Guidance on information requirements and chemical safety assessment : Appendix R7-1 for nanomaterials applicable to Chapter R7a Endpoint specific guidance : Version 4.0. December 2022.
Date: 2022-12-22
Institutions: European Chemicals Agency
Subjects: chemical product, chemicals legislation, environmental risk prevention, health risk, industrial hazard, nanotechnology, technical regulations, toxic substance, user guide

Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint 
specific guidance) 
Version 4.0 – December 2022 

1 

G U I D A N C E  

Guidance on information requirements and chemical safety 
assessment   
Appendix R7-1 for nanomaterials applicable to 
Chapter R7a Endpoint specific guidance 

Version 4.0 
December 2022  

 
 
 
 
 
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Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint 
specific guidance) 
Version 4.0 – December 2022 

LEGAL NOTICE 

This  document  aims  to  assist  users  in  complying  with  their  obligations  under  the  REACH 
Regulation.  However,  users  are  reminded  that  the  text  of  the  REACH  Regulation  is  the  only 
authentic  legal reference  and  that  the  information  in  this  document  does  not  constitute  legal 
advice.  Usage of the information remains under the sole responsibility of the user. The European 
Chemicals Agency does not accept any liability with regard to the use that may be made of the 
information contained in this document. 

Guidance on information requirements and chemical safety assessment   
Appendix R7-1 for nanomaterials applicable to Chapter R7a - Endpoint specific guidance 

Reference: ECHA-22-G-02-EN 
Cat. Number: ED-04-22-321-EN-N 
ISBN: 978-92-9468-254-3 
DOI: 10.2823/496606 
Publ.date: December 2022 
Language: EN 

© European Chemicals Agency, 2022 

Permission to reproduce extracts from BS ISO 9276-6:2008 is granted by BSI. No other use of 
these extracts is permitted. British Standards can be obtained in PDF or hard copy formats from 
the  BSI  online  shop:  www.bsigroup.com/Shop  or  by  contacting  BSI  Customer  Services  for 
hardcopies only: Tel: +44 (0)20 8996 9001, Email: cservices@bsigroup.com. 

If you have questions or comments in relation to this document please send them (indicating 
the  document  reference,  issue  date,  chapter  and/or  page  of  the  document  to  which  your 
comment  refers)  using  the  information  request  form.  The  information  request  form  can  be 
accessed via the Contact ECHA page at: http://echa.europa.eu/contact 

European Chemicals Agency 
Mailing address: P.O. Box 400, FI-00121 Helsinki, Finland 
Visiting address: Telakkakatu 6, 00150 Helsinki, Finland 

 
 
 
 
 
 
 
 
 
 
 
Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint 
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Version 4.0 – December 2022 

3 

DOCUMENT HISTORY 

Version 

Changes 

Version 1 

First edition 

Date 

April 2012 

Version 2 

•  New advisory note (section 1.1) on testing for ecotoxicity and 

May 2017 

Version 3 

October 2021 

fate, to   provide   overall   advice   for   conducting   ecotoxicity   
and environmental fate testing for nanomaterials  

•  Update of section 1.2.1on aquatic pelagic toxicity, to clarify that 
high insolubility cannot be used as a waiver and to include 
further recommendations on the text to be performed for this 
endpoint 

•  Update of section 1.2.2. on Toxicity for sediments organisms to 

provide advice on spiking methods and include applicability of 
available OECD guidelines 

•  Update of section 1.2.3 on degradation/ biodegradation to clarify 
that waivers for hydrolysis and degradation simulation testing 
are not applicable as sole evidence, provide advice on 
photocatalytic degradation and general advice on performing the 
tests 

Please note that the numbering of the sections has changed, the 
section numbers above refer to the updated numbering of the 
guidance sections. 

•  Create a Glossary section with definitions 
•  Section 3.1.1. becomes 2.1.1. General advisory note on 
testing with nanoforms and sampling strategy and 
sample preparation for human health endpoints: 
content updated and restructured: 

o  Update Fig 1. and added a disclaimer. 
o  Section 2.1.1.1. Considerations on solubility and 

dissolution rate 

o  Section 2.1.1.2. Available methods for solubility and 

dissolution testing 

o  Section 2.1.1.3. Test material characterisation and 

reporting and sample preparation 
o  Section 2.1.1.4. Biological sampling 
o  Section 2.1.1.5. Use of Non-Animal Testing 

Approaches 

•  Section 3.2.2. becomes 2.2.2. Repeated dose toxicity: 

update regarding: 

o  The mandatory status of some parameters (e.g. 
dissolution rate in water and relevant biological 
media). 

o  The conditions under which an older study can be 

used. 

o  The doses to be used in repeated dose inhalation 

studies.  

o  The consequences of the dose on agglomeration for 
designing and interpreting repeated dose toxicity 
studies (RDT).  

o  The mechanism of toxicity for fibres 
o  The type of toxicokinetic investigations that can be 

done under RDT studies  

o  Section 2.2.2.1. The concept of lung overload: 
updated with the most recent references.  

o  Section 2.2.2.2. Indirect genotoxicity 

 
 
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Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint 
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Version 4.0 – December 2022 

•  Section 3.2.3. becomes 2.2.3. Mutagenicity and 

Carcinogenicity updated and restructured: 

o  2.2.3.1. Bacterial (Ames) mutagenicity assays are 

not recommended 

o  2.2.3.2. Recommended approach for gathering 

mutagenicity information on nanomaterials according 
to REACH 

o  2.2.3.3. Recommendations for the in vitro tests 

▪ 

▪ 

▪ 

2.2.3.3.1. Nanomaterials characterization in 
the test medium 
2.2.3.3.2. Verification of uptake into target 
cells 
2.2.3.3.3. Recommendations to avoid 
interference with uptake or endpoint analysis 

o  2.2.3.4. In vivo test and exposure of target tissue 

Version 4 

•  Update of the advisory note on testing and sampling 

2022 

strategy and sample preparation for 
ecotoxicological endpoints: 
o  Update and changing of Section 1.1 and sub sections with 

1.1.1. Characterisation of test materials, 1.1.2 Sample 
preparation of test materials and 1.1.3 General 
considerations for Fate and (Eco)-Toxicological testing 

o  Update of Glossary Section 

•  Update of the section 1 for water solubility, 
granulometry, Kow, adsorption/desorption: 
o  Addition of sub-section for Section 1.2.1 Water solubility with 

waivers and dissolution rate, 

o  Addition of sub-section for Section 1.2.2 Partition coefficient 
n-octanol/water, with waivers and dispersion stability, 
Addition of the sub-section for Section 1.2.4 Dustiness, 

o  Addition of sub-section for Section 1.2.5 

Adsorption/Desorption with waivers and alternative methods 
to Koc 

 
 
 
 
 
Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint 
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PREFACE 

Three  appendices  (appendices  to Information Requirements  and  Chemical  Safety  Assessment 
Guidance Chapters R7a, R7b and R7c) specifying information requirements have been developed 
to  provide  advice  for  registrants  when  preparing  REACH  registration  dossiers  that  cover 
nanoforms [1].The advice provided in this document focuses on specific recommendations for 
testing materials that are nanoforms of substances1. As most of the guidelines and publications 
are referring to nanomaterials or nanoparticles, also the terms “nanomaterial” and “nanoparticle” 
are used. Annex VI of REACH defines the terms “nanoform” and “set of similar nanoforms”2 and 
establishes the requirements for characterisation of the identified nanoforms of the substance. 
Advice on substance identification and registration of nanoforms can be found in the guidance 
“Appendix  for  nanoforms  applicable  to  the  Guidance  on  Registration  and  Substance 
Identification” and shall be applied when registering nanoforms of a substance under REACH [2]. 
A glossary is available to define the terms used in this guidance. As this appendix is specifically 
addressing REACH information requirements, nanoform is the preferred term and used whenever 
possible.  

Part  of  the  provided  advice  is  not  strictly  nanoform  specific  and  may,  for  instance,  also  be 
applicable  to  other  particulate  forms  of  substances  (e.g.  where  dissolution  rate  is  relevant). 
However, advice has been included when it is considered especially relevant for nanoforms and 
should therefore be part of the nanoform specific guidance. If such nanospecific advice is not 
available  no  additional  guidance  for  the  information  requirement  has  been  included  in  this 
appendix because of one of the following reasons a) the endpoint is not relevant for nanoforms, 
b)  the  parent  guidance  is  considered  equally  applicable  to  nanoforms  or  c)  more  research  is 
needed to develop nanospecific advice. This appendix is providing advice specific to nanoforms 
and does not supersede the applicability of the general principles given in Chapter R.7a [3], i.e. 
the  parent  guidance  which  is  applicable  in  case  of  the  absence  of  nanospecific  advice  in  this 
appendix. Please note that this document and its parent guidance provides specific guidance for 
meeting  the  information  requirements set  out in  Annexes  VII  to  XI  to the  REACH Regulation. 
General information for meeting the information requirements such as collection and evaluation 
of  available  information,  and  adaptation of information  requirements,  is  available  in Chapters 
R.2 to R.5 of Guidance on IR&CSA. Moreover, when considering the use of data already available, 
“Guidance on information requirements and chemical safety assessment  – Appendix R.6-1 for 
nanoforms applicable to the Guidance on QSARs and Grouping of Chemicals” [4] may be useful 
as  it  provides  how  to  approach  read-across  for  hazard  data  between  nanoforms,  as  well  as 
nanoforms and the non-nanoform of the same substance.  

1 See Annex VI of the REACH Regulation (EU) 1907/2006, as amended by Commission Regulation (EU) 2018/1881 on 
how to address nanoforms of substances.  

2 In this document the term “set of nanoforms” is used equivalent to  “set of similar nanoforms” as defined in Annex VI.  

 
 
 
 
 
 
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Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint 
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Version 4.0 – December 2022 

Table of Contents 
1. 

RECOMMENDATIONS FOR PHYSICO-CHEMICAL PROPERTIES .................................... 10 

1.1  General remarks .............................................................................................. 10 

  Characterisation of test materials ................................................................. 10 

  Sample preparation of test materials ............................................................. 11 

  General considerations for Fate and (Eco)-Toxicological testing ........................ 13 

1.2  Specific advice for endpoints .............................................................................. 15 

  Water solubility .......................................................................................... 15 

  Partition coefficient n-octanol/water .............................................................. 20 

  Granulometry ............................................................................................. 25 

  Dustiness................................................................................................... 31 

  Adsorption/desorption ................................................................................. 35 

RECOMMENDATIONS FOR TOXICOLOGICAL INFORMATION REQUIREMENTS for 

2. 
NANOMATERIALS ........................................................................................................ 38 

2.1  General advisory notes ..................................................................................... 38 

  General advisory note on testing with nanoforms and sampling strategy and 
sample preparation for human health endpoints ........................................................ 38 

  Advisory note on the consideration of assay interference ................................. 47 

2.2  Specific advice for individual endpoints ............................................................... 49 

  Acute toxicity ............................................................................................. 49 

  Repeated dose toxicity ................................................................................ 50 

  Mutagenicity and Carcinogenicity .................................................................. 59 

Appendix 1 

: Models for fate and exposure of nanomaterials .......................................... 63 

References ................................................................................................................. 66 

Table of Tables 
Table 1: Methods of measuring airborne dispersed or nebulised particles............................ 27 

Table 2: Test methods to measure dustiness of bulk materials that contain or release nano-
objects or submicrometer particles ................................................................................ 33 

Table 3: Potential sources of nanomaterials interference with commonly used assays .......... 48 

Table 4: Overview of some models for fate for nanomaterials ........................................... 63 

Table of Figures 
Figure 1: Scheme of the testing required to fulfill Annex VII, Section 7.7 information 
requirement of nanoforms ............................................................................................ 17 

Figure 2: Scheme of the testing required to fulfill Annex VII, Section 7.8 information 
requirement of nanoforms ............................................................................................ 21 

Figure 3: Schematic representation of exemplified dispersion stability results from an extended 
test (extracted from OECD TG 318) ............................................................................... 23 

Figure 4: Decision process for selecting the applicable guidance document ......................... 38 

Figure 5: Schematic representation of the relationship between retained lung burden and 
duration of exposure leading to the phenomenon of lung overload. .................................... 55 

 
 
 
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Glossary 3 

Accumulation:  
In (eco)toxicology, accumulation is the gradual build-up over time of a compound, in this case 
nanoparticles and their metabolites, in a whole organism or a tissue or organ, also defined as 
bioaccumulation.  In  the  environment  accumulation  is  the  gradual  build-up  over  time  of 
compounds, in this case nanoparticles and their degradation products, in a defined (or part of 
a) environmental compartment. 

ADME: Absorption, distribution, metabolism, excretion. 

Agglomerate: A collection of weakly bound particles or aggregates where the resulting external 
surface area is similar to the sum of the surface areas of the individual components [2], [5], [6] 
and [7]. 

Agglomeration: Process of contact and adhesion whereby dispersed particles are held together 
by weak physical interactions ultimately leading to enhanced sedimentation by the formation of 
particles (agglomerates) of larger than colloidal size [8]. The process occurs when two particles, 
i  and  j,  collide  and  attach  to  one  another  to  form  an  agglomerate.  The  agglomeration  rate 
ij,provides a quantitative description of the speed of the agglomeration process and 
constant, kagg
i,j and the probability for favourable attachment upon 
depends on the collision rate constant, kcoll
collision, described by the attachment efficiency 𝛼 [9].  

Aggregate:  A  particle  comprising  of  strongly  bound  or  fused  particles  [2].  In  contrast  to 
agglomeration, aggregation occurs where particles are held by strong bonds like sinter bridges. 

Bronchoalveolar  lavage  (BAL):  The  sample  containing  cells,  particles,  and  secretions, 
obtained by flushing the small airways and alveoli of the lungs with saline while the animal is 
anaesthetised. 

BALF: Bronchoalveolar lavage fluid. 

Bioavailability : The amount of a substance accessible to an organism for uptake or absorption 
across its cellular membrane. In toxicology this is measured as the proportion of a substance in 
the systemic circulation compared with the total amount of a substance that has been ingested 
or inhaled (modified from [10]). In ecotoxicology it is also measured as the amount taken up 
via cell surfaces (e.g. gills for fish) from the aqueous, sediment and/or soil compartment. 

Biodegradation:  Degradation  of  a  substance  resulting  from  interaction  with  the  biological 
environment [11]. 

Biodurability: The ability to resist chemical and biochemical alteration through dissolution and 
enzymatic  biodegradation  or  chemical  disintegration  within  biological  media  (modified  from 
[11]).  Biodurability  (dissolution  and  biodegradation)  is  measured  using  in  vitro  acellular  and 
cellular tests. 

Biopersistence:  The  ability  of  a  material  to  persist  in  the  body  due  to  its  biodurability  and 
resistance to physiological clearance [11]. It is determined using in vivo methods. 

Biotransformation: Alteration of a substance resulting from interaction with biological systems 
[11].  

3 As most of the guidelines and publications are referring to nanomaterials or nanoparticles, also the terms 
‘nanomaterial’ and ‘nanoparticle’ are used in addition to “nanoform”. Nanoform, is a recent REACH regulatory term 
which was not used in the scientific studies or authority reports mentioned in this guidance. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Clearance: (1) In (eco)toxicology, the volume of blood or plasma or mass of an organ effectively 
cleared of a substance by elimination (including metabolism and excretion) divided by the time 
of elimination. Total clearance is the sum of the clearances of each eliminating organ or tissue 
for  a  given  substance.  (2)  In  pulmonary  toxicology,  the  volume  or  mass  of  the  lung  cleared 
divided  by  the  time  of  elimination  is  used  qualitatively  to  describe  removal  of  any  inhaled 
substance which deposits on the lining surface of the lung [12]. 

Dissolution: Dissolution, as used in this guidance, is the process by which a nanomaterial in an 
aqueous medium or biological environment is dissolving into their constituent ions or molecules 
[7]. 

Dissolution half-life/half-time: A time interval in the dissolution process that corresponds to 
a concentration decrease by a factor of 2 for the nanomaterials and a corresponding increase of 
its ions or molecular forms [11]. 

Dispersion: Microscopic multi-phase system in which discontinuities of any state (solid, liquid 
or gas: discontinuous phase) are dispersed in a continuous phase of a different composition or 
state [11]. Dispersion may also refer to the “act of” dispersion. 

Heteroagglomeration:  Agglomeration  of  particles  (here  nanomaterials) with  other  particles, 
including  other  nanomaterials  and  non-nanomaterials  (synthetic  or  natural),  that  differ  in 
composition or size, for example Suspended Particulate Matter (SPM) [11], [8] and [13]. 

hetero: Heteroagglomeration attachment efficiency. 

Homoagglomeration: A form of agglomeration describing the agglomeration of the same type 
of particles, e.g. the nanoparticles with each other [13] and [14]. 

Impaired clearance: A continuously increasing prolongation of lung clearance of poorly soluble 
particles  or  fibres  when  the retained  lung  burden exceeds a  certain  threshold  (modified  from 
[12]). It can be caused by toxicity (impairment of alveolar macrophages function or cytotoxicity), 
or by overload of alveolar macrophages. 

Lung burden: The amount of test chemical that can be analytically measured in the lung at a 
given time point (modified from [12]). 

Lung overload: A phenomenon of impaired clearance in which the deposited dose of inhaled 
poorly soluble particles of low toxicity (PSLT) in the lung overwhelms clearance from the alveolar 
region leading to a reduction in the ability of the lung to remove particles. Lung particle overload 
results  in  an  accumulation of  particles  greater  than  that expected  under  normal  physiological 
clearance. This definition is relevant for all species (not just rat). This definition is independent 
of  the  underlying  mechanism(s)  (e.g.  macrophage  mobility  impairment).  A  key  issue  is  that 
increased particle retention due to large lung burdens needs to be differentiated from that due 
to high cytotoxicity particles (e.g. quartz) [15]. 

Nanoform: A form of a natural or manufactured substance containing particles, in an unbound 
state or as an aggregate or as an agglomerate and where, for 50% or more of  the particles in 
the number size  distribution,  one  or  more  external  dimensions  is in the size  range  1  nm-100 
nm, including also by derogation fullerenes, graphene flakes and single wall carbon nanotubes 
with one or more external dimensions below 1 nm [2]. 

Nanomaterial: A natural, incidental or manufactured material consisting of solid particles that 
are  present,  either  on  their  own  or  as  identifiable  constituent  particles  in  aggregates  or 
agglomerates, and where 50% or more of these particles in the number-based size distribution 
fulfil at least one of the following conditions: 
(a) one or more external dimensions of the particle are in the size range 1 nm to 100 nm;  

 
 
 
 
 
 
 
 
 
 
 
 
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(b)  the  particle  has  an  elongated  shape,  such  as  a  rod,  fibre  or  tube,  where  two  external 
dimensions are smaller than 1 nm and the other dimension is larger than 100 nm; 
(c) the particle has a plate-like shape, where one external dimension is smaller than 1 nm and 
the other dimensions are larger than 100 nm. 
In the determination of the particle number-based size distribution, particles with at least two 
orthogonal external dimensions larger than 100 µm need not be considered. A material with a 
specific surface area by volume of <6 m2/cm3 shall not be considered a nanomaterial [5]. 

Nanoparticle: A particle with one or more external dimensions in the size range of 1 nm to 100 
nm. This covers all particles with any external dimension on the nanoscale including “nanofibres” 
(two  external  dimensions  in  the  nanoscale)  and  “nanoplates”  (one  external  dimension  in  the 
nanoscale). 

NOM: Natural Organic Matter. 

Particle: A minute piece of matter with defined physical boundaries [2]. 

PEO: Post-exposure observation. 

Poorly  soluble  particle  (PSP):  Solid  aerosol  particles  deposited  in  the  lung  that  do  not 
undergo rapid dissolution and clearance [12]. The definition is restricted to lung and to aerosols. 
A  PSP  is  generally  understood  as  having  a  solubility  of  less  than  0.1  g  dissolved  in  100  ml 
dissolvent within 24 hours [12]. Examples of solvents are the simulated biofluids which include 
artificial lung lining fluid that contains salts and proteins or in an acidic environment that mimics 
the lysosomal fluid of macrophages. Specific criteria determining a PSP were recently elaborated 
[9]. 

Poorly  soluble  particles  of low  toxicity  (PSLT):  A  PSP which  does  “not cause  more  than 
minimal  and  transient  granulocytic  inflammation up  to a  lung  burden  causing  overload in  the 
rat” [12]. 

QSAR: Quantitative structure–activity relationship. 

Set  of  similar  nanoforms:  Under  the  REACH  Regulation,  it  is  a  group  of  nanoforms 
characterised in accordance with section 2.4 of REACH where the clearly defined boundaries in 
the parameters in the points 2.4.2 to 2.4.5 of the individual nanoforms within the set still allow 
to  conclude  that  the  hazard  assessment,  exposure  assessment  and  risk  assessment  of  these 
nanoforms can be performed jointly4 [2]. 

Simulated body fluid: A solution with an ion concentration close to that of a physiological fluid. 

Solubility: The proportion of a solute that dissolves in a given quantity of solvent, at a given 
temperature under equilibrium conditions, i.e. in a saturated state [16]. 

4  A justification shall  be  provided  to  demonstrate  that  a  variation  within  these  boundaries  does  not  affect  the  hazard 
assessment, exposure assessment and risk assessment of the similar nanoforms in the set. A nanoform can only belong 
to one set of similar nanoforms. 

 
 
 
 
 
 
 
 
 
 
 
 
 
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Version 4.0 – December 2022 

1.  RECOMMENDATIONS FOR PHYSICO-CHEMICAL 

PROPERTIES  

1.1 

General remarks 

This guidance provides advice on the testing of physico-chemical properties of the nanoforms of 
a substance as per REACH information requirements. This information is relevant to fulfill the 
information requirements set in REACH and are applicable to nanoforms. 

Many of these physico-chemical properties of the nanoforms will depend on the test conditions, 
i.e. they may present themselves distinctively variable depending on the specific surrounding, 
e.g.  a  nanoform  may  easily  dissolve  in  one  medium,  while  it  remains  a  particle  in  another 
medium. This is reflected, for instance, in the requirement that for nanoforms apart from water 
solubility, information on the dissolution rate in relevant biological and environmental media is 
required. 

The information requirements addressed in R.7a are therefore also highly relevant for the design 
and justification of the environmental and toxicological testing required under Annex VII to X of 
REACH, including characterisation of the test material and sample preparation to allow toxicity 
testing.  Further  guidance  is  provided  below  on  the  relevance  of  the  determination  of  these 
physico-chemical properties and methods for (eco)toxicological and fate testing. 

Moreover, Appendix R6-1 for nanoforms applicable to the Guidance on QSARs and Grouping of 
Chemicals [4] provides an approach on how to justify the use of hazard data between nanoforms 
(and  the  non-nanoform)  of  the  same  substance.  The  Guidance  is  describing  additional 
parameters that may be required for assessing whether the available hazard and fate data are 
valid for different nanoforms of a substance or not. The registrant will have to characterise the 
test material according to these parameters to be able to follow the above-mentioned guidance. 
For example, baseline information on solubility,  dissolution rate and dispersion stability of the 
nanoforms to be grouped is needed for the development of grouping hypothesis [4]. 

  Characterisation of test materials 

The fate and hazard posed by all possible forms of the substance, including nanoforms, covered 
by  a  registration,  must  be  addressed  with  the  provided  toxicological,  ecotoxicological  and 
environmental  fate  information  in  the  registration  dossier.  In  order  to  show  that  the  test 
material(s)  are  representative  for  the  assessed  nanoforms,  specific  information  has  to  be 
reported in the endpoint study record under the test material information field in IUCLID (see 
below). 

Recital 12 of the REACH amended Regulation (EU 2018/1881) [17] for nanoforms stipulates: “to 
allow  for  adequate  assessment  of  the  relevance  of  any  physicochemical,  toxicological  and 
ecotoxicological  information  for  the  different  nanoforms,  the  test  material  should  be 
appropriately characterised. For the same reasons, test conditions documented and a scientific 
justification for the relevance and adequacy of the utilised test material as well as documentation 
for the relevance and adequacy of the information obtained from means other than testing for 
the different nanoforms should be provided.” 
Consequently, the following parameters have to be provided in line with Annex VI Section 2.4 
requirements for the tested nanoform: 

•   Name(s) or other identifiers of the nanoform of the substance. 

•  Number  based  particle  size  distribution  with  indication  of  the  number  fraction  of 

constituent particles in the size range within 1 nm – 100 nm. 

 
 
 
 
 
 
 
 
 
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•  Description  of  surface  functionalisation  or  treatment  and  identification  of  each  agent 

including IUPAC name and CAS or EC number. 

•  Shape, aspect  ratio and  other  morphological characterisation:  crystallinity,  information 
on  assembly  structure  including,  e.g.  shell-like  structures  or  hollow  structures,  if 
appropriate. 

•  Surface area (specific surface area by volume, specific surface area by mass or both). 

Besides this information data from the test material including purity and, if technically feasible, 
quantities  of  identified  contaminants  and  impurities  must  be  analysed  prior  to  the  start  of  a 
study and provided in the registration dossier, in order to prevent errors in the interpretation of 
results due to impurities of the particles. 

  Sample preparation of test materials 

The following section focuses on the preparation of the sample for testing. Sample preparation 
is widely recognised as one of the most critical steps towards reliable testing of nanoforms and 
requires  monitoring  of  the  nanoforms  stability.  There  are  many  variables  to  consider  when 
establishing a method for sample preparation. Common issues to be considered include storage 
(see  for  instance  ACEnano  [18]),  colloidal  and  chemical  stability  of  the  tested  nanoform,  the 
media  composition,  characterisation  of  stock  dispersions  and  characterisation  of  samples 
(prepared from stock dispersions) prior to administration/testing [19]. 
When considering aqueous media, it can be difficult to distinguish between a dispersed and a 
dissolved nanoform due to its small particle size in aqueous media. It is important to recognise 
that solubility and dispersibility are two distinct phenomena. Solubility is the degree to which a 
material  (the solute)  can  be  dissolved  in  another  material  (the  solvent)  resulting  in a  single, 
homogeneous, stable phase and it is relevant for solids, liquids and gases. Dispersibility is the 
degree  to  which  a  particulate  material  can  be  uniformly  distributed  in  another  material  (the 
dispersing medium or continuous phase). Historically, the term “dissolved” was defined as the 
ipart  of  a  liquid  sample  that  had  passed  through  a  0.45  μm  filter.  However,  dispersions  of 
nanoparticles  will  pass  through  such  filters.  Therefore,  “dispersed”  is  the  term  to  use  when 
particles are present in the aqueous media, while the use of the term “dissolved” is restricted to 
solutions in the strict sense of the definition ( [19], [20]). In the context of sample preparation 
next to information on dissolution, knowledge about the dispersion of a nanoform and how stable 
it is under given conditions is important.  

To  avoid  misinterpretation  of  the  results  of  a  dissolution  study  by  not distinguishing  between 
dispersed and truly dissolved fractions of the nanoform, a thorough characterisation of all used 
stock dispersions and their stability, is required, i.e. the original dispersion, as received from a 
supplier or as prepared from a powder prior to testing, the nanoform stock dispersion prepared 
in the respective test medium and all potential intermediate nanoform dispersions required for 
a dilution series to reach the desired test concentration. In this context, an adequate analytical 
method should be applied for particle detection and the choice of method should be justified. 

Information on the stability of the dispersion of a nanoform is central for the interpretation of 
the characterisation results and the actual testing of nanoforms (“as received”, “as used”, “as 
dosed/as exposed”) and is a key factor during sample preparation. It is further noted that the 
stability  of  the  dispersion  and  the  level  of  agglomeration  may  be  different  for  the  different 
doses/concentrations  and  is  affected  by  dilution.  Thus,  detailed  information  on  the  dilution 
factors ha to be provided. 

The  stability  of  a  dispersion  is  determined  by  intermolecular  forces  involving  particle-particle 
interactions  as  well  as  those  between  the  particles  and  their  surrounding  matrix.  Due  to 
attractive forces (e.g. van der Waals interactions) particles tend to agglomerate unless stabilised 

 
 
 
 
 
 
 
 
 
 
 
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by  surface  charge  or  steric  effects.  In  liquid  media,  modifications  in  pH,  ionic  strength  and 
concentrations of molecular constituents can significantly alter the particle dispersion. 

The  stability  of  a  dispersion  is  also  typically  assessed  using  comparative  particle  size 
measurements and requires a reliable method of measuring the baseline particle size distribution 
of  the  material.  To  assess  the  initial  agglomeration  and/or  aggregation  state  a  number  of 
parameters  are  recommended  including  the  effective  cross  section  and  can  be  measured  via 
aerodynamic/light scattering or by electron microscopy ( [21], [22]). 
For  example,  by  comparing  changes  in  particle  size  distribution,  a  qualitative  assessment  or 
proxy  measure  of  the  state  of  dispersion  can  be  made.  Examples  of  methods  applicable  for 
spherical  particles  are  Zeta  potential  measurement,  combined  with  Dynamic  Light  Scattering 
(DLS)  or  Ultra-violet  visible  (UV-VIS)  spectroscopy  to  monitor  the  stability  of  nanoparticle 
dispersions in stock or test media and to gain a qualitative understanding of the agglomeration 
process. Other  methods  [23],  when suitable,  e.g.  particle  tracking analysis, can also  be  used 
[24]. For aquatic and sediment toxicity testing, the Guidance Document (GD) OECD GD 317 also 
includes analytical techniques to characterize the  as-produced or as-received test material, as 
well as the test material in stock and test dispersions. 

Several documents are available to assist registrants in the sample preparation process of test 
material  for  nanomaterial  testing.  Initially  the  following  reports  were  provided  as  guiding 
instructions: 

•  Sample Preparation and Dosimetry for the Safety Testing of Manufactured Nanomaterials 

OECD GD 36 [19] 

•  Ecotoxicology and Environmental  Fate  of  Manufactured  Nanomaterials: Test  Guidelines 

[25] 

However, most recently the advice provided in guidance document OECD GD 317 [26] has to be 
followed to the extent possible. 

To  sum  up  the  following  important  aspects  need  to  be  considered  for  sample  preparation  of 
nanoforms: 

•  Characterization  of  the  physicochemical  properties  of  nanoforms  (e.g.  particle  size 
distribution, shape, composition, specific surface area, surface chemistry and impurities) 
as 
of 
in 
agglomeration/sedimentation/dissolution where applicable). 

test  medium 

produced 

(degree 

present 

and 

the 

as 

Sample  preparation  needs  to  be  controlled,  consistent,  relevant,  reliable  and  robust.  The 
different  test  preparation  stages  may  include  e.g.  the  use  of  powder  and/or  dispersions 
depending on the respective endpoint and required exposure concentrations which carries the 
risk  that  the  test  item  undergoes  distinct  physico-chemical  changes  already  during  these 
different preparation stages. Monitoring of changes is necessary for an accurate description of 
the actual test item instead of the assumptive, pristine nanoform. 

•  The preparation of the nanoform dispersion (including stability) and composition of the 
test  medium  (such  as  pH,  organic  matter,  salts  etc.)  are  key  characteristics  and 
interactions between the nanoform and the test medium influence their physico-chemical 
properties  ultimately  determining  their  fate  and  behaviour  and  consequently  potential 
adverse (eco)toxicological effects. 

•  The applied sample preparation protocol detailing procedures for controlled, reliable, and 
robust processes need to be scientifically justified and reported in the study summary. 

ISO 14887:2000 [27] outlines procedures for the preparation of good dispersions from various 
powder/liquid  combinations  for  particle  size  analysis  of  substances  in  general.  Suggested 
dispersion procedures for a range of nanoforms are also emerging in the scientific literature e.g., 

 
 
 
 
 
 
 
 
 
 
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[28] [29]. 

In relation to sample preparation, it is necessary to be aware that aggregates and agglomerates 
of  nanomaterials  can  be  formed  in  the  dispersion  (powder  and  aerosol  forms)  and  that  their 
presence is influenced by  several factors including method of synthesis, storage and handling 
conditions.  For  aerosolised  powders,  the  situation  can  be  even  more  complex  as  the 
concentration and  diffusion  characteristics of  the aerosol  can  cause  the  state  of  dispersion  to 
change over time. 

The procedures should be carefully assessed to determine if they are adequate for the respective 
test  material.  Modifications  may  be  required  for  different  materials  in  the  context  of 
(eco)toxicological  testing.  For example, for  testing  inhalation  toxicity,  standards are  available 
that  outline  procedures 
the 
evaporation/condensation  method  (ISO  10801:2010  [29]  and  support the  characterisation  of 
nanoforms in inhalation exposure chambers [29]. 

the  generation  of  metal  nanoparticles  using 

for 

Another important component of sample preparation is the reliability of the sampling itself which 
means  that  the  test  aliquot  represents  the  physical  and chemical  characteristics  of  the  entire 
sample.  The  characterisation  of  particle  properties  like  size,  form  and  specific  surface  area 
requires  very  careful  sampling  and  sample  aliquoting,  as  well  as  considerate  storage  if  the 
sample is  not  processed  immediately  to  prevent  transformation  of  the nanoform  in  question. 
ISO  14887:2000  [27]  specifies  methods  for  obtaining  a  representative  test  aliquot  with  a 
specified  confidence  level  from  a  defined  sample  of  particulate  material  (powder,  paste, 
suspension or dust) for the measurement of particle size, size distribution and surface area. 

General considerations for Fate and (Eco)-Toxicological testing 

The  interactions  between  nanoforms  and  their  surroundings  need  to  be  accounted  for,  when 
testing fate and (eco-)toxicity, as this will influence the overall (eco)toxicity and fate processes. 
In particular, the interaction of nanomaterials with the environmental or biological media has to 
be considered in case dissolution occurs. The test design and more specifically the test conditions 
(e.g. composition of test media, agitation, light, exposure method, temperature) influence the 
dissolution of nanomaterials affecting then the test results [19] [30]. 
If a nanoform is well soluble in water (see Sections 1.2.1 and 2.1.1 for thresholds) and has a 
high  dissolution  rate  in  relevant  biological  (see  Section  2.1.1)  or  environmental  (see  Section 
1.2.1) media, then it is likely  that the nanoform is rapidly, increasingly and finally completely 
present in its molecular or ionic form. Therefore, it can be expected to behave similarly and elicit 
the same response as the non-nanoform of the substance. This could be the case for the metal 
ions  released from  the nanoform,  where e.g.  the  metal  salts  (“ionic  form”) can  be  used  as a 
positive  control, compared  to  the  nanoform.  If, however,  the  nanoform  under  investigation  is 
poorly  or  only  partially  soluble  with  a  low  dissolution  rate  in  biologically  or  environmentally 
relevant test media, then it will likely be present in the test system in a particulate form. In this 
case,  the  advice  provided  in  Appendices  for  nanoforms  applicable  to  Chapters  R.7a  (this 
document),  R.7b  and  R.7c  would  apply  including  the  considerations  for  partially  soluble 
nanoforms where both the dissolved and particulate forms need to be accounted for. 

In addition to Section 1.1.2. (Sample preparation of test materials) the following requirements 
for (eco)toxicological and fate testing must be fulfilled: 

fate 

•  Any  (eco)toxicological  and 

testing  must  be  accompanied  with  suitable 
characterisation  methods  to  monitor  the  exposure  situation,  such  as  exposure 
concentration  and  form  of  the  tested  nanoform  (dissolved  and/or  particulate  form). 
Concerning dose metrics for nanoforms using exclusively chemical analysis to determine 
mass based concentrations/metrics is not sufficient as further explained in the last bullet 
point, (for aquatic and sediment toxicity testing see OECD GD 317 [26], Section 4). 

 
 
 
 
 
 
 
 
 
 
 
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•  Monitoring  of  dissolution  and  dispersion  stability  must  be  conducted  at  least  at  the 
beginning and at the end of the test but depending on the results additional intermediate 
monitoring  may  be  required  if  a  high  dissolution  rate  or  reduced  stability  of  the  test 
dispersion is observed. 

•  Since the most appropriate dose metrics may not be known, the use of other dose metrics 
than mass-based, such as surface area and particle counts, are a favourable addition to 
the mass metrics. These measurements will allow for a conversion from mass to particle 
counts  and/or  to  surface  area  and  are  considered  essential.  While  diminishing  the 
uncertainty related to the conversion when the metrics are used independently it will also 
consequently reduce the amount of required testing. 

As fate and behaviour of a nanoform can differ from that of another nanoform and/or their non-
nanoform,  appropriate  analytical  methods  are  needed  for  fate  and  (eco-)  toxicity  testing  of 
nanomaterials. These also need to consider how the dissolution and the stability of the dispersion 
of  the  investigated  nanomaterial  influence  each  other.  With  respect  to  aquatic  and  sediment 
toxicity testing OECD flowcharts outlining strategies for preparing, controlling and maintaining 
test dispersions are given in OECD GD 317 [26], Section 5. OECD GD 318 [8] Sections 2 to 4 
are advising how solubility, dissolution rate and dispersion stability testing informs further fate 
and  ecotoxicological  testing. Characterising  the  dissolution  rate  and  dispersion stability in  the 
respective  test  medium  does  not  only  help  to  find  the  appropriate  testing  strategy  and  test 
conditions,  but  also  amplifies  the  correct interpretation  of  the results. General information  on 
dissolution and dispersion stability for nanoforms is beneficial when building grouping and read 
across hypothesis [31]. 

1.1.3.1  Considerations of impurities and contaminants for (eco)toxicological 

testing 

Complementary  to  the  considerations  listed  in  Section  1.1.2  (Sample  preparation)  and  1.1.3 
(Fate  and  (eco)toxicological  testing)  the  influence  of  contaminants  (including  biological 
contaminants) and impurities on (eco)toxicological test results need to be taken into account. 
For  example,  metallic  impurities  such  as  Cobalt  and  Nickel  catalysts  used  in  the  production 
process of certain nanomaterials were shown to inhibit hatching in zebrafish embryos (e.g. [32]). 
Likewise metal impurities of MWCNT can contribute to their inhalation toxicity in rodents (e.g. 
[33] and [34]). 

Also of particular concern is the adverse impact  on test results of endotoxin contamination in 
nanomaterial samples. Endotoxin (lipopolysaccharide) is a constituent of the outer cell wall of 
gram-negative bacteria and as such is found ubiquitously within the environment. Endotoxin can 
however generate a range of toxic effects either at the whole organism level causing responses 
such  as  fever,  “endotoxin  shock”  and  death,  or  at  the  cellular  level  via  the  triggering  of 
inflammatory cascades leading to the secretion of pro-inflammatory mediators. 

Due to this potent response an endotoxin contaminated test sample may lead to confounding 
results  (including  a  potential  false  positive)  in  biological  assays.  Therefore,  establishing  the 
presence  and  levels  of  endotoxin  in  a  test  sample  is  an  important  step  during  the  sample 
preparation  for  (eco)toxicological  testing.  Endotoxin can  be  measured  using  in vitro  methods 
such as the macrophage activation test (MAT), validated by European Committee on Validation 
of Alternative Test Methods [35] or the Recombinant Factor C (rFC) test  [36]. Both rFC assay 
and MAT assay have been adopted by the European Pharmacopoeia  [37], [38] and [39]. With 
ISO  29701:2010  an  international  standard,  the  limulus  amebocyte  lysate  (LAL)  test,  for 
analysing endotoxin contamination in nanomaterial samples is available [40]. Although issues 
regarding contamination are not nanospecific, the increased relative surface area of nanosized 
systems compared to other particles means that the possible amounts of adsorbed endotoxin 
(e.g.  grams  adsorbed  endotoxin  per  gram  of  material)  are  significant  and  deserve  therefore 
special attention [41]. 

 
 
 
 
 
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15 

1.2  Specific advice for endpoints 

  Water solubility 

Annex  VII,  Section  7.7  of REACH  requires  the  determination of solubility  and,  additionally for 
nanoforms, the determination of the dissolution rate in relevant media. 

Water solubility is covered in Section R.7.1.7 of the parent guidance.  There is no specific test 
guideline  to  test  water  solubility  of  nanomaterials.  However,  OECD  GD  318  [8]  describes 
applicability and limitations of OECD TG 105 [42] and OECD GD 29 [43] for testing solubility and 
dissolution of nanomaterials in simulated environmental media. 

For  water solubility,  both  OECD  TG  105  and  GD  29  adapted  for  nanomaterial  testing  [8] are 
applicable. 
The  flask  method  presented  in  OECD  TG  105  states  to  measure  solubility  after  a  24  hour 
equilibration period. Analysis is to be done after a separation step. However, the recommended 
method in OECD TG 105 is only considered to be adequate when using ultra-filtration. 
OECD  GD  29  [43]  allows  to  test  dissolution and  transformation  of  metals  with  test  durations 
varying  between  1  up  to  28  days  (common  duration  of  7  days).  The  OECD  GD  29  protocol 
provides advice on how to determine the transformation or dissolution and provides knowledge 
to which extent metals and poorly soluble metal compounds can produce soluble ionic forms and 
other metal bearing species in aqueous media, at different pHs. It should be noted that if the 
recommendations provided under OECD GD 318 are not considered, the method as described in 
OECD  GD  29  is  not  applicable  to  nanoform  testing  (e.g.  0.20  µm  filtration  is  not  adequate). 
Nevertheless, with appropriate adaptations (in particular for the filtration step) the method can 
be applied for nanoform testing. Ultra-filtration using a 3-10 kDa cut-off membrane is considered 
an applicable separation method for nanomaterials [16]. 
OECD GD 318 provides further guidance on the application of  OECD GD 29 and other TGs for 
measuring  water  solubility  and  dissolution  rate  of  nanomaterials  in  environmental  relevant 
conditions,  including  the  applicability  of  the  static  batch  mode  (screening  test,  adapted  from 
OECD GD 29) and the dynamic mode (based on ISO Technical Report (TR) 19057:2017 [44]). 

Overall, solubility measurements should be performed after 24 hours, or up to equilibrium, and 
must  consider  different  pHs,  as  per  OECD  GD  29  recommendations,  and  an  appropriate 
separation method i.e. ultrafiltration with 3-10 kDa cut-off membrane. 
Under these conditions, a nanoform is considered highly soluble i.e. no detectable particles are 
present, if its water solubility is above 100 mg/L (see Figure 1). The threshold of 100 mg/L for 
solubility in water is set based on the work done in support of OECD GD 29 [43] and considered 
as  a  conservative  approach  with  prioritising  environmental  safety.  The  duration  of  solubility 
studies  to  identify  highly  soluble  nanoforms  should  be  set  to  24  hours,  longer  tests  will  not 
improve  significance  and  value  of  ecotoxicological  test  design,  their  results  or  their 
interpretation. The analytical method should be sufficient to measure reliably the water solubility 
hence, the limit of quantification (LOQ) of the analytical method must be justified. 

For  nanoforms  REACH  regulation  (Section  7.7  of  REACH  Annex  VII)  requires  in  addition  to 
solubility also the determination of the dissolution rate in water and relevant media. Based on 
this  guidance,  this  must  be  considered  for  nanoforms  with  a  water  solubility  of  100  mg/L  or 
below. 

For  nanoforms,  it  is  necessary  to  consider  that  the  dissolution  rate  may  be  affected  by  the 
specific properties of the nano-sized materials. Properties such as particle size and shape, impact 
the materials’ surface area and the specific surface area and surface coating, will influence the 
nanoform-solute interactions. Therefore, the impact of such properties on the determination of 
the dissolution rate must be considered. Further information regarding the dissolution rate can 
be found in Section 1.2.1.1 below. 

 
 
 
 
 
 
 
 
 
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Providing information on dissolution is needed to determine the potential release to the aquatic 
environment and therefore the aquatic exposure.  
The  fate and  behaviour  of  nanoforms is affected  by  their  dissolution  rate  and  their  degree of 
dispersion. Distinction between a dispersed nanoform (where both solid and liquid fractions are 
present) and a dissolved nanoform (where no detectable solid fraction is present) is important 
as it has implications on the testing and characterisation strategies, as illustrated in Figure 1. 

This situation  is not  unique  to  nanoforms, and  indeed  the  parent  guidance  already  highlights 
that  “measurement  of  the  solubility  of  sparingly soluble  compounds  requires  extreme care  to 
generate  saturated  solutions  of  the  material  without  the  introduction  of  dispersed  material”. 
However,  this  problem  is  further  amplified  in  the  case  of  sparingly  soluble  nanoforms.  It  is 
important  to  ensure  that  no  undissolved  material  contributes  to  what  is  being  measured  as 
dissolved material, meaning that an appropriate separation method is used. 

For  environmental  endpoints  covering  fate  and  ecotoxicity  the  choice  of  nanospecific  testing 
and/or  following  the  parent  guidance  must  be  duly  justified  and  documented  based  on  the 
presence  of soluble  and/or solid  fraction.  Where  partial  dissolution  is  observed,  the impact  of 
each fraction must be considered (i.e. data on non-nanoforms of the substance may inform on 
the hazards of the solubilised fraction). 

 
 
 
 
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Consider NF characterisers provided under Annex VI of REACH (e.g. shape, size, surface  

coating) to decide on appropriate methodology and to set the test conditions  

  Determination of concentration  
dependent solubility in water  

Water solubility >100mg/L  

within 24 hours 

Yes 

No 

NF is considered to behave as a non - 
nanoform and no nano - specific testing  
is required (including dispersion stability  
test)  

Determination of dissolution rate in water and  
relevant media 

(Dynamic dissolution test as per OECD GD 318 ) 

Follow parent guidance for  
fate and ecotoxicological  
testing 

Nano - specific testing required  for solid  
fraction 

AND 

Impact of soluble fraction (i.e. based on  
parent guidance) must be addressed 

Figure  1:  Scheme  of  the  testing  required  to  fulfill  Annex  VII,  Section  7.7  information 
requirement of nanoforms of a substance and their impact on testing strategies with respect to 
environmental  fate  and  ecotoxicity.  Solubility  in  water  defines  whether  determination  of 
dissolution rate is required and subsequently nanospecific testing has to be performed for fate 
and ecotoxicity assessment of those nanoforms with a solubility limit ≤100 mg/L within 24 hours 
or whether the nanoform is considered to behave as a soluble substance and the parent guidance 
applies. 

With respect to solublity testing that can inform on human toxicity testing, information can be 
found under section 2.1.1. 

 
 
 
 
 
  
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1.2.1.1  Dissolution rate 

Annex  VII,  section  7.7  of  REACH  states  that:  “For  nanoforms,  in  addition  the  testing  of 
dissolution  rate  in  water  as  well  as  in  relevant  biological  and  environmental  media  shall  be 
considered.”. Thus, information on the dissolution rate has to be provided as part of the solubility 
endpoint  and  should  be  investigated  in  relevant  media,  such  as  the  ones  used  in  the 
(eco)toxicological tests. For nanoforms with a water solubility above 100 mg/L this information 
requirement is considered of low value. 

Dissolution rates in the different biological and environmental media used for (eco)toxicological 
and  fate  tests  are  variable  and  may  affect  the  bioavailability  of  substances.  Knowledge  on 
dissolution rates will also help to predict the toxicokinetic behaviour of particles.  

Currently,  no  test  guideline  is  available  for  determining  the  dissolution  rate  of  nanomaterials 
although  some  projects  are  working  on  developing  a  specific  TG  for  dissolution  rate  of 
nanomaterials in water and biological media (OECD Working Group of the National Coordinators 
for  the  Test  Guidelines  Programme(WNT)  1.5  project  on  “Determination  of  Solubility  and 
Dissolution  Rate  of  Nanomaterials  in  Water  and  Relevant  Synthetic  Biological  Media”)  and  in 
environmental media (OECD WNT projects 3.10 “Dissolution rate of nanomaterials in the aquatic 
environment”).  

Nevertheless, OECD GD 318 [8] provides guidance on how to determine dissolution, dispersion 
and agglomeration of certain nanoforms in environmental media, based on available TGs. Such 
methods  are  also  considered  relevant  to  determine  nanoform  dissolution  rates  in  water  as 
required under Section 7.7 of REACH. Where the nanoform is outside the applicability domain of 
the available TGs and GDs, the registrant is required to adapt such an information requirement 
with a robust technical and scientific justification for the inapplicability of the used test methods, 
and the lack of an adopted relevant guidance and/or test guideline. 

Parameters such as pH, ionic strength or suspended particulate matter influence the dissolution 
rate of nanoforms and should be carefully evaluated and reported. 
The following paragraphs are mainly focussed on environmental testing. Nevertheless, many of 
the statements may be relevant for water and biological media as well. 

To  test  dissolution,  a  static  batch  mode  (screening  test,  adapted  from  OECD  GD  29)  and  a 
dynamic flow-through mode (based on ISO TR 19057) for environmental relevant conditions are 
described in OECD GD 318. 

As  discussed  above  the  determination  of  the  dissolution  rates  depends  on  an  appropriate 
separation of particulates from the dissolved fraction and suitable time resolution. However, this 
can be a drawback for a screening test based on the static batch mode, especially for rapidly 
and  fully  dissolving  nanoforms.  Information  on  appropriate  separation  techniques  for 
nanomaterials can be found in ISO TR 19057. Based on OECD GD 318 the recommended method 
is centrifugal ultrafiltration as it promotes a suitably rapid separation allowing for calculation of 
dissolution rates. Still, care should be taken, when choosing filter cut-off to ensure no passage 
of smaller solid fractions below the filter cut-off value. Also, build-up of ions should be considered 
when  small  filter  cut-off  values  are  chosen  and similarly  caution should  be  taken when using 
NOM as it can block filters with smaller cut-off sizes. 

For slowly dissolving nanoforms, the applicability of the batch method is mostly dependent on 
the  analytical  power  as  the  solubility  limit  may  not  be  within  the  resolution  of  the  analytical 
method, i.e. staying under the limit of quantification or even limit of detection. 

The dynamic method is based on a flow-through system and comprises the use of a test medium 
delivered  at  constant  flow  rate  through  a  compartment  which  entraps  the  nanoparticles,  i.e. 

 
 
 
 
 
 
 
 
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using ultrafiltration membranes. Sensitivity can be partially compensated by adapting the low 
flow rate and nanoform concentration. 

For  both  methods,  static  batch  mode  and  dynamic  flow  through  mode,  their  applicability  for 
testing non-metallic nanoforms depends on the accuracy of the analytical method. In general, 
most  of  the  nanoforms  tested  are  inorganic  materials  (mainly  metal  and  metal  oxide  forms). 
Other nanoforms such as organic or organometallic and carbon-based nanoforms have been less 
tested and analytical methods may lack precision for quantification. The development of various 
analytical  methods  is  ongoing  (including  methods  for  organic  nanoforms)  to  increase  the 
accuracy and sensitivity of the measurements and their applicability for measuring dissolution 
rates  of  nanoforms  in  water.  Further  specific  considerations  to  the  adaptation  of  analytical 
methods to nanoform testing (e.g. quantification of solid or dissolved fractions) is not yet fully 
developed. 

OECD GD 318 also provides a formula to calculate the dissolution rate. 

Dissolution rate can  be  determined  from  a  graph showing  solubility (as  ionic  concentration in 
mg/L) as a function of time. Most nanomaterials follow (pseudo-) first order kinetics and can be 
determined as the loss of solid material over time. The parameters impacting the results are the 
initial  mass  of  nanomaterial  in  the  test,  its  specific  surface  area  and  solubility,  and  the  test 
conditions  (e.g.  shaken,  stirred,  not  agitated).  For  comparison  purpose,  the  dissolution  rate 
should be normalised by surface area and can be calculated based on Noyes-Whitney equation: 

where D is the diffusion coefficient of the dissolved species in the medium, A is the surface area 
of the nanomaterial, h is the thickness of the diffusion layer, c s is the saturation concentration 
(solubility limit), and c is the concentration of dissolved ions in the test medium. 

OECD  GD  318  also  provides  advice  on  how  both  static  batch  and  dynamic  methods  can  be 
adapted  to  measure  dissolution  rate in  natural  or  artificial  environmental  media.  According  to 
OECD GD 318, the applicability of the dynamic method based on ISO TR 19057 for toxicological 
test media, i.e. in lung and gastro fluids, has already been demonstrated in  [45] and [46]. In 
addition,  the  OECD  WNT  1.5.  project  will  provide  a specific  guidance  document  to  determine 
dissolution in biological media and water [47]. While this is ongoing work, further toxicological 
considerations and  advice  on information  regarding solubility and  dissolution  of nanoforms  in 
biological media are described under Section 2.1.1 of this guidance and are to be followed. 

Alternatively,  OECD  TG  105  is  also  considered  to  be  potentially  adaptable  to  determine 
dissolution rates. The use of the column elution method with continuous measurements of the 
dissolved  fraction  would  allow  the  determination  of  a  nanoform  dissolution  rate.  To  do  so, 
nanoform adsorption to the substrate must be warranted. 

With respect to dissolution rate testing that can inform on human toxicity testing, information 
can be found under section 2.1.1.1. 

1.2.1.2  Waiving of water solubility 

Annex VII, Section 7.7 of REACH states in column 2 : “The study does not need to be conducted if — 
the  substance  is  hydrolytically  unstable  at  pH  4,  7  and  9  (half-life  less  than  12  hours),  or  —  the 
substance is readily oxidisable in water. If the substance appears “insoluble” in water, a limit test up 
to  the  detection  limit  of  the  analytical  method  shall  be  performed.  For  nanoforms  the  potential 
confounding effect of dispersion shall be assessed when conducting the study.” 

 
 
 
 
 
 
 
 
 
 
 
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In the parent guidance Section R.7.1.7.1, it is noted that water insolubility is used as a regulatory 
trigger for waiving certain physicochemical and ecotoxicological endpoints. 

Considering the nanospecific properties and constraints in assessing the solubility of nanoforms, 
waiving the information requirement based on their apparent insolubility as quoted in Annex VII, 
Section  7.7,  column  2:  “must  always  be  accompanied  with  robust  technical  and  scientific 
justification comprising information on dissolution and dispersion stability of the nanoform(s)” 
(see Section 1.2.1.1 & 1.2.2.2). 

Partition coefficient n-octanol/water 

1.2.2.1  Applicability of partition coefficient n-octanol/water 

The  n-octanol/water  partition  coefficient  (Kow)  is  defined  as  the  ratio  of  the  equilibrium 
concentrations  of  a  dissolved  substance  in  a  two-phase  system  consisting  of  the  largely 
immiscible  solvents  n-octanol  and  water.  In  a  two-phase  system,  nanoparticles  behave 
differently  from  organic  molecules.  Particles  do  not  form  solutions  but  colloidal  dispersions, 
which  are  multiphase  systems  and  thermodynamically  unstable.  Therefore,  the  fate  of 
undissolved  nanomaterials  cannot  be  predicted  by  equilibrium  partitioning  [48],  [49]  as 
nanomaterials  cannot  reach  thermodynamic  equilibrium  by  distributing  between  two  phases, 
water and n-octanol, due to their particulate nature. 

OECD  TGs  recommended  in  the  Section R.7.1.8.3  of  the  parent  ECHA  Guidance  for  partition 
coefficient n-octanol/water, i.e. OECD TG 107 [50], OECD TG 117 [51] and OECD TG 123 [52], 
are in most cases not applicable to nanoforms [21], [20], [53]. Results will be impacted by the 
presence  of  a  colloidal  suspension,  which  could  be  present  if  the  manufactured  nanomaterial 
does not completely dissolve [19], [20], [31]. 

If  it  is  shown  that  the  nanoform  has  a  water  solubility  above  100  mg/L,  as  explained  under 
Section 1.2.1, the impact of particles may be neglected, and the parent guidance applies. 

If on the other hand, it is shown that the nanoform  has a water solubility equal or below 100 
mg/L, dispersion stability is an information requirement under Annex VII, Section 7.8 of REACH. 
Hence,  nanospecific  testing  is  needed  for  the  fate  and  ecotoxicity  assessment  of  the  solid 
fraction. Information on Kow may still be of value for the dissolved fraction of organic nanoforms 
and nanoforms with an organic coating. This is illustrated in Figure 2 and further detailed under 
section 1.2.2.2 and 1.2.2.3. 

 
 
 
 
 
 
 
 
 
 
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Figure  2:  Scheme  of  the  testing  required  to  fulfill  Annex  VII,  Section  7.8  information 
requirement of nanoforms of a substance and their impact on testing strategy. 

Where the water solubility is below or equal to 100 mg/L determining the dispersion stability is 
required for assessing the solid fraction, along with the Kow for assessing the dissolved fraction. 

1.2.2.2  Dispersion stability 

According to Annex VII, section 7.8, determination of dispersion stability in water is required for 
nanoforms. 

Furthermore, information on dispersion stability in relevant environmental media is  needed to 
help predict fate and transport of the nanoforms in the environment, as illustrated in Figure 2. 
However, information on  dispersion stability  can  not  be seen  as  a one-to-one  replacement  of 
KOW and additional properties need to be considered as well. 
Dissolution  (rate),  agglomeration,  aggregation,  deposition  and  attachment  are  considered 
informative  properties  to  predict  the  environmental  behaviour  of  the  nanomaterials  in  the 
environment and organisms [48], [54], [55], [56].  
In  this  line,  OECD  GD  318  [8]  includes  recent  developments  on  dispersion  stability 
measurements,  considering  both  homoagglomeration,  as  per  OECD  TG  318  [57],  and 
heteroagglomeration. These agglomeration parameters can be provided in addition to dispersion 
stability in order to justify behaviour and fate of the nanoforms and consequently some choice 
in the testing strategy. 

Dispersion  stability  is  a  relevant  fate  descriptor  to  also  inform  on  further  testing  strategies 
related to aquatic fate and hazard testing while adsorption/desorption informs on test strategy 
on soil and sediment. Providing information on dispersion stability (as per OECD TG/GD 318) is 
needed to determine the potential exposure to the aquatic environment. Adsorption/desorption 
(mobility  potential  in  soil)  as  per  OECD  GD  342  [58]/TG  312  [59]  will  complement  the 
information on the environmental behaviour of nanomaterials. 

Dispersion stability test, as per OECD TG 318 and OECD GD 318 recommendations  (initial concentration in particle number basis = 1012particles/L)YesWater solubility >100mg/LNoKowFor solid fraction, dispersion stability needed to inform further fate and ecotox testing on nanoforms (see ECHA Guidance R.7.b and R.7.c)Impact of soluble fraction on fate and ecotoxicity  (i.e. based on parent guidance and considering Kow) must be addressed 
 
 
 
 
 
 
 
 
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Dispersion stability based on homoagglomeration 

Agglomeration is the process by which two particles interact. In case the two particles are of the 
same kind, this process is called homoagglomeration. The OECD TG 318 [57] defines dispersion 
stability, accounting for agglomeration and sedimentation. As such, it can be used to  design a 
testing  strategy  to  determine  homoagglomeration;  i.e.  particle-particle  attachment  of 
nanomaterials in ecotoxicological test media. 

OECD TG 318 proposes (screening) testing for dispersion stability based on homoagglomeration 
(nanoparticle-nanoparticle interaction) under environmental conditions by a screening test and 
an extended test. For the screening test, the media is stabilised with NOM and, for comparison 
purposes,  the  tests  are  performed on  particle number  concentration  basis.  The  test  guideline 
provides  information  on  the  quantity  or  relative  percentage  of  the nanomaterial  that  remains 
dispersed in the aquatic medium tested in a given time frame.  

Testing in other environmental conditions is described in OECD GD 318 [8] where feasibility of 
testing in other test media, such as natural waters, is also explained. 

The dispersion stability test follows a 2-tiered approach based on the particles remaining in the 
water phase after homoagglomeration and sedimentation during a given time frame (described 
in OECD TG 318 and under Section 3 of OECD GD 318): 

•  Tier 1 (screening test): measurements undertaken at two time points (0 and 6h, which 
represents test start and end) at relevant pH conditions, with natural organic matter (10 
mg/L dissolved organic matter) and a range of electrolyte concentrations (0, 1 and 10 
mM  Ca(NO3)2).  An  additional  measurement  is  done  at  the  end  of  the  test,  after 
centrifugation  of  the  sample.  The  centrifugation  parameter  is  calculated  in  order  to 
achieve a particle cut off value of 1 µm. 

•  Tier 2 (extended test): in addition to the condition range described in the screening test 
i.e. pH and electrolyte range, presence and absence of natural organic matter is tested. 
When  no  organic  matter  is  added,  sodium  bicarbonate  (5  mM)  has  to  be  added  as  a 
buffering agent. The stability of the dispersion is measured hourly with sub-sampling over 
the test duration of 6 hours). 

Based  on  the  results  of  the  screening  test,  i.e.  the  percentage  of  nanoforms  remaining  in 
dispersion under all test conditions, nanoforms can be qualified as of: 

•  High stability - if ≥90% of the initial test concentration remains in dispersion for all test 

conditions or 

•  Low stability – if ≤10% of the initial test concentration remains in dispersion for all test 

conditions. 

Where the measured concentration is within 10 and 90% of the initial test concentration,  i.e. 
intermediate  stability,  at  any  of  the  test  conditions,  the  extended  test  (Tier  2)  needs  to  be 
performed.  The  extended  test  allows  a  differentiation  of  settling  behaviour  (which  is  due  to 
certain  nanomaterial  properties,  e.g.  density),  over  time.  As shown in Figure  3,  the  red  line 
represents a nanoparticle agglomerating but not settling due to low density whereas the green 
line  may represent either  high  density  nanoparticles  that  agglomerate and  settle  quickly  or  a 
mixed sample composed of an unstable, high density, fraction and a stable one. 

 
 
 
 
 
 
 
 
 
 
 
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Figure 3: Schematic representation of exemplified dispersion stability results from an extended 
test  (extracted  from  OECD  TG  318).  The  hourly  measurements  revealing  the  sedimentation 
behaviour of the nanomaterial. 

Overall, the applicability of OECD TG 318 for nanoforms should be considered based on: 

•  Material density (i.e. >1g/cm3). 

•  Sensitivity of the analytical method allowing quantification of the nanoforms from 100 to 
10% of the particles present in the water column, i.e. LOQ lower or equal to 10% of initial 
mass concentration. 

When  applying  the  test  with  natural  waters,  careful  removal  of  natural  particles/colloids  and 
microorganisms via filtration over a membrane of pore size ≤ 0.1 µm or via ultracentrifugation 
should be pursued in order not to compromise the test. Complex samples such as natural waters 
are  considered  to  have  a  very  specific  hydro-chemical  composition  and  if  no  pre-filtration  or 
centrifugation  is  performed  the  results  of  the  test  will  be  rather  representative  of  hetero-
agglomeration (see heteroagglomeration considerations below). 
To  ensure  data  comparability  media  constituents  interfering  with  agglomeration  have  to  be 
reported (e.g. divalent cations and anions, pH and type of organic matter). The elements to be 
characterised are further discriminated under Section 3.0 and 3.1 of OECD GD 318. 

OECD GD 318 provides further consideration when testing nanoform dispersion stability namely: 

•  Although  NOM-nanoparticle  interactions  vary  with  particle  properties  (e.g.  surface 
charge),  NOM  is  known  to  form  nanoparticle  “coronas”,  i.e.  surface  coverage,  which 
highly influences the dispersion behaviour of the test material. 

•  Use  of  stabiliser  aids  dispersing  extremely  hydrophobic  materials  will  impair  the 

evaluation of the stability of the pristine material. 

•  A  6-hour  long  test  performed  with  a  fixed  particle  number  concentration  (e.g.  1012 
particles/L, as per OECD TG 318) is recommended, to generate comparable results. It is 
advised  to  take  size  distribution  (instead  of  simple  average  diameter) into  account, in 
addition  to  density  for  the  conversion  of  nanomaterial  mass  concentration  to  particle 
number concentration. Nevertheless, average  diameter should  still  produce acceptable 
data, i.e. within the methods sensitivity range (e.g. one order magnitude difference). 

•  The sedimentation velocity of particles depends on several factors such as the relative 
density  of  the  starting  material  and  its  agglomerates,  the  3D  structure  of  the 
agglomerate,  its  surface  chemistry  and  therefore  interaction  with  the  surrounding 
medium. To reduce the impact of density on determining agglomeration, it is required to 
proceed with sample centrifugation, as described in OECD TG/GD 318. 

 
 
 
 
 
 
 
 
 
 
 
 
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Regarding quantification of dispersion stability, it is noted that the applicable analytical methods 
for  nanoforms  are  limited  only  by  their  sensitivity.  Inductively  coupled  plasma  mass 
spectrometry (ICP-MS) and optical emission spectroscopy (ICP-OES) are recommended in the 
test  guideline  however limitations  (e.g.  with regards  to  the  presence  of  dissolved  material  or 
non-detectable elements) are also noted there. 

Furthermore,  the  OECD  GD  318  provides  further  considerations  on  alternative  methods 
(including  qualitative  and  (semi)quantitative  ones).  UV/VIS  photometry,  for  instance,  can  be 
used  as  a  quantitative  method.  However,  careful  considerations  of  its  applicability  based  on 
material  properties  is  needed.  This  method  provides indication of  the maintenance  of  particle 
size and number in suspension therefore, nanoform stability can to some extent be monitored. 
Additionally,  non-quantitative  measurements  such  as  electrophoretic  mobility,  i.e.  Zeta 
potential,  can  also  be  used  as  an  indicator  of  particle  (in)stability.  Further  information  on 
methods applicability can be found in “NanoDefine Technical Report D7.6” [23]. 

Dispersion stability based on heteroagglomeration 

Heteroagglomeration, i.e. the interaction between two particles of different nature, is considered 
the most relevant type of agglomeration process to be investigated for fate evaluation, as it is 
performed under test conditions closer to natural settings. 
A test guideline to determine attachment efficiency of heteroagglomeration is not yet available 
but OECD GD 318 provides guidance on how a heteroagglomeration test could be performed. 
The  heteroagglomeration  kinetics  depend  on  number  ratios  of  nanoparticles  and  suspended 
particulate matter (SPM) in the system, as well as the collision rate constant, which depends on 
particle  size,  density  and  velocity  gradient.  Agglomeration  rate,  sedimentation  or attachment 
efficiency provide information on the heteroagglomeration behaviour of nanomaterials. Actually, 
heteroagglomeration  attachment  efficiency  has  been  shown  to  be  the  most  suitable 
measurement  aiding  in  fate  evaluation,  as  it  measures  the  fraction  of  collisions  resulting  in 
attachment.  Therefore  it  provides  a  better  proxy  to  set  the  study  design  for  fate  (e.g. 
bioaccumulation) and ecotoxicological test conditions. 

1.2.2.3 

 Waiving of partition coefficient n-octanol/water for nanoforms 

Annex VII, Section 7.8 of REACH states in column 2 that: “For nanoforms, whether of inorganic 
or organic substances, for which the partition coefficient n-octanol/water is not applicable the 
study of dispersion stability shall be considered instead”. 

There are currently constraints in assessing the n-octanol/water partition coefficient (Kow) of the 
nanoforms,  as  explained  in  Section  1.2.2.1.  Therefore,  waiving  n-octanol/water  partition 
coefficient  information  requirement  should  always  be  accompanied  by  a  robust  technical  and 
scientific justification on the applicability of the used test method (e.g. showing absence of water 
solubility or a low dissolution rate of the nanomaterial). 
In case  of  partial  solubility  and  dissolution  rate  of  the nanoforms,  Kow can  be considered  and 
measured for the soluble fraction, at the same time than dispersion stability  for the particulate 
fraction (see Figure 2). 

As explained in the parent guidance (Section R7.1.8.1.) the n-octanol/water partition coefficient 
(KOW)  is  used  in  numerous  estimation  models  and  algorithms  for  environmental  partitioning, 
sorption,  bioavailability,  bioaccumulation  and also  human toxicity and ecotoxicity.  To  prevent 
erroneous interpretation of such models, where the nanoforms is not fully dissolved information 
on the dispersion stability may provide useful information instead of Kow (see Section 1.2.2.2 for 
information on dispersion stability). Nevertheless, information on dispersion stability should not 
be  seen  as  a one-to-one  replacement  of  KOW.  Furthermore,  the use  of Kow  is  not  sufficient to 
waive  the  generation  of  further  data  on  nanoform  fate  (e.g.  column  2  adaptation  of 
bioaccumulation information requirement) on its own (see ECHA Guidance R7c). 

 
 
 
 
 
 
 
 
 
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Currently there are no standardised methods for fate descriptors to predict the behaviour and 
transport  of  nanoforms  in  the  environment  and  biological  media  as  alternatives  to  n-
octanol/water partition coefficient [48], [49]. Environmental fate descriptors for nanoforms are 
further  discussed  in  Section  1.2.5  on  adsorption/desorption,  where  state-of-the-art  on 
attachment efficiency of nanomaterials is reported (e.g. OECD GD 342 [58]). Also, there is a list 
of the models and specific parameters under development, suggested as alternative methods to 
Kow and Koc as alternative fate descriptors, but sufficient validation is still pending (available in 
Appendix 1). 

Granulometry 

The  data  requirement  in  accordance  with  REACH  Annex  VI  section  2.4.2  for  “number  based 
particle size distribution with indication of the number fraction of constituent particles in the size 
range within 1 nm – 100 nm” applies for each registrant of a joint submission whereas the data 
requirement in accordance with REACH Annex VII section 7.14 for “granulometry” applies jointly 
for the members of the joint submission. As REACH Annex VI requires that “information shall be 
reported in such a manner that it is clear which information in the joint submission pertains to 
which nanoform of the substance”, in practice the registrant submitting the Annex VII-X dataset 
corresponding to a nanoform or a set of nanoforms submits the granulometry data. 

The size distribution of constituent particles as well as aggregates and agglomerates may have 
an  impact  on  the  selection  of  the  most  appropriate  route  of  exposure,  on  the  intake  of  the 
particles within cells or organisms and on sample preparation. Therefore, the granulometry data 
should  provide  any  additional  information  on  the  particle  size  distribution  of  the  nanoform 
necessary  to  carry out  the hazard  assessment  on  the  actual  test  material  (“as  received”,  “as 
used”,  “as  dosed/as  exposed”).  Thus,  it  is  recommended  to  provide  as  a  minimum  the 
granulometry  information  of  (all)  the  test  material(s)  used  in  tests  to  fulfil  Annex  VII-X 
information requirements. 

The  different  characterisation  parameters  of  nanoforms, such  as  the  constituent  particle  size, 
the  shape  of  the  particles  and  the  surface  treatment,  may  have  a  significant  impact  on  the 
granulometry  of  those  nanoforms.  Therefore,  when  the  granulometry  data  generated  on  one 
nanoform  is  used  to  fulfil  the  data  requirement  for  another  nanoform,  the  differences  in  the 
characterisation  parameters  must  be  addressed  in  the  read-across  justification.  The  same 
applies equally to read-across between a nanoform and a non-nanoform. 

As with non-nanoforms, information on granulometry is relevant for the assessment of exposure 
to airborne particles/dusts, as well as for the performance of toxicity studies via the inhalation 
route. For the purpose of performing inhalation toxicity studies, the most relevant parameter to 
measure and to report is the aerodynamic diameter of the particles. The aerodynamic diameter 
is defined as diameter of a spherical particle with a density of  1000 kg/m3 that has the same 
settling velocity as the particle in question. The mass median aerodynamic diameter (MMAD) is 
generally reported, and is the aerodynamic diameter where 50% of the particles by mass are 
below that size, and 50% of the particles are above that diameter. Note that this guidance is not 
intended  to  describe  how  to  generate  or  characterise  exposure  conditions  for  the  purpose  of 
inhalation  toxicity  studies  for  nanomaterials.  Some  guidance  on  this  subject  can  be  found  in 
ISO/TR  (Technical  Report)  19601:2017  [60]:  The  generation  of  aerosols  for  the  purpose  of 
inhalation toxicity studies. 

For  reaching  a  conclusion  on  granulometry,  it  has  to  be  taken into  account  that  the  potential 
release of particles into the workplace or environment is an important consideration in the design 
and operation of many industrial processes and safe handling of substances. Release of particles 
may  present  hazard  and  could  cause  adverse  health  effects  to  humans  and  affect  the 
environment. It is therefore important to obtain data about the propensity of substances to be 
released as particles, allowing risks to be evaluated, controlled and minimised. Measurement of 

 
 
 
 
 
 
 
 
 
 
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the  release  of  particles  from  powdered  substances  has  similarities  to  the  conventional 
measurement of the dustiness of a powder, but with significant differences in the methods and 
instrumentations suited to different particle size ranges. 
A number of methods are available for determining the particle size fractions (Section 1.2.3.1) 
which are then used to assess the possible health effects resulting from inhalation of airborne 
particles in the workplace. A number of methods covering different ranges of particle sizes are 
available though none of them is applicable to the entire size range. Multiple techniques should 
be used where possible in order to formulate a complete understanding of the particle properties, 
and the optimum set of required techniques should be selected based on the specific substance 
and  form  under  investigation.  Furthermore,  also  the  other  characterisation  parameters  of  the 
particles such as shape may impact the applicability of a method for the particle size distribution 
measurement. 

1.2.3.1  Test methods for granulometry 

The  characterisation  of  particles  requires  very  careful  sampling  and  sample  fractionation 
practises  to  be  followed.  ISO  14488:2007  [61]  specifies  methods  for obtaining  a  test  aliquot 
from a defined sample of particulate material (powder, paste, suspension or dust) that can be 
considered to be representative with a defined confidence level. Further information is available 
in Section 1.1.2 of this appendix on Sample Preparation. 

The  methods to  measure  the  particle  size  distribution  of  the  constituent  particles,  aggregates 
and  agglomerates  and/or  mass  median  aerodynamic  diameter  must  be  such  that  they  are 
applicable for nanoforms. The methods specified in the OECD TG 125 for particle size and particle 
size distribution of nanomaterials [62] and the method listed in Table 1 can be used to measure 
the  particle  size  and  size  distribution  of  nanoforms  to  fulfil  the  Granulometry  endpoint.  Also 
Table 2 in Section 1.2.4 can be consulted for some methods to measure particles and fibres. 

 
 
 
 
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Material and size range 

Data type 

Particles in an aerosol 

Size range: ~1 nm  – 1 µm 

Size distribution based 
on number counted 
(number count per size 
interval). From the 
distribution, MMAD can 
be calculated, with 
knowledge of the 
density of the particles. 

Table 1: Methods of measuring airborne dispersed or nebulised particles 

Method and details 
Scanning Mobility Particle Sizer (SMPS) (ISO 15900:2020 [63]; ISO 10808:2010 [29]; ISO 
28439:2011 [64]) 

SMPS operates by charging particles and fractionating them based on their mobility when passing 
between electrodes. This method combines a Differential Mobility Analyser (DMA) and an Optical 
Particle Counter (OPC). SMPS detects and counts particles, and enables measurement of the particle 
size distribution and count median diameter of nano aerosols, up to 108 particles/cm³. This method 
also allows evaluation of particle surface area, mass dose, composition and dispersion to support 
effective analysis of inhalation toxicity testing results. SMPS also has useful application in relation to 
exposure estimation. 

Measurement with SMPS is the only currently available method that meets all of the following 
requirements in the size range below 100 nm: i) measurement of particle size distribution during 
particle exposures in a continuous manner with time resolution appropriate to check stability of 
particle size distribution and concentration; ii) measurement range of particle sizes and 
concentrations covers those of the particle aerosols exposed to the test system during the toxicity 
test; iii) particle size and concentration measurements are sufficiently accurate for particle toxicity 
testing and can be validated by ways such as calibration against appropriate reference standards; iv) 
resolution of particle sizing is sufficiently accurate to allow conversion from number-weighted 
distribution to surface area-weighted or volume-weighted distribution. 

However, SMPS is relatively slow and requires a scanning approach to measure different size 
intervals in series. This method is restricted to ambient temperatures below 35 °C (due to 
evaporation of butanol in the CPC) and requires aerosolisation of the sample. SMPS cannot 
distinguish between agglomerates and primary particles. For non-spherical particles (e.g. high 

 
 
 
 
 
 
 
 
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Method and details 
aspect ratio nanomaterials), estimation of diameter and mass concentration by SMPS can result in 
significant error. Assembling data of measurements from SPMS and OPC to provide a whole picture 
of particle size distribution is not appropriate, due to the different principles employed by the two 
methods [65]. It is important to know the stability of the source, since rapid changes of the size 
distribution, particle concentration, or both, can affect measurement of the size distribution.  This is 
relevant to consider for nanomaterials, which have a high tendency to agglomerate in the 
atmosphere 

Material and size range 

Data type 

Fast Mobility Particle Sizer (FMPS) 

Particles in an aerosol Size 
range: ~5 - 560 nm   

FMPS enables determination of the size distribution of sub-micrometre aerosol particles, up to 107 
particles/cm³ (depending on particle size). Measurements can be made with a time resolution of 
one second or less, enabling visualization of particle size distributions in real time. However, FMPS is 
typically less sensitive than the SMPS at low particle concentrations. 

Diffusion batteries 

The operation of diffusion batteries is based on the Brownian motion of the aerosol particles. 
Depositional losses through diffusion are a function of particle diameter. By measuring diffusion 
based deposition rates through systems with varying geometries, it is possible to determine particle 
size distribution. The deposition systems are usually placed together in series to form a diffusion 
battery. The diffusion battery can be designed for determination of particle sizes as low as 2 nm 
depending upon instrument setup. This method has useful application in relation to exposure 

Particles in an aerosol 

Size range: 0.005 – 0.1 μm 

Size distribution based 
on number counted 
(number count per size 
interval). From the 
distribution, MMAD can 
be calculated, with 
knowledge of the 
density of the particles 

Particle number in 
intervals according to 
diffusion diameter, 
from which the median 
diffusion diameter can 
be determined with 
knowledge of the 
density of the particles. 

 
 
 
 
 
 
 
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Method and details 
estimation. 

Material and size range 

Data type 

The primary property measured is the diffusion coefficient of the particles and this has to be 
converted to particle diameter. The instrument needs to be operated with a particle counter 
(typically a continuous flow Condensation Particle Counter) in order to determine the number 
concentration before and after each diffusion stage. Inversion of the raw data to real size 
distribution is complex and the solutions of the equations do not give unambiguous results in the 
case of polydisperse aerosol size distributions. 

ISO/TR 27628:2007 [66] provides an informative description of this method. 

Optical Particle Counter (OPC) 

OPC is a widely used method for detecting and counting aerosolised particles, and operates across a 
wide temperature range (0 – 120 °C). Enables agglomerates/aggregates of primary particles to be 
measured and counted. OPC has useful application in relation to exposure estimation. 

However, OPC is insensitive to particles smaller than approximately 100-300 nm in diameter and 
provides insufficient coverage of potential primary particle. Assembling data of measurements from 
SPMS and OPC to provide a whole picture of particle size distribution is not appropriate, due to the 
different principles employed by the two methods [65]. 
ISO/TR 27628:2007 [66] provides an informative description of this method. 

Particles in an aerosol 

Size range: 0.3 – 17 μm 

Particle number 
concentration 

Laser scattering/diffraction 

In general, the scattering of the incident light gives distinct pattern which are measured by a 
detector. This technique is particle property dependent – i.e. material has unique scattering and 

Particles of all kind 
Size range: 0.06 - 100 μm 

Particle size/size 
distribution, from 
which mass median 
diameter can be 

 
 
 
 
 
 
 
 
 
 
 
 
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diffraction properties which are also particle size dependent. It is important to calibrate the 
instrument with similar material (of the same size range as the material to be measured). Laser 
scattering techniques are suitable for geometric particles, viz spheres, cubes and monocrystals. 
Particle size will be established optically. The MMAD can be calculated by means of a calculation 
correction. 
The method is suitable to determine the distribution of particles of respirable and inhalable size. 
Laser diffraction assumes a spherical particle shape. Test products should therefore have no 
extreme aspect ratios, with a restriction of 3:1 for non-spherical particles. This method has limited 
applicability really suitable in the sub-100 nm range. In the range below several microns, results 
strongly depend on optical constants of particles. 

Material and size range 

Data type 
calculated, with 
knowledge of the 
density of the particles. 

Light scattering aerosol spectrometer (LSAS) 

Particles in an aerosol 
Size range: 0.06 - 45 μm 

LSAS is a type of light scattering instrument, applicable for measuring the size, number 
concentration and number/size distribution of particles suspended in a gas. LSAS can be used for 
the determination of the particle size distribution and particle number concentration at relatively 
high concentrations of up to 1011 particles/m3. The large measurement range of LSAS may result in 
high uncertainty in nanoscale measurements. 

Measurements may be dependent on the reflectivity of particles. Laser diffraction assumes a 
spherical particle shape. Test products should therefore have no extreme aspect ratios, with a 
restriction of 1:3 for non-spherical particles. This method has limited applicability really suitable in 
the sub-100 nm range. In the range below several microns, results strongly depend on optical 
constants of particles. 

Particle size/size 
distribution, from 
which mass median 
diameter can be 
calculated, with 
knowledge of the 
density of the 
particles 

 
 
 
 
 
 
 
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The  particle  detection  methods  in  Table  1  can  be  used  to  characterise  the  distribution  of 
aerosolised particles. These methods are preferred since they measure particles in the air and 
as  such  the  mass  median  aerodynamic  diameter  (MMAD)  and  geometric  standard  deviation 
(GSD), but are subject to limitations. All instruments to determine particle size are limited to 
specific ranges of particle size depending on the principle of operation. Therefore, more than one 
type of instrument is often used with overlapping size ranges. Often depending on the material, 
these size distributions may not match exactly, because different measuring principles deliver 
different equivalent diameters. Moreover, the lower sizes of 1nm to 3 nm cannot be accurately 
measured in aerosol measurement instrumentation because of diffusion losses in tubes or at the 
inlet  of  the 
for  particle  size  distribution 
characterisation  also  results in  a  degree of  artificiality if  the engineering  set-up  introduces an 
upper limit on the aerosol size as a result of the operational conditions (e.g. flow rate and exit 
orifice).  The  upper  size  limit  can  be  predicted  using  Stoke’s  equation.  Other  methods  that 
measure inhalable fractions only or that give no detailed distributions are detailed in Table 2. 

instruments.  Aerosolisation  of  substances 

1.2.3.2  Published data on granulometry 

Information on granulometry of nanoforms has been published in peer-reviewed literature. Some 
electronic  databases  exist  collecting  published 
information  on  properties  of  specific 
nanomaterials, including information on granulometry. However, registrants need to ensure that 
the data available is relevant for the specific nanoforms in their dossiers before using this for 
the  purpose  of REACH  registrations.  This  includes  ensuring  that  the  test  material  is  identified 
and well characterised and that the used method is described in detail and, where applicable, 
the  test  is  carried  out  in  accordance  with  the  test  guidelines.  It  should  be  also  clear  if  the 
measured  particle  size  corresponds  to  the  constituent  particle  size  and/or  agglomerates  and 
aggregates. 

Dustiness 

Annex VII , Section 7.14 bis of REACH includes “dustiness” as an information requirement only 
for nanoforms. 

1.2.4.1  Type of property 

Dustiness may be defined as the propensity of a powder to form airborne dust by a prescribed 
mechanical stimulus. It depends on a number of factors such as physicochemical properties of 
the  particles  (e.g.  size,  shape,  relevant  density,  type  of  coating),  the  environment  (e.g. 
moisture,  temperature),  the  type  of  process  (e.g.  energy  applied),  the  interaction  between 
particles  during  agitation  (e.g.  friction  shearing)  and  the  sampling  and  measurement 
configuration. 

Dustiness  is  of  considerable  importance  for  the  exposure  and  risk  assessment  of  particulate 
materials as: 

• 

• 

It is important when considering the potential workplace exposure; 

It  is  used  as  an  input  parameter  for  control  banding  and exposure  modelling  tools  for 
nanomaterials; 

•  Knowledge on dustiness can be used to improve the product characteristics (e.g. create 
less dusty products) and help users of the products to choose products that potentially 
may lead to lower exposures for consumers. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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1.2.4.2  Test methods for dustiness 

There are currently no standardized methods for dustiness at OECD level. However, the OECD 
WNT  project  1.8.  [47]  will  provide  a  specific  TG/GD:  “Determination  of  the  dustiness  of 
manufactured nanomaterials”. While this is ongoing work, CEN has published 5 standards (EN 
17199: 1-5) for the testing of dustiness of materials that release or contain nanomaterials. EN 
17199-1  [67]  gives  advice  on  the  methodology  and  provides  guidance  to  choose  the  most 
adequate test method. The other 4 standards, EN 17199-2 to 5 [68], [69], [70], [71], provide 
further details on the different test methods. 

•  Rotating drum (EN 15051-2 and also included in EN 17199-2) [68] 

•  Small rotating drum (EN 17199-4) [70] 

•  Continuous drop (EN 15051-3 and also included in EN 17199-3) [69] 

•  Vortex shaker (EN 17199-5) [71] 

 
 
 
 
 
 
 
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Table 2: Test methods to measure dustiness of bulk materials that contain or release 
nano-objects or submicrometer particles 

Material and 
size range 

Dry 
powders/granula
tes/friable 
products 

Size range: 0.01-
10,000 μm 

MMAD 

MMAD (mass 
median 
aerodynamic 
diameter) can be 
determined via an 
appropriate 
coupled analytical 
technique. 

Dry 
powders/granula
tes/friable 
products 
Size range: 0.5-
10,000 μm 

MMAD can be 
determined via an 
appropriate 
coupled analytical 
technique. 

 Method and details 

Rotating drum method (EN 15051-2; EN 17199-2) [72] , [65] and 
small rotating drum method (EN 17199-4) [67] 

The rotating drum and small rotating drum methods involve the 
continuous multiple dropping of a sample of the bulk material in 
a slow horizontal winnowing current of air. The dust released 
from dropping bulk material is conducted by the airflow to a 
sampling section where aerosol real-time instruments measure 
time-resolved particle concentrations and time-resolved size 
distributon of the aerosol generated. In addition, airborne nano-
objects and strutures can be collected for off-line (analytical) 
electron microscopy analysis. 

For the small rotating drum, a respirable dust cyclone collects the 
dust fractions onto a suitable media for gravimetric analysis. 

For the rotating drum, the determination of the inhalable, 
thoracic and respirable mass fractions of the released dust is 
carried out sparetlely according to EN 15051-1 [73] and EN 
15051-2 [72]. 

The small rotating drum requires smaller amounts of bulk 
material for testing (2 to 6 g) compared to the rotaling drum 
method. 

Continuous drop method (EN 15051-3) [74] 

This method is based on the size selective sampling of an 
airborne dust cloud produced by the continuous single dropping 
of material in a slow vertical air current. The dust released by 
dropping material is conducted by the airflow to a sampling 
section where it is separated into the inhalable and respirable 
fractions. 
This method is suitable to determine the distribution of particles 
of respirable or inhalable size. 
The continuous single-drop method requires a total amount of 
500 g for the required five single test runs. It has been 
highlighted that such large amounts of test material may not be 
practical if very toxic and/or costly materials are to be tested and 
there is a need for test systems that can be operated under 
controlled atmospheric environments using much smaller 
amounts of material [75]. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Material and 
size range 

MMAD 

Dry 
Powders 

Size range: 10 
nm-1000 nm 

 Method and details 

Vortex shaker (EN 17199-5) [71] 

The vortex shaker method consists of especially designed 
cylindrical container that is continuously shaken according to a 
circular orbital motion, and in which a small volume (0.5 mL) of 
the test sample is placed. The released aerosol is transferred to 
the sampling and measurement section. The aerosol real time 
instruments measures time-resolved particle concentrations and 
the time-resolved size distribution of the aerosol generated 
within the vortex shaker. In addition, airborne nano-objects5 and 
airborne nano-structures can be collected for off-line (analytical) 
electron microscopy analysis. A respirable cyclone collects the 
dust fractions onto a suitable media for gravimetric analysis. 

It  is  recommended  to  choose  the  methods  most  relevant  to  simulate  the  operations/tasks 
expected to be performed. The first three methods are intended to simulate workplace scenarios 
where handling involves dropping and differ in the intensity and duration  of the treatment for 
the  material.  The vortex  shaker intends  to  simulate a  worst  case scenario,  where  the  higher 
energy is applied to the material.  

Each  of  the  standards  details  the  methodology  for  dustiness  testing  including  sample 
preparation,  determination  of  moisture  content  etc.  The  standards  propose  a  number  of 
measurands  of  dustiness  and  in  addition,  they  establish  test  methods  that  characterise  the 
aerosol  from  its  particle size  distribution and  the  morphology  and  chemical composition  of its 
particles. It is recommended to test one of the following measurands: 

•  Respirable dustiness mass fraction (mg of airborne respirable particles/per of kg 

tested materials) 

•  Number based dustiness index from 10 nm to 1 µm (particles per milligram) 

•  Number based emission rate (particles per milligram/s) 

5 Nano-object: material with one, two or three external dimensions in the nano scale [source EN 17199-1:2019] 

 
 
 
 
 
 
 
 
 
 
 
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1.2.4.3  Exposure based waiver for dustiness 

Annex  VII  of  REACH  provides  in  column  2  the  following  specific  rules  for  adaptation  of  the 
standard  information  requirement  for  dustiness:  “The  study  does not  need  to  be conducted  if 
exposure to granular form of the substance during its life-cycle can be excluded.” 

Granular can be interpreted to mean "particles". All nanoforms, by definition, will have at least 
50%  of  their  particles  by  number  below  100  nm,  and  all  nanoforms  will  be  "granular"  when 
available as a dry powder. However, some nanoforms are available only in suspensions, or are 
incorporated  into  a  matrix  throughout  their  entire  lifecycle.  In  this  case,  there  may  be  no 
exposure to the dry powder, or the granular form in general. 

Exposure based waiving should be accompanied by scientific support to show that exposure to 
the dry powder or the granular form can be excluded during the entire lifecycle of the nanoforms. 

Adsorption/desorption 

In the parent guidance, the methods for determining this endpoint are shown in Table R.7.1-14 
“Methods for the measurement of adsorption”. Adsorption/desorption measurements are used 
in fate modelling to indicate which compartment in the environment will be exposed the most or 
might  need  to  be  considered  in  hazard  and  risk  assessment.  These  measurements  help  to 
determine  in  which  environmental  compartment  i.e. soil, sediment  or water,  the substance  is 
most  likely  to  end  up  and  whether  it  is  likely  to  be  mobile  or  immobile.  For  instance,  high 
adsorption  to  soil  would  show  that  both  soil  and  sediment  are  highly  relevant  environmental 
compartments to be considered in hazard assessment. 

Adsorption  is  temporary  (reversible)  or  permanent  bonding  between  the  substance  and  a 
surface. With regards to nanoforms, the distribution coefficient between solid phase and a liquid 
phase (Kd) very often must be based on actual testing since estimations of Kd derived from the 
organic carbon-water partition coefficient (Koc) and the octanol-water partition coefficient (Kow) 
is not applicable for most nanoforms, i.e. non-highly dissolving nanoforms (see Section 1.2.1). 
Kd measurement is based on the assumption of a thermodynamic equilibrium between a liquid 
and a solid phase. Equilibrium partitioning does not apply to undissolved   nanomaterials [53], 
[48], [49], [21] as described in Section 1.2.2. Nanomaterial dispersions can be kinetically stable 
for a long period of time (typically through electrostatic or steric stabilization) but they will never 
reach  thermodynamic equilibrium  and consequently cannot  be  equilibrated  with  an  additional 
phase [49], [76] [77]. 

Therefore, nanomaterials strive to reduce their surface energy by attaching to other particles in 
the system. This attachment can be: 

•  homoagglomeration/aggregation 

between 

the 

particles 

of 

the 

same 

nanomaterial/nanoform, or 

•  heteroagglomeration/aggregation with other particles, or 

• 

to the interface between phases (deposition or attachment). 

Because of the inability to accurately quantify the physico-chemical forces contributing to particle 
attachment,  this  step  is  typically  described  by  an  empirical  parameter  termed  the  particle 
attachment efficiency () that needs to be determined in agglomeration (heteroagglomeration) 
or deposition experiments [48], [77]. 

OECD  TG  106  Adsorption  –  Desorption  Using  Batch  Equilibrium  Method  [78]  is  partially 
inadequate  when  the  substance  in  question  has  a  low  dissolution  rate,  i.e.  is  present  as  a 
dispersion,  because  it  is  currently  not  possible  to  differentiate  between  adsorbed  or 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
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aggregated/agglomerated particles settled during the centrifugation step, and a new TG needs 
to be developed ( [20], [79]). However, if it is shown that a nanomaterial has a high dissolution 
rate,  it  can  be  assessed  in  the  same  way  as  non-nanoforms  of  substances  and  the  parent 
guidance will apply (as explained under Section 1.1.2.1 and Section 1.2.1). 

1.2.5.1  Relevant method to measure adsorption/desorption of nanoforms  

OECD TG 312 Leaching in Soil Columns [59] allows to study the mobility and leaching of the test 
substance into deeper soil layers or ground water. While it is agreed that the OECD TG 312 is 
generally applicable for the testing of nanoforms, a GD using this TG to test nanomaterials was 
established and published on 28 July 2021 OECD GD 342 [58]. 

This guidance provides the information and methods to measure the soil adsorption behaviour 
of nanoforms (mobility and retention) so that such tests can be applied to assess quantitively 
the mobility of nanomaterials in soils and deduce attachment efficiencies. 

OECD TG 312 implies thermodynamic processes where non-nanoforms of substances often will 
reach  an  equilibrium  [59].  As  such  processes  are  not  applicable  for  nanoforms,  attachment 
efficiency for heteroagglomeration (hetero) can be calculated instead ( [58]). 

hetero expresses the probability that nanomaterials will attach when they collide with the soil 
grain surface and takes into account random effects caused by the way the soil matrix happens 
to  be  structured  (  [80]).  A  quantitative  estimation  of    can  be  obtained  where  a  continuous 
nanomaterial input is applied into the column transport test and the nanomaterial concentration 
is  monitored  over  time in  the  eluate as  it  is  the case  within  TG  312.  However,  it  needs  to  be 
noted that the determination of hetero is based on the “clean bed” assumption, which is valid 
only during the early stages of the deposition process, when low particle loadings are applied 
and no significant repulsion between particles and the porous medium is present. Outside these 
settings more complex mechanisms can influence the particle transport (e.g. blocking, ripening) 
and  with  hetero  not  being  able  to  accurately  describe  the  system  anymore,  leading  to 
misinterpretation and misuse of the data. 

To reliably report on nanomaterial transport in soils using  OECD GD 342, the overall recovery 
(mass balance) of the nanomaterial should be reported. In accordance with GD 342 on the OECD 
TG 312, a recovery (for non-labelled nanomaterials) of at least 70% should be considered, but 
it is acknowledged that this strongly depends on many different variables. Thus, the test set up 
needs to be reviewed individually considering all the parameters and test set up (such as particle 
type, the choice of application, spiked amount and used soil), when the suggested recovery of 
70% is not reached. 

The selection  of  test soils  has  to  relate  to  environmental  relevance  rather than  to  properties, 
and at least two soils differing either in pH, organic carbon content, clay content and/or texture 
should  be considered. Generally,  soils  with  high  clay content are  to  be  avoided  because  here 
particle transport occurs predominantly in macropores  [81] making experiments with saturated, 
stacked soil cores unrepresentative for nanoform transport rates. 
This reduction of number of soils to two from the parent TG is based on reasons of practicability. 
Because  soils  with  high  clay  content  (soil  1  in  OECD  TG  312)  tend  to  block  during  leaching 
(nanoforms strongly attach to the clay minerals preventing break through [82], [83], [84]) and 
sandy soils with high carbon content (soil 5 in OECD TG 312) are only of limited availability. In 
case  natural  soils  are  used  a  control  experiment  with  soils  not  previously  exposed  to 
nanomaterials  has  to  be  conducted  to  determine  the  background  of  naturally  occurring 
nanoforms. 

To account for more realistic conditions of nanomaterial mobility in soils for which a considerably 
longer residence time is expected, flow rates of 2-3 L·m-2·h-1 are to be used, avoiding an artificial 
break through and posing as a realistic worst-case scenario. As a leaching solution mimicking 

 
 
 
 
 
 
 
 
 
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artificial  rain  fall,  aqueous  solutions  of  0.005  M  KCl  or  NaCl  should  be  used.  For  nanoforms 
reacting  with  chloride  (e.g.  silver  that  would  precipitate  and  render  them  unavailable  for  the 
chemical analysis), other suitable anions such as NO3 (KNO3) have to be used. Furthermore, the 
use of divalent salts such as CaCl2 will not provide a worst case scenario test as nanomaterials 
homoaggregates are generally less mobile in the presence of Ca2+. The chosen monovalent salt 
should not react with the nanomaterial, e.g. accelerating its dissolution and the stability of the 
nanomaterial  suspension  should  be  measured  prior  to  the  column  test.  To  mimic  conditions 
where the soil pore water is dominated by divalent cations a solution of 0.005 M CaCl2 can be 
used according to GD 342. However, it has to be taken into account that this will not work for 
nanomaterials  reacting with  chloride such  as  silver.  Furthermore,  it  does not  present a  worst 
case with respect to mobility. 

The  advice  provided  here  is  not  applicable  to  nanomaterials  with  high  dissolution  rates  (see 
Section  1.1.2.,  1.2.1).  For  those  nanomaterials,  it  might  result  in  testing  the  adsorption 
behaviour of the dissolved fraction and not the nanoform. The advice is also not applicable for 
nanomaterials with low dispersion stability in aqueous media (with definitions given in OECD TG 
318  (2017)  [57]).  Testing  of  such  nanomaterials  will  result  in  a  not-representative  and  not-
reproducible  addition  of  test  material  to  the  soil  column.  Guidance  on  how  to  determine 
dispersion stability/dissolution rate of the nanoforms in environmentally relevant conditions can 
be  gained  from  OECD  TG  318.  Information  on  the  use  of  the  data  for  further  environmental 
testing and assessment strategies can be found in GD 318 [8]. Testing of these parameters has 
to be performed before applying OECD TG 312, GD 342. 

1.2.5.2  Alternative fate descriptors to adsorption/desorption of nanoforms  

A list of available models to predict alternative fate descriptors for nanomaterials is available in 
Error! Reference source not found.. The OECD published results of a study where 10 nanospecific 
environmental exposure tools and models were tested for functionality and accuracy. Three of 
the models mentioned in Appendix 1 – namely nanoFATE, simplexbox4nano and nanoDUFLOW 
are part of this report and details on the functional assessment as well as the outcome of the 
statistical analysis can be found there [85]. 

These  models are still under  development  and  further  validation  is  needed,  in  particular with 
regard to the uncertainties and applications. Such validation is especially important when these 
models are used for exposure of the environmental compartment and organisms. When they are 
thoroughly  validated  models,  they  will  be  recommended  as  an  option  to  provide  suitable 
alternative information on the sorption and agglomeration/aggregation of nanomaterials. 

1.2.5.3  Waiving of adsorption/desorption to nanoforms  

Annex  VIII  of  REACH,  Section  9.3.1.  states  in  column  2:  “  For  nanoforms,  use  of  any 
physicochemical property (e.g. octanol-water partition coefficient) as a reason for waiving the 
study shall include adequate justification of its relevance to low potential for adsorption.” 

It is necessary to take into account the nanoform specific properties and constraints in assessing 
the adsorption/desorption properties of nanoforms by currently available methods, based on Kd 
derived from the organic carbon-water partition coefficient (Koc) and the octanol-water partition 
coefficient  (Kow),  such  as  OECD  TG  106  or  with  the  use  of  particle  attachment  efficiency  () 
specifically  developed  for  nanomaterials.  Consequently,  waiving  the  information  requirement 
based  on  low  adsorption/desorption  similarly  to  Kow  and  dissolution  rate  should  always  be 
accompanied by a robust technical and scientific justification on the applicability of the used test 
method with further justifications on nanoforms behaviour in soil and sediment (e.g. nanoform 
being water soluble or having a high dissolution rate as detailed under Section 1.2.1 or not being 
dispersed as detailed under Section 1.2.2). 

 
 
 
 
 
 
 
 
 
 
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2.  RECOMMENDATIONS FOR TOXICOLOGICAL 

INFORMATION REQUIREMENTS for NANOMATERIALS  

2.1 

General advisory notes  

General advisory note on testing with nanoforms and sampling 
strategy and sample preparation for human health endpoints 

These advisory notes do not describe any detailed protocol but, instead, aim to provide useful 
advice with regard to specific aspects that are particularly important for nanoform testing, and 
references to relevant resources. For a test material fulfilling the definition of a nanoform, the 
testing strategy (Fig. 5) is dependent on its solubility and/or dissolution rate in water. Dissolution 
rate in relevant biological fluids is an important parameter in toxicokinetics evaluation, and may 
also  be  used  for  grouping  and  building  of  the  read-across  or  to  substantiate  the  weight  of 
evidence6 in the cases where existing studies with nanoforms are available. 

The  figure  below  shows  a  decision  tree  that  can  be  used  to  determine  whether  nanospecific 
advice  should  be  used,  or,  due  to  the  conclusions  on  the  nanoform’s  properties,  the  advice 
provided by the parent guidance can be followed instead. 

Figure 4: Decision process for selecting the applicable guidance document 
*The  given  values  [16],  [86],  may  be  updated  when  more  validated  information  becomes 
available with regard to testing in relevant biological media. 

2.1.1.1  Considerations on solubility and dissolution rate 

The Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) has stated 
that  many  nanomaterials  have  considerable  solubility  and  that  for  “these  materials  the 
interaction with living systems remains close enough to the bulk chemical agent to justify the 
use of well-established toxicological testing procedures and approaches” [87]. Water solubility 
may give a first indication on the (none) biopersistence of a nanomaterial [88]. EFSA Guidance 
on Technical Requirements [16] considers, a solubility equal to or higher than 33.3 g/L as a high 
solubility in water, in line also with the SCCS Guidance (2019) [89] and with the categories for 
degrees of solubility proposed by JECFA  [86] and the European and US Pharmacopoeias [16]. 
Based on a pragmatic and consistent approach among different regulatory bodies, a similar value 

6  https://echa.europa.eu/documents/10162/17250/practical_guide_how_to_use_alternatives_en.pdf/148b30c7-c186-
463c-a898-522a888a4404 

Does the test material fulfil the definition of a nanoform?Is the nanoform highly soluble in water (>33.3 g/L) and has a half-life of water dissolution      min.?* See parent guidance (Chapter R. 7a)See Appendix R. 7-1 (this document)NONOYESYES 
 
 
 
 
 
 
 
 
 
 
 
 
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for water solubility is recommended also here to decide whether this guidance is applicable (see 
Figure 4). In the context of risk assessment in occupational settings, BAuA [90] proposed that 
nanomaterials with a water solubility above 100 mg/L could be considered as soluble7 and thus 
not biopersistent. 

The latest approaches for the risk assessment of nanomaterials recommend a strategy in which 
the dissolution rate and equilibrium in water is a first key element [91], [16]. Dissolution rate is 
a kinetic process which informs not only whether a substance dissolves but also on how long it 
takes to dissolve. Dissolution rate  depends on the size and surface properties of the particles 
and fibres, and also on the nature of media where the release of ions occurs, on the temperature, 
the  pH  and  the  presence  of  substances  interacting  with  the  nanoform’s  surface.  The  pH  and 
temperature should be specified and relate to biologically relevant conditions. Another important 
factor in the dissolution of nanoparticles is aggregation [92]. Aggregation decreases the available 
external surface area thereby reducing the extent of dissolution [93]. It has been observed that 
the dissolution rate decreases as the aggregation state of nanoparticles increases. 

To determine the dissolution rate in water, the protocol proposed by EFSA [16] is recommended. 
In the EFSA Guidance on Technical Requirements [16] a high dissolution rate in water is defined 
as a half-life of 10 min or less corresponding to a dissolved fraction equal to or higher than 88% 
in 30 min [16]. In this context EFSA Guidance on Technical Requirements refers to ‘concentration 
corresponding  to  exposure  at  maximum  use  level’.  A  similar  threshold  for  dissolution  rate  in 
water is proposed in this guidance (see Fig. 5). 

Annex  VII,  section  7.7.  column  1,  of  the  REACH  Regulation  specifies,  that  (beside  the  water 
solubility testing) “for nanoforms, in addition the testing of dissolution rate in water as well as 
in relevant biological and environmental media shall be considered” (see section 1.2.1). 

The determination of the dissolution rate (k) as well as the dissolution half-life in biological fluids 
provides an insight on how a certain particle may interact with its biological environment  [92] 
and  allows  an  informed  decision for  the  design  of  the  repeated  dose studies.  The  dissolution 
rates are important parameters in toxicokinetics evaluation, which can also be used for grouping 
and  read-across.  Recently  a  method  and  thresholds  [45]  have  been  proposed  for  grouping 
nanoparticles based on dissolution rates in phagolysosomal simulant fluid. 

Until dissolution occurs, the kinetics of nanomaterials are governed by their particulate nature, 
whereas  after  dissolution  the  (dissolved)  ions  or  molecules  determine  the  toxicokinetics  [94]. 
Thus,  if  the  dissolution  rate  is  not  very  high,  uptake  of  particles  may  occur.  A  very  high 
dissolution  rate  is  considered  close  to  instantly  dissolved  [91],  and  means  that  the  material 
dissolves quickly at the site of entry, so that cellular uptake in the lung or gastrointestinal tract 
occurs almost exclusively as the dissolved species. When particles remain present for a longer 
duration,  they  can  be  taken  up  by  macrophages  or  translocated  across  the  epithelium  as 
particles. Effects due to release of ions or molecules after distribution to cells/tissues (i.e. the 
so-called  "Trojan-horse"  mechanism,  in which nanoparticles  are internalized  within  cells,  then 
release  high  levels  of  toxic  ions),  should  be  investigated,  as  the  local  dissolution  in  the 
macrophages and in tissues may lead to a different toxicity profile as compared to the solute 
itself.  For  example,  although  the  toxicity  of  ZnO  nanoforms  is  predominantly  driven  by  the 
release of Zn2+ ions, in vivo studies showed that ZnO nanoparticles, but not the soluble ions, 
triggered  the  recruitment  of  eosinophils  to  the  lungs  [95].  In  addition,  the  exposure  to  ZnO 
nanoparticles caused chronic effects that lasted up to four weeks. No aqueous extract caused 
such sustained inflammation, probably because soluble ions are rapidly cleared from the lungs 
[96]. Most probably the observed toxicity with ZnO nanoparticles is the result of the combined 
effects of the nanoparticles and of Zn2+ ions [97]. 

7 Please note this value is only used as an indication for (non) biopersistence and should not be used as a threshold for 
solubility/insolubility in other contexts (such as triggering a waiver for insolubility for environmental endpoints)  

 
 
 
 
 
 
 
 
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The  dissolution  of  a  nanomaterial  is  a  time-dependent  process  (depending  on  the  rate  of 
solubilisation and the surface area) and is directly related to its in vitro or in vivo biopersistence 
and biodurability that decreases with increasing dissolution rate [88]. In contact with a biological 
environment,  the  nanoparticles  may  undergo  changes  involving  dissolution,  re-precipitation, 
protein coating and agglomeration. Such events are part of biotransformation. Biotransformation 
could  impact  clearance  and  can  also  contribute  to  the  biokinetics  of  nanoparticles  [98]  and 
potentially  to  their  toxicity  [99].  Therefore,  biotransformation  is  an  important  element  to 
investigate in the context of toxicokinetics. A method to assess biotransformation was recently 
described  [98].  However,  as  no  standard  method  applicable  to  all  nanoforms  is  currently 
available, the method used must be justified and thoroughly documented. The information on 
biotransformation  is  not  a  mandatory  requirement  but  it  is  considered  important  for  the 
implementation of grouping and read-across using dissolution rates [98]. 

Dissolution studies in relevant biological fluids can also be performed to substantiate the weight 
of evidence6 in cases were studies are already available and fulfill the criteria described in Section 
2.2.2. OECD [100] lists as examples of biological media the artificial pulmonary interstitial fluid 
(Gamble’s) balanced electrolyte solution (neutral) and alveolar lysosomal fluid (ALF) (acidic) as 
well as gastric (acidic) and intestinal (neutral) fluids.  Different biological media may influence 
both the kinetics of dissolution and the saturation concentration depending in particular on pH 
and/or enzymatic action [101]. In addition, some water insoluble nanomaterials may be non-
biodurable  in  biological  fluids  and  this  can  be  assessed  from  data  on  the  dissolution  rate.  In 
general, the most appropriate route of administration, having regard to the likely route of human 
exposure, determines which biological media are relevant [91]. 

The  OECD  Guidance  documents  on  inhalation  toxicity  studies  [102]  states  that  “In  order  to 
decide whether your material is poorly soluble, the solubility of a solid material may be assessed 
by measuring solubility in a simulated biofluid. A poorly soluble material is generally understood 
as having a solubility of less than 0.1 g dissolved in 100 ml dissolvent within 24 hours.” Examples 
of dissolvent are the simulated biofluids. For the inhalation route of exposure, the simulated lung 
lining fluid and (macrophage) phagolysosomal simulant fluid are relevant for the assessment of 
dissolution  via  this  route  of  exposure  [102].  The  data  in  the  simulated  lung  lining  fluid  are 
important as this medium simulates the extracellular environment. Assessment of the stability 
in  lysosomal  conditions  is  important  for  the  investigation  of  biopersistence  and  intracellular 
accumulation [92]). Within the cell, the nanomaterials generally distribute to lysosomes, where 
degradation  can  occur  due  to  the  acidic  conditions  and  the  presence  of  enzymes.  Using  an 
optimized abiotic flow-through method for 24 (nano)forms of 6 substances, Koltermann-Jülly et 
al. 2018 [45] proposed a grouping based on the dissolution rates in phagolysosomal fluid (pH 
4.5).  For  example,  a  high  dissolution  rate  (k>100  ng/cm2/h)  for  a  non-persistent  nanoform 
corresponds  to  an  abiotic  dissolution  ranging  from  30%  to  100%  after  7  days.  Indeed,  a 
dissolution rate of 30-100% within 7 days is in line with a complete clearance during 21 days 
post-exposure. 

It  is  important  to  assess  the  dissolution  rate  in  both,  simulated  lung  lining  fluid  and 
phagolysosomal  fluid.  For  example, if  dissolution only  occurs  in  the  lysosomal  fluid  but  not in 
the simulated lung lining fluid, uptake of particles by lung macrophages as well as translocation 
across the lung epithelium can occur, as the lung lining fluid has been reported to stimulate the 
nanoparticle  uptake  by  human  alveolar  epithelial  cells  [103].  Following  translocation,  the 
nanomaterials may relocate at a different location than the lung. In this case the read-across to 
the solute is not possible, since the dissolution of the nanoform (and release of the solute) takes 
place at a different location, which may lead to a different distribution profile. Comparisons of 
the  distribution  profiles  for  particles and solutes would  help  to assess whether  read-across  to 
the soluble form is possible. 

For  the  oral  route  of  exposure,  dissolution  of  nanomaterials  in  simulated  gastric  fluid  and 
macrophage  phagolysosomal  fluid  is  relevant.  The  macrophage  phagolysosomal  fluid  may 
provide  insight  on  the  persistence  of  the  nanoform  after  cellular  uptake.  A  nanomaterial  is 

 
 
 
 
 
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considered to dissolve quickly or to have a high dissolution rate if 12% or less of the material 
(mass  based)  remain  as  particles  after  30  min  of  in  vitro  digestion  in  gastrointestinal  fluid 
compared to the particulate concentration at the beginning of the dissolution test [104]. If the 
nanoform  does not  dissolve  quickly  in  the  gastrointestinal fluid,  then  dissolution  in  lysosomal 
fluid should be performed. A half-life of ca. 24 hours is considered indicative of high dissolution 
rate in lysosomal fluid. This would result in 12% or less of the material (mass based) remaining 
at 72 hours compared to the particulate concentration at the beginning of the dissolution test 
[104]. 
For dermal exposure, the dissolution rate in artificial sweat could be used.  

2.1.1.2  Available methods for solubility and dissolution testing 

Different  analytical  techniques/methods  and  their  suitability  to  measure  solubility  of 
nanomaterials have been reviewed [105]. The determination of the solubility of nanomaterials 
in water should be done according to OECD TG 105 with specific considerations for nanoforms 
[106]. The pH and temperature should be specified for solubility testing and relate to biologically 
relevant conditions [107]. 

In  the  solubility/dissolution  kinetics  experiments,  the  solid  phase needs  to  be separated  from 
the  liquid  phase  in  order  to  avoid  artefactual  high  solubility  values  by  also  analysing  solid 
materials. Presence of particles can be checked qualitatively by e.g. Tyndall analysis and solid 
nanoparticles can be removed by ultracentrifugation or ultrafiltration [105]. 

In  water,  so  far,  no  specific  OECD  testing  guideline  (TG)  is  available  for  determination  of 
solubility and dissolution rate for nanomaterials but  some may be adapted e.g. OECD TG 105 
[42], [101], [105] and ISO 19057 [108].  

Dissolution studies on particle systems are often carried out by inductively coupled plasma mass 
spectrometry (ICP-MS), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and 
atomic  absorption  spectroscopy  (AAS),  which  are  a  few  of  the  most  employed  methods  for 
quantitative  analysis.  Real-time  dissolution  kinetics  measurements  can  be  performed  using 
Dynamic Light Scattering (DLS), UV/vis spectrometry, fluorescence spectroscopy and confirmed 
using inductively coupled plasma mass spectrometry (ICP-MS) [109].  

In  a  complex  matrix,  it  is  acknowledged  that  measuring  dissolution  is  highly  challenging.  At 
present there is no universal robust, rapid test method for regulatory testing that is applicable 
for  all  types  of  nanomaterials  and  all  types  of  matrices  [110].  During  the  NANoREG  project, 
several  methods  have  been  identified  for  future  use  (e.g.  ultrafiltration  UF-ICP-MS/AES, 
ultracentrifugation UC-ICP-MS/AES, single particle sp-ICP-MS, and colorimetry). UF-ICP-MS/AES 
was considered a relatively easy and highly robust method that can be used for a rather broad 
range of nanomaterials if no nanomaterial-matrix interactions take place. If these interactions 
do take place, sp-ICP-MS is considered a good choice for measurement, given that the material 
is not below the size detection limit. Nevertheless, to select the best suitable method, knowledge 
of physical-chemical properties of the nanomaterial is crucial and it is recommended to use a 
combination of techniques [110]. 
Due to the fact that the assessment of dissolution in biological media is currently performed only 
by non-validated methods, the used protocols need to be well documented and justified. 

Albeit  not  validated for  nanomaterials,  information from  bioelution  studies could  be  useful  for 
read-across for metallic compounds. An ESAC Opinion on Scientific Validity of the Bioelution Test 
Method was finalised in July 2020 [111]. 

ENV/JM/MONO(2018)11 [112] compiled the available information on the determination of the 
biodurability  (through  dissolution)  of  different  NMs  included  in  the  OECD  Sponsorship 
Programme for the Testing of Manufactured Nanomaterials as indicator for their toxicity. With 
cellular and acellular in vitro tests, the dissolution of NMs is determined using static and dynamic 

 
 
 
 
 
 
 
 
 
 
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(such as flow-through) protocols in the presence of different biological and environmental media 
with  different  pH  values  and  chemical  compositions.  New  studies  found  that  dynamic  abiotic 
dissolution  systems  adequately  predicted  the  overall  pulmonary  biopersistence  of  several 
nanoforms  [45],  [98].  A  recent  OECD  Guidance  document  [106]  provides  guidance  for  the 
methods addressing dissolution rate and dispersion stability for nanomaterials, in particular with 
the development of the dynamic testing or flow through system that could be applied to estimate 
the  dissolution  in  biological  media.  There  is  also  ongoing  work  on  the  WNT  project  1.5  on 
“Determination  of  Solubility  and  Dissolution  Rate  of  Nanomaterials  in  Water  and  Relevant 
Synthetic Biological Media” aiming to provide harmonised approaches for testing solubility and 
dissolution rate of nanomaterials. In addition, development of an OECD Guidance document on 
"Integrated  in  vitro  approach  for  intestinal  fate  of  orally  ingested  nanomaterials"  is  under 
developement. It is based on a two-step approach in which the dissolution behavior of nanoform 
in  simulated  digestion  environment  is  coupled  with  a  biological  evaluation  of  nanoparticles 
uptake/passage in a cellular model of intestinal barrier. 

As long as no standard methods are available, detailed description and justification of the method 
including adequate controls (and reference materials, if possible) is required. 

2.1.1.3  Test material characterization and reporting and sample preparation 

The introductory text of Annexes VII-X of the REACH Regulation (EC) No 1907/2006 amended 
for  nanomaterials,  provides  that:  "Without  prejudice  to  the  information  submitted  for  other 
forms, any relevant physicochemical, toxicological and ecotoxicological information shall include 
characterisation  of  the  nanoform  tested  and  test  conditions.  A  justification  shall  be  provided 
where  QSARs  are  used  or  evidence  is  obtained  by  means  other  than  testing,  as  well  as  a 
description of the range of the characteristics/properties of the nanoforms to which the evidence 
can be applied." 

Prior  to  toxicological  testing,  the  sample  characterization  and  preparation  including  special 
considerations  on  dispersion  and  dosimetry,  should  be  performed,  as  advised  in  the  OECD 
Guidance  on  Sample  Preparation  and  Dosimetry  for  the  Safety  Testing  of  Manufactured 
Nanomaterials  [19],  and as specified  in  Sections  1.1.1  and  1.1.2  of  this  Appendix.  Additional 
useful  information  can  be  found  in  the  report  of  the  OECD  expert  meeting  on  the  physical 
chemical  properties  of  manufactured  nanomaterials  and  test  guidelines  [113].  A  harmonized 
preparation  of  the  test  sample  will  enable  the  comparison  of  the  data  and  their  further  use. 
Information on the characterisation of test material serves multiple purposes: 

a)  enables  linking to  the  identity  of  the nanoform/set  of nanoforms  being  covered  in  the 

dossier and therefore supports data relevance.  

b)  provides  general  information  on  the  material’s  properties  'as  test  sample'  to  support 

handling/storage and repeatability/reproducibility of results, and 

c)  facilitates the comparability and use of toxicological data for grouping of the nanoforms 
of  a  substance  or  justifying  read-across  between nanoforms,  and  between nanoforms 
and  the  non-nanoforms  (For  further  information  see  Appendix  R.6-1  for  nanoforms 
applicable to the Guidance on QSARs and Grouping [31]). 

Sections  1.1.2  and  1.1.3  of  this  Appendix  explain  in  detail  the  importance  of  these  physico 
chemical  parameters  for  toxicological  testing  and  also  gives  information  on  how  these 
parameters  can  be  determined.  A  critical  review  of  the  methods  used  to  characterize  the 
manufactured nanomaterials to assess their  health and safety risks, describing the limitations 
and accessibility of each method is also available [114]. 

The  chemical  composition,  the  physicochemical  properties,  and  the  interaction  of  the 
nanomaterials  with  biological  systems  may  in  some  cases  influence  aspects  of  exposure, 
deposition (lung), systemic uptake and bioaccumulation, dissolution, toxicokinetics and toxicity 
[114].  The  hazards  posed  by  all  possible  forms  of  the  substance  covered  by  a  registration, 
including  nanoforms/set  of  nanoforms,  must  be  addressed  by  the  toxicological  and 

 
 
 
 
 
 
 
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ecotoxicological information provided in the registration dossier. In order to demonstrate that 
the results of a study are relevant for the hazard of a specific nanoform or set of nanorforms in 
the dossier, some information on the tested substance should be reported in the endpoint study 
record  under  the  test  material  information  field  in  IUCLID  (please  see  more  under  “How  to 
prepare 
[115] 
(https://echa.europa.eu/documents/10162/22308542/howto_prepare_reg_dossiers_nano_en.
pdf/5e994573-6bf9-7040-054e-7ab753bd7fd6) 
following parameters must be provided: 

in  https://echa.europa.eu/manuals).  The 

nanoforms” 

registration 

substances 

dossiers 

with 

for 

•  Chemical composition (as described in the ECHA Guidance for identification and 

naming of substances under REACH and CLP); 

•  Characterisation  parameters  of  the  test  material  (as  described  in  the  ECHA 
Guidance Appendix for nanoforms applicable to the Guidance on Registration and 
Substance Identification 

o  Number  based  particle  size  distribution  with  indication  of  the  number 
fraction of constituent particles in the size range within 1 nm – 100 nm; 

o  Shape and aspect ratio; 
o  Description of surface functionalisation or treatment; 
o  Specific surface area. 

Moreover, Appendix R6-1 for nanoforms applicable to the guidance on QSARs and Grouping of 
Chemicals  [31]  provides  an  approach  on  how  to  justify  the  use  of  hazard  data  between 
nanoforms  (and  the  non-nanoform(s))  of  the  same  substance.  The  Guidance  details  some 
(additional) parameters that may be required to be able to assess whether the available hazard 
data are applicable for different nanoforms of a substance. The registrant may wish to consider 
taking into account such parameters when characterising the test material, in order to be able 
to follow the above-mentioned guidance. For example, the dissolution rate, surface chemistry 
and dispersability have been reported as a founding basis for the grouping of the nanomaterials 
( [31], [116]). 

Nanoforms characterization (e.g. size, dissolved ion fraction, shape) in the exposure system at 
least at the start and end of the in vitro experiments is important to confirm their presence in 
the test system and to observe potential changes that they may undergo. This characterisation 
should also include the assessment of the state of agglomeration. This is an important parameter 
to investigate also for in vivo studies, and characterisation of aerosols is recommended in OECD 
412/413. These TGs also state that “scanning and/or transmission electron microscopy should 
always be used periodically (e.g., monthly) for monitoring and qualitative confirmation of particle 
size and shape for all particulates, not just nanomaterials.” 

Suitable  suspension-based  techniques  include  Dynamic  Light  Scattering  (DLS),  analytical 
ultracentrifugation  (AUC)  or  asymmetrical  flow  field-flow  fractionation  (AF4)  coupled  with 
inductively coupled plasma mass spectrometry (AF4/ICP-MS). 

DLS  has  been  used  in  many  European  projects  (e.g.  NANoREG,  NanoReg2)  in  order  to 
characterize nanomaterials in the medium. However, DLS is suitable to investigate the particle 
size  distribution  and  works  well  with  monodisperse,  non-agglomerated  and  spheroidal 
nanomaterials.  DLS  is  therefore,  more  appropriate in studies  with  more  simple  environments 
such as the ones used in dissolution studies. DLS cannot detect the presence of small aggregates 
and it is not able to resolve small modifications of the particle size distributions (PSDs) happening 
under physiological conditions [117]. 

Analytical  ultracentrifugation  (AUC)  is  one  of  the  most  accurate  techniques  for  the 
characterization  of  nanoparticles  in  the  liquid  phase  because  it  can  resolve  PSDs  with  high 
resolution and detail also in the sub-nm range [118]. AUC combines separation, concentration 
and detection steps into one single measurement improving total analysis times and reducing 
experiment complexity [117]. 

 
 
 
 
 
 
 
 
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AF4 is the most commercialized flow field-flow (FFF) fractionation technique and by far the most 
widely used FFF technique for nanomaterials. It shows good selectivity and size resolution. By 
showing differences in PSDs, AF4 is also very useful for monitoring particle modifications, such 
as  the  adsorption  of  macromolecules  on  the  surface,  or  dissolution  or  aggregation  processes 
[119].  The  online  coupling  of  AF4  with  multiangle  light  scattering  (MALS),  UV,  differential 
refractive index (dRI) or fluorescence detection, or offline combination with TEM or UV, allows 
for detailed characterization of the physicochemical properties of different populations within a 
polydisperse  sample  with  respect  to size,  molar  mass  or  concentration,  shape, conformation, 
structure, and aggregation state [120]. 

The  capability  of  multi-element  analysis  is  an  added  beneﬁt  when  coupling  AF4  with  mass 
spectrometry.  AF4/ICP-MS  offer  substantial  advantages  for  detecting  nanoparticles  and 
assessing many parameters in complex matrices over traditional characterization methods such 
as microscopy, light scattering, and filtration [121]. AF4-ICP-MS has a good size resolution and 
can detect a very small nanoparticle size (up to 2 nm). 

ICP-MS is a suitable method to quantify the dissolved fraction in the culture media, especially 
for metal nanomaterials. Single particle spICP-MS is a new promising method derived from using 
the  inductively  coupled  plasma  mass  spectrometer  (ICP-MS)  in  the  time-resolved  mode.  The 
coupling  of  FFF  to  spICP-MS,  allows  nanoparticle  dimensions  to  be  determined  at  very  low 
concentration  levels,  by  choosing  one  or  two  specific  chemical  elements  with  the  common 
instruments,  or  multi-elements  with  fast  scanning  quadrupoles  or  TOF  (time-of-flight)  mass 
analyzers  [119].  Within  the  work  alongside  OECD  projects  to  support  and  accelerate  the 
development of Test Guidelines and Guidance Documents related to nanomaterial test methods,  
NanoHarmony project Task 1.4 (Determination of concentrations of ENMs in biological samples 
using  spICP-MS)  focuses  on  the  optimisation  and  standardisation  of  spICP-MS  but  other 
techniques will also be addressed. 

An  integrated  assessment  of  residual  solids  with  respect  to  transformations  of  shape,  size 
distribution, and crystallinity by protocols for preparation, analysis and statistical image analysis, 
using TEM, optionally supported by XPS and EDX is available  [98]. However, XPS is currently 
not  widely  available.  SEM  and  TEM  are  the  “gold standard”  techniques  for  nanoparticle size-, 
shape-, crystallinity- and composition- characterisation. While TEM is a highly valuable technique 
for the size analysis of nanomaterials, simple TEM sample preparation methods (by evaporation 
of the solution) do not distinguish between agglomerates formed during the drying of the sample 
on  the  support (grid)  and  aggregates  already  present  in  the NP  suspension.  Cryo-TEM  might 
help to overcome this drawback by an ultra-fast conversion of the NP suspension into a vitrified 
film on the grid, allowing direct morphological visualization of nanomaterials at near native state, 
and  thus,  reducing  the  risk of aggregation  and  of  the introduction  of  artefacts  due  to  sample 
preparation [122]. However, the technique needs laborious sample preparation and particles or 
aggregates larger than 300 nm cannot be imaged. 

The information on transformation is not a mandatory requirement but it is considered important 
for the implementation of grouping and read-across. 

An  important  step  in  the  sample  preparation  for  in  vitro  or  in  vivo  studies  is  dispersion.  A 
generally  applicable  standard  operation  procedure  (SOP)  is  currently  not  available  for  the 
dispersion of a dry powder in liquids. Since the use of a certain protocol or analytical technique 
depends  on  the  type  of  nanomaterial  and  medium,  the  dispersion  efficiency  of  the  applied 
protocol  and  the  stability  of  the  dispersion  should  be  tested  and  properly  documented.  A 
dispersion  protocol  can  be  considered  effective  if  it  yields  samples  which  consist  as  much  as 
possible  of  non-agglomerated/non-aggregated  particles  [16].  Reference  materials  or  self-
generated and properly characterised and documented test materials are essential for controlling 
and  comparing  the  performance of  analytical  methods  used  for  nanomaterial characterisation 
and  in  their  validation  [104].  Dispersion  stability  must  be  verified  periodically.  ISO/TR 

 
 
 
 
 
 
 
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13097:2013  (Guideline  for  the  characterization  of  dispersion  stability)  provides  technical 
guidance on the characterisation of dispersion stability [123]. 

Several  dispersion  protocols  have  been  developed  and  published  [106],  [124],  [125],  [126], 
[127],  [128],  [129],  [130],  [131].  Dispersion  protocols  for  toxicological  studies  have  been 
developed by different international organisations and in various research projects, in particular 
EU NanoGenoTox8, EU NANoREG9. While dispersion protocols are available, their applicability 
domain  for  toxicological  testing  needs  to  be  considered.  For  example,  the  NanoDefine  project 
[132],  developed  an  integrated  empirical  approach,  which  allows  identifying  a  material  as  a 
nanomaterial or not a nanomaterial according to the EC Recommendation. Nevertheless, care 
should be taken as the additives added to the dispersion medium may only be conditionally/not 
compatible with toxicological testing. 
As long as no test guidelines are available, detailed description and justification of the method 
including adequate controls (and reference materials, if possible) is required. 

Before  in vivo administration  of  a nanoform,  the  possible interactions with  the  administration 
vehicle need also to be determined and reported. For example, the nanoforms may adsorb on 
the  walls  of  the  delivery  system and  may  no  longer  be  available,  thus affecting  the  delivered 
dose [133]. The check of lab-ware retention capability is essential in order to make experiments 
reliable and improve the reproducibility of in vivo studies. 

The “aging” of nanoforms during the storage may also influence the outcome of a toxicity study. 
For example, the biological action of freshly prepared and aged silver nanoparticles is strongly 
different due to the different amounts of released ions [134]. 

To help to determine the concentration of nanoforms in biological samples, data from the OECD 
WPMN project “Guidance on the determination of concentrations of NPs in biological samples for 
(eco)toxicity studies” is expected to become available by the end of 2021. 

2.1.1.4 

 Biological Sampling 

Two OECD test guidelines, OECD TG 412 (Subacute Inhalation Toxicity: 28-day Study) and OECD 
TG 413 (Sub chronic Inhalation Toxicity: 90-day Study) have been updated to enable the testing 
and characterisation of effects of nanomaterials and are currently available. The OECD guidance 
document  GD  39  on  inhalation  toxicity  testing  has  also  been  updated  and  validated  for 
nanomaterial testing and should be consulted in the design of the studies [102]. 
The biological samples (e.g.organs and tissues, blood cells, body fluids) to be collected in the in 
vivo  toxicological studies  are  specified  in  the  relevant test  guidelines. However, if there is  an 
indication  that  the nanomaterials  would  be  distributed in  other  tissues not listed in  the OECD 
TGs,  then  the  collection  of  these  additional  tissues  (including  the  sampling  method)  is 
recommended. 
It  is  advised  to  handle  and  store  the  samples  adequately  to  allow  the  performance  of  later 
analysis (e.g. storage by chemical or physical tissue fixation for microscopy  [135], freezing for 
burden analysis ( [136], [137]. Caution should be exercised to use the most appropriate storage 
method  for  specific  tissues  as  the  available  information  is  based  on  a  limited  number  of 
nanomaterials and tissues. 

2.1.1.5  Use of Non-Animal Testing Approaches10 

Article  25  of  the  REACH  regulation  specifies  that  testing  on  vertebrate  animals  should  be 

8 http://www.nanogenotox.eu 
9 https://www.rivm.nl/en/international-projects/nanoreg 
10  This  advice  is  applicable  for  all  endpoints  relevant  for  human  health,  i.e.,  not  only  to  those  having  a  nanospecific 
entry in this document.  

 
 
 
 
 
 
 
 
 
 
 
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conducted only as a last resort, i.e. only when all other scientific available methods have been 
evaluated. Therefore, in order to fill any data gaps, there is a legal obligation to evaluate all the 
existing data and data from non-animal methods of hazard assessment before considering any 
new  tests  using  vertebrates.  Registrants  are  advised  to  keep  informed  about  ongoing 
developments and validation efforts of the OECD and the European Union Reference Laboratory 
for alternatives to animal testing (EURL ECVAM), as well as on the regulatory acceptance of new 
methods by ECHA [138]. Implementation of non-animal approaches for nanomaterials requires 
the  prior  consideration  of  all  available  information,  including  nanomaterial  characterisation, 
which is a critical requirement for grouping and read-across and quantitative structure–activity 
relationships  (QSARs).  In  addition,  relevant  and  reproducible  in  vitro  systems  may  be  used. 
Adverse  Outcome  Pathways  (AOPs)  relevant  to  nanomaterials  are  under  development  at  the 
OECD and offer new approaches to integrated assessment (https://aopwiki.org and and OECD 
EAGMST  https://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-
screening-and-toxicogenomics.htm). 

In silico models and read-across 

Regarding  the  use  of non-testing  data for nanomaterials, it is necessary  to  take  into account 
that: 

•  The  use  of in silico  models (e.g.  QSARs)  for  nanomaterials still needs to  be  validated. 
Thus, the use of such models for nanomaterials in deriving an assessment of hazard for 
humans  must  be  scientifically  justified  and  applied  on  a  case-by-case  basis  only. 
However, results from non-testing methods can be useful information in the context of 
weight  of  evidence6  or can  provide  essential  information  for  the  planning  of an  animal 
test. A range of in silico models, such as those used to determine nanomaterial kinetics, 
QSARs and physiologically based pharmacokinetic (PBPK) models have been developed 
for nanomaterials [139], [140], [141] [142], [143]. In silico modeling could be also useful 
to provide an initial prediction of a deposited and retained dose in the lung (e.g. the Multi-
Path  Particle  Dosimetry  (MPPD)  model).  As  with  any  in  silico  model,  care  must  be 
employed with regard to the applicability domains of these models. 

•  The  use  of  grouping  and  read-across  approaches  is  another  step  to  consider  before 
performing animal testing. Annex VI of the REACH Regulation as amended stipulates that 
“where technically and scientifically justified, the methodologies set out in Annex XI.1.5 
shall be used within a registration dossier when two or more forms of a substance are 
“grouped” for the purposes of one, more or possibly all the information requirements”. 
In this respect, it is advised to consider the ECHA guidance Appendix R.6-1 for nanoforms 
applicable to the Guidance on QSARs and Grouping of the Chemicals [31] when data on 
other (nano)forms11 of the same substance are available. Regarding read-across and/or 
grouping between (nano)forms of different substances the advice provided in the  ECHA 
Guidance Chapter R.6 on QSARs and Grouping of the Chemicals [4] and its nanospecific 
appendix [31] must be considered. Lamon et al. 2018 reviewed the available approaches 
and case studies on the grouping of NMs to read-across hazard endpoints. The grouping 
frameworks identified  hazard  classes  depending  on  PC  properties,  hazard  classification 
modules in control banding (CB) approaches, and computational methods, that can be 
used  for  grouping  for  read-across  [144].  Grouping  frameworks  are  currently  being 
developed, e.g. by the EU project GRACIOUS, to substantiate nanospecific read-across 
and grouping also for regulatory purposes (https://www.h2020gracious.eu/). 

In vitro studies 

This section discusses the possible contribution of in vitro data to the adaptation possibilities. 

In accordance with Article 13(1) of the REACH regulation, “Information on intrinsic properties of 

11 The term (nano)form intends to cover nanoforms and non-nanoforms of the substance 

 
 
 
 
 
 
 
 
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substances may be generated by means other than tests, provided that the conditions set out 
in Annex XI are met”. The information from in vitro tests should always be considered before 
performing an animal test. 
It  has  been  shown  that  in  vitro  assays  (e.g.  [145],  [146],  [147])  that  are  not  yet  formally 
validated but can be demonstrated to be scientifically valid, may be useful on a case-by-case-
basis for the assessment of nanomaterials. They can be used as part of a weight of evidence 6 
approach  that  involves  thorough  physicochemical  characterisation  of  NMs,  in  vitro  screening 
tests, ‘-omics’, the use of non-testing approaches (in silico models, read across) and the use of 
OECD  and  EURL  ECVAM  validated/ approved  in vitro  methods. References  [148],  [89],  [149] 
include provisions for the acceptance of data from in vitro studies. 

According  to  OECD  [150]  for  in  vitro  testing  the  “Characterisation of  the  materials  should  be 
undertaken  in  the  cell  culture  medium  used  both  at  the  beginning  of  treatment  and,  where 
methodologies exist, after treatment. The intent when applying nanomaterials to a cell culture 
medium is to create conditions that are comparable, to the extent possible, with the biological 
and  physiological  conditions  within  the  in  vivo  system”.  Considerations  for  in  vitro  testing  of 
NMs, which should lead to increased reliability and relevance are given in the scientific literature 
[151]. Drasler et al. 2017 [151], concluded that the most important criteria to produce reliable 
and  robust  data  from  in  vitro  nanotoxicological  assays  are:  (i)  detailed  NM  characterization, 
including physicochemical properties before, during and after the testing, (ii) use of comparable 
and realistic dose metrics and test conditions, and (iii) implementation of chemical positive and 
negative controls and reference NMs allowing for comparison between studies, both intra- and 
interlaboratory. 

An  analysis  of  the  applicability  of  in  vitro  methods  to  NMs  and  recommendations  for  the 
adaptations needed is available [152]. 

Advisory note on the consideration of assay interference 

Various nanomaterials  have  on  occasion  been found  to interfere with  several  commonly used 
assays  for  determining  their  cellular  or  toxic  effects.  For  example,  some  nanomaterials  may 
contribute to the absorbance or fluorescence of colorimetric or fluorometric assays. In addition, 
due  to  their  large  surface  area,  nanomaterials  may  bind  to  assay  components  including  the 
substrates  (e.g.  CNT  with  the  reagent  in  MTT  2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-
tetrazolium bromide assays; [153], Fe3O4 with WST-1, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-
(2,4-disulphophenyl)-2H-tetrazolium  )  [154]  or  the  biomarker  being  measured,  (e.g.  lactate 
dehydrogenase  (LDH)  and  cytokine  proteins;  see  for  example  [155]).  Please  note  that  other 
factors such as coatings or impurities may also have an influence on the assay. 
A summary list of potential sources of nanomaterial interference with commonly used  in vitro 
assays has been developed by Kroll et al. [156] and is reproduced in the table below. 

 
 
 
 
 
 
 
 
 
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Table 3: Potential sources of nanomaterials interference with commonly used assays 

Cytotoxicity 
assay 

Detection principle 

Nanomaterials 
interference 

Altered 
readout 

Nanomaterials 
type 

Cell viability  

MTT/WST-1 

Colorimetric  
detection of  
mitochondrial  
activity  

Adsorption of  
substrate  

Reduced  
indication of 
cell  
viability  

Carbon  
nanomaterials 

LDH  

Colorimetric  
detection of LDH  
release  

Inhibition of 
LDH  

Reduced  
indication of  
necrosis  

Trace metal-
containing  
nanomaterials   

Annexin V/ 
Propidium  
iodide  

Fluorimetric  
detection of 
phosphatidylserine 
exposure (apoptosis  
marker)  
Propidium iodide 
staining of DNA  
(necrosis marker)  

Ca2+-depletion  

Dye adsorption  

Reduced  
indication of  
apoptosis  

Reduced  
indication of  
necrosis  

Carbon  
nanomaterials  

Neutral red  

Colorimetric  
detection of intact  
lysosomes  

Dye  
adsorption  

Reduced  
indication of 
cell viability  

Carbon  
nanomaterials 

Caspase  

Fluorimetric  
detection of  
Caspase-3 activity 
(apoptosis marker)  

Inhibition of  
Caspase-3  

Reduced  
indication of  
oxidative 
stress  

Carbon  
nanomaterials  

Stress response 

Dichlorofluorescein 
(DCF)  

Fluorimetric  
detection of ROS  
production  

Fluorescence  
quenching  

Reduced  
indication of  
oxidative 
stress 

Carbon  
nanoparticles  

Inflammatory response  

ELISA (enzyme-
linked 
immunosorbent 
assay) 

Colorimetric  
detection of  
cytokine  
secretion  

Cytokine 
adsorption  

Reduced  
indication of  
cytokine  
concentration  

Carbon  
nanoparticles  
Metal oxide  
nanoparticles  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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It  should  be  noted  that  the  above  list  is  not  exhaustive  and  the  potential  for  inhibition  or 
enhancement  of  test  results  should  always  be  investigated.  The  agglomeration,  dispersion 
and/or dose may influence the outcome of the test. 

Assay interference is not limited to colorimetric assays. Interference with medium components 
such as medium depletion or corona formation by adsorption of essential nutrients or interaction 
with buffer salts, resulting in increased disssolution of nanoparticles was reported [109], [157]. 
Within some standard methodologies, the method requires the use of spiked samples (addition 
of a known reference/control sample) to test for inhibition or enhancement of the spiked control. 
This is evaluated by assessing the measured value against the expected value, which should be 
a cumulative value of the spike and of the sample. 

Assay  interference  should  always  be  investigated  wherever  possible,  irrespective  of  standard 
method requirements. In case it would not be possible, this has to be justified. Furthermore, for 
many of the studies reported, it is not possible to ascertain whether the assays were adequately 
controlled to assess for interferences. Thus, if other methods for assessing interferences are not 
available, as a general precaution, it is reccommended to use more than one assay to assess 
the studied endpoint or effect, as for example advised by Landsiedel et al. [158] for genotoxicity 
studies. The potential for inhibition or enhancement of the test result may impact the validity of 
relevant in vitro test methods. For example carbon nanotubes are suspected to interfere with 
the MTT assay [159] or WST-1 assay [154] and this may cause issues with tests such as OECD 
TG 431/EU B.40 bis Human Skin Model tests (EPISKIN™, EpiDerm™) which uses the MTT assay. 
However,  knowledge  on nanomaterial  assay  interference  is incomplete  and  so  precautions  to 
ensure the validity of an assay, such as the above-mentioned use of control spikes  should be 
used. 

Due to the potential for interference resulting in misleading results in numerous assays, utmost 
care should be taken in testing for such interference and appropriate controls must be used for 
the assays. 

2.2  Specific advice for individual endpoints 

Acute toxicity 

The parent guidance Chapter R.7a Section 7.4 [3] provides the general testing strategy for acute 
toxicity.  The  advice  provided  in  the  parent  guidance  should  be  followed  together  with  the 
recommendations given in this section. 
According to the revised REACH Annex VII, Section 8.5.1 Column 2: “For nanoforms, a study by 
the oral route shall be replaced by a study by the inhalation route (8.5.2), unless exposure of 
humans via  inhalation is  unlikely,  taking  into  account  the  possibility  of  exposure  to aerosols, 
particles or droplets of inhalable size”. 

Hence, when the dossier covers nanoforms,  testing by a route other than inhalation needs to 
be  justified.  The inhalation route  is appropriate if  exposure  of  humans  via inhalation is likely 
taking into account the possibility of exposure to aerosols, particles or droplets of an inhalable 
size. Guidance on the interpretation of physico-chemical data regarding respiratory absorption 
can be found in Table R.7.12-2 in parent guidance Chapter R.7c [160]. The information derived 
from  in  vitro  models  ranging  from  cell  lines  grown  in  monoculture  to  lung-on-a  chip  (LOC) 
microphysiological systems and  in vitro airway models aiming to characterise the cellular and 
molecular mechanisms responsible for the effects of inhaled toxicant exposure  [161] could be 
useful in a WoE approach and provide an initial insight in the toxicity of the substance. 

In the event there is no exposure of humans via the inhalation route then the oral route (as for 
most substances) is the default first choice of administration at Annex VII.  

 
 
 
 
 
 
 
 
 
 
 
 
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The current acute inhalation test guidelines employ death as an endpoint (OECD TG 403 and TG 
436) or replace lethality as an endpoint with evident toxicity as ﬁxed concentration procedure 
(FCP) (OECD TG 433). It is however, acknowledged that none of these test guidelines contain 
provisions specific for nanomaterials. Nevertheless, the recently updated OECD GD 39 provides 
some  advice  applicable  for  the  acute  inhalation  in  case  of  testing  nanomaterials  [102].  As 
sometimes for low density materials it is not feasible to reach the top dose, the physico-chemical 
properties including tested material density should be taken into account when dosing for the 
acute studies. The OECD GD 39 emphasizes that “Knowledge of dustiness and particle size for 
solid  test  chemicals  will  allow  for  selection  of  the  ideal  testing  approach  and  starting 
concentration that will enhance respirability (e.g., through the use of micronization)” and that 
“The particle size distribution should be determined at least once during a single exposure study 
for each concentration level using an appropriate method of measurement”. OECD GD 39 also 
states  that  “Aerosol  testing  at  greater  than  2  mg/L  should  only  be  attempted  if  a  respirable 
particle size can be achieved.” For nanomaterials, the degree of agglomeration increases with 
dose. The degree of agglomeration affects the aerodynamic diameter and thereby the deposition 
in the lung. For oral exposure, a higher degree of agglomeration can reduce the absorption. The 
consequences  of  the  dose  on  agglomeration  should  therefore  be  taken  into  account  when 
designing and interpreting acute toxicity tests.  

Repeated dose toxicity 

Annexes VIII-X, Section 8.6.1. and Annexes IX-X, Section 8.6.2., Column 2 of REACH Regulation 
(EC) No 1907/2006 amended (underlined text) for nanoforms’ testing, provide that: 
“For nanoforms toxicokinetics shall be considered including recovery period and, where relevant, 
lung clearance.”  

and under “Further studies shall be proposed by the registrant or may be required by the Agency 
in accordance with Articles 40 or 41 in case of:” third indent: 

“indications of an effect for which the available evidence is inadequate for toxicological and/or 
risk  characterisation.  In  such  cases  it  may  also  be  more  appropriate  to  perform  specific 
toxicological  studies  that  are  designed  to  investigate  these  effects  (e.g.  immunotoxicity, 
neurotoxicity, and in particular for nanoforms indirect genotoxicity),”. 

In contrast to acute toxicity testing, the information requirements for repeated dose testing of 
nanoforms  do  not  prescribe  inhalation  as  default  route.  However,  Column  2  of  Annex  VIII, 
Section 8.6.1. and of Annex IX, Section 8.6.2. specify that “Testing by the inhalation route is 
appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure 
of  the  substance  and/or  the  possibility  of  exposure  to  aerosols,  particles  or  droplets  of  an 
inhalable size”. Therefore, inhalation can, in most situations, be seen as the most likely route of 
exposure to nanomaterials, and thus the following focuses on inhalation testing. 

The  testing  protocols  to  be  followed in repeated  dose  testing  by  inhalation are  OECD  TG 412 
[162] and TG 413 [163] together with the guidance provided in OECD GD 39 [102]. When testing 
poorly soluble solid aerosols that are likely to be retained in the lungs, Option B in the Annex of 
OECD TG 412/413 should be used. OECD GD 39 defines the poorly soluble particles (PSPs) as 
“Solid aerosol particles deposited in the lung that do not undergo rapid dissolution and clearance” 
and states that “A poorly soluble material is generally understood as having a solubility of less 
than 0.1 g dissolved in 100 ml dissolvent within 24 hours.” [102]. A recent expert group [15] 
on the hazards and risks of poorly soluble particles of low toxicity, concluded that  PSPs can be 
defined  as  particles  for  which  their  alveolar  macrophage-mediated  clearance  rate  is  not 
shortened  by  their  dissolution  rate  in  the  lung.  For  chemicals  that  accumulate  in  the lung  or 
translocate into specific accumulating organs following repeated exposures, toxicokinetic shall 
be considered, as the accumulated dose is partly a function of clearance.  Therefore, for these 
nanoforms the lung clearance investigation is considered relevant. This is also in agreement with 

 
 
 
 
 
 
 
 
 
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OECD TG 412/413, which require the measurements of lung burden when a range-finding study 
or other information demonstrates that a poorly soluble aerosol is likely to be retained in the 
lung. For PSPs, the rat lung burden is an important issue to consider in the toxicological outcome 
and therefore a special chapter within this section is included.  

For fibre-like particles, in addition to the overload of macrophages, it should be considered that 
the  pathogenicity  of  the  fibres  may  play  a  role in  their  toxicity  [164]  and  that  they  may  also 
cause impaired lung clearance below overload. Fibre pathogenicity is characterised by frustrated 
phagocytosis and/or migration of fibres into the pleural, leading to activated macrophages, which 
stimulate an amplified proinflammatory cytokine response from the adjacent pleural mesothelial 
cells [165]. This mechanism of toxicity is not addressed further in this guidance. The OECD GD 
39  specifies  that  for  chemicals  with  fibre  geometry,  additional  specific  observations,  such  as 
interstitial and pleural detection and possibly quantification, depending on fibre dimensions, may 
be needed when performing an OECD TG 412/413 study [102]. 
When  considering  the  testing  strategy  for  repeated  dose  toxicity  (Section  7.5.6.  of  REACH 
Guidance R.7.a. [3]) of nanoforms it should be noted that: 

•  Especially  for  workers  (and  in  some  cases  for  consumers,  e.g.  in  case  of  sprayable 
products)  inhalation  is  the  most  likely  route  of  exposure  to  nano(particles),  nano 
aerosols and  dust.  Hence,  the  repeated  dose  toxicity studies should  be  performed via 
inhalation,  unless  there  is  convincing  information  (e.g.  uses,  dissolution  rate)  that 
justifies another route. Any modification of the protocols described in OECD TG 412 and 
413 ( [162] and [163]) should be justified; 

•  When dose range finding studies or repeated dose studies are performed, for PSPs, it is 
recommended to collect additional toxicokinetic data as described in Appendix R7-2 for 
nanomaterials  applicable  to  Chapter  R7c  Endpoint  specific  guidance).  In  addition,  to 
make full use of the test, if there is a particular concern it is recommended to address it 
within the study design (e.g. accumulation, specific organ toxicity, etc.).  

•  When testing PSPs, it may be necessary for a range-finding study to be longer than 14 
days to allow for a robust assessment of test chemical solubility and lung burden [102]. 
Dissolution data in simulated lung fluid and lysosomal fluid are important for an informed 
decision  before  starting  the  animal  studies.  Annex  VII,  section  7.7.  column  1,  of  the 
REACH  Regulation  specifies,  beside  the  water  solubility  data  requirement,  that  “for 
nanoforms,  in  addition  the  testing  of  dissolution  rate  in  water  as  well  as  in  relevant 
biological and environmental media shall be considered.”. Therefore the information on 
dissolution must in principle be available before the in vivo testing. 

•  Exposure concentrations should be selected to cover the entire range of lung burdens, 
i.e., concentrations ranging from those which do not delay clearance to those which do 
delay clearance. Similarly, post-exposure periods should not be markedly shorter than 
one elimination half-time of the particulate test material [102]. 

•  The  doses  to  be  used  in  repeated  dose  inhalation  studies  should  not  exceed  the 
maximum tolerated concentration. However, the doses in hazard tests needs to cover a 
range  of  doses  to  be  able  to  detect  any  potential  hazard.  This  means  the  inhalation 
studies with PSPs should include  an exposure concentration high enough to produce a 
toxic effect or an overload of particle clearance inducing lung inflammation, as well as 
exposures not causing lung particle overload. A similar approach was proposed for the 
design of chronic inhalation studies [15]. OECD TG 412 [162] and TG 413 [163] provide 
advice  on  doses  to  be used.  This  includes  the  provision  that  the  highest  dose should 
elicit  unequivocal  toxicity  without  causing  undue  stress  to  animals  or  affecting  their 
longevity. 

 
 
 
 
 
 
 
 
 
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•  For  nanomaterials,  the  degree  of  agglomeration  increases  with  dose.  The  degree  of 
agglomeration affects the aerodynamic diameter and thereby the deposition in the lung. 
For  oral  exposure,  a  higher  degree  of  agglomeration  can  reduce  the  absorption.  The 
consequences of the dose on agglomeration should therefore be taken into account for 
designing and interpreting repeated dose toxicity tests. In silico modeling could be useful 
to provide an initial prediction of deposited and retained dose in the lung. For instance, 
the Multi-Path Particle Dosimetry  (MPPD)  model   can  be  used  to assess  the  influence 
that the characteristics of the aerosols (MMAD, GSD, solubility, density, hygroscopicity) 
can have on the dose [166]. 

• 

Information from the dose–range finding studies and lung burden data are useful for the 
interpretation of a study outcome.  Knowing the retained lung burden during exposure 
and  at  different  time  points  post-exposure,  the  following  can  be  determined: 
nanomaterials lung clearance and retention kinetics, critical dosimetric comparisons to 
clearance  and  retention  data  of  well-established  benchmark  materials  (allowing 
comparative Dose - Response relationships to be analysed) [167]. The selection of PSP 
benchmark materials should be justified. Humans and rodents differ significantly in their 
biokinetic functions so the effects of nanomaterials observed in rodents do not directly 
translate to humans. Nevertheless, the lung burden data cannot be used to exclude the 
relevance of toxicological findings in the experimental animal for human risk assessment 
[162], [163].  A  detailed  discussion  on  the concept  of  rat lung  burden of  PSPs and its 
impact in the toxicological assessment is provided in section 2.2.2.1. 

•  Although lung burden measurement is mandatory at only one post-exposure observation 
(PEO)  period  in  Option  B  (at  PEO-1)  of  OECD  TGs  412/413,  the  TGs  provide  that  a 
minimum of two lung burden measurements are necessary when investigating clearance 
kinetics. If the use of two post-exposure time points is considered sufficient, lung burden 
measurements may be performed at PEO-1 (main study) and at PEO-2 (recovery group) 
only, if timing for evaluation of recovery and lung clearance can be aligned. Lung burden 
should  be  measured  for  all  concentrations  [102].  Lung  burden  measurement  at  three 
time  points  allow  curve  fitting  on  post-exposure  clearance  kinetics.  The  need  for 
additional post-exposure observations, the duration of the post-exposure interval and the 
timing of  the  post-exposure  observations  (PEOs) are  determined  by  the study  director 
based upon results from, among others, the range-finding study. 

•  The data on lung burden and clearance are also important in the context of read-across 
of  the  hazard  data  from  studies  using  different  nanoforms  of  the  same  substance.  
Incorporating measurement of retained lung burden in the study design allows a coupling 
of  subchronic  and  subacute  inhalation  data  with  other  appropriate  test  data  (from 
shorter-term inhalation,and cellular and  acellular assays)  which can  facilitate  grouping 
and may result in saving animals [167]. For example for nanoforms with different particle 
sizes, the same external concentrations can result in differences in retained dose [167]. 
Conversely,  different  external  concentrations  can  result  in  the  same  retained  dose  for 
different particle sizes. 

•  Assessment  of  organ  burden  in  potential  target  tissues  other  than  lung  should  be 
considered  to  estimate  the  systemic  availability.  As  a  first  indication  of  translocation, 
detection and burden measurements in local lymph nodes may be determined in addition 
to lung burden measurements. To monitor the fate and effects of PSPs in the body it is 
recommended to collect the samples at several time points and/or from different organs 
and tissues. Data from range-finding studies, if proven robust, could be used to determine 
the  appropriate  sampling  times  in  order  to  investigate  these  toxicokinetic  parameters 
(i.e. nanomaterials  burden  in secondary  organs).  If  satellite animals  are  used for lung 
burden,  the  same  animals  should  be  used  for  any  other  organ  burden  assessment 
deemed necessary by the study director. In ISO TR 22019 [94] the liver, spleen, lung, 
brain,  kidney,  lymph  nodes  at  the  organ  of  entry,  and  bone  marrow  are  considered 

 
 
 
 
 
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relevant organs for the toxicokinetics of NPs. Other relevant tissues include reproductive 
organs (uterus and testis) and adipose tissue [168]. The issue of bioaccumulation and 
organ  burden  is  further  discussed  in  section  2.1.3.  of  Appendix  R7-2  to  Chapter  R.7c 
[169]. 

•  Since  the  lower  respiratory  tract  (i.e.,  the  alveoli)  is  the  primary  site  of  deposition 
(depending on agglomerate size) and retention for inhaled nanoparticles, bronchoalveolar 
lavage (BAL) analysis is a useful complementary technique to histopathology predict and 
quantitatively estimate pulmonary inflammation and damage (for further information on 
BAL parameters please see OECD TGs 412 and 413). BAL analysis allows dose-response 
and  time-course  changes  of  alveolar  injury  to  be  suitably  investigated.  Therefore,  for 
nanomaterials testing, it is mandatory to include BAL analysis (further details are given 
in  Section R.7.5. (repeated  dose  toxicity)  of  Chapter R7.a  of  the  parent  guidance [3]. 
BAL fluid should be analysed for all the concentrations [102].  

• 

It  is  strongly  advised  to  use  more than  one  dose-describing  metric  and  to  include  the 
mass metric. The choice of method(s) selected should be justified, as also explained in 
Section 2.2.2.1.1. 

OECD  TG  412,  TG  413,  and  GD  39  should  be  consulted  when  designing  developmental  and 
reproductive toxicity studies using TG 422, TG 421, TG 443 and TG 414 with exposure by the 
inhalation route. 

With  regard  to  the  ‘old’  studies  performed  with  nanoforms  prior  to  the  update  of  OECD  TG 
412/413,  several  conditions  need  to  be  fulfilled  in  order  for  these  studies  to  be  considered 
appropriate: 

•  The  test  material  is  well  characterised  (i.e.  size  distribution,  shape  and  aspect  ratio, 
surface treatment and specific surface area is known) and ensure the representativeness 
for the registered NF/set of NF, and  

•  The  parameters  from  the  testing  guidelines  valid  at  the  time  when  the  study  was 
performed are covered. In case that only the respiratory system has been investigated 
and not all the organs listed for histopathology by the testing guidelines, the study cannot 
be seen as equivalent to an OECD 412 or 413. 

If the study fulfils the conditions above, then it can be considered for the use under WoE. 
The updated  testing  guidelines specify  for  the  substances  likely  to  be  retained in  the lung 
that  BAL  and  lung  burden  measurements  at  specified  PEO  should  be  performed.  For  the 
studies  performed  prior  this  update,  for  which  this  information is  missing,  the information 
must be substantiated using weight of evidence6 approach with further data:  

•  dissolution studies in artificial lung lining fluid and lysosomal fluid.  
• 

in cases where no toxicity is seen in an old repeated dose study, the lack of toxicity seen 
in  vivo  can  be  substantiated  with  data  from  studies  assesing  cytotoxicity  and 
inflammation  markers  with  the  registered  nanoform  in  relevant  cell  lines.  Overall,  the 
cytotoxicity  assays  should  always  consider  the  cell  type  and  nanomaterials  used, 
therefore a generalized recommendation cannot be given  [151].  Various factors could 
have  contributed  to  the  “no  effects”  outcome  seen  in  the  old  in  vivo  study  such  as 
agglomeration,  dispersion  protocol,  adherence  of  the  tested  substance  to  the  delivery 
system etc. 

For the oral studies (OEC 407, OECD 408, OECD 421/422) the same principles apply. If a proper 
characterisation  of  the  test  material  (nanoform)  is  provided  (i.e.  size  distribution,  shape  and 
aspect ratio, surface treatment and specific surface) and ensure the representativeness for the 
registered NF/set of NF, and if the study has been conducted according to the testing guidelines 
(including analysis of all the organs listed in the TG) then the study can be considered for the 

 
 
   
 
 
 
 
 
 
 
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use of WoE.  The information from such a study must be substantiated with further data such 
as  dissolution studies  in  artificial  gastrointestinal  fluid and lysosomal  fluid.  In cases  where  no 
toxicity is seen in the repeated dose study, cytotoxicity studies in relevant  mammalian cell lines  
could substantiate the lack of intrinsic toxicity seen in vivo. If an available oral study complies 
with all the criteria listed above, it can be used only for the relevant route of exposure, i.e. oral. 

2.2.2.1  Advisory note on the consideration of lung burden within inhalation 

toxicity assessment 

This  section  describes  the concept  of  rat  lung  burden  of  PSPs  and  the associated effects,  the 
differences  between  species  and  the  extrapolation  of  the  results  to  humans.  Care  should  be 
taken  when  interpreting  lung  burden  in  the  context  of  human  risk  assessment.  Lung  effects 
observed in animals exposed to PSPs by inhalation should be considered relevant for the hazard 
in humans unless it can be clearly substantiated otherwise. 

Results  from  inhalation  studies  in  rats  have shown  that  the  PSPs  can  induce  serious  adverse 
pulmonary  effects  if  inhaled  in  high  concentrations  due  to  material  accumulation,  as  lung 
clearance mechanisms are not able to remove materials at the same time or at a higher rate as 
the dose is delivered. This condition named “lung overload”, occurs when the retained particle 
burden in the lung exceeds a certain threshold [170]. A recent expert group [15] on the hazards 
and  risks  of  poorly  soluble  particles  with  low  toxicity  (PSLT)  proposed  the  following  expert 
consensus  definition  “Lung  Particle  Overload  is  a  phenomenon  of impaired  clearance  in which 
the deposited dose of inhaled PSLT in the lung overwhelms clearance from the alveolar region 
leading to a reduction in the ability of the lung to remove particles. Lung Particle Overload results 
in an accumulation of particles greater than that expected under normal physiological clearance. 
This  definition  is  relevant  for  all  species  (not  just  rat).  This  definition  is  independent  of  the 
underlying mechanism(s) (e.g. macrophage mobility impairment). A key issue is that increased 
particle  retention  due  to  large  lung  burdens needs  to  be  differentiated  from  that  due  to high 
cytotoxicity particles (e.g. quartz).”During prolonged exposure of rats to PSPs, the lung burden 
of particles increases until equilibrium is reached between deposition and clearance of particles 
[171] as shown by the curves A, B and C in Figure 5 This equilibrium can be reached very fast 
or may take up to many days. Below the lung overload threshold, particles are cleared via normal 
mechanisms at a constant clearance rate, in general generating little or only a minor or reversible 
response (exposure concentrations in curves A and B). 

 
 
 
 
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Figure 5: Schematic representation of the relationship between retained lung burden 
and duration of exposure leading to the phenomenon of lung overload. 

Curves A, B, and C are associated with progressively increasing exposure doses. If the exposure 
level  is  sufficiently high  and  the  duration  of  exposure sufficiently  long,  alveolar  macrophage-
mediated clearance of particles can be overwhelmed. When this occurs, the retained lung burden 
increases linearly with further exposure (curve C*), reproduced from [171]. 
Once the threshold has been reached, the clearance mechanisms of the lung become overloaded. 
This  is  typified  by  a  progressive  impaired  particle  clearance  from  the  deep  lung,  reflecting  a 
breakdown in alveolar macrophage (AM)-mediated dust removal due to the loss of AM mobility 
[170].  This  is shown  in  the C* curve  of  Figure  5 whereby  at  the  point  of  threshold,  particle 
retention increases rather than an equilibrium being established (as demonstrated by the dashed 
line).  
The result of this net increase in particle accumulation is persisting lung inflammation, cessation 
of alveolar-mediated clearance and an increase in accumulation of particle laden macrophages 
and/or  free  (non-phagocyted)  particles  within  the  lung  alveoli.  The  potential  progression  of 
inflammatory  reactions  toward  a  granulomatous  type  in  rats  was  found  to  depend  on  the 
exposure duration and the level of the particle (surface) burden in the lung [172] as well as of 
the volumetric load of alveolar macrophages [173]. 

The situation of lung burden is most commonly associated with repeated inhalation exposure of 
rats to PSPs but it can also occur after single or repeated instillation of PSPs into the lung (due 
to a high deposition fraction as a result of direct instillation) or possibly as a result of a single 
massive inhalation exposure  [174]. Since this phenomenon occurs at relatively high exposure 
levels of respirable PSPs it is often argued that the observed adverse effects are a product of 
the  lung  burden  caused  by  experimental  conditions  and  not  always  a  true  reflection  on  the 
intrinsic toxic potential of the particles to cause inflammation, fibrosis and cancer. Exposure to 
highly reactive or toxic particles may cause inflammation, fibrosis and cancer at lower exposure 
levels (non-overload conditions) due to intrinsic properties of the particles themselves. 

Care should be taken when interpreting lung burden in the context of human risk assessment. 
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for  the  hazard  in humans  unless  it can  be clearly substantiated  otherwise. When  designing  a 
new  study,  the  doses  to  be  used  in  repeated  dose  inhalation  studies  should  not  exceed  the 
maximum tolerated dose. However, the doses in hazard tests needs to cover both lower doses 
(seen  as  a  valid  approach  to  determine  the  lung  overload  together  with  the  time-course 
measurement of actual lung particle burdens [15]) as well as higher doses which enable to detect 
any potential hazard so that the study can fulfill its regulatory purposes. OECD TGs 412 [162] 
and 413 [163] provide advice on dosages to be used. This includes the provision that the highest 
dose should elicit unequivocal toxicity without causing undue stress to animals or affecting their 
longevity. 

In  the  studies  performed  with  PSPs  the  measurement  of  changes  in  lung  burden  over  post-
exposure time(s) provides essential information on lung clearance and allows clarification of  the 
deposited vs the exposed particle amount. Different imaging techniques may also be used for a 
semi-quantitative assessment of the PSPs in the tissue [175]. The assessment of the dissolution 
potential as an indicator for biopersistence can also be done using in vitro systems [92]. 
Information on clearance and biopersistance is important in the context of toxicokinetics, read-
across and weight of evidence. 

Rat  is  currently  considered  the  most  sensitive  species  for  inhalation  toxicity  testing  for 
nanoparticles  [176].  However,  as  it  can  be  difficult  to  interpret  the  findings  of  overload  of 
alveolar  macrophages  in  rat  studies,  a  better  understanding  of  the  rat  lung  burden  and  its 
relevance to humans is needed. A recent study that analysed the issues of impaired clearance 
and the relevance for human health hazard and risk assessment [177] pointed out that while 
rats  and  humans  have  a clear  difference  in  lung  clearance  kinetics  of PSPs,  the  fact  that  the 
appropriate  dose  metric  often  is  unknown  and  the  level  of  clearance  impairment  is  seldom 
assessed  makes  it  difficult  to  relate  the  effects  observed  to  an  impaired  clearance.  Several 
studies have assessed the responses to lung overload in different species, and the relevance of 
this data for humans. For instance, in a comparative study assessing the long-term pulmonary 
response of rats, mice and hamsters to inhalation of ultrafine grade titanium dioxide [137], the 
same air concentrations caused overload effects in rats and mice but not in hamsters. Also, the 
inflammatory  and  pathological  responses  were  less  severe  in  mice  than  in  rats  and  they 
diminished with time irrespective of the similar lung burdens  [137], [178]. However, in relation 
to  the  relevance  of  animal  data  for  humans,  other  studies  have  pointed  out  that  the  lung 
responses  to high lung  burdens  of PSP  of low  toxicity can  be  qualitatively  similar  in  rats  and 
humans [179]. Based on experience with exposure of coal miners, a primate-specific interstitial 
particle sequestration compartment is hypothesed [180]. Borm et al [173] discuss whether this 
mechanism could explain why humans, in contrast to rats, seem not to have an increased risk 
of  lung  cancer  under  lung  overload  conditions  [173].  Nevertheless,  there  seems  to  be  some 
conditional  evidence  for  particle  overload  associated  with  impaired  clearance  in  coal  miners 
[173].  While  lung  inflammation  after  PSPs  exposure  does  not  necessarily  lead  to  tumour 
formation  in  humans  and  carcinogencity  [15],  fibrosis  is  however,  a  response  seen  upon 
exposure  to  PSPs  in  both  rats  and  humans,  though  not  in  mice  [177].  Independent  of  the 
underlying mechanism(s) (e.g. macrophage mobility impairment), the concept of lung overload 
is relevant for all species (not just rat) and under overload conditions, the rat was considered 
“not to be unique in its inflammatory, hyperplastic, and fibrotic responses to PSLT” albeit the rat 
is more sensitive [15]. 

Therefore, the use of existing rodent data, obtained after exposure to high doses of PSPs, cannot 
automatically be dismissed as irrelevant for humans in the context of risk assessment and the 
interpretation of such data should be approached with caution. In the case of adverse effects 
observed in animals under overload conditions, the relevance for humans has to be assumed a 
priori; any claimed non-relevance for humans must be supported by data. 
For further information, Bos et al. 2019 [177] and Driscol and Borm, 2020 [15] provide a detailed 
analysis on the pulmonary toxicity in rats following inhalation exposure to PSPs and discuss the 
relevance of rat hazard studies for human health and risk assessment. There are also several 
other review articles covering the subject of lung overload such as Miller  [171], who provided 

 
 
 
 
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an  in-depth  discussion  of  particle  deposition,  clearance  and  lung  overload.  Borm  et  al  [181] 
discussed the importance of overload in the context of risk assessment whereas in an editorial 
of  Borm  et  al,  [173]  the  state  of  the  art  concerning  lung  particle  overload  concepts  is 
summarized.  These  reviews  also  present  different  views  on  how  to  assess  lung  overload  and 
how to interpret the data and emphasize the fact that the topic is still under debate. 
In  conclusion,  based  on  the  current  understanding,  lung  effects  observed  in  experimental 
animals  exposed  to  PSPs  by  inhalation  should  be  considered  relevant  for  humans.  When 
designing a new study, the doses in hazard tests needs to cover both, lower doses as well as 
higher doses to enable to detect any potential hazard and to fulfill its regulatory purposes. 

2.2.2.1.1  Metrics 

The question of which dose metric best describes the association between deposited dose in the 
lung,  and  subsequent  inflammation  and  impaired  clearance  function  is  particularly  relevant. 
There have been several suggested metrics, but volumetric load of AM and surface area appear 
to  be  the  most  relevant  [173]  in  interpreting  lung  overload-related  as  well  as  other  adverse 
effects and in establishing limit concentrations. Morrow et al. [170] hypothesised that overload 
begins when the particulate volume exceeds approximately 60  μm3/AM (which produces a 6% 
increase in the average alveolar macrophage volume) and that total cessation of AM-mediated 
clearance occurs when the particulate volume exceeds 600 μm3/AM (producing a 60% increase 
in the average alveolar macrophage volume). Extending the Morrow concept, Pauluhn ( [182], 
[183]) modelled a generic particle displacement volume threshold for agglomerated PSPs. 
Oberdoerster et al. [184] suggested that the particle surface area better correlates the overload 
with impaired clearance. Several studies suggest that, particle surface area correlates well with 
induced pathogenic events in lung ( [181], [185], [186]). In a study by Tran et al. [186] data 
from a series of chronic inhalation experiments on rats with two poorly soluble dusts (titanium 
dioxide and barium sulphate) was analysed. The results indicated that when lung burden was 
expressed as particle surface area, there was a clear relationship with the level of inflammation 
and  translocation  to  the  lymph  nodes.  Most  usefully,  based  on  the  shape  of  the  statistical 
relationship for lung response to particles, the authors suggested the presence of a threshold at 
approximately 200–300 cm2 of lung burden for “low-toxicity dusts” in rats. 

Whilst some studies indicate mass as a less sensitive indicator of lung overload [187], the mass 
concentration is still important because there is already a large body of data and research on 
the exposure to and toxicity of particles using the mass-based metric. Therefore, for the sake of 
comparison(s), the mass concentration should always be reported. 

Other studies ( [188]) found that the particle number or the number of functional groups in the 
surface of nanoparticles ( [189], [190]) was the best dose metric.  

The most relevant dose metric seems to vary depending on the specific nanoparticle in question. 
Surface areas, mass, and the particle number should be reported in order to establish the dose 
metric  that  best  describes  the  association  between  deposited  dose  in  the  lung,  overload 
conditions and the subsequent pathogenic effects and in order to establish the dose metric most 
relevant for risk assessment.   

It is therefore vital to fully characterise test materials, so that the measured response can be 
retrospectively  correlated  with  multiple-dose  metrics,  without  the  need  for  repeat  testing.  In 
general, the more metrics are reported the better. 
In conclusion, it is strongly advised to use more than one different dose-describing metric and 
to  justify  the choice  of  the selected  methods.  However, since  all  regulatory  limits  and  effects 
values are based on mass concentration, this is still default and needs to be reported. 

 
 
 
 
 
 
 
 
 
 
 
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2.2.2.1.2  Main recommendations for lung burden 

•  Data from existing studies performed with high doses of PSPs showing adverse effects 

cannot automatically be dismissed as irrelevant for humans. 

•  When  planning/performing,  new  studies,  the  use  of  excessively  high  doses  should  be 
avoided  (in  order  not  to  exceed  the  maximum  tolerated  dose).  However,  the  doses in 
hazard  tests should  include  an exposure concentration high  enough  to produce  a  toxic 
effect  or  an  overload  of  particle  clearance  inducing  lung  inflammation,  as  well  as 
concentrations not causing lung particle overload. 

•  Lung burden data provide useful information on the pulmonary (retained) dose as well 
as  on  clearance  behaviour  and  may  support  the  read-across  and  weight  of  evidence 
approaches. Lung burden and BALF must be measured for all concentrations [102]. 
•  The most relevant metric should be used and mass metric should always be included. It 

is strongly recommended to use more than one metric. 

2.2.2.2  Indirect genotoxicity 

The term 'indirect genotoxicity' is mentioned in the REACH section on repeated dose studies (i.e. 
Annex VIII section 8.6.1, and Annex IX section 8.6.2): “Further studies shall be proposed by the 
registrant or may be required by the Agency in accordance with Articles 40 or 41 in case of […] 
- indications of an effect for which the available evidence is inadequate for toxicological and/or 
risk  characterisation.  In  such  cases  it  may  also  be  more  appropriate  to  perform  specific 
toxicological  studies  that  are  designed  to  investigate  these  effects  (e.g.  immunotoxicity, 
neurotoxicity, and in particular for nanoforms indirect genotoxicity),”. 

Formally,  primary  genotoxicity  and secondary  genotoxicity can  be  distinguished, as  described 
by several authors [191], [192], [193]. Primary genotoxicity is a genotoxic effect caused by a 
substance  that  interacts  with  or  is internalised in the  target  cell.  Primary  genotoxicity  can  be 
either  1)  direct  genotoxicity,  where  the  substance  enters  the  cell  nucleus  of  the  target  cell, 
interacts  directly  with  DNA  (or  disturbs  the  mitotic  apparatus)  to  produce  DNA  lesions  (or 
chromosomal modifications); or 2) indirect genotoxicity, where the substance does not directly 
interact with DNA of the target cell, but generates reactive oxygen/nitrogen species (ROS/RNS) 
inducing  oxidative DNA  damage,  or  induces  damages  to  the  mitosis  apparatus  or  to enzymes 
involved in e.g. DNA replication or DNA repair, or antioxidant activity. Secondary genotoxicity 
involves mechanisms in which the substance does not interact directly with the target cell but 
produces a particle-induced inflammatory response in neighbouring cells/tissues that generates 
ROS/RNS and induces a (secondary) genotoxic effect in the target cell. 

Both primary and secondary mechanisms (as defined above) were reported to play a role in the 
genotoxic effects induced in vivo by nanomaterials [194], [195], [196], [197]). 

In the scope of REACH column 2 of Annex VIII, 8.6.1 and of Annex IX, 8.6.2, the term “indirect 
genotoxicity” refers to oxidative DNA damage (which results from ROS/RNS generated either in 
the  target  cell  or  by  inflammatory  response  in  neighbouring  cells).  The  observed  indirect 
genotoxicity  may  thus  be  due  to  both  primary  and  secondary  genotoxicity  mechanisms  (as 
defined above). 

The  most  frequently  described  mechanism  to  explain  the  genotoxicity  of  nanomaterials  is  an 
indirect  mechanism,  i.e.  via  generation  of  oxidative  species  or  indirect  consequences  of 
inflammation [198], [158], [199], [200], [201]. The specific toxicological studies to investigate 
the  indirect  genotoxicity  (i.e.  DNA  damage  due  to  high  levels  of  reactive  oxygen/nitrogen 
species)  could  be  triggered  by  available  robust  data  showing  that  the  nanoform  induces  a 
significant inflammation and/or the generation of high levels of oxidative species by the tested 
nanomaterial  after  repeated  dose  administration  (e.g.  28-day  study  at  Annex  VIII).  The 
observed inflammation would be considered as relevant to trigger specific genotoxicity studies 
only  in case  it  is  persistent. In case  an  in vivo  genotoxicity  test  required  to address  the  data 

 
 
 
 
 
 
 
 
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requirement under section 8.4 of REACH show positive results that are relevant for classification 
and labelling for mutagenicity hazard, such 'specific toxicological studies' would not be needed. 
The comet assay appears to be a suitable test to assess such genotoxic effects, because 1) the 
standard  comet  assay  can  detect  some  DNA  damages  induced  by  oxidative  stress  and  2)  its 
sensitivity can be increased by using endonuclease (e.g., OGG1, FPG) to detect oxidised DNA 
bases  that are  not  detected  in  the  standard comet  assay  [201].  It  is noted  that  the  modified 
comet assay to detect oxidative DNA damages is not yet validated and the current OECD TG 489 
(2016) for the standard comet assay does not provide recommendations for the modified comet 
assay. Moreover, the influence of inflammation on the comet assay results needs to be further 
investigated. 

Mutagenicity and Carcinogenicity 

The parent guidance12
 Section R.7.7 provides the general testing strategy for mutagenicity and 
carcinogenicity. The advice provided in the parent guidance should be followed together with the 
recommendations given in this section. The text below provides advice only on the mutagenicity 
endpoint. 
The assessment of mutagenicity/genotoxicity generally relies on the investigation of in vitro (and 
possibly  in  vivo)  effects  for  three  major  endpoints:  i.e.  gene  mutation,  clastogenicity  and 
aneugenicity.  It  is  now  widely  accepted,  based  on  international  collaborative  studies  and  the 
large databases available, that no single assay can detect all genotoxic substances and that a 
battery of tests should be implemented. 
Different groups have published several reviews on the genotoxic assessment of nanomaterials 
[202], [203], [200], [204] and the most recent comprehensive review [201] extended previous 
works  with  critical  analysis  of  published  data.  All  these  reviews  agreed  on  a  number  of 
recommendations  that  are  described  in  this  section  (and  also  mentioned  in  recent  guidance 
documents from EFSA [104] and SCCS [89]). 

2.2.3.1  Bacterial (Ames) mutagenicity assays are not recommended 

The bacterial reverse mutation (Ames) test (OECD TG 471 [205]/EU B.12/13: Bacterial reverse 
mutation test (in vitro)) detects point mutations in Salmonella typhimurium and Escherichia coli 
(  [206],  [207];  [208]).  However,  the  strains  of  bacteria  used  in  the  standard  assays  do  not 
appear  to  have  the  ability  to  take  up/internalise  nanoparticles,  as  they  lack  mammalian 
mechanisms  of  endocytosis,  pinocytosis,  and  phagocytosis  [209], [210],  [158],  [211], [201]. 
The updated text of Annex VII of REACH quoted in section below reflects the agreement by the 
scientific community that the standard bacterial Ames assay test is usually not adequate to be 
part  of  the  battery  of  mutagenicity  tests  for  ‘poorly  soluble’  particles  (  [158],  [202],  [209], 
[203], [200], [204], [201]). In 2014, OECD 43 [150] already stated that 'The use of the Ames 
test  (TG  471)  is  not  a  recommended  test  method  for  the  investigation  of  the  genotoxicity  of 
nanomaterials'. 

2.2.3.2  Recommended  approach  for  gathering  mutagenicity  information  on 

nanomaterials according to REACH 

The  revised  Annex  VII of REACH,  for  section  ‘8.4.1  In  vitro  gene  mutation study in  bacteria’, 
column 2 is now reflecting the agreement not to recommend the Ames test and, for the endpoint 
8.4.1 In vitro gene mutation study in bacteria, column 2 reads “The study does not need to be 
conducted for nanoforms where it is not appropriate. In this case other studies involving one or 
more in vitro mutagenicity study(ies) in mammalian cells (Annex VIII, sections 8.4.2. and 8.4.3 
or other internationally recognised in vitro methods) shall be provided”. 
In order to assess the in vitro mutagenicity of nanomaterials, the Ames test must not be used, 
unless there is evidence that the nanomaterial penetrates the cell wall of the bacteria or that 

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indirect genotoxic effects due to generation of reactive oxygen species outside the bacteria. In 
most cases, the Ames test is not appropiate and one or more in vitro test(s) on mammalian cells 
must be performed at Annex VII instead. The registrant must provide at least one of the following 
tests: 

1.  one test detecting gene mutation: the in vitro mammalian cell gene mutation test using 
the thymidine kinase gene (according to OECD TG 490, latest updated in 2016) or the in 
vitro  mammalian cell  gene  mutation  test  using  the  Hprt  and  xprt  genes  (according  to 
OECD TG 476, latest updated in 2016). 

2.  one  test  detecting  chromosomal  aberration:  the  in  vitro  mammalian  cell  micronucleus 
test  (according  to  OECD  TG  487,  latest  updated  in  2016),  or  the  in  vitro  mammalian 
chromosomal aberration test (according to OECD TG 473, latest updated in 2016). 
Given that the standard REACH data requirement at Annex VII (Ames test) investigates gene 
mutation, in case of a nanoform registered at Annex VII, ECHA considers appropriate that the 
registrant  provides  data  from  an  in  vitro  test  detecting  also  gene  mutation,  i.e.  performed 
according to OECD TG 490 or OECD TG 476. At Annexes VIII to X, the registrant should provide 
data from two in vitro tests, one detecting gene mutation (OECD TG 490 or OECD TG 476) and 
one detecting chromosomal aberration (OECD TG 487 or OECD TG 473). 

2.2.3.3  Recommendations for the in vitro tests 

During the OECD/WPMN13 expert meeting on the Genotoxicity of Manufactured Nanomaterials 
in  Ottawa, Canada  in  November  2013  [150],  several  consensus  statements  were  agreed  and 
recommended to investigate the genetic toxicity of nanomaterials. These recommendations are 
further supported by more recent scientific literature (e.g. see reviews by Magdolenova  et al. 
[200], Pfuhler et al. [203], Doak et al. [209]; Dekkers et al [91]; Elespuru et al. [201]). 

2.2.3.3.1 

Nanomaterials characterization in the test medium 

When  investigating  nanomaterials,  one  must  have  or  generate  detailed  data  on  the  intrinsic 
properties  of  the  studied  nanomaterial  as  described  in  section  2.1.1.3  (e.g.  size,  shape, 
crystallinity, surface treatment) as well as on its properties under the test condition. For in vitro 
genotoxicity  test,  the  “characterization  of  the  test  material  should  be  undertaken  in  the  cell 
culture  medium  used  during  the  test,  both  at  the  beginning  of  treatment  and,  where 
methodologies  exist,  after  treatment”  [201].  The  recent  JRC  report  describes  relevant 
characterisation  methods  and  apply  them  for  gold,  silica  and  silver  nanoparticles  [212].  Such 
characterisation helps to monitor the dispersion of the nanomaterial and to ensure that the cells 
are exposed to a sufficient effective dose under in vitro conditions. 

2.2.3.3.2 

Verification of uptake into target cells 

The  verification  of  cellular  uptake  by  appropriate  methods  is  highly  advised  for  in  vitro 
genotoxicity  tests.  The  importance  of  cell  uptake  was  pointed  out  by  the  reviews  mentioned 
above, the Nanogenotox report [213], the OECD report [150], and recent guidance documents 
from EFSA [104] and SCCS [89]. Several parameters (e.g. agglomeration and protein coating) 
can influence cell uptake. 
In vitro tests should be performed on cell lines that have demonstrated the ability to take up 
nanoparticles. A recent JRC report [214] showed that several cell lines (Caco-2, A549, CHO, V79 
and  TK6)  demonstrated  their  ability  to  internalise  the  tested  nanomaterials  (gold,  silica  and 
silver  nanoparticles).  If  a  test  is  performed  on  a  cell  line  for  which  the  uptake  capacity  is 
unknown, the test data should demonstrate the uptake ability of the studied cell line. Such data 
can  be  generated  using  optical-  and  electron-based  microscopy  techniques  (e.g.  fluorescence 
microscopy, transmission electron microscopy) ( [215], [216], [217], [218], [214]). It is noted 
that  the  fluorophore  used  for  labelling  may  either  influence  the  uptake  or  detach  from  the 
nanomaterial (which would complicate the interpretation of the analysis).  The measurement of 

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intracellular nanomaterial can be more or less challenging depending on the nature of the studied 
nanomaterial. 

2.2.3.3.3 

Recommendations to avoid interference with uptake or endpoint 
analysis 

An  in  vitro  mutagenicity  test  usually  includes  an  experiment  in  the  absence  of  metabolic 
activation system (S9mix) and another experiment in the presence of S9mix. The use of S9 mix 
in in vitro studies can affect the outcome of the tests: like for any other tested chemical, S9 can 
induce the formation of mutagenic metabolites (in case the nanomaterial can be metabolised); 
also, the addition in the culture medium of proteins (contained in S9) can modify the cellular 
uptake of nanomaterials ( [200], [209] and [203]). It it thus recommended to perform in vitro 
tests in the presence of S9 only for the NMs composed of organic materials. 

Cytotoxicity  measurements  described  in  relevant  test  guidelines  (e.g.  relative  population 
doubling,  cloning  efficiency  or  relative  total  growth)  are  appropriate  to  determine  the  top 
concentration  that  should  be used  for nanomaterials  in  vitro  tests.  Moreover,  the cytotoxicity 
should  be  assessed  concurrently  with  the  genotoxicity  (not  in  separate  experiments)  [150] 
[201]. Since a variety of proliferaton or cytotoxicity assays employ colourimetry or fluorometry, 
dye interference should be checked beforehand (section 2.1.2 above). 
“The intent when applying nanomaterials to a cell culture medium is to create conditions that 
are comparable, to the extent possible, with the biological and physiological conditions within 
the in vivo system” [150]. 

According  to  Annex  VIII  8.4.2  of  REACH,  a  micronucleus  test  (OECD  TG  487  [219])  or  a 
chromosomal  aberration  test  (OECD 473  [220])  is required.  Nanogenotox  concluded  that  the 
guideline for the testing of chemicals in vitro mammalian cell micronucleus test (OECD TG 487) 
is "applicable  for nanomaterials  but  may  need  some adaptation in  order  to  provide  predictive 
results in vivo” [213]. 

A project on the adaptation of the in vitro mammalian cell micronucleus assay (TG 487 [219]) 
for nanomaterials testing is ongoing in the OECD WPMN rolling work plan (Project 4.95: Guidance 
Document on the Adaptation of In Vitro Mammalian Cell Based Genotoxicity TGs for Testing of 
Manufactured Nanomaterials). The project focuses on the physico-chemical characterisation of 
nanomaterials  and  protocol  modifications  (selection  of  cell  type  with  respect  to  uptake 
mechanisms,  use  of  cytochalasin  B,  timing  of  exposure  to  nanomaterials,  specification  of 
controls,  dose  ranges  and  dose  metrics).  Two  intermediary  reports  studying  gold,  silica  and 
silver  nanoparticles  were  produced  by  this  project  and  recently  published:  in  2018,  on  the 
physicochemical characterisation in water and in serum-containing cell culture media [212]; in 
2020, on the in vitro cytotoxicity and cellular uptake in five different cell lines [214]. 
At  this  time, it is recommended  to conduct  genotoxicity  tests  for nanoforms  according  to  the 
available  OECD  test  guidelines,  as  for  any  other  forms  of  a  substance,  with  the  exception  of 
specific methods adaptations required for nanomaterials, as mentioned here and in the OECD 
guidance on Sample Preparation and Dosimetry [19]. For instance, if the in vitro micronucleus 
assay is performed using a mitosis blocking agent (cytochalasin B), it is recommended to add 
this agent post-treatment or to use a delayed co-treatment protocol, in order to ensure a period 
of  exposure  of  the cell  culture  system  to  the  nanomaterial  in  the  absence  of  cytochalasin  B  ( 
[150], [104], [89], [201]). Moreover, cell lines derived from the expected target tissue(s) should 
be utilised, if available and possible. 

2.2.3.4  In vivo test and exposure of target tissue 

Prior to conducting an in vivo genotoxicity study, there is a need to have relevant toxicokinetic 
data or to conduct toxicokinetic investigations to assess whether the nanomaterial reaches the 
target tissue, where the target tissue is not the site of contact. An in vivo test is not applicable 

 
 
 
 
 
 
 
 
 
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for detecting the genotoxicity of a nanomaterial if the nanomaterial does not reach the target 
tissue [150]. 
In the absence of toxicokinetic information demonstrating systemic availability and/or exposure 
of target tissue(s), it is recommended to investigate the genotoxic effects in the site of contact 
tissue(s). The basis for selecting the route of administration for testing should be to consider the 
route most applicable to human exposure(s) [150]. Currently inhalation is considered the most 
likely  route  for  human  exposure  to  nanoforms  -  at  least  for  workers  -  (See  parent  Guidance 
Chapter R.7a: Section R.7.5.6 [3]). The selected route of administration should be justified and 
the issue of exposure of target tissues should be addressed.

 
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63 

Appendix 1 

: Models for fate and exposure of nanomaterials 

There is on-going research and development of modelling tools to assess the fate of nanomaterials. The list of methods given below is not 
exhaustive and includes methods based on attachment efficiency and dissolution rate of nanomaterials. More information on these methods 
that may be used to predict fate and transport of nanomaterials in the environment and organisms can be found at [221]. 
Further information on the models and their validation status can be found in the referenced publications for each model. 

Table 4: Overview of some models for fate for nanomaterials 

Model 

Overview 

Output 

SimpleBox4nano 
(SB4N): 
Classical 
multimedia mass 
balance 
modelling system 

NanoDUFLOW: 
Spatiotemporally 
explicit 
hydrological 
model 

The model expresses engineered nanoparticles (ENP) 
transport and concentrations in the environmental 
compartments (air, water, soil, etc.) accounting processes 
such as aggregation, attachment, and dissolution. The model 
solves simultaneous mass balance equations. 

Feedbacks between local flow conditions and engineered 
nanoparticles (ENPs) fate processes, such as homo- and 
heteroaggregation, resuspension and sedimentation, are 
modelled. 

The output is mass 
concentrations of ENPs as free 
dispersive species, 
heteroaggregates with natural 
colloids, and larger natural 
particles in each compartment 
in time and at steady state. 

The outputs are the 
concentrations of all ENP 
forms and aggregates in water 
and sediment in space and 
time, and retention. 

Link to the model 
tools 

References 

[222] 

http://www.rivm.nl/si
mplebox 

[223] 

DUFLOW Modelling 
Studio (v3.8.7) 
software package with 
a set of specific 
processes defined by 
the user via the 
NanoDUFLOW 
submodel. 

 
 
 
 
 
  
64 

Appendix R7-1 for nanomaterials applicable to Chapter R7a (Endpoint specific guidance) 
Version 4.0 – December 2022 

Link to the model 
tools 

As a first case study in 
Praetorius et al., [56] a 
river model was used. 

References 

[56] 

Example of 
application: 
[224] 

Model 

Overview 

Output 

Steady-state 
distribution 
model 

Multimedia model was developed using nanospecific 
process descriptions such as homo- and heteroaggregation, 
dissolution and sedimentation to estimate the steady-state 
distribution 

The output is 
nanoparticle/mass 
concentrations in water and 
sediment, and its distance 
from the source. 

Considers a wider range of ENM processes, including 
emissions to air, water (freshwater and marine), and soils 
(urban, agricultural, undeveloped) from their manufacturing, 
use, and disposal; advection in and out of main 
environmental compartments; rate-limited transport across 
compartments; resuspension to air and attachment to 
aerosols; transformation into other ENMs or compounds; in 
natural waters aggregation, sedimentation, dissolution, 
filtration, and sorption to suspended particles and the 
subsequent deposition to sediment; considers long-term 
accumulation of NPs and dissolved metal ions; allows 
inclusion of key transformation processes (e.g. oxidation, 
sulfidation, adsorption of NOM, loss of primary coating) 

Water-Soil-Organism model, a complex multimedia 
spatiotemporal model predicts the fate and bio-uptake, 
across space and in time, of nanomaterials entering the soil 
and aquatic environments. It works by coupling submodels 
for environmental compartments: soils, rivers, bed 
sediments, lakes, estuaries and the sea, and simulating the 
transport of nanomaterials between these compartments of 
nanomaterials in different forms and states; useful for 
identifying accumulation hotspots and studying the 
temporal dynamics of NM concentrations. 

NanoFATE 

NanoFASE 

Nanoparticle/mass 
concentrations in different 
environmental compartments; 
long term 
concentrations/releases 

https://nanofate.eu/  

Spatiotemporal distribution of 
nanomaterials (NM) across 
multiple environmental 
compartments, making it 
distinct from lower-tier 
screening level models, such 
as SimpleBox4nano, 

http://nanofase.eu/sh
ow/element_268  

 
 
 
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specific guidance) 
Version 4.0 – December 2022 

Model 

Overview 

Output 

LOTOS-Euros 

A long-range (regional scale) spatiotemporal atmospheric 
substance transport and deposition model; open source 

Wide range of applications 
such as air quality forecast, 
emissions, depositions etc.  

65 

References 

Open source, 
see link; 
[225] 

Link to the model 
tools 

https://lotos-
iieuros.tno.nl/publicati
ons/model-
documentation/ 

 
 
 
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66 

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