Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance : elements to consider.
Date: 2016-04-01
Institutions: European Chemicals Agency, Joint Research Centre
Subjects: data collection, data processing, nanotechnology, pharmaceutical industry, pharmaceutical product, regulation (EU), technology, toxic substance, toxicology

Usage of (eco)toxicological data for 
bridging data gaps between and grouping 
of nanoforms of the same substance

Elements to consider

Nanomaterials  based  solutions  have  a  lot  to  offer  for  a  wide  range  of  industry  sectors  from 
pharmaceuticals  to  surface  technologies.  Nanomaterials  may  be  manufactured  in  several  different 
shapes, sizes  and/or  with  a  wide  range of surface treatments. Changes made to the particles may 
not  only  provide  a  new  function  for  the  nanomaterial  but  also  influence  its  (eco)  toxicological 
behaviour. 

Therefore, a  need was  identified  in  2014 to develop a pragmatic approach  for how to ensure safe 
use  of  the  potentially  numerous  nanoforms  of  the  same  substance  under  REACH.  A  nanoform  is 
defined  in  this  document  as  a  composition  of  a  substance  that  meets  the  requirements  of  the  EC 
definition of a nanomaterial. Different nanoforms may differ with regard to size distributions, shape 
and/or surface chemistry.  

Together  with  RIVM  and  JRC,  ECHA  initiated  and  led  a  project  over  2015  to  develop  a  scientific 
reference paper to explore the scientific aspects of justifying when and how to use test data from an 
(eco)toxicity study on one nanoform to cover other nanoforms of the same substance.  

This work resulted in this scientific reference paper consolidating the current state of play in science 
across several FP7 projects. In the process of drafting the paper, ECHA Nanomaterial Working Group, 
was  consulted  twice  as  well  as  industry.  This  scientific  reference  paper  will  be  a  cornerstone  in 
future works in the field in the EU as well as a contribution to international discussions at OECD. 

Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the 
same substance  
Elements to consider 

ISBN:  
Cat. Number:  
DoI:  
Publ.date:  
Language:  

978-92-9247-810-0 
ED-02-16-228-EN-N 
10.2823/982046 
March 2016 
EN 

© European Chemicals Agency, Joint Research Centre, Dutch National Institute for Public Health and 
the Environment, 2016 

© Cover page: European Chemicals Agency, Fotolia 

This publication is solely intended for information purposes and does not necessarily represent the 
official opinion of the Dutch National Institute for Public Health and the Environment (RIVM), Joint 
Research  Centre  (JRC)  or  The  European  Chemicals  Agency  (ECHA).  RIVM,  JRC  and  ECHA  are  not 
responsible for the use that may be made of the information contained in this document. 

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

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table of Contents 

1. 

2. 

2.1 

2.2 

Introduction ......................................................................................................................... 3 

Nanospecific considerations for read-across and grouping ................................................ 9 

Physicochemical characterisation of different (nano)forms ............................................... 9 

Physicochemical  parameters  and  their  influence  on  (eco)toxicological  and/or 
environmental fate ............................................................................................................ 11 

2.3 

Changes through the life cycle of a nanomaterial ............................................................ 14 

3. 

Justifying grouping and read-across of nanoforms ........................................................... 14 

3.1 

A strategy for using data between nanoforms ................................................................. 15 

3.1.1  Step 1: Identification of the nanoforms ............................................................................ 16 
3.1.2  Step 2: Initial grouping of the nanoforms ......................................................................... 17 
3.1.3  Step 3: Identification of available data and data gaps ...................................................... 17 
3.1.4  Step 4: Identification of potential source nanoforms ....................................................... 17 
3.1.5  Step 5: Substantiate hypothesis ........................................................................................ 19 
3.1.6  Step 6: Assess new data .................................................................................................... 21 
3.1.7  Final considerations ........................................................................................................... 22 

4. 

References ......................................................................................................................... 23 

Annex I: 

Glossary of commonly used terms .................................................................................... 26 

 
Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

3 

1. Introduction 

1.1  Aim 

In general, application of grouping of substances and read-across between substances is recognised 
as  a  valuable  approach  in  regulatory  frameworks  e.g.  to  fill  potential  data  gaps  in  the  hazard 
characterisation, based on availability of adequate data from similar substances. Grouping and read-
across,  together  with  e.g.  weight  of  evidence  approaches,  are  considered  as  ‘adaptations’,  i.e. 
alternative ways to minimise e.g. animal testing to meet the regulatory information requirements, 
while  fulfilling  the  ultimate  aim  of  REACH  (Regulation  (EC)  No  1907/2006  concerning  the 
Registration, Evaluation, Authorisation and Restriction of Chemicals) (EC, 2006), which is to protect 
human health and the environment thus ensuring safe use of chemicals. It is expected that also for 
nanomaterials  grouping  and  read-across  approaches  will  be  an  important  means  of  addressing 
identified data gaps. 

The European  Commission  issued  a  Recommendation on the definition of the term ‘nanomaterial’ 
(EU, 2011), which is the definition that will be used in this document; of special importance is Article 
2  stating  that:  “‘Nanomaterial’  means  a  natural,  incidental  or  manufactured  material  containing 
particles [...] one or more external dimensions is in the size range 1 nm-100 nm.”.  

The term ‘nanoform’ is used in this document to distinguish forms of a substance that fulfil the EC 
Recommendation  on  the  definition  of  the  term  ‘nanomaterial’  but  differ  with  regard  to  size 
distributions, shape and/or surface chemistry. 

Due to the numerous possible nanoforms with the same chemical identity (i.e. covered by the same 
registration  dossier)  but  with  differences  regarding  other  physicochemical  properties  (e.g.  surface 
modification,  size  distribution  and  particle  shape),  there  is  a  need  for  alternative  approaches  that 
would allow predicting  hazard  properties by reading across between nanoforms and/or from non-
nanoforms to nanoforms of the same substance to minimise testing, in particular, on animals. Under 
REACH,  each  nanoform  may  be  seen  as  being  manufactured/imported  for  a  specific  use  of  that 
registered  substance,  and  thus  the  data  submitted  to  fulfil  the  information  requirements  for  the 
substance should be demonstrated to be representative also for each nanoform.  

The  aim  of  this  document  is  to  consolidate  existing  information  and  develop  approaches  that  a 
registrant  can  use  to  scientifically  justify  that  certain  (eco)toxicological  studies undertaken  on one 
nanoform  of  a  substance  (or  the  non-nanoform)  can  be  used  to  predict  the  hazard  properties  of 
(an)other  form(s)  of  the  same  substance.  The  approaches  will  form  a  cornerstone  in  further 
discussions  with  the  European  Commission,  Member  States,  Industry  and  Non-Governmental 
Organisations whose outcome will form a basis for ECHA’s processes for developing guidance. 

Work on this subject has already been done for conventional substances under REACH such as the 
development by ECHA of “Guidance on information requirements and chemical safety assessment. 
Chapter  R.6:  QSARs  and  grouping  of  chemicals”  (ECHA,  2008)  and  “Read-Across  Assessment 
Framework” (ECHA, 2015). Earlier initiatives included ECHA’s “Background paper An Introduction to 
the Assessment of Read-Across” in ECHA’s Experts Workshop on Read-Across Assessment with the 

 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

active  support  of  Cefic  LRI1.  2-3  October  2012”2;  and  ECHA  “Summary  of  Workshop  on  the  Read-
Across  Assessment  Framework  (RAAF)  (2-3  October  2014)”3.  Furthermore,  the  OECD  revised  its 
guidance on grouping of chemicals (OECD, 2014a), whose first edition dates back to 2007. 

For nanomaterials, Industry (ECETOC) (e.g. Arts et al., 2014; Arts et al., 2015a; Arts et al., 2015b) and 
Member  States  (e.g.  RIVM-Arcadis  project,  see  Sellers  et  al.,  2015)  as  well  as  several  research 
projects  in  FP7  (e.g.  MARINA  (Oomen  et  al.,  2015),  NanoREG  and  GUIDEnano4)  and  individual 
researchers (e.g. Gebel et al., 2014; Walser and Studer, 2015) have started proposing methodologies 
for grouping and read-across and illustrating their ideas with case studies.  

The OECD guidance on grouping of chemicals (OECD, 2014a) also mentions nanomaterials, however 
noting that “Principles and guidance  for  grouping nanomaterials for the  purpose of  assessing  their 
(eco)toxicological and fate properties are under development.” No concrete guidance is provided for 
nanomaterials  in  that  document,  as  accepted  principles  are  not  established  in  scientific  literature 
yet.  To  facilitate  the  discussion  on  this  subject,  OECD  organised  an  “Expert  Meeting  on 
Categorization  of  Manufactured  Nanomaterials”  in  Washington  in  September  2014  (OECD,  2016) 
and is organising a second event under the heading “Expert Meeting on Grouping and Read Across 
for the Hazard Assessment of Manufactured Nanomaterials” in Brussels in April 2016. 

It is crucial to harvest the outcome from these studies to integrate the important experiences gained 
and  have  a  broad  basis.  Furthermore,  emphasis  should  be  put  on  finding  a  workable  balance 
between scientific uncertainty and regulatory needs. 

Fulfilling  the  standard 
information  requirements  under  REACH  by  means  of  read-across 
encompasses predicting the unknown properties of a certain substance using test data generated on 
a substance  that is  considered  to  be  similar based on structural considerations or mode of action. 
This is different  from  predicting  the  unknown properties of one or more nanoform(s) of the  same 
substance  from  known  properties  of  (an)other  nanoform(s).  In  particular,  the  type  and  amount of 
supporting  evidence  needed  to  support  a  claim  of  ‘similarity’  between  nanoforms  of  the  same 
substance  may  differ.  Therefore,  the  main  challenge  is  to  devise  an  approach  that  enables  an 
acceptable  level  of  confidence  that  test  data  generated  on  one  or  more  nanoform(s)  (or  on  non-
nanoforms) apply to other nanoform(s) without compromising the hazard assessment.  

Finally,  the  aim  of  this  document  is  to  illustrate  a  number  of  core  principles  (i.e.  elements  to 
consider),  which  are  based  on  the  current,  albeit  incomplete,  scientific  understanding.  This  would 
lead  to  a  structured  approach  to  guide  registrants  and  regulators  on  how  to  apply  grouping  and 
read-across  concepts  to  nanoforms.  The  guidance  should  provide  some  generic  guidelines,  which 
should be combined with the fact that the specific physicochemical and (eco)toxicological properties 
of  a  substance  and  its  nanoforms  affect  how  filling  data  gap(s)  may  be  scientifically  justified  on  a 

1   LRI: Long Range Initiative 
2  Available 

at: 
http://echa.europa.eu/documents/10162/5649897/ws_raa_20121003_background_paper_an_introductio
n_to_the_assessment_of_read-across_in_echa_en.pdf 

3  Available at: https://echa.europa.eu/documents/10162/13628/workshop_summary_raaf_en.pdf. 
4  More  information  on  these  projects  can  be  found  on  their  respective  websites:  www.marina-fp7.eu, 

www.nanoreg.eu, and www.guidenano.eu. 

 
                                                           
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5 

case-by-case  basis.  The  decision  on  whether  a  data  gap  exists  for  a  specific  nanoform  as  well  as 
whether the data gap can be filled by using information from another nanoform, and consequently 
the scientific justification for it, remains the responsibility of the registrant. 

1.2  Legal basis 

The objectives of  REACH  are  set out  in its preamble, including recitals 1 and 38, and in Article 13, 
which  all  underline  the  need  to  generate  data  for  hazard  assessment  by  means  other  than  tests 
whenever  this  is  possible.  Annex  XI,  Section  1.5  of  REACH  sets  the  conditions/criteria  for  using 
grouping and read-across approaches to fulfil the information requirements for substances. 

If  the  read-across  approach  is  adequate,  testing  to  fulfil  the  information  requirement  for  that 
endpoint is unnecessary. A read-across approach and subsequent development of a robust scientific 
justification  may  also  support  a  conclusion  for  a  certain  hazard  endpoint  by  using  a  weight-of-
evidence approach. 

1.2.1  What is grouping of substances? 

Grouping  describes  the  general  approach  to  assessing  more  than  one  chemical  at  the  same  time 
(ECHA,  2008;  OECD,  2014a).  Substances  that  are  structurally  similar  and  have  physicochemical, 
toxicological, ecotoxicological and/or environmental fate properties, which are likely to be similar or 
to follow a regular pattern, may be considered as a group of substances. These similarities may be 
based on a number of factors in accordance with REACH Annex XI, section 1.5: 

•  Common functional group (i.e. chemical similarity within the group); 

•  Common precursors and likely common breakdown products via physical and/or biological 

processes which result in structurally-similar chemicals; 

•  A constant pattern in the changing of the potency of the properties across the group (i.e. of 

physicochemical parameter and/or biological properties). 

Usually,  the  terms  category  approach  and  analogue approach  are  used  to  describe  techniques  for 
grouping  chemicals  (ECHA,  2008;  OECD,  2014a).  The  term  analogue  approach  is  used  when  the 
grouping  is  based  on  a  very  limited  number  of  chemicals,  where  trends  in  properties  are  not 
apparent (ECHA, 2008; OECD, 2014a). In a category approach, more members are generally present, 
enabling the detection of trends across endpoints (ECHA, 2008; OECD, 2014a). 

For registration of a substance under REACH, the information requirements have to be met. Within a 
group of substances, a data gap might be filled in by using several techniques including read-across 
(as described in section 3.1). 

Grouping  of  nanomaterials  often  addresses  different  forms  of  the  same  substance  rather  than 
grouping  nanoforms  of  different  substances.  Nevertheless,  approaches  proposing  the  grouping  of 
nanoforms  of  different  substances  (i.e.  different  nanomaterials)  have  also  been  published  (Arts  et 
al., 2014; 2015a). 

 
 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

1.2.2  What is read-across between substances? 

Read-across  is  an  established  technique  for  predicting  endpoint  information  from  one  or  more 
source  substance(s)  to  a  target  substance  by  using  data  concerning  the  same  endpoint.  This 
prediction  should  be  based  on  a  robust  scientific  justification  (OECD,  2014a).  Consequently,  the 
read-across approach  has to  be  considered on an endpoint by endpoint basis due  to the different 
complexities (e.g. key parameters, biological targets) of each endpoint. 

In  an  analogue  approach,  read-across  is  employed  within  a  group  of  a  very  limited  number  of 
substances for which trends are not apparent: i.e. the simplest case is read-across from one source 
substance to one target substance. 

In  a  category  approach,  read-across  is  used  within  a  group  of  a  number  of  substances  for  which 
trends are apparent. 

In all cases, read-across must be justified scientifically and documented thoroughly. Several pieces of 
evidence may be used to justify the read-across, with the aim of strengthening the case. 

When comparing with the grouping approach presented above, it is seen that read-across between 
nanomaterials differs from read-across between substances. Target and source nanomaterials used 
in read-across are generally different forms of the same substance rather than different substances. 

1.2.3 

Consideration for whenever read-across is used 

Annex  XI,  Section  1.5  of  REACH  requires  that  whenever  read-across  is  used  all  of  the  following 
conditions should be fulfilled: 

a.  “Be  adequate  for  the  purpose  of  classification  and  labelling  and/or risk  assessment”  –  If the 
read-across data on the source substance is used as a key study, the data shall be adequate, 
reliable  and  robust  enough  to  enable  the  registrant  and  the  evaluator  to  decide  on  the 
appropriate classification and labelling for the target chemical. Similarly, if the data from the 
source substance is used as a key study, it shall provide a sufficiently reliable dose descriptor, 
i.e. point of departure for the risk assessment.  

b.  “Have adequate and reliable coverage of the key parameters addressed in the corresponding 
test  method  referred  to  in  Article  13(3)”  –  Test  methods  referred  to  in  Article  13(3)  are 
regularly revised to reflect progress in science, and thus, the revised test methods may include 
investigations on additional important parameters. Covering these key parameters is essential 
to ensure that the level of information gathered by testing the source substance is equivalent 
to that expected from a new study performed according to the most current test method. 

c.  “Cover  an  exposure  duration  comparable  or  longer  than  the  corresponding  test  method 
referred to in Article 13(3) if exposure is a relevant parameter” – As an example: a sub-chronic 
repeated dose toxicity (90-day) study can be used to cover the information requirements for a 
sub-acute repeated dose toxicity (28-day) study but not vice versa. 

d.  “Adequate  and  reliable  documentation  of  the  applied  method  shall  be  provided.”  –  The 
documentation  provided  must  be  sufficient  to  allow  an  independent  assessment  of  the 

 
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7 

adequacy  and  the  scientific  validity  of  the  read-across  approach  (see  also  section  3.1.).  The 
following elements are considered essential to adequately document a read-across approach: 

1)  a read-across hypothesis; 

2)  a justification for the read-across hypothesis; 

3)  a list of all the substances included in the approach; 

4)  detailed substance identity information of all substances included in the approach; 

5)  a list of the endpoints that are to be read-across; 

6)  a data matrix; 

7)  a conclusion on the applicability of the proposed read-across approach. 

1.3  Differences between “non-nanoforms” and nanoforms 

REACH  is  based  on  the  principle  “that  industry  should  manufacture,  import  or  use  substances  or 
place them on the market with such responsibility and care as may be required to ensure that, under 
reasonably foreseeable conditions, human health and the environment are not adversely affected”. 

This means that  all  forms of  a  substance shall be covered by the hazard information submitted to 
demonstrate safe use of the registered substance. 

The question of how to register nanoforms under REACH is currently under discussion. In principle, 
there are two scenarios to consider: 

i.  A nanoform that has a corresponding non-nanoform, i.e. a form of the substance with a 
particle  size  distribution  that  does  not  meet  the  requirements  of  the  EC 
Recommendation  on  the  definition  of  the  term  ‘nanomaterial’  (EU,  2011)  (e.g.  silver 
ingots  vs.  silver  nanoparticles).  Multiple  different  nanoforms  and  non-nanoforms  may 
exist for a given substance, depending on variations in key physicochemical parameters 
(e.g. surface treatment, shape). 

ii.  A  nanoform  that  has  no  corresponding  non-nanoform  (e.g.  single  walled  carbon 
nanotubes).  Multiple  different  nanoforms  may  exist  for  a  given  substance,  depending 
on variations in key physicochemical parameters (e.g. surface treatment, shape) 

Even  for  substances  that  do  not  exist  in  nanoforms,  it  is  acknowledged  that  the  differences  in 
particle size, shape and surface treatment, as well as other physicochemical parameters may lead to 
differences  in  the  hazard  properties  specified  in  the  REACH  standard  information  requirements  of 
Annexes  VII-X.  Therefore,  the  different  nanoforms  should  be  assessed  individually  in  order  to 
determine whether the difference(s) in e.g. size, shape and/or chemical composition due to surface 
treatment  will  result  in  different  hazard  profiles.  Although  this  need  for  a  separate  assessment  of 
nanoforms is not stated explicitly in REACH, ECHA considers this to be implicit in the legal text. This 
practice of performing a separate assessment of each individual nanoform in a registration dossier 
covering  multiple  form(s)  of  a  substance,  should  serve  as  a  general  guiding  principle  in  this 
document. 

 
 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

The general rules for adaptation of the standard information requirements specified in Annex XI of 
REACH also apply to nanoforms of a registered substance. As described in Section 1.2.3, read-across 
according  to  REACH  Annex  XI,  Section  1.5.  is  regarded  as  a  technique  for  predicting  endpoint 
(hazard) information for one target substance by using data from the same endpoint from another 
substance(s)  (source  substance(s)).  These  conditions  will  be  slightly  different  for  nanoforms,  as  all 
nanoforms of a substance are normally considered to be the same substance for REACH registration 
purposes. Grouping of nanoforms based on similarity and read-across between nanoforms within a 
registration dossier may be a useful tool for demonstrating safe use for all nanoforms of a registered 
substance. 

By  analogy  to  REACH  Annex  XI,  Section  1.5,  the  application  of  the  grouping  concept  to  different 
nanoforms  of  the  same  substance  requires  that  (eco)toxicological  effects  can  be  predicted  by 
interpolation  of  data  from  source  nanoform(s)  (and/or  non-nanoform(s))  of  a  substance  to  target 
nanoform(s)  of  that  same  substance.  Based  on  the  same  principle,  the  relevant  (eco)toxicological 
properties of the target nanoform(s) in a group could be predicted from the properties of the source 
nanoform(s)  within  that  group.  This  prediction  may  be  a  result of observing  a constant  pattern of 
change of properties across each group. 

Generally,  the  correct  application  of  REACH  Annex  XI,  Section  1.5  requires  sufficient  knowledge 
about  the  identity  of  the  source  and  target  substance(s).  Insufficient  knowledge  of  substance 
identity  (e.g.  inadequate  knowledge  of  the  chemical  composition)  can  lead  to  challenges  for 
regulators  in  assessing  and  thus  accepting  a  proposed  grouping  (category)  and/or  read-across 
approach. By analogy, when applying grouping and read-across approaches to nanoforms within the 
same registration dossier, there is a need to have a sufficiently clear description of the identities of 
the source and target nanoform(s) so that both industry and regulators know “what the nanoform 
is”. However, adequate knowledge of the “what a nanoform is” may require additional information 
that is not part of the standard identity information for substances. To apply the principles outlined 
in  REACH  Annex  XI,  Section  1.5,  adequate  information  on  “what  the  nanoform  is”  should  also  be 
given in the registration dossier. 

Therefore,  the  document  at  hand  also  presents  details  on  the  type  of  information  needed  to 
determine  “what  the  nanoform  is”  (see  below  in  Section  1).  It  is  not  only  a  prerequisite  for 
successful  use  of  the  principles  outlined  in  REACH  Annex  XI,  Section  1.5,  but  also  the  basis  for 
scientifically sound grouping of nanoforms. 

Decreasing  the  size  and  surface  modification(s)  of  a  particle  can  change  its  characteristics  in 
sometimes  startling  ways,  affecting  fundamental  behaviours  such  as  solubility,  reactivity, 
environmental  transport,  and  toxicokinetics,  which  in  turn  can  affect  its  (eco)toxicological  effects 
and  the  fate  in  the  environment.  In  consequence,  the  Group  Assessing  Already  Registered 
Nanomaterials (GAARN) has noted that (ECHA, 2013) 

“When  considering  reading  across  to  another  nanoform  or  a  counterpart  bulk  material,  a 
solid  scientific  justification  should  be  provided  in  the  IUCLID  dossier  of  the  registered 
substance.  It  is  insufficient  to  justify  the  use  of  data  for  read-across  based  only  on  the 
chemical  composition  of  a  nanomaterial,  and  further  physicochemical  parameters  such  as 
aspect  ratio,  shape,  form,  solubility,  surface  area,  charge,  surface  treatment  etc.  should 

 
Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

9 

provide  a  reliable  dataset  to  support  a  sound  scientific  interpretation  of  the  similarities  or 
differences among (nano)forms.” 

Of note, in the “Guidance for identification and naming of substances under REACH and CLP” (ECHA, 
2014)  shape  and  surface  area  are  indicated  as  ‘other’  identification  parameters,  particularly  for 
minerals.  In  addition,  a  revision  of  the  REACH  Annexes  and  related  standard  information 
requirements  for  substances  that  addresses  possible  amendments  for  nanomaterials  is  currently 
(2016) on-going. 

2. Nanospecific considerations for read-across and grouping 

As  described  in  Chapter  1,  application  of  read-across  requires  the  proper  characterisation  of  each 
nanoform. The spectrum of physicochemical parameters to be considered is provided in Section 2.1 
Subsequently,  Section  2.2  summarises  the  current  state  of  knowledge  on  how  the  specific 
characteristics  of  nanoforms/nanomaterials  may 
(eco)toxicological  and/or 
environmental fate properties. 

influence 

their 

Section  2.3  discusses  the  transformation  of  nanomaterials  throughout  their  life  cycle.  As  for  any 
chemical,  an  important  recognition  is  that  relevant  physicochemical  properties  of  a  nanomaterial 
may  change  during  its  life  cycle  or  in  different  steps  of  its  biological  pathways  (Arts  et  al.,  2014; 
Oomen  et  al.,  2014;  Arts  et  al.,  2015a).  The  OECD  published  a  “Guidance  Manual  towards  the 
Integration  of  Risk  Assessment  into  Life  Cycle  Assessment  of  Nano-enabled  Applications”  (OECD, 
2015) that may provide relevant background considerations in this context. 

Furthermore,  when  considering  possible  trends  in  behaviour,  it  should  be  realised  that  several 
physicochemical  parameters  are  closely  related  and  may  directly 
impact  each  other. 
Physicochemical  parameters  may  work  together  enhancing  their  “individual”  effects (e.g.  reducing 
size  and  increasing  relative  surface  area  may  both  lead  to  a  higher  reactivity),  or  they  may 
counteract changing the overall impact on the behaviour of a nanoform (e.g. reducing size may lead 
to  higher  reactivity  but  also  to  a  higher  agglomeration  rate,  which may  reduce  exposure  and  may 
result  in  a  lower  toxicity).  Some  physicochemical  parameters  may  also  influence  the  toxicokinetic 
behaviour of a nanomaterial whereas other parameters impact aspects of its toxicity. 

2.1  Physicochemical characterisation of different (nano)forms

The information currently required under REACH in Annex VI5 may in some cases not be sufficient to 
identify  and  characterise  nanoforms  (SCENIHR,  2009;  JRC,  2011)  thus  additional  information  on 
particle characteristics is essential to enable proper hazard assessment of nanoforms. 

Efforts have been made worldwide to establish a set of physicochemical endpoints that would allow 
adequate characterisation of a nanomaterial for (regulatory) safety (risk) assessment (Oberdörster et 
al., 2005; SCENIHR, 2009; OECD, 2010; Stone et al., 2010; JRC, 2011; OECD, 2012, 2014b) and it is still 
subject  to  some  discussion.  There  is  general  agreement  that,  in  addition  to  the  standard  data 
requirements for physicochemical properties, some further properties to be routinely considered for 

5  Based  on  consolidated  version  of  22  August  2014,  available  at:  http://eur-lex.europa.eu/legal-

content/EN/TXT/PDF/?uri=CELEX:02006R1907-20140822. 

 
 
 
                                                           
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

studies of nanoforms include: specific surface area, particle size and particle size distribution, surface 
chemistry, agglomeration and aggregation, crystalline phase, shape and aspect ratio, photo-catalytic 
properties,  porosity  and  pour  density,  dustiness,  dispersibility,  zeta  potential  and  reactivity  (redox 
potential,  radical  formation).  The  relevance  of  these  properties  would  depend  on  the  individual 
nanomaterial. 

1.  Chemical composition comprises crystal structure and crystalline phase. 
2.  Surface characteristics, which include coating chemistry, functionalisation (e.g. capping agents), surface charge (e.g. zeta potential). 
3.  Surface area includes porosity. 
4.  Solubility includes water equilibrium solubility and rate of dissolution in relevant media. 
5.  Hydrophobicity for nanoforms is dependent on e.g. van der Waals energy, Hamaker constant, zeta potential. Analytical determination of the 

hydrophobicity of nanoforms is still under development, e.g. sessile drop contact angle, dye adsorption. 

6.  Dispersibility refers to the relative number or mass of particles in a suspending medium, and relates to stability (Sellers et al., 2015), aggregation and 

agglomeration in relevant media, and is dependent on e.g. van der Waals energy, Hamaker constant, zeta potential. 

7.  Physical hazards comprise explosiveness, flammability, and autoflammability. 

Figure 1. 

Key  properties  that  characterise  a  nanoform  (adapted  from  Sellers  et  al.,  2015), 
arranged  under  headings  from  ITS-NANO  (Stone  et  al.,  2014).  These  properties  can 
affect exposure, toxicokinetics, fate and/or (eco)toxicological behaviour and thus the 
possible  risk  posed  by  nanoforms.  These  constitute  the  basic  information  needed 
(based  on  current  knowledge)  to  implement  the  assessments  described  in  Figure  3. 
The information on the chemical and physical identity (“What they are”) can be used 
for a first assessment on the possibility to apply read-across for a given nanoform6. 

For  physicochemical  characterisation  of  a  nanoform,  a  distinction  can  be  made  between  intrinsic 
material properties (such as chemical composition, primary particle size, shape, and water solubility) 
and system-dependent properties defined by the surroundings in which the nanoform is placed (e.g. 

6  Note that i) some properties may not always be relevant for each read-across case, for example dustiness 
may  only  apply  to  powders;  ii)  some  properties  (for  example  dispersibility  and  solubility)  are  system-
dependent, iii) the key physicochemical properties are derived mostly from studying nanoforms of metals, 
metal  oxides  or  carbon  based  materials,  and  iv)  differences  in  “what  they  are”  may  not  have  to  exclude 
read-across  per  se,  as  “where  they  go”  and  “what  they  do”  may  be  more  important  in  read-across 
justification. 

 
 
                                                           
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11 

dissolution  rate  in  biological  media,  surface  reactivity  and  dispersibility).  The  current  level  of 
knowledge does not allow deducing possible correlations between intrinsic material properties and 
apical toxic effects. It is therefore important to consider both the intrinsic properties of a nanoform 
and the available knowledge with regard to system dependent properties, bio-physical interactions 
and in vitro effects to justify read-across. In this context, detailed information on the nanoform and 
its  production  process  may  also  be  valuable  to  better  understand  the  behaviour  and  evaluate  the 
validity of read-across. 

The  key  physicochemical  parameters  for  nanoform  characterisation  (that  includes  both  REACH 
requirements for substances and additional nanospecific endpoints) listed in Figure 1 should be seen 
as a minimal set to be addressed when considering grouping or read-across of nanoforms. 

2.2  Physicochemical  parameters  and  their  influence  on  (eco)toxicological 

and/or environmental fate 

As  described  in  Section  2.1,  for  nanoforms  in  particular  both  the  intrinsic  properties  as  well  as 
system dependent properties affect (eco)toxicological and/or environmental fate. Table 1 includes a 
summary of  the  different parameters  presented  in Figure  1 and their relevance for environmental 
and  human  health  endpoints.  A  more  extensive  overview  of  the  current  understanding  of  the 
potential influence of these different physicochemical parameters on the toxicological properties of 
a  nanoform  can  be  found  in  the  scientific  literature  (e.g.  Oberdörster  et  al.,  2005;  Sellers  et  al., 
2015). On a case-by-case basis, additional parameters not included here may be relevant for certain 
nanomaterial  and  endpoints,  e.g.  for  acute  inhalation  toxicity  of  fibre-like  materials,  rigidity 
(stiffness), hardness and aspect ratio of the material may play an important role in hazard and safety 
assessment (Tran et al., 2008). 

Table 1: 

Summary of key physicochemical parameters to be considered for grouping and read-
across  of  nanoforms  and  their  relevance  for  human  health  and  environmental 
endpoints 

What they are (Chemical identity) 

Chemical composition, including crystalline structure 

Detailed information on chemical composition is fundamental for determining human health and environmental 
effects of nanoforms, as is the case for non-nanoforms. However, size, shape and surface characteristics of a 
nanoform may cause the nanoform to exhibit a different behaviour compared to the non-nanoform of a material 
with the same composition. 
Crystalline structure may for some nanoforms influence other properties of the material (e.g. reactivity, zeta 
potential, Hamaker constant) in a way that affects human and environmental toxicity. Decreasing size of particles 
may introduce crystallographic changes in the material (contraction of the crystalline lattice or deformation). 
Based on the present understanding of nanoparticle behaviour, changes to the crystalline structure seems 
particularly relevant for metals, metal-oxides or carbon based nanomaterials. 

Impurities 

As for non-nanoforms, impurities can substantially contribute to the human and environmental toxicity of 
nanoforms. 

 
 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

Surface characteristics, including coating chemistry, functionalisation (e.g. capping agents), surface 
charge density (e.g. zeta potential) 

Considering human health endpoints, the surface chemistry of a nanoform affects its reactivity and systemic 
absorption. Surface modification(s) may determine which biomolecules adhere to the nanoform, its distribution 
and cellular uptake, and its toxic effects. Surface charge may influence systemic distribution and cellular uptake 
of a nanoform, and ultimately its toxicity. 
In the environment surface characteristics will influence sorption to environmental or biological media and the 
reactivity of a nanoform. In addition, similar to considerations for human health endpoints, the surface chemistry 
of a nanoform affects its reactivity and systemic absorption. Surface modification(s) may determine the 
biomolecules that adhere to the nanomaterial, its distribution and cellular uptake, and its toxic effects. Surface 
charge may influence systemic distribution and cellular uptake of a nanomaterial, and ultimately its toxicity. 

What they are (Particle characteristics) 

Particle size / range 

The size of the nanoform affects other physicochemical parameters, such as crystallinity, zeta potential and 
specific surface area, and may determine exposure, and whether the nanoparticle can be internalised into an 
organism. Once internalised, particle size may also affect the distribution within the body, and the toxicity at both 
the point of entry and distally. Size distribution is not a static parameter; it may also change during the course of 
(environmental) toxicity testing (as well as during the life cycle of the material) due to e.g. partial dissolution, 
interaction with test media or preferential absorption of smaller particles. 

Shape 

Particle shape may affect the internalisation of a nanoform (e.g. the ability of a nanoform to penetrate into a cell) 
and its (environmental) toxicity. In inhalation studies, particle shape may influence nanoform deposition within the 
lungs and may also influence its persistence in the lungs and probably in other sites. Shape may also influence  
mode-of-action like for high aspect ratio materials, as well as other parameters, such as zeta potential. 

Surface area, including porosity 

The increase of relative surface area with decreasing particle size may increase the reactivity of a nanoform 
relative to its mass. Furthermore, as a consequence of the increased surface to volume ratio, porosity may affect 
the crystalline structure. 

Where they go (Fundamental behaviour) 

Solubility: Rate of dissolution / Equilibrium solubility 

The rate of dissolution depends on the chemical composition, particle size, coating, stability, manufacturing 
process, and biological environment; for substances that have a high rate of dissolution, the ion(s) may be 
dictating the toxicity also of the nanoforms, which will be an important aspect of the evaluation. ‘Water solubility’ 
is an intrinsic material property, but in most cases the system-dependent property ‘dissolution rate in relevant 
biological media’ will be more relevant as this fundamentally affects the bioavailability of substances in the 
(biological) environment. The relevance of the different media is depending on the actual route of exposure 
and/or the environmental compartment under evaluation. 

Hydrophobicity 

Hydrophobicity for nanoforms is dependent on e.g. Van der Waals energy, Hamaker constant and zeta potential. 
Analytical determination of the hydrophobicity of nanoforms is still under development, e.g. sessile drop contact 
angle, dye adsorption. While these parameters can influence agglomeration and sorption, as well as ‘dispersibility 
in biological media’ and dustiness, currently the exact relationships between them are not clear 

Dispersibility 

This parameter can influence the degree of environmental transport and (environmental) exposure. Furthermore, 
this parameter may influence the degree of internal exposure (particularly by the oral route; however particle 
dispersibility also affects nanomaterial mobility within the lung and hence its potential for systemic uptake). 

 
Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

13 

Dustiness 

This parameter is mainly relevant for exposure via air (particularly by inhalation) and transport through air. 
In the environment this parameter is not relevant to aquatic/sediment exposures and only to a limited extent for 
soil exposures. 

What they do (reactivity) 

Physical hazards: Flammability / Autoflammability / Explosiveness 

These parameters are relevant in assessing the risk of injury in occupational settings, but are not primary 
parameters characterised in (environmental) toxicity studies. 

Biological (re)activity (or surface reactivity) 

The biological (re)activity or surface reactivity of a nanoform of a substance appears to generate reactive oxygen 
species (ROS) which induce inflammation, and thus elicit cellular toxicity. 

Photoreactivity 

Photoreactivity may increase with decreasing particle size. In human toxicity testing, this parameter may be 
particularly relevant when considering dermal exposure, but it may also play a role in other exposure routes. 
In the environment this parameter may be particularly relevant when considering the aquatic compartment, but it 
may also play a role in other compartments. 

In  the  environment,  processes  that  influence  the  transport  behaviour  include  adsorption  and 
desorption  processes 
to  suspended  matter,  aggregation  and  agglomeration  processes, 
sedimentation  and  re-suspension,  dissolution,  dispersion,  biodegradation  (of  coatings),  interaction 
with  organic  biomolecules  at  the  nano-bio  interface7,  interaction  with  contaminants,  interaction 
with living organisms, and transfer via the food chain. Apart from physicochemical parameters of the 
nanomaterial these processes are influenced by environmental parameters, including temperature, 
pH,  ionic  strength  (in  particular,  of  divalent  ions)  and  conductivity,  presence  and  type  of  natural 
organic  matter,  dispersants  and  proteins.  Interactions  at  the  nano-bio  interface  are  clearly 
influenced by the type of biomolecules (proteins, exudates, etc.) that are excreted/secreted by the 
organism under consideration. These processes are also relevant for non-nanomaterials, but as the 
ecotoxicological  and/or  environmental  fate  of  a  nanoform  depends  both  on  its  chemistry  and 
particle  characteristics  the  influence  of  these  processes  are  particularly  important  for  nanoforms 
(Oberdörster  et  al.,  2005;  Sellers  et  al.,  2015).  In  particular,  the  dispersion  method  and  the 
composition  of  the  aqueous  media  can  influence  the  results  when  determining  ecotoxicological 
endpoints for nanoforms (see section 2.3). 

In (human) organisms, information on the (main) route(s) of exposure (inhalation, dermal, oral) is a 
first  step  in  understanding  the  toxicokinetic  profile  of  a  nanomaterial.  For  example,  for  the 
inhalation  route  of  exposure,  specific  deposition  in  the  lungs  needs  to  be  considered.  The 
toxicokinetic  profile  of  a  nanomaterial  provides  information  on  the  absorption  and  subsequent 
exposure  of  target  organs/tissues  over  time.  Toxicokinetics,  in  a  traditional  sense,  encompasses 
absorption,  distribution,  metabolism,  and  excretion  (ADME).  For  nanomaterials  toxicokinetics  are 
further  complicated  by  changes  in  the  physicochemical  properties  of  the  material  that  may  occur 

7  The ‘nano–bio’ interface comprises the dynamic physicochemical interactions, kinetics and thermodynamic 
exchanges  between  nanomaterial  surfaces  and  the  surfaces  of  biological  components  (for  example 
proteins, membranes, phospholipids, endocytic vesicles, organelles, DNA and biological fluids) (Nel et al., 
2009). 

 
 
                                                           
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

during these different ADME processes. The specific toxicokinetic profile of a nanomaterial depends 
on  several  different  physicochemical  parameters  of  the  nanoform,  e.g.  composition,  size,  shape, 
agglomeration/aggregation state, surface properties (including surface charge), hydrophobicity, and 
dissolution. 

Furthermore, and parallel to the description above of the influence of environmental parameters on 
the  fate  and  behaviour  of  a  nanomaterial,  the  toxicokinetic  profile  in  a  (human)  organism  also 
depends on the temperature, pH and ionic strength of the biological fluid in which the nanomaterial 
is taken up (e.g. serum, saliva, blood). Hence, ‘system-dependent properties’ (i.e. dissolution rate in 
biological media, surface reactivity and dispersibility), biomolecules present and interactions at the 
nano-bio  interface  of  cells  at  the  target  site  can  provide  relevant  information  on  the  likelihood  of 
distribution  and  potential  for  accumulation.  Information  from  (available)  in  vivo  studies  including 
data  on  internal  exposure  and  elimination  over  time  provide  further  relevant  information  on  the 
toxicokinetic behaviour. 

2.3  Changes through the life cycle of a nanomaterial

While  considering  available  data  and  evaluating  the  applicability  of  data  from  one  nanoform  for 
another nanoform,  it  is  important  to  realise that throughout  its  life cycle, a nanoparticle is due to 
change  because  of  e.g.  encounters  with  other  particles  and  constituents  (e.g.  ageing  process, 
interaction  with  environmental  media  (e.g. 
agglomeration,  aggregation,  corona  formation), 
dissolution,  corona  formation,  aggregation  or  disaggregation,  chemical  reactions,  transformation), 
and degradation (e.g. loss of the coating). This implies that nanoforms can change during their life 
cycle. Any change may affect one or more of the physicochemical parameters described in Table 1 
and may potentially affect the activity, reactivity, fate, toxicokinetics and toxicity in a significant way, 
which could lead to a different behaviour. Therefore, any change in the physicochemical parameters 
during the life cycle may justify a careful assessment of its impact on (eco)toxicological properties to 
ensure  that  data  from  one  nanoform  may  still  be  used  for  another  nanoform.  This  may  raise  the 
question  on  whether  source  and  target  nanoforms  behave  similarly  in  the  environment  from  the 
moment of emission to actual exposure and inside the organism. It also impacts on the way data on 
a  form  are  obtained,  raising  questions  on  how  the specific  nanoform  behaves  in  the  test  medium 
and  whether  the  form  tested  is  representative  for  the  form  to  which  an  organism  or  the 
environment is exposed. 

Similar  processes  of  change  may  take  place  inside  organisms  as  well  as  during  the  absorption, 
distribution, metabolism and excretion processes (ADME). 

3. Justifying grouping and read-across of nanoforms  

This  chapter  presents  different  steps  to  group  nanoforms  and  to  decide  on  read-across  of  data 
between  nanoforms  taking  into  account  the  provisions  and  definitions  summarised  in  Chapter  1. 
Most importantly, the use of hazard data from a source nanoform to the target nanoform must be 
justified  and  scientifically  substantiated.  Therefore,  the  present  chapter  addresses  the  issues 
relevant for building and substantiating a hypothesis for such justification specifically for nanoforms. 
It  should  be  noted  that  a  different  hypothesis  may  apply  and  may  have  to  be  developed  per 
information requirement under REACH for which a data gap is identified. 

 
 
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15 

Guidance on the approach for read-across under REACH is developed for chemicals and outlined in 
ECHA Guidance Chapter R.6 (ECHA, 2008). Further nanospecific considerations are indicated in the 
current  document  and  ECHA  Guidance  (ECHA,  2012a,  b,  c).  A  matrix  could  serve  to  obtain  a 
comprehensive  overview  of  the  available data and how data gaps have been addressed, either by 
generating new data or by using adaptations such as read-across. In the case of a registration dossier 
under  REACH  including  nanoforms,  the  matrix  should  identify  the  different  forms  covered  by  that 
specific  registration  dossier.  Where  relevant,  the  matrix  could  furthermore  illustrate  the  possible 
grouping of nanoforms, and could support in the justification used to define the boundaries of each 
group. 

Taking note of the ECETOC decision-making framework for the grouping of nanomaterials (Arts et al., 
2015a),  information  on  the  water  solubility,  dissolution  rate,  the  aspect  ratio,  biophysical 
interactions and cellular effects, likelihood of biopersistency, and the reactivity of the nanoform may 
support a first formation of group(s) of nanoforms with a similar chemical identity for the purpose of 
read-across.  A  hypothesis  for  read-across  may  accordingly  be  developed  for  these  group(s)  of 
nanoforms  rather  than  for  each  individual  nanoform.  During  the  development  of  this  hypothesis, 
one may come to the conclusion that i) the initial grouping is well suited to address the data gap(s) 
identified, or ii) there is reason to redefine the group(s) (possibly into subgroups) to address the data 
gap(s) or iii) no grouping is possible. 

3.1  A strategy for using data between nanoforms

This  document  focuses  on  a  strategy  for  read-across,  i.e.  for  using  data  from  one  or  more  source 
nanoforms to a target nanoform of the same substance to fill gaps in the hazard data. 

The strategy is building on six different steps, which are further clarified in sections 3.1.1–3.1.7: 

1.  Identification  of  the  nanoforms.  This  involves  the  identification  of  the  nanoforms  based  on 

their basic physicochemical parameters (see “what they are” in Figure 1). 

2.  Initial grouping of the nanoforms. Based on similarities in those physicochemical properties 
that  influence  “what  they  are”  and  “where  they  go”  (Figure  1),  grouping  of  the  identified 
nanoforms in the dossier may be considered. The boundaries of each group should be clearly 
defined. 

3.  Identification of available data and data gaps. This involves making an inventory (possibly in 
matrix  form  as  described  above)  of  the  information  available  per  hazard  endpoint  required 
under REACH for the nanoforms in each group, and consequently identifying where the data 
gaps  are  i.e.  the  target  nanoform(s)  and  the  endpoint(s)  that  need  to  addressed  via  read-
across. 

4.  Identification  of  potential  source  nanoforms.  For  each  data  gap,  this 

involves  the 
identification of source nanoforms in the group from which information may be used for read-
across to the target nanoform(s). This also involves (hypothesis based) scientific justification of 
the appropriateness of the identified source nanoforms. 

5.  Substantiate  hypothesis.  This  involves  information  gathering  to  substantiate  the  hypothesis 
for read-across. When a group of nanoforms is considered for read-across it may be necessary 

 
 
 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

to  re-evaluate  the  initial  grouping.  If  applicable,  a  testing  strategy  can  be  built  that  may 
(partly) cover multiple data gaps. 

6.  Assess any new data for the impact on the hypothesis. In an iterative process, interpret the 
information that  becomes available  to evaluate  if  it sufficiently substantiates the  hypothesis 
and builds justification for read-across (or not). 

Substance

Identification of the nanoforms
•  The different nanoforms in one REACH registration dossier 

are  individually characterised by their basic physico-
chemical parameters (define “what they are”)

Initial grouping of nanoforms
•  Find similarities on “what they are”
•  Find similarities on “where they go”

Information requirements
REACH (tonnage)

Identification of available data and data gaps
•  Data available on the (group of) nanomaterial(s)
•  Identification of data gaps

Identification of potential source materials
•  Hypothesis building to justify use of source materials

•  Consider similarities in physicochemical parameters
•  Consider relationships between physicochemical 

parameters and toxicokinetics and hazard

•  Identify information needed to substantiate the hypothesis

Consider all endpoints 
required under REACH in 
an overall testing strategy

Substantiate hypothesis
•  Information gathering
•  Consider data needed to improve argumentation
•  Build testing strategy

Assess new data
•  Hypothesis sufficiently substantiated?

YES

Fill data gap

1

2

3

4

5

6

NO

Figure 2. 

Strategy for using data between nanoforms. 

3.1.1 

Step 1: Identification of the nanoforms 

The  starting  point  is  well  characterised  nanoforms  that  are  uniquely  defined  by  chemical  and 
physical  properties  but  share  the  same  substance  identity  within  one  REACH  registration  dossier. 
When necessary, additional parameters may be needed to provide a proper characterisation of the 
identified nanoforms. In particular the chemical and physical parameters that define the nanoforms 
(i.e.  chemical  and  physical  identity  parameters  under  the  heading  “what  they  are”  in  Figure  1) 
should be used as a starting point in identifying nanoforms. 

 
 
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17 

3.1.2 

Step 2: Initial grouping of the nanoforms 

As  with  non-nanoforms,  in  case  some  nanoforms  are  grouped  based  on  similarities  in  those 
physicochemical properties that influence “what they are” and “where they go” (see Figure 1), it is 
essential to characterise such a group and provide clear boundaries to define the scope of the group. 
This may include characterising every single nanoform in the group, or a (scientific) justification that 
the  group  can  be  characterised  differently  (cf.  Arts  et  al.,  2014;  Arts  et  al.,  2015a;  Oomen  et  al., 
2015),  for  example  by  characterising  the  output  of  a  well-defined  production  process.  The 
boundaries of a group should be defined by (a set of) specific physicochemical properties, e.g. by a 
detailed description of the production process, by the water equilibrium solubility of the nanoform 
that is similar to that of the non-nanoform, or by a specific (high) aspect ratio (cf. Arts et al., 2015a). 
It should be noted that there may be a limit to the diversity of nanoforms within a group, as a too 
diverse  group  might  reduce  the  possibilities  for  read-across  or  strongly  hamper  a  clear  scientific 
justification for read-across. 

As  outlined  above  in  the  introduction  of  Chapter  3,  in  further  steps  in  the  read-across,  additional 
gathering of data may initiate reconsideration of group boundaries, or subgroups of the initial group 
may need to be defined. 

3.1.3 

Step 3: Identification of available data and data gaps 

Information  requirements  under  REACH  also  apply  to  nanoforms.  As  nanoforms  with  the  same 
substance  identity  may  behave  differently,  there  is  a  need  to  carefully  assess,  for  each  data 
requirement  under  REACH,  if  the  data  available  on  the  substance  are  also  representative  for  the 
registered  nanoforms.  Data  gaps  need  to  be  identified  and  addressed.  Motivated  grouping  of 
nanoforms (as addressed above in Section 3.1.2) may optimise the use of available data. 

3.1.4 

Step 4: Identification of potential source nanoforms 

When  a  data  gap  is  identified  for  a  target  nanoform8,  the  first  step  in  read-across  is  to  assess 
whether available data for (an)other (nano)form(s) in the same group could be used to fill this data 
gap.  This  evaluation  should  be  based  on  a  hypothesis,  meaning  that  hypothesis  formulation  and 
source material identification are two closely connected processes. The hypothesis should be fit to 
guide justification on which  material(s) may be used as a source (non-)nanoform(s) to fill  the data 
gap  of  the  target  nanoform,  under  which  conditions  or  assumptions  the  identified  data  on  source 
form(s)  might  be  used,  and  which  information  (if  any)  should  be  generated  to  substantiate  the 
hypothesis  and  validate  the  actual  use  of  this  data.  A  group  approach  (see  Section  3.1.2)  may 
provide a useful starting point for such hypothesis formulation, as well as e.g. knowledge about the 
production process, the mode-of-action, and the functionality of the nanoform or its uses. Further, 
the hypothesis should combine the chemical and physical identity (i.e. physical and chemical identity 
parameters  described  in  Figure  1  and  Section  3.1.1)  with  information  regarding  fundamental 
behaviour and reactivity to identify potential source form that are sufficiently similar to the target 
nanoform to be used for read-across. 

8  See description of read-across (Annex I) for further explanation of target and source forms. 

 
 
                                                           
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

Obviously, multiple data gaps may be present per nanoform or per group of nanoforms. To address 
these  data  gaps,  hazard  data  from  the  same  source  nanoform  may  not  be  available.  There  are 
different possibilities to evaluate and scientifically justify the use of data from a source nanoform to 
fill a data gap. 

Justifying  read-across  should  aim  at  arguing  that  the  source  and  target  nanoforms  are  sufficiently 
similar  to  share  data  for  a  given  endpoint  under  REACH.  This,  however,  also  implies  developing  a 
rationale for a scenario that maximises safety: 

1.  the  target  nanoform  is  equally  or  less  hazardous  than  the  source  nanoform  (hazard 

argument), and 

2.  very similar or smaller amounts of the target nanoform reach the target site compared to the 

source nanoform (toxicokinetics/environmental fate argument). 

This argumentation can be based on available knowledge on relationships between physicochemical 
parameters, toxicokinetics and hazard characterisation. 

In  general,  it  is  assumed  that  the  more  similarities  are  identified  between  the  physicochemical 
parameters of source and target nanoforms, the more likely it is that read-across can be scientifically 
justified. 

Consideration  of  source  forms  preferably  include  the  assessment  of  benchmark  materials9  to 
establish  when  “similar”  is  “similar  enough”  for  the  purpose  of  read-across.  However,  the  current 
state of knowledge does not yet allow the establishment of benchmark materials. Systematic testing 
and  the  evaluation  of  testing  results  should  aim  for  a  better  understanding  of  nanoforms  and 
especially  the  potential  correlation  between  physicochemical  characteristics  and  toxicity, 
understanding of toxicokinetic behaviour and fate, which may eventually result in the identification 
of  benchmark  materials.  Arts  et  al.  (2014;  2015a)  presents  first  thoughts  on  a  possible  approach 
towards benchmark materials, which may inspire further thinking on how to evaluate “similar” for 
the  different  elements  presented  in  Figure  1  in  the  evaluation  of  read-across  potential  and  the 
identification of source materials. 

When drafting a hypothesis, the following questions may further aid in the identification of potential 
source forms10: 

•  Is it a single nanoform under consideration or multiple nanoforms of the same substance that 

may be placed in one group? 

•  Is the nanomaterial organic or inorganic? 

•  Does the nanomaterial have a coating? 

•  What is the water solubility and/or dissolution rate of the nanoform? 

9  Benchmark  materials  are  source  (nano)materials,  which  have  been  tested  and  evaluated  according  to 

standard criteria and to which target materials may reliably be compared for grouping purposes. 

10  This list of questions is not intended as an exhaustive list. 

 
                                                           
Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

19 

•  Based on knowledge of the manufacturing process and/or based on analytical data, does the 
nanomaterial contain impurities that are of (eco)toxicological concern in relevant amounts? 

•  Is there a non-nanoform of the material? 

•  Does the manufacturer make any claims regarding special properties of this material that are 
related  to  its  purpose  but  may  also  be  relevant  in  a  read-across  or  grouping  approach  (e.g. 
transparency, reactivity, antibacterial activity)? 

•  Is any information available about how this nanomaterial and its properties change as it ages 

that may be considered in (eco)toxicological and environmental fate assessment? 

For the potential source materials the following information should be available: 

-  Hazard and/or toxicokinetic data related to the specific endpoint 

-  Sufficient information to assess the data quality and reliability (see Section 3.1.6) 

Currently, it is  considered  necessary  that source nanoform hazard data are obtained  via  the same 
exposure route as the identified data gap for the target nanoform. This is based on the fact that the 
different  routes  of  exposure  may  correspond  to  different  exposure  media  that  affect  the 
toxicokinetic behaviour of nanomaterials. The specific effect of the exposure media on toxicokinetic 
behaviour of nanoforms, however, is not yet sufficiently understood to allow the extrapolation for 
nanoforms of data from one route to another, which sometimes can currently be applied for non-
nanoforms provided that it can be justified. 

The  justification  for  using  the  data  from  the  source  material  for  the  target  material  should  then 
describe the evidence and assumptions feeding into the rationale for using the same data also for 
the  target  nanoform.  The  justification  should  furthermore  describe  what  information  should 
preferably be gathered to support, or falsify, the hypothesis. It is important to note that building the 
hypothesis should  be  seen  as  an  iterative process. Based on the development of further insight in 
the potential to use data of the source material for the target material, the original hypothesis may 
have to be adjusted or another source material has to be identified. 

3.1.5 

Step 5: Substantiate hypothesis 

Based  on  the  hypothesis  and  the  information  needed  to  substantiate  or  falsify  the  hypothesis  the 
required  information  should  be  gathered  and/or  a  testing  strategy  should  be  developed.  This 
strategy may follow a tiered approach, focussing first on information that could be obtained based 
on  the  physicochemical  parameters,  i.e.  in  silico  or  general  insights.  If  needed,  a  second  tier  for 
substantiating read-across could then include in vitro testing. In vivo testing should be considered as 
the highest tier and should (currently) only be used to generate the REACH relevant data when read-
across cannot be substantiated based on in silico or in vitro data. 

This approach on information  gathering is  usual for read-across applied to chemicals, and  the  first 
steps in the tiered approach proposed by Arts et al.  (2014; 2015a) are in line with it  where  Tier 1 
involves  testing  of  the  intrinsic  material  (i.e.  ‘what  they  are’  in  Figure  1),  and  Tier  2,  the  ‘system-
dependent properties’ (i.e. ‘where they go’ in Figure 1). Based on the outcome of their Tiers 1 and 2 
Arts  et  al.  (2015a)  foresee  further  in  vivo  testing  in  Tier  3.  When  in  vivo  testing  is  considered 

 
 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

necessary to fill a data gap, this should be done for the REACH relevant endpoint, preferably in such 
a way that results can be used for a broader group of nanoforms. 

Similar to building the hypothesis, it should be noted that the testing strategy potentially has to be 
adjusted  after  each  test  result  obtained  which  may  involve  reconsidering  the  applicability  of  the 
source material. 

Also  a  hypothesis  based  on  assuring  that  less  of  the  target  nanoform  reaches  the  target  site 
(toxicokinetics  argument),  and  the  target  nanoform  is  less  hazardous  (hazard  argument),  requires 
scientific substantiation. This is illustrated in Figure 3 and further explained below. 

For toxicokinetics  (i.e. ‘where they  go’  in Figure 1), it may be possible to  scientifically substantiate 
the  argument  that  less  of  the  target  material  is  transferred  across  a  portal  of  entry  (skin, 
gastrointestinal tract, lung epithelium), deposited in the lung or distributed to target tissues, or that 
the  target nanoform  is  better  cleared/less persistent and  thus less likely to accumulate over time. 
Such  information  can  be  obtained  by  physicochemical  information  (e.g.  solubility)  or  biophysical 
testing  (e.g.  dissolution  rate  in  physiologically  relevant  media),  in  silico  (e.g.  multiple  path  particle 
dosimetry modelling for lung deposition) and in vitro testing (e.g. skin permeation). It may also be 
necessary  to  obtain  physicochemical  parameters  as  input  for  e.g.  in  silico  tools.  For  example, 
information on the aggregation/agglomeration state and aerodynamic diameter of a material in air is 
needed  for  modelling  to  estimate  lung  deposition.  Obviously,  the  level  of  confidence  that  can  be 
derived from the data of these different approaches to substantiate the hypothesis is different and 
this needs to be taken into consideration. 

Figure 3. 

Different aspects of hypothesis substantiation per endpoint based on assuring that 
less of the target material reaches the target site (toxicokinetics argument), and the 
target material is less hazardous (hazard argument) (adapted from Oomen et al., 
2015). 

 
 
Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

21 

For  hazard  characterisation  (i.e.  ‘what  they  do’  in  Figure  1),  it  may  be  possible  to  scientifically 
substantiate  the  argument  that  the  target  nanoform  is  equally  or  less  hazardous  than  the  source 
nanoform, e.g. by in silico methods and in vitro testing (e.g. reactivity, formation of reactive oxygen 
species).  Knowledge  on  the  mechanism  of  toxicity  can  help  to  select  suitable  tests  and  build  the 
argumentation. The in silico methods such as quantitative structure-activity relationships are in the 
initial phases of development, see e.g. Burello and Worth (2011). 

For  both  in  vitro  and  in  vivo  toxicokinetic  and  hazard  data,  the  testing  conditions  should  be 
considered,  and  especially  their  relevance  for  the  target  nanoform.  For  example,  it  should  be 
evaluated if the testing medium and dispersion method used to obtain the in vivo data of the source 
material  are  also  relevant  for  the  target  nanoform;  and  if  it  can  be  anticipated  that  the  testing 
conditions  result  in  differences  in  behaviour  between  target  and  source  nanoform,  e.g.  that  one 
nanoform may aggregate whereas the other does not. 

3.1.6 

Step 6: Assess new data 

As a final step, the toxicokinetics and hazard arguments are combined into an overall assessment of 
whether  the  data  of  a  source  nanoform  can  be  used  for  hazard/risk  assessment  of  the  target 
nanoform. Application of source data is only deemed possible if both the toxicokinetics and hazard 
arguments show that less or similar amounts of the target material reaches the target site and is less 
or  equally  hazardous.  The  uncertainty  related  to  the  different  pieces  of  information  needs  to  be 
considered in the overall assessment. 

Ultimately, the information gathered in Step 5 should be used to scientifically justify the read-across 
hypothesis.  This  justification  should  at  least  address  the  potential  influences  of  physicochemical 
parameters and any differences between the source and target nanoforms as illustrated in chapter 
1. This approach is illustrated in Figure 3. 

In summary, any nanoform needs to be properly characterised by chemical and physical composition 
to  enable  identification  (cf.  Section  2.1  including  Figure  1  and  Table  1  as  well  as  Section  3.1.1). 
Ideally,  there  should  be  no  relevant  differences  in  the  impurity  profile  between  the  actual  test 
material and the form with an identified data gap. If any differences are identified, their impact on 
the prediction should be assessed. 

It  is  important  that  a  hazard  characterisation  of  nanomaterial(s)/nanoform(s)  contains  sufficient 
(eco)toxicological  data,  including  scientifically  sound  justification  for  grouping  and  read-across  and 
supporting information to enable adequate risk assessment. All hazard data should be presented in 
conjunction with a specific argument to what extent these data support the hazard characterisation 
of that nanomaterial. 

The generation of an overview matrix of available data and data gaps identified by the registrant (cf. 
Section 3.1.3) may be especially helpful to organise the information on the different forms covered 
by  the  registration  dossier  under  REACH,  and  to  illustrate  how  these  have  been  grouped  and  the 
justification used to delimit each group (cf. Sections 3.1.2, 3.1.4 and 3.1.5). The approach used for 
read-across  should  follow  the  one  developed  for  chemicals  as  outlined  in  ECHA  Guidance  Chapter 
R.6 (ECHA, 2008) with nanospecific considerations as indicated in the current document and ECHA 
Guidance (ECHA, 2012a, b, c). 

 
 
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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

All supporting  information  that  is  relevant to the assessment of the nanoform(s) under evaluation 
should  be  reported.  This  includes  any  additional  studies  conducted  to  support  the  grouping  and 
read-across approach. All data provided must also be accompanied with a detailed description of the 
materials (i.e.  full  characterisation, cf.  Sections  2.1 and  3.1.1) and of the methods used, as well as 
appropriate statistical indicators of the quality and reliability of the results. 

An  analysis  of  the  source  study  used  for  the  prediction  of  a  property  needs  to  be  conducted.  The 
requirements regarding the results of the read-across method demands that the source study (and 
details of test set-up) meet all requirements for a key study used as stand-alone information to meet 
an  information  requirement  under  REACH  (including  the  default  REACH  requirements  regarding 
adequacy, relevance and reliability). 

3.1.7 

Final considerations 

The underlying aim of this read-across assessment is to minimise testing as much as possible whilst 
maintaining a high level of protection of man and the environment, through compliance with REACH. 
When building the testing strategy, the overall goal of REACH, which is to protect human health and 
the environment thus ensuring safe use of chemicals, should be fulfilled. 

At present, the knowledge on the interplay between physicochemical parameters, (eco)toxicological 
properties  and  environmental  fate  is  rapidly  developing.  This  provides  an  avenue  for  better 
understanding  of  nanomaterials’  specific  properties  and  behaviour  and  can  subsequently  support 
progress in development of guidance to enhance the current risk assessment framework. 

Nevertheless,  the  present  approach  for  read-across between  nanoforms  as  outlined  above,  points 
towards the need for data on physicochemical parameters of each nanoform as the crucial starting 
point  to  obtaining  a  better  understanding  on  the  behaviour,  fate,  toxicokinetics  and  toxicity  of 
nanomaterials, which is the cornerstone in developing a scientific, robust justification for grouping 
or  the  use  of  data  for  read-across.  Furthermore,  the  data  quality  is  critical  and  monitoring  of 
physicochemical  parameters  during  testing 
is  therefore  a  key  element.  This  also  requires 
harmonisation  and  standardisation  of  regulatory  test  methods  for  physicochemical,  toxicokinetics 
and hazardous endpoints. 

The  approach  in  this  document  uses  physicochemical  parameters  of  each  nanoform  as  the  crucial 
starting point, and is limited to read-across for hazard endpoints. In the future it may be possible to 
explore the possibilities for read-across for physicochemical properties as well. 

Each  potential  read-across  option,  including  additional  (time,  money  and  animals  for)  potential 
testing needed for its scientific justification, should thus be weighed against the actual testing of the 
target material for REACH relevant endpoints. 

 
 
 
Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

23 

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Usage of (eco)toxicological data for bridging data gaps between and grouping of nanoforms of the same substance 

Annex I: 
CLP 

Nanoform 

Glossary of commonly used terms 

Regulation (EC) No 1272/2008 on classification, labelling and packaging of 
substances and mixtures. 

The  term  ‘nanoform’  is  used  in  this  document  to  distinguish  forms  of  a 
substance that fulfil the EC Recommendation on the definition of the term 
‘nanomaterial’  but  differ  with  regard  to  size  distributions,  shape  and/or 
surface chemistry. 

Group (or category) 

are 

that 

substances 

Under  REACH, 
similar  with 
physicochemical, toxicological, ecotoxicological and/or environmental fate 
properties that are likely to be similar or to follow a regular pattern may be 
considered as a group of substances. Within a group of substances, a data 
gap might be filled by read-across, as described below. 

structurally 

Read-across 

REACH 

Toxicokinetics 

is  a  technique  for  predicting  endpoint 
Under  REACH,  read-across 
information  for  one  substance  (target  substance),  by  using  data  from  the 
same  endpoint 
(source  substance(s)). 
(an)other  substance(s), 
Consequently,  the  read-across  approach  has  to  be  considered  on  an 
endpoint-by endpoint basis.  

from 

Regulation  (EC)  No  1907/2006  concerning  the  Registration,  Evaluation, 
Authorisation and Restriction of Chemicals (REACH). 

The relationship between systemic exposure of a substance/nanoform and 
its toxicity. Four main processes exist: absorption, distribution, metabolism 
and excretion (ADME). 

 
E
D

-
0
2
-
1
6
-
2
2
8
-
E
N
N
–
D
o

-

.

I
:
1
0
2
8
2
3
/
9
8
2
0
4
6
–
I
S
B
N

:
9
7
8
-
9
2
-
9
2
4
7
-
8
1
0
-
0