquid sedimentation ISO 14488
distribution Particle si ze Nanoparticle tracking analysis ISO 14887
: ISO 15900
Graphical representation, Raman spectroscopy
dis tribution
e.g. histogram, and/or Laser-induced incandescence ISO 16700

values for statistical Confocal laser scanning microscope (CLSM) ISO/TS 19590
parameters such as mean, Single particle ICP-MS ISO 20998-1
median, and/or mode
Tangential flow filtration for nanomaterial ISO 21501 series
separation followed by appropriate
detection, e.g. ICP-MS (ISO 10993-14) ISO 22412
Particle size Analysis o f (cryo-)SEM or (cryo-)TEM image
Number of aggregate/ Angle dependent scattering at different
agglomerate wavelengths
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number o f primary Small angle X-ray scattering ISO/TR 13097
Aggregation/ particles X-ray di ffraction (XRD) ISO/TS 12025
agglomeration Number o f primary X-ray absorption spectroscopy (XAS) ISO 13322-1
state particles in the
aggregate/ Small angle neutron scattering
agglomerate Rheology methods
Distribution of number Centrifugal liquid sedimentation
o f primary particles per Laser diffraction
aggregate/agglomerate Nanoparticle tracking analysis
Size-independent Analysis o f SEM, TEM, atomic force
descriptors of shape microscopy (AFM), or SPM images ISO 16700
Shape Distribution of values Scattering techniques ISO 13322-1
of the size-independent spICP-MS
shape descriptors
ISO 15901-1
Methods based on gas or liquid ISO 15901-2
adsorption isotherms [Brunauer-
Surface area Volume- and/or Emmett-Teller (BET) theory] ISO 15901-3
mass-specific sur face area Liquid porosimetry ISO 18757
Image analysis ISO 13322-1
Laser-induced incandescence ISO 9277

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Table 2 (continued)
Relevant
C haracteris tic Meas urand E xample meas urement methods I SO guidance on
methodolo gy
Inter ferometry
Reflectometry
Scanning probe microscopy (SPM) and
Surface Size and geometry atomic force microscopy (AFM) ISO 25178
nanostructures Scanning tunnelling microscopy (STM)
Contact profilometry
Non-contact profilometry
Auger electron spectroscopy
X-ray photoelectron spectroscopy (XPS) ISO/TR 14187
Elemental and Secondary ion mass spectrometry (SIMS) ISO 18115
molecular abundance
Surface 3D atom probe tomography ISO 24236
chemistry Reactivity (chemical Energy dispersive X-ray spectrometry ISO 15471
reaction rate) Electron energy loss spectrometry (EELS) ISO 18118
Low energy ion spectroscopy ISO/TR 19319
Raman and other molecular ISO 17973
spectroscopies
Net number of positive and Isoelectric point
negative charges per unit Electrophoretic light scattering
particle surface area Electrophoresis ISO 20998
Surface charge Electro-osmosis ISO 13099
Zeta potential Electric sonic amplitude
Colloidal vibration current
Solubi lity:
Maximum mass or
concentration of the solute
that can be dissolved in There are no specific methods for
a unit mass or volume of assessing the solubility o f nano-objects.
a solvent at specified (or
standard) temperature or Tangential flow filtration for
pressure nanomaterial separation followed by ISO 20998
appropriate detection, e.g. ICP-MS
(ISO 10993-14) ISO 13099
Solubility/ Methods to assess dispersibility o f
dispersibility
D is persibility:
Maximum mass or nano-objects are based on particle size/
concentration of the size distribution and aggregation/
dispersed phase present agglomeration state (see particle size
in a unit mass of the above).
dispersing medium
(solvent) or unit
volume of the dispersion
(solvent plus dispersed
phase) at specified (or
standard) temperature
and pressure
The information given in Table 2 is based on ISO/TR 13014. There are other documents available
providing recommendations on nanomaterial/nano-object characterization (e.g. ISO/TS 17200).
Most of these parameters and methods listed are relevant for the characterization of nanoparticles. It
should be noted that other forms/shapes o f nanomaterials can be used, e.g. nanofibres and nanoplates,

ISO/TR 1 0993 -2 2 : 2 01 7(E)
for the production of medical devices. Some parameters or methods listed might not be applicable to
other forms/shapes of nanomaterials.
The methods listed can each have their limitations; there fore, the methodology used should be
demonstrated to be appropriate for the nanomaterials investigated.
For certain nanomaterials (e.g. nanosilver, nanoscale zinc oxide), dissolution or ion shedding can
occur. The dissolution o f nanomaterials in di fferent physiological environments can be assessed
using tangential flow filtration which is a method to separate nanomaterials from their solubilized
counterparts. The separated fractions can then be quantified using inductively coupled plasma mass
spectrometry (ICP-MS) [63 ] .
5 .3 Use of reference materials
The availability o f positive and negative control samples o f nanomaterials is o f key importance for
reproducibility and reliability o f nanomaterial sa fety testing. However, positive and negative control
samples for nanomaterials are lacking. There fore, the use o f “representative test materials” has been
proposed [65 ] . ISO/TC 229 has defined a representative test material as “material, which is su fficiently
homogenous and stable with respect to one or more specified properties, and is implicitly assumed to
be fit for its intended use in the development o f measurement and test methods that target properties
other than those for which homogeneity and stability have been demonstrated” (ISO/TS 16195).
This approach is currently applied in the OECD Working Party on Manu factured Nanomaterials
collaborative project for nanomaterial sa fety testing using materials from the European Commission
Joint Research Centre repository o f representative nanomaterials. Guidance on generic requirements
for representative test materials for development of methods for characteristic testing, performance
testing and sa fety testing o f nanoparticle and nanofibre powders is provided in ISO/TS 16195.
General guidance for the use and preparation of reference materials in the biological evaluation of
medical devices isGet
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available for nanomaterial properties other than size. Reference materials for nanomaterial particle size
assessments are increasingly available from national and community agencies, including the National
Institute o f Standards and Technology (NIST, United States), the Joint Research Centre o f the European
Commission (JRC, European Union) and the Federal Institute for Materials Research and Testing
(BAM; Germany). BAM, in cooperation with ISO/TC 229, maintains a database o f all currently available
nanoscale reference materials worldwide, available at http://www.nano -refmat.bam .de/en/ . Currently
available nanoscaled reference materials include titanium dioxide, colloidal silica, gold and single
walled carbon nanotubes. A number of new reference materials are reported to be under development
by these organizations, as well as commercial sources, but the initial focus o f development has been
on nanoscale reference materials for calibration of instrumentation, rather than on development of
re ference materials for benchmarking biological responses.
It should be noted that the use o f appropriate standard re ference materials (SRM) is significant only
in the context o f standardized protocols for size and shape metrology. Protocols should work across
a range o f imaging technologies and require high statistical certainty with minimal human bias.
In addition, reference materials can also be used as internal and/or external reference. For example
external standards can be used to calibrate the length scale of imaging tools for subsequent imaging,
while internal standards allow the inclusion o f a size standard within the unknown.
Development o f a set o f commonly accepted re ference materials, including consensus on suitable
positive and negative control nanoparticles for di fferent testing systems, has been identified as a critical
need for the risk assessment o f nanomaterials [65 ] . Although the need for such reference materials is
well recognized, development continues to be slow as a result o f practical di fficulties including lack
o f consensus on which materials and properties to characterize, significant technical challenges, and
economic factors [64] [65 ] [66 ] .

ISO/TR 1 0993 -2 2 : 2 01 7(E)
6 Sample preparation
Sample preparation is a critical process in the characterization and biological testing of medical devices
and the materials used in their fabrication. Important considerations for testing of medical devices in
their final product form include representative sampling o f the device; preparation o f product extracts,
and storage and stability o f the prepared test material. General guidance on these topics is provided in
ISO 10993-12.
Special considerations may be necessary for the sample preparation o f medical devices containing or
consisting of nanomaterials (e.g. the use of nano-object dispersions instead of extracts).
NOTE Specific guidance for nanomaterials on sample preparation and dosing methods is available in
ISO/TR 16196.
6.2 Special considerations for nanostructured materials
ISO 10993-12 is applicable to medical devices having internal nanostructures and/or surface
nanoscale structures. A nanostructured surface increases the total surface area of a device. The
use of the recommended surface areas in ISO 10993-12 underestimates the actual surface area of a
nanostructured device, if measured at the external dimensions. However, this could be considered as a
conservative approach of the extraction process used for sample preparation, but it should be realized
that the patient is exposed to the total surface.
In ISO 10993-12, it is stated that the standard surface area can be used to determine the volume of
extraction vehicle needed, and that this area includes the combined area of both sides of the sample
and excludes indeterminate surface irregularities. The same approach should be used for material with
nanostructured surfaces.
In line with ISO 10993-12, other surface-area-to-volume extraction ratios of nanoporous materials can
be used i f they simulate the conditions during clinical use or result in a measure o f the hazard potential.
The extraction techniques as described in ISO 10993-12 will probably not result in the release o f
embedded nanomaterials and/or nanomaterials on the sur face o f a material due to the fixation in the
matrix or on the sur face. Only when there is a relatively weak bonding on the sur face extraction o f the
nano-objects can be expected.
6.3 Special considerations for nano-obj ects
For nano-objects, some of the sample preparation requirements described in ISO 10993-12 might
not be applicable and additional aspects (e.g. particle aggregation/agglomeration) might need to be
considered. For example, it might be that by using extracts, only the e ffects o f residues/contaminants
are evaluated. In addition, the nanoscale size allows the nanomaterials to be dispersed in a liquid for
most assays needed to be per formed for a biological evaluation and risk assessment o f nanomaterials.
Nano-objects may be dispersed in a liquid allowing most biological evaluations to be per formed,
thereby allowing a direct risk assessment o f the nano-object. Polar and non-polar media are generally
considered for extraction per ISO 10993-12. However, a nano-object dispersion should be selected using
media based on clinical relevance and dispersability. The dispersion is optimized to deliver the nano-
object in a specific test system and most nanodispersions are prepared and used in a polar solution.
The small size and potentially altered physicochemical characteristics o f nano-objects present
significant challenges for sample preparation when compared to testing o f bulk (non-nanoscaled)
materials or chemicals. Contributing factors include surface properties of nano-objects that increase
their reactivity; formation o f aggregated or agglomerated objects; trans formation o f nano-objects in
dispersions via hydration, partial dissolution or other processes and the potentially strong impact o f
low-level contaminants on the physicochemical properties and toxicological properties o f nano-objects.
As with other types o f test articles, there is a possibility for nano-objects to adsorb to container sur faces.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Also, the rate o f delivery to cells in culture can be a ffected by di ffusion and gravitational settling (see
9.2 ). It is important to veri fy number concentrations o f the nano-objects used in test samples.
Sample preparation protocols have to be developed in tandem with metrology methods ( Table 2).
Aggregation and agglomeration limit the capability o f determining small changes in particle size
distribution with high throughput automated metrology. At the same time, it is metrology o f large
populations o f particles or fibers that allows for the determination o f the extent o f agglomeration and
aggregation. An awareness o f these issues is necessary for development o f reliable sample preparation
protocols for nano-objects, devices that contain nano-objects, or devices generating or releasing nano-
objects. Resolving these issues can require significantly increased e ffort directed toward development
of sample preparation and handling strategies compared to devices using conventional materials.
An important consideration in the sample preparation of nanomaterials is the distinction between
solubility and dispersibility o f a test item or test item component. Some nanoparticles are partly or
slowly soluble. The distinction between solubility and dispersibility is important because a dispersion
o f particulate material can elicit a response that di ffers from the molecular or elemental toxicity
predicted from the chemical composition [67 ] . A nano-object that is soluble or partly/slowly soluble in
biological media is likely to present to the test system in molecular form. Sparingly soluble or insoluble
nanomaterials will likely be presented to the test system as a dispersion o f nano-objects. In practice,
care ful analysis can be required to determine whether a particular nano-object is fully dispersed,
partially dissolved (e.g. in the case o f some metals) or fully dissolved under specified laboratory
conditions. These dissolved nano-objects can elicit a response similar to that of non-nanoscale
solubilized materials. When applicable, the dissolution rate should also be considered in addition to the
solubility itsel f.
Nano-objects can be very sensitive to techniques used in sample preparation as a result o f their unique
sur face properties. The dispersion o f nano-objects is influenced by interactions between nano-objects
and by interactions o f nano-objects with their environment. Dispersed nano-objects are not necessarily
only in the primary Getform.
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bonded or fused objects) and agglomerates (collections o f weakly bound objects, aggregates or mixtures
o f the two) can form in solution, powder and aerosol forms o f nano-objects unless stabilized by sur face
charge or steric effects. As a result, the state of dispersion and object size distribution of nano-objects
in a sample can change over time. This behaviour has important implications for preparation of extracts
and/or stock and dosing dispersions, where slight modifications o f pH, ionic strength or presence o f
molecular constituents can significantly alter nano-object dispersion. For this reason, determination
o f test article stability is a critical factor in obtaining representative and reproducible results during
biological evaluation.
Sample preparation of nano-objects can encompass characterization of as-produced or vendor-supplied
material and preparation o f stock and dosing solutions for animal or in vitro studies. The details of
preparation can vary depending on the dosing route and method o f delivery. Common issues in test
sample preparation and administration include:
— identity, storage and stability o f test materials including batch-to-batch variability;
— chemical composition of the test medium;
— selection of appropriate dose metric;
— characterization o f samples prepared from stock dispersions prior to dose administration.
Additional information on these topics is presented below. The sample preparation protocols and
rationale should be care fully documented to enable replication o f test samples as needed.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
6.4 Identity, storage and stability of stock nanomaterials
Following are some of the factors that should be considered for dispersion and storage of nanomaterial
preparations.
— Stock dispersions should be prepared according to the processes used to prepare the nanomaterials
in the final product. Alternative preparation techniques may be appropriate to prepare dispersions
for biological evaluation. Stock dispersions are ideally characterized by uni form object size and
predictable polydispersity [68] .
— The methods used to disperse nano-objects are based on their applications and include sonication,
stirring, use of solvents and use of stabilizing agents such as surfactants. For instance, chemical
moieties such as polyvinylpyrrolidone, citrate or tannic acid can be placed on nano-objects to
prevent agglomeration/aggregation or increase dispersion in solution. Small chain polymers such
as polyethylene glycol can be conjugated to nano-objects to prevent recognition by the mononuclear
phagocyte system (MPS) and thus increase circulation time within the body[69 ] [ 70 ] . Proteins or
carbohydrates can be added to nano-objects to target certain cells or tissues within the body. One
should be cognizant of the chemicals/coatings which are attached to the nanomaterial of interest
since they are a key to identi fying biological e ffects, and can also be a source o f contamination.
— The procedures used to disperse nano-objects should be care fully chosen as they have the potential
to alter the physicochemical characteristics and toxicity o f nano-objects. Possible mechanisms
for altered toxicity include fragmentation o f nano-objects by grinding and alteration o f sur face
properties by use o f dispersing agents, desorption o f sur face coatings, and changes in oxidation
state, among others. Dispersion methods should be e valuated for such e ffects on a case-by-case basis.
E ffects observed during evaluation o f the dispersion method should be controlled or quantified
when there is evidence that the toxicity o f the nanomaterial and/or nano-object preparation is
altered.
— Nanomaterials should be stored per an appropriate defined, controlled procedure. General
considerations include avoiding extremes of temperature, light, and moisture.
— Storage and handling procedures should take into account the reactivity o f the nanomaterial.
— Nanomaterials used in medical devices should be analyzed for the presence o f contaminants and
impurities. The presence o f low-level contaminants or impurities can significantly impact the
physicochemical and toxicological properties o f nanomaterial preparations and can con found
analytical and experimental determinations. Examples o f contaminants found in nanomaterials
include bacterial endotoxin, sur factants, residual solvents, metals and catalysts. Contamination
can occur during manufacture, handling and dispersion of nanomaterials; therefore, nanomaterial
preparations might need to be analyzed for contamination at more than one stage during the sample
preparation process.
— Stability o f nanomaterial preparations should be evaluated to determine whether:
— the nanomaterial dissolves, degrades, or transforms over time (see Clause 7); and
— the particle size distribution or sur face charge changes over time. Stock solutions should be re-
made and re-characterized as necessary.
6.5 Description of chemical composition of stock and dosing dispersions
Dispersions of nano-objects used in biological evaluation studies should be compatible with

physiologic conditions. For example, dosing solutions for in vitro mammalian cell assays should be
isotonic, adjusted to a pH of 7,4 and useable in the presence of divalent ions and protein mixtures [71] .
The chemical composition used to create physiologic conditions can have unintended e ffects on the
physicochemical characteristics o f nanomaterials including the degree o f nano-object agglomeration
and/or aggregation. Also, the nano-objects themselves can have an effect on the culture media used.
It is important to characterize and document the composition of test media and dosing solutions to

ISO/TR 1 0993 -2 2 : 2 01 7(E)
ensure reproducibility o f experimental conditions. The following parameters constitute a starting list
for characterization of media and dosing solutions used for in vitro and in vivo testing [68 ] :
— ionic strength;
— calcium and magnesium concentration and anion used as source (e.g. MgSO 4 or MgCl 2 );
— pH and composition o f pH bu ffering system;
— organic additives (e.g. serum, bovine serum albumin, antibiotics);
— identity and concentration o f dispersing agents.
6.6 Characterization of stock dispersions
The methods used to create stock dispersions should be documented in detail to enable reproducibility.
The following parameters constitute a starting list for characterization o f stock dispersions [68 ] :
— information from the manufacturer;
— analytical data for physicochemical properties as outlined in Clause 5;
— measured mass concentration in the stock dispersion; (also to be expressed as sur face area, and
number concentration)
— for metal-based nanomaterials, measured concentration of dissolved metal ion(s);
— identity and (where feasible) measured concentrations o f impurities and contaminants;
— details o f preparation including shape, volume, and material type o f the vessel used to create the
stock dispersion;
— stability (shel f li fe) o f the stock dispersions.
Characterization o f other parameters may be needed on a case-by-case basis.
Steps taken to prevent contamination (e.g. use o f ultrapure water and reagents) should be described. The
possibility o f contamination occurring from deterioration o f the probe tip or container sur face should
be considered when preparations are prepared by ultrasonication o f suspensions. Also, contamination
from other sources should be considered.
Guidance on the preparation and characterization o f nanomaterials for inhalation toxicity testing is
provided in ISO 10801 and ISO 10808.
6.7 Characterization of dosing solutions prepared from stock dispersions
The methods used to create dosing dispersions should be documented in detail to enable reproducibility
and to aid in the interpretation o f study results. The following parameters constitute a starting list for
characterization of dosing solutions prior to administration to test animals or in vitro systems [68 ] :
— recommended parameters for characterization o f stock dispersions as described in 6.5 and 6.6;
— description of medium used to prepare dosing/sample solution and volume prepared;
— pH o f dosing/sample solution and description o f pH bu ffering system;
— description of dispersion procedure(s) applied, including details such as sonication/vortexing time
and/or energy input;
— details of dose/sample administration, including volume administered, mixing procedure (for in
vitro tests), and elapsed time since sonication or mixing of the sample dispersion;

ISO/TR 1 0993 -2 2 : 2 01 7(E)
— details o f any re-analysis o f subsamples from stock or sample/dosing dispersions to veri fy properties
at the conclusion o f dosing or a fter modifications to the dosing solution.
Steps taken to improve dispersion and/or dispensability o f the dosing solution should be described in
detail and justified.
Detailed recommendations for preparation of nano-object dosing dispersions for oral, inhalation, and
dermal toxicology studies are provided in ISO/TR 16196 and Re ference [68 ].
6.8 Dose metrics
Dose levels for toxicology studies are conventionally expressed on a mass concentration basis. However,
there are multiple attributes o f nanomaterials that can influence their toxicological properties. It is
generally accepted that in addition to mass concentration, other parameters including sur face area
and particle number concentration should be used to fully characterize the dose o f nanomaterials.
Adequate characterization details should be provided to enable the end user to interconvert various
dose metrics, including mass concentration, number of particles, and surface area[72 ] . However, it is
not always possible to accurately measure sur face area and/or number concentrations especially when
agglomeration occurs. In addition, sur face area measurements are currently limited to determinations
of nanomaterials in powder form while such measurements for liquid nanodispersions are still under
development. Unless the particle number concentration or sur face area concentration can be directly
measured, they are likely to have higher uncertainties than the mass concentration [73 ] .
There can be situations where mass concentration is appropriate for describing the dose of a
nanomaterial. For example, when toxicity is mediated by ions released by the nano-objects, the most
appropriate dose metric might be the mass of soluble ions. Also, for inhalation exposure (aerosolization)
other dose metrics might apply.
The possibility o f fractional deposition should be considered when determining the toxicologically
relevant dose in in vitro studies of nanomaterials [72 ] [74] [75 ] . The contact of small nano-objects (e.g.
hydrodynamic diameter <40 nm) with cultured cell layers is primarily determined by di ffusion and
convection forces. Larger nano-objects and aggregates of nano-objects formed in the cell culture
medium settle more rapidly because o f the additional influence o f sedimentation forces. These factors,
as well as interaction with proteins and other constituents o f the culture medium, can influence the
number o f nano-objects that directly contact cultured cells.
Parameters that influence the deposition mechanisms [such as density and the bivariate (length and
width) size distribution], the concentration that reaches the cell layer sur face (deposited dose), and
the amount o f NMs taken up by the cells (cellular dose) can give additional in formation to interpret
the observed biological responses [72 ] [76 ] [118 ] . It has to be noted that in practice it can be di fficult to
measure deposition and cellular uptake.
6.9 Additional considerations
6.9.1 Endotoxin
Bacterial endotoxin, or lipopolysaccharide (LPS), a cell wall component o f gram-negative bacteria, is

by far the predominant type o f pyrogen. Endotoxin is omnipresent in the environment and has been
identified as a common contaminant o f nanomaterials [77 ] .
The presence of endotoxin as a contaminant in nanomaterials can confound biological evaluation tests
leading to incorrect conclusions about biocompatibility [77 ] [ 78 ] [79 ] . Therefore, it is important to evaluate
the endotoxin contamination which is usually done at the stock solution and/or source material stage.
Traditional quantification assays for endotoxin might not work reliably for nanomaterials because their
properties can inter fere with the reagents and/or detection methods used in these assays [80 ] [81 ] . In the
commonly used Limulus amebocyte lysate (LAL) assay, for example, nano-objects can inter fere with
the reactivity o f endotoxin, the LAL reaction, or detection o f the reaction products [81 ] . Such interference
can result in either overestimation or underestimation o f endotoxin in the sample. Additionally, the

ISO/TR 1 0993 -2 2 : 2 01 7(E)
presence and composition of phospholipids in the test solution can be determined to exclude the
presence of endotoxin.
The choice of an appropriate method to assess the endotoxin contamination in medical devices should
be made on case-by-case basis. Adapted LAL assay protocols for use with nanomaterials are available.
A decision tree to aid in selection of an appropriate LAL format for a particular nanomaterial has been
proposed and is use ful for understanding the potential issues associated with testing di fferent types o f
nanomaterials [83 ] . Alternative methods and their advantages and disadvantages compared to the LAL
assay are reviewed in detail in Re ferences [82 ]and [83 ].
ISO 29701 describes the application of a test using LAL reagent for the evaluation of nanomaterials
intended for cell-based in vitro biological test systems. The test is suitable for use with nano-object
samples dispersed in aqueous media, e.g. water, serum or reaction medium, and to such media incubated
with nano-objects for an appropriate duration at 37 °C. ISO 29701 is restricted to test samples for in
vitro systems, but the methods can also be adapted to nano-objects to be administered to animals by
parenteral routes.
Another method is the monocyte activation test (MAT) which is a validated quantitative method that
exploits the natural human fever mechanism and can be used to test for endotoxins associated with
nanomaterials, as well as other biological pyrogens (e.g. yeast, parasitic and viral pyrogens) [84] [85 ] [86 ] .
While methods exist to remove endotoxin from nanomaterials, the procedures can result in
agglomeration or other undesirable changes. A recommended approach to management of endotoxin
contamination is development of practices that prevent contamination during manufacture of
nanomaterials [77 ] .
It is important to note that endotoxin is not inactivated by most o f the sterilization methods [82 ] .
However, it can be inactivated by at least 30 min o f dry heat at ≥250 °C (European Pharmacopeia 8.0,
section 2.6.14). Also, European Pharmacopeia 8.0, section 5.1.2, indicates that dry heat at temperatures
higher than 220 °CGetis fmore
requently
FREE used for sterilization
standards and depyrogenation
from Standard Sharing Groupoandf glassware.
our chatsIn this case,
demonstration o f ≥3-log reduction in heat-resistant bacterial endotoxin can be used as a replacement
for biological indicators. The methods for endotoxin inactivation mentioned above can potentially
modi fy or degrade nanomaterials.
6.9.2 Sterilization
Sterilization is an important consideration for the pre-clinical biological evaluation of medical devices
and use o f the finished product.
— In testing, addition of non-sterile nano-object dispersions to media containing proteins and
nutrients can result in growth of contaminating microorganisms such as bacteria, fungi, and
viruses. Sterilization of nano-object preparations is required to prevent microbial contamination
that can inter fere with test systems and con found test results.
— Sterilization o f the finished product is o ften necessary to eliminate potential microbial cont amination.
Available methods for sterilization of nanomaterials are reviewed in References [87 ] and [88 ] which
include:
— autoclaving;
— sterile filtration;
— gamma irradiation;
— ethylene oxide.
High hydrostatic pressure has been proposed as an additional method o f sterilization [87 ] , but
improvements are needed to increase the ability o f this method to eliminate bacterial spores.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
The commonly used methods for sterilization listed above have the ability to modi fy or degrade
nanomaterials. Potential modifications resulting from sterilization are material-specific and can
include:
— decomposition;
— alterations in size and shape;
— aggregation state;
— type and concentration o f impurities;
— degradation profile;
— stability;
— biocompatibility profile o f the material;
— and/or functional behaviour.
For example, heat sterilization by autoclaving can alter the mechanical properties o f polymers having a
glass transition and/or melting point below 120 °C [87 ] . Sterilization with gamma irradiation or ethylene
oxide, methods that can be applied to heat-sensitive materials, can generate free radicals that can
produce toxic degradants [88 ] . Characterization of nanomaterials or nanomaterial-containing products
should be per formed a fter sterilization to account for any changes caused by the sterilization process.
Sterile filtration, an alternative method for heat- and chemical-sensitive nanomaterials [87 ] , involves
filtration through membrane filters with pore diameters o f 0,22 µm (220 nm). As such, it is not
applicable to nanomaterials containing fractions with a particle diameter o f approximately 220 nm or
greater. In general, sterile filtration cannot be used for sterilization o f highly viscous suspensions. Also,
nano-objects can absorb or adhere to the fibres o f the filter, thus reducing the concentration o f the
nano-objects.
Because the e ffects o f sterilization are likely to be material-specific, impacts o f sterilization on
nanomaterial properties need to be evaluated on a case-by-case basis. Testing o f several methods might
be needed to select the optimal mode of sterilization for a particular nanomaterial.
The changes induced by sterilization o f a nanomaterial can have prolonged e ffects. For example, free
radicals formed during sterilization using gamma irradiation or ethylene oxide can be transient, but
can also initiate cascading reactions that lead to formation of degradants and/or alteration in product
function. However, addition o f free radical scavengers (e.g. histidine or phenylalanine) can protect
against biomaterial degradation as was demonstrated for alginate based biomaterials [89 ] . A stability
program suitable for the detection of changes during the shelf life of the sterilized nanomaterial and/or
finished product should be established to determine whether such changes occur.
In some cases, it can be di fficult to identi fy a compatible sterilization method for nanomaterials.
Manufacture under sterile conditions can be a solution in such cases.
7 Release of nano-obj ects from medical devices
A comprehensive identification and characterization o f potentially released nano-objects might be

necessary under physiological conditions similar to the intended conditions o f use. The release kinetics,
quantity, migration and bioaccumulation o f the nano-objects in biological environments should be
evaluated.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
7.2 Degradation products
I S O 10 9 9 3 -9 provide s a fra mework and s ta r ti ng p oi nt to add re s s the identi fic ation and qua nti fic ation
of potential degradation products from medical devices. According to ISO 10993-9:2009, Annex A,
degradation studies should be considered if
a) the device is designed to be absorbable,
b) the device i s i ntende d to b e i mpl ante d for longer than 3 0 days , or
c) an i n forme d con s ideration o f the materi a l(s) s ys tem i nd ic ate s that toxic s ub s tance s c an b e rele as e d
du ri ng b o dy contac t.
ISO 10 9 9 3 -1 3 , ISO 10 9 9 3 -14 and ISO 10 9 9 3 -1 5 cover genera l as p e c ts o f de gradation o f p olymers ,
cera m ic s , and me ta l s a nd a l loys , re s p e c tively.
In conjunction with the release of ions, the occurrence of corrosion can also result in the release of
f
p a r ticle s at the na no s c a le . For cer tai n na nomateri a l s , it i s known th at new na no - obj e c ts c an b e orme d
from the released ions (e.g. nanosilver [90 ). ]
7.3 Release of nano-obj ects by wear
With many medical devices , there i s p o tential for the device to wear over ti me duri ng i ntended us e and
releas e nano - obj e c ts (e. g. p ar ticles , fibres , flakes , fragments) into its envi ronment. T hi s i s o f p ar ticu lar
concern given the known bio -p ers i s tence o f cer tain nano - obj e c ts which can bi nd biological elements with
great e fficienc y due to their nano s cale s i ze, large s p eci fic s ur face are a and high reac tivity [91 . Therefore,
]
releas e o f nano - obj ec ts by we ar should b e prop erly addres s ed i f the fol lowi ng conditions apply:
a) the device is a nanomaterial;

b) the device is coated with a nanomaterial;
c) the device, under its normal condition of use, generates friction with biological tissue or is subjected
to friction between its components or with cement or composites; or
d) if the manufacturing residues can include incidental nanomaterials.
Me tho d s for s a mpl i ng we a r p a r ticle s generate d by j oi nt rep lacement i mpla nts in human s a nd in
j oi nt s i mu lators are de s crib e d i n I S O 178 5 3 . I t s p e c i fie s the app aratu s , re agents a nd te s t me tho d s to
i s ol ate and cha rac teri ze b o th p olymer and me ta l we a r p ar ticle s from s a mple s o f ti s s ue s e xci s e d from
around the j oi nt replacement i mplant, ob tai ne d at revi s ion s u rger y or p o s t mor tem, a nd from s ample s
o f j oi nt s i mu lator te s t flu id s . S ome o f the s e pro ce du re s cou ld cer tai n ly b e adap te d for i s olation and
charac teri z ation o f p ar ticle s from hu man biolo gic a l flu id s (e . g. s ynovi a l fluid) .
I n the conte x t o f th i s do c u ment, it shou ld b e no te d that me d ic a l device s (e . g. i mpla nts , denta l fi l l i ngs)
can generate nano-objects due to wear even when nanomaterials are not used in the manufacturing of
these medical devices.
7.4 In situ processing
Mechanical treatment (e.g. polishing, grinding) of certain medical devices in situ , such as performed in
f
denti s tr y, ha s the p o tenti a l to generate materi a l s at the nano s c a le, i rre s p e c tive o whe ther the origi na l
medical device contains nanomaterials [92 ]

. T h i s s hou ld b e add re s s e d i n the ri sk a s s e s s ment.
8 Toxicokinetics
T he maj or ri s k a s s o ci ate d with nanomateri a l s i s con s idere d to b e relate d to the pre s ence or rele as e o f
free nano-objects, ions, or components that comprise the individual nanomaterials. In medical devices,
ISO/TR 1 0993 -2 2 : 2 01 7(E)
nanomaterials can be present as free, fixed or embedded materials each with their potential for release
o f the nano-objects in the body.
The kinetic properties o f nano-objects can be described by absorption, distribution, metabolism and
excretion/elimination (ADME) as potentially sequential processes. Toxicokinetic studies need to be
considered as part o f the toxicological risk assessment o f medical devices containing nanomaterials.
A toxicokinetic study is only required i f the nanomaterial has the potential to be released from a
medical device and become absorbed, distributed, metabolized, and/or excreted. ISO 10993-16
provides a framework on how to carry out toxicokinetic studies. Overall, the standard is applicable
to nanomaterials but some adjustments might have to be made such as labelling techniques, animal
model, study duration, dosing strategy and analysing techniques.
Factors such as route of administration, size of the nano-object or its aggregates/agglomerates, surface
properties (chemistry and charge), animal species, dose and dosing methods have all been reported to
influence the toxicokinetics in animal models [69 ] [70 ] [93 ] [94] [95 ] . When nanomaterials are used in test
systems, one has to be aware that some o f the properties that need to be determined can be a ffected
and are largely dependent on the surrounding environment (e.g. tissue culture media, blood/serum,
protein presence). Such interactions with the environment can result in a temporal evolution of the
nanomaterials themselves e.g. by obtaining/shedding a protein coating, the formation o f nano-
object agglomerates/aggregates and other changes in the nanomaterials. Such changes can affect the
nanomaterial characteristics, which can impact the toxicological profile o f a nanomaterial.
There fore, i f the risk assessment concludes that a toxicokinetic study is required; factors that can
influence the study design, the per formance o f the study and interpretation o f the results should be
addressed.
8 . 2 Fa c to r s i n fl u e n c i n g th e to x i c o ki n e ti c s
8.2 .1 Physicochemical properties
Several studies have shown that properties such as size and size distribution, shape, charge,
agglomeration and aggregation, hydrophilicity, and sur face structure a ffect the ADME behaviour.
For example, while smaller nanoparticles (10 nm to 15 nm) have been reported to have a widespread
distribution, larger nanoparticles tend to accumulate in organs o f the mononuclear phagocyte system
(MPS) notably liver and spleen [69 ] [95 ] organs known to play an important role in filtering aged or
damaged red blood cells (RBCs) and other particles from the blood [97 ] . It has been shown that particles
larger than 200 nm are more likely to accumulate in the spleen and not in the liver due to the size o f the
inter-endothelial cell slits (approximately 200 nm in width). In a study conducted on gold nanoparticles,
with a size ranging from 1,4 nm to 200 nm, a similar finding has been reported in the liver where
accumulation increased with increasing particle size[95 ] . At the cellular level, it has been reported
that nanoparticles larger than 100 nm can be taken up by cells via di fferent endocytic pathways, such
as clathrin- or caveolae-mediated endocytosis [98 ] . Larger nanoparticles are less likely to have skin
penetration, while their smaller counterparts may be able to access the deeper epidermis, dermis, and
lower cell layers [96 ] .
The physicochemical properties o f nanomaterial sur faces play a crucial role in determining the
interaction with biomolecules [99 ] and biological systems. Sur face properties such as hydrophilicity and
hydrophobicity and charge are known to influence protein adsorption, the stability and strength o f
their adsorption, as well as the nature of adsorbed biomolecules (see 8.2.2). So called capping agents can
be used to stabilize nano-objects by binding to the nano-object sur face via covalent bonds or by non-
covalent chemical interactions. These capping agents are meant to prevent nano-object aggregation.
Examples o f capping agents include small organic molecules, water-soluble polymers, polysaccharides,
lipids, proteins, and sur factants. The capping agents can a ffect the binding stability and/or toxicity.
Some physicochemical properties were found to a ffect toxicokinetics. Nanoparticles with sizes at
or below 10 nm have a faster excretion through urine [100 ] and a more widespread distribution [93 ] .
Hydrophilic coating [e.g. polyethylene glycol (PEG)] increases the blood circulation time [69 ] [ 70 ] . Also,
for 20 nm silica nanoparticles, the amount in liver and spleen was found to be higher compared to
80 nm silica nanoparticles [101 ] . Both the 20 nm silica nanoparticles and 80 nm particles were excreted
ISO/TR 10993-22:2017(E)
via the urine with the excretory rate for 80 nm being higher than for the 20 nm silica nanoparticles [102 ] .
It has been reported that objects less than 12 nm in diameter may cross the blood–brain barrier [103 ] . In
addition, the surface charge controls the corona formation (see 8.2.2) as well as the interaction with the
cell membranes which also may a ffect the toxicokinetics. So far, however, it is an open question which
parameter is most significant in influencing the ADME.
8.2.2 Biomolecular adsorption

Nanomaterials in biological environments are subject to rapid protein adsorption on their surfaces,
forming what is known as the protein “corona”. Coronas have been reported to be dual-layered
systems composed o f an inner core o f strongly bound proteins and an outer layer o f rapidly exchanging
molecules [104] . It should be noted that the protein corona is not static and can change depending on the
direct environment of the nano-object. In addition, other biomolecules, such as lipids, can also adhere
to the nanomaterial sur face. The formation o f a serum protein “corona” enhances recognition and
uptake by cells o f the mononuclear phagocyte system [99 ] [104 ] [105 ] . This generally results in a rather
rapid clearance o f the nano-objects from the blood, typically into liver and spleen which are key organs
o f the mononuclear phagocyte system [70 ] [93 ] [94] [105 ] [106 ] [107 ] [108 ] [109 ] [110 ] .
The structure and composition o f the protein corona depends on the synthetic identity o f the
nanomaterial (i.e. material-intrinsic properties such as size, shape, composition, charge and
hydrophobicity [111 ] ), the nature o f the physiological environment (e.g. blood, interstitial fluid, cell
cytoplasm), and the duration o f exposure [112 ] .
The protein corona alters the size and interfacial composition of a nanomaterial, giving it a biological
identity that is distinct from its synthetic identity. As a result, physiological responses including
signalling, kinetics, transport, accumulation and toxicity are influenced by the acquired biological
identity [104 ] [105 ] [113 ] . It is important to note that depending on tissues and cellular compartments,
significant di fferences exist in biochemical composition, ionic strength and acidity o f biological
fluids. The nature Get and morequantity o f proteins
FREE standardsadsorbed on nano-objects,
from Standard and the
Sharing Group andsurrounding
our chats biological
compartment might contribute to the nature and extent of cellular responses including nano-object cell
recognition, internalization (e.g. by macrophages) and subsequent outcomes.
Plasma and serum media are use ful for identi fying which proteins absorb onto specific nanomaterials.
The use o f simulant fluids, relevant to the biological compartment, could help assess the fate o f nano-
objects under physiological conditions. Simulant fluids can be used to determine the biomolecules
that could adsorb on the nanomaterial sur face and evaluate the li fe cycle trans formations o f
nanomaterials [114] [115 ] [116 ] [117 ] . In formation on the use o f simulant fluids has also been included in
the OECD dossiers from the sponsorship programme. For example, Osmond-McLeod et al. [117 ] used
simulated biological fluid (Gamble’s solution) to assess the durability o f di fferent types o f carbon
nanotubes.
As the nano-object interacts with its environment, a vehicle (e.g. suspending solution) can influence
the toxicokinetics o f the nano-objects. Some vehicles a ffect nano-object agglomeration altering the size
distribution whereas other vehicles a ffect the binding o f substances to the sur face. Pre ferably, the vehicle
should be the same as used for other toxicity tests. In short, it is crucial to carry out appropriate material
characterization both o f the pure nanomaterial as well as the nanomaterial used in the test system.
Surface nanostructures can have an effect on nanomaterial-protein interaction/adsorption, their
circulation/distribution and ultimately on their toxicokinetic profile. The sur face chemistry and
structure o f nanomaterials are particularly important as they determine the nature and extent o f
primary interaction with biological systems, and subsequent cascade o f events. Sur face molecules
can be inherent to the native structure o f nanomaterials or intentionally added moieties intended to
alter sur face properties o f nanomaterials to provide them with specific functionalities. For example,
surface coatings can be utilized to alter surface properties of nanoparticles to prevent aggregation or
agglomeration, promote pre ferential protein adsorption or enhance specific tissue or cell targeting [118 ] .

ISO/TR 1 0993 -2 2 : 2 01 7(E)
8.2 .3 Exposure route
The route o f entry is important as it can change the sur face o f the nanomaterials/nanoparticles and
the biodistribution o f nano-objects in the body [94] [104 ] [105 ] [119 ] [120 ] [121 ] . Depending on the site of
application, further kinetics o f a released nano-object can be a ffected by adherence o f molecules to
the surface of a nanomaterial. In this respect, the formation of a serum protein corona is suggested to
enhance recognition and uptake by cells o f the mononuclear phagocyte system (MPS) [99 ] [104 ] [105 ] . This
generally results in a rather rapid clearance o f the nanoparticles from the blood, typically into liver
and spleen which are key organs o f the MPS [70 ] [93 ] [94] [106 ] [107 ] [108 ] [109 ] [110 ] . Surface treatment, for
example PEGylation o f nanomaterials, has been found to delay the blood clearance o f IV-administerered
nanomaterials [69 ] [70 ] . For most exposure routes, other than intravenous, the majority o f nano-objects
remain at the entry point in nearby tissues, or the local draining lymph node. A fter local release, nano-
objects can enter the systemic circulation directly or indirectly via the lymph drainage.
Translocated Au nanoparticles (1,4 nm) after intratracheal administration showed an organ distribution
pattern with a higher uptake in the kidney compared to the liver whereas the liver is the predominant
target organ after intravenous administration [122 ] . Accordingly, nano-objects released from a medical
device can be covered by di fferent biomolecules depending on exposure route (e.g. intravenous blood
catheters versus intratracheal intubation). Current data in the literature suggest that systemic uptake o f
nano-objects through skin is limited [123 ] [124 ] [125 ] . Dermal uptake depends on nanoparticle properties,
on the anatomical site (skin thickness) and the skin barrier integrity [125 ] . Also, mechanical movements
and chemicals can enhance absorption. If the lung is exposed to nano-objects, the distribution in the
lung will depend on the properties of the nano-objects, where nano-object size is an important factor.
When the nasal cavity in rodents was exposed to nano-objects, transport o f nano-objects to brain via
sensory nerves were observed [126 ] [127 ] [128 ] .
Nanomaterials have been found in almost all types o f organs and tissues, which makes prediction
o f target organs di fficult. The level o f nanomaterials found in di fferent organs is o ften low making
identification and quantification o f nanomaterials challenging.
8.2 .4 Dose
I f the doses administered exceed the clearance capacity, accumulation in the body occurs. Nano-objects
deposited in the alveoli are primarily phagocytized by alveolar macrophages which are ultimately
transported to the mucociliary escalator and cleared mechanically via the airways. I f the dose exceeds
clearance, overload/saturation o f clearance occurs and the kinetics are changed [129 ] . In blood, nano-
objects are mainly cleared via uptake by phagocytic cells in organs o f the MPS. However, high or
repeated nano-object doses injected into the bloodstream can, for example, overwhelm the phagocytic
cells in the liver and spleen, in which case the particles would be redistributed to other organs [130 ] [131 ] .
Also, it has been reported that toxicokinetics o f nano-objects in the bloodstream is a ffected by the time
interval between doses [132 ] .
8.2 .5 Species and gender
The physiology and anatomy di ffer between species and genders, and there fore, it cannot be excluded
that this results in modification o f the toxicokinetics for nano-objects. Results from toxicokinetic
studies on gender differences have shown that female rats are more susceptible to accumulation of
silver nano-objects and their derivatives in kidneys than are male rats [133 ] [134 ] . Also, toxicokinetic
differences between species have been observed. When mice, rats and hamsters were exposed to
titanium dioxide nano-objects via inhalation, a disturbance in clearance of nano-objects from lung was
observed in mice and rats. As a result, the burden of nano-objects in the lung of mice and rats was
higher than for hamsters [135 ] . When Brown et al [136 ] compared clearance of particulate matter in the
.
lungs between rats and humans, they found a faster clearance rate for rats than for humans. Even i f
the target organ for a nano-object is the same, the localization within the organ can be different [137 ] .
The species and gender in which the toxicokinetics is studied support the systemic toxicity studies, so
alignment o f toxicokinetic and systemic toxicity studies should be considered when selecting species
and gender. In view of the limited information on gender aspects, this issue should be further studied.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
8.2 .6 Measurement techniques
In order to measure nanomaterials in tissue and organs, the nanomaterial or its elemental components
have to be detectable from the biological background. To aid in this, the nano-object can either be
labelled with radioactive or fluorescent labels or alternatively determined by its elemental composition,
using analytical methods such as inductively coupled plasma-mass spectrometry (ICP-MS). Analysis
that relies on the elemental composition has the disadvantage that it does not inform the user of the
nature of the nanomaterial (i.e. whether it is present as a nano-object or degraded into elemental
form). This limitation could be overcome by using single particle ICP-MS (spICP-MS) [138 ] . spICP-MS is
a relatively new technique that is extremely use ful for characterizing and identi fying nanoparticles
because it is able to provide insight and information into their elemental chemical composition, size,
size distribution and number concentration. Furthermore, spICP-MS has the ability to provide critical
in formation to assess the risk level o f devices which contain or leach nano-objects by discriminating
between elemental ions and nanoparticles which can present different toxicities.
Labelling techniques also have disadvantages. For example, a label can be released from a nano-object
and/or it can alter the interaction of the nano-object with its environment, which in turn will affect
toxicokinetics. When selecting a labelling technique, the stability o f the bond between the label and
the nano-object should be considered along with its detection limit. Methods with low detection limits
might not be able to detect very low levels o f nano-objects in tissues and organs without reprocessing.
For some analytical techniques (e.g. ICP-MS), additional processing o f the test samples can be necessary
in order to increase the sensitivity. I f the dose is increased or i f repeated dosing is used to assure
detectability o f nano-objects in tissue/organs, the MPS system can become saturated and hence, the
toxicokinetic behaviour modified (as discussed in 8.2.4).
9 Toxicological evaluation
9.1 Get more FREE standards from Standard Sharing Group and our chats
General considerations
Since it is known that nanomaterials display di fferent physicochemical properties (e.g. mechanical,
chemical, magnetic, optical or electric) from that o f their bulk forms, it would be logical to expect
nanosized materials to affect the biological behaviour responsible for different effects occurring at the
cellular, subcellular and biomolecular levels (e.g. genes and proteins), including cellular uptake. As a
result, a di fferent toxicological profile from that induced by conventional materials can be expected
after exposure to nanomaterials.
Nano-objects have the potential to translocate throughout the body and be taken up by tissues that are
downstream from the site of administration (see Clause 8 on toxicokinetics). They have the potential
to cross not only the cellular membrane but also the intracellular structural membranes o f the nucleus
and mitochondria to interact or interrupt DNA synthesis and other cellular functions [139 ] .
When in contact with the biological environment, nano-objects interact with proteins at quantitative
and qualitative levels that are dictated by the nature o f the physiological environment (e.g. blood,
plasma, cytoplasm, etc.) and nanomaterial characteristics. Similarly, when exposed to testing media,
nano-objects are expected to interact and/or interfere with the environment depending on their
inherent nature and the conditions o f exposure; they can then display di fferent behaviour from
corresponding bulk materials. It is there fore necessary to specifically validate any testing method
designed for biological device evaluation. Similar to medical devices, the choice of endpoints would be
dependent on time and site of contact. However, the choice of the actual testing method can also depend
on the nanomaterial characteristics as these can interfere with various testing methods.
There are several known pit falls in toxicity testing o f nanomaterials that should be avoided (see 9.2 to
9.8 for specific tests). Also, in the future, more testing pitfalls might come to light as the knowledge o f
the toxicity and fate o f nanomaterials improve. There fore, it is important to stay up to date with regard
to the state of the art on testing of nanomaterials.
Potential biological interaction is not directly dependent on the concentration or number o f molecules,
but on the nanomaterial/nano-object itsel f. The dose-response relationship in nanotoxicology might not

ISO/TR 1 0993 -2 2 : 2 01 7(E)
be the traditional unit of mass or concentration but rather nano-object numbers or nanomaterial/nano-
object total surface area.
In addition to characterization, detailed description of experimental conditions should be documented,
including the following.
— for the dose description of the nano-object used in the test, at least three parameters should be
recorded: mass, number of particles and surface area, allowing switching between or calculating
various dose metrics (ISO/TR 13121);
— suspension/reconstitution medium;
— testing medium (e.g. % serum);
— characteristics of the medium (pH, salt, surfactant, etc.) ;
— a ny add itive s to the me d iu m a nd thei r e ffe c ts on the na no - obj e c ts s uch a s d i s p ers ion, aggre gation,
etc. should be listed;

— ion rele a s e i n a s p e c i fic me d iu m s hou ld b e cha rac teri ze d;
— suspension status should be documented in the reconstitution/suspension medium and testing

medium;
— container material.
It should be realized that in several areas, alternative testing methods are being developed to replace,
in vivo
re duce a nd refi ne an i ma l a s s ays . Whenever p o s s ible, s uch as s ays , when va l idate d, shou ld a l s o b e
considered for the evaluation of nanomaterials used in or released from medical devices.
ISO/TR 16197 describes toxicological screening methods for manufactured nanomaterials.
9.2 In vitro cytotoxicity testing
9.2 .1 General considerations
I n genera l, c yto toxic ity i s regarde d as the i mp ai rment o f cel lu lar fu nc tion s i nclud i ng but no t l i m ite d to
the d i s r up tion o f plas ma membrane i ntegrity, i nter ference with organel le func tion and d i s rup tion o f
the c yto s kele ton . T he damage dep end s on the concentration o f the agents . E va luation o f c yto toxicity
related to nanomaterials used in medical devices involves incubation of cultured cells with a device
and/or f
ex trac ts o a device either d i re c tly ff
(d i re c t contac t) or th rough di u s ion (i nd i re c t contac t) .
The in vitro tests are designed to determine the biological response of mammalian cells and potential
c yto toxic e ffe c ts o f me d ic a l device s a nd materia l s i n thei r comp o s ition s . C yto toxicity o f nano - obj e c ts
c an b e s ele c ti ve dep end i ng on cel l s en s itivity to the toxic ant, pre s ence o f s p e c i fic re cep tors , or up ta ke
me chan i s m s . T here fore, when as s e s s i ng na nomateria l c yto toxic ity, s p e ci a l attention s hou ld b e given to
the in vitro te s ts cho s en . I n add ition to cho o s i ng the relevant cel l- c u ltu re mo del, p a rame ters s p e ci fic to
na no - obj e c ts s hou ld b e ta ken i nto account.
ISO 10993-5 describes accepted and standardized test methods for assessing the in vitro c yto toxicity
of me d ic a l device s . T he s e i nclude a s s ays s uch as neutra l re d up ta ke, colony formation, MTT
[3 - (4, 5 - d i me thylth i a z ol-2 -yl) -2 , 5 - d iphenylte tra z ol iu mbrom id] and XTT {2 , 3 -bi s[2 -me thox y- 4 -n itro -
5 - s u l fophenyl] -5 -[( phenyla m i no) c arb onyl] -2 H -te tra z ol iu m hyd roxide}, that as s e s s the fol lowi ng as
i nd ic ators o f c yto toxicity:
— cel l up ta ke;
— cel l lys i s;
— cell growth inhibition;

— cel l colony formation;

ISO/TR 1 0993 -2 2 : 2 01 7(E)
— metabolic activity; and

— other e ffects on the cells (e.g. morphology, membrane lesions, etc.).
Although these methods have only been accepted for devices with conventional materials, these can be
and have been applied to nanomaterials given that their physico-chemical properties are considered.
Testing strategies have been proposed for both the in vitro testing [140 ] including evaluation of existing
data for their relevance to the expected exposure scenario [141 ] .
Nano-objects are generally taken up by the cells in in vitro studies.Once inside the cells, nano-objects
can interact with the biological components and disturb the cellular functions. The intracellular
localization o f the nano-objects inside the cells would depend on their physicochemical properties,
size and dose. As in vivo , nano-objects tend to end up mainly in cells o f the MPS, in vitro cytotoxicity
testing in both phagocytic (e.g. RAW264,7 murine macrophages, THP-1 human cells as di fferentiated
macrophages) and non-phagocytic cell lines (e.g. 3T3 murine fibroblast, L929 murine fibroblasts, HaCaT
human keratinocytes) can be considered (ISO 10993-5, ISO 19007:—, Annex A).
One mechanism reported to be involved in cell toxicity is oxidative stress [142 ] [143 ] that triggers
activation o f signalling pathways that are sensitive to redox potential, which would ultimately culminate
in production o f cytokines and chemokines involved in pro-inflammatory responses. Especially when
nanomaterials are inducing cytotoxicity via reactive oxygen species (ROS), the nano-objects do not
necessarily have to enter the cells. A similar situation can exist for nanomaterials that are inducing
cytotoxicity via ion release such as ZnO and Ag nano-objects.
Some parameters are considered critical while conducting in vitro cytotoxicity assays. Examples are
indicated below.
9 .2 .2 Consideration of nanomaterial interference with the assays
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nanomaterials. A wide variety o f inter ferences is possible when evaluating cytotoxicity o f nanomaterials
in vitro [150 ] . Due to electric charges and optical properties, nanomaterials can potentially inter fere with
test protocols relying on colorimetric assays and/or fluorescent agents. Interaction with nutrients, e.g.
adsorption, might be another cause o f false positive outcomes o f a cytotoxicity assay.
Specifically, nano-objects have been shown to interact with dyes used in assays such as MTT, XTT, lactate
dehydrogenase (LDH) and dichlorofluorescin (DCF) [151 ] [152 ] [153 ] [154 ] . Moreover, some nano-objects
can themselves disperse/absorb light and therefore interfere with the measurements in colorimetric
assays. These aspects need to be considered when using colorimetric methods. In addition to direct
inter ference, it should be noted that nano-objects can directly bind/adsorb to biological mediators
leading to altered measurements) [150 ] [152 ] [153 ] [157 ] [158 ] [159 ] . Incorporation of appropriate controls
and removal o f nano-objects via centri fugation be fore reading the assay can reduce the variations in
data generated for the same nano-objects) [155 ] [156 ] . Corroboration of several test results from different
methodologies might be required for a scientifically sound interpretation.
9.2 .3 Consideration of relevant dose and dose metrics
When using in vitro methods, it is important that the doses of nano-objects to be tested include a
broad range for evaluation o f the cytotoxicity (ISO 19007). Additionally, it is important to realize the
existence o f di fferent dose metrics and to use a relevant dose metric to express any dose response
relationship. Defining the appropriate dose metric o f nano-objects represents a challenging issue for
cytotoxicity evaluation in cell culture models. Mass, sur face area, and number o f nanoparticles have all
been proposed as important dose metrics and are considered at the OECD level [72 ] [144] [145 ] [146 ] .
9.2 .4 Consideration of nano-obj ect kinetics
In in vitro cell culture models, dissolved compounds reach the cells by di ffusion, while nano-objects
can get into contact with cells by sedimentation [aggregated nanoparticles (NPs)] and by di ffusion
(single particles). It has been reported that these processes are predominantly size-dependent [147 ] .

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Aggregation is also influenced by the culture media and conditions, as well as the physicochemical
properties of nano-objects that dictate their interaction with their environment [148 ] .
The e ffective size and density o f particles in cell culture systems are a function o f their
agglomeration/aggregation state, packing density and shape. These properties can have pro found
impact on the rate o f delivery to cells in culture by di ffusion and gravitational settling [148 ] . On the other
hand, the agglomeration state has been reported to affect the oxidative stress-mediated dose–response
profiles in vitro .
Nano-objects characterized by their small mass can remain in suspension due to relatively low
sedimentation forces [72 ] . This limits their contact with cells and the dose of delivered nano-objects in
culture models where the cells adhere to the bottom surface of culture dishes. Therefore, consideration
should be given to the unique kinetics o f nano-objects in solution which are subject to media density
and viscosity, particle size, shape, charge and density, etc.
Depending on these factors, the nano-objects can then di ffuse, settle, or agglomerate, influencing their
level of contact and transport in the cells [72 ] . So, depending on the system/model utilized, the nominal
and e ffective dosimetry (i.e. mass or number or sur face area dose o f particles that a ffect the cells) o f
nano-objects can have great differences, leading to differences between the assessed effect and the real
effect of nano-objects [149 ] . Such issues can be addressed by applying in silico modelling, such as in vitro
sedimentation, di ffusion and dosimetry (ISDD) and multiple-path particle dosimetry (MPPD) models, to
generate dose profiles for the deposited NMs.
9.3 Genotoxicity, carcinogenicity and reproductive toxicity
ISO 10993-3 specifies strategies for hazard identification and tests on medical devices for the following
biological aspects: genotoxicity, carcinogenicity, and reproductive and developmental toxicity. In
general, ISO 10993-3 is applicable for evaluation of a medical device or its components whose potential
for genotoxicity, carcinogenicity or reproductive toxicity has been identified or is unknown. The
possibility exists that a nanomaterial can have di fferent genotoxicity, carcinogenicity or reproductive
toxicity profiles as compared to the corresponding bulk material. Specific considerations for
nanomaterials are discussed in the paragraphs below. There are two major types o f genotoxic damage;
mutagenic and clastogenic, both of which should be assessed.
Specific strategies for the genotoxicity testing for nanomaterials have been described [160 ] . Conflicting
results regarding the genotoxicity o f nanomaterials have been reported. There fore, possible
nanoparticle genotoxic e ffects should be assessed on a case-by-case basis. However, when some
studies show a positive genotoxic outcome whereas other studies do not, for the risk assessment, the
nanoparticle should be considered as potentially genotoxic. Some o f the negative studies were not
relevant as DNA exposure was not demonstrated (e.g. negative Ames test).
Certain nano-objects can cross the cell membrane, enter the nucleus and interact with nuclear
DNA and proteins. In addition, direct contact between nano-objects and DNA can also occur during
cell division when the nuclear envelope disappears. Metallic nano-objects appear to be of high
concern for genotoxicity and carcinogenicity due to their reactive behaviour. Genotoxicity was
demonstrated for several metallic nanoparticles, e.g. Ag-NP [157 ] [161 ] [162 ] [163 ] , Au-NP [164] [165 ] ,
and nickel nanoparticles [139 ] [166 ] [167 ] [168 ] . However, negative genotoxicity results were also
reported [167 ] [168 ] [169 ] [182 ] . Experimental studies in rats by Hansen et al. [170 ] showed the development
o f rhabdomyosarcoma following intramuscular implantation o f metallic nickel particles that were o f
nano-size as well as o f fine-size (micrometer scale).
Nano-objects can generate free radicals following interaction with cell constituents of the same scale
and can induce DNA lesions or affect chromosome segregation during mitosis, resulting in perturbation
o f cell division and disorganization o f cell tra fficking. In contrast, it also has been reported that SWCNT
can mitigate ultrasonication-induced DNA damage [172 ] .

ISO/TR 1 0993 -2 2 : 2 01 7(E)
The genotoxic effect of nanomaterials can also be the result from indirect mechanisms, involving pro-
oxidative effects or DNA repair inhibition.
It is thought that oxidative stress followed by an inflammatory response and abnormal cellular
apoptosis are some o f the non-genotoxic events that can be elicited by nano-objects [139 ] . These events
can predispose cells to a carcinogenic outcome. Evaluations of these cellular responses in in vitro and
in vivo models can be performed as indicators for the potential to induce indirect DNA damage [139 ] .
Di fferentiated responses o f two types o f TiO 2 (anatase and rutile) were observed that were related to
the production o f reactive oxygen species (ROS) [173 ] . The most comparative outcome was the production
o f ROS in human epithelial and lung fibroblast cells. The results in Re ference [174 ] suggest that there
is a di fference in reactivity o f the di fferent TiO 2 particles based on the composition o f the crystalline
structure (anatase or rutile) versus non-crystalline TiO 2 , and sizes larger than 10 microns [174 ] .
In a study by Park et al. [171 ] , researchers showed that nanoparticles composed of zinc, silver and
aluminium showed similar toxicity in human alveolar cells. In addition, human epidemiology studies
suggest a relationship between metallic nanoparticle exposure by inhalation and the onset o f Hodgkins
lymphoma [139 ] . With the exception o f tumor induction by chronic inflammation and the situation in
lung overload (see 9.3.4), there is little data on carcinogenesis by nanoparticles.
In general, a negative result in a genotoxicity assay has to be considered care fully. To exclude a direct
genotoxic effect, an important issue is the potential exposure of the cellular target compartment
(nucleus) for in vitro and/or tissue for in vivo studies. However, it should be noted that also indirect
e ffects (e.g. due to ROS induction) can be responsible for genotoxicity.
9 .3 .2 In vitro genotoxicity tests
The bacterial reverse mutation test (ISO 10993-3) used in genotoxicity testing might not be appropriate
for mutagenicity testing o f nano-objects due to the uncertainty o f nano-object uptake and thus DNA
exposure [57 ] [58 ] [175
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in the Ames bacterial reverse mutation assay [176 ] . Weak gene mutations in Salmonella strains TA102,
TA104, and YG3003 have been caused by C60 in polyvinylpyrrolidone irradiated by visible light[177 ] .
The problem in the bacterial mutation assays is the interpretation (and conclusions) when a negative
result is obtained.
For the genotoxicity testing o f nano-objects, mammalian cell systems are recommended because o f
their potential to take up the nano-objects. The following tests are commonly used to assess in vitro
genotoxicity o f nanomaterials.
— In vitro gene mutation test in mammalian cells (the mouse lymphoma tk assay, MLA); or the
hypoxanthine-guanine phosphoribosyltrans ferase (HPRT) mutation assay. The MLA has the
capability for detection o f both gene mutations and chromosomal damage (formation o f large and
small colonies).
— In vitromicronucleus assay for detecting cytogenetic damage in conjunction with a gene mutation
assay (e.g. HPRT) or the mouse lymphoma assay detecting small scale sequence alterations (point
mutations).
— Comet (single cell gel electrophoresis assay) assay for detecting various types o f DNA damage
(e.g. single and double DNA strand breaks, oxidized bases and DNA crosslinks) depending on the
conditions o f the assay, e.g. double strand breaks are neutral comet; single strand breaks are
alkaline comet; oxidative damage depends on the addition o f enzymes.
For the in vitro genotoxicity testing, similar pitfalls might be present as described in 9.2. In addition,
there are some specific issues to consider.
The value of adding liver S9 metabolic fractions is dependent on the base material, coating and other
additives present in the nanomaterial. While inorganic particles may not be subject to metabolic
activation, other materials may be. For testing o f some inorganic nano-objects (e.g. metal oxide nano-
objects), there might not be value in adding liver S9 metabolic fractions to the in vitro assays because
the chemical components o f the nano-object may not be metabolized. However, for nanomaterials which

ISO/TR 1 0993 -2 2 : 2 01 7(E)
contain organic components or chemical entities (e.g. polymer carbon-based nanomaterials), metabolic
transformations can occur. In addition, the presence of S9 or other proteins, e.g. in serum, can cover the
sur face o f nano-objects thereby influencing their uptake and reactivity. Thus, the inclusion o f liver S9
metabolic fraction should be considered.
The micronucleus test can be used for nanomaterial testing. When the protocol requires using
cytochalasin B (cyt-B) to identi fy cells that have divided, one should determine whether endocytosis
and/or exocytosis o f nanomaterials is not a ffected by cyt-B, or use a sequential version o f the assay
where cyt-B is added a fter the exposure period and potential uptake. It is known that cyt-B can a ffect
the cellular uptake o f nano-objects [178 ] [179 ] .
9.3 .3 In vivo genotoxicity tests
When in vivo testing is indicated, appropriate methods should be used to establish that the tested nano-
object reaches the target organ. If this cannot be demonstrated, a second in vivo test in a different target
organ, for which uptake can be demonstrated, might be needed to confirm the absence o f genotoxicity
in vivo . In vivo subacute and/or subchronic toxicity studies can be used to determine the distribution
of the nano-objects. This information on the tissue distribution can guide the selection of the in vivo
genotoxicity test to ensure the tested organ is relevant to the distribution and accumulation o f the
nano-objects. A pitfall of the in vivo genotoxicity assay can be that the target organ is not exposed to the
nano-objects investigated.
Proposals for combining genotoxicity testing with other in vivo tests have been described [180 ] [181 ] .
For each study protocol, the route o f exposure in the intended human population should be considered
and exposure o f the target organs should be demonstrated. Results from the toxicokinetics studies can
be used to demonstrate organ exposure.
The following tests are commonly used to assess in vivo genotoxicity:
— micronucleus test in rodent erythrocytes or bone marrow;
— chromosomal analysis in rodent bone marrow
— DNA strand break analysis (in vivo Comet assay).
The choice o f the appropriate test system should be justified and documented.
When other in vivo test systems are used to obtain additional in formation on genotoxicity, the decision
should be justified and documented using evidence-based rationale.
For any in vivo genotoxicity assay such as the single cell gel assay (Comet assay), it is recommended to
identi fy target organs based on toxicokinetic studies and/or subchronic in vivo studies.
Several transgenic animal models are available for determination of DNA damage after in vivo exposure
(OECD TG 488, 2013). One of those models is the Lac-Z model transgenic mouse model, which has been
used to evaluate the genotoxicity o f TiO 2 nano-objects in vivo [182 ] . The model consists of C57Bl/6 mice
harboring the pUR288 plasmid (containing the LacZ reporter gene) inserted in head-to-tail sequences
homozygously on both chromosomes 3 and 4[182 ] [183 ] . This mouse model allows assessment of
mutagenicity in several organs, making it a valuable in vivo model for investigating genotoxic effects
and repairing mechanisms after exposure to chemical agents.
Other transgenic models mentioned in the OECD TG 488 for which su fficient data are available to
support their use in this TG are: lacZ bacteriophage mouse (Muta™Mouse 2)); lacZ plasmid mouse; gpt
2) Muta™Mouse is a trademark o f HRP Inc. This information is given for the convenience o f users o f this document
and does not constitute an endorsement by ISO o f the product named. Equivalent products may be used i f they can
be shown to lead to the same results.
ISO/TR 1 0993 -2 2 : 2 01 7(E)
delta (gpt and Spi−) mouse and rat; lacI mouse and rat (Big Blue® 3)). The cII positive selection assay
can be used for evaluating mutations in the Big Blue® and Muta™Mouse models. Mutagenesis in the
transgenic rodent models is normally assessed as mutant frequency; i f required, however, molecular
analysis o f the mutations can provide additional in formation (OECD 488).
9.3 .4 Carcinogenicity
The human genome is continuously exposed to DNA-damaging agents such as reactive oxygen species,
ultraviolet light, and genotoxic chemicals [184] . Numerous in vitro and in vivo studies have found
that nanomaterials induce DNA damage and mutations. The link between genotoxicity and cancer
is well recognized, so the results o f these studies are use ful for predicting the carcinogenicity o f
nanomaterials [185 ] . Chronic inflammation as suggested for rod-like nano-objects [192 ] [193 ] might play a
key role because it could lead to elevated oxidant levels. Although a considerable amount o f in formation
concerning genotoxicity is available (see 9.3.1 ), including contradicting results for many nanomaterials,
the level o f understanding o f nanomaterial carcinogenicity is limited.
It is well known that genotoxicity and chronic inflammation can lead to carcinogenicity. It has
also been reported that biopersistence and induction o f chronic inflammation has been known to
induce tumors in the lung[186 ] [187 ] . These effects may be due to what is designated as an “overload”
dosing[188 ] [189 ] [190 ] [191 ] . Biopersistence is mainly determined by the chemical composition o f the
nano-objects’ size and shape (e.g. fibers). Some nanomaterials such as nano TiO2 and carbon nanotubes
(CNTs) have been indicated to be involved in tumor development in animal models [193 ] . Possible
mechanisms involve DNA lesions and ROS production during inflammation.
An evaluation o f the carcinogenic risk should be considered i f human exposure is high or chronic. The
most common in vivo tests to assess the carcinogenic potential o f chemicals are the carcinogenicity
test as described in EC B.32 and OECD 451 and the combined chronic toxicity/carcinogenicity test
as described in EC B.33 and OECD 453. For medical devices, carcinogenicity testing is described in
ISO 10993-3. In addition, transgenic
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It should be noted however that the use o f such tests should be evaluated on a case-by-case basis, as
their suitability has not been demonstrated for nano-objects.
9.3 .5 Reproductive toxicity
While data on reproductive toxicity o f nanomaterials remain scarce, there is an increasing interest
in exploring their potential e ffects on the reproductive apparatus, cells o f the germ line, embryonic
development and offspring. Because nanoparticles are able to penetrate through biological barriers,
there is a possibility o f crossing the reproductive system barriers (e.g. blood-testis barrier and placental
barrier) with a potential impact on sperm vitality and function as well as embryo development.
Some nano-objects have been shown to cross the biological barrier of the reproductive tissues such
as the blood-testis barrier and the placental barrier depending on their type and experimental
conditions/models [196 ] . In a recent review, it was concluded that it is plausible that NP may translocate
from the respiratory tract to the placenta and fetus, but also that adverse e ffects can occur secondarily
to maternal inflammatory responses [197 ] . Damage to genetic material through interaction with DNA
molecules can also lead to mutations and affect reproduction and development of the next generations.
Determination o f whether reproductive toxicity testing is required can be made based on the exposure
and use o f a medical device as included in a risk assessment. I f required based on a risk assessment o f a
medical device, assessment o f reproductive toxicity o f the nanomaterials used should be considered in
compliance with the requirement of ISO 10993-3.
3) Big Blue® is a trademark o f Stratagene. This information is given for the convenience o f users o f this document
ISO/TR 1 0993 -2 2 : 2 01 7(E)
When su fficient and adequate evidence demonstrates that a nanomaterial or its metabolites do not
reach the reproductive system organs, no reproductive toxicity testing is necessary. This evidence can
be based on data from absorption, distribution, metabolism and excretion (ADME) studies.
When no evidence is available to rule out contact with the reproductive system organs, testing should
be considered, especially in the following cases:
a) prolonged or permanent-contact medical devices likely to come into direct contact with
reproductive tissues, embryos or foetus;
b) energy-depositing medical devices;
c) absorbable or containing leachable nanomaterials/nanoparticles. for which complete elimination
has not been demonstrated.
It should be noted that the use o f some exposure methods are not recommended as they can inter fere
with prenatal development. For example, intraperitoneal administration can cause the tested nano-
objects to be directly injected in the uterus itsel f or pass through the wall o f the uterus and directly
a ffect the developing embryos/ foetuses. Also, “nose only” inhalation exposure might not be appropriate
for pregnant females due to the fact that the animals are kept under stress ful conditions and have no
access to food and water [59 ] .
In addition to ISO 10993-3, OECD 421 can be used to gather initial information on possible reproductive
toxicity e ffects o f a nanomaterial. The results o f the test can also be used for initial hazard assessment
and can be help ful in the decision-making process as to whether additional tests are required or not. I f
additional tests are considered necessary, in view o f the outcome o f the screening test, they should be
performed in accordance with OECD 414, OECD 415, OECD 416 or OECD 422, as appropriate.
9.4 Immunotoxicity, irritation and sensitization
9.4.1 General considerations
ISO/TS 10993-20 presents an overview o f immunotoxicology with particular re ference to the potential
immunotoxicity o f medical devices.
ISO 10993-10 describes the procedure for the assessment of medical devices and their constituent
materials with regard to their potential to produce irritation and skin sensitization. This document
includes
— pretest considerations for irritation, including in silico and in vitro methods for dermal exposure,
— details of in vivo (irritation and sensitization) test procedures, and
— key factors for the interpretation o f the results.
Inflammation and allergic/autoimmune reactions can result from exposure o f the immune system to
nano-objects. The extent and type o f reactions depend on the antigenic characteristics o f nano-objects,
their adjuvant potency, inflammatory e ffect and their ability to activate the complement system.
Immune response can then either be stimulated or suppressed.
9 .4.2 Immunotoxicity
Most nanomaterials studied to date are nano-objects which upon entering the systemic circulation
end up in the MPS cells (e.g. macrophages, dendritic cells, or Langerhans cells) which play a central
role in the immune system. There fore, the potential immunotoxicity o f nanomaterials needs specific
consideration. In general, immunotoxicity is evaluated during repeat dose toxicity testing (e.g. 28 d
or 90 d) during which the first indications for immunosuppression and/or immunostimulation can
be detected. General considerations for immunotoxicity testing o f medical devices are described in
ISO/TS 10993-20. For medical devices, immunotoxicity investigations typically involve prolonged
implantation studies (ISO 10993-6 and ISO 10993-11). For nano-objects, alternative routes of exposure
ISO/TR 1 0993 -2 2 : 2 01 7(E)
need to be considered like intravenous administration for systemic exposure, e.g. as it has been
reported for nanosilver in Reference [198 ].
Because o f the complexity o f immune system, in vitro models provide a reliable and preferred method of
studying the immune cell function. The impact o f nanomaterials on immune cell function can be studied
by evaluating signaling pathways, such as the nuclear factor kappa B pathway, in specific immune
cell lines [199 ] [200 ] . A number of in vitro assays have been published by the National Cancer Institute’s
Nanotechnology Characterization Laboratory to specifically suit nanoparticle studies and their e ffect
on imunne cells (http://ncl .cancer.gov/ working _assay-cascade .asp ). These assays are intended for
assessing parameters such as phagocytosis, chemotaxis, and nitric oxide production by macrophages in
addition to many other endpoints.
Several reviews are available on e ffects o f nanomaterials on the immune system [82 ] [207 ] [208 ] [ 209 ] .
Nano-objects have been used as haptens or hapten carriers, which indicates that they are capable o f
exerting an adjuvant activity a ffecting the immune system [201 ] [202 ] [203 ] [204 ] . For silver nanoparticles,
the e ffects on the immune system were found to be the most sensitive parameter o f systemic toxicity
after intravenous administration for 28 d[198 ] [205 ] .
An issue o f Methods in 2007 was specifically dedicated to animal models for the evaluation o f
immunotoxicity o f chemicals [206 ] . WHO has published three Environmental Health Criteria documents
within the International Program on Chemical Sa fety (ICPS) dealing with immunotoxicity (EHC
180), sensitization (EHC 212) and autoimmunity (EHC 236). An EHC document on immunotoxicity o f
nanomaterials is in preparation.
9 .4.3 Sensitization
Nano-objects and nanomaterials might lead to sensitization; however, their sensitization potential
is relatively unknown. Additionally, nano-object interaction with proteins leads to the formation o f
nano-object/protein complexes,
Get more FREE which as a secondary
standards e ffect can
from Standard result Group
Sharing in sensitization [210 ] . In addition,
and our chats
possible immune reactions against the protein as part of the nano-object/protein complex might need
to be considered. See also 8.2.2 regarding the formation of a protein corona.
So far, the sole type o f (hyper)sensitivity response that has been documented for nano-objects is
pseudoallergy based on complement activation (CARPA; see 9.5.2).
The currently available literature describes mainly lack o f sensitization capacity for
nanoparticles [211 ] [212 ] [213 ] [214] [215 ] [216 ] [217 ] . Various assays described in ISO 10993-10 were used
including the Buehler test (BT), guinea pig maximization test (GPMT), local lymph node assay (LLNA),
human patch test (HPT), and a modified GPMT (GPMT with sur face application). Included in these
assays were gold nanoshells (150 nm), silver nanoparticles (10 nm), zinc oxide nanoparticles (20 nm),
titanium dioxide nanoparticles (20 nm to 100 nm), polystyrene latex beads (50 nm), C60 fullerenes
(1 nm), silicon dioxide nanoparticles (7 nm to 10 nm), and carbon nanotubes (1,8 nm to 60 nm).
The BT, LLNA, HPT, and modified GPMT sensitization tests might not be e ffective for many nanomaterials
due to the barrier function o f skin [218 ] . The target cells and organs for sensitization, dendritic cells in
the skin and the draining lymph node, might not be reached by the nano-objects. There fore, a negative
outcome cannot be interpreted as the nanomaterials having no sensitizing properties; this negative
result can be due to the inability o f the nano-objects to penetrate the skin sur face. In the modified
GPMT, the test substances were not injected concomitantly with the Freunds complete adjuvant (FCA)
but topically applied at the (inflammatory) site in which the FCA was injected [58 ] [219 ] . More research is
required to explore this issue. I f confirmed, then new nanomaterial-specific sensitization assays might
need to be developed.
There are a number of in vitro sensitization assays that were developed in response to the ban in Europe
to per form animal testing on cosmetic ingredients and the REACH legislation specifically addressing
non animal testing methods. Among those that have been the most extensively tested are the direct

ISO/TR 1 0993 -2 2 : 2 01 7(E)
peptide reactivity assay (DPRA), the human cell line activation test (h-CLAT), KeratinoSens 4) and
SenCeeTox 5) . At this time, it is unclear if these in vitro assays are capable o f assessing the sensitization
potential of engineered nanomaterials.
9.4.4 Irritation
In compliance with ISO 10993-1, irritation tests (including intracutaneous reactivity) should be
considered to estimate the irritation potential of medical devices, materials and/or their extracts,
using an appropriate site for application such as skin, eye and mucous membrane in a suitable model.
This requirement is also applicable to nanomaterials. The test(s) performed should be appropriate for
the route (skin, eye, mucosa) and duration o f exposure or contact as per ISO 10993-10.
Various properties o f nano-objects can a ffect their uptake a fter skin, eye, or mucosa exposure;
these properties include (but are not limited to) size, shape, surface area, surface charge, surface
energy/activity, solubility, aggregation state, polydispersity and ion dissolution kinetics [218 ] . Larger
nano-objects are less likely to have skin penetration, while their smaller counterparts can be able
to access the deeper epidermis, dermis, and lower cell layers [96 ] [220 ] [221 ] . In addition, the effective
size (i.e. hydrodynamic radius) o f functionalized nano-objects should also be considered, as that will
determine their interactions with keratinocytes that may a ffect penetration a fter exposure. Shape can
also have an e ffect on skin penetration, with spherical nano-objects showing a higher permeation than
elongated nano-objects [222 ] .
Chemical composition o f the main nanomaterial can also a ffect potential irritation caused by
nanomaterials. Some research has shown that Au nano-objects penetrated into the deeper skin
layers, while Ag[223 ] and TiO 2 nanoparticles stayed in the stratum corneum [224 ] . There is some
evidence that specific nano-objects (e.g. carbon-based dendrimers) are capable o f penetrating the
skin to some degree [225 ] . However, single-walled carbon nanotubes were found to have minimal skin
and eye irritation when tested in vivo per OECD TG 404 and OECD TG 405, respectively [216 ] . Surface
composition o f the nano-object is also important for potential skin penetration. For example, sur face
charge has been reported to a ffect quantum dot penetration and cytokine release [226 ] . In fact, the
sur face functionalization or derivatization o f the nano-object coating can be a cause for toxicity, rather
than the nano-object itself[173 ] [227 ] ; there fore, care should be taken when designing test methods and
interpreting test results.
Most published data suggest that intact and even sunburned skin is a good barrier against
penetration o f nano-objects and that skin penetration beyond the epidermal layers does not
occur[228 ] [229 ] [230 ] [231 ] [232 ] [233 ] . However, hair follicles, sweat glands, and compromised surfaces
due to mechanical injury (e.g. cuts, abrasions), dermatological conditions (e.g. dermatitis, psoriasis,
eczema), pathological conditions (e.g. in fection, inflammation), and photodamage (e.g. sunburn) might
be more susceptible to nanomaterial uptake through the skin. In addition, the use o f certain cosmetic
ingredients can increase skin penetration [234 ] .
For materials or devices with patient contact other than intact skin, the intracutaneous reactivity
test should be considered to assess the localized reaction o f tissue to nanomaterials. This test may
be applicable where the determination o f irritation by dermal or mucosal tests is inappropriate (e.g.
where medical devices are implanted or have blood contact). The intracutaneous introduction of the
nano-objects bypasses the stratum corneum, and thus nano-objects may have a higher irritation
potential for fibroblasts than dermal exposure [235 ] [236 ] [237 ] [238 ] . This test might also be useful where
nanomaterials are hydrophobic (see ISO 10993-10). For other clinical routes o f exposure, irritation
might be assessed by topical application o f nanomaterials.
4) KeratinoSens is a trademark o f Cyprotex. This in formation is given for the convenience o f users o f this document
5) SenCeeTox is a trademark o f Cyprotex. This information is given for the convenience o f users o f this document
ISO/TR 1 0993 -2 2 : 2 01 7(E)
Under the current scheme of ISO 10993-10, mucosa irritation tests are listed under Annex B as special
f
i rritation te s ts . C hoice o the s e te s t me tho d s ne e d to b e rationa l i z e d b a s e d on the s p e ci fic u s e cond ition s
and exposure routes of the materials and devices. Studies on several nano-objects have shown that
ocular irritation tests are comparable or more sensitive than dermal irritation tests in detecting
p o tentia l ff
de tri menta l e e c ts c au s e d [216 239 . Little information is available in
b y the s e materia l s ] [ ]
published literature on the use of other mucosa irritation methods to test nano-objects.
Various attempts have been made to evaluate and validate in vitro ski n i rritation mo del s a s a lternative
f
me tho d s to te s t nanomateri a l s . H owever, many con s ideration s have to b e made i n the s ele c tion o mo del s
f in vitro and dermal irritation

and s tudy de s ign . Wh i le s ome s tud ie s showe d e quiva lent re s u lts rom
mo del s , o thers rep or te d p o tentia l ar te fac ts c aus e d b y i nter ference due to nano - obj e c t i nterac tion with
te s t re agents or i nterleu ki n s . T here i s a l s o a lack o f s tud ie s to comp are the e ffe c tivene s s o f the d i fferent
typ e s o f s ki n mo del s i n a s s e s s i ng nanomateri a l s [239 240 241 .

] [ ] [ ]
T he b ovi ne corne a l op acity a nd p erme abi l ity as s ay (B C OP) a nd E piO c u la r™

6) eye i rritation te s t (E I T )
were u s e d to eva luate s evera l N M s for thei r eye i rritati ng c ap acity [242 ]
. S ome i rritati ng ac tivity was
ob s er ve d on ly for nano s i lver i n the E piO c u lar™ eye i rritation te s t (E I T ) wh i le i n the B C OP, va riable
re s u lts were no te d . S evera l d r y p owder nanomateri a l s [me ta l oxide s (Z nO, TiO 2 , CeO 2 ), amorphous SiO 2
a nd M WC N Ts] , th re e orga nic pigments , qua r tz , and ta lc were negative i n b o th a s s ays .
9.5 Haemocompatibility
E va luation o f h aemo comp atibi l ity shou ld b e p er forme d on me d ic a l device s contai n i ng nanomateria l s
in direct or indirect contact with blood. Moreover, even for devices which are not intended to be blood-
contac ti ng , i f the toxicoki ne tic s tudy reve a l s a p o tenti a l tran s lo c ation of fre e na no s i ze d p a r ticle s
origi nate d from the me d ic a l device i nto the s ys tem ic blo o d ci rc u lation, then haemo comp atibi l ity s hou ld
also be considered.Get more FREE standards from Standard Sharing Group and our chats
ISO 10993-4 provides general requirements for evaluating the interactions of medical devices with
blood. It describes:
a) a categorization of medical devices that are intended for use in contact with blood, based on the
i ntende d u s e a nd duration o f contac t as defi ne d i n I S O 10 9 9 3 -1 ;
b) the fundamental principles governing the evaluation of the interaction of devices with blood;
c) the rationa le for s truc ture d s ele c tion o f te s ts accord i ng to s p e c i fic c ate gorie s , to ge ther with the
pri nc iple s and s c ienti fic b as i s o f the s e te s ts .
B lo o d i nterac tion s are clas s i fie d i nto s evera l c ategorie s b as e d on the pri mar y pro ce s s or s ys tem b ei ng
me a s ure d: h aematolo g y, haemolys i s , th romb o s i s , co agu l ation, pl atele t ac tivation a nd complement
s ys tem ac tivation .
T he s e p a rame ters also apply to haemo comp atibi l ity a s s e s s ment for nanomateri a l s , to ge ther with
s p e c i fic con s ideration s a s d i s c u s s e d b elow. D ue to thei r s i z e, nano - obj e c ts m ight b e able to m igrate and
re ach the blo o d ci rc u lation s ys tem, where they c an i nduce pro th romb o tic e ffe c ts a nd platele t ac tivation .
H aemo comp atibi l ity a s s e s s ment shou ld there fore b e con s idere d for nano s truc tu re d materia l s , device s
containing nanomaterials, and nano-objects released from devices in direct or indirect contact with
circulating blood.
M any o f the re s p on s e s to me d ic a l device s that ca n o cc u r i n blo o d relate to the device s u r face are a com i ng
into contact with the blood. Therefore, the ratio of the device’s surface area to the volume of whole
blood exposed (cm 2 f f
/m l whole blo o d, WB) i s an i mp or ta nt ac tor or as s e s s i ng hemo comp atibi l ity. O ther
fac tors i n fluenci ng i nterac tion s with blo o d i nclude ge ome tr y and s u r face chem i s tr y. D o s e re s p on s e
6) E p iO cular™ is a trademark o f M atTek C o rp o ratio n. This in fo rmatio n is given fo r the co nvenience o f us ers o f this
do cument and do es no t co ns titute an endo rs ement by I S O o f the p ro duct named. E quivalent p ro ducts may b e us ed
i f they can b e s hown to lead to the s ame res ults .

ISO/TR 1 0993 -2 2 : 2 01 7(E)
testing should be considered with nanomaterials using appropriate in vitro and in vivo models to gauge
sa fety for the appropriate blood interaction categories mentioned above. See also ISO 10993-4.
9.5 .2 Complement system activation
The complement system is involved in the innate immune de fences against non-sel f-entities via
opsonization o f materials to allow recognition and uptake by macrophages. It has been reported that the
interaction between nano-objects and the complement system is regulated by several factors including
size, morphology and sur face [128 ] . Abnormal increases in complement system activation due to the
presence o f nanoscale materials in blood can induce significant inflammatory reactions. There fore,
complement activation should be included as part o f a biological risk assessment o f nanomaterials
especially i f they are intended to come into direct contact with circulating blood, or i f there is a potential
migration o f free nanosized particles into the systemic blood circulation.
Complement activation can result in an acute hypersensitivity reaction [243 ] . This specific type o f
hypersensitivity syndrome is called C-activation related pseudoallergy (CARPA). There has been a
number o f reports o f this type o f complement cascade activation being induced by polyethylene glycol
(PEG)ylated liposomes and other nanosized substances. Such CARPA inducers include the following:
liposomal drugs, micellar solvents, radio contrast agents, carbon nanotubes, liposome- and polymer-
based nanomedicines, PEG-coated vesicles, and other lipid-based and phospholipid-methoxyPEG
conjugate stabilized preparations [244] [245 ] .
9.5.3 Specific considerations for haemocompatibility testing
Haemocompatibility is a key consideration for blood contacting nanomaterials. This is because
proteins from blood are immediately adsorbed on the sur face o f nano-objects, leading to a cascade
o f events that would ultimately result in success or failure o f a medical device. Depending on their
size/surface area and other inherent or acquired surface characteristics, nano-objects can cause
variable haemocompatibility outcomes. Indeed, size and sur face roughness have been reported to play
important roles in the extent of blood serum protein adsorption, platelet adhesion and activation, and
whole blood clotting kinetics [246 ] . See also 8.2.2.
While materials with nano-structures on their sur face area can reasonably be directly evaluated using
the conventional methods described in ISO 10993-4, the haemocompatibility evaluation o f free nano-
objects can be much more challenging. Due to their higher sur face/volume ratio than bulk materials,
considerably more serum protein may readily adsorb onto free nano-objects distorting the subsequent
cascade o f reactions occurring in the blood as soon as foreign bodies enter systemic circulation. Also,
interactions with platelets, coagulation factors and endothelial cells might be altered due to potential
aggregation/agglomeration of free nano-objects once in contact with blood. In light of these potential in
vitro test inter ferences, specific attention should be paid to the reproducibility, reliability and sensitivity
o f the methods used be fore arriving at any conclusions regarding the haemocompatibility o f free nano-
objects. O f note, a standard test method for the analysis o f haemolytic properties o f nanoparticles was
published by the American Society for Testing and Materials (ASTM E2524) adapted from the ASTM
Standard Practice F756 usually followed to evaluate haemolytic properties o f bulk materials.
It should be noted that pro-inflammatory and pro-coagulant factors (e.g. TNF-a, IL-6, IL-8, MCP-1, tissue
factor) from endothelial cells can be induced by NPs [247 ] . Both the pro-coagulant and pro-inflammatory
potential o f NPs could be detected in an endothelial cells and monocytes co-culture model [248 ] [249 ] .
So, it is recommended that in addition to the basic hemocompatibility screening, also the activation o f
the endothelial cells and/or monocytes as well as the interaction o f endothelial cells, monocytes with
NPs should be determined (e.g. by evaluation o f markers for cell adhesion molecules like CD54/CD106/
CD62E, pro-inflammatory cytokines like TNF-α, IL-6, IL-8, MCP-1, and pro-coagulant factors).
9.6 Systemic toxicity
The systemic toxicity profile o f nanomaterials cannot be predicted by the systemic toxicity profile
o f the corresponding bulk materials. This is especially due to their ability to translocate in the body
and reach organs fairly inaccessible to conventional materials. Nano-objects have been described as

ISO/TR 1 0993 -2 2 : 2 01 7(E)
potentially crossing all protective barriers including the nuclear membrane, blood-brain and foeto-
placental barriers. Systemic toxicity o f nano-objects should there fore be considered.
One key parameter to consider when addressing the systemic toxicity potential o f nanomaterials is
their solubility profile. Soluble nanomaterials will dissolve when they contact tissues or fluids just
like soluble bulk materials in medical devices do. However, with poorly soluble nanomaterials, the
body’s clearance capacity and de fence mechanisms can be quickly overwhelmed, leading to long-term
systemic accumulation which can cause adverse e ffects. The biopersistence o f insoluble nanomaterials
could lead to changes in lysosomal permeability and enzymatic activity and macrophage apoptosis.
Depending on their clinical use and the intrinsic properties of the nanomaterials, the evaluation of their
potential systemic toxicity should consider several exposure durations (see ISO 10993-11).
Because nano-objects can potentially be distributed throughout the body, the selection o f tissues/organs
intended for histopathological analyses (ISO 10993-11) should be considered on a case-by-case basis
with special emphasis on the MPS (e.g. notably liver, spleen), kidneys, brain, bone marrow and others,
depending also on the route of administration and intended clinical use.
Special attention should be paid to the administered dose; the dose metric of mass or concentration
usually applicable to bulk materials may not be suitable for nanomaterials whose systemic toxicity
will mainly depend on the particle itsel f interacting with a biological system. The particle number
administered and/or the resulting surface area to which a patient was exposed might be better
parameters to describe a dose response relationship.
However, mass is a convenient dose metric in terms o f dosing. When all metric parameters are known,
the dose in mass can easily be translated to dose per number o f particles and/or dose per sur face area.
Also, the size o f the dose and the dosing frequency can influence the outcome o f a systemic toxicity
test. Exposure to nano-objects can saturate or stimulate uptake in organs, especially organs o f the
MPS. Studies on rodents where carbon and poly(lactide-co-glycolide) nano-objects were intravenously
injected have shown Getthat
morephagocytic cells in liver
FREE standards fromand spleen Sharing
Standard can be saturated,
Group andwith redistribution o f
our chats
nano-objects to other organs as a result [130 ] [ 131 ] . When Biozzi et al. injected carbon nano-objects
[132 ]
to the blood o f rats, they observed that the uptake in the liver was enhanced by repeated dosing. They
suggested that the change in behaviour was related to an adaptation of the MPS to the new conditions
especially to the level o f nano-objects in blood [132 ] . In other studies where nano-objects as carbon
colloids were injected intravenously in a dose that caused blockage o f particle uptake to liver, the
phagocytic activity o f the liver went back to normal a fter 3 d to 6 d[250 ] [251 ] .
9.7 Pyrogenicity
Like any material sur faces, nanomaterials can be coated with bacterial endotoxins or lipopolysaccharide
(LPS), especially when the nanomaterials are produced under non-sterile conditions or in the presence
o f water. The presence o f endotoxins on nanomaterial sur faces may inter fere with the interaction
between nanomaterials and the biological systems and a ffect assessment results (e.g. non-specific
inflammation). It should be noted that terminal sterilization (dry heat, moist heat, ethylene oxide,
gamma and electron beam radiation) does not inactivate bacterial endotoxin. The US Pharmacopeia
recommends treating materials for su fficient time and temperature to achieve a ≥3-log reduction in the
activity o f endotoxin (typically at least 250°C for at least 30 min) (USP 1995) (See 6.9.1).
ISO 29701 describes the limulus amebocyte lysate (LAL) test for the determination o f endotoxin in
nanomaterial samples for use in cell based in vitro biological systems. Another method is the monocyte
activation test (MAT) which is a validated quantitative method that exploits the natural human fever
mechanism and can be used to test for endotoxins associated with nanomaterials as well as other
biological pyrogens (e.g. yeast, parasitic and viral pyrogens) [84][85 ][86 ] . Information relevant for
endotoxin testing of nanomaterials is also included in 6.9.1 and ISO 10993-11. Additional resources
which may be help ful include USP 85, USP 151, and ANSI/AAMI ST72. In addition to endotoxin-mediated
pyrogenicity, nanomaterial-mediated pyrogenicity should be considered as part o f the biological
evaluation of nanomaterials, per ISO 10993-11.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
9.8 Implantation
I mpl antation te s ts for me d ic a l device s are de s c rib e d i n I S O 10 9 9 3 - 6 . D ep end i ng on the typ e o f me d ic a l
device, various implantation sites can be considered (e.g. subcutaneous, intramuscular, intracranial,
etc.) . For free nano-objects, direct injection into the appropriate tissue should be considered.
Sp e c i fic attention shou ld b e fo c u s e d on m igration o f the nano - obj e c ts i nto the lo c a l d rai n i ng lymph
nodes regarding possible release of nano-objects from a medical device.

When an i mplantation te s t i s u s e d to eva luate p o tentia l s ys tem ic toxicity, the re qu i rements o f b o th
ISO 10993-6 and ISO 10993-11 should be considered.

I t shou ld b e re a l i z e d that a control materi a l i s com mon ly u s e d i n i mpla ntation te s ts . S ome cer ti fie d
reference nanomaterials are available that are standardized for size measurements. In addition,
so c a l le d “repre s entative” nanomateria l s are now avai lable wh ich a re widely used com merci a l
nanomaterials; see 5.3.

1 0 Presentation of characterization and test results
The test report containing data on the characteristics and biological effects of nanomaterials should
include, where applicable, but is not limited to, the following information:
a) device and na nomateri a l de s crip tion a nd de ta i l s u s i ng the appropri ate phys ico chem ic a l
characteristics noted herein:

1) identi fic ation o f na nomateria l s , i nclud i ng chem ic a l comp o s ition, and s tr uc tu re;
2) size;
3) mor pholo g y;
4) agglomeration and aggregation rate/state;

5) s olubi l ity;
6) s u r face cha rge, s u r face are a and chem i s tr y;
7) description of intended use, nature and duration of biological contact/interaction;

8) evidence o f pu rity o f the na nomateria l i n the fi na l device i f appl ic ab le, and identi fic ation a nd
quanti fic ation o f any le achable and/or chem ic a l re s idue s;
9) a na lytic a l data to s upp or t s tabi l ity o f the na nomateri a l (e . g. for na no - obj e c ts , th i s m ight i nclude
aggregation rate/s tate u s i ng s to ck s a mp le s and va riou s do s i ng s olution s u s e d i n the va riou s
a s s ays) ;
b) sample preparation (e.g. extracts, direct contact) and testing conditions (nanomaterial
concentration and me d iu m, conta i ner f
materia l) i nclud i ng identi fic ation o s tandard pro to col s
applied;
c) s ta nda rd ana lytic a l me tho d s , i f appl ic able;
d) de s crip tion o f ana lytic a l te s t me tho d s , i nclud i ng quanti fic ation l i m its;
e) assessment of medical device material degradation and potential nano-object release including
rationa le to s upp or t f
the ade quac y o e xp eri menta l cond ition s to the device’s i ntende d use
environment;
f) description of medical device material degradation and potential nano-object release test methods,
test conditions, test materials and procedures, including controls;

ISO/TR 1 0993 -2 2 : 2 01 7(E)
g) identification and quantification o f degradation products (e.g. form and condition o f degradation
products, their stability and controls used);
h) evidence o f test validation, including use o f appropriate controls and supportive justifications,
where applicable;
i) summary o f results;
j) interpretation, discussion of results and conclusions;
k) statement o f compliance to appropriate good laboratory practices and/or to quality management
systems for test laboratories (e.g. ISO/IEC 17025).
When developing and validating new methods, the testing performed should contain the information in
accordance to ISO/TR 13014.
1 1 Risk assessment
1 1 .1 General considerations
Although the general principles o f chemical risk assessment are considered applicable to
nanomaterials [55 ] [59 ] [252 ] [253 ] , nanomaterials present some special challenges, including unique
physicochemical properties, greater compositional uncertainty, changing properties in biological
systems, exposure measurement di fficulties, and appropriate dose metric decisions that might require
specific guidance. There fore, transparency is important during the risk assessment process where
inclusion and exclusion o f data, assumptions, variability and uncertainties should be discussed. In
absence o f essential data, the risk assessor will be unable to complete the risk assessment. Hence, it
should be clear from the assessment on how in formation has been taken into account when the final
risk assessment is Get
determined.
more FREE It should be noted
standards fromthat the biological
Standard Sharingrisk assessment
Group and our ochats
f nanomaterials
comprises the assessment of both the soluble and insoluble fractions of the nanomaterial. It is
recognized that critical in formation needed for risk assessment o f nanomaterials can o ften be lacking,
leading to significant uncertainty. Such uncertainties are expected to diminish as relevant new data
become available from nanomaterial testing and research. Periodic reassessment o f the risk associated
with use of a nanomaterial should be considered as new information becomes available.
Potential biopersistence/bioaccumulation o f nanomaterials presents a specific challenge to the
risk assessment relative to chemical risk assessment. In many cases, the body can be incapable o f
metabolizing or actively breaking down the nanomaterial which results in a potential indefinite
persistence within tissues or cells. The persistence within tissues or cells has been demonstrated
for various types o f nanomaterials including silver [90 ] [ 254 ] , zinc oxide, titanium dioxide [255 ] , gold
nanoparticles [237 ] [238 ] , and carbon nanotubes [239 ] . Also, for TiO 2 nano-objects, prolonged persistence
was reported [259 ] . The specific biological effects associated with this persistence are not well
understood, yet there is some evidence to suggest that this phenomenon can result in a constant state
o f inflammation that may eventually lead to carcinogenesis [192 ] [260 ] . In addition, one should consider
the release of nano-objects as a result of wear/age/use of medical devices manufactured without the
use of nanomaterials.
The risk management process for biological evaluation o f medical devices is described in
ISO 10993-1:2009, Annex B. ISO 10993-1 does not define terms such as risk assessment, risk analysis
and risk evaluation but follows the wording o f ISO 14971 and the guidance document ISO/TR 15499.
However, in ISO 10993-1:2009, Annex B, the risk analysis is extended and also comprises risk evaluation
and risk control. ISO/TR 15449 provides more detailed advice on carrying out biological risk evaluations
within a risk management process than ISO 10993-1. An overview o f the risk management process is
provided in the flowchart in Figure 1 , but the scope o f this document is limited to risk assessment,
which comprises risk analysis and risk evaluation. The risk is described in ISO 14971 as being the
product o f two independent factors: the probability o f harm and the severity o f that harm.
The risk assessment process for nanomaterials is evolving and many tools are under development
to assist in the evaluation o f nanomaterial risks such as nanoin formatics in silico techniques [261 ] ,

ISO/TR 1 0993 -2 2 : 2 01 7(E)
quantitative nanoparticle activity relationship (QNAR) [262 ] [263 ] [264 ] , exposure models [265 ] [266 ] ,
physiologically based pharmacokinetic (PBPK) modelling [248 ] [249 ] , systemic toxicology testing
and high throughput screening [269 ] . See ISO/TR 16197. At present, these tools are used in frequently
to support regulatory submissions for nanomaterials. However, their importance may grow in the
future. Weight o f evidence is another term that is o ften mentioned in risk assessment documents on
nanomaterials and may be use ful[271 ] [272 ] . OECD provides a summary o f important issues to consider
in the risk assessment o f nanomaterials that also are applicable for medical devices [252 ] .
Figure 1 provides an overview o f the entire risk management process as based upon ISO 10993-1, but
the scope o f this document is limited to risk assessment that is presented in Figure 1 above the shaded
area. The shaded area in Figure 1 indicates risk control as part o f the risk management.

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Figure 1 — O verview of the risk management process
1 1 .2 Exposure assessment
T he prob abi l ity o f ha rm i s mo s t o ften ch arac teri z e d b y e xp o s ure as s e s s ment, cl i nic a l e xp erience or
comparison to similar applications. For nanomaterials used in medical devices, this is associated
with the potential release and formation of nano-objects from the device. Exposure assessment aims
to e s tabl i s h how much nanomateria l the p atient i s e xp o s e d to, and i f the exp o s u re ma ke s the nano -
obj e c ts ava i l able for lo c a l ti s s ue re s p on s e s a nd/or s ys tem ic exp o s u re . T here fore, it b e come s i mp or tant

ISO/TR 1 0993 -2 2 : 2 01 7(E)
to describe the exposure scenario and establish release, migration and formation of nano-objects from
the medical devices by selecting accurate test methods. Consider for the exposure assessment:
— intensity, frequency and duration o f contact;
— route o f exposure (e.g. tissue/blood, dermal, oral or respiratory);
— intake or uptake rates;
— bioavailability.
Initially, the focus is on the estimation o f the likelihood and extent o f absorption/distribution o f
nano-objects into the body either by simulating the exposure, measuring actual exposure, or making
assumptions o f exposure. The systemic uptake can, however, vary between di fferent exposure
conditions, exposure routes, health conditions o f the patient, and bioavailability. Exposures via
inhalation, intravenous injection or tissue implantation are often of greater concern than oral or dermal
exposure.
A challenge during the exposure assessment is the characterization of the nanomaterial. It can be
di fficult in vivo , especially in tissue and body fluids, due to limited availability o f analytical methods
but also due to low concentrations. Another limitation is that chemical analysis does not always provide
information about the particulate nature of the nanomaterial. The characterization, therefore, might
have to confirm i f the nanomaterial is in a solid, solubilized, degraded form. It has been shown that
some nanomaterials (e.g. nanosilver) can dissolve or shed metal ions, that may precipitate to new
nanomaterials with a different chemical composition [90 ] [272 ] . The analytical methods should be
described and justified. The exposure assessment selected should take into account aspects such as:
— suitability;
— sensitivity;
— robustness;
— accuracy;
— detection limits;
— material form (solid, soluble or degraded);
— sources o f variability and uncertainty.
11.3 Biological hazard identification

A hazard is a potential source o f harm. All known and reasonably foreseeable hazards associated with
the nanomaterial in both normal and reasonable fault conditions should be identified. Nevertheless, to
cause harm, a hazardous situation has to occur which o ften involves a sequence o f events. Identification
and characterization o f these events are important when risks are to be estimated.
When risk assessors are conducting the hazard identification/characterization step, they should
consider the following:
— results of literature review;
— results of material characterization;
— samples preparation;
— dose selection and dose description;
— test methods;
— toxicokinetics;

ISO/TR 10993-22:2017(E)
— description of the hazard, including dose response and mode of action.

For some types o f biological e ffects, it is not possible to derive a typical toxicological dose-response
relationship. In these cases, other methods should be used to characterize the hazard and classi fy the
severity o f harm. Here, special attention should be paid as to how hazardous situations arise and the
nature o f their outcome. Examples o f such biological e ffects include foreign body reactions to implanted
nanomaterials and blood coagulation due to nanomaterial surface structure and design. Appropriate
documentation o f these other types o f biological e ffects is necessary to assure transparency and
traceability.
11.4 Risk estimation

The principles for risk estimation are considered to be applicable for nanomaterials, but might be more
complex due to the currently limited understanding o f how nanomaterials interact with test systems
and the human body. Weight o f evidence approaches and principles on how to derive tolerable intake
values described in ISO 10993-17 can be useful in this process. ISO 10993-17 provides guidance on
how to use uncertainty factors and set a tolerable intake value for leaching substances and may also
be use ful for nanomaterials that may become available for systemic exposure by release, degradation,
wear or by its constitution.
All available and appropriate information from literature reviews, biological evaluation, clinical use,
and exposure assessment should be used to estimate the health risk posed by the nanomaterial.
Furthermore, the quality, reliability and relevance o f in formation should also be taken into account.
Data used for the risk estimation should be evaluated for reliability based on:
— confirmation o f the identity and physicochemical properties o f the material tested in relation to the
material being evaluated;
— whether or notGetthemore
dataFREE
have been generated according Sharing
to an accepted testing or measurement
approach; standards from Standard Group and our chats
— whether the applied approach is accepted for use in testing nanomaterials.

Identified and characterized risks are compared to the exposure or the probability o f a hazard to cause
harm. A risk arises i f there is a probability for harm. When it is not possible to determine the exposure
(the probability o f harm), the risk assessment, a fter assuming a worst-case exposure scenario, might
have to depend on the severity o f the hazard alone. Nevertheless, since risk is a function o f exposure,
and frequency and duration o f use are both exposure factors, the probably o f harm will rise when either
of these two factors increase.
11.5 Risk evaluation
Risk evaluation is a process in which estimated risks are evaluated against predefined acceptance
criteria to determine i f risk control is necessary. This process is identical for all medical devices
whether or not they contain nanomaterials. As with all devices, a qualitative risk evaluation might be
necessary when data available to per form a quantitative risk evaluation are insu fficient. The risk level
depends not only on the potential severity o f the hazard and the probability o f harm ful exposure, but
also on the device’s intended use.
12 Biological evaluation report
The biological evaluation report should contain the items as described in ISO 10993-1, including more
specific in formation regarding the use o f nanomaterials. Expert assessors who have the necessary
knowledge and experience should determine and document:
a) the strategy and program content for the biological evaluation o f the medical device that contains,
generates or is composed of nanomaterials/nano-objects;

ISO/TR 1 0993 -2 2 : 2 01 7(E)
b) the criteri a for de term i n i ng the accep tabi l ity o f the ri sk relate d to the nanomateria l(s) for the
i ntende d pu rp o s e, i n l i ne with the ri sk ma nagement pl an;
c) the ade quac y o f the materi a l ch arac teri z ation o f the me d ic a l device and o f the na nomateria l s;
d) the rationa le for s ele c tion and/or wa ivi ng o f te s ts and the degre e o f uncer ta i nty i n the i nterpre tation
of those tests;
e) the interpretation of relevant existing data and results of testing relevant to the medical device;
f) a ny add itiona l d ata col le c te d to comp le te the biolo gic a l eva luation;
g) overa l l biolo gic a l s a fe ty conclu s ion s for the me d ic a l device a nd a ny degre e o f u ncer ta i nty i n com i ng
to these conclusions.

ISO/TR 10993-22:2017(E)
Bibliography
[1] I S O 9 2 76 (a l l p ar ts) , Representation of results of particle size analysis
[2 ] I S O 9 2 7 7, Determination o f the specific surface area o f solids by gas adsorption — BET method
[3 ] I S O 10 8 01 , Nanotechnologies — Generation of metal nanoparticles for inhalation toxicity testing
using the evaporation/condensation method
[4] Nanotechnologies — Characterization of nanoparticles in inhalation exposure chambers

I S O 10 8 0 8 ,
for inhalation toxicity testing
[5 ] ISO 11 3 6 0 , Nanotechnologies — Methodology for the classification and categorization o f

nanomaterials
[6] I S O/ T S 1 2 02 5 , Nanomaterials — Quantification o f nano-object release from powders by generation

of aerosols
[7 ] Surface chemical analysis — Secondary-ion mass spectrometry — Calibration of the

I S O 1 3 0 8 4,
mass scale for a time-o f-flight secondary-ion mass spectrometer
[8 ] I S O/ T R 1 3 0 9 7, Guidelines for the characterization of dispersion stability
[9] I S O 1 3 0 9 9 (a l l p a r ts) , Colloidal systems
[10] ISO 131 21 , Nanotechnologies — Nanomaterial risk evaluation
[1 1] ISO 1 3318 (a l l p a r ts) , Determination of particle size distribution by centrifugal liquid

Getmethods
sedimentation more FREE standards from Standard Sharing Group and our chats
[1 2 ] ISO 13320, Particle size analysis — Laser diffraction
[1 3 ] I S O 1 3 3 2 2 (a l l p a r ts) , Particle size analysis -- Image analysis methods
[14] I S O/ T R 141 8 7, Surface chemical analysis — Characterization of nanostructured materials
[1 5 ] ISO 14 4 8 8 , Particulate materials — Sampling and sample splitting for the determination of
particulate properties
[16] I S O 14 8 8 7, Sample preparation — Dispersing procedures for powders in liquids
[17 ] I S O 1 5 471 , Surface chemical analysis — Auger electron spectroscopy — Description of selected
instrumental performance parameters
[1 8 ] Biological evaluation of medical devices -- Guidance on the conduct of biological

I S O/ T R 1 5 49 9,
evaluation within a risk management process
[19] Biological evaluation of medical devices — Part 33: Guidance on tests to evaluate
I S O/ T R 1 59 0 0 ,
genotoxicity — Supplement to ISO 10993-3
[2 0] I S O/ T R 1 5 9 01 (a l l p a r ts) , Nanotechnologies — Methodology for the classification and categorization

of nanomaterials
[2 1] I S O/ T S 1619 5 : 2 01 3 , Nanotechnologies — Guidance for developing representative test materials
consisting o f nano-objects in dry powder form
[2 2 ] Nanotechnologies — Compilation and description of sample preparation and dosing

I S O/ T R 1619 6 ,
methods for engineered and manufactured nanomaterials
[2 3 ] Nanotechnologies — Compilation and description of toxicological screening methods

I S O/ T R 1619 7,
for manufactured nanomaterials

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[2 4] I S O/ T S 16 5 5 0 , Nanotechnologies — Determination of silver nanoparticles potency by release of
muramic acid from Staphylococcus aureus
[2 5 ] I S O 1670 0 , Microbeam analysis — Scanning electron microscopy — Guidelines for calibrating image
magnification
[2 6 ] I S O/I E C 1702 5 , General requirements for the competence of testing and calibration laboratories
[2 7 ] I S O/ T S 17 2 0 0 , Nanotechnology — Nanoparticles in powder form — Characteristics and

measurements
[2 8 ] ISO 178 5 3 , Wear o f implant materials — Polymer and metal wear particles — Isolation and
characterization
[2 9] ISO 179 7 3 , Surface chemical analysis — Medium-resolution Auger electron spectrometers —

Calibration of energy scales for elemental analysis
[3 0] I S O 1 81 1 5 (a l l p ar ts) , Surface chemical analysis —- Vocabulary
[3 1] I S O 1 81 1 8 , Surface chemical analysis — Auger electron spectroscopy and X-ray photoelectron
spectroscopy — Guide to the use of experimentally determined relative sensitivity factors for the
quantitative analysis of homogeneous materials
[3 2 ] I S O 1 814 4, Environmental tobacco smoke — Estimation o f its contribution to respirable suspended
particles — Method based on solanesol
[3 3 ] Fine ceramics (advanced ceramics, advanced technical ceramics) — Determination of

I S O 1 8 75 7,
specific surface area o f ceramic powders by gas adsorption using the BET method
[3 4] ISO 19 0 0 7, Nanotechnologies — In vitro MTS assay for measuring the cytotoxic e ffect o f
nanoparticles
[3 5 ] Surface chemical analysis — Fundamental approaches to determination of lateral

I S O/ T R 19 3 19,
resolution and sharpness in beam-based methods
[3 6] I S O 19 5 9 0 , Nanotechnologies — Size distribution and concentration of inorganic nanoparticles in
aqueous media via single particle inductively coupled plasma mass spectrometry
[3 7 ] I S O 2 0 9 9 8 (a l l p ar ts) , Measurement and characterization of particles by acoustic methods
[3 8 ] ISO 21 5 01 (a l l p a r ts) , Determination of particle size distribution — Single particle light

interaction methods
[3 9] ISO 2 2 3 0 9, Microbeam analysis — Quantitative analysis using energy-dispersive spectrometry
(EDS) for elements with an atomic number o f 11 (Na) or above
[4 0] I S O 2 2 41 2 , Particle size analysis — Dynamic light scattering (DLS)
[41] I S O 2 2 4 8 9, Microbeam analysis — Electron probe microanalysis — Quantitative point analysis for
bulk specimens using wavelength dispersive X-ray spectroscopy
[42 ] ISO 2 417 3 , Microbeam analysis — Guidelines for orientation measurement using electron
backscatter diffraction
[43 ] I S O 2 42 3 6 , Surface chemical analysis — Auger electron spectroscopy — Repeatability and

constancy of intensity scale
[4 4] I S O 2 5178 , Geometrical product specifications (GPS) — Surface texture: Areal
[45 ] I S O 2 762 8 , Workplace atmospheres — Ultrafine, nanoparticle and nano-structured aerosols —
Inhalation exposure characterization and assessment

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[46] ISO 29701, Nanotechnologies — Endotoxin test on nanomaterial samples for in vitro systems —
Limulus amebocyte lysate (LAL) test
[47] ISO/TS 80004-1:2015, Nanotechnologies — Vocabulary — Part 1: Core terms
[48] ISO/TS 80004-2:2015, Nanotechnologies — Vocabulary — Part 2: Nano-objects
[49] ISO/TS 80004-6:2013, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
[50] AAMI/ST 72, Bacterial endotoxins — Test methods, routine monitoring, and alternatives to
batch testing
[51] ASTM E2524, Standard Test Method for Analysis o f Hemolytic Properties o f Nanoparticles
[52] C urtis A.S.G., Dalby M.J., G adegaard N. Cell signaling arising from nanotopography:
implications for nanomedical devices. Nanomed. 2006, pp. 67–72
1
[53] K er E.D., N ain A.S., Weiss L.E., Wang J., S uh an J., A mon C.H. Bioprinting of growth factors
onto aligned sub-micron fibrous sca ffolds for simultaneous control o f cell di fferentiation and
alignment. Biomaterials. 2011, pp. 8097–8107
32
[54] D e P eppo G.M., Agheli H., K arlsson C., E ks trö m K., B risby H., L enner å s M. Osteogenic
response o f human mesenchymal stem cells to well-defined nanoscale topography in vitro. Int. J.
Nanomedicine. 2014, pp. 2499–2515
9
[55] H ris tozov D.R., G ottardo S., C ri tto A., M arcomini A. Risk assessment o f engineered
nanomaterials: a review o f available data and approaches from a regulatory perspective.
Nanotoxicology. 2012, pp. 880–898
6
[56] H ansen S.F., L arsen B.H., O lsen S.I. Baun ACategorization framework to aid hazard
identification
Geto fmore
nanomaterials. Nanotoxicology.
FREE standards pp. 243–250
2007, Sharing
from Standard 1 Group and our chats
[57] EFSA. Guidance on the risk assessment o f the application o f nanoscience and nanotechnologies
in the food and feed chain. EFSA J. 2011, 9 p. 2140
[58] SCCS/1484/12, GUIDANCE ON THE SAFETY ASSESSMENT OF NANOMATERIALS IN
COSMETICS. European Commission, Brussels, Belgium, 2012. http://ec .europa .eu/ health/
scientific _committees/consumer _sa fety/docs/sccs _s _005 .pdf
[59] SCENIHR. Guidance on the Determination o f Potential Health E ffects o f Nanomaterials Used in
Medical Devices. European Commission, Brussels, Belgium 2014. http://ec .europa .eu/ health/
scientific _committees/emerging/docs/scenihr_o _045 .pdf
[60] C raw f ord R.J., & Webb H.K. Sur face topographical factors influencing bacterial attachment.
Adv. Colloid Interface Sci. 2012, 179 -182 pp. 142–149
[61] Webb H.K., & T ruong V.K. Roughness parameters for standard description of surface
nanoarchitecture. Scanning. 2012, pp. 257–263
34
[62] L insinger T.P.J., Roebben G., S ol ans C., R amsch R. Reference materials for measuring the size
o f nanoparticles. TrAC Trends in Analytical Chemistry. 2011, 30 pp. 18–27
[63] M aurer E.I., S h arm a M., S chl ager J.J., H ussain S.M. Systematic analysis o f silver nanoparticle
ionic dissolution by tangential flow filtration: toxicological implications. Nanotoxicology. 2014,
8 pp. 718–727
[64] E lder A., Lynch I., G rieger K. Human health risks o f engineered nanomaterials: critical
knowledge gaps in nanomaterials risk assessment. In: Nanomaterials: Risks and Benefits,
(L inkov I., & S tee vens J. eds.). Springer, Dordrecht, 2009, pp. 3–29.
[65] Roebben G., R asm ussen K., K es tens V., L insinger T.P.J., R auscher H., E mons H. Reference
materials and representative test materials: the nanotechnology case. J. Nanopart. Res. 2013,
15 pp. 1–13
ISO/TR 1 0993 -2 2 : 2 01 7(E)
[66] S te fani ak A.B., H ackle y V.A., Roebben G., E h ara K., H ankin S., P ostek M.T. Nanoscale
re ference materials for environmental, health and sa fety measurements: needs, gaps and
opportunities. Nanotoxicology. 2013, 7 pp. 1325–1337
[67] ECHA E urope an C hemic als Agenc y. Guidance on information requirements and chemical
sa fety assessment. Appendix R7-1 Recommendations for nanomaterials applicable to Chapter
R7a Endpoint specific guidance. ECHA-12-G-03-EN, Helsinki. 2012
[68] OECD. 2012. Guidance on sample preparation and dosimetry for the sa fety testing o f
manu factured nanomaterials. Series on the sa fety o f Manu facture Nanomaterials No. 36.
JM/MOMO, Paris, NV, 2012, pp. 40.
[69] N iidome T., Yam agata M., O kamoto Y. PEG-modified gold nanorods with a stealth character
for in vivo application. J. Control. Release. 2006, 114 pp. 343–347
[70] L ankveld D.P.K., R ayavarapu R.G., K rys tek P., O omen A.G., Verh aren H.W., Van L eeu wen
T.G. Blood clearance and tissue distribution o f PEGylated and non-PEGylated gold nanorods
after intravenous administration in rats. Nanomedicine (Lond.) . 2011, pp. 339–349
6
[71] S ch ulze C., K roll A., L ehr C.-M., S ch ä f er U.F., B ecker K., S chnekenburger J. Not ready to
use - overcoming pit falls when dispersing nanoparticles in physiological media. Nanotoxicology.
2008, pp. 51–61
2
[72] T eeguarden J.G., H inderli ter P.M., O rr G., T hrall B.D., P ounds J.G. Particokinetics in vitro:
dosimetry considerations for in vitro nanoparticle toxicity assessments. Toxicol. Sci. 2007, 95
pp. 300–312
[73] P etersen E.J., D i amond S.A., K ennedy A.J., G oss G.G., H o K., L e ad J. Adapting OECD
Aquatic Toxicity Tests for Use with Manu factured Nanomaterials: Key Issues and Consensus
Recommendations. Environ. Sci. Technol. 2015, pp. 9532–9547
49
[74] C ho E.C., Z h ang Q., X i a Y. The e ffect o f sedimentation and di ffusion on cellular uptake o f gold
nanoparticles. Nat. Nanotechnol. 2011, pp. 385–391
6
[75] L ison D., & H uau x F. In vitro studies: Ups and downs o f cellular uptake. Nat. Nanotechnol.
2011, pp. 332–333
6
[76] C lippinger A.J., A hlu wali a A., A llen D., B onner J.C., C ase y W., C astranova V. Expert
consensus on an in vitro approach to assess pulmonary fibrogenic potential o f aerosolized
nanomaterials. Arch. Toxicol. 2016, pp. 1769–1783
90
[77] C rist R.M., G rossm an J.H., Patri A.K., S tern S.T., D obrovolskai a M.A., A disesh ai ah
P.P. Common pit falls in nanotechnology: lessons learned from NCI’s Nanotechnology
Characterization Laboratory. Integr. Biol. 2013, pp. 66–73
5
[78] E sch R.K., H an L., Foarde K.K., E nsor D.S. Endotoxin contamination of engineered
nanomaterials. Nanotoxicology. 2010, pp. 73–83
4
[79] Vallhov H., Qin J., J oh ansson S.M., A hlborg N., M uh ammed M.A., S che yni us A. The
importance of an endotoxin-free environment during the production of nanoparticles used in
medical applications. Nano Lett. 2006, pp. 1682–1686
6
[80] D obrovolskai a M.A., N eun B.W., C logston J.D., D ing H., L jubimova J., M c N eil S.E.
Ambiguities in applying traditional Limulus amebocyte lysate tests to quanti fy endotoxin in
nanoparticle formulations. Nanomedicine (Lond.) . 2010, pp. 555–562
5
[81] N eun B.W., & D obrovolskai a M.A. Detection and quantitative evaluation of endotoxin
contamination in nanoparticle formulations by LAL-based assays. Methods Mol. Biol. 2011, 697
pp. 121–130

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[82] D obrovolskai a M.A., & M c N eil S.E. Endotoxin and Engineered Nanomaterials. Pages 77-110
in Dobrovolskaia MA, McNeil SE.(Eds). Handbook o f Immunological Properties o f Engineered
Nanomaterials. World Scientific Publishing Co, Singapore. 2013
[83] G i annakou C., G eertsm a R.E., D e J ong W.H., Van L overen H., Vandebriel R.J., Park M.V.D.Z.
Immunotoxicity testing o f nanomedicinal products: possible pit falls in endotoxin determination.
Current Bionanotechnology. 2016, 2 pp. 95 -102
[84] D obrovolskai a M.A., N eun B.W., C logs ton J.D., G rossm an J.H., M c N eil S.E. Choice of
method for endotoxin detection depends on nanoformulation. Nanomedicine (Lond.) . 2014, 9
pp. 1847–1856
[85] H artung T., & S abbioni E. Alternative in vitro assays in nanomaterial toxicology. Wiley
Interdiscip. Rev. Nanomed. Nanobiotechnol. 2011, pp. 545–573
3
[86] S chindler S., von Aulock S., D aneshi an M., H artung T. Development, validation and
applications o f the monocyte activation test for pyrogens based on human whole blood. ALTEX.
2009, pp. 265–277
26
[87] Vauthier C., & B ouchem al K. Methods for the Preparation and Manu facture o f Polymeric
Nanoparticles. Pharm. Res. 2009, 26 pp. 1025–1058
[88] S ubb arao N. Impact o f Nanoparticle Sterilization on Analytical Characterization. Pages 53-71
in Dobrovolskaia MA, McNeil SE. (Eds). Handbook o f Immunological Properties o f Engineered
Nanomaterials. World Scientific Publishing Co, Singapore. 2013
[89] U lset A.S., M ori H., D alheim M.Ø., H ara M., C hristensen B.E. Influence o f amino acids,
bu ffers, and ph on the γ-irradiation-induced degradation o f alginates. Biomacromolecules. 2014,
15 pp. 4590–4597
[90] Van D er Z ande M., Vandebriel R.J., Van D oren E., K ramer E., H errera R i vera Z., S errano -
Rojero C.S. Distribution, Elimination, and Toxicity o f Silver Nanoparticles and Silver Ions in
Rats a fter 28-Day Oral Exposure. ACS Nano. 2012, pp. 7427–7442
6
[91] S anchez V.C., P ietruska J.R., M iselis N.R., H urt R.H., K ane A.B. Biopersistence and potential
adverse health impacts o f fibrous nanomaterials: what have we learned from asbestos? Wiley
Interdiscip. Rev. Nanomed. Nanobiotechnol. 2009, pp. 511–529
1
[92] B ogdan A., B uckett M.I., J apuntich D.A. Nano-sized aerosol classification, collection and
analysis–method development using dental composite materials. J. Occup. Environ. Hyg. 2014,
11 pp. 415–426
[93] D e J ong W.H., H agens W.I., K rystek P., B urger M.C., S ips A., G eertsm a R.E. Particle
size-dependent organ distribution of gold nanoparticles after intravenous administration.
Biomaterials. 2008, pp. 1912–1919
29
[94] L ipka J., S emmler -B ehnke M., S perling R.A., Wenk A., Takenaka S., S chleh C.
Biodistribution o f PEG-modified gold nanoparticles following intratracheal instillation and
intravenous injection. Biomaterials. 2010, pp. 6574–6581
31
[95] H irn S., S emmler -B ehnke M., S chleh C., Wenk A., L ipka J., S ch ä ff ler M. Particle size-
dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous
administration. Eur. J. Pharm. Biopharm. 2011, 77 pp. 407–416
[96] S onavane G., Tomoda K., S ano A., O hshim a H., T erada H., M akino K. In vitro permeation
o f gold nanoparticles through rat skin and rat intestine: E ffect o f particle size. Colloids Sur f. B
Biointerfaces. 2008, pp. 1–10
65
[97] M oghimi S.M., H unter A.C., M urray J.C. Long-circulating and target-specific nanoparticles:
theory to practice. Pharmacol. Rev. 2001, 53pp. 283–318

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[98] H ill aire au H., & C ou vreur P. Nanocarriers’ entry into the cell: relevance to drug delivery.
Cell. Mol. Life Sci. 2009, pp. 2873–2896
66
[99] N el A.E., M adler L., Velegol D., X i a T., H oek E. M/V., Somasundaran P., Klaessig F., Castranova
V., Thompson M., Understanding biophysicochemical interactions at the nano-bio inter face. Nat.
Mater. 2009, pp. 543–557
8
[100] Tang S., C hen M., Z heng N. Sub-10-nm Pd Nanosheets with Renal Clearance for E fficient Near-
In frared Photothermal Cancer Therapy. Small. 2014, 15 pp. 3139–3144
[101] X ie G., S un J., Z hong G., S hi L., Z h ang D. Biodistribution and toxicity o f intravenously
administered silica nanoparticles in mice. Arch. Toxicol. 2010, pp. 183–190
84
[102] X ie G.P., S un J., Z hong G.R. Tissular Localization and Excretion o f Intravenously Administered
Silica Nanoparticles o f Di fferent Sizes. J. Nanopart. Res. 2012, 14 pp. 671–680
[103] A lkilan y A.M., & M urph y C.J. Toxicity and cellular uptake o f gold nanoparticles: what we have
learned so far? J. Nanopart. Res. 2010, 12 pp. 2313–2333
[104] Lynch I., C edervall T., L undqvist M., C abaleiro -L ago C., L inse S., D awson K.A. The
nanoparticle–protein complex as a biological entity; a complex fluids and sur face science
challenge for the 21st century. Adv. Colloid Inter face Sci. 2007, 13 4 –135 pp. 167–174
[105] Lynch I., S alvati A., Dawson K.A. Protein-nanoparticle interactions: What does the cell see?
Nat. Nanotechnol. 2008, pp. 546–547
4
[106] L ankveld D.P., O omen A.G., K rys tek P., N eigh A., T roost- de J ong A., N oorl ander C.W. The
kinetics o f the tissue distribution o f silver nanoparticles o f di fferent sizes. Biomaterials. 2010,
31 pp. 8350–8361
[107] D emoy M., G ib aud S., A ndreu x J.P., Weingarten C., G ouri tin B., C ou vreur P. Splenic
trapping o f nanoparticles: complementary approaches for in situ studies. Pharm. Res. 1997, 14
pp. 463–468
[108] G ibaud S., D emoy M., A ndreu x J.P., Weingarten C., G ouri tin B., C ou vreur P. Cells involved
in the capture o f nanoparticles in hematopoietic organs. J. Pharm. Sci. 1996, 85 pp. 944–950
[109] L enaerts V., N agelkerke J.F., Van B erkel T.J., C ou vreur P., G risl ain L., Rol and M. In
vivo uptake o f polyisobutyl cyanoacrylate nanoparticles by rat liver Kup ffer, endothelial, and
parenchymal cells. J. Pharm. Sci. 1984, 73 pp. 980–982
[110] S adauskas E., Wallin H., S toltenberg M., Vogel U., D oering P., L arsen A. Kupffer cells are
central in the removal of nanoparticles from the organism. Part. Fibre Toxicol. 2007, p. 10 4
[111] L undqvist M., S tigler J., E li a G., Lynch I., C edervall T., D awson K.A. Nanoparticle size
and surface properties determine the protein corona with possible implications for biological
impacts. Proc. Natl. Acad. Sci. USA. 2008, 105 pp. 14265–14270
[112] Walke y C.D., & C h an W.C. Understanding and controlling the interaction of nanomaterials
with proteins in a physiological environment. Chem. Soc. Rev. 2012, 41 pp. 2780–2799
[113] M onopoli M.P., A berg C., S alvati A., D awson K.A. Biomolecular coronas provide the biological
identity o f nanosized materials. Nat. Nanotechnol. 2012, 12 pp. 779–786
[114] M wilu S.K., E l B adawy A.M., B radh am K., N elson C., T hom as D., S checkel K.G. Changes
in silver nanoparticles exposed to human synthetic stomach fluid: e ffects o f particle size and
sur face chemistry. Sci. Total Environ. 2013, 447 pp. 90–98
[115] Pal T., S au T.K., J ana N.R. Reversible Formation and Dissolution of Silver Nanoparticles in
Aqueous Surfactant Media. Langmuir. 1997, pp. 1481–1485
13

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[116] Rogers K.R., B radh am K., Tol aym at T., T hom as D.J., H artm ann T., M a L. Alterations in
physical state o f silver nanoparticles exposed to synthetic human stomach fluid. Sci. Total
Environ. 2012, 42 0 pp. 334–339
[117] O smond -M c L eod M.J., P ol and C.A., M urph y F., Waddington L., M orris H., H awkins S.C.
Durability and inflammogenic impact o f carbon nanotubes compared with asbestos fibres. Part.
Fibre Toxicol. 2011, p. 15
8
[118] O berd ö rster G., M aynard A., D onaldson K., C astranova V., F i zpatric J., Ausm an K.
Principles for characterizing the potential human health effects from exposure to nanoparticles:
elements o f a screening strategy. Part. Fibre Toxicol. 2005, p. 8
2
[119] F u C., L iu T., L i L., L i u H., C hen D., Tang F. The absorption, distribution, excretion and toxicity
of mesoporous silica nanoparticles in mice following different exposure routes. Biomaterials.
2013, pp. 2565–2575
34
[120] L i C.H., S hen C.C., C heng Y.W., H uang S.H., Wu C.C., K ao C.C. Organ biodistribution, clearance,
and genotoxicity o f orally administered zinc oxide nanoparticles in mice. Nanotoxicology. 2012,
6 pp. 746–756
[121] H uang X., Z h ang F., Z hu L., C hoi K.Y., G uo N., G uo J. E ffect o f injection routes on the
biodistribution, clearance, and tumor uptake o f carbon dots. ACS Nano. 2013, 23 pp. 5684–5693
[122] S emmler -B ehnke M1. Kreyling WG, Lipka J, Fertsch S, Wenk A, Takenaka S, Schmid G, Brandau
W. Biodistribution of 1.4- and 18-nm gold particles in rats. Small. 2008, pp. 2108–2111
4
[123] C rosera M., B ovenzi M., M aina G., A dami G., Z anette C., F lorio C. Nanoparticle dermal
absorption and toxicity: a review o f the literature. Int. Arch. Occup. Environ. Health. 2009, 82
pp. 1043–1055
[124] DEPA. Danish Ministry o f the Environment, Environmental Protection Agency. Dermal
absorption of nanomaterials. Environmental Project no. 1504. 2013
[125] C ampbell C.S., C ontreras -Rojas L.R., D elgado -C h arro M.B., G u y R.H. Objective assessment
o f nanoparticle disposition in mammalian skin a fter topical exposure. J. Control. Release. 2012,
162 pp. 201–207
[126] E lder A., G elein R., S ilva V., F eikert T., O panash uk L., C arter J. Translocation o f inhaled
ultrafine manganese oxide particles to the central nervous system. Environ. Health Perspect.
2006, 114 pp. 1172–1178
[127] O berd ö rster G., E lder A., R inderknech t A. Nanoparticles and the brain: cause for concern?
J. Nanosci. Nanotechnol. 2009, pp. 4996–5007
9
[128] Z h ang L., B ai R., L i u Y., M eng L., L i B., Wang L. The dose-dependent toxicological effects
and potential perturbation on the neurotransmitter secretion in brain following intranasal
instillation o f copper nanoparticles. Nanotoxicology. 2012, pp. 562–575
6
[129] O berd ö rs ter G. Toxicokinetics and e ffects o f fibrous and nonfibrous particles. Inhal. Toxicol.
2002, pp. 29–56
14
[130] H alpern B.N., B enacerra f B., B iozzi G. Quantitative study o f the granulopectic activity o f the
reticulo-endothelial system. I. The e ffect o f the ingredients present in India ink and o f substances
a ffecting blood clotting in vivo on the fate o f carbon particles administered intravenously in
rats, mice and rabbits. Br. J. Exp. Pathol. 1953, 3 4 pp. 426–440
[131] Panagi Z., B eletsi A., E vangel atos G., L i vaniou E., I th akissios D.S., Avgoustakis K. Effect
o f dose on the biodistribution and pharmacokinetics o f PLGA and PLGA-mPEG nanoparticles.
Int. J. Pharm. 2001, 2 21 pp. 143–152
[132] B iozzi G., B enacerra f B., H alpern B.N. Quantitative study o f the granulopectic activity o f
the reticulo-endothelial system. II. A study o f the kinetics o f the R. E. S. in relation to the dose

ISO/TR 1 0993 -2 2 : 2 01 7(E)
o f carbon injected; relationship between the weight o f the organs and their activity. Br. J. Exp.
Pathol. 1953, pp. 441–457
34
[133] K im Y.S., S ong M.Y., Park J.D., S ong K.S., Ryu H.R., C h ung Y.H. Subchronic oral toxicity o f
silver nanoparticles. Part. Fibre Toxicol. 2010, p. 20
7
[134] S ung J.H., J i J.H., Park J.D., Yoon J.U., K im D.S., J eon K.S. Subchronic inhalation toxicity o f
silver nanoparticles. Toxicol. Sci. 2009, 108 pp. 452–461
[135] B ermudez E., M angum J.B., Wong B.A., A sgh ari an B., H e xt P.M., Warhei t D.B. Pulmonary
responses o f mice, rats, and hamsters to subchronic inhalation o f ultrafine titanium dioxide
particles. Toxicol. Sci. 2004, pp. 347–357
77
[136] B rown J.S., Wilson W.E., G rant L.D. Dosimetric comparisons of particle deposition and
retention in rats and humans. Inhal. Toxicol. 2005, pp. 355–385
17
[137] D emoy M., A ndreux J.P., Weingarten C., G ouri tin B., G uillou x V., C ou vreur P. Spleen
capture o f nanoparticles: influence o f animal species and sur face characteristics. Pharm. Res.
1999, pp. 37–41
16
[138] P eters R.J., R i vera Z.H., van B emmel G., M arvin H.J., Weigel S., B ou wmees ter H.
Development and validation of single particle ICP-MS for sizing and quantitative determination
o f nano-silver in chicken meat. Anal. Bioanal. Chem. 2014, pp. 3875–3885
406
[139] M agaye R., & Z h ao J. Recent progress in studies o f metallic nickel and nickel-based nanoparticles’
genotoxicity and carcinogenicity. Environ. Toxicol. Pharmacol. 2012, 3 4 pp. 644–650
[140] S h atkin J.A., & O ng K.J. Alternative Testing Strategies for Nanomaterials: State o f the Science
and Considerations for Risk Analysis. Risk Anal. 2016, 3 6 pp. 1564–1580
[141] S h arm a M., S h atkin J.A., C airns C., C anady R., C lippinger A.J. Framework to Evaluate
Exposure Relevance and Data Needs for Risk Assessment o f Nanomaterials using in Vitro Testing
Strategies. Risk Anal. 2016, 3 6 pp. 1551–1563
[142] X i a T., Kovochich M., N el A. The role o f reactive oxygen species and oxidative stress in
mediating particulate matter injury. Clin. Occup. Environ. Med. 2006, 5 pp. 817–836
[143] Z h ang H., J i Z., X i a T., M eng H., L ow-K am C., L iu R. Use of metal oxide nanoparticle band gap
to develop a predictive paradigm for oxidative stress and acute pulmonary inflammation. ACS
Nano. 2012, 22 pp. 4349–4368
[144] M onteiller C., T ran L., M ac N ee W., Fau x S., J ones A., M iller B. The pro-inflammatory
e ffects o f low-toxicity low solubility particles, nanoparticles and fine particles, on epithelial cells
in vitro: The role of surface area. Occup. Environ. Med. 2007, pp. 609–615
64
[145] O berdorster G., O berdorster E., O berdors ter J. Nanotoxicology: An emerging discipline
evolving from studies o f ultrafine particles. Environ. Health Perspect. 2005, 113 pp. 823–839
[146] D elm aar C.J., P eijnenburg W.J., O omen A.G., C hen J., d e J ong W.H., S ips A.J. A practical
approach to determine dose metrics for nanomaterials. Environ. Toxicol. Chem. 2015, 34
pp. 1015–1022
[147] Kong B., S eog J.H., G rah am L.M., L ee S.B. Experimental considerations on the cytotoxicity o f
nanoparticles. Nanomedicine (Lond.) . 2011, pp. 929–941
6
[148] S h arm a G., Kodali V., G a ff re y M., Wang W., M inard K.R., K arin N.J. Iron oxide nanoparticle
agglomeration influences dose rates and modulates oxidative stress-mediated dose-response
profiles in vitro. Nanotoxicology. 2014, pp. 663–675
8
[149] L ison D., T hom assen L.C.J., R abolli V., G onz alez L., N apierska D., S eo J.W. Nominal and
E ffective Dosimetry o f Silica Nanoparticles in Cytotoxicity Assays. Toxicol. Sci. 2008, 10 4
pp. 155–162
ISO/TR 1 0993 -2 2 : 2 01 7(E)
[150] G uadagnini R., H al amoda Kenz aoui B., C artwrigh t L., Pojana G., M agdolenova Z.,
B il anicovaD. 2013. Toxicity screenings o f nanomaterials: challenges due to inter ference with
assay processes and components o f classic in vitro tests. Nanotoxicology. 2015, 9 () pp. 25–32
[151] C ase y A., H erzog E., D avoren M., Lyng F.M., B yrne H.J., C h ambers G. Spectroscopic analysis
confirms the interactions between single walled carbon nanotubes and various dyes commonly
used to assess cytotoxicity. Carbon. 2007, 45 pp. 1425–1432
[152] Wö rle -K nirsch J.M., P ulskamp K., K rug H.F. Oops they did it again! Carbon nanotubes hoax
scientists in viability assays. Nano Lett. 2006, 6 pp. 1261–1268
[153] M onteiro -R i viere N.A., I nm an A.O., Z h ang L.W. Limitations and relative utility o f screening
assays to assess engineered nanoparticle toxicity in a human cell line. Toxicol. Appl. Pharmacol.
2009, 23 4 pp. 222–235
[154] L upu A.R., & P opescu T. The noncellular reduction o f MTT tetrazolium salt by TiO 2 nanoparticles
and its implications for cytotoxicity assays. Toxicol. In Vitro. 2013, 2 7 pp. 1445–1450
[155] X i a T., H amilton R.F., B onner J.C., C randall E.D., E lder A., Fazloll ahi F. Interlaboratory
evaluation o f in vitro cytotoxicity and inflammatory responses to engineered nanomaterials:
the NIEHS Nano GO Consortium. Environ. Health Perspect. 2013, 121 pp. 683–690
[156] O ng K.J., M ac C orm ack T.J., C l ark R.J., E de J.D., O rtega V.A., F eli x L.C. Widespread
nanoparticle-assay inter ference: implications for nanotoxicity testing. PLoS One. 2014, 9
p. e90650
[157] Park M.V.D.Z., N eigh A.M., Vermeulen J.P., d e l a Fon te yne L.J.J., Verh aren H.W., B riede
J.J. The e ffect o f particle size on the cytotoxicity, inflammation, developmental toxicity and
genotoxicity o f silver nanoparticles. Biomaterials. 2011, 3 2 pp. 9810–9817
[158] Val S., H ussain S., B oland S., H amel R., B aez a-S quiban A., M arano F. Carbon black and
titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells:
need for multiparametric evaluation due to adsorption artifacts. Inhal. Toxicol. 2009, () 21
pp. 115–122
[159] Wilhelmi V., F ischer U., van B erlo D., S ch ulze -O stho ff K., S chins R.P.F., A lbrech t C.
Evaluation o f apoptosis induced by nanoparticles and fine particles in RAW 264 Macrophages:
facts and artefacts. Toxicol. In Vitro. 2012, pp. 323–334
26
[160] P f uhler S., E lespuru R., A ardem a M.J., D oak S.H., M ari a D onner E., H onm a M. Genotoxicity
o f nanomaterials: Refining strategies and tests for hazard identification. Environ. Mol. Mutagen.
2013, pp. 229–239
54
[161] A sh a R ani . P.V., Mun, G.L.K., Hande, M.P., and Valiyaveettil, S. Cytotoxicity and Genotoxicity o f
Silver Nanoparticles in Human Cells. ACS Nano. 2009, pp. 279–290
3
[162] M ei N., Z h ang Y.B., C hen Y., G uo X.Q., D ing W., A li S.F. Silver nanoparticle-induced mutations
and oxidative stress in mouse lymphoma cells. Environ. Mol. Mutagen. 2012, 53 pp. 409–419
[163] B utler K.S., P eeler D.J., C ase y B.J., D air B.J., E lespuru R.K. Silver nanoparticles: correlating
nanoparticle size and cellular uptake with genotoxicity. Mutagenesis. 2015, 30 pp. 577–591
[164] Paino I.M.M., M arangoni V.S., d e O li veira R.D.S., A n tunes L.M.G., Z ucolotto V. Cyto and
genotoxicity o f gold nanoparticles in human hepatocellular carcinoma and peripheral blood
mononuclear cells. Toxicol. Lett. 2012, 215 pp. 119–125
[165] S ch ulz M., M a-H ock L., B rill S., S trauss V., T reum ann S., G roters S. Investigation on the
genotoxicity o f di fferent sizes o f gold nanoparticles administered to the lungs o f rats. Mutat. Res.
2012, 745 pp. 51–57
[166] A h amed M., & A lh adl aq H.A. Nickel nanoparticle-induced dose-dependent cyto-genotoxicity
in human breast carcinoma MCF-7 cells. Onco Targets Ther. 2014, pp. 269–280
7

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[167] L andsiedel R., K app M.D., S chulz M., Wiench K., O esch F. Genotoxicity investigations on
nanomaterials: methods, preparation and characterization of test material, potential artifacts
and limitations–many questions, some answers. Mutat. Res. 2009, 681 pp. 241–258
[168] D oak S.H., M anshi an B., J enkins G.J., S ingh N. In vitro genotoxicity testing strategy for
nanomaterials and the adaptation of current OECD guidelines. Mutat. Res. 2012, 745pp. 104–111
[169] B arnes C.A., E ls aesser A., A rkusz J., S mok A., Palus J., L e ś ni ak A. Reproducible comet assay
o f amorphous silica nanoparticles detects no genotoxicity. Nano Lett. 2008, pp. 3069–3074
8
[170] H ansen T., C lermont G., A lves A., E loy R., B rochh ausen C., B outrand J.P. Biological
tolerance o f di fferent materials in bulk and nanoparticulate form in a rat model: sarcoma
development by nanoparticles. J. R. Soc. Inter face. 2006, pp. 767–775
3
[171] Park S., L ee Y.K., J ung M., K im K.H., C h ung N. Cellular toxicity o f various inhalable
nanoparticles on human alveolar epithelial cells. Inhal. Toxicol. 2007, pp. 59–65
9
[172] P etersen E.J., T u X., D i zdaroglu M., Z heng M., N elson B.C. Protective roles of single-wall
carbon nanotubes in ultrasonication-induced DNA base damage. Small. 2013, pp. 205–208 9
[173] S ayes C.M., Wahi R., Kuri an P.A., L i u Y., Wes t J.L., Ausm an K.D. Correlating nanoscale titania
structure with toxicity: a cytotoxicity and inflammatory response study with human dermal
fibroblasts and human lung epithelial cells. Toxicol. Sci. 2006, 92 pp. 174–185
[174] Warhei t D.B., Webb T.R., R eed K.L., F rerichs S., S ayes C.M. Pulmonary toxicity study in rats
with three forms o f ultrafine-TiO 2 particles: differential responses related to surface properties.
Toxicology. 2007, 230 pp. 90–104
[175] D oak S.H., M anshi an B., J enkins G.J.S., S ingh N. In vitro genotoxicity testing strategy for
nanomaterials and the adaptation of current OECD guidelines. Mutat. Res. 2012, 745pp. 104–111
[176] C li f t M.J.D., R aem y D.O., E ndes C., A li Z., L ehm ann A.D., B randenberger C. Can the Ames
test provide an insight into nano-object mutagenicity? Investigating the interaction between
nano-objects and bacteria. Nanotoxicology. 2013, pp. 1373–1385
7
[177] S era N., Toki wa H., M i yata N. Mutagenicity o f the fullerene C60-generated singlet oxygen
dependent formation of lipid peroxides. Carcinogenesis. 1996, pp. 2163–2169
17
[178] D oak S.H., G ri ff i ths S.M., M anshi an B., S ingh S., Willi ams P.M., B rown A.P. Confounding
experimental considerations in nano(geno)toxicology. Mutagenesis. 2009, 24pp. 285–293
[179] S erda R.E., G u J., B h avane R.C., L i u X., C hi appini C., D ecuzzi P. The association of silicon
microparticles with endothelial cells in drug delivery to the vasculature. Biomaterials. 2009,
30 pp. 2440–2448
[180] FDA. 2012. “International Con ference on Harmonisation; guidance on S2(R1) Genotoxicity
Testing and Data Interpretation for Pharmaceuticals intended for Human Use; availability.
Notice.” Fed Regist 77(110): 33748-33749
[181] Roth f uss A., , & H onm a M et al. “Improvement o f in vivo genotoxicity assessment: combination
o f acute tests and integration into standard toxicity testing.” Mutat Res. 2011, 72 3 (2): 108-120
[182] L ouro H., Tavares A., Vi tal N., C osta P.M., A lverc a E., Z wart E. Integrated approach
to the in vivo genotoxic effects of a titanium dioxide nanomaterial using LacZ plasmid-based
transgenic mice. Environ. Mol. Mutagen. 2014, pp. 500–509
55
[183] G ossen J.A., M artus H.-J., Wei J., Vijg J. Spontaneous and x-ray induced deletion mutations in
a LacZ plasmid-based transgenic mouse model. Mutat. Res. 1995, 3 31 pp. 89–97
[184] J ackson S.P., & B artek J. The DNA-damage response in human biology and disease. Nature.
2009, 461 pp. 1071–1078

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[185] Kum ar A., & D h awan A. Genotoxic and carcinogenic potential of engineered nanoparticles: an
update. Arch. Toxicol. 2013, pp. 1883–1900
87
[186] B orm P.J., & K re yling W. Toxicological hazards of inhaled nanoparticles–potential implications
for drug delivery. J. Nanosci. Nanotechnol. 2004, 4 pp. 521–531
[187] P ott F., & Roller M. Carcinogenicity study with nineteen granular dusts in rats. Eur. J. Oncol.
2005, 10pp. 249–281
[188] O berd ö rs ter G. Lung particle overload: implications for occupational exposures to particles.
Regul. Toxicol. Pharmacol. 1995, pp. 123–135
21
[189] Valberg P.A., B ruch J., M c C unne y R.J. Are rat results from intratracheal instillation o f 19
granular dusts a reliable basis for predicting cancer risk? Regul. Toxicol. Pharmacol. 2009, 54
pp. 72–83
[190] Roller M. Di fferences between the data bases, statistical analyses, and interpretations o f lung
tumors o f the 19-dust study – Two controversial views. Exp. Toxicol. Pathol. 2007, 58 pp. 393–405
[191] M or f eld P., A lbrech t C., D rommer W., B orm P.J.A. Dose response and threshold analysis
o f tumour prevalence a fter intratracheal instillation o f six types o f low and high sur face area
particles in a chronic rat experiment. Inhal. Toxicol. 2006, pp. 215–225
18
[192] P ol and C.A., D u ff in R., K inloch I., M aynard A., Wall ace W.A., S e aton A. Carbon nanotubes
introduced into the abdominal cavity o f mice show asbestos-like pathogenicity in a pilot study.
3
[193] D onaldson K., M urph y F.A., D u ff in R., P ol and C.A. Asbestos, carbon nanotubes and the
pleural mesothelium: a review o f the hypothesis regarding the role o f long fibre retention in the
parietal pleura, inflammation and mesothelioma. Part. Fibre Toxicol. 2010, p. 5
7
[194] S torer R.D., F rench J.E., D onehower L.A., G ulezi an D., M i tsumori K., R ecio L.IWGT
Working Group. Transgenic tumor models for carcinogen identification: the heterozygous
Trp53-deficient and RasH2 mouse lines. Mutat. Res. 2003, 5 40 pp. 165–176
[195] B overho f D.R., C h amberl ain M.P., E lcombe C.R., G onz alez F.J., H e f lich R.H., H ern á ndez
L.G. Benthem Jv, Gollapudi BB. Transgenic animal models in toxicology: historical perspectives
and future outlook. Toxicol. Sci. 2011, 121 pp. 207–233
[196] Austin C.A., U mbrei t T.H., B rown K.M., B arber D.S., D air B.J., F rancke -C arroll S.
Distribution o f silver nanoparticles in pregnant mice and developing embryos. Nanotoxicology.
2012, pp. 912–922
6
[197] H ougaard K.S., C ampagnolo L., C h avatte -Palmer P., Tarrade A., Rousse au -R alli ard D.,
Valentino S. A perspective on the developmental toxicity o f inhaled nanoparticles. Reprod.
Toxicol. 2015, pp. 118–140
56
[198] D e J ong W.H., Van D er Ven L.T.M., S leij ff ers A., Park M.V.D.Z., J ansen E.H.J.M., Van L overen
H. Systemic and immunotoxicity o f silver nanoparticles in an intravenous 28 days repeated dose
toxicity study in rats. Biomaterials. 2013, 34 pp. 8333–8343
[199] S h arm a M., S alisbury R.L., M aurer E.I., H ussain S.M., S ulentic C.E. Gold nanoparticles
induce transcriptional activity o f NF-κB in a B-lymphocyte cell line. Nanoscale. 2013, 5
pp. 3747–3756
[200] T sai C.Y., L u S.L., H u C.W., Yeh C.S., L ee G.B., L ei H.Y. Size-dependent attenuation of TLR9
signaling by gold nanoparticles in macrophages. J. Immunol. 2012, 188 pp. 68–76
[201] I shii N., F i tril awati F., M anna A., A ki yam a H., Tam ada Y., Tam ada K. Gold Nanoparticles
Used as a Carrier Enhance Production o f Anti-Hapten IgG in Rabbit: A Study with Azobenzene-
Dye as a Hapten Presented on the Entire Sur face o f Gold Nanoparticles. Biosci. Biotechnol.
Biochem. 2008, 72 pp. 124–131
ISO/TR 1 0993 -2 2 : 2 01 7(E)
[202] M aquieira Á., B run E.M., G arc é s -G arc í a M., P uch ades R. Aluminum Oxide Nanoparticles
as Carriers and Adjuvants for Eliciting Antibodies from Non-immunogenic Haptens. Anal. Chem.
2012, pp. 9340–9348
84
[203] D ykm an L.A., S taroverov S.A., B ogat yre v V.A., S hch yogole v S.Y. Adjuvant properties of
gold nanoparticles. Nanotechnol. Russ. 2010, pp. 748–761
5
[204] D wi vedi P.D., T ripathi A., A ns ari K.M., S h anker R., D as M. Impact of Nanoparticles on the
Immune System. J. Biomed. Nanotechnol. 2011, pp. 193–194
7
[205] Vandebriel R.J., Tonk E.C.M., D e L a Fonte yne -B l ankesti jn L.J., G remmer E.R., Verh aren
H.W., Van D er Ven L.T. Immunotoxicity o f silver nanoparticles in an intravenous 28-day
repeated-dose toxicity study in rats. Part. Fibre Toxicol. 2014, 11 p. 21
[206] D e J ong W.H., & Van L overen H. Guest Editor’s Introduction. Animal models in
immunotoxicology. Methods. 2007, 41 pp. 1–2
[207] D obrovolskai a M.A., G ermolec D.R., We aver J.L. Evaluation o f nanoparticle immunotoxicity.
4
[208] B oraschi D., C ostantino L., I tali ani P. Interaction of nanoparticles with immunocompetent
cells: nanosa fety considerations. Nanomedicine (Lond.). 2012, pp. 121–131
7
[209] Farrera C., & Fadeel B. It takes two to tango: Understanding the interactions between
engineered nanomaterials and the immune system. Eur. J. Pharm. Biopharm. 2015, 95 pp. 3–12
[210] Z olnik B.S., G onz alez -F ernandez A., S adrieh N., D obrovolskai a M.A. Minireview:
Nanoparticles and the Immune System. Endocrinology. 2010, 151 pp. 458–465
[211] G ad S.C., S h arp K.L., M ontgomery C., Payne J.D., G oodrich G.P. Evaluation o f the Toxicity
o f Intravenous Delivery o f Auroshell Particles (Gold–Silica Nanoshells). Int. J. Toxicol. 2012, 31
pp. 584–594
[212] K im J.S., S ong K.S., S ung J.H., Ryu H.R., C hoi B.G., C ho H.S. Genotoxicity, acute oral and dermal
toxicity, eye and dermal irritation and corrosion and skin sensitisation o f silver nanoparticles.
Nanotoxicology. 2013, 7 pp. 953–960
[213] SCCS. 2012 OPINION ON Zinc oxide (nano form), COLIPA S 76. http://ec .europa .eu/ health/
scientific _committees/consumer _sa fety/docs/sccs _o _103 .pdf
[214] SCCS. 2013. Opinion on Titanium Dioxide (Nano form). Colipa No. S75. Scientific Committee on
Consumer Sa fety. European Commission. SCCS/1516/13. Luxembourg. pp. 31-33. Available at:
http://ec .europa .eu/ health/ scientific _committees/consumer _sa fety/docs/sccs _o _136 .pdf
[215] Park Y.H., J eong S.H., Yi S.M., C hoi B.H., K im Y.R., K im I.K. Analysis for the potential o f
polystyrene and TiO 2 nanoparticles to induce skin irritation, phototoxicity, and sensitization.
Toxicol. In Vitro. 2011, pp. 1863–1869
25
[216] E m a M., M atsuda A., Kobayashi N., N aya M., N akanishi J. Evaluation o f dermal and eye
irritation and skin sensitization due to carbon nanotubes. Regul. Toxicol. Pharmacol. 2011, 61
pp. 276–281
[217] E m a M., M atsuda A., Kobayashi N., N aya M., N akanishi J. Dermal and ocular irritation
and skin sensitization studies o f fullerene C60 nanoparticles. Cutan. Ocul. Toxicol. 2013, 32
pp. 128–134
[218] B aroli B. Penetration o f nanoparticles and nanomaterials in the skin: fiction or reality? J.
Pharm. Sci. 2010, 99 pp. 21–50
[219] ECHA. 2014. Guinea pig maximization test o f multi-walled carbon nanotubes (MWCNT),
synthetic graphite in tubular shape. European Chemicals Agency Registered Substances

ISO/TR 1 0993 -2 2 : 2 01 7(E)
Database. EC List No. 936-414-1. Bayer MaterialScience AG, et al. Available at: https://echa
.europa .eu/information-on-chemicals/registered-subs tances
[220] Rouse J.G., Yang J., Rym an -R asm ussen J.P., B arron A.R., M onteiro -R i viere N.A. Effects of
mechanical flexion on the penetration o f fullerene amino acid-derivatized peptide nanoparticles
through skin. Nano Lett. 2007, pp. 155–160
7
[221] H oet P.H., B rü ske -H ohl f eld I., S al ata O.V. Nanoparticles – known and unknown health risks.
J. Nanobiotechnology. 2004, p. 122
[222] Rym an -R asm ussen J.P., R i viere J.E., M onteiro -R i viere N.A. Penetration o f intact skin by
quantum dots with diverse physicochemical properties. Toxicol. Sci. 2006, 91 pp. 159–165
[223] S amberg M.E., O ldenburg S.J., M onteiro -R i viere N.A. Evaluation o f silver nanoparticle toxicity
in skin in vivo and keratinocytes in vitro. Environ. Health Perspect. 2010, 118 pp. 407–413
[224] M onteiro -R i viere N.A., & F ilon F.L. Skin. In: Adverse E ffects o f Engineered Nanomaterials.
Elsevier, 2012., 10.1016/B978-0-12-386940-1.00011-8
[225] O gunsol a O.A., K raeling M.E., Z hong S., P och an D.J., B ronaughd R.L., R agh avan S.R.
Structural analysis o f “flexible” liposome formulations: new insights into the skin-penetrating
ability o f so ft nanostructures. So ft Matter. 2012,40 pp. 10226–10232
[226] Rym an -R asmussen J.P., R i viere J.E., M onteiro -R i viere N.A. Surface coatings determine
cytotoxicity and irritation potential o f quantum dot nanoparticles in epidermal keratinocytes. J.
Invest. Dermatol. 2007, 1 27 pp. 143–153
[227] Wang S., L u W., Tovm achenko O., R ai U.S., Yu H., R ay P.C. Challenge in Understanding Size
and Shape Dependent Toxicity o f Gold Nanomaterials in Human Skin Keratinocytes. Chem. Phys.
Lett. 2008,Get more
463 pp. 145–149
FREE standards from Standard Sharing Group and our chats
[228] K aradzovska D., B rooks J.D., M onteiro -R i viere N.A., R i viere J.E. Predicting skin
permeability from complex vehicles. Adv. Drug Deliv. Rev. 2013, 65pp. 265–277
[229] L ee H.A., I mran M., M on teiro -R i viere N.A., C olvin V.L., Yu W.W., R i viere J.E. Biodistribution
o f quantum dot nanoparticles in per fused skin: Evidence o f coating dependency and periodicity
in arterial extraction. Nano Lett. 2007, pp. 2865–2870
7
[230] M onteiro -R i viere N.A., & R i viere J.E. Interaction o f nanomaterials with skin: Aspects o f
absorption and biodistribution. Nanotoxicology. 2009, 3 pp. 188–193
[231] M onteiro -R i viere N.A., Wiench K., L andsiedel R., S ch ulte S., I nm an A.O., R i viere J.E.
Sa fety Evaluation o f Sunscreen Formulations Containing Titanium Dioxide and Zinc Oxide
Nanoparticles in UVB Sunburned Skin: An In Vitro and In Vivo Study. Toxicol. Sci. 2011, 1 2 3
pp. 264–280
[232] P row T.W., M on teiro -R i viere N.A., I nm an A.O., G rice J.E., C hen X., Z h ao X. Quantum dot
penetration into viable human skin. Nanotoxicology. 2012, 6 pp. 173–185
[233] Z h ang L.S.W., & M onteiro -R i viere N.A. Use o f con focal microscopy for nanoparticle drug
delivery through skin. J. Biomed. Opt. 2013, 18 p. 6
[234] J atana S., C all ah an L.M., P entl and A.P., D e L ouise L.A. Impact of cosmetic lotions
on nanoparticle penetration through ex vivo C57BL/6 hairless mouse and human skin: A
comparison study. Cosmetics. 2016, 3 p. 6
[235] D ing L., S tilwell J., Z h ang T., E lboudwarej O., J i ang H., S elegue J.P. Molecular
Characterization o f the Cytotoxic Mechanism o f Multiwall Carbon Nanotubes and Nano-Onions
on Human Skin Fibroblast. Nano Lett. 2005, 5 pp. 2448–2464
[236] S ayes C.M., G obin A.M., Ausm an K.D., M endez J., Wes t J.L., C olvin V.L. Nano-C60 cytotoxicity
is due to lipid peroxidation. Biomaterials. 2005, pp. 7587–7595
26

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[237] Papageorgiou I., B rown C., S chins R., S ingh S., N e wson R., D avis S. The effect of nano- and
micron-sized particles o f cobalt–chromium alloy on human fibroblasts in vitro. Biomaterials.
2007, 28 pp. 2946–2958
[238] Z h ang Y., H u L., Yu D., G ao
C. Influence o f silica particle internalization on adhesion and
migration o f human dermal fibroblasts. Biomaterials. 2010, 31 pp. 8465–8474
[239] K ishore A.S., S urekh a P., M urth y P.B. Assessment of the dermal and ocular irritation
potential o f multi-walled carbon nanotubes by using in vitro and in vivo methods. Toxicol. Lett.
2009, 191 pp. 268–274
[240] Warhei t D.B., H oke R.A., F inl ay C., D onner E.M., R eed K.L., S ayes C.M. Development of
a base set o f toxicity tests using ultrafine TiO 2 particles as a component o f nanoparticle risk
management. Toxicol. Lett. 2007, 171 pp. 99–110
[241] Park Y.H., K im J.N., J eong S.H., C hoi J.E., L ee S.H., C hoi B.H. Assessment o f dermal toxicity o f
nanosilica using cultured keratinocytes, a human skin equivalent model and an in vivo model.
Toxicology. 2010, pp. 178–181
2 67
[242] Kolle S.N., S auer U.G., M oreno M.C., T eubner W., Wohlleben W., L andsiedel R. Eye
irritation testing o f nanomaterials using the EpiOcular™ eye irritation test and the bovine
corneal opacity and permeability assay. Part. Fibre Toxicol. 2016, 13 p. 18
[243] S zebeni J., M uggi a F., G abizon A., B arenholz Y. Activation o f complement by therapeutic
liposomes and other lipid excipient-based therapeutic products: Prediction and prevention. Adv.
Drug Deliv. Rev. 2011, pp. 1020–1030
63
[244] A balde S.L., & F ernandez A.G. Nanostructures and Allergy. In: Handbook o f Immunological
Properties of Engineered Nanomaterials, (D obrovolskai a M.A., & M c N eil S.E. eds.) . World
Scientific Publishing Company, Ltd, Hackensack, New Jersey, 2013, pp. 528–9.
[245] Z h ang X.-Q., Xu X., B ertrand N., P ridgen E., S wami A., Farokhz ad O.C. Interactions of
nanomaterials and biological systems: implications to personalized nanomedicine. Adv. Drug
Deliv. Rev. 2012, pp. 1363–1384
64
[246] S mi th B.S., Yori ya S., G rissom L., G rimes C.A., P opat K.C. Hemocompatibility o f titania
nanotube arrays. J. Biomed. Mater. Res. A. 2010, 95 pp. 350–360 [Erratum in: J Biomed Mater
Res A. 96, 608, 2011]
[247] L iu X, & S un J. Endothelial cells dys function induced by silica nanoparticles through oxidative
stress via JNK/P53 and NF-kB pathway. Biomaterials. 2010, 31 pp. 8198–8209
[248] L iu X, Xue Y, D ing T, S un J Enhancement o f proinflammatory and procoagulant responses to
silica particles by monocyte-endothelial cell interactions. Part. Fibre Toxicol. 2012, 9 p. 36
[249] L i u X., & S un J. Potential proinflammatory e ffects o f hydroxyapatite nanoparticles on
endothelial cells in a monocyte–endothelial cell coculture model. Int. J. Nanomedicine. 2014, 9
pp. 1261–1273
[250] K elly L.S., D obson E.L., F inne y C.R., H irsch J.D. Proli feration o f the reticuloendothelial
system in the liver. Am. J. Physiol. 1960, 198pp. 1134–1138
[251] Parker H.G., & F inne y C.R. Latent period in the induction o f reticuloendothelial blockade. Am.
J. Physiol. 1960, 198 pp. 916–920
[252] OECD (Organization for Economic Co-operation and Development). Important Issues on
Risk Assessment o f Manu factured Nanomaterials. Series on the Sa fety o f Manu factured
Nanomaterials No. 33. ENV/JM/MONO(2012)8. 2012
[253] M ac P h ail R.C., G rulke E.A., Yokel R.A. Assessing nanoparticle risk poses prodigious
challenges. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2013, 5 pp. 374–387

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[254] L ee J.H., K im Y.S., S ong K.S., Ryu H.R., S ung J.H., Park J.D. Biopersistence o f silver nanoparticles
in tissues from Sprague-Dawley rats. Part. Fibre Toxicol. 2013, 10 p. 36
[255] C ho W.S., K ang B.C., L ee J.K., J eong J., C he J.H., S eok S.H. Comparative absorption,
distribution, and excretion of titanium dioxide and zinc oxide nanoparticles after repeated oral
administration. Part. Fibre Toxicol. 2013, p. 9
10
[256] M a X., Wu Y., J in S., T i an Y., Z h ang X., Z h ao Y. Gold Nanoparticles Induce Autophagosome
Accumulation through Size-Dependent Nanoparticle Uptake and Lysosome Impairment. ACS
Nano. 2011, pp. 8629–8639
5
[257] S impson C.A., S alleng K.J., C li ff el D.E., F eldheim D.L. In vivo toxicity, biodistribution, and
clearance of glutathione-coated gold nanoparticles. Nanomedicine. 2013, pp. 257–263
9
[258] E lgrabli D., F lori ani M., A bella-G all art S., M eunier L., G amez C., D el al ain P.
Biodistribution and clearance of instilled carbon nanotubes in rat lung. Part. Fibre Toxicol.
2008, p. 20
5
[259] G eraets L. OOmen AG, Krystek P, Jacobsen NR, Wallin H, laurentie M, Verharen HW, Brandon
EFA, De Jong WH. Tissue distribution and elimination a fter oral and intravenous adminisrtation
of diffenrent titanium dioxide nanoparticles in rats. Part. Fibre Toxicol. 2014, p. 30
11
[260] Rym an -R asmussen J.P., C esta M.F., B rody A.R., S hiple y-P hillips J.K., E veri tt J.I., T e wksbury
E.W. Inhaled carbon nanotubes reach the subpleural tissue in mice. Nat. Nanotechnol. 2009, 4
pp. 747–751
[261] Panneerselvam S., & C hoi S. Nanoin formatics: emerging databases and available tools. Int. J.
Mol. Sci. 2014, pp. 7158–7182
15
[262] P uz yn T., R asule v B., G aje wicz A., H u X., D asari T.P., M ich alkova A. Using nano-QSAR to
predict the Get o f metal oxide nanoparticles. Nat. Nanotechnol. 2011, pp. 175–178
more FREE standards from Standard Sharing Group and our chats
cytotoxicity 6
[263] X i a X.R., M onteiro -R i viere N.A., R i viere J.E. An index for characterization o f nanomaterials
in biological systems. Nat. Nanotechnol. 2010, pp. 671–675
5
[264] Winkler D.A., M ombelli E., P ietroi us ti A., T ran L., Worth A., Fadeel B. Applying
quantitative structure-activity relationship approaches to nanotoxicology: current status and
future potential. Toxicology. 2013, 313 pp. 15–23
[265] ICRP (I n ternational C ommi ttee on R adiologic al P rotec tion ). Human Respiratory Tract
Model for Radiological Protection. ICRP Publication 66. Ann. ICRP. 1994, (1-3). Available at:
24
http://www.icrp .org/publication .asp?id= ICRP %20Publi cation%2066
[266] H o f m ann W. Modelling particle deposition in human lungs: modelling concepts and comparison
with experimental data. Biomarkers. 2009, 14 () pp. 59–62
[267] L i M., A l -J am al K.T., Kostarelos K., R eineke J. Physiologically based pharmacokinetic
modeling of nanoparticles. ACS Nano. 2010, pp. 6303–6317
4
[268] C arl ander U., L i D., J olliet O., E mond C., J oh anson G. Toward a general physiologically-
based pharmacokinetic model for intravenously injected nanoparticles. Int. J. Nanomedicine.
2016, pp. 625–640
11
[269] N el A., X i a T., M eng H., Wang X., L in S., J i Z. Nanomaterial Toxicity Testing in the 21st
Century: Use o f a Predictive Toxicological Approach and High-Throughput Screening. Acc. Chem.
Res. 2012, pp. 607–621
46
[270] Z uin S., M icheletti C., C ritto A., P ojana G., J ohnston H., S tone V. Weight of evidence
approach for the relative hazard ranking o f nanomaterials. Nanotoxicology. 2011, pp. 445–458
5

ISO/TR 1 0993 -2 2 : 2 01 7(E)
[2 71] H ristozov D.R., Z abeo A., Foran C., I sigonis P., C ri tto A., M arcomini A. A weight of
evidence appro ach for ha z ard s cre eni ng o f engi ne ere d nanomateri a l s . N ano toxicolo g y. 2 01 2 , 6
pp. 880–898
[2 7 2 ] Tseng M.T., F u or K., F ernandez otran G.R., D eng Z.B., G rah am U. Persistent Hepatic
Q. , L -B
Structural Alterations Following Nanoceria Vascular Infusion in the Rat. Toxicol. Pathol. 2014,
42 pp. 984–996

ISO/TR 1 0993 -2 2 : 2 01 7(E)
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Biological evaluation of medical

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1 Scope .................................................. ................................................... ................................................... ................................................... ...................... 1

2 Normative references .................................................. ................................................... ................................................... .............................. 2

3 Terms and definitions .................................................. ................................................... ................................................... ............................. 2

9.3 .4 C arcino genicity .................................................. ................................................... ................................................... ....... 32

iv © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved v

vi © ISO 2017 – All rights reserved

Biological evaluation of medical devices —

© ISO 2017 – All rights reserved 1

ISO 14971, Medical devices — Application of risk management to medical devices

2 © ISO 2017 – All rights reserved

Note 2 to entry: See “ultrafine particle” in ISO/TR 27628:2007, 2.21.

[SOURCE: ISO/TS 80004-2:2015, 4.4]

© ISO 2017 – All rights reserved 3

4 © ISO 2017 – All rights reserved

4.1 General considerations

© ISO 2017 – All rights reserved 5

4.2 Biological evaluation of nanomaterials

4.3 Categorization of nanomaterials

6 © ISO 2017 – All rights reserved

Table 1 — Considerations for biological evaluation of medical devices

C ategory a Type of nanomaterial Considerations in addition to the biological

— Consider potential cellular or tissue effects due to direct

© ISO 2017 – All rights reserved 7

C ategor y a Type of nanomaterial Cons iderations in addition to the biolo gical

— Consider characterization o f physicochemical properties

4.4 Nanomaterial equivalence

Proper identification and characterization o f the nanomaterial is essential. For nanomaterials,

Knowledge o f the physicochemical properties o f nanomaterials is essential to understanding their

— How does it interact with the surrounding environment?

8 © ISO 2017 – All rights reserved

i n I S O 10 9 9 3 -9, I S O 10 9 9 3 -1 3 , I S O 10 9 9 3 -14 a nd I S O 10 9 9 3 -1 5 . D e tai le d guidance s p e c i fic a l ly for the

feed, cosmetic ingredients and medical devices [57 58 59 . ] [ ] [ ]

— object size and size distribution;

— d i s p ers ibi l ity.

con s idere d on a c a s e -b y- c a s e b a s i s i nclude:

— radical formation potential;

1) For details, see http://www.iso .org/iso/home/store/catalogue_tc/cata logue_tc_browse.htm?commid= 381983 .

Devices incorporating nanostructured surfaces can require characterization of morphological features

© ISO 2017 – All rights reserved 11

e.g. histogram, and/or Laser-induced incandescence ISO 16700

12 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 13

5 .3 Use of reference materials

14 © ISO 2017 – All rights reserved

6.1 General considerations

© ISO 2017 – All rights reserved 15

16 © ISO 2017 – All rights reserved

6.4 Identity, storage and stability of stock nanomaterials

6.5 Description of chemical composition of stock and dosing dispersions

Dispersions of nano-objects used in biological evaluation studies should be compatible with

© ISO 2017 – All rights reserved 17

6.6 Characterization of stock dispersions

18 © ISO 2017 – All rights reserved

6.8 Dose metrics