Alternative testing strategies : replacing, reducing and refining use of animals in research.
Date: 2008-04-11
Institutions: Directorate-General for Research and Innovation
Subjects: animal experimentation, medical research, research method, research policy, research programme

Genomics & Biotechnology for Health

Replacing, reducing and refining use of animals in research

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EUR 22846

 
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ALTERNATIVE TESTING STRATEGIES

Replacing, reducing and refining use of animals in research

Edited by
Charles Kessler

2008

Life Sciences, Genomics and Biotechnology for Health

EUR 22846

Directorate-General for Research

NEW THERAPIES

ALTERNATIVE TESTING STRATEGIES

TABLE OF CONTENTS

INTRODUCTION 5

ACuteTox
Optimisation and pre-validation of an in vitro test 
strategy for predicting human acute toxicity  11

ARTEMIS 
In  vitro  neural  tissue  system  for  replacement 
of  transgenic  animals  with  memory/learning 
17
deficiencies  

carcinoGENOMICS 
Development  of  a  high  throughput  genomics-
based 
for  assessing  genotoxic  and 
carcinogenic properties of chemical compounds 
19
in vitro  

test 

COMICS 
Comet assay and cell array for fast and efficient 
22
genotoxicity testing 

EXERA 
in  vitro  models  of 
Development  of  3D 
estrogen-reporter  mouse 
the 
pharmacotoxicological  analysis  of  nuclear 
25
receptors-interacting compounds (NR-ICs) 

tissues 

for 

Invitroheart 
Reducing  animal  experimentation 
in  drug 
testing by human cardiomyocyte in vitro models 
29
derived from embryonic stem cells 

LIINTOP 
Optimisation of liver and intestine in vitro models 
for  pharmacokinetics  and  pharmacodynamics 
33
studies  

MEMTRANS 
Membrane  transporters:  in  vitro  models  for  the 
study of their role in drug fate  
37
PREDICTOMICS 

2

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Short-term in vitro assays for long-term toxicity 
39

ReProTect 
Development  of  a  novel  approach  in  hazard 
and  risk  assessment  of  reproductive  toxicity  by 
a combination and application of in vitro, tissue 
44
and sensor technologies  

Sens-it-iv 
Novel testing strategies for in vitro assessment of 
49
allergens  

TOXDROP 
Innovative  ‘cell  on  chip’  technology  to  screen 
chemicals for toxicity, using cultured cells within 
53
tiny ‘nanodrops’ of culture fluid  

VITROCELLOMICS 
Reducing  animal  experimentation  in  preclinical 
predictive drug testing by human hepatic in vitro 
models derived from embryonic stem cells   58

BBMO 
Biosensors based on membrane organisation to 
61
replace animal testing  

CONAM 
Consensus  networking  on  alternatives  within 
64
Europe  

ForInViTox 
Forum  for  researchers  and  regulators  to  meet 
67
manufacturers of toxicology test methods  

InViToPharma 
Workshop  on  the  need  of  in  vitro  toxicity  tests 
69
within the pharmaceutical industry  

 
ALTERNATIVE TESTING STRATEGIES

NHR DevTox 
A  prospective  analysis  of  the  mechanisms  of 
nuclear  hormone  receptors  and  their  potential 
as  tools  for  the  assessment  of  developmental 
71
toxicity 

RAINBOW 
Research  on  animal  and  in  vitro  studies  and 
numerical  methods:  bridging  opportunities 
73
through a workshop  

RETHINK 
Mini-pigs as models for the toxicity testing of new 
medicines and chemicals: impact assessment  76

SCARLET 
Structure-activity  relationships  leading  experts 
78
in mutagenicity and carcinogenicity  

FP7 PROJECTS 

 81

ESNATS 
Embryonic  stem  cell-based  novel  alternative 
83
testing strategies 

NANOTEST 
Development  of  methodology  for  alternative 
testing  strategies  for  the  assessment  of  the 
toxicological  profile  of  nanoparticles  used  in 
86
medical diagnostics  

OPENTOX 
Promotion,  development,  acceptance  and 
implementation of QSARs (quantitative structure-
88
activity relationship) for toxicology  

PREDICT-IV 
Profiling the toxicity of new drugs: a non animal-
integrating  toxico-dynamics 
based  approach 
90
and biokinetics  

START-UP 
in  3Rs 
technological 
Scientific  and 
alternatives  research  in  the  process  of  drug 
92
development and Union politics  

issues 

INDEX OF PROJECTS 

 94

INDEX OF 
COORDINATORS 

 96

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

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4

ALTERNATIVE TESTING STRATEGIES

INTRODUCTION

‘Alternative testing strategies’ refers to ef-

forts to replace animals in experimenta-
tion as part of the “Three Rs” approach of 
replacement,  reduction  and  refinement.  Devel-
oping robust and effective in vitro tests has been 
a priority for EU research programmes since the 
1980s.  The  challenge  is  to  develop  alternative 
methods  which  can  be  validated  according  to 
international  standards,  be  recognised  by  regu-
latory authorities and be adopted by industry. A 
particular application of the methods now is the 
EU’s registration, evaluation and authorisation of 
chemicals policy (REACH).

The  purpose  of  this  compilation  of  information 
on EU-supported research in alternative testing 
strategies  is  to  demonstrate  the  range  of  ac-
tivities  undertaken  and  initial  results  obtained 
in  this  field  during  the  course  of  the  EU’s  Sixth 
Framework  Programme  for  Research  (2002-
2006),  notably  under  the  heading ‘Applications 
of knowledge and technologies in the field of ge-
nomics and biotechnology for health’ in the ‘Life 
sciences, genomics and biotechnology for health’ 
thematic priority. Information is also provided on 
projects being supported following the first call 
for proposals made by the Health priority of the 
newly-launched Seventh Framework Programme 
(2007-2013);  this  indicates  that  research  efforts 
towards  alternative  testing  are  continuing,  and 
further  support  to  the  area  is  envisaged  as  the 
programme develops.

The  projects  described  fall  into  three  groups: 
high-throughput techniques, specific toxicity as-
says and projects focusing on specific scientific, 
social  or  ethical  aspects  and  taking  the  form  of 
fora  and  workshops. The  projects  are  placed  in 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

5

ALTERNATIVE TESTING STRATEGIES

INTRODUCTION

alphabetical order in the book and a summary of the main topics covered by the activities is shown in 
schematic version in the figure below:

EU Alternative Testing Strategies Research (FP6)

High throughput
techniques

Cell
chips

Carcinogenicity

Cell array
genotoxicity

Specific toxicity
assays 

3D model
estrogen

Liver,
intestine
toxicity

Reproductive
toxicity

Neurotoxicity

Cardiotoxicity
 hESC

Skin 
respiratory
toxicity

Acute
toxicity

Hepato
Toxicity
 hES cells

Liver, 
kidney
toxicity

Membrane 
transport

Fora and
workshops

in vitro 
in Pharma

QSARs 

3Rs 
EU
network

Bio
sensors

Stakeholder 
forum

Mini pigs
In 3Rs

New 
end points
(NHR)

Different colours refer to different calls for proposals. Shapes: oval – integrated projects; rectangle – specific targeted 
research project; circle – specific support action. 

Main topics of alternative testing strategies research projects described in this review.

The  aim  of  the  projects  described  here  was  to 
create  technological  platforms  for  the  develop-
ment of alternative testing strategies. To achieve 
this,  projects  were  built  around  five  different 
funding schemes:

Integrated  projects  (IP).  These  are  the  largest 
size  projects  and  integrate  a  range  of  different 
activities,  such  as  research,  demonstration  and 
training. They also permit projects to take a multi-
disciplinary approach, to link underlying biology 
and tool development and enable scientists, cli-

nicians and other stakeholders to work together 
to achieve their deliverables.

Specific  Targeted  Research  Projects  (STREP). 
These  are  smaller  projects  which  focus  on  spe-
cific  research  issues. They  may  have  an  applied 
focus  but  are  less  multi-disciplinary  and  wide-
ranging than the Integrated Projects.

SME-Specific Targeted Research Projects. Tar-
geted Research Projects designed to encourage 
research  and  innovation  efforts  of  small  and 

6

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

medium-sized enterprises (SMEs) and where re-
search-led SMEs play a leading role.

Specific  Support  Actions  (SSA)  for  training, 
conferences or prospective studies in support of 
the programme.

European  research  projects  are  encouraged  to 
be  open  towards  the  general  public  and  to  en-
gage with stakeholders and interest groups. This 
is  particularly  important  in  the  field  of  alterna-
tive testing which is of considerable public inter-
est.  Accordingly  many  projects  organise  public 
meetings  and  dialogue  and  set  up  websites  on 
the consortium, the research and results. 

Website  addresses  are  given  in  the  details  of 
each project and provide more up-to-date infor-
mation than this publication.

The number of projects supported by the differ-
ent  funding  schemes  and  the  EC  financial  con-
tribution to them is shown in the table below. It 
can  be  seen  that  during  the  Sixth  FP  overall  21 
projects  were  supported  with  an  EC  contribu-
tion of over EUR 63 million; this was distributed 
among  about  255  research  teams,  almost  55  of 
which  are  SMEs.  In  the  Seventh  FP,  five  projects 
are under contract negotiation and envisage an 
EC contribution of around EUR 30 million.

Funding scheme

Sixth FP

Integrated project

Specific Targeted Research 
Project

SME-Specific Targeted 
Research Project

Specific Support Action

Total

Seventh FP (First Call)

Number of 
projects

EC financial contribution (million €)

4

4

5

8

21

5

39.5

8.7

13.0

2.1

63.3

30.4 (planned)

Numbers of projects supported and EC financial contribution to alternative testing strategies research

It is clear from this compilation that alternative testing strategies is an extremely active area of re-
search for European scientists both from the public and private sectors. These efforts in the health 
programme are complemented by related research undertaken by the nanosciences, nanotechnolo-
gies, materials & new production technologies programme and by the environment programme of 
the Seventh FP.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

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INTRODUCTION

While  the  desire  to  avoid  the  use  of  animals  in 
safety  testing  is  straightforward  and  easily  ex-
pressed,  making  this  a  reality  is  complex.  The 
projects described in this book concern the first 
step in the process, namely the development of 
new tools, techniques and methods that can be 
used in safety testing in pharmaceutical, chemi-
cal, cosmetic or other industries. This step is es-
sentially methodological, is a key one and is one 
dependent on scientific innovation. However to 
get  these  new  methods  adopted,  they  need  to 
be  scientifically  validated  by  comparison  with 
existing standard methods. This task is undertak-
en by the European Centre for the Validation of 
Alternative  Methods  (http://ecvam.jrc.it),  which 
is  a  partner  in  some  of  the  projects.  Even  once 
validated, there are still hurdles to be overcome 
for  the  new  tests  to  become  standard  practice. 
For  instance,  different  countries  have  different 
regulatory requirements and industry has to in-
vest in setting up the new methods. This step is 
facilitated through the European Partnership for 
Alternative Approaches to Animal Testing (http://
ec.europa.eu/enterprise/epaa/index_en.htm), 
which is a joint initiative from the European Com-
mission  and  a  number  of  companies  and  trade 
federations  active  in  various  industrial  sectors 
whose purpose is to promote the development 
of  new “Three  Rs”  methods  as  modern  alterna-
tive  approaches  to  safety  testing.  Authorities 
in  charge  of  implementation  and  enforcement 
have a particular role in ensuring that validated 
new techniques are used.

In conclusion, it can be seen that replacement of 
animals in safety testing involves many steps and 
different sectors, and that nothing would be pos-
sible in this field without scientific innovation.

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Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

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Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

ACUTETOX

Optimisation and pre-validation of an in vitro test strategy for predicting human 
acute toxicity

Contract No 
Project type 

LSHB-CT-2004-512051
Integrated Project 

EC contribution  e 9 000 000

Starting date   1 January 2005
Duration  60 months

Website  www.acutetox.org

Background and objectives:

The  ACuteTox  project  aims  to  develop  and 
prevalidate  a  simple  and  robust  in  vitro  testing 
strategy  for  the  prediction  of  human  acute  sys-
temic toxicity. This has the potential to replace the 
animal acute toxicity tests that are currently used 
for regulatory purposes. The extensive amount of 
work  performed  since  the  1970s  has  led  to  the 
large number of existing in vitro models for acute 
toxicity testing. Many studies have shown good 
correlation  between  in  vitro  basal  cytotoxicity 
data and rodent LD50 values. Moreover, the MEIC 
(Multicentre  Evaluation  of  In  vitro  Cytotoxicity) 
programme showed a good correlation (around 
70%) between in vitro basal cytotoxicity data and 
human  lethal  blood  concentrations.  However,  a 
certain  number  of  misclassifications  will  occur 
when the existing tests are used. ACuteTox aims 
to identify factors that can optimise the in vitro/
in vivo correlation for acute systemic toxicity. 

The project is divided into 9 scientific Work Pack-
ages (WPs): 
•	

WP1:	 The	generation	of	a	high	quality	in 
vivo database;
WP2:	 The	generation	of	a	high	quality	in 
vitro database;
WP3:	 Iterative	amendment	of	the	testing	
strategy;
WP4:	 New	 cell	 systems	 and	 new	 end-
points;

•	

•	

•	

•	

•	

•	

•	

•	

WP5:	 Alerts	 and	 correctors	 in	 toxicity	
screening (I): Role of ADE;
WP6:	 Alerts	 and	 correctors	 in	 toxicity	
screening (II): Role of metabolism;
WP7:	 Alerts	 and	 correctors	
in	 toxicity	
screening (III): Role of target organ toxicity;
WP8:	 Technical	 optimisation	 of	
the	
amended test strategy;
WP9:	 Pre-validation	of	the	test	strategy.	

The  overall  scientific  objectives  of  the  project 
are:
1. 

Compilation,  critical  evaluation  and  gen-
eration of high quality in vitro and in vivo 
data  for  comparative  analysis  (WP1  and 
WP2);
Identification of factors (absorption, distri-
bution,  excretion,  metabolism  and  organ 
specificity)  that  influence  the  correlation 
between  in  vitro  toxicity  (concentration) 
and in vivo toxicity (dosage), and the defi-
nition  of  an  algorithm  that  accounts  for 
this (WPs 5, 6, 7.1, 7.2, 7.3);
Exploration  of  innovative  tools  and  cel-
lular  systems  to  identify  new  end-points 
and strategies to better anticipate animal 
and human toxicity (WP4);
Design of a simple, robust and reliable in 
vitro  test  strategy  amenable  for  robotic 
testing,  associated  with  the  prediction 
model for acute toxicity (WPs 3, 8, and 9).

2. 

3. 

4. 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

11

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

ACUTETOX

Approach and methodology:

Known outliers of available in vitro/in vivo corre-
lations are being evaluated in order to introduce 
further parameters that might improve the cor-
relation,  including  absorption,  distribution  and 
excretion,  metabolism  and  organ  specificity. 
Reference  chemicals  selected  mainly  from  pre-
vious  studies  (MEIC  and  ECVAM/ICCVAM  valida-
tion,  for  example)  are  being  tested  in  different 
in  vitro  and  in  silico  assays.  This  allows  for  the 
integration  of  alerts  in  a  prediction  algorithm 
which, together with a robust implementation of 
medium-throughput  approaches,  would  enable 
the creation of a new testing strategy with better 
prediction  for  toxicity  classification. The  project 
is  being  carried  out  over  a  five-year  period;  the 
testing  strategy  will  be  prevalidated  in  the  last 
two years. 

Expected outcome:

The  formal  validation  of  the  definitive  testing 
strategy will lead to regulatory approval and its 
incorporation  into  the  set  of  standardised  test 
guidelines  for  chemical  hazard  assessment. The 
proposed  testing  strategy  could  potentially  re-
place EU methods B.1bis and B.1tris in Annex V 
of  Dir  67/548  EEC  and,  subsequently,  the  corre-
sponding OECD Test Guidelines 420, 423 and 425.

Main findings:

The  work  in  WP1  and  WP2  has  been  finalised. 
In  WP1,  animal  and  human  data  for  the  97 
ACuteTox reference compounds were compiled. 
The  database  contains  LD50  values  from  2  206 
animal studies, as well as human data from 2 902 
case reports, including acute sub-lethal and lethal 
blood concentration data. Descriptive summaries 
containing  physico-chemical  data,  LD50  values, 
human 
data,  pharmacokinetics-/ 
toxicokinetics-  data,  metabolism,  toxicological 
mechanisms,  and  target  organs  for  all  97 
reference chemicals have been recorded. 

toxicity 

The testing of the ACuteTox reference chemicals 
in  six  basal  cytotoxicity  tests  (WP2)  has  been 
completed.  Data  for  most  of  the  97  reference 
compounds are available for the Fa32/NR uptake, 
Fa32/protein  content,  3T3/NR  uptake  and  NHK/
NR uptake systems. Data are available for half of 
the  compounds  in  the  HepG2/protein  content 
and  HL60/ATP  content  systems.  Data  from WP1 
and WP2, as well as from WP4 to WP7, are stored 
in AcuBase, a database developed in WP3 to facil-
itate Standard Operating Procedures (SOPs) stor-
age, data transfer from all partners and statistical 
analysis of larger data sets. 

The  data  from  WP1  and  WP2  have  been  com-
pared in some preliminary multivariate analyses 
with  the  aim  of  identifying  additional  outliers 
that will be tested by the Partners before the fi-
nal selection of methods that will enter the pre-
validation phase. One of the aims of the project 
is  to  adapt  the  methods  of  the  testing  strategy 
to  high-throughput  screening  (HTS).  In  WP3,  a 
number of test assays such as the 3T3/NR uptake 
and  HepG2/MTT  assays  have  successfully  been 
adapted  to  two  commercially  available  HTS  ro-
botic platforms. 

In WP4 through WP7, the testing of the first 2 sets 
of  reference  chemicals  (no.  1-46)  has  continued 
and the results are reported in AcuBase. This test-
ing will continue until month 30 when the data 
sets will be integrated in the in vitro/in vivo com-
parisons  with  the  aim  of  selecting  methods  for 
improving  the  correlation  and  thus  becoming 
candidates for inclusion in the testing strategy.

The aim of WP4 is to provide an alternative way 
for improving the prediction of acute toxicity by 
incorporating  more  specific  end-point  param-
eters, and/or cell models from  the haematopoi-
etic system in the testing strategy. Effects on the 
in vitro production of cytokines in whole human 
blood cultures as well as effects on the CFU-GM 
and  progenitors  of  megakaryocytes  have  been 
measured  for  20  chemicals  and  the  results  are 

12

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

now  being  analysed.  The  miniaturising  of  the 
Cytomic  Panels  for  Cytotoxicity  and  Oxidative 
Stress Screening, as well as the testing of 20 ref-
erence  chemicals,  have  been  finalised  and  the 
results show good correlation with human blood 
LC50 values. 

The  most  crucial  parts  of  the  kinetic  behaviour 
have been studied in WP5. For this purpose, the 
determination  of  kinetic  parameters  is  being 
performed  either  by  experimental,  in  vitro  tests 
or  computer-based  kinetic  modelling.  Neural 
network  methodologies  that  are  useful  for  es-
timating  oral  absorption  and  blood  brain  bar-
rier passage have been developed. Results from 
three variants of the Caco-2 model for prediction 
of  oral  absorption,  used  in  three  different  labo-
ratories, have been compared. So far, the results 
show  good  agreement  between  the  different 
variants. Toxicity  studies  and  permeability  stud-
ies  using  in  vitro  BBB  models  have  been  per-
formed  for  22  and  19  compounds  respectively, 
showing relatively good correlation with in vivo 
data.  Another  aim  of  WP5  is  to  investigate  the 
partitioning behaviour of a number of polycyclic 
aromatic hydrocarbons (PAHs) to different com-
ponents of a typical in vitro assay by using solid 
phase microextraction. This technique was prov-
en to accurately measure the free concentration 
of  compounds  such  as  PAHs,  being  easy  to  use. 
Finally,  in  vitro  plasma  protein  binding  of  refer-
ence compounds has been performed. The data 
obtained from WP5 are now the basis for further 
biokinetic modelling. 

In order to evaluate if toxicity is dependent on me-
tabolism, the effects of 21 reference compounds 
are being been compared between a metabolic 
competent  model  (primary  hepatocytes)  and 
a non-metabolising cell type (HepG2) by use of 
MTT (WP6). By comparing the concentration-tox-
icity  curves  of  each  compound  in  both  models 
it is possible to ascertain whether the molecule 
elicits toxic effects preferentially on hepatocytes, 
suggesting that a bioactivation of the xenobiotic 

is  required.  Recombinant-defective  adenoviral 
vectors  encoding  major  CYP  genes  involved  in 
foreign  compound  metabolism  have  been  de-
veloped as a way to overcome the problem with 
non-metabolising cell systems. 

For  Tamoxifen,  Cyclosporin  A,  and  to  some  ex-
tent  also  Tetracyclin,  which  toxicity  is  depend-
ent on 3A4-dependent biotransformation, toxic-
ity  was  observed  only  in  HepG2  cells  that  were 
infected  with  adenovirus  encoding  for  CYP450 
3A4. Another aim of WP6 was to investigate how 
METEOR and DEREK software performs in predic-
tion of metabolic fate of compounds with known 
biotransformation. Fourteen ACuteTox reference 
compounds were tested, indicating that METEOR 
appears to be an interesting alternative for in sili-
co prediction of metabolism. Furthermore, it was 
concluded  that  even  if  the  DEREK  programme 
does not predict acute toxicity per se, important 
information  regarding  the  toxic  profile  of  the 
tested substances can be gained. 

In  the  neurotoxicity  Work  Package,  WP  7.1, 
26  chemicals  (half  of  them  neurotoxic)  have 
been  studied  in  native  or  differentiated  human 
neuroblastoma  SH-SY5Y  cells,  primary  cultures 
of  mouse  or  rat  neurons,  and  mature  re-
aggregated  rat  brain  cells  by  using  more  than 
20  different  endpoints  (such  as  mitochondrial 
membrane  potential,  GABAA  receptor  function, 
[3H]GABA  uptake,  GAD  activity,  [3H]aspartate 
uptake,  glutamate  release,  AChE  activity,  ChAT 
activity NMDA-glutamate receptor, acetylcholine 
esterase  activity,  uptake  of  [3H]noradrenalin, 
depolarisation-  and  carbachol-evoked  changes 
in  CMP,  intracellular  free  Ca2+  concentrations, 
gene  expression,  ROS,  2-deoxyglucose  uptake, 
[3H]uridine 
[35S]methionine 
incorporation, 
incorporation, GS activity, and CNP activity). 

The results show that the broad collection of as-
says  could  predict  the  neurotoxic  compounds. 
However,  the  challenge  is  to  find  more  general 
assays  that  could  identify  several  different  neu-

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

13

ALTERNATIVE TESTING STRATEGIES

ACUTETOX

rotoxic mechanisms of action. For the measure-
ment of nephrotoxicity in WP7.2, transepithelial 
resistance  (TEER)  was  chosen  as  the  functional 
assay  and  the  LLC-PK1  cell  line  as  the  test  sys-
tem. The  REMS  automated  device  was  selected 
for measurement of TEER. Twenty-one reference 
chemicals  (of  which  5  are  nephrotoxic)  were 
tested and the overall results show that the TEER 
model using renal epithelial cells is a promising 
model for detection of nephrotoxicity.

The  main  goal  of  WP7.3  is  to  identify  a  set  of 
markers characteristic of acute liver toxicity that 
could  be  of  use  in  high  throughput  screening. 
Metabolic  competent  cells  (rat  hepatocytes), 
non-competent  hepatic  cells  (HepG2)  and  non 
hepatic  cells  (3T3  fibroblasts)  were  exposed  to 
the 21 selected compounds using the MTT assay. 
Also,  the  following  biochemical  functions  were 
examined in the cells: cellular ATP levels, forma-
tion of reactive oxygen species (ROS) as an index 
of  oxidative  stress,  cellular  protein  content  and 
mitochondrial membrane potential. 

The results were compared with the MTT assays 
and suggest that neither ATP levels, ROS forma-
tion or protein content, when measured at early 
times  (5  hours  of  incubation)  or  late  times  (24 
hours  of  incubation)  allowed  a  better  discrimi-
nating effect than the one obtained by the MTT 
test. Another objective of WP7.3 is to develop an 
in vitro strategy to assess the effects of chemicals 
on the impairment of hepatocyte bile acids and 
bilirubin transport. This is done by generating a 
cell  bank  expressing  the  various  hepatocellular 
transport systems and establishing assays as well 
as to characterise the interaction of chemicals in 
vitro with individual transporters. 

As  a  result  of  these  efforts,  a  protocol  outlining 
a  test  system  to  identify  (alert)  for  substances 
which  are  potentially  hepatotoxic  because  of 
their  capability  to  impair  hepatic  transport  is 
expected.

14

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Coordinator 

Leila Risteli 
Oulu University
P.O. Box 8000
90014 Oulu, Finland
E-mail: lristeli@oulu.fi

Scientific coordinator

Cecilia Clemedson
Expertrådet AB
Högklintavagen 7
Sundbyberg, Sweden
E-mail: cecilia.clemedson@expertradet.se

Partners 

José Castell, Maria Jose Gomez-Lechon 
University Hospital La Fe
Valenia, Spain

Pilar Prieto, Thomas Cole, Sandra Coecke
EVCAM
Ispra, Italy 

Joan-Albert Vericat 
NeuroPharma
Madrid, Spain

Bas Blaauboer 
Utrecht University
Utrecht, Netherlands

Per Garberg 
Biovitrum
Stockholm, Sweden 

Richard Clothier 
University of Nottingham
Nottingham, UK 

José-Enrique O´Connor 
University of Valencia
Valencia, Spain 

ALTERNATIVE TESTING STRATEGIES

Laurence Lafanechere 
Centre de Griblage de Molécules Bio-actives
Grenoble, France

Beatriz Albella
CIEMAT
Madrid, Spain

Pedro Buc Calderon 
Université Catholique de Louvain
Louvain-la-Neuve, Belgium

Ana Castro and Cristina Sunol 
Consejo Superior de Investigaciones Cientificas
Barcelona, Spain 

Thaly Lakhanisky 
Institute of Public Health
Brussels, Belgium

Myriam Fabre
Advanced In vitro Cell Technologies
Barcelona, Spain

Anna Forsby 
Stockholm University
Stockholm, Sweden

Hans-Jürgen Ahr 
Bayer AG
Wuppertal, Germany

Gabrielle Hawksworth 
University of Aberdeen
Aberdeen, UK

Robert Rudowski 
University of Warsaw
Warsaw, Poland

Paul Honegger 
University of Lausanne
Lausanne, Switzerland

Elisabeth Hooghe-Peters 
Free University of Brussels
Brussels, Belgium

Mónica Betanzos 
GAIKER
Zamudio, Spain

Christina Rudén 
Royal Institute of Technology
Stockholm, Sweden

Michael P. Ryan 
University College Dublin
Dublin, Ireland

Michael Sjöström 
Umeå University
Umeå, Sweden

Annalaura Stammati 
Istituto Superiore di Sanità
Rome, Italy

Bruno Stieger 
University Hospital
Zurich, Switzerland

Inge Mangelsdorf
Fraunhofer
Hannover, Germany

Jitka Ulrichova 
Palacky University
Olomous, Czech Republic

Albrecht Wendel 
STZ INPuT
Konstanz, Germany

Per Artursson 
Uppsala University
Uppsala, Sweden

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

15

ALTERNATIVE TESTING STRATEGIES

ACUTETOX

Roméo Cecchelli 
University of Artois
Lens, France

Ada Kolman 
Swedish Research Fund without Animal Experiments
Stockholm, Sweden

Victor Rimbau 
University of Barcelona
Barcelona, Spain

16

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

ARTEMIS

In vitro neural tissue system for replacement of transgenic animals with memory/
learning deficiencies

Contract No 
Project type 

LSHB-CT-2007-037862 
SME-Specific Targeted Research Project 

EC contribution  e 1 984 900

ARTEMIS

Starting date   1 March 2007
Duration  36 months

Website  www.artemisproject.eu

Background and objectives:

Approach and methodology:

The  ARTEMIS  project  seeks  to  design,  develop 
and  optimise  an  in  vitro  system  to  replace  the 
use  of  animals  in  transgenics  and  toxicology  ex-
periments, and in studies related to the effects of 
genes, chemicals, and neural tissue structure and 
function,  such  as  memory  and  learning.  From  a 
scientific perspective, the ARTEMIS partners target 
the  development  of  a  three-dimensional  neural 
tissue-like construct that is formed by the synaptic 
connections developed among neurons that are 
produced from mouse embryonic stem cells. 

For  its  operational  goal,  the  consortium  seeks 
to replace transgenic animals with memory and 
learning deficiencies with the in vitro developed 
neural  tissues.  ARTEMIS  will  develop  the  tissue 
in vitro from embryonic cell lines that have the 
genes involved in memory and learning “switched 
off”. The consortium will assess and compare the 
ability of the transgenic tissue to memorise elec-
trical stimuli with that of the normal tissue. 

ARTEMIS  is  targeting  the  most  critical  network 
function  of  neural  tissue:  memory  storage  and 
learning  capability.  The  functions  concern  the 
ability  of  the  in vitro  neuronal  network  to  store 
in its pattern of connectivity the memory of one 
or  two  spatiotemporal  electrical  signals,  and 
identify  the  difference  between  these  signals 
and other stimuli in later stimulation (following 
memory storage).

The  system  in  question  would  provide  a  broad 
technological  basis,  upon  which  a  number  of 
application-specific in vitro systems could be cre-
ated. A neural tissue offering possibilities for the 
implementation of several types of in vitro tests 
is being developed. It is also significant that the 
in vivo system can provide a long-term and more 
direct  assessment  of  toxic  or  pharmacological 
effects  on  neuronal  development,  i.e.  learning 
impairment  rather  than  end-point  biochemical 
markers of RNA.

Assessing the role of genes in memory and learn-
ing  in  vitro  would  provide  preliminary  informa-
tion at tissue level, so as to determine the design 
of  the  transgenic  animals  towards  the  optimal 
ones with a higher probability of having altered 
phenotypes.  This  would  effectively  reduce  the 
number of transgenic animals that are currently 
produced  by  trial-and-error  methods.  The  pro-
posed  in  vitro  system  can  effectively  integrate 
biochemical damages to behavioural ones. 

ARTEMIS  will  use  the  system  in  tests  aimed  at 
providing information on the biochemical mech-
anisms  of  memory  defects  in  neural  tissue  pro-
duced from transgenic cell lines, for which flaws 
in earlier in vivo experiments had failed to refine 
the  mechanisms.  This  system  will  also  be  used 
in neurotoxicity tests so as to measure the same 
system  toxicity  endpoints  at  different  levels  of 
the  neural  tissue  organisation,  such  as  cellular 
and synaptic network levels.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

17

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

ARTEMIS

The design of the system refers not only to the 
network structure, but also to the process of the 
network development. It targets stimulus-specif-
ic  connectivity  patterns  and  includes  structural 
and functional designs of the in vitro process of 
the  neuronal  network  development.  The  pat-
terns’  features  are  not  static;  they  evolve  as  the 
network is exposed to stimuli.

Expected outcome:

The proposed methodology of ARTEMIS, in con-
nection with the scientific and technical advance-
ments related to the project, will be effective in 
making  feasible  the  training  of  a  neuronal  net-
work  that  can  respond  specifically  to  electrical 
stimuli. Moreover, existing protocols of the neu-
ronal  differentiation  of  mouse  embryonic  stem 
cells provide evidence of suitability in the provi-
sion of neurons expressing genes involved in the 
formation of a functional synaptic network.

The consortium will draw up a proposal for for-
mal prevalidation studies in order to ensure the 
validation  and  use  of  this  system,  and  would 
guarantee  that  animals  not  be  used  in  the  pre-
diction of behavioural changes.

Coordinator

Petros Lenas
University Complutense of Madrid 
Madrid, Spain
E-mail: petros.lenas@opt.ucm.es

18

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Partners

Janusz M. Rosiak
Technical University of Lodz
Lodz, Poland

Daniel Horák
Institute of Macromolecular Chemistry
Academy of Sciences of the Czech Republic
Prague, Czech Republic

Giovanni Verreschi
Electronic Technology Team (eTT)
Rome, Italy

Andras Dinnyes
BioTalentum Ltd
Gödöllő, Hungary

George, Szekeres
Histopathology Ltd
Pecs, Hungary

Thomas Hartung
Joint Research Centre
Brussels, Belgium

Wolfram Riedel
Bioservice Scientific Laboratories GmbH
Planegg, Germany

Ernest Arenas
Karolinska Institute
Stockholm, Sweden

Sulev Kõks
Quattromed
Tartu, Estonia

José Fernando F. Mendes
University of Aveiro
Aveiro, Portugal

ALTERNATIVE TESTING STRATEGIES

carcinoGENOMICS

Development of a high throughput genomics-based test for assessing genotoxic 
and carcinogenic properties of chemical compounds in vitro

Contract No 
Project type 

LSHB-CT-2006-037712
Integrated Project 

EC contribution  e 10 440 000

Starting date   1 November 2006

Duration  60 months

Website  www.carcinogenomics.eu

Background and objectives

Over  a  5-year  period,  the  carcinoGENOMICS 
project will develop in vitro methods to test the 
carcinogenic properties of compounds as an al-
ternative to the chronic rodent bioassays that as-
sess  chemical  genotoxicity  and  carcinogenicity. 
The major goal is to develop a series of mecha-
nism-based in vitro tests that are representative 
of  various  modes  of  carcinogenic  action. These 
tests  will  refer  to  a  number  of  major  target  or-
gans for carcinogenic action, e.g. liver, lungs and 
kidneys. 

The novel assays will be based on the application 
of  genomics  technologies  (i.e.,  genome-wide 
transcriptomics and metabonomics) in tests us-
ing  robust  in  vitro  systems  (rat/human).  They 
will  explore  stem  cell  technology  and  generate 
genomic  responses  from  a  well-defined  set  of 
model compounds for genotoxicity and carcino-
genicity. The project partners will assess pheno-
typic markers in order to anchor gene expression 
modulations and metabolic profiles. 

Through extensive biostatistics, literature mining, 
and  analysis  of  molecular  expression  datasets, 
the  carcinoGENOMICS  network  will  identify  dif-
ferential  genetic  pathways  with  the  capacity  to 
predict mechanisms of chemical carcinogenesis 
in  vivo.  Transcriptomic  and  metabonomic  data 
will  be  integrated  into  a  holistic  understanding 
of  systems  biology,  and  then  used  to  build  an 
iterative in silico model of chemical carcinogen-

esis.  Subsequently,  predictive  genetic  pathways 
will be used as the scientific basis for developing 
high-throughput  technology  for  accelerating 
analysis  of  genomics  responses  in vitro,  indicat-
ing human carcinogenic risk, by a factor of 100. 
This will enable the efficient assessment of high 
numbers of compounds for genotoxicity and car-
cinogenicity as required under the REACH (Reg-
istration, Evaluation and Authorisation of Chemi-
cals) initiative, while reducing in vivo testing.

In  developing  toxicogenomics-based  tests  for 
chemical  safety,  carcinoGENOMICS  addresses 
a  crucial  area  within  the  LifeSciHealth  Priority, 
namely ‘the development of new in vitro tests to 
replace  animal  experimentation’. With  reference 
to the Three R Principle (Replace, Reduce, Refine), 
as  highlighted  in  the  LifeSciHealth  Priority,  the 
project is directed towards replacing chronic ro-
dent bioassays for assessing chemical genotoxic-
ity and carcinogenicity. 

It  reflects  the  requirement  of  the  LifeSciHealth 
Priority  that  alternative  methods  are  to  be  de-
veloped  and  validated  based  on  international 
standards. In doing so, these methods can obtain 
regulatory approval and then be applied in the 
industrial sector, as well as regulatory establish-
ments and elsewhere. 

The project also relates to the overall aim of the 
LifeSciHealth  Priority,  namely  ‘To  build  on  the 
sequencing  of  the  human  genome  and  many 
other  genomes  with  the  result  of  improving 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

19

 
 
 
 
 
 
  
 
carcinoGENOMICS

ALTERNATIVE TESTING STRATEGIES

to  human  carcinogens  in  vitro,  based 
on  discriminative  genetic  pathways 
for  human  genotoxicity  and  chemical 
carcinogenesis.

Expected outcome:

The  outcome  of  the  work  foreseen  under  car-
cinoGENOMICS will be a mature protocol on tox-
icogenomics-based in vitro screens predictive for 
genotoxic and carcinogenic properties of chemi-
cals which has been through pre-validation and 
which will be submitted to the European Centre 
for  the  Validation  of  Alternative  Methods  (EC-
VAM)  for  formal  validation  according  to  inter-
national standards. This will lead to subsequent 
regulatory  acceptance  and  world-wide  applica-
tion  in  industry,  regulatory  establishments  and 
elsewhere.  This  will  be  facilitated  by  including 
ECVAM as a partner in the management scheme 
of carcinoGENOMICS.

Combining  pathway-associated  gene  expres-
sion  with  metabolic  profiles  generated  in  vitro, 
as is foreseen in carcinoGENOMICS, represents a 
highly  innovative  approach  possibly  leading  to 
in silico models that may be used to predict the 
carcinogenic  potential  of  a  compound  in  vivo. 
Furthermore, toxicogenomics-based assays may 
outperform currently available tests for genotox-
icity, as well as for genotoxic and non-genotoxic 
carcinogenicity,  without  using  animals. These  in 
vitro  methods  may  therefore  play  a  major  role 
under the new system of the Community regard-
ing the REACH initiative. 

human  health  and  to  stimulate  industrial  and 
economic  activity’.  This  may  be  translated  into 
practical terms by improved exposure standards 
in  EU  chemical  policymaking,  thereby  reducing 
human health risks, and upgrading quality of life 
and environment at the European level.

Approach and methodology:

The  major  research  hypothesis  underlying  this 
project is that it is possible to generate transcrip-
tomic  and  metabonomic  profiles  from  a  set  of 
well-defined genotoxic and non-genotoxic carci-
nogenic  compounds  in  in vitro  cellular  systems, 
which reliably predict genotoxic and carcinogen-
ic events in vivo. In order to test this hypothesis, 
the project has comprised different components 
in such a way so as to fully exploit the latest in-
novations in cell technology, genomics analysis, 
and bioinformatics: 
•	

Component 1: innovative cell technology. 
Exploratory research will be conducted on 
human embryonic stem cells (hES) to de-
termine whether it is possible to generate 
a robust and unlimited source of compe-
tent human cells. This represents an inno-
vative approach and its feasibility will be 
investigated  in  the  case  of  hES-derived 
hepatocytes,  with  a  special  emphasis  on 
their propagation and differentiation.
Component 2: novel combination of tran-
scriptomic  and  metabonomic  analyses 
of  carcinogen-exposed  cellular  systems. 
Both  transcriptomics  and  metabonomics 
are  established  genomic  technologies, 
which  have  been  shown  to  yield  profiles 
capable of discriminating between classes 
of chemical agents.
Component 3: novel applications for bio-
informatics, with respect to standardising 
the  infrastructure  for  high-quality  data 
storage and mining. 
Component  4:  dedicated  novel,  high-
throughput  technologies  for  speeding 
up  the  analysis  of  genomics  responses 

•	

•	

•	

20

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
ALTERNATIVE TESTING STRATEGIES

Coordinator

Jos Kleinjans 
Maastricht University 
Department of Health Risk Analysis and Toxicology 
6200 MD Maastricht, Netherlands
E-mail: J.Kleinjans@grat.unimaas.nl

Partners 

José Castell
University Hospital La Fe 
Valencia, Spain

Vera Rogiers
Vrije Universiteit Brussel
Brussels, Belgium

Petter Björquist
Cellartis
Gothenburg, Sweden

Leon Mullenders
Leiden University Medical Centre
Leiden, Netherlands

Michael Ryan
University College Dublin
Dublin, Ireland

Edwin Roggen
Novozymes
Bagsvaerd, Denmark

Hector Keun
Imperial College
London, UK

Myriam Fabre
ADVANCELL
Barcelona, Spain

Susanna-Assunta Sansone
European Molecular Biology Laboratory
European Bioinformatics Institute 
Heidelberg, Germany

Rob Stierum
TNO Quality of Life 
Delft, Netherlands

Ralf Herwig
Max Planck Institute for Molecular Genetics
Munich, Germany

Jens Linge
Centre for the Validation of Alternative Methods
Brussels, Belgium

Bernward Garthoff
European Consensus Platform for Alternatives
Brussels, Belgium

Paul  Carmichael
Unilever 
London, UK

Paul Jennings
Innsbruck Medical University
Innsbruck, Austria

Jochen Koenig
Genedata 
Basel, Switzerland

Christoph Chesné
Biopredic International
Rennes, France

Edward Lock
John Moores University 
Liverpool, UK

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

21

COMICS

Comet assay and cell array for fast and efficient genotoxicity testing

ALTERNATIVE TESTING STRATEGIES

Contract No 
Project type 

LSHB-CT-2006-037575
SME-Specific Targeted Research Project

EC contribution  e 3 189 385

Starting date   1 January 2007
Duration  36 months

Website  http://comics.vitamib.com

Background and objectives:

Approach and methodology:

A battery of reliable and validated in vitro assays 
is  needed  to  test  for  genotoxic  and  cytotoxic 
effects of chemicals, without resorting to animal 
experiments.  The  comet  assay,  a  sensitive 
indicator  of  DNA  damage,  will  be  combined 
in  this  project  with  the  Cell  Array  system  in 
order  to  establish  and  validate  high  capacity 
assays  suitable  for  chemical  testing.  Up  to  800 
cell  samples  will  be  processed  for  comets  on  a 
single microscope slide. Arrays will use cells with 
different  metabolic  capabilities,  and  data  on 
cytotoxicity will be obtained in parallel with DNA 
damage.  A  medium-throughput  assay  will  also 
be developed. 

Comet  analysis  by  differential  staining  of  dam-
aged/undamaged  DNA  using  established  and 
novel  dyes  combined  with  automated  image 
analysis will be faster and more reliable than at 
present. A crucial aspect of the cellular response 

Figure 1. The comet assay: examples of comets from cells 
with increasing levels of DNA damage (0-4). The images are 
produced by embedding cells in agarose on a glass slide, 
lysing to remove membranes and soluble proteins, including 
histones, and conducting electrophoresis at high pH. DNA 
breaks relax supercoiled DNA loops which are then able to 
extend towards the anode and form the ‘comet tail’. The % 
of DNA in the tail quantitatively reflects the number of DNA 
breaks. [Reproduced, by permission of Humana Press, from 
Molecular Biotechnology 26 (2004) 249-261]

22

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

Figure 2. The principle of the ‘padlock probe’. The probe 
(left panel) has at its ends sequences complementary to 
adjacent parts of the target DNA. After hybridisation, these 
ends are ligated and the probe thus locked onto the target. 
These probes can be designed to lock onto defined gene 
sequences, and so permit an examination of damage and 
repair in those specific genes. [Figure provided by Prof. Mats 
Nilsson.]

to  DNA  damage  is  DNA  repair;  variations  be-
tween people can affect cancer risk, while geno-
toxic chemicals can act by interfering with repair. 
COMICS will compare two methods for measur-
ing  repair,  one  based  on  the  comet  assay  and 
the  other  using  a ‘Repair  Chip’  approach.  In  ad-
dition, the project partners will apply fluorescent 
probes  which  lock  onto  the  DNA  after  hybridi-
sation  (‘padlock  probes’)  to  study  gene-specific 
DNA repair. 

COMICS will also assess the modified comet as-
say  methods  for  reproducibility  and  sensitivity 
in an inter-laboratory prevalidation trial, using a 
coded set of standard chemicals, to satisfy regu-
latory  bodies,  as  well  as  industrial  users  of  the 
technology.  The  result  will  be  robust,  validated, 
high-throughput  in vitro  genotoxicity  tests. This 
proposal  brings  together  academic  partners 
and  SMEs,  together  with  a  large  industrial  con-
cern, and the European Centre for the Validation 
of  Alternative  Methods  (ECVAM).  Dissemination 
through  professional  and  trade  publications, 
regulatory  channels,  and  scientific  conferences 
will  lead  to  widespread  adoption  of  the  new 
methods.

The comet assay for DNA damage is widely used 
but  has  a  major  drawback  as  it  is  labour-inten-
sive,  and  therefore  can  be  used  only  in  studies 
in  which  the  numbers  of  samples  are  relatively 
small. The development of high-throughput vari-
ants will increase its applicability in both geno-

•	
•	

•	

toxicity  testing  and  population  biomonitoring. 
DNA  damage  is  a  good  marker  of  exposure  to 
genotoxic  chemicals;  measuring  cellular  repair 
of  this  damage  gives  additional  information  on 
the mode of action of genotoxic agents and on 
individual susceptibility to carcinogens.

The  overall  objective  is  to  develop  reliable  and 
tested genotoxicity and cytotoxicity assays that 
will, when combined, reduce the need for animal 
experiments in assessing the safety of chemicals. 
Specific aims include the following:
•	

increasing	 the	 throughput	 of	 the	 comet	
assay by up to 20 times;
developing	 further	 the	 cell	 array	 system	
as a parallel assay for cytotoxicity;
seeking	optimal	cell	types	for	use	in	geno-
toxicity and cytotoxicity testing; 
increasing	the	speed	of	scoring	of	comets;
using	lesion-specific	enzymes	and	inhibi-
tors  to  measure  different  kinds  of  DNA 
damage;
developing	 and	 comparing	 methods	 for	
measuring DNA repair activity; 
devising	 an	 approach	 to	 measure	 gene-
specific DNA damage and repair; 
validating	 the	 comet	 assay	 in	 its	 various	
forms;
establishing	reference	and	internal	stand-
ards for use in the comet assay; 
making	 the	 various	 innovative	 products	
available  for  use  by  companies  and  re-
searchers investigating DNA damage and 
repair.

•	

•	

•	
•	

•	

•	

•	

•	

•	

Role of SMEs:

The SMEs target the following actions:
•	

devising	new	software	for	image	analysis	
as required by the novel methods of scor-
ing of comets; 
providing	suitable	cell	lines;	
offering	 custom-made	 DNA-damaging	
chemicals for calibration purposes; 
providing	dyes	for	differential	staining	of	

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

23

ALTERNATIVE TESTING STRATEGIES

COMICS

•	

intact and damaged DNA; 
pre-market	 product	 development	 and	
testing.

Gunnar Brunborg
Norwegian Institute of Public Health
Oslo, Norway

Expected outcome: 

COMICS  seeks  to  develop  validated  assays  for 
DNA  damage  and  repair,  that  will  be  accepted 
by regulatory authorities for use in genotoxicity 
testing and will thereby minimise reliance on ex-
perimental animals.

Potential applications:

Commercial genotoxicity testing, biomonitoring 
in  human  population  studies,  basic  research  in 
DNA damage and repair.

Scientific Coordinator 

Andrew Collins
University of Oslo
Department of Nutrition
PB 1046 Blindern
0316 Oslo, Norway
E-mail: a.r.collins@medisin.uio.no

Partners

Béatrice Schaack
Commissariat à l’Energie Atomique
Grenoble, France

Françoise Soussaline
IMSTAR SA
Paris, France

Thomas Hartung
Joint Research Centre
Ispra, Italy

24

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Christiane Guillouzo
Institut National de la Santé et de la Recherche Médicale
Rennes, France

Katarina Volkovova
Slovak Medical University
Bratislava, Slovakia

Jon Eigill Johansen
Chiron AS
Trondheim, Norway

Neven Zoric
TATAA Biocenter
Göteborg, Sweden

Elizabeth Martin
AstraZeneca
Macclesfield, UK

Galen Milne
Thistle Scientific Ltd
Glasgow, UK

Andrew Smart
Severn Biotech Ltd
Kidderminster, UK

Christophe Chesne
Biopredic International
Rennes, France

Mats Nilsson
Uppsala University
Uppsala, Sweden

Karel Angelis
Institute of Experimental Botany
Prague, Czech Republic

ALTERNATIVE TESTING STRATEGIES

EXERA

Development of 3D in vitro models of estrogen-reporter mouse tissues for the pharmaco-
toxicological analysis of nuclear receptors-interacting compounds (NR-ICs) 

Contract No 
Project type 

LHSB-CT-2006-037168
Specific Targeted Research Project

EC contribution  e 2 173 492

Starting date   1 October 2006
Duration  36 months

Website  www.altaweb.eu/exera

Background and objectives:

•	

•	

Industries from different fields (pharmaceuticals, 
chemicals,  cosmetics,  foods  and  toxicologicals) 
need reliable, fast and economic in vitro models, 
which are alternative to animal testing and can 
provide predictive data on the actions of NR-ICs, 
and  in  particular  ER-ICs  (estrogen  receptors-
interacting  compounds),  on  animal  and  human 
health. These needs are increasing for a number 
of reasons: 
•	

more	 new	 drugs	 with	 endocrine	 action	
are  being  produced  (mainly  estrogens/ 
antiestrogens  for  women),  and  80%  of 
the marketed drugs is directed to women: 

birth control, cancer therapy and preven-
tion, hormone replacement therapies; 
the	recognition	that	a	wide	and	increasing	
number  of  xenocompounds  that  come 
from  the  industrial  production  and  that 
contaminate  the  environment  are  active 
endocrine  structures  (for  both  men  and 
women);
research	evidence	is	increasingly	empha-
sising the limitations of several non-mam-
malian models for a correct estimation of 
the physiological consequences (risks and 
benefits)  linked  to  the  exposure  of  hu-
mans to ER-ICs. 

Regulators of estrogen receptors activity

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

25

 
 
 
 
 
 
EXERA

The  need  for  appropriate  in  vitro  models  that 
can reproduce features and reactivity of specific 
mammalian target tissue/organs to ER-ICs is thus 
becoming  an  imperative  research  priority.  The 
scientifically, economically, socially and ethically 
relevant stakes are considerable. 

The  tissue-  and  organ-specific  in  vitro  models 
that have been built so far have several 
serious limitations: 
1. 

Most  of  the  available  cell  lines  of  mam-
malian  origin  are  derived  from  tumours 
or  have  a  transformed  phenotype.  Their 
functional  and  structural  features  do  not 
mirror the original tissue, thus giving rise 
to an altered response to various endog-
enous and exogenous factors with respect 
to  the  in  vivo  situation  (Joung  K,  2003; 
Lewis JB, 2000; Van Den Belt K, 2004).
When mammalian-derived in vitro models 
are  available,  they  consist  of  primary  cell 
cultures or in isolated tissue slices: their in 
vitro  survival  is  limited  (time-course  and 
dose-response  studies  are  very  difficult) 
and their behaviour is strictly dependent 
on the variability among individuals (their 
performance  cannot  be  standardised). 
Moreover,  if  of  human  origin,  they  have 
the disadvantage of depending on regular 
supplies from available clinical sources.
2-Dimensional culture conditions may be 
not  optimal  for  tissue-like  organisation 
and cellular functions (e.g. polarised cells 
of  a  parenchymal  tissue,  which  normally 
require complex cellular interactions, can-
not  behave  physiologically  when  adher-
ing  to  solid  substrates,  as  in  the  case  of 
conventional culture conditions).
Conventional  cell  cultures  often  do  not 
express  suitable  easy-to-assay  quantifi-
able  markers  or  they  need  transfection 
procedures  that  increase  the  variability 
of the data. The most commonly used cell 
cultures  to  study  endocrine  actions  are 
strongly biased toward female-specific ef-

2. 

3. 

4. 

ALTERNATIVE TESTING STRATEGIES

fects,  being  mostly  composed  of  female 
cells  (i.e.  endocrine  responsive  cancers) 
(Kojima  M,  2005).  The  recent  knowledge 
of the presence of high concentrations of 
estrogen receptors in male tissues makes 
these  biological  materials  obsolete  for 
some aspects. 
The  systems  used  for  the  in  vitro  and  in 
vivo  analysis  of  NR-ICs  (mainly  estrogens 
and  androgens)  are  generally  composed 
of cells derived from reproductive tissues. 
The recent knowledge of the widespread 
distribution  of  nuclear  receptors  (in  par-
ticular steroid receptors) in all the tissues 
of the organism and their involvement in 
several diseases, makes the available sys-
tems  inadequate  to  assess  the  effects  of 
NR-ICs  on  the  whole  physiology  (Villa  R, 
2004). 
The available models do not easily provide 
information on the effects of compounds 
at different developmental stages. 

5. 

6. 

To overcome the above-mentioned deficiencies 
of  inadequate  in  vitro  models,  seven  industry 
representatives have decided to join their efforts 
with those of three public research organisations 
in order to  develop novel in vitro 3D models of 
estrogen-reporter mouse tissues for the Pharma-
co-toxicological  analysis  of  ER-ICs  for  liver,  skin, 
bone  (non  reproductive  systems),  and  ovaries 
and testis (sex-specific reproductive systems).

Approach and methodology:

EXERA’s  approach 
evidence:  

is  based  upon  powerful 

Use  of  the  transgenic  MOUSE-1:  This  estrogen-
reporter mouse model represents a new strategy 
that allows studying estrogen receptors-mediat-
ed gene regulation in vivo and in derived in vitro 
systems.  The  numerous  studies  performed  on 
this  animal  model  in  several  laboratories  have 
demonstrated its reliability and suitability to the 

26

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
ALTERNATIVE TESTING STRATEGIES

study  of  molecule  acting  through  estrogen  re-
ceptors. The mouse was generated by Partner 2 
(Maggi A, 2005; Ciana P, 2004; Di Lorenzo D, 2002) 
and is the object of a patent application (PCT N° 
WO  02/01949  A3  date  10.01.2002)  extended  to 
several extra-European States.

3D  cultures:  Cell-cell  and  extracellular  matrix-cell 
interactions play a fundamental role in maintain-
ing  the  function  of  numerous  organ  systems. 
Hence, tissue engineering represents a good way 
to  overcome  limits  of  monolayer  cultures  and  to 
maintain tissue-like architecture and functionality.

Techniques and methodologies: The steps to reach 
the proposed objectives will involve several com-
plementary techniques and partner expertise: cell 
isolation, conditional immortalisation, cell banking, 

CELL SYSTEMS THAT WILL BE DEVELOPED
from two reproductive and
 three non reproductive tissues

Estrogen effects:
Metabolism
Rigeneration
Response to xenobiotics

LIVER
(BRHSP)

Targets of ER-ICs

SKIN
(BRHSP)
Estrogen  Effects:
Collagen synthesis
Wound healing
Maintenance of:
skin moisture and
hyaluronic acid
levels

BONE
(UTURKU)
Estrogen  Effects:
Maintenance of 
bone mass

OVARY 
(BSV)
Estrogen  Effects:
Occytes maturation 
     control, etc

TESTICULE
(BSV)
Estrogen  
Effects:
Development
Fertility

Cell lines
(organotypic cultures)

3D-cultures, whole genome expression profiles, in 
vitro imaging, in vivo imaging, and application of 
3D-cultures devices (RCCS Technology).

Cell  isolation  from  tissues  of  MOUSE-1:  Reliable 
protocols  available  inside  the  partnership  will 
be  applied  so  as  to  isolate  well  differentiated 
cells  from  liver,  skin,  bone,  testis  and  ovaries  of 
MOUSE-1 and establish cell cultures with “physi-
ological”  estrogen-dependent  phenotypes  for 
immortalisation.  Cell  cultures  will  be  constantly 
controlled and characterised with respect to the 
in vivo situation with specific markers (luciferase 
enzymatic assay and in vitro cell imaging, endog-
enous  markers  of  tissue  health  and  differentia-
tion)  and  by  gene  expression  profiles.  Cells  will 
be isolated either from male and female mice. 

Conditional Immortalisation: This step will be per-
formed by transfection methodologies in 3D and 
by using suitable commercially available vectors, 
made inducible by specifically modified antibiot-
ics devoid of hormonal actions. The immortalis-
ing gene will be switched on for cell production, 
and off for characterisation and testing. 

Constitution of a cell bank: Immortalised cell cul-
tures  that  will  satisfy  the  following  parameters 
will be expanded and controlled for banking: (1) 
Immortalisation  must  not  modify  the  estrogen-
dependent pathways. This will also be controlled 
by gene expression profiles; (2) Tissue health and 
homeostasis. Cells must show a healthy differen-
tiated phenotype with respect to tissue specific 
markers.

3D-cultures  adapted  to  grow  cells  with  an  un-
altered  estrogen-dependent  phenotype:  Estro-
gen-dependent  pathways  will  be  characterised 
in  3D-cultures.  Comparison  between  hormone 
stimulation in vitro and in vivo will be performed 
with the use of the transgenic marker (luciferase) 
and gene expression profiles. Data on the activity 
of selected ER-ICs will be produced in each spe-
cific cell line with respect to: (1) agonism/antago-

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

27

ALTERNATIVE TESTING STRATEGIES

EXERA

nism  of  estrogenic  actions;  (2)  tissue  specificity 
and gender specificity.

Assessment of the 3D-culture systems for the phar-
maco-toxicological characterisation of ER-ICs.

Expected outcome:

•	

•	
•	

the	application/adaptation	of	new	3D-cul-
ture technologies to cell cultures devoted 
to the study of ER-ICs; 
the	constitution	of	a	cell	bank;
a	 battery	 of	 differentiated	 3D	 cell-based	
systems  derived  from  estrogen-reporter 
mice  for  basic  and  applied  research  (e.g. 
pharmacology and toxicology), public use 
and industrial use.

Major publications:

Penza,  M.,  Montani,  C.,  Romani,  A.,  Vignolini,  P., 
Ciana,  P.,  Maggi,  A.,  Pampaloni,  B.,  Caimi,  L.,  Di 
Lorenzo,  D., ‘Genistein  accumulates  in  body  de-
pots  and  is  mobilized  during  fasting,  reaching 
estrogenic levels in serum that counter the hor-
monal actions of estradiol and organochlorines’, 
Toxicol Sci, 2007, Mar 3.

Penza, M., Montani, C., Romani, A., Vignolini, P., Pam-
paloni, B., Tanini, A., Brandi, M.L., Alonso-Magdale-
na,  P.,  Nadal,  A.,  Ottobrini,  L.,  Parolini,  O.,  Bignotti, 
E.,  Calza,  S.,  Maggi,  A.,  Grigolato,  P.G.,  Di  Lorenzo, 
D., ‘Genistein affects adipose tissue deposition in 
a  dose-dependent  and  gender-specific  manner’, 
Endocrinology, 2006, Dec;147(12):5740-51. 

Coordinator

Diego Di Lorenzo
Laboratorio di Biotecnologie 
Ospedale Civile di Brescia
P.le Spedali Civili 1
25123 Brescia
E-mail: dilorenzodiego@yahoo.it

28

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Partners

Adriana Maggi
University of Milan
Center of Pharmacology and Biotechnology
Milan, Italy

Kalervo Väänänen 
University of Turku
Turku, Finland

Paul Tomkins
Bioserv Ltd & CBBR
School of Science 
AIT, Athlone, Ireland

Richard Fry 
CELLON SA 
Bereldange, Luxembourg

Mikko Unkila
Hormos Medical Pharmacity
Turku, Finland

Jean-Pol Detiffe
DNAVision 
Charleroi, Belgium

Eunan McGlinchey
Archport Ltd
Dublin, Ireland

Manfred Hennecke
Berthold Technologies GmbH & Co. KG
Bad Wildbad, Germany

Aldo Tagliabue
ALTA
Siena, Italy

  
 
ALTERNATIVE TESTING STRATEGIES

Invitroheart

Reducing animal experimentation in drug testing by human cardiomyocyte in 
vitro models derived from embryonic stem cells

Contract No 
Project type 

LSHB-CT-2007-037636
Specific Targeted Research Project

EC contribution  e 2 701 611

Starting date   1 January 2007
Duration  36 months

Website  http://er-projects.gf.liu.se/~invitroheart

Background and objectives:  

Invitroheart  seeks  to  establish  stable  cell  lines 
that reflect human cardiomyocyte properties in 
a  reliable  manner,  through  the  development  of 
models  derived  from  human  embryonic  stem 
(hES)  cells.  Its  aim  is  to  deliver  reliable  in  vitro 
models  that  the  pharmaceutical  industry  could 
use  to  replace  experimental  animals,  in  the  fol-
lowing  instances:  (1)  investigations  on  pharma-
cological toxicity and safety of compounds in the 
drug discovery and development processes; and 
(2)  the  testing  of  toxic  effects  of  chemicals  ac-
cording to the new system of the Community on 
the Registration, Evaluation and Authorisation of 
Chemicals (REACH). 

In  the  pharmaceutical  industry,  reliable  in  vitro 
cell  models  would  contribute  in  replacing  cur-
rent  techniques  with  animal  experimentation 

in  the  selection  and  optimisation  of  lead  com-
pounds, and in the documentation of a selected 
drug candidate before it enters clinical phases. In 
the  toxicity  testing  of  chemical  substances,  the 
replacement  of  animal  testing  methods  can  be 
attained  as  well.  The  means  to  accomplish  the 
objective, in addition to new stable hES cell-de-

The QT-screen technology for assessment  of electro-
physiological effects

In the left panel, the transition from the 60 electrode 
Multi-Electrode-Array to a single electrode in a well of a 
96 well plate is shown. The middle panel aligns an actual 
QT-Screen recorded field potential from this technology 
with illustrations of corresponding action potential and 
electrocardiogram measurements, under control condi-
tions and with prolonged QT interval. The right panel 
shows an automated system for high throughput QT pro-
longation screening. This QT-screen technology can thus 
replace telemetry/electrocardiogram studies on dogs 
and other animals, in order to assess effects on cardiac 
repolarisation and arrhythmogenic activity of drugs (QT-
screen technology from Multi Channel Systems GmbH).

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

29

 
 
 
 
 
 
Invitroheart

ALTERNATIVE TESTING STRATEGIES

rived cardiomyocytes, are as follows: 
•	

state-of-the-art	methods	for	electrophysi-
ological cardiac cell monitoring;
optical	micro-sensor	monitoring	in	micro-
cultivation systems for in vitro screening;
for	
a	 multi-micro-bioreactor	 platform	
high-throughput  screening  of  drugs  and 
chemicals.

•	

•	

Approach and methodology:

Invitroheart will carry out comparative studies of 
cardiomyocytes  derived  from  hES  cells  with  es-
tablished in vitro models in order to validate the 
new  models  and  methods. The  outcome  of  the 
project is new efficient in vitro prevalidation mod-
els that will significantly reduce the use of animal 
experimentation  for  cardiotoxicity  testing  by 
60% to 80%. Furthermore, it will strengthen the 
possibility  for  the  participating  SMEs  to  market 
new potential products in the areas of in vitro as-
say methods and in vitro compound screening. 

Studies  of  toxicity  and  safety  pharmacology  in 
general and cardiotoxicity in particular are key ac-
tivities throughout drug discovery programmes 
in  the  pharmaceutical  industry.  Such  activities 
are initiated to detect detrimental compound ef-
fects. For example, electrophysiological changes 
such as QT prolongation, which leads to delayed 
ventricular repolarisation and cardiac arrhythmia, 
are induced by a vast range of chemical entities 

30

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Cell culture plates with optical sensor system for oxygen 
and pH monitoring

Monitoring of the important physiological parameters 
pH and oxygen is essential for assessing the physiologi-
cal condition of a cell culture. Consequently, this also 
becomes a very efficient means for detection of toxic 
effects on the culture. Optical micro-sensors for pH and 
oxygen offer many advantages over other sensor systems, 
such as non-invasive and non-destructive measurement 
from outside, through the transparent wall of a small bio-
reactor or cell culture plate. Optical sensors can also easily 
be combined with other biosensor methods. The panels 
show culture plates equipped with OxoDishTM and 
HydroDishTM technology for oxygen and pH monitor-
ing respectively, and on the right a SensorDishReaderTM 
monitoring device (technologies from PreSens GmbH).

for multiple therapeutic areas. Unintended QT ef-
fects of new drugs are the most common cause 
of drug withdrawal from the market and delays 
in or lack of regulatory approval for marketing. 

Clinical cardiotoxic effects are defined as symp-
toms of clinical heart failure, and subclinical car-
diotoxic effects as cardiac abnormalities detect-
ed in asymptomatic persons by means of various 
methods. One of the problems is the availability 
of  preclinical  models  capable  of  rapidly  screen-
ing  a  large  library  of  substances  and  of  provid-
ing rational bases for clinical trials. A significant 
bottleneck  in  the  development  of  novel  assays 
has  been  the  lack  of  research  purposes.  Animal 
models have been invaluable for risk assessment 
of compound safety; however, critical limitations 
remain in these models for robust prediction of 
certain toxic outcomes in humans. 

ALTERNATIVE TESTING STRATEGIES

Specific  technology-related  objectives  are  as 
follows:
•	

to	establish	relevant	hES	cell-derived	car-
diomyocytes  cultures  that  allow  a  more 
predictable  preclinical  lead  testing  pro-
gramme to be carried out; 
to	develop	a	real-time	sensor-based	in vit-
ro model for short-term studies of cardiac 
side-effects, that mimics the function and 
complexity of the cardiomyocyte tissue in 
vivo; 
to	develop	a	real-time	sensor-based	in vit-
ro model for long-term studies of cardio-
toxicological effects, that mimics the func-
tion and complexity of the cardiomyocyte 
tissue in vivo; 
to	 establish	 a	 versatile	 cell	 lab	 platform	
based  on  the  developed  cell  lines,  and 
cultivated  in  advanced  miniaturised  bio-
reactor  systems  with  non-invasive  meas-
urement techniques for in vitro testing of 
cardiotoxicity. 

Aim: 

The overall aim of the project can be summarised 
as follows:
•	

•	

•	

•	

to	 replace	 animals	 with	 human	 cell	 cul-
ture  systems,  in  preclinical  pharmaceuti-
cal development and chemical substance 
toxicity testing; 
to	 support	 the	 predictability	 of	 the	 drug	
discovery  and  development  process  of 
cardiovascular pharmaceuticals by allow-
ing  more  reliable  and  relevant  testing  in 
the  preclinical  phase,  and  hinder  weak 
lead  candidates  from  entering  clinical 
phases with innovative human cardiomy-
ocyte cell systems;
to	 deliver	 an	 in  vitro  testing  system  with 
adjacent methodology pertinent for vali-
dation  in  GLP/SOPs  environment  for  car-
diac safety;
to	deliver	in vitro testing systems with adja-
cent methodology pertinent for chemical 
substance toxicity testing within REACH;
to	reduce	or	even	totally	abolish	(this	be-
ing  the  ultimate  aim)  the  use  of  animals 
in  drug  and  chemical  substance  testing, 
refine the model system under considera-
tion  and  replace  the  animal  models  cur-
rently used.

•	

•	

•	

•	

A monitoring platform for high-throughput screening

An important aim of Invitroheart is to integrate several 
bioassay technologies, such as QT-screen and optical sen-
sors, into one monitoring platform with hES cell-derived 
cardiomyocytes. This platform technology will enable 
assessment of cardiac toxicity at a far greater speed as 
compared with conventional animal models currently 
used in the pharmaceutical industry.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

31

Invitroheart

Role of SMEs:

Out of nine partners in Invitroheart, four are SMEs 
(Cellartis,  Multi  Channel  Systems,  Pharmacelsus 
and  PreSens)  and  their  share  of  the  requested 
funding from the Commission amounts to 59%. 
The SMEs’ contribution is crucial to the project as 
regards  the  generation  of  cells  and  assay  tech-
nologies, and as experts in life science and micro-
sensor  technologies,  they  are  the  key  providers 
of state-of-the-art technology. 

Expected outcome:

Successful  results,  if  adopted  by  the  European 
pharmaceutical community, will lead to:
•	

the	preservation	of	significant	numbers	of	
experimental animals;
carried	 out	 during	
reduced	 work	
pharmaceutical  drug  discovery  and 
development  in  the  preclinical  and  early 
clinic phases;
improved	 predictability	 of	 quality	 of	 lead	
candidates, 
increasing  the  chances  for 
passing the entire clinical trials process;
reduced	total	development	time	of	leads;
attainment	of	better	quality	and	safety	in	
documentation filed to regulatory bodies.

•	

•	

•	
•	

ALTERNATIVE TESTING STRATEGIES

Scientific coordinator 

Carl-Fredrik Mandenius
Linköping University, IFM
581 83, Linköping, Sweden
E-mail: cfm@ifm.liu.se

Partners

Peter Sartipy
Cellartis AB
Gothenburg, Sweden

Thomas Meyer
Multi Channel Systems GmbH
Reutlingen, Germany

Christine Batzl-Hartmann
Pharmacelsus GmbH
Saarbruecken, Germany

Achim Stangelmeyer
PreSens GmbH
Regensburg, Germany

Morten Laursen
Lundbeck A/S
Department of Safety Pharmacology
Valby, Denmark

Anders Lindahl
Sahlgrenska University Hospital
Department of Clinical Chemistry/Transfusion Medicine
Gothenburg, Sweden

Elmar Heinzle
Saarland University
Department of Biochemical Engineering
Saarbruecken, Germany

Susanne Bremer
ECVAM 
Joint Research Centre, Institute for Health 
and Consumer Protection
Ispra, Italy

32

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

LIINTOP

Optimisation  of  liver  and  intestine  in  vitro  models  for  pharmacokinetics  and 
pharmacodynamics studies

Contract No 
Project type 

LSHB-CT-2006-037499
SME-Specific Targeted Research Project

EC contribution  e 2 933 291

Starting date   1 January 2007
Duration  36 months

Website  www.liintop.cnr.it

Background and objectives:

The main aim of LIINTOP is to optimise and pro-
vide  established  protocols  and  experimental  in 
vitro  models  for  testing  intestinal  and  liver  ab-
sorption, metabolism and toxicity of molecules of 
pharmacological interest. The added value of the 
project, with respect to the existing experimental 
approaches in this field, is to provide optimised 
sequential  procedures  that  are  easily  amenable 
to validation studies for the screening and test-
ing  of  new  drugs,  possibly  by  miniaturised  and 
automated  technology. The  direct  participation 
of SMEs in the research activities will assure that 
those procedures will meet the requirements of 
industrial application.

The project, therefore, comprises a number of dif-
ferent approaches, which will integrate at various 
levels. A first basic approach will be the optimisa-
tion of in vitro liver and intestinal models for their 
use  in  the  transport  and  toxicity  of  structurally 
diverse reference drugs, chosen with the help of 
a steering committee of relevant stakeholders. A 
parallel approach will deal with the identification 
of  the  transport  and  metabolic  pathways,  and 
possible cytotoxic effects of these drugs, in order 
to  develop  appropriate  monitoring  procedures. 
Newly  advanced  technologies  (genomics,  pro-
teomics, metabolomics) will be used to develop 
high-throughput  models  related  to  the  specific 
area  of  intestine-liver  absorption  and  biotrans-
formation. Each approach will address the relia-
bility of the protocols involved and the relevance 

of the whole procedure to the in vitro/in vivo ex-
trapolation of drug effects. To this end, a unit in 
charge  of  computer-based  studies  will  support 
and pilot the project throughout its course.

In  Europe,  three  directives  regulate  the  testing 
of  chemicals:  Council  Directive  67/548/EEC  and 
its  subsequent  amendments;  Council  Directive 
88/379/EEC  and  subsequent  amendments,  and 
Council  Directive  76/769/EEC.  Moreover,  Coun-
cil Regulation No. 793/93 on the evaluation and 
control  of  risk  of  existing  substances  also  deals 
with the same topic. The main new aspect is that 
a  distinction  has  been  made  between  the  new 
substances identified since 1981, and those iden-
tified before then. For the latter group (containing 
about 100 000 substances), it has been estimated 
that  insufficient  data  are  available  concerning 
their safety. Thus, the White Paper proposes a har-
monisation of testing requirements for new and 
existing  substances,  by  introducing  a  new  sys-
tem for the Registration, Evaluation and Authori-
sation of new and existing Chemical Substances 
(REACH) (COM-2001/88 Final; COM-2003/644).

This  implies,  on  the  one  hand,  a  cumbersome 
plan  of  testing,  and  on  the  other  hand,  the  use 
of an extensive number of animals. The Europe-
an  Centre  for  validation  of  Alternative  Methods 
(ECVAM)  has  already  addressed  the  possibility 
of  using  alternative  methods,  based  on  the  3Rs 
model, in order to reduce this number, or at least 
to  reduce  the  animal  suffering  associated  with 
certain kind of tests. The EU has addressed this is-

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

33

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

optimised sequential procedures for the screen-
ing  and  testing  of  new  drugs  will  meet  the  re-
quirements  of  industrial  application.  This  pro-
posal  addresses  an  issue  of  great  relevance  in 
the  drug  discovery  process,  i.e.  absorption  and 
enterohepatic  bio-disposition  of  drugs,  which 
presently  still  relies  on  in  vivo  experimentation. 
The  development  of  integrated  in  vitro  predic-
tive  tools  capable  of  addressing  these  issues 
—  possibly  sustained  by  miniaturised  and  au-
tomated technology — will have a positive out-
come in the competitiveness and success of the 
pharmaceutical  industry,  since  decisions  taken 
on partially characterised compounds (or those 
with questionable human relevance) may lead to 
great economic losses at later stages. 

Expected outcome:   

LIINTOP  seeks  to  provide  standardised  cellular 
models  of  human  hepatocytes  and  enterocytes 
reliable  for  the  prediction  of  drug  absorption, 

General Strategy for
in vitro scaling

LIINTOP

sue in Directive 86/609. The use of non-validated 
alternatives  has  also  been  suggested,  based  on 
the ‘weight of evidence’, i.e. widely used and well-
consolidated  procedures.  This  project  may  well 
contribute  to  those  aspects,  by  providing  new 
procedures submitted to optimisation. 

A successful outcome of LIINTOP, in fact, will have 
a  strong  and  diversified  impact  on  social  and 
economic issues.  The main effect will be related 
to  the  expanded  use  of  in vitro  systems,  and  to 
meeting existing expectations from the public at 
large and from industrial enterprises. In vitro sys-
tems meet with the demand for reducing animal 
experiments, thus satisfying a widely shared ethi-
cal  concern.  Furthermore,  they  offer  economic 
advantages in terms of reduced time consump-
tion and lower costs in the assessment of safety 
for novel drugs.

From  the  scientific  point  of  view,  the  LIINTOP 
project represents a unique effort to link, by an 
in vitro approach, different systems 
(i.e. gastrointestinal tract and liver) 
and  pathways  involved  in  vivo  in 
the  absorption  and  metabolism 
of  orally  ingested  substances. This 
kind  of  approach  is  important  for 
further  development  of  tests  for 
chronic  exposure  in  which  the  in-
terrelation  between  different  or-
gans  is  a  key  factor,  and  where  at 
present  nearly  no  in vitro  data  are 
available.  Such integrated models 
will  be  developed  to  the  point  of 
entering pre-validation procedures 
in order to be submitted to regula-
tory boards. 

In vitro experiment

In vitro modelling

In vitro scaling

Various systems 
and conditions

‘Pure’ kinetic measures

Scaling factors

Role of SMEs: 

The direct participation of SMEs in 
the research activities (particularly 
on  transporters  and  metabolites 
identification)  will  ensure  that  the 

In vitro modelling

Mechanism-Based 
models

In vitro prediction

Clearance, WBPBPK model, 
plasma-conc time profiles

34

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

metabolism and toxicity. Sequential procedures, 
easily amenable to validation studies, possibly by 
miniaturised and automated technology, will be 
developed.  Based  on  these  models,  the  project 
partners  will  obtain  a  database  concerning  in 
vitro  absorption,  metabolism  and  toxicity  of  se-
lected  drugs,  including  the  characterisation  of 
the regulation of relevant genes. They will devel-
op prediction mathematical models of pharma-
cokinetics and pharmacodynamics, based on the 
available in vivo data and on the in vitro data from 
the cellular models used in the project.

Best case scenario:
•	

transferring	 the	 best	 hepatocyte	 and	
enterocyte  models  and  relevant  testing 
procedures  to  the  industrial  setting,  for 
high-throughput  applications  in  the  de-
velopment of new drugs;
establishing	 the	 relevance	 of	 the	 pro-
posed in vitro models to the human in vivo 
situation.

•	

Human HepaRG hepatocytes differentiated in cellchip 
system ( from C.Guguen-Guillouzo, Inserm U522, Rennes, 
France)

Potential applications:

Optimised  sequential  procedures  and  refer-
ence  standards  concerning  in  vitro  models  of 
hepatocytes  and  enterocytes,  easily  amenable 
to  validation  studies,  will  allow  a  new  molecule 
of pharmacological activity to be tested for the 
following:
•	

potential	toxicity	at	the	level	of	the	intesti-
nal mucosal barrier;
intestinal	absorption;
metabolic	 modification	 in	 the	 intestinal	
cells;
absorption	 and	 metabolism	
hepatocytes;
hepatotoxicity;
optimal	pharmacokinetic	behaviour.

the	

in	

•	
•	

•	

•	
•	

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

35

ALTERNATIVE TESTING STRATEGIES

Vera Rogiers
Vrije Universiteit Brussel
Departmenet of Toxicology
Brussels, Belgium

Jouko Uusitalo
Novamass Analytical Limited 
Oulu, Finland 

Sjeng Horbach 
NV Organon
DMPK & Safety
Department of Pharmacology
Oss, Netherlands

Karen Rowland-Yeo
Simcyp Limited Blades Enterprise Centre
Sheffield, UK

Ugo Zanelli
SienaBiotech SpA
Siena, Italy

Myriam Fabre
Advancell 
Barcelona, Spain

Peter Krajcsi
SOLVO
Budaörs, Budapest, Hungary

LIINTOP

Scientific coordinator 

Flavia Zucco
CNR, Istituto di Neurobiologia e Medicina Molecolare
via del Fosso di Fiorano 64 
00143 Rome, Italy
E-mail: f.zucco@inmm.cnr.it 

Partners

Pascale Anderle
Ente Ospedaliero Cantonale / Istituto Oncologico 
Svizzera Italiana (EOC/IOSI)
Bellinzona, Switzerland

Jose V. Castell
Dep.de Bioquímica Fac. Medicina/
Centro de Investigacion
Hospital Universitario La Fe
Valencia, Spain

Christophe Chesne 
BIOPREDIC International
Rennes, France

André Guillouzo
INSERM U62O
Faculté de Pharmacie
Université de Rennes 1
Rennes Cedex, France

Jouni Hirvonen
University of Helsinki
Faculty of Pharmacy
Helsinki, Finland

Brian Houston
University of Manchester
School of Pharmacy and Pharmaceutical Sciences
Manchester, UK

Maria Laura Scarino
Istituto Nazionale di Ricerca per gli Alimenti
 e la Nutrizione (INRAN)
Rome, Italy

36

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
ALTERNATIVE TESTING STRATEGIES

MEMTRANS

Membrane transporters: in vitro models for the study of their role in drug fate

Contract No 
Project type 

LSHB-CT-2006-518246
Specific Targeted Research Project

EC contribution  e 1 900 000
Starting date   1 April 2006

Duration  36 months

Background and objectives:

MEMTRANS  seeks  to  optimise  and  prevalidate 
in vitro cultured cell models so as to predict oral 
absorption  and  pharmacokinetics  of  efflux  sys-
tems  substrates  (e.g.  P-gp,  MRP2,  BCRX),  as  well 
as  to  establish  an  in  vitro  cut-off  for  the  risk  of 
secretion-associated problems and to study the 
structure-affinity-transport  relationships.  These 
actions would allow the following:
•	

and 

a	 reduction	 in	 the	 number	 of	 animal	 ex-
periments to study efflux processes;
the	in vitro study of drug-drug (including 
drug-food 
phytopharmaca) 
interactions 
secretion 
transporters; 
the	 gathering	 of	 useful	 information	 for	
modelling transporter interactions in oth-
er normal and transformed tissues (blood 
brain barrier, lung mucosa, tumours).

related  with 

•	

•	

The objectives of the project will be achieved by 
using a systems biology approach that involves 
modelling and simulating the complex dynamic 
interactions between proteins (transporters), me-
tabolites (i.e. substrates) and cells (lipoidal barri-
ers). Computational and mathematical predictive 
models will be generated from the data, based on 
the system parameters and on the drug charac-
teristics. The experimental data generated in the 
project will be analysed from a mechanistic point 
of view in order to split all the individual steps in-
volved in transport. The mathematical models, in 
combination with the right physiological, physi-

cochemical  and  chemical  information,  could  be 
applied to predict drug transport in a wide range 
of membranes in the organism (blood brain bar-
rier, liver, kidney, tumours, etc.), but especially in 
the gastrointestinal tract.

Approach and methodology:

The  MEMTRANS  partners  target  the  following 
items.
•	

Prevalidation	of	different	methods	to	pre-
dict  gastrointestinal  absorption  of  secre-
tion transporters substrates. 
Standard	 operation	 procedures	 for	 main-
taining  and  working  with  the  developed 
models  in  order  to  reduce  inter-labora-
tory  variability  and  to  allow  exchange  of 
data obtained in different laboratories. This 
might  lead  to  a  reduction  of  animal  and 
human studies, as shown by the validation 
of  cell  culture  models  for  gastrointestinal 
absorption, which is accepted by the FDA 
in its guidance about biowaivers based on 
the  Biopharmaceutical  classification  sys-
tem.  These  concepts  are  also  included  in 
the EMEA bioequivalence guidelines.
Mathematical	 models	 to	 predict	 from	 in 
vitro data the in vivo intestinal efflux char-
acteristics and its impact in drug ADME.

•	

•	

Expected outcome: 

With  this  improved  validated  in  vitro  methodol-
ogy,  avoiding  extensive  animal  experimentation 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

37

 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

MEMTRANS

in preclinical phases to study efflux processes and 
to prevent failures of drug candidates due to poor 
absorbability would be possible. At the same time, 
an appropriate in vitro model with a level expres-
sion of P-glycoprotein — similar to that found in 
experimentation animals and with less interlabo-
ratory variability — would allow the combination 
of  data  obtained  in  different  laboratories.  In  this 
way, it will improve the statistical signification of 
the  established  relationships.  All  these  facts  will 
be  reflected  in  the  faster  development  of  new 
drugs for patients. 

The oral route for drug administration is the most 
convenient  and  preferred  by  patients,  and  the 
prediction of drug absorption from the GI tract is 
a key issue for a potential new medication in the 
process  of  drug  design  and  development.  The 
early  prediction  of  favourable  pharmacokinetic 
properties is the new paradigm in the screening 
of drug candidates. Current in vitro models for the 
predictions  of  drug  transport  across  biological 
membranes  include  cell  cultures  that  reproduce 
physiological  characteristics  of  different  barriers, 
such as the intestine, the blood-brain barrier or the 
kidney and liver. These models have shown good 
performance for compounds that are transported 
by  passive  diffusion.  However,  for  drugs  with  a 
carrier-mediated mechanism, the predictions are 
less accurate, mainly due to the differences in ex-
pression levels and affinity for the carriers in vitro 
versus the in vivo situation. 
It is of paramount importance to know in the early 
stages  of  drug  discovery,  whether  a  new  com-
pound is or is not a substrate and/or an inhibitor 
of an efflux pump, in order to prevent absorption 
problems, drug-drug, and drug-food interactions, 
and to minimise pharmacological resistance.

The in vitro cell cultures currently used (as Caco-2 
cells) for high-throughput screening of absorbabil-
ity-distribution of new drugs show good perform-
ance with passively absorbed drugs, but in the case 
of  compounds  absorbed  by  active  mechanisms 
the predictability is still poor. The expression level 

of  the  transporters  differs  between  clones  and 
laboratories, and it is highly dependent on the cul-
ture conditions. Standardised procedures and the 
use of quality controls to verify the functionality of 
the transporters are essential for achieving good 
predictive performance. On the other hand, stable 
cell lines with good expression of the transporter 
of interest are scarcely available. 
The MEMTRANS team will collaborate with the BI-
OSIM Network of Excellence in the dissemination 
tasks. Therefore, the MEMTRANS results will be dis-
seminated within the BIOSIM as a complementary 
channel.

Coordinator 

Marival Bermejo
University of Valencia
Facultad de Farmacia 
Departamento Farmacia y Tecnologia Farmaceutica 
Av. Vicente A. Estelles sn Burjassot 
46100 Valencia, Spain
E-mail: mbermejo@uv.es

Partners 

Miguel Borras
Intervalence Biokinetics
Valencia, Spain

Eleonore Haltner
Across Barriers GmbH
Saarbrücken, Germany

Peter Krajcsi
SOLVO
Budaörs, Budapest, Hungary

Pranas Japertas
Aukstieji Algoritmai
Vilnius, Lithuania

Balazs Sarkadi
Hungarian Academy of Sciences/NMC
Budapest, Hungary

38

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

PREDICTOMICS

Short-term in vitro assays for long-term toxicity

Contract No 
Project type 

LSHB-CT-2004-504761
Specific Targeted Research Project

EC contribution  e 2 259 754

Starting date   1 September 2004

Duration  39 months

Website  www.predictomics.com

Background and objectives:

Over  30%  of  drugs  under  development  have 
failed  in  the  last  decade  because  of  mid-term 
toxicities in animals, or as a result of unexpected 
adverse events in early clinical studies that could 
not be detected in preclinical studies. The predic-
tion of toxicity of this class of compounds would 
be more efficient if good quality and human-rel-
evant toxicological data could be made available 
at earlier development stages.

While acute toxicity can be reasonably detected 
during  the  early  preclinical  studies,  long-term 
toxicity is more difficult to predict, relying almost 
exclusively on animal experiments that frequent-
ly are not predictive enough for humans. Animal 
experimentation of this kind is expensive, time-
consuming,  raises  ethical  issues  and  does  not 
necessarily imply toxicological relevance to man.

Despite past efforts to develop predictive in vitro 
tests for specific organ toxicity (i.e. isolated/per-
fused  organs,  tissue  slices,  primary  cultures,  cell 
lines, organotypic cultures, etc.), their reliability in 
anticipating chronic toxicity is still very limited.

There  is  a  clear  need  for  well-standardised  and 
robust  in  vitro  models  suitable  for  the  screen-
ing of toxic and sub-chronic toxicity induced by 
xenobiotics. The  underlying  concept  in  PREDIC-
TOMICS  was  that  chronic  toxicity  phenomena 
may  be  identified  even  in  short-term  assays  if 
proper  indicators  are  identified  and  monitored. 

•	

•	

•	

•	

•	

This Specific Targeted Research Project aimed to 
develop  an  in  vitro  strategy  predictive  of  drug-
elicited kidney and liver chronic toxicity, by com-
bining  emerging  technologies  and  advanced 
culture models.

As  a  combined  basic  and  targeted  research 
project, PREDICTOMICS aimed for the following:
•	

a	better	understanding	of	the	mechanisms	
of chronic toxicity to identify relevant and 
early changes induced by chronic toxins;
the	 development	 of	 advanced	 and	
innovative  cell  culture  models  for  liver 
and  kidney  cells, 
including  targeted-
directed cell transformation and stem cell 
technology;
optimisation	 and	 technological	 improve-
ment of genomic, proteomic and cytomic 
tools  to  increase  their  sensitivity  and  ac-
curacy for these types of toxicity studies;
(mechanisti-
identification	 of	 primary	
cally linked) and secondary biomarkers of 
chronic  toxicity,  based  on  combined  ge-
nomic proteomic and cytomic analysis of 
cells exposed to model hepatotoxins and 
nephrotoxins;
based	on	the	identified	primary	and	sec-
ondary biomarkers, the establishment of a 
hierarchical  flow  chart  and  a  mathemati-
cal  model  so  as  to  anticipate  a  potential 
risk of causing chronic liver and/or chronic 
toxicity of new drug candidates;
prevalidation	of	the	screening	platform.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

39

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

PREDICTOMICS

Approach and methodology:

The strength of this project lies in the combina-
tion  of  a  comprehensive  analysis  of  cell  effects 
induced  by  toxicants  via  Cytomics/  Genomics/ 
Proteomics  (i.e.  PREDICTOMICS),  and  a  deeper 
mechanistic knowledge of drug-induced chron-
ic  toxicity.  Outstanding  among  these  were  the 
identification  of  endpoints/pathways  affected 
by the early toxic compounds treatment, and the 
identification of primary and secondary biomar-
kers  sets,  classified  according  to  their  relevance 
to the mechanism of chronic toxicity.

Main findings:

The following publishable results were obtained 
in Years 1 and 2 of the project, and are sustained 
by papers, either published or submitted.

1. 

2. 

The suitability of different hepatocyte cul-
tures for predictomics analysis was inves-
tigated. Collagen cultures were also exam-
ined, concerning their ability to maintain 
long-term  cultures,  and  simultaneously 
to meet the requirements for further ge-
nomic analysis. Studies on the role of gene 
expression  and  regulation  of  hepatocyte 
adult  phenotype  were  conducted  using 
trichostatin  analogues  and  vectors  en-
coding  for  key  transcription  factors  (P1, 
P3). Primary rat hepatocytes treated with 
HDAC  inhibitors  better  retain  the  adult 
phenotype in culture, and may constitute 
a  suitable  strategy  to  improve  perform-
ance of primary cultured hepatocytes.
The role of transcription factors (HNF4), ac-
tivators, as well as repressors (SRC1, SRC2, 
PGC1  alpha,  PCAF)  were  investigated  in 
detail, to understand the lack of expression 
of CYP’s in the human hepatoma HepG2. 
Adenoviral  expression  vectors  were  con-
structed  allowing  a  detailed  analysis  of 
these factors. Expression of PGC1 alpha in 
hepatoma  cells  resulted  in  the  upregula-

3. 

4. 

5. 

6. 

7. 

tion of key hepatic genes, including CYPs.
Hepatic  progenitor  cells  were  success-
fully isolated from the human liver in or-
der to derive hepatocytes suitable for the 
purpose of the project. In an appropriate 
differentiation  culture  medium,  the  dif-
ferentiation  of  progenitor  cells  towards 
the  two  lineages  (ductal,  parenchymal) 
was  achieved.  Cells  acquire  many  of  the 
biochemical  features  displayed  by  adult 
hepatocytes,  but  showed  limited  prolif-
eration/passage stability. 
A methodology to examine multiparamet-
ric hepatocyte metabolism indicators was 
set up and adapted for rapid FACS analysis 
of hepatocytes treated with drugs.
It  was  possible  to  reproduce  in  vitro  the 
esteatosis induced by drugs. The genomic 
identification  a 
analysis  allowed  the 
number  of  genes  and  proteins  that  are 
consistently upregulated as a consequence 
of the lipid accumulation in hepatocytes. 
A  cytomic  analysis  was  also  established, 
enabling  an  accurate  measurement  of 
lipid content in hepatocytes treated with 
steatosic  drugs.  The  combination  of  the 
three 
technologies  appeared 
to  be  a  suitable  integrated  strategy  to 
identify  this  class  of  hepatotoxicants, 
in  accordance  with  the  PREDICTOMICS 
principles.
To  approach  the  identification  of  drugs 
with cholestasic potential, new fluorescent 
bile acid derivatives were synthesised and 
successfully adapted to cytometric analy-
sis.  Initial  studies  with  model  cholesta-
sic  drugs  revealed  effects  that  could  be 
monitored with this assay. Effects on gene 
expression in hepatocytes were also con-
ducted;  however,  no  conclusive  results 
have yet been obtained, as the number of 
genes altered is small.
Optimised,  stable,  and  well-characterised 
renal  mono-  and  co-cultures  for  in  vitro 
nephrotoxicity  studies  were  established, 

‘-omics’ 

40

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

8. 

9. 

and  standard  operating  procedures  pre-
pared).  A  perfusion-based  kidney  culture 
model was developed and optimised The 
characterisation  of  rat  primary  proximal 
tubular cells and the cell line NRK-52E, as 
compared  to  rat  kidney  in vivo,  was  con-
ducted  by  using  gene  expression  profil-
ing).  Close  proximity  indirect  filter-based 
renal  co-culture  systems  with  epithelial/
endothelial and epithelial/fibroblast part-
ner cells were developed .
A wide number of cytomic assays (includ-
ing matrix metalloproteinase secretion, tis-
sue inhibitor of matrix metalloproteinase 
secretion, IL-6 secretion, RANTES secretion 
(P5), ATP production, inner mitochondrial 
membrane  potential  (JC-1),  ROS  produc-
tion, LDH release, lactate production, DNA 
synthesis  and  resazurin  reduction)  have 
been examined for their usefulness for in 
vitro toxicity testing. Three assays (LDH re-
lease  for  cytotoxicity,  BrdU  incorporation 
for DNA synthesis and resazurin reduction 
related to viable cell number) were select-
ed, based on their ease of application, glo-
bal toxicological relevance and sensitivity, 
which will be utilised to identify sub-lethal 
concentrations  of  the  test  compounds. 
Cytomic  data  were  generated  from  HK-2 
cells  intoxicated  with  ochratoxin  A,  cy-
closporine  A,  FK506,  rapamycin  and  cad-
mium chloride .
Gene  array  experiments  were  conducted 
comparing plastic grown HK-2 cells, filter 
grown HK-2 cells in monoculture and HK-
2 cells cultured in co-culture with microv-
ascular endothelial cells . Gene expression 
profile experiments for ochratoxin A (OTA) 
were conducted using HK-2 cells, human 
primary proximal tubular cells and rat pri-
mary  proximal  tubular  cells.  Comparison 
of different renal culture models was com-
pleted and a consensus reached as to the 
model  of  choice  for  further  testing  and 
toxicogenomic  profiling.  This  model  was 

10. 

11. 

HK-2 cells cultured on plastic supports in 
monoculture.
Identification  of  altered  gene  expression, 
generated  following  exposure  to  CsA, 
FK506 and rapamycin (P6) and ochratoxin 
A.  Gene  expression  profile  experiments 
for  CsA,  CdCl2,  diquat  dibromide,  FK506, 
and rapamycin were conducted using the 
HK-2 cell model. A number of genes were 
identified  that  are  considered  to  be  po-
tentially related to the cell injury process 
triggered  by  nephrotoxin  exposure.  This 
list will be expanded once the data for all 
nephrotoxins  has  been  completed  and 
statistically analysed.
Preliminary  proteomic  analysis  using  CsA 
as a test compound, has revealed a number 
of  differentially  expressed  proteins  that 
could serve as potential markers.

Major publications:

Martinez-Jimenez, C.P., Gomez-Lechon. M.J., Cas-
tell,  J.V.,  Jover,  R., ‘Under-expressed  coactivators 
PGC1alpha and SRC1 impair HNF4alpha function 
and promote dedifferentiation in human hepato-
ma cells’, J Biol Chem, 2006, 28:29840-29849.

Castell,  J.V.,  Jover,  R.,  Martinez-Jimenez,  C.P., 
Gomez-Lechon, M.J., ‘Hepatocyte cell lines: their 
use,  scope  and  limitations  in  drug  metabolism 
studies’,  Expert  Opin  Drug  Metab  Toxicol,    2006, 
Apr;2(2):183-212.

Abberger, Th., Jennings, P., Mirlach, A., Prajczer, S., 
Pfaller,  W., ‘Application  of  a  population  balance 
model to a perfusion in vitro toxicity system’, Toxi-
col. In vitro, 2006, 20(7):1213-24.

Bijuklic,  K.,  Sturn,  D.H.,  Jennings,  P.,  Kountchev, 
J., Pfaller, W., Wiedermann, C.J., Patsch, J.R., Joan-
nidis, M., ‘Mechanisms of Neutrophil Transmigra-
tion  Across  Renal  Proximal  Tubular  HK-2  Cells’, 
Cell Physiol Biochem, 2006, 17, 233-244.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

41

ALTERNATIVE TESTING STRATEGIES

PREDICTOMICS

DE-DIFFERENTATION and PHENOTYPE LOSS of HEPATOCYTES

LIVER

Lack of key transcription 
factors or increased repressors

Chromalin remodelling

methylation

(Trichostatine)
Histone
Deacetylase

Hypo-acet/lated 
histories

Gene are not expressed !

Lack of expression 
of key coactivators or
increased corepressors

Gene are not expressed !

Gene are not expressed !

Elaut, G., Henkens, T., Papeleu, P., Snykers, S., Vink-
en, M., Vanhaecke, T., Rogiers, V., ‘Molecular mech-
anisms underlying the dedifferentiation process 
of  isolated  hepatocytes  and  their  cultures’,  Cur-
rent Drug Metabolism, 2006, 7(6), 629-660.

Slattery, C., McMorrow, T.,  Ryan, M.P., ‘Overexpres-
sion of E2A proteins induces epithelial-mesenchy-
mal  transition  in  human  renal  proximal  tubular 
epithelial cells suggesting a potential role in renal 
fibrosis’, FEBS Lett, 2006, Jul 24; 580(17):4021-30.

De Haij, S., Woltman, A.M., Trouw, L.A., Bakker, A.C., 
Kamerling, S.W., van der Kooij, S.W., Chen, L., Kro-
zcek, R.A., Daha, M.R. van Kooten, C., ‘Renal tubu-
lar epithelial cells modulate T cell responses via 
ICOS-L and B7-H1’, Kidney Int, 2005, 68:2091-2102.

Slattery, C., Campbell, E., McMorrow, T., Ryan, M.P., 
‘Cyclosporine A-induced renal fibrosis: a role for 
epithelial-mesenchymal  transition’,  Am  J  Pathol, 
2005, Aug;167(2):395-407.

42

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

Armin Wolf
Novartis Pharma AG
Integrated Compound & Product Profiling
Basel, Switzerland

Laureano Simón
Progenika 
Derio, Vizcaya, Spain

Carmen Cuevas
Pharmamar
Madrid, Spain

Jan Snoeys
Johnson & Johnson 
Pharmaceutical Research & Development
Toxicology and Pathology Department
Beerse, Belgium

Bernward Garthoff
Ecopa
European Consensus Platform on 3-R Alternatives
Brussels, Belgium

Pilar Prieto and Thomas Hartung
ECVAM
Joint Research Centre
Institute for Health and Consumer Protection (IHCP)
Ispra, Italy

Coordinator

José V. Castell 
University Hospital La Fe
Unit of Experimental Hepatology
Valencia, Spain
E-mail: info@predictomics.com 

Partners 

Walter Pfaller
Innsbruck Medical University
Division Physics
Department of Physiology and Medical Physics
Innsbruck, Austria

Vera Rogiers
Vrije Universiteit Brussel
Faculty of Medicine and Pharmacy 
Department of Toxicology
Brussels, Belgium

Patrick Maurel
INSERM U128
Liver Unit
Montpellier, France

Moh R. Daha
Leiden University Medical Center
Department of Nephrology
Leiden, Netherlands

Michael Ryan
University College Dublin
Conway Institute of Biomolecular and Biomedical 
Research
Department of Pharmacology
Belfield, Dublin, Ireland

Heidrun Ellinger-Ziegelbauer
Bayer AG (Business Group Pharma)
Research Toxicology
Wuppertal, Germany

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

43

ReProTect

Development  of  a  novel  approach  in  hazard  and  risk  assessment  of  reproductive 
toxicity by a combination and application of in vitro, tissue and sensor technologies

ALTERNATIVE TESTING STRATEGIES

Contract No 
Project type 

LSHB-CT-2004-503257
Integrated Project 

EC contribution  e 9 100 000
Starting date   1 July 2004
Duration  60 months

Website  www.reprotect.eu

Background and objectives: 

The  development  of  reliable  testing  strategies 
for industrial chemicals, biocides, pesticides and 
cosmetic  ingredients  is  not  only  relevant  for 
reducing  the  cost  of  testing  and  animal  experi-
mentation, but it also serves as a sound basis for 
the adequate risk assessment of chemicals. 

It has been estimated that the detection of repro-
ductive toxicants under the Registration, Evalua-
tion and Authorisation of Chemicals (REACH) ini-
tiative will have a very great impact on the use of 
animals  for  regulatory  safety  testing.  Under  the 
new legislation, it is estimated that around 5 000 
chemicals  will  require  testing  for  reproductive 
toxicity and developmental toxicity. By following 
the present regulatory safety testing guidelines, 
millions of animals will be necessary just to de-
tect reproductive toxicants for the evaluation of 
existing  chemicals  in  Europe.  In  addition  to  the 
huge  number  of  animals  required  by  the  exer-
cise, the costs are very high. The testing costs for 
the development toxicity study and for the two-
generation study will represent up to 60% of the 
total testing costs of REACH. 

In order to reduce the number of animals, as well 
as  the  testing  costs,  we  need  intelligent  testing 
strategies  which  both  make  use  of  existing  in-
formation  and  also  integrate  alternative  infor-
mation sources such as in vitro tests, QSARs and 
read-across approaches. In certain areas of repro-
ductive toxicity testing, a number of useful and 

promising  in  vitro  models  are  already  available, 
but  they  need  to  be  converted  into  tests  with 
predictive  power  for  toxicological  safety  test-
ing.  ReProTect  is  aiming  to  optimise  these  tests 
in  order  to  prepare  them  for  formal  validation 
studies. In vitro test batteries will provide a more 
detailed understanding of the main chemical tar-
get tissues or targeted biological mechanisms in 
the  reproductive  cycle,  such  as  gametogenesis, 
steroidogenesis,  embryogenesis,  etc.,  and  will 
thereby support regulatory decisions.

Approach and methodology: 

The reproductive cycle can be broken down into 
fundamental  elements,  as  shown  in  the  Work 
Packages structures of ReProTect. However, due 
to its pioneering function and its strategic com-
ponents,  the  project’s  role  lies  far  beyond  the 
field  of  reproductive  toxicity.  For  the  first  time, 
complex  tiered  test  strategies  will  be  devised 
and  integrated  in  a  conceptual  framework  for 
safety assessment. This entirely new approach for 
hazard and risk assessment will have to be fully 
evaluated as to its relevance and reliability. This 
framework is to be expanded during a a series of 
consensus workshops. ReProTect is based on sev-
eral innovative technologies in order to develop 
the test strategy (e.g. genetically engineered cells 
and reporter genes).
In the area of prenatal development, murine and 
human  embryonic  stem  cells  will  be  used.  The 
establishment  of  human  embryonic  stem  cells 
in 1998 raised hopes in many research areas, in-

44

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

cluding the development of alternatives to ani-
mal experiments. ReProTect is using human em-
bryonic stem cell lines for embryotoxicity testing 
in vitro, which is a unique approach.

Furthermore, some of the toxicological endpoints 
are based on novel technologies, including pro-
teomics,  genomics  and  sensor  technologies. 
The systematic analysis of these techniques in a 
defined  toxicological  area  will  help  to  estimate 
the  value  of  these  new  technologies.  However, 
the  main  innovation  of  the  project  remains  the 
development  and  prevalidation  of  toxicologi-
cal  test  strategies  based  on  in vitro  and  in silico 
methods, and their integration into a conceptual 
framework.

Expected outcome: 

Several  toxicological  endpoints  that  can  assess 
the hazards of chemicals to male and female fer-
tility  will  be  covered  by  in vitro  tests  included  in 
the ‘Fertility’ research area. A reliable test battery 
for  testicular  toxicity,  which  explores  various  as-

Structure of the project

pects, such as sperm motility, sperm DNA damage 
and toxicity on Sertoli and Leydig cells, is expected 
soon.  Moreover,  the  toxicity  of  chemicals  on  fe-
male fertility is assessed using a multiparametric 
test  on  folliculogenesis  and  a  preliminary  series 
of  tests  for  female  germ  cell  toxicity.  Optimised 
in vitro tests are now available, and formal valida-
tion of the established tests is expected after an 
independent statistical evaluation on the data is 
produced. 

In July 2006, a new research area emerged, follow-
ing the selection of new project participants. Re-
search area 2, called ‘Implantation’ aims to estab-
lish  cell  culture  models  and  explants  specifically 
for the creation of the endometrium, and to iden-
tify in vitro models for the assessment of placental 
toxicity. The  development  of  in  vitro  tests  based 
on  human  endometrial  endothelial  and  stromal 
cells, as well as models using explants tissue, is ex-
pected. Furthermore, the feasibility of cell and tis-
sue culture models specific for assessing adverse 
effects on the process of placentation, is foreseen.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

45

ALTERNATIVE TESTING STRATEGIES

ReProTect

Moreover, further development of the murine em-
bryonic  stem  cell  tests  (EST  —  already  validated 
for detecting chemical effects during cardiac cell 
differentiation) is to be expected. New endpoints 
able to assess effects on neural and skeletal tissues, 
as well as a metabolic system, will be included. 

In  order  to  avoid  interspecies  variations  in  drug 
testing,  the  adaptation  of  the  murine  system  to 
human  embryonic  stem  cells  is  currently  being 
evaluated and is expected soon. Furthermore, ul-
tra-sensitive, differential protein expression analy-
sis  of  differentiating  mouse  and  human  ES  cell 
cultures exposed to embryotoxic drugs and other 
chemicals is expected to identify specific protein 
expression profiles. 

Research  area  4,  ‘Cross-cutting’,  has  been  estab-
lished in order to support the other three research 
areas.  QSAR  tests  on  barrier  functions  (placenta/ 
blood/ testis), as well as QSARs tests on metabolic 
systems are already developed and promising re-
sults  are  now  available.  In  this  research  area,  the 
development  of  in  vitro  methods  for  detecting 
endocrine disrupting (ED) function of chemicals is 
anticipated. Advanced test methods (PALM, MELN, 
ER-CALUX,  AR-CALUX)  and  receptor  binding  as-
says  are  now  optimised,  and  the  assessment  of 
the  transferability  of  the  models  is  in  place. The 
receptor binding optimisation is also being con-
ducted under the umbrella of OECD as part of the 
international  collaboration  with  the  USA  and  Ja-
pan. Due to the advanced stage of these methods, 
an in vitro testing battery for ED is expected in the 
near future. 

Main findings: 

During the first two years, ReProTect explored the 
predictive power of a range of pioneering in vitro 
tests. It is envisaged that all tests will provide infor-
mation on the mechanistic basis of a standard op-
eration procedure (SOP), and an assessment of the 
intra-laboratory reproducibility of the test, accord-
ing  to  the  ECVAM  Modular  Approach  of  valida-

tion.  More  than  150  peer-reviewed  reproductive 
toxicants with different toxicological mechanisms 
have been selected, in order to support the opti-
misation process of test protocol development. 

ReProTect has successfully identified, developed 
and  optimised  11  tests  that  could  be  incorpo-
rated into a reproductive toxicity testing battery. 
These  include  mechanistic  endpoints  directed 
at effects on leydig/sertoli cells, folliculogenesis, 
germ cell maturation, the motility of sperm cells, 
steroidogenesis,  fertilisation,  and  the  embryo 
preimplantation. 

ReProTect  is  the  first  project  that  explores  the 
potential of human embryonic stem cell (hES) re-
search  upon  neuronal  systems,  and  builds  upon 
murine (mES) research on differentiation, includ-
ing  neural,  cardiac  and  skeletal  cells. Various  ap-
proaches, such as the identification of protein bi-
omarker signatures, as well as the monitoring of 
selected markers by using histochemical and mo-
lecular biological parameters after exposure with 
teratogenic compounds, are being followed. A ro-
bust SOP for the maintenance of the hES cells and 
a preliminary protocol for neuronal differentiation 
is now available within the project. To ascertain in-
ter-species  variability,  a  comparative  assessment 
of hES and mES is being conducted. 

In addition, five new partners have been recruited 
in order to analyse toxic effects upon implantation. 
This research area is divided into two Work Pack-
ages,  one  concerning  the  regular  preparation  of 
the uterus for implantation as a target for chemi-
cal insult, the other addressing placental functions. 
Several ex vivo models and in vitro models, such as 
chorionic villi explants, trophoblast cells, pericyte/
endothelial primary cultures, as well as the placen-
tal perfusion system, will be developed in the next 
two years. With respect to ED hazard assessment, 
six tests for screening for putative (anti) estrogenic 
and (anti) androgenic compounds have been op-
timised, and are now being analysed for their pre-
dictive power.  

46

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ALTERNATIVE TESTING STRATEGIES

Coordinator  

Michael Schwarz 
University of Tuebingen
Institute of Pharmacology and Toxicology
Department of Toxicology
Wilhelmstr. 56  
72074 Tuebingen, Germany
E-mail: michael.schwarz@uni-tuebingen.de

Scientific coordinator 

Susanne Bremer and Cristian Pellizzer
ECVAM Joint Research Centre
Ispra, Italy 

Partners

Axel Peter and Nico Themmen
Erasmus MC
Rotterdam, Netherlands

Bart van der Burg
BioDetection Systems
Amsterdam, Netherlands

Aldert Piersma
National Institute of Health and the Enviroment 
Bilthoven, Netherlands

Johan Smitz
Vrije Universiteit Brussel
Brussels, Belgium

Margino Steemans
Johnson & Johnson
Beerse, Belgium

Giovanna Lazzari
Laboratorio di Tecnologie della Riproduzione
Cremona, Italy

Alberto Mantovani
Istituto Superiore di Sanita
Rome, Italy

Ilpo Huhtaniemi
Imperial College London
London, UK

Anne Tilloy-Ellul
Pfizer
Amboise, France

Horst Spielmann 
Federal Institute for Risk Assessment
Berlin, Germany

Hans Ahr
Bayer
Wuppertal, Germany

Andre Schrattenholz
ProteoSys
Mainz, Germany

Heinz Nau
School of Veterinary Medicine Hannover
Hannover, Germany

Lennart Dencker
University of Uppsala
Uppsala, Sweden

Eva Brittebo
University of Uppsala
Uppsala, Sweden

Matts Olovsson
University of Uppsala
Uppsala, Sweden

Ovanes Mekenyan
University of Bourgas
Bourgas, Bulgaria

Alois Jungbauer
University of Natural Resources 
and Applied Life Sciences Vienna
Vienna, Austria

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

47

 
ALTERNATIVE TESTING STRATEGIES

Kirsi Vähäkangas
University of Kuopio
Kuopio, Germany

Luana Ricci Paulesu
University of Siena
Siena, Italy

ReProTect

Andrea Galli
Istituto Lazzari Spallanzani
Rivolta d’Adda, Italy

Mark Cronin
University of Liverpool
Liverpool, UK

Rita Cortvrindt
EggCentris
Zellik, Belgium

Ine D.H. Waalkens-Berendsen
TNO
Zeist, Netherlands

Marcello Spanò
ENEA
Rome, Italy

Hilda Witters
VITO
Boeretang, Belgium

Elisabeth Knudsen
University of Copenhagen
Copenhagen, Denmark

Thomas Hartung
Joint Research Centre
Ispra, Italy

Nicolas Fabre
In vitro Testing Industrial Platform
Madrid, Spain

Albrecht Wendel
University of Konstanz
Konstanz, Germany

Wolfgang Shäfer
University of Freiburg
Freiburg, Germany

48

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ALTERNATIVE TESTING STRATEGIES

Sens-it-iv

Novel testing strategies for in vitro assessment of allergens

Contract No 
Project type 

LSHB-CT-2006-018681
Integrated Project 

EC contribution  e 10 999 700

Starting date   1 October 2005
Duration  60 months

Website  www.sens-it-iv.eu

Background and objectives:

With some forms of allergy on the rise, massive 
resources are being invested worldwide to inves-
tigate which compounds are the culprits and why 
some  otherwise  harmless  compounds  do  elicit 
adverse  immune  responses.  The  need  for  non-
animal alternatives is now all the more important 
because of existing and pending EU regulations. 
The 7th Amendment to the Cosmetics Directive 
(Directive  76/768/EEC)  will  completely  ban  all 
animal testing for cosmetic ingredients by 2009 
at  the  latest.  The  ban  covers  skin  sensitisation. 
Conversely, the new EU legislation on chemicals 
(REACH)  will  require  a  great  deal  of  additional 
chemical  testing.  It  is  estimated  that  skin  sensi-
tisation testing is among those human health ef-
fects that require large numbers of animals.

Therefore, 28 groups from academia and indus-
try, as well as special interest organisations, have 
joined  the Sens-it-iv consortium to develop non-
animal tests and testing strategies to assess aller-
genic potential. The overall goal of Sens-it-iv is to 
develop strategies to replace animal experimen-
tation  by  in vitro  assays  for  identifying  skin  and 
respiratory sensitisers. This is seen in relation to 
the use of safe ingredients by the chemical, cos-
metic and pharmaceutical industry. 

•	

•	

Approach and methodology:

Sens-it-iv  will  build  on  and  exploit  our  increas-
ing  understanding  of  the  mechanisms  through 

which  chemicals  and  proteins  induce  allergy  to 
develop  novel,  realistic  and  accurate  methods 
for safety assessment and product development. 
Sens-it-iv aims to acquire a solid understanding 
of the processes occurring in vivo when tissue is 
challenged by a potential sensitiser, and to com-
pare  this  with  molecular  indicators  on  the  cells 
involved in these reactions. Based on this under-
standing, the partners will develop assay systems 
that  model  sensitisation,  rather  than  irritation 
and toxicity of chemicals and proteins. 

Scientific objectives

The  scientific  objectives  of  Sens-it-iv  are  as  fol-
lows.
•	

Use	 of	 functional	 genomics,	 proteomics,	
and  immunohistology  before  and  after 
the chemical challenge of tissue slices, so 
as  to  address  ex  vivo  phenotypic  charac-
teristic changes of human epithelial cells 
(EC),  antigen  presenting  dendritic  cells 
(DC) and effector T cells.
Establishment	 of	 in  vitro  conditions  sup-
porting  an  in  vivo-like  crosstalk  between 
EC, DC and T cells, and the cascade of cellu-
lar and molecular events triggered in such 
a complex system by a test-compound.
Description	 of	 the	 chemical	 features	 re-
lated to the intrinsic stability of allergens 
in  relation  to  the  metabolic  capacity  of 
cells  in  the  target  tissue.  Chemical  struc-
tures  and  peptide  sequences  involved  in 
hapten  formation  will  be  characterised. 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

49

 
 
 
 
 
 
Sens-it-iv

The  processes  of  bio-activation  and  hap-
ten formation will be studied by advanced 
metabonomic and proteomic technology.

Technological objectives

Sens-it-iv  has 
objectives:
•	

the 

following 

technological 

set-up	 of	 an	 inductive	 database	 for	 the	
acquired scientific data, including all avail-
able literature information to allow queries 
for data patterns and predictive models;
creation	 of	 prototypes	 of	 cell-based	 pre-
dictive  assays  through  the  implementa-
tion of the cellular and excreted markers 
suggested by bio-informatics to represent 
key mechanisms of sensitisation; 
refinement	and	optimisation	of	these	as-
says for prevalidation. 

•	

•	

Expected outcome:

The  coordinated  and  extensive  characterisation 
of the impact of compounds on cellular-molecu-
lar  interactions  will  identify  key  mechanisms  of 
sensitisation. The  development  of  a  technology 
platform will allow queries for the following: 
•	

data,	 regularities,	 patterns	 and	 models	
within the data;
predictive	models,	and	new	methods	and	
strategies  for  the  design  of  safer  chemi-
cals and drugs. 

•	

The  ultimate  goal  of  Sens-it-iv  is  the  establish-
ment of in vitro assays that are ready for prevali-
dation  and  approval  by  ECVAM,  the  European 
Centre for Validation of Alternative Methods.

Main findings:

In its first 12 months , the Sens-it-iv project made 
significant progress towards its goals: 

•	

A	learning	panel	of	9	chemicals	(including	
3 skin sensitisers, 3 respiratory sensitisers 

50

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ALTERNATIVE TESTING STRATEGIES

•	

•	

•	

•	

•	

•	

•	

and 3 controls) and 2 proteins was identi-
fied, stored and distributed from identical 
batches to all participants
Precision	 Cut	 Lung	 Slices	 (PCLS)	 technol-
ogy for the testing of lung sensitisers was 
refined, resulting in a standard operation 
procedure for ex vivo profiles of lung epi-
thelial cells (EC), antigen presenting den-
dritic cells (DC) and effector T cells.
A	variety	of	human	cell	lines	representing	
the major players in allergy is being stud-
ied in a coordinative effort, with standard-
ised  culture-  and  test-conditions  already 
emerging. 
A	 unique	 catalogue	 of	 gene	 expression	
profiles  for  EC  and  DC  of  different  ori-
gins  was  established.  The  transcriptome 
responses  by  A549  alveolar  cells  to  pro-
teases were analysed, revealing a number 
of new markers and pathways of potential 
interest to this project.
An	 epidermal	 equivalent	 model	 with	
normalised  expression  of  defined  in  vivo 
markers was exposed in pilot experiments 
to the learning set of chemicals.
Protocols	for	generating	distinct	allergen-
specific  effector/memory  T  cells,  for  per-
formance of DC – T cell co-cultures, were 
established.  The  development  of  a  sus-
tainable adaptive immune response mod-
el for assessing the allergenic potential of 
compounds was initiated. Various  in vitro 
T cell priming assays were assessed, and a 
new protocol of polyclonal ‘cell amplifica-
tion’ was established.
Proteome	 expression	 profiles	 of	 primary	
human  keratinocytes  and  human  HaCat 
cells  were  analysed  by  two-dimensional 
electrophoresis  and  preliminary  differen-
tial experiments with selected compounds, 
and are now ready for comparison.
Hapten	 formation	 as	 a	 metabolic	 route	
was  addressed  using  radiochemical  and 
immunochemical  methods  for  detection 
of  protein  adducts  in  order  to  elaborate 

ALTERNATIVE TESTING STRATEGIES

•	

pathways of bio-activation and bio-inacti-
vation.  Emphasis  was  placed  on  the  use 
of a cutaneous cytochrome P 450 cocktail 
to mimic cutaneous oxidative metabolism;
A	central	repository	for	Sens-it-iv	data	has	
been  created. The  basic  infrastructure  in-
cludes the following: 
(i)  a  version  control  system,  that  tracks 
the progress of the project and can re-
store previous stages of the project;
(ii)  multiple  redundant  backup  systems 
to guarantee the integrity of the sub-
mitted data;

(iii) a web interface for the submission and 

retrieval of Sens-it-iv data.

A  monthly  newsletter  was  established  on  the 
Sens-it-iv  web  site  to  inform  the  public  of  up-
coming  activities  involving  the  project,  and  re-
cent major breakthroughs.

Coordinator

Erwin L. Roggen
Novozymes A/S
Smoermosevej 11  2880 
Bagsvaerd, Denmark
E-mail: elro@novozymes.com

Partners 

Herman-Josef Thierse 
Ruprecht-Karls-University of Heidelberg 
Heidelberg, Germany 

Silvia Casati
Institute for Health and Consumer Protection 
Ispra, Italy 

Ian Kimber
Syngenta Central Toxicology Laboratory 
Cheshire, UK 

Silvia Teissier
L’Oréal
Paris, France 

David Basketter
Unilever UK Central Resources Limited
Sharnbrook, UK 

Carl Borrebaeck
Lunds Universitet 
Lund, Sweden 

Federica Salusto
Institute for Research in Biomedicine 
Bellinzona, Switzerland 

Sue Gibbs
VU University Medical Centre 
Amsterdam, Netherlands 

Kevin Park 
University of Liverpool
Liverpool, UK 

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51

ALTERNATIVE TESTING STRATEGIES

Sens-it-iv

Julie McLeod
University of the West of England
Bristol, UK

Armin Braun
Fraunhofer Institute of Toxicology and Experimental 
Medicine 
Hannover, Germany 

Joan-Albert Vericat 
In vitro Testing Industrial Platform 
Rotterdam, Netherlands

Helma Hermans
Scientific Writing & Consultancy S.C
Pedreguer, Spain 

Marina Marinovich
Università degli Studi di Milano
Milan, Italy 

Hans Merk 
Rheinisch-Westfälische Technische Hochschule Aachen 
Aachen, Germany

Jean-Pierre Lepoittevin
Université Louis Pasteur 
Strasbourg, France 

Angel Garcia Martin
Proteomika S.L.
Derio, Spain

Christoph Helma
Albert-Ludwigs-University Freiburg
Freiburg, Germany

Stefan Martin
Universitätsklinikum Freiburg
Freiburg, Germany

Vera Rogiers 
VUB representing Ecopa
Brussels, Belgium 

Inge Nelissen 
Vlaamse Instelling voor Technologisch Onderzoek 
Mol, Belgium 

Mario Assenmacher
Miltenyi Biotec
Bergisch Gladbach, Germany 

Andrea Cavani 
Provincia Italiana della Congregazione dei Figli 
dell’Immacolata Concezione
Rome, Italy

Hans-Ulrich Weltzien
Max-Planck-Institut für Immunbiologie 
Freiburg, Germany

Enrico Maggi
Università di Firenze 
Firenze, Italy

Monique Marrec Fairley
The European Cosmetic Toiletry and 
Perfumery Association 
Brussels, Belgium

Pierre Aeby 
Cosmital SA
Marly, Switzerland

52

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ALTERNATIVE TESTING STRATEGIES

TOXDROP

Innovative  ‘cell  on  chip’  technology  to  screen  chemicals  for  toxicity,  using 
cultured cells within tiny ‘nanodrops’ of culture fluid

Contract No 
Project type 

SP22-CT-2004-513698
Specific Targeted Research Project

EC contribution  e 1 615 887.89
Starting date   1 January 2005
Duration  24 months

Website  http://toxdrop.vitamib.com

Background and objectives:

The  TOXDROP  project  set  out  to  develop  low-
cost, rapid and unambiguous toxicological assay 
screening while respecting the Good Cell Culture 
Practice.

Traditionally,  in  vitro  toxicology  assays  are  per-
formed  in  Petri  dishes  or  microtiter  plates  with 
over 10 000 cells per well. This amount of cells is 
inadequate if each cell needs to be wholly phe-
notyped. To  streamline  the  manipulation  of  mi-
crotiter plates, and to reduce the number of cells 
per assay, the TOXDROP project partners recom-
mended using the alternative method ‘DropChip’, 
where cells are cultured in nanodroplets on a mi-
croscope glass slide. Using this method, the part-
ners anticipated carrying out multiparametric as-
says and significantly increasing the accuracy in 
measuring the cell response to toxic addition. 

TOXDROP, which was carried out over a period of 
2  years,  allowed  successful  in  vitro  toxicological 
studies using nanodrop cultures, and placed this 
innovative  concept  in  the  context  of  the  devel-
opment of ‘cell on chip’ devices.

Approach and methodology:

The drops were maintained at a defined position 
throughout the experiments using a hydrophilic/
hydrophobic  interface  which  enabled  stabilisa-
tion and individualisation of the drops for high-
throughput  capabilities.  The  culture  conditions 

on  chip  were  set  so  as  to  obtain  approximately 
100  cells  at  fixation  time  (day  4)  in  the  non-ex-
posed  cultures,  which  was  enough  to  provide 
statistical  analysis  significance  while  avoiding 
growth inhibition and stress due to excessive cell 
density. In order to develop a proactive strategy 
and  record  the  toxicology  of  various  chemicals 
using ‘cell on chip’, the partners used this reduced 
number of 100 cells per drop assay and realised a 
high level of cell behaviour characterisation. 

Using  the  metallic  mesh  embedded  in  the  chip 
for  precise  positioning, TOXDROP  performed  au-
tomated  smart  capture  using  IMSTAR’s  (partner 
5) Pathfinder™ system. The Pathfinder™ imaging 
platform enables a fully automated capture of the 
whole chip with intelligent image-data manage-
ment (IDB, patent n° 01921459.2) for each spot at 
0.6µm  resolution  compatible  with  multispectral 
fluorescence single cell detection and multipara-
metric cellular characterisation. Its multiplex capa-
bility makes the platform a good tool for complex 
phenotyping in large scale toxicology. 

The  partners  also  set  up  culture  conditions  and 
protocols  for  cell  viability  and  differentiation  of 
hepatotoxicity  for  a  new  human  hepatoma  cell 
line HepaRG in drop culture. A new specific fluo-
rescent  oxidative  stress  assay  based  on  EGFP  re-
porter gene induction was engineered in HepaRG 
cells under the control of the hsp22 promoter; it 
allows  a  selective  induction  of  the  construct  re-
stricted to differentiated hepatocyte colonies and 
to oxidative stress, especially heavy metal related.

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53

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

TOXDROP

Main findings: 

TOXDROP  aimed  at  providing  an  original  ap-
proach  for  automated  High-Throughput  High-
Content Screening in toxicity assays using a rev-
olutionary  format  of  cellular  nanodrops  formed 
on a glass slide. 
Two individual approaches were tested in order 
to facilitate toxicity analysis within these cellular 
nanodrops: on the one hand, fluorescence micro-
scopy  was  used  to  identify  fluorescent  proteins 
(fluorimetric  assay),  while  on  the  other  hand,  2- 
and  3-dimensional  mass  spectrometric  analysis 
of cells was performed (ToF-SIMS analysis). New 
software tools were developed for the analysis of 
the images provided by both approaches.

Nanodrops  are  realised  through  conventional 
piezo  dispensing  or  by  using  a  microfluidic  de-
vice  (CellJet),  developed  through  collaboration 

TOXDROP  developed  a  fluorometric  cell-based 
assay  for  global  toxicity  monitoring  using  the 
“stress  promoter  technology”.  For  that  purpose, 
several cell lines have been generated by partner 
3 (UCBL). They were chosen as models of organs 
that are particularly sensitive to toxic chemicals, 
such  as  liver  cells  (HepG2),  lung  bronchial  epi-
thelium cells (16HBE), keratynocytes (HaCat) and 
differentiated  hepatocytes  (HepaRG,  in  collabo-
ration with partner 2, INSERM), and which could 
express  Enhanced  Green  Fluorescent  Protein 
(EGFP) upon induction of heat shock family stress 
promoters . Such engineered cell lines are able to 
respond to the presence of a chemical stress by 
increasing  the  expression  of  the  fluorescent  re-
porter protein EGFP (Figure 1).

Figure 1: EGFP reporter gene expression is induced in the 
presence of increasing concentration of toxic chemicals. 
The Hsp induction is monitored by the green EGFP signal, 
cell nucleus is stained in blue by Hoechst and actin cell 
cytoplasm is stained in red by Phalloïdin.

between  partners  1  and  4  (CEA  and  EPFL). This 
device is based on impedance detection of cells 
within  the  chip  and  allows  ejection  of  one  cell 
per  drop  to  obtain  100  cells  containing  drop-
assays.  Additionally,  specific  substrates  for  the 
Nanodrops were engineered.

One  key  development  of  the  “stress  promoter 
technology” is the development of Hepatocytes 
HepaRG  cells.  This  cell  type  displays  active  bi-
otransformation  capacities  and  was  transfected 
by a specific vector by partner 3 (UCBL) and part-
ner 2 (INSERM). Selection of individual clones was 
performed  3  days  after  transfection  and  4  days 

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Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
ALTERNATIVE TESTING STRATEGIES

after DMSO treatment. It allows a selective induc-
tion  of  the  construct  by  oxidative  stress,  which 
particularly relates to heavy metals, restricted to 
differentiated hepatocyte colonies.

In  order  to  perform  the  fluorimetric  assay,  the 
engineered  cell  lines  were  provided  to  Partner 
1 (CEA) in order to set up dispensing conditions, 
and  to  allow  their  culture  on  ‘cell  on  chip’  de-
vices. The high level of proliferation allowed the 
network  to  obtain  sufficient  cell  density  to  test 
several chemical stress conditions. A ‘cell on chip’ 

Figure 2: HepaRG. Differentiation appears after 4 days of 
DMSO treatment.

Cellular analysis and multiplex phenotypic char-
acterisation was performed using IMSTAR (part-
ner 5) Pathfinder™ leading technologies for HCS 
analysis. Using this patented technology, an au-
tomated and quick detection of cell borders, as 
well  as  fluorimetric  response  of  cells  were  pos-
sible.  Thus,  the  enormous  amount  of  data  ob-
tained  by  both  the  fluorimetric,  as  well  as  the 
mass spectrometric approach developed within 

device  was  used  to  analyse  400  independent 
nanodrops.  Each  nanodrop  contained  100  indi-
vidual  engineered  HepG2  cells  and  was  spiked 
with an individual species and concentration of a 
toxic substance. The results obtained showed an 
increasing  fluorescence  signal  upon  increasing 
the concentration of the toxic substance (figure 
3; published in PLoS).

Figure 3: Multiplexed toxicity assay in drops. 2a. Cell 
dispensing with sciFlexarrayer robot arrayer. 2b. Zoom on 
the assembled mosaic of images corresponding to HepG2 
hsp22-EGFP clone after 6h exposure to 10 doses of arsenate 
in quintuplicate measurements (columns); the Hsp induction 
is monitored by the green EGFP signal, cell nucleus is stained 
in blue by Hoechst and cell cytoplasm is stained in red by 
Phalloïdin. 2c. Heterogeneity in cell response is illustrated by 
an example of Hsp response to 5.10-5M arsenate exposure. 
Scale bar represents 500 µm. Fully automated image capture 
with a 10× objective and dedicated image analysis were 
performed using the same detection protocols by IMSTAR 
Pathfinder™ Cellscan system.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

55

TOXDROP

ALTERNATIVE TESTING STRATEGIES

Figure 4: ToF-SIMS assay and application of Pathfinder™ 
technology: Detection of a fluorophor on an identical 
sample position by fluorescence microscopy (left) and 
ToF-SIMS Imaging (middle). Cell border detection by 
IMSTAR Pathfinder™ technology on a fluorimetric assay 
nanodrop (right).

Lemaire,  F.,  Mandon,  C.A.,  Reboud,  J.,  Papine,  A., 
Angulo,  J.,  Pointu,  H.,  Diaz-Latoud,  C.,  Lajaunie, 
C.,  Chatelain,  F.,  Arrigo,  A.P.,  Schaack,  B., ‘Toxicity 
assays  in  nanodrops  combining  bioassay  and 
morphometric  endpoints’,  PLoS  ONE,  2007,  Jan 
17;2:e163. 

Cerec, V., Glaise, D., Garnier, D., Morosan, S., Turlin, 
B.,  Drenou,  B.,  Gripon,  P.,  Kremsdorf,  D.,  Guguen-
Guillouzo,  C.,  Corlu,  A.,²  ‘Transdifferentiation  of 
hepatocyte-like  cells  from  the  HepaRG  cell  line 
through  bipotent  progenitor’,  Hepatology,  2007 
Apr;45(4):957-67.

Patent: 
A new HepaRG cell line carrying a marker of he-
patic oxidative stress French and English.

the TOXDROP project, could be evaluated. Given 
the large amount of information to be processed, 
a shared database was initiated to facilitate data 
storage and implementation of statistical tools.

Pathfinder™  technology  created  a  bridge  be-
tween  these  two  approaches  in  order  to  finally 
provide  a  comprehensive  platform  integrating 
all  characterisation  data  with  full  compatibility 
and transparency for the user, although the phys-
ical nature of image fluorimetry and mass spec-
trometry  is  very  different.  It  is  to  highlight  that 
IMSTAR developed integration software to make 
the Pathfinder™ technology capable of reading 
Tascon mass spectrometry data and to organise 
them within the Pathfinder™ database.

Major publications:

Mandon,  C.A.,  Diaz,  C.,  Arrigo,  A.P.,  Blum,  L.J.,  
‘Chemical  stress  sensitive  luminescent  human 
cells: molecular biology approach using inducible 
Drosophila  melanogaster  hsp22  promoter’,  Bio-
chem Biophys Res Commun , 2005, 335(2):536-44.

Mandon,  C.A.,  Diaz-Latoud,  C.,  Arrigo,  A.P.,  Blum, 
L.J.,    ‘Dithiocarbamate  fungicide  thiram  detec-
tion:  Comparison  of  bioluminescent  and  fluo-
rescent  whole-cell  bioassays  based  on  hsp22 
stress promoter induction’, J Biotechnol, 2006, Jul 
13;124(2):392-402. 2006.

56

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

Coordinator

Béatrice Schaack
CEA
Laboratoire Biopuces, bat 40-20
38054 Grenoble, Cedex 9, France
E-mail: beatrice.schaack@cea.fr

Partners 

Christiane Guillouzo
INSERM
Rennes, France

André-Patrick Arrigo
University of Lyon-Ezus
Lyon, France

Philippe Renaud
Ecole Polytechnique Fédérale de Lausanne
Lausanne, Switzerland

Françoise Soussaline
IMSTAR
Paris, France

Birgit Hagenhoff
TASCON GmbH
Munster, Germany

Olivier de Bardonnèche
VITAMIB
Meylan, France

Sandra Coecke and Maurice Whelan
Joint Research Centre
Ispra, Italy

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

57

 
VITROCELLOMICS

Reducing  animal  experimentation  in  preclinical  predictive  drug  testing  by 
human hepatic in vitro models derived from embryonic stem cells

ALTERNATIVE TESTING STRATEGIES

Contract No 
Project type 

LSHB-CT-2006-018940
Specific Targeted Research Project 

EC contribution  e 2 942 000

Starting date   1 January 2006
Duration  36 months

Website  http://er-projects.gf.liu.se/~vitrocellomics

Background and objectives:

The objective of VITROCELLOMICS is to establish 
stable cell lines that reliably reflect human hepatic 
properties  through  the  development  of  models 
derived  from  human  embryonic  stem  cells.  The 
aim is to deliver reliable in vitro models that could 
be used by the pharmaceutical industry to replace 
experimental animals in investigations on human 
drug metabolism, uptake and efflux properties of 
compounds  in  the  drug  discovery,  and  develop-
ment  processes.  In  the  pharmaceutical  industry, 
reliable in vitro cell models would replace current 
techniques  and  animal  experimentation  in  the 
selection and optimisation of lead compounds, as 
well as in the documentation of a selected drug 
candidate before it enters clinical development.

Studies  of  metabolism  and  pharmacokinetic 
properties  have  become  a  key  activity  in  the 
early drug discovery screening programmes. This 
is mainly driven by the fact that as many as 40% 
of new chemical entities failed in the late clinical 
phases  because  of  pharmacokinetic  problems. 
Moreover, adverse drug reactions, most of which 
are pharmacokinetic-based, are the 4th-6th lead-
ing  cause  of  death  in  hospitalised  patients  in 
the USA. Therefore, there is an urgent need for in 
vitro tools to predict pharmacokinetics and pos-
sible  toxic  reactions  of  new  compounds  at  an 
early stage in drug discovery, in order to be able 
to select high quality compounds that could be 
developed  into  drugs  that  are  safe  and  easy  to 
administer. 

58

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Pharmaceutical companies have made major in-
vestments to screen for relevant metabolic prop-
erties early in the drug discovery process. A major 
part  of  current  human  related  in  vitro  methods 
are based on fractionated tissue of human origin 
(usually waste material from operations), primary 
cells, expressed enzymes, hepatoma cell lines, etc. 
However, a major problem is still the poor predic-
tive power in the in vitro tools that are available. 
Consequently,  the  pharmaceutical  industry  still 
relies  heavily  on  animal  models  and  allometric 
scaling to predict human pharmacokinetics. Re-
liable in vitro cell models would replace current 
techniques  and  animal  experimentation  in  the 
selection  and  optimisation  of  lead  compounds, 
and  in  the  documentation  of  a  selected  drug 
candidate before it enters clinical development. 
In vitro cell models that could reliably predict hu-
man  metabolism  and  disposition  would  reduce 
the need for animal experimentation for this pur-
pose by 60-80%.

Approach and methodology:

The means to accomplish the objectives of VIT-
ROCELLOMICS are, in addition to new stable hu-
man embryonic stem (hES) cell derived hepato-
cytes, the following:
•	

3D-hepatic	 cell	 culture	 and	 co-culture	
methods;
microcultivation	 monitoring	 systems	 for	
in vitro screening;
genomic	 and	 metabolomic	 character-
isation;

•	

•	

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

•	

a	 multi-micro-bioreactor	 platform	 for	
high-throughput 
screening  of  drug 
candidates.

•	

Comparative  studies  of  hepatocytes  derived 
from  hES  cells  with  established  in  vitro  models 
will  be  carried  out  in  order  to  validate  the  new 
models and methods.

dation  in  GLP/SOPs  environment  for  ab-
sorption, metabolism, and toxicity;
reducing,	 or	 even	 totally	 abolishing,	 the	
use  of  animals  in  drug  testing,  refining 
the  model  system  under  consideration, 
and replacing the animals currently used, 
which is the ultimate aim.

The overall objectives of VITROCELLOMICS are:
•	

replacing	 animals	 in	 preclinical	 pharma-
ceutical development by human cell cul-
ture systems; 
supporting	the	predictability	of	the	drug	
discovery  and  development  process  by 
allowing  more  reliable  and  relevant  test-
ing  in  the  preclinical  phase,  and  hinder-
ing weak lead candidates to enter clinical 
phases  with  innovative  human  hepatic 
cell systems;
delivering	 an	 in vitro  testing  system  with 
adjacent methodology pertinent for vali-

•	

•	

As measurable and verifiable specific objectives 
for  the  project,  the  following  4  technology-re-
lated objectives are defined: 
1. 

establishing  relevant  hES  cell  derived 
hepatocyte  cultures  that  allow  a  more 
predictable  preclinical  lead  testing  pro-
gramme to be carried out; 
developing a 3-dimensional in vitro model 
for long-term studies of drug metabolism 
and  toxicology  that  mimics  the  in  vivo 
tissue  or  organ  cyto-architecture  and 
function;
establishing  a  versatile  cell  lab  platform 

2. 

3. 

Part of the platform: a
 4-compartment artificial liver 
bioreactor with three independent 
capillary bundles (red and blue: 
culture medium perfusion; yellow: 
oxygenation). Liver cells are located 
in the extra-capillary space.

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

59

ALTERNATIVE TESTING STRATEGIES

VITROCELLOMICS

based on the developed cell lines and culti-
vated in advanced miniaturised bioreactor 
systems  with  non-invasive  measurement 
techniques  for  in  vitro  testing  of  metabo-
lism, toxicology and absorption;
quantifying  metabolism  by  in  vitro  assay 
methods.

4. 

Expected outcome:

The expected outcome of the project is new, effi-
cient in vitro prevalidation models, which will sig-
nificantly reduce the use of animal experimenta-
tion for prediction of human drug metabolism by 
60-80%. In addition, the models will also increase 
safety and quality of compounds chosen as can-
didates in the different stages of the drug discov-
ery  and  development  process.  Furthermore,  it 
will strengthen the possibility for SMEs to market 
new potential products in the areas of cell assays 
and in vitro compound screening.

The clinical expertise in the project is manifested 
by the involvement of 2 renowned European uni-
versity  hospitals,  one  SME  founded  by  another 
well-known  European  university  hospital,  and 
the  partnership  with  one  of  the  leading  Euro-
pean pharmaceutical companies. Also, the coor-
dinator has managerial background from an in-
ternational  pharmaceutical  company,  while  the 
project  leader  is  from  one  of  the  SME  partners. 
Three partners represent a solid reputable bioen-
gineering background. Other important partners 
are  one  SME,  which  is  focused  on  the  develop-
ment  of  standardised  assay  conditions  for  drug 
testing,  and  the  European  Centre  for Validation 
of Alternative Methods (ECVAM). Links to animal 
care institutes are also comprised in the project.

Coordinator

Carl-Fredrik Mandenius
Linköping University
Division of Biotechnology, IFM
581 83 Linköping, Sweden
E-mail: cfm@ifm.liu.se

Partners

Elmar Heinzle
Saarland University
Saarbruecken, Germany

Inger Johansson
Karolinska Institute
Stockholm, Sweden

Manuel Carrondo
Instituto de Biología Experimental e Tecnológica (IBET)
Oeiras, Portugal

Görg Gerlach
Charité University Hospital
Berlin, Germany

Petter Björquist
Cellartis AB
Gothenburg, Sweden

Christine Hartmann-Batzl
Pharmacelsus GmbH
Saarbruecken, Germany

Tommy B. Andersson
AstraZeneca AB Mölndal 
DMPK and Bioanalytical Chemistry
Mölndal, Sweden

Sandra Coecke
European Centre for Validation of Alternative 
Methods (ECVAM) 
 Joint Research Centre
Institute for Health and Consumer Protection
Ispra, Italy

60

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

  
ALTERNATIVE TESTING STRATEGIES

BBMO

Biosensors based on membrane organisation to replace animal testing

Contract No 
Project type 

LSSB-CT-2005-005199
Specific Support Action

EC contribution  e 443 878

Starting date   1 January 2005
Duration  30 months

Website  www.chem.umu.se/bbmo

Background and objectives:

Approach and methodology:

The  ultimate  goal  of  the  BBMO  project  is  the 
development and validation of alternative tech-
niques to replace animal testing in drug screen-
ing  and  environmental  control  protocols.  This 
Specific  Support  Action  was  organised  as  the 
first step towards uniting fragmented industrial 
and academic research groups to form a power-
ful platform in biophysical chemistry and biosen-
sor  technology.  Since  the  programme’s  launch, 
smaller working and expert groups have focused 
on  the  study  of  relevant  research  areas. The  re-
sults  of  this  work  were  discussed  at  a  Strategic 
Meeting during the course of the project.
•	

The	 first	 objective	 was	 to	 initiate	 and	
carry  out  five  illustration  projects  within 
the project’s timeframe, in order to facili-
tate the integration between the research 
groups in the consortium. 
The	 second	 objective	 was	 the	 organisa-
tion  of  a  Strategic  Meeting  focusing  on 
particular  tasks:  synthesis,  film  formation 
and  characterisation,  and  modification 
with functional molecules. 
The	third	objective	is	to	finalise	a	prospec-
tive  analysis  that  supports  future  devel-
opments in membrane-based biosensing 
technology,  validated  by  comprehensive 
inter-calibration techniques. 
The	fourth	objective	is	to	produce	a	Work	
Programme  consisting  of  a  set  of  Work 
Packages (WPs).

•	

•	

•	

A  kick-off  meeting  was  arranged  in  Sweden  at 
the very beginning of the project, where all the 
partners  met  and  presented  their  own  compe-
tences, thoroughly discussing how each respec-
tive  partner  could  contribute  to  the  project.  It 
was  possible  to  identify  five  smaller  clusters  of 
people in good accordance with what was out-
lined  in  the  original  proposal.  Furthermore,  an 
intense  discussion  through  mailing  and  web-
conferences followed, which resulted in the fol-
lowing  five  illustration  projects  that  have  been 
running  throughout  the  project,  where  several 
partners are involved. 
1. 

Langmuir  Blodgett  films  on  cellulose 
acetate. 
Investigations  of  perfluorooctane  sul-
fonate 
(pfos),  perfluorooctanoic  acid, 
(pfoa)  and  fluorotelomer  alcohol  at  lipid 
membrane systems. 
Peptide interaction with lipid layers.
Hydrophilic repellent films on gold.
Development  of  a  glucolipid  membrane 
surfaces from achyloplasma bacteria. 

2. 

3. 
4. 
5. 

The main idea was to find ways to integrate re-
search  between  the  various  laboratories  with 
the  small  resources  that  were  at  hand,  so  that 
the  goals  could  be  met.  Several  papers  have 
been submitted for publication or are in manu-
script. Three  web  courses  were  planned  during 
the  project  and  ran  with  modest  participation 
of  some  of  the  partners  or  their  PhD  students. 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

61

 
 
 
 
 
 
BBMO

ALTERNATIVE TESTING STRATEGIES

Scanning electron microscopy image of 
a LB monolayer of cellulose membrane 
deposited on a solid support (indium 
tinoxide). Result of a collaboration 
project between Prof. Mandler, HUJI, 
Jerusalem and Prof. Vadgama, QM 
London.

Full paper in preparation from 
electrochemical characterisation of the 
biomimetic film

Overall, several new and unique research projects 
have  been  initiated  throughout  the  project  pe-
riod, although the project did not have any sup-
port  for  research.  The  main  reason  behind  the 
success is the great enthusiasm among the many 
partners that brought in PhD students and post 
docs in the illustration projects, but also the coor-
dinator’s efforts, travelling to all partners, e-mail-
ing and arranging web-conferencing.

As this is not a research project, the approach to 
these courses is introductory and not of exami-
nation  character.  The  three  web  courses  are  as 
follows.
•	

‘Electrochemical	 Impedance	 with	 focus	
on  Lipid  systems.’  A  podcasting  course 
was prepared by Britta-Lindholm-Sethson 
at  UmU.  During  this  course,  some  of  the 
experimental outcomes from the illustra-
tion project were included.
‘Evince	web	course’	run	by	Umbio	AB	and	
made  available  on  their  homepage  in 
2005.

•	

‘Example  of  Fourier  Transform 
in  Electro-
chemistry’  run  by  Magnus  Rosvall  at  Rosvall 
Instruments.

62

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

Coordinator

Britta Lindholm-Sethson
Umeå Universty
Department of Chemistry
901 87 Umeå, Sweden
E-mail: Britta.Sethson@chem.umu.se

Partners

Renata Bilewicz,
Warsaw University
Faculty of Chemistry 
Warsaw, Poland

Daniel Mandler
The Hebrew University of Jerusalem
Department of Inorganic and Analytical Chemistry
Jerusalem, Israel

Pankaj Vadgama
Queen Mary and Westfield College University of London
IRC in Biomedical Materials
London, UK

Josep Galceran
ETSEA
Departament de Quimica
Lleida, Spain

L. Andrew Nelson
University of Leeds
Mathematics and Physical Sciences 
Leeds, UK

Brian McCraith
Dublin City University
The National Centre for Sensor Research
Dublin, Ireland

Günter Gauglitz
University of Tübingen
Institute for Physical and Theoretical Chemistry
Tübingen, Germany

Paul Geladi
SLU Röbäcksdalen
Unit of Biomass Technology and Chemistry
Umeå, Sweden

Inger Vikholm-Lundin
VTT Information Technology 
Tampere, Finland

Jan van Deventer
Luleå University of Technology 
Division of Computer Science and Electrical Engineering 
/ EISLAB
Luleå, Sweden

Roger Koeppe
University of Arkansas
Department of Chemistry and Biochemistry
Fayetteville, USA

Fredrik Pettersson
UmBio AB
Umeå, Sweden

Magnus Rosvall
Rosvall Instruments 
Surte, Sweden

Philip Judson
LHASA Ltd 
Leeds, UK

Christoph Methfessel
BTS-PT-AS Biophysics
Leverkusen, Germany

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

63

 
 
 
 
CONAM

Consensus networking on alternatives within Europe

ALTERNATIVE TESTING STRATEGIES

Contract No 
Project type 

LSSB-CT-2004-504776
Specific Support Action

EC contribution  e 150 000

Starting date   1 March 2004
Duration  36 months

Website  www.ecopa.eu

Background and objectives:

the NCPs hucopa and polcopa, respectively. 

The CONAM project was proposed by ecopa (eu-
ropean consensus-platform for alternatives), the 
only  quadripartite  not-for-profit  organisation  at 
EU  level  promoting  the  3Rs  strategy  for  the  re-
placement, reduction, and refinement of animal 
experiments  in  research  and  regulatory  testing. 
Consensus means that the four parties concerned, 
namely  animal  welfare,  industry,  academia,  and 
governmental  bodies,  are  represented  in  ecopa 
as  well  as  in  the  individual  National  Consensus 
Platforms (NCPs), which form the building stones 
of the umbrella organisation.

The objective of CONAM was to build a solid ex-
tensive  consensus  network  on  3R-alternatives, 
ideally including all European countries (existing 
and new EU countries, as well as candidate coun-
tries), and with links to 3R-relevant organisations 
and  institutes.  One  partner,  VUB/ecopa,  was  in-
volved in this project. Ecopa groups 16 national 
consensus platforms, of which 13 are full mem-
bers and 3 are associates. 

Expected outcome: 

•	

With  regards  to  the  creation  of    new  platforms, 
ecopa organised, together with ECVAM, a stake-
holder meeting in Prague in June 2004 to inform 
the new EU Member States about the 3Rs, as well 
as the validation of alternative methods and the 
work and objectives of ecopa. Thanks to this con-
tact  building,  Hungary  and  Poland  established 

64

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Already  existing  organisations  adapted  their 
structure  according  to  the  ecopa  rules,  so  as  to 
have  representatives  from  the  four  parties  con-
cerned,  e.g.  Germany  with  the  Stiftung  set,  and 
the UK with the Boyd group. New platforms were 
also  launched,  in  Norway,  Denmark  and  Ireland, 
for example, resulting in a current total number 
of  16  NCPs  (Belgium,  the  Czech  Republic,  Den-
mark, Germany, Ireland, Spain, Italy, Hungary, the 
Netherlands,  Austria,  Poland,  Finland,  Sweden, 
UK, Switzerland and Norway). France is expected 
to join in 2007, and is now in the phase of build-
ing a NCP according to ecopa’s rules.

Main findings:

CONAM has succeeded in doing the following:
•	

The	 ecopa	 website	 was	 improved,	 ex-
panded  from  a  simple  webpage  to  a  full 
website,  complete  with  news  and  events 
sections, databases and links, an archives 
section,  information  pages  about  educa-
tion and legislation, and a forum. 
The	 ecopa	 newsletter	 was	 created:	 10	 is-
sues of the ‘ecopa messenger’ have been 
sent  out  to  date,  and  they  are  also  avail-
able on the ecopa website.

For the first time in Europe and working through 
ecopa,  CONAM  has  created  an  information  ex-
change system on alternative method develop-
ment, that almost in real time, supplies interested 

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

organisations,  institutions  and  individuals  with 
key information. 

An  ethics  working  group  was  formed;  it  organ-
ised a consensus training course in Ljubljana in 
June 2005. The report is available on the website. 
A  workshop  on ‘The  Use  of  Human  Tissue’  was 
organised in Brussels in November 2006 and the 
report is made available on the project website.

An educational workgroup was created and the 
Spanish  platform  REMA,  being  part  of  it,  organ-
ised  2  training  courses  on  alternative  method-
ologies in 2006. A similar initiative was taken on 
behalf  of  the  Italian  platform,  IPAM,  in  Rome  in 
February 2007.

A  workgroup  on  ‘Chemical  Policy,  REACH’  was 
created. Besides the organisation of several work-
shops, (e.g. in Brussels in November 2006, where 
discussions focused on the limitations and prob-
lems  of  REACH as regards the high numbers of 
animal use, and the implementation problems of 
alternatives), the workgroup also prepared a re-
port on the impact of  REACH on animal use. 

During  the  run  of  CONAM,  several  workshops 
were organised.
•	

On	 an	 annual	 basis,	 ecopa	 organised	 its	
plenary  workshop  in  Brussels  in  Novem-
ber/December.  These  workshops  always 
offered a forum for exchange of initiatives 
and experience with respect to alternative 
methods  development.  Each  time,  a  spe-
cial session was foreseen for young scien-
tists to show their newest research results 
and the technologies involved.
Within	CONAM,	the	workshops	‘ecopa	Sci-
ence  Initiative  (eSI)’  were  organised  on  2 
occasions  in  Pueblo  Acantilado  in  Spain, 
in  October  2004  and  September  2006. 
The  idea  to  organise  these  workshops 
was  developed  during  the  annual  ecopa 
workshop  in  November  2003:  ecopa  had 
performed a literature analysis to find out 

•	

how  many  original  research  ideas  in  the 
field of the 3Rs were developed within the 
last  five  years.  It  was  concluded  that  the 
field had an urgent need of new and fresh 
initiatives.  Therefore,  young  and  promis-
ing  scientists  were  brought  in  contact 
with established researchers to discuss in-
novative technologies and their potential 
applications  in in vitro  research.  Different 
scientific disciplines were combined in or-
der to let scientists think “about” and “for” 
alternative methods. Some fruitful collab-
orative projects — focused on the further 
development and optimisation of alterna-
tive methods — were initiated, thanks to 
these workshops.

In  order  to  stay  updated  with  respect  to  devel-
oping  research  within  the  3Rs,  the  workgroup 
on ‘3Rs-research’ was created within the CONAM 
project.  As  such,  participation  in  several  FP6 
projects could be realised, e.g. in PREDICTOMICS, 
ReProTect,  A-CUTE-TOX,  Sens-it-iv,  Biosim,  Car-
cinogenomics, ForInvitox and Liintop.

Within  the  CONAM  project,  contact  building 
with relevant bodies and third parties has been 
realised, e.g. with IVTIP, OECD, Colipa, CEFIC, ESAC, 
SusChem and the epaa initiative, among others.

Also  running  is  an  EU  study,  initiated  by  ecopa 
in collaboration with P&G and the Eurogroup for 
Animal Welfare, on the availability of funding of 
alternative  methods  within  the  Member  States. 
This will provide a comprehensive picture of EU 
research in this area, and will be of great assist-
ance  in  planning  an  EU-coordinated  research 
programme. A computer programme, ‘the Animal 
use Calculator’, on the realistic number of animals 
used within the context of REACH was elaborat-
ed, and can be downloaded free of charge on the 
ecopa website (available since 1 February 2006).

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

65

 
ALTERNATIVE TESTING STRATEGIES

Coordinator

Vera Rogiers 
Vrije Universiteit Brussel
Laarbeeklaan 103 
1090 Jette, Belgium
E-mail: vrogiers@vub.ac.be

CONAM

Major publications: 

Rogiers,  V.,  ‘ECOPA:    The  European  Consensus 
Platform  on  three  Rs  alternatives’,      Alternatives 
to Laboratory Animals (ATLA), 2004, 32, 349-353. 
Rogiers, V., ‘Recent developments in the way for-
ward  for  alternative  methods:  formation  of  na-
tional consensus platforms in Europe’. Toxicology 
and  Applied  Pharmacology,  2005,  207  (suppl.  2) 
S408-S413. 

Rogiers, V., Pauwels, M., ‘Good science must be the 
key factor in the development and use of alterna-
tive methods for safety assessment of cosmetics’, 
Altex - Alternativen zu Tierexperimenten, 2005, 22 
(Special Issue), 259

Rogiers, V., Pauwels, M., ‘Good science must be the 
key factor in the development and use of alterna-
tive methods for safety assessment of cosmetics’, 
Altex - Alternativen zu Tierexperimenten, 2006, 23 
(Special Issue) 346-352. 

In press
Rogiers, V., ‘Replacement, reduction, refinement,’ 
Public  Service  Review:  European  Union,  2007,  13, 
UK, March Edition

66

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ALTERNATIVE TESTING STRATEGIES

ForInViTox

Forum for researchers and regulators to meet manufacturers of toxicology test 
methods

Contract No 
Project type 

LSSB-CT-2007-037779
Specific Support Action

EC contribution  e 288 850

Starting date   2007 (date to be confirmed) 

Duration  24 months

Background and objectives:

Approach and methodology:

With  the  support  of  the  European  Commission, 
researchers  and  the  industry  in  recent  years 
have conducted research on in vitro replacement 
tests. This  has  resulted  in  an  important  number 
of  scientifically-sound  methods  and  new  strat-
egies,  but  the  transfer  of  these  inventions  to 
potential  users  has  been  much  slower  than  ex-
pected,  mainly  due  to  difficulties  encountered 
in the transferability, official approval, as well as 
the production of test kits under conditions that 
meet the requirements of good laboratory prac-
tice (GLP).

The  project  is  structured  into  6  Work  Packages 
(WPs).  Three  are  devoted  to  retrieving  the  rel-
evant  information  required:  availability  of  in-
ventions  (WP1);  present  and  presumably  future 
needs of users (WP2); and ability of currently ex-
isting manufacturers (WP3) to meet the required 
demands.  This  will  be  followed  by  an  analysis 
of  the  outcome  of  these  exercises,  with  the  aid 
of  experts  (WP4). The  ultimate  goal  is  to  set  up 
a  forum  where  the  interested  parties  can  meet 
(WP5). A general management Work Package is 
also anticipated (WP6).

In order to ensure that  research has the desired 
socioeconomic impact, the present gap between 
inventions  and  potential  users  needs  to  be 
bridged.  The  purpose  of  the  ForInViTox  project 
is to establish a forum where representatives of 
manufacturers, research projects and regulatory 
agencies  continuously  get  a  chance  to  discuss 
how to speed up the process of making in vitro 
methods available for end users.

The following activities will take place:
•	

a	retrieval	of	existing,	not	properly	exploit-
ed knowledge; 
an	analysis	of	the	needs	of	toxicity	tests;	
an	 inventory	 of	 the	 producers	 of	 in  vitro 
toxicity tests.

•	
•	

Expected outcome:

ForInViTox  will  summarise  the  data  from  these 
reviews  and  identify  the  most  urgent  testing 
needs. The project partners will prepare an anal-
ysis  --  complete  with  input  from  a  highly  quali-
fied expert group. It will include suggestions for 
stimulating research, technology transfer, devel-
opment of smaller manufacturers or other steps 
to further increase the use of in vitro tests. 

At  the  end  of  the  project,  a  forum  event,  with 
representatives  from  the  different  stakeholders, 
will take place. An interactive forum website for 
discussion,  information  and  education  around 
issues  concerning  implementation  and  use  of 
alternative  toxicity  tests,  as  well  as  presenta-
tions  of  innovations  and  manufacturers  will  be 
developed. The  outcome  of  the  forum  will  be  a 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

67

 
 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

ForInViTox

White book, recommending actions to meet the 
increased needs for alternative toxicity tests. 

Coordinator

Cecilia Clemedson
Expertrådet Environmental Competence Ltd
Rådan 3
Sollentuna, Sweden
E-mail: cecilia.clemedson@expertradet.se

Partners 

Eugenio Vilanova
Red Española de Métodos Alternativos
Elche-Alicante, Spain

Christiane Buta
Stiftung zur Einschränkung von Tierversuchen
Frankfurt am Main, Germany

Joan Albert Vericat
In vitro Testing Industrial Platform
Rotterdam, Netherlands

Erica Toft
Sliverdal Science Park
Sollentuna, Sweden

68

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

InViToPharma

Workshop  on  the  need  of  in  vitro  toxicity  tests  within  the  pharmaceutical 
industry

Contract No 
Project type 

LSSB-CT-2007-037814
Specific Support Action

EC contribution  e 578 000

Starting date   2007 (date to be confirmed)

Duration  24 months

Background and objectives:

A workshop where representatives of manufactur-
ers of toxicity tests, researchers developing new in 
vitro  pharmaceutical  toxicity  tests,  the  European 
pharmaceutical  industry  and  regulatory  authori-
ties  participate  makes  it  possible  for  stakehold-
ers to discuss the supply and demand for in vitro 
pharmaceutical toxicity tests, new and old. 

The  workshop  will  also  increase  business  op-
portunities  for  the  manufacturers  of  in  vitro 
pharmaceutical toxicity tests, often SMEs, as the 
participants will have ample time to sit down and 
confer in smaller groups and one-on-one.

development of new in vitro toxicity tests, 
high-throughput  screening  tests,  and 
tests monitoring the toxicological mecha-
nism in detail, etc.;
to	 identify	 and	 present	 academic	 model	
systems  that  are  suitable  for  use  in  the 
pharmaceutical industry;
to	 identify	 and	 present	 in-vitro	 toxicity	
tests manufactured by enterprises;
to	 analyse	 the	 correspondence	 between	
the available in vitro toxicity tests and the 
test strategies used in the pharmaceutical 
industry.

•	

•	

•	

Approach and methodology:

A dialogue between the stakeholders within the 
area of pharmaceutical toxicity testing is crucial 
for  achieving  a  more  general  use  of  alternative 
toxicity tests. More efficient toxicity tests are also 
an important factor in making it possible for the 
European  pharmaceutical  industry  to  boost  its 
competitiveness. The increasing amount of sub-
stances  passing  the  drug  development  process 
needs to be tested with efficient methods as ear-
ly as possible for new pharmaceuticals. This de-
mands more robust and cost-efficient methods, 
which are not always available today.

The  InViToPharma  project  will  start  with  a  gen-
eral analysis of the current situation: This is Work 
Package 1 (WP1) and it will precede a planning 
meeting with the Advisory committee, involving 
representatives from the stakeholders. The infor-
mation  that  will  form  the  basis  for  the  analysis 
will be retrieved from questionnaires presented 
to  pharmaceutical  companies.  The  outcome  of 
the compiled answers and a general analysis of 
the  tendencies  will  be  discussed  at  the  second 
Advisory  committee  meeting,  and  will  form  the 
base for the workshop programme. 

The main objectives of this project are as follows:
to	 identify	 the	 need	 within	 the	 pharma-
•	
ceutical industry for in vitro toxicity tests, 
e.g. what kind of tests are required, which 
toxicity areas are of most concern for the 

A two-day workshop on the subject ‘What is the 
need of in vitro toxicity tests within the pharma-
ceutical industry’ will take place at Silverdal Sci-
ence Park in Stockholm, Sweden. The workshop 
will  comprise  both  introductory  lectures  and 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

69

 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

InViToPharma

smaller working groups focused on different is-
sues, defined after the general analysis described 
in  WP1.  The  outcome  from  the  working  group 
meetings will be reported at a session for all par-
ticipants, followed by a general discussion. A final 
report from the workshop will be produced and 
distributed.

Expected outcome:

The  project  will  bring  together  stakeholders 
within  the  area  of  pharmaceutical  toxicity  test-
ing, researchers in the field of in vitro toxicology, 
the pharmaceutical industry, manufacturers of in 
vitro toxicity tests for pharmaceuticals and regu-
latory  authorities.    Drawing  together  all  these 
players has the aim of attaining an effective in-
terplay, for safer and more efficient and drug de-
velopment, in line with the 3 Rs principle, within 
the European pharmaceutical sector.

Coordinator

Erica Toft
Silverdal Science Park
Rådan 3
191 40 Sollentuna, Sweden 
E-mail: Erica.Toft@expertradet.se

Partners

Cecilia Clemedson
Expertrådet AB
Sollentuna, Sweden

70

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

NHR DevTox

A  prospective  analysis  of  the  mechanisms  of  nuclear  hormone  receptors  and 
their potential as tools for the assessment of developmental toxicity

Contract No 
Project type 

LSSB-CT-2004-512513
Specific Support Action

EC contribution  e 144 000

Starting date   1 January 2005
Duration  11 months

Background and objectives:

The  aim  of  NHR  DevTox  was  to  analyse  mecha-
nistic  approaches  that  could  be  applied  for  the 
assessment  of  developmental  toxicity,  thus  po-
tentially  leading  to  an  innovative  non-animal 
testing methodology. 

As  all  normal  processes  of  biological  develop-
ment are ultimately controlled at the molecular 
level, the NHRDevTox project proposed a unique 
approach for developmental toxicology by stud-
ying the nuclear receptors that are fundamental 
to these processes. Recent and emerging techni-
cal advances in the fields of genomics and pro-
teomics, among others, enable the study of such 
processes  and  of  the  mechanisms  underlying 
their  perturbation  by  exposure  to  xenobiotics. 
Accordingly, the prospective analysis focused on 
the role of nuclear hormone receptors (NHRs) in 
developmental toxicity. 

Approach and methodology:

The methodology proposed for this SSA involved 
convening  two  workshops  in  which  a  consorti-
um of scientists from leading European research 
teams  active  in  the  area  of  molecular  and  cell 
biology,  genomics  and  developmental  toxicol-
ogy,  complemented  by  external  experts,  would 
review the state of the science of NHRs, and their 
role in developmental and reproductive biology. 
The options for further study and research were 
reviewed and discussed, so as to identify the pri-

ority areas of research that should be carried out 
to advance the scientific knowledge of reproduc-
tive  toxicology,  and  attain  the  ultimate  goal  by 
highlighting  the  different  technologies  needed 
to deliver success.

The  first  workshop  was  designed  to  review  the 
regulatory  background,  to  describe  the  state  of 
the  science,  and  to  identify  the  research  tools 
currently  available  for  studying  developmental 
toxicity  at  the  molecular  level,  with  particular 
reference  to  the  role  of  NHRs.  Discussion  was 
encouraged to highlight the most promising as-
pects  of  research  that  would  deliver  the  objec-
tives  set  out  in  the  SSA,  particularly  within  the 
context of evaluating chemical hazards. 

The second workshop was designed to develop a 
cohesive plan of research (with input from invited 
experts) which would deliver a greatly improved 
understanding  of  the  role  of  nuclear  hormone 
receptors  in  developmental  biology,  particu-
larly within the context of their perturbation by 
chemicals  causing  toxicity.  Workshop  2  applied 
the  generic  paradigm  developed  in  Workshop 
1  to  the  review  and  discussion  of  specific  areas 
considered to be most promising for study. 

It is recommended that research is conducted to 
further elucidate the role of NHR mechanism de-
velopmental toxicity, based on the paradigm and 
schematics  designed  in  this  SSA.  Such  research 
should consider both upstream and downstream 
signalling  pathways,  as  well  as  co-regulators.  It 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

71

 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

NHR DevTox

would  initially  focus  upon  a  particular/specific 
toxicological  endpoint  that  is  capable  of  being 
modelled in an animal-based system. 

The mechanisms elucidated in such an approach 
should then be tested for their relevance to hu-
mans  though  the  use  of  in  vitro  systems.  If  the 
resultant mechanism of action(s) determined by 
the  approach  is  relevant  to  humans,  then  cell-
based systems that are capable of reporting on 
the ability or otherwise of chemicals to influence 
those mechanisms should be developed. 

Expected outcome:

The  major  scientific  outcome  of  this  work  is  a 
generic  paradigm  on  how  the  developed  ap-
proaches can be used to determine a mechanism 
of action of reproductive toxicity, and how they 
might be applied to future research projects. This 
is  a  novel  approach  for  the  development  of  in 
vitro  technologies,  particularly  in  that  many  ex-
isting  approaches  use  imperial  methods,  rather 
than starting from a mechanistic understanding 
of the toxicological phenomena for which a test 
system is intended. 

Coordinator

Mike Penman 
ExxonMobil Petroleum and Chemical 
Hermeslaan 2
1831 Machelen, Belgium
E-mail: michael.g.penman@exxonmobil.com

Partners

Cliff Elcombe 
CXR Biosciences 
Dundee, UK

Alexi Sedo 
Charles University 
Prague, Czech Republic

Margarita Hadzopoulous-Cladaras
Aristotle University
Thessaloniki, Greece

Jaroslaw Dastych 
International Institute of Molecular and Cell Biology
Lodz, Poland

Purkyne Military Medical Academy
Czech Republic 

Jiri Stulik 
Medical Research Centre 
Hradec Kralove, Poland

Monica Puzianowska-Kuznicka 
Medical Research Centre Polish Academy of Sciences 
Warsaw, Poland 

Francis Carpanini
FCT Consult
Crowthorne, UK

Konrad Rydzynski, Maciej Stepnik
Nofer Institute
Lodz, Poland

72

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
ALTERNATIVE TESTING STRATEGIES

RAINBOW

Research  on  animal  and  in  vitro  studies  and  numerical  methods:  bridging 
opportunities through a workshop

Contract No 
Project type 

LSSB-CT-2005-018695
Specific Support Action

EC contribution  e 145 950

Starting date   1 March 2006
Duration  12 months

Website  www.rainbow-project.eu 

Background and objectives:

The  RAINBOW  project  organised  the  RAINBOW 
workshop, held in Milan, Italy from 11 to 13 De-
cember 2006, which addressed the integration of 
in vivo, in vitro and computer-based methods (in 
silico) for studying the toxic properties of chemi-
cals. The workshop targeted regulators, academic 
and industrial scientists and stakeholders, includ-
ing  chemical  industries,  animal  welfare  experts 
and public organisations. Around 50 participants 
from 14 EU and non-EU countries attended the 
workshop,  which  helped  break  down  barriers 
between scientific and societal areas. The partici-
pants  discussed  the  implications  of  the  REACH 
initiative on the integration of methodologies.

There  is  an  urgent  need  for  test  systems  that 
can  fill  the  enormous  data  gap  for  untested  or 
insufficiently  tested  substances,  as  efficiently 
as  possible.  The  recent  approval  of  the  REACH 
regulation  by  the  European  Parliament  is  an 
important step towards addressing this societal 
request. An efficient test strategy must consider 
the limitations in economic resources and testing 
capacity.  It  must  also  be  in  line  with  the  aim  of 
reducing  the  use  of  animals  in  toxicological 
testing.  By  2009,  the  7th  amendment  to  the 
Cosmetics  Directive  will  prohibit  the  use  of 
animals  for  premarketing  toxicity  testing  for 
cosmetic products in the EU (Directive 2003/15/
EC, Official Journal L66:26-35).

Individual  toxicological  and  ecotoxicological 
tests can be described in terms of their cost, va-
lidity, reliability, and sensitivity. It should be not-
ed that there is no such thing as the perfect test. 
If  researchers  had,  for  all  important  endpoints, 
tests that fulfil the criteria of low cost, sufficient 
sensitivity  and  high  validity  and  reliability,  then 
the  scientific  uncertainties  inherent  in  testing 
and risk assessment could be reduced substan-
tially. In reality, every test is a trade-off between 
these requirements. 

Since every test represents a trade-off between 
these aspects, researchers face the challenge of 
combining  tests  with  different  strengths  and 
weaknesses,  and  converting  them  into  scien-
tifically well-founded and resource-efficient test 
systems,  where  the  tests  compensate  for  each 
other’s weaknesses as much as possible. The in-
tegration  of  the  various  possibilities  offered  by 
in vivo, in vitro and in silico methods is the most 
mature solution to the knowledge gap, which is 
huge. Integration can take advantage of the pos-
sibilities of each approach. 

In regulatory applications, toxicological tests are 
combined  into  test  systems.  A  test  system  con-
tains rules for when and in what order the differ-
ent tests should be performed. With the resources 
presently available, it would be necessary to use 
tiered systems in which relatively simple tests are 
performed for all chemicals that are up for assess-
ment, and the outcomes of these simple tests are 
used to prioritise substances for further, more re-

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

73

 
 
 
 
 
 
RAINBOW

source-intensive testing. Furthermore, efforts to-
wards integration should address a more flexible 
scheme beyond the classical tiered approach, in 
which in silico screening is the first step, followed 
by in vitro and finally in vivo. A feedback mecha-
nism may offer advantages, and multiple parallel 
inputs may increase understanding. 

In future, a large number of data, from in vivo, in 
vitro and in silico studies, will become available. A 
strategy  to  integrate  the  different  methods  has 
to allow for the evolving situation.

Main findings:

The  discussion  at  the  workshop  addressed  the 
barriers  to  more  efficient  integration  and  the 
needs to solve current limitations, including lan-
guage, concepts, formalism and subjectivism.
The  discussion  identified  areas  to  be  evaluated 
in  order  to  improve  the  integration  between  in 
vivo, in vitro and in silico methods. Some of these 
efforts should clarify certain aspects of individu-
al methods, allowing a better dialogue between 
the methods; other efforts are required to better 
plan the overall integrating scheme.
•	

Qualify	 and	 quantify	 uncertainties.	 Each	
method (in vivo, in vitro and in silico) should 
produce  a  result  (for  instance,  a  toxicity 
value or class) with a given defined uncer-
tainty. If such an uncertainty is not defined 
it will be very difficult to use the result of 
a given method for the final chemical as-
sessment. Also, the current practice is not 
satisfactory in this aspect.
Improve	transparency.
Improve	standardisation,	achieving	a	com-
mon metric.
Evaluate	 advantages	 and	 disadvantages	
of each method: in vivo, in vitro and in silico. 
It has not been possible, within the work-
shop,  to  explicitly  list  a  series  of  features 
characterising  the  different  approaches. 
Some common features apply to the same 
category  of  methods,  but  this  is  not  al-

•	
•	

•	

ALTERNATIVE TESTING STRATEGIES

•	

•	

•	

•	

•	

•	

•	

•	

ways the case. Pros and cons of individual 
methods  are  related  to  the  target  of  the 
integrated system, which may differ.
Know	how	results	obtained	from	this	test	
relate  to  effects  on  the  target  system,  i.e. 
humans,  and  the  ecosystems  that  the 
regulation intends to protect, so as to de-
termine  the  predictive  value  of  any  test. 
This, however, is seldom possible. At best, 
researchers  can  compare  the  simple  test 
to a more advanced one, and even this is 
a  far  from  perfect  approximation.  Even  a 
state-of-the-art  test,  such  as  a  long-term 
animal test, provides in its turn only an es-
timate of the effects in humans; 
Codify	 in	 mathematical	 terms	 the	 com-
mon scheme for risk assessment.
Define	 the	 purposes	 of	 the	 integrated	
system.
Define	a	clearer	and	more	formally	correct	
decision  analysis,  identify  criteria  and  ac-
ceptability of decisions.
Identify	the	inputs,	outputs	and	scope	of	
the  overall  scheme.  Different  strategies 
are  necessary  for  the  different  scopes. 
For  instance,  if  the  target  of  the  integra-
tion  is  for  regulatory  purposes,  attention 
has to be given to false negatives and the 
thresholds for them. If the purpose of the 
integration  is  drug  development,  false 
positives have to be minimised.
Identify	a	reference	for	an	integrated	sys-
tem. Bodies, such as OECD and ECVAM, ad-
dress  individual  methods,  but  the  evalu-
ation  of  the  integrated  system  requires 
advanced efforts.
Identify	features	for	the	evaluation	of	the	
integrated  system,  such  as  cost,  friendli-
ness and possibility of automatism.
Develop	 integrated	 in  silico  tools,  which 
will  address  the  overall  chemical  assess-
ment,  targeting  ecosystems  and  improv-
ing the current approach in which in silico 
models  mimic  the  existing  tests  focused 
on specific species.

74

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

Coordinator 

Emilio Benfenati
Istituto di Ricerche Farmacologiche “Mario Negri”
Laboratory of Environmental Chemistry and Toxicology
via Eritrea 62
20157 Milan, Italy
E-mail: benfenati@marionegri.it

Partners 

Giuseppina Gini 
Politecnico di Milano
Department of Electronics and Information
Milan, Italy  

Jan-Ake Gustafsson
Karolinska Institutet
Stockholm, Sweden

Robert Luttik
Rijksinstituut voor Volksgezondheid en Milieu
Bilthoven, Netherlands

Wilhelmina Hermans
In vitro Testing Industrial Platform
Rotterdam, Netherlands

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

75

 
RETHINK

Mini-pigs  as  models  for  the  toxicity  testing  of  new  medicines  and  chemicals: 
impact assessment

ALTERNATIVE TESTING STRATEGIES

Contract No 
Project type 

LSSB-CT-2005-018776
Specific Support Action

EC contribution  e 200 000 

Starting date   1 March 2006
Duration  24 months

Website  www.rethink-eu.dk

Background and objectives:

The objective of RETHINK is to provide an assess-
ment of the mini-pig as an alternative in toxicity 
testing, and of the potential contribution of the 
mini-pig to the 3Rs, i.e. replacement, refinement, 
and  reduction  in  animal  testing.  Mini-pigs  are 
strains of domestic pigs that are markedly small-
er  than  farmyard  varieties,  and  are  thus  better 
adapted to laboratory housing.

The pig closely resembles man in many features 
of  its  anatomy,  physiology,  biochemistry  and 
lifestyle. In particular, the cardiovascular system, 
skin  and  digestive  tract  are  considered  to  be 
very  good  models  of  their  human  equivalents. 
Because of these similarities, the toxic effects of 
chemicals  and  drugs  in  pigs  may  resemble  the 
effects  in  man  more  closely  than  some  other 
commonly used laboratory animals. 

If the pig really is a good model for man, then pig 
and mini-pig cells and tissues could be especially 
useful for in vitro tests and alternative methods. If 
the mini-pig provides a better predictive model 
of  toxicities  to  humans  than  traditionally  used 
animal models, then there could be a significant 
public health benefit. 

Approach and methodology:

The  RETHINK  partners  have  assembled  groups 
of  experts  from  around  Europe  to  participate 
in  expert  study  groups.  Guided  by  the  project 

76

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

steering  group,  these  study  groups  are  care-
fully reviewing the available knowledge base on 
pigs and mini-pigs. The expert groups are cover-
ing different areas and are answering questions 
about the utility, validity added value and ethics 
of mini-pigs in toxicity testing.   

If the mini-pig is to provide a valid and realistic 
alternative  approach  for  use  in  the  regulatory 
testing  of  new  medicines  and  chemicals,  while 
contributing  to  the  3Rs,  many  questions  need 
to  be  asked.  These  questions  cover  5  different 
areas:
•	
•	
•	

welfare	and	animal	care;	
ethical	issues;
development	 of	 new	 medicines	 and	
chemicals;
safety	testing;
genomics	 and	 other	 emerging	 techno-
logies.

•	
•	

The  agenda  has  been  prepared  for  the  expert 
groups, defining the relevant questions that each 
group must answer. In addition, they must sum-
marise the state of the art in their area, identify 
gaps  in  the  knowledge  where  further  research 
is  required,  point  out  technical  gaps  that  may 
hinder progress, summarise the advantages and 
disadvantages of the mini-pig, and formulate rec-
ommendations regarding the use of mini-pigs in 
regulatory toxicology and their potential contri-
bution to the 3Rs.

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

Expected outcome:

The result of the project will be a detailed report, 
which  will  provide  concrete  advice  on  several 
points: 
•	

ensuring	 that	 the	 welfare	 needs	 of	 mini-
pigs are met in laboratories;
the	scope	for	the	application	and	develop-
ment of the 3Rs through the use of mini-
pigs;
the	potential	role	and	best	deployment	of	
mini-pigs in toxicology testing strategies;
the	validity	and	added	value	of	mini-pigs	
in regulatory toxicology;
proposals	 for	 initiatives	 to	 fill	 gaps	 in	
knowledge or ‘technical gaps’;
implications	for	the	development	of	new	
medicines and chemicals.

•	

•	

•	

•	

•	

Coordinator: 

Roy Forster
Centre International de Toxicologie
BP 563 27005 
Evreux, France 
E-mail: coord@rethink-eu.dk

Partners:

Lars Ellegaard
Ellegaard Göttingen Mini-pigs
Dalmose, Denmark

Jan Willem van der Laan
Rijksinstituut voor Volksgezondheid en Milieu 
Bilthoven, Netherlands

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

77

 
 
SCARLET

Structure-activity 
carcinogenicity

relationships 

leading  experts 

in  mutagenicity  and 

ALTERNATIVE TESTING STRATEGIES

Contract No 
Project type 

LSHB-CT-2007-44166
Specific Support Action

EC contribution  e 112 840 
Starting date   1 June 2007

Duration  12 months

Background and objectives:

The  relationship  between  structure  and  toxic 
activity  in  chemical  mutagens  and  carcinogens 
has  been  widely  investigated.  Structure-activity 
concepts  have  also  been  exploited  to  develop 
safer  chemicals.  While  studies  based  on  biased 
data  sets  may  provide  useful  information  for 
academic purposes, the extensive regulatory use 
of (Q)SARs (Quantitative Structure Activity Rela-
tionships)  foreseen  by  various  legislative  initia-
tives, including REACH, requires that the models 
fulfil severe quality criteria. 

Unfortunately,  this  ideal  situation  is  not  a  com-
mon  occurrence  in  toxicology.  This  project  is 
aimed  at  organising  a  discussion  and  a  work-
shop so that leading experts can critically review 
mutagenicity  and  carcinogenicity  in  the  field 
of  (Q)SARs,  while  identifying  important  issues 
that  require  further  investigation.  The  SCARLET 
project  is  multidisciplinary  and  multisectorial, 
and targets 40 participants; regulators, scientists, 
problem holders, animal welfare experts, citizen 
organisations,  and  the  chemical  industry  will 
participate.  Registration  will  be  free  in  order  to 
secure  a  wider  participation.  Moreover,  five  fel-
lowships  will  be  awarded  to  young  researchers. 
The  workshop  will  be  useful  in  overcoming  the 
barriers between science and the general public. 

SCARLET  will  discuss  a  framework  for  compar-
ing  and  integrating  individual  research  activi-
ties.  Coordinators  of  EC  projects  will  be  invited, 

and their results will be disseminated, effectively 
raising the profile of European research. This will 
also  avoid  repetition  of  work,  making  optimal 
use of public money and identifying synergisms. 
The EC projects will be a way to exploit recom-
mendations from the workshop. Verifiable results 
will include the workshop, the internet site, a list 
of  software  and  databases,  and  the  final  report, 
which will contain recommendations on the use 
of  computer-based  methods  in  carcinogenicity, 
mutagenicity and genotoxicity. A paper summa-
rising the workshop discussion will be submitted 
to a scientific journal.

The main objective is to organise an internation-
al  scientific  workshop  with  leading  experts  on 
QSARs for genotoxicity and carcinogenicity. This 
workshop  will  combine  the  experience  of  the 
various  scientific  communities  working  in  this 
area; evaluate the current state of the art; identify 
open  research  issues;  harmonise  collaborations 
within the EC to solve them; and provide scien-
tific guidance for the implementation of genoto-
xicity  and  carcinogenicity  QSARs  for  regulatory 
purposes.

SCARLET originates from a deep need in society 
to  improve  risk  assessments  for  an  increasing 
number of chemical compounds that is used in 
the  modern  world.  More  specifically,  it  will  ad-
dress the requirements of the 7th amendment of 
the Cosmetics Directive 76/768/EEC and the re-
cent  legislative  initiative  REACH,  to  characterise 
the toxicological properties of a large number of 

78

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES

chemicals. The determination of carcinogenic and 
genotoxic  properties  requires  at  present  in vivo 
(carcinogenicity,  some  genotoxicity  endpoints) 
and in vitro experiments that are time consuming, 
expensive and require a large number of experi-
mental animals. QSARs are an attractive alterna-
tive because they are fast, cheap and require no 
biological experiments. In fact, QSARs are already 
routinely used for the risk assessment of chemi-
cals by the regulatory agencies of the USA, Can-
ada, Japan, and some EU Member States, and the 
OECD has developed guidelines for their use. 

In addition, it is likely that the stakeholders  will 
require  additional  criteria  for  the  acceptance  of 
QSAR models. For this reason, the main scientif-
ic  and  technical  objective  will  be  to  specify  the 
OECD and stakeholders’ requirements, as well as 
to establish unambiguous and objective accept-
ance  criteria  for  genotoxicity  and  carcinogenic-
ity predictions. Based on the requirements of the 
stakeholders,  it  is  likely  that  further  scientific  or 
technical topics will have to be addressed, which 
may include:
•	

The application of QSARs in a regulatory setting is 
still controversial. Therefore, the SCARLET project 
partners  have  identified  the  following  specific 
objectives for the proposed workshop:
•	

identification	 of	 requirements	 for	 QSAR	
models  for  genotoxicity  and  carcino-
genicity  from  the  perspective  of  various 
stakeholders  (scientists,  regulators,  end 
users and industries);
evaluation	 of	 existing	 QSAR	 models	 and	
techniques  in  respect  to  these  require-
ments and to the OECD principles for vali-
dating QSAR models;
identification	of	open	research	issues	and	
harmonisation of efforts to solve them;
preparation	 of	 a	 document	 with	 recom-
mendations  for  the  regulatory  applica-
tion  of  genotoxicity  and  carcinogenicity 
QSARs  containing,  for  example,  available 
databases, proposed protocols, criteria for 
performance  acceptability  and  suitable 
software characteristics.

•	

•	

•	

These  societal  objectives  generate  a  series  of 
scientific objectives. The key issues have already 
been addressed by the ‘OECD Principles for the 
Validation of (Quantitative) Structure-Activity Re-
lationship  Models  for  Regulatory  Purposes’  but 
the guidelines do not provide objective accept-
ance or rejection criteria for QSAR models. 

proposing	 a	 platform	
for	 organising	
knowledge  about  databases,  algorithms 
and  programmes  that  are  suitable  for 
modelling genotoxicity and carcinogenic-
ity endpoints; 
proposing	a	framework	for	the	integration	
of  multiple  QSAR  studies  that  deal  with 
their complexity and redundancy;
evaluating	 the	 role	 of	 QSAR	 tools	 within	
the  framework  of  an  intelligent  testing 
strategy  for  a  decision  support  system 
(e.g.  by  integrating  in vivo,  in vitro  and  in 
silico data with mechanistic knowledge);
identifying	 unresolved	 research	
issues	
within  this  area  and  to  coordinate  re-
search efforts.

•	

•	

•	

These  objectives  will  be  addressed  within  the 
workshop, in the preparatory discussions of the 
advisory  committee,  and  by  the  use  of  a  ques-
tionnaire  for  the  stakeholders.  The  verifiable 
results will be the workshop, the internet site, a 
list of suitable software and databases for geno-
toxicity and carcinogenicity predictions, the final 
document  and  the  published  papers.  This  out-
come  also  represents  the  dissemination  of  the 
results of this project.

The  new  EC  chemicals  legislation  REACH  iden-
tifies  the  need  for  increased  information  on 
the  properties  of  chemicals,  in  order  to  ensure 
the  appropriate  management  of  their  risks  and 
hazards.  To  minimise  animal  testing,  REACH  is 
pursuing  a  smart  and  targeted  testing  strategy 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

79

SCARLET

where  predictions  from  QSAR  models  can  play 
an important role in filling data gaps and provid-
ing information for targeted bioassays. Similarly, 
the  7th  amendment  of  the  Cosmetics  Directive 
76/768/EEC foresees a ban on the testing of cos-
metic ingredients on animals, if a validated alter-
native method is available. 

The  proposed  workshop  will  contribute  to  in-
creased information on industrial chemicals and 
cosmetics, as well as to a reduced number of ani-
mal experiments without compromising on pub-
lic health issues.

Expected outcome:

The expected achievements are: 
an	international	workshop;
•	
a	document	summarising	the	final	results	
•	
from the workshop; 
a	 list	 of	 suitable	 databases	 and	 software	
for the issues addressed; 
a	 series	 of	 papers	 sent	 to	 a	 recognised	
journal. 

•	

•	

Coordinator 

Emilio Benfenati
Istituto di Ricerche Farmacologiche
via Eritrea 62 20157 
Milan, Italy
E-mail: benfenati@marionegri.it

Partners 

Christoph Helma
In Silico Toxicology
Freiburg, Germany

Paolo Mazzatorta
NESTEC
Lausanne, Switzerland

Romualdo Benigni
Istituto Superiore di Sanità
Rome, Italy 

80

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
FP7 PROJECTS

82

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES – FP7
ESNATS

Embryonic stem cell-based novel alternative testing strategies

 Grant Agreement No  HEALTH-F5-2007-201619

Project type 

Integrated Project 

EC contribution  e 11 895 577

Starting date   1 April 2008 (planned)

Duration  60 months

Breakdown and objectives:

ESNATS  aims  to  develop  a  novel  toxicity  test 
platform  based  on  embryonic  stem  cells  (ESC), 
especially human ESC (hESC), to accelerate drug 
development, reduce research and development 
(R&D)  costs  and  propose  a  powerful  alternative 
to animal tests (3Rs). ESNATS will address the fol-
lowing current drug-testing shortcomings:
•		

testing	 takes	 place	 late	 in	 the	 develop-
ment cycle;
animal	test	systems	bear	the	risk	of	non-
prediction due to inter-species variation;
	 non-ESC	 assays	 rely	 on	 primary	 cells	 or	
cells of malignant origin that are hard-to-
standardise and limited in regard to quan-
tity, homogeneity and genetic diversity;
existing	 assay	 systems	 based	 on	 primary	
animal cell lines do not reliably represent 
the physiological situation.

•		

•	

•		

up of hESC culture. The predictivity, quality  and 
reproducibility of ESNATS will be evaluated in a 
proof of concept study. ESNATS aims towards the 
following benefits: increase safety due to better 
predictivity of human test systems; reduce, refine 
and replace animal tests; lower testing cost; and 
support medium-, high-throughput testing.

Expected outcome:

ESNATS objectives will be achieved in a five-year 
multi-disciplinary  collaboration  of  leading  Eu-
ropean  researchers  in  alternative  testing,  toxi-
cology,  ESC  research,  genomics,  modelling,  and 
automation.  The  consortium  will  also  include 
representatives  from  regulatory  bodies,  the 
pharmaceutical industry and ethical advisors to 
provide guidance to ensure rapid applicability of 
the developed test systems.

Approach and methodology:

ESNATS  will  develop  a  battery  of  toxicity  tests 
using hESC lines subjected to different standard-
ised culture protocols. Tests will cover embryoid 
bodies  in  different  developmental  stages  and 
differentiated derivatives, including gamete and 
neuronal lineages, complemented with test sys-
tems for hepatic metabolism. Predictive toxicog-
enomics and proteomics markers will be identi-
fied. The individual tests will be integrated into an 
‘all-in-one’ test system. To enable future industrial 
use, ESNATS will prepare automating and scaling 

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

83

 
 
 
 
ESNATS

Coordinator

Jürgen Hescheler
Klinikum der Universität zu Köln 
62 Kerpener Str.
D-50924  Cologne, Germany
E-mail: j.hescheler@uni-koeln.de

84

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

Partners

Susanne Bremer
Joint Research Centre
Ispra, Italy

Karim Nayernia
University of Newcastle
Newcastle upon Tyne, UK

Karl-Heinz Krause
Université de Genève 
Geneva, Switzerland

Jan Hengstler
Forschungsgesellschaft für 
Arbeitsphysiologie und 
Arbeitschutz e.V. (IfADo)
Dortmund, Germany

Vera Rogiers
European Consensus Platform on 3R 
Alternatives to Animal Experimentation
Brussels, Belgium

Rosemary Drake
The Automation Partnership (Cambridge) Ltd
Royston, UK

Jaak Vilo
OÜ Quretec 
Tartu, Estonia

André Schrattenholz
ProteoSys AG
Mainz, Germany

Luc Grobet
Université de Liège
Liège, Belgium

ALTERNATIVE TESTING STRATEGIES – FP7

Giovanna Lazzari and Cesare Galli
Consorzio per l’Incremento Zootecnico SRL
San Miniato, Italy

Raimund Strehl
Cellartis AB
Gothenburg, Sweden

Benjamin Reubinoff
Cell Cure Neurosciences Ltd
Jerusalem, Israel

Luc Stoppini
Capsant Neurotechnologies Ltd
Romsey, UK

Marcel Leist
Universität Konstanz
Konstanz, Germany

Glyn Stacey
National Biological Standards Board 
Potters Bar, UK

Florence Salmon
TNO
Delft, Netherlands

Paul De Sousa
The University of Edinburgh
Edinburgh, UK

Vera Rogiers
Vrije Universiteit Brussel
Brussels, Belgium

Andreas Nüssler
Technische Universität München
Munich, Germany

Emmanuelle Da Silva
ARTTIC SAS
Paris, France

Andreas Kortenkamp
University of London
The School of Pharmacy
London, UK

Annamaria Rossi
N.V. Organon
Oss, Netherlands

Ulla Wändel Liminga
Läkemedelsverket / 
Medical Products Agency
Uppsala, Sweden

Nina Ostenfeldt
H. Lundbeck A/S
Copenhagen, Denmark

Joan-Albert Vericat
In vitro Testing Industrial Platform
Tres Cantos, Spain

Richard Vogel
Bundesinstitut für Risikobewertung
Berlin, Germany

Donald Bruce
Edinethics Ltd
Edinburgh, UK

Beat Schmid
Swissmedic
(Swiss Agency for Therapeutic Products)
Bern, Switzerland

Ludwig Hothorn
Gottfried Wilhelm Leibniz -
Universität Hannover
Hannover, Germany

Nicole Clemann
F. Hoffmann-La Roche, Ltd
Basel, Switzerland

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

85

NANOTEST

Development  of  methodology  for  alternative  testing  strategies  for  the  assess-
ment of the toxicological profile of nanoparticles used in medical diagnostics

 Grant Agreement No  HEALTH-F5-2007-201335

Project type 

Specific Targeted Research Project 

EC contribution  e 3 933 271

Starting date   1 April 2008 (planned)

Duration  42 months

Website  www.nanotest.org

As NP action is likely to involve oxidative stress, the 
consortium will focus on the cross-cutting areas 
of inflammation, cellular toxicity, immunotoxicity, 
genotoxicity and related endpoints. Following the 
development of standard operating procedures 
and  the  generation  of  a  common  database, 
in  parallel  with  in  silico  assays  (QSAR,  PBPK 
modeling), NANOTEST will evaluate toxic effects 
and interactions of NP used in nanomedicine. 

Expected outcome:

Results  will  be  validated  in  an  experimental 
ethically  approved  in  vivo  model.  The  most 
advanced  and  standardised  techniques  will 
be  adapted  for  automation  and  prepared  for 
validation  by  ECVAM.  Separate  WPs  will  be 
dedicated  to  dissemination  and  to  effective 
administrative and scientific management.

Breakdown and objectives:

Nanoparticles  (NP)  have  unique,  potentially 
beneficial properties, but their possible impact on 
human health has not been adequately assessed. 
The  main  goal  of  NANOTEST  is  to  develop 
alternative  high-throughput  testing  strategies 
using  in  vitro  and  in  silico  methods  to  assess 
the  toxicological  profile  of  NP  used  in  medical 
diagnostics.  The  consortium’s  specific  aims  are 
to: (1) define NP properties and fully characterise 
NP  to  be  used;  (2)  study  NP  interactions  with 
molecules, cells and organs and develop in vitro 
methods  to  study  the  toxicological  potential  of 
NP; (3) validate in vitro findings in short-term in 
vivo models and study particle effects in animals 
and  (ex  vivo)  in  humans  to  assess  individual 
susceptibility  to  NP;  and  (4)  develop  in  silico 
models of NP interactions.

Approach and methodology:

Experimental  work  is  structured  in  four  Work 
Packages  (WPs)  to  address  NP  characterisation 
and key elements in the evaluation of NP uptake, 
exposure  and  toxicology.  NANOTEST  integrates 
the 
investigation  of  toxicological  properties 
and  effects  of  NP  in  several  target  systems  by 
developing  a  battery  of  in  vitro  assays  using 
cell  cultures,  organotypic  cell  culture  and  small 
organ fragments (ex vivo) derived from different 
biological  systems:  blood,  vascular,  liver,  lung, 
placenta, digestive and central nervous systems. 

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Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES – FP7

Partners

Lang Tran
Institute of Occupational Medicine
Edinburgh, UK 

Lucienne Juillerat-Jeanneret
Centre Hospitalier Universitaire Vaudois
Lausanne, Switzerland

Francelyne Marano
University  Paris Diderot
Paris, France

Margaret Saunders
United Bristol Healthcare NHS Trust 
Bristol, UK

Thomas Hartung
Joint Research Centre (ECVAM)
Ispra, Italy

C. Housiadas
National Centre for Scientific Research ‘Demokritos’
Athens, Greece

Katarina Volkovová
Slovak Medical University
Bratislava, Slovakia

Lisbeth Knudsen
University of Copenhagen
Copenhagen, Denmark

José Castell
Advancell
Barcelona, Spain

Antonio Marcomini
Università Ca’ Foscari di Venezia
Venice, Italy

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

87

Cooordinator

Maria Dusinska
Norwegian Institute for Air Research
18 Instituttveien
P.O. Box 100
NO - 2027  Kjeller, Norway
E-mail: mdu@nilu.no

OPENTOX

Promotion,  development,  acceptance  and 
(quantitative structure-activity relationship) for toxicology

implementation  of  QSARs 

 Grant Agreement No  HEALTH-F5-2007-200787

Project type 

Specific Targeted Research Project 

EC contribution  e 2 975 360

Starting date   1 April 2008 (planned)

Duration  36 months

Website  www.opentox.org/projects/

  opentox/wiki/OpenTox

OpenTox  will,  however,  go  beyond  REACH  and 
long-term  effects  because  create  models  for 
other  endpoints  (e.g.  sensitisation,  liver-toxicity, 
cardio-toxicity, ecotoxicity).

Expected outcome:

The  proposed  framework  will  support  the  de-
velopment of new QSAR models and algorithms 
by automating routine tasks, providing a testing 
and  validation  environment,  and  allowing  the 
easy  addition  of  new  data.  For  this  reason,  the 
project  partners  expect  that  OpenTox  will  lead 
to QSAR models for further toxic endpoints and 
generally improve the acceptance and reliability 
of (Q)SAR models.

Breakdown and objectives:

The goal of the OpenTox project is to develop a 
predictive  toxicology  framework  with  a  unified 
access to toxicological data, QSAR. It will provide 
tools  for  the  integration  of  data  from  various 
sources (public and confidential), for the genera-
tion and validation of QSAR models, libraries for 
the  development  and  integration  of  new  QSAR 
algorithms, and validation routines. OpenTox will 
attract toxicological experts without QSAR exper-
tise, as well as model and algorithm developers. It 
will move beyond existing attempts to solve indi-
vidual research issues by providing a flexible and 
user-friendly framework that integrates existing 
solutions and new developments.

OpenTox  will  be  relevant  for  REACH  as  it  gives 
risk assessors simple access to experimental data, 
QSAR models and toxicological information that 
adheres  to  European  and  international  regula-
tory requirements.

OpenTox  will  be  published  as  an  open  source 
project  to  allow  a  critical  evaluation  of  its  algo-
rithms, to promote dissemination, and to attract 
external  developers.  Facilities  for  the  inclusion 
of  confidential  in-house  data  and  for  accessing 
commercial prediction systems will be included.

OpenTox will contain high-quality data and QSAR 
models for chronic, genotoxic and carcinogenic 
effects. These are the endpoints with the greatest 
potential to reduce animal testing. The impact of 

88

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

 
 
 
 
 
 
ALTERNATIVE TESTING STRATEGIES – FP7

Partners

Christoph Helma
In Silico Toxicology
Freiburg, Germany

Nina Jeliazkova
Ideaconsult Ltd
Sofia, Bulgaria

Romualdo Benigni
Instituto Superiore di Sanita’ 
Rome, Italy

Stefan Kramer
Technical University of Munich
Munich, Germany

Andreas Karwath
Albert Ludwigs Universität Freiburg 
Freiburg, Germany

Haralambos Sarimveis
National Technical University of Athens
Athens, Greece

David Gallagher
Congresbury, Bristol, UK

Vladimir Poroikov
Institute of Biomedical Chemistry of Russian Academy of 
Medical Sciences
Moscow, Russia

Indira Ghosh
Bioinformatics Center, University of Pune
Pune, India

I. Mangelsdorf
Fraunhofer Gesellschaft
Institut Toxikologie und Experimentelle Medizin
Hannover, Germany

Coordinator

Barry Hardy 
Douglas Connect 
14 Bärmeggenweg
CH - 4314 Zeiningen, Switzerland
E-mail: barry.hardy@douglasconnect.com

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

89

PREDICT-IV

Profiling the toxicity of new drugs: a non animal-based approach integrating 
toxico-dynamics and biokinetics

 Grant Agreement No  HEALTH-F5-2007-202222

Project type 

Integrated Project 

EC contribution  e 11 330 907

Starting date   1 April 2008 (planned)

Duration  60 months

Breakdown and objectives:

The overall aim of PREDICT-IV is to develop strat-
egies to improve the assessment of drug safety 
in  the  early  stage  of  development  and  late  dis-
covery  phase  by  an  intelligent  combination  of 
non  animal-based  test  systems,  cell  biology, 
mechanistic  toxicology  and  in-silico  modelling 
– in a rapid and cost effective manner. A better 
prediction  of  the  safety  of  an  investigational 
compound  in  early  development  will  be  deliv-
ered. Margins-of-safety will be deduced and the 
data generated by the proposed approach may 
also  identify  early  biomarkers  of  human  toxic-
ity  for  pharmaceuticals. The  results  obtained  in 
PREDICT-IV  will  enable  pharmaceutical  compa-
nies to create a tailored testing strategy for early 
drug safety.

The project will integrate new developments to 
improve and optimise cell culture models for tox-
icity testing and to characterise the dynamics and 
kinetics of cellular responses to toxic effects in vit-
ro. The target organs most frequently affected by 
drug toxicity will be taken into account, namely 
liver and kidney. Moreover, predictive models for 
neurotoxicity  are  scarce  and  will  be  developed. 
For each target organ the most appropriate cell 
model will be used. The approach will be evalu-
ated using a panel of drugs with well-described 
toxicities and kinetics in animals and partly also 
in humans. 

90

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

This  approach  will  be  highly  advantageous,  as 
it will allow a direct comparison between the in 
vivo and in vitro data. A parallel analysis of several 
dynamic and kinetic models with a broad spec-
trum of endpoints should allow for the identifi-
cation of several relevant biomarkers of toxicity. 
Inter-individual susceptibilities will be taken into 
account  by  integrating  the  polymorphisms  of 
the  major  drug  metabolising  enzymes  and  cor-
relating  the  observed  effects  in  the  human  cell 
models  with  their  genotype.  Environmental  in-
fluences on cellular toxicity to these compounds 
will  also  be  evaluated  using  hypoxic  stress  as  a 
relevant test model.

Coordinator

Wolfgang Dekant
Julius-Maximilians-Universität Würzburg 
2 Sanderring
D-97070 Wuerzburg, Germany
E-mail: dekant@toxi.uni-wuerzburg.de

 
 
 
 
ALTERNATIVE TESTING STRATEGIES – FP7

Partners

Stefan Mueller
Merck KGaA
Institute of Toxicology
Darmstadt, Germany

Walter Pfaller and Paul Jennings
Innsbruck Medical University
Department of Physiology and Medical Physics
Innsbruck, Austria

Emanuela Testai
Istituto Superiore di Sanità
Environment and Primary Prevention Department
Mechanisms of Toxicity Unit
Rome, Italy

Frédéric Bois
Institut National de L’Environnement 
Industriel et des Risques 
Experimental Toxicology Laboratory
Verneuil-En-Halatte, France

Laura Gribaldo
Institute for Health and Consumer Protection
ECVAM, Joint Research Centre
Ispra, Italy

Bernd Mayer
Emergentec Biodevelopment GmbH
Vienna, Austria

Armin Wolf
Novartis
Basel, Switzerland

Lysiane Richert
KaLyCell
Besançon, France

André Guillouzo
University of Rennes I
INSERM U620
Rennes, France

Martin Leonard
University College Dublin, Conway Institute
Dublin, Ireland

Bas Blaauboer
University of Utrecht
Institute for Risk Assessment
Utrecht, Netherlands

Amin Rostami
SimCyp Ltd
Sheffield, UK

Knut Reinert
Freie Universität Berlin
Department of Mathematics and Computer Science
Berlin, Germany

Christian Huber
Universität des Saarlandes
Saarbrücken, Germany

Paul Honegger
University of Lausanne
Department of Physiology
Lausanne, Switzerland

Romeo Cecchelli
Université d’Artois
Blood Brain Barrier Laboratory
Lens, , France

Annette Kopp-Schneider
Deutsches Krebsforschungszentrum
Heidelberg, Germany

Dieter Weiss
Universität Rostock
Department of Animal Physiology, 
Rostock, Germany

Olaf Schröder
NeuroProof GmbH
Rostock, Germany

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

91

START-UP

Scientific and technological issues in 3Rs alternatives research in the process of 
drug development and Union politics

 Grant Agreement No  HEALTH-F5-2007-201187

Project type 

Specific Support Action 

EC contribution  e 317 964

Starting date   1 March 2008
Duration  24 months

Breakdown and objectives:

Approach and methodology:

The START-UP project aims to identify, and make 
proposals  to  eliminate,  bottlenecks  in  the  3Rs 
approach  in  pharmaceutical  discovery  and  de-
velopment. To this end, the project will organise 
three workshops to determine the state of the art 
of the 3Rs in the EU, assess strengths and gaps in 
the 3Rs in Europe, and identify rate limiting steps 
on  a  scientific  and  technological  level;  this  will 
lead  to  a  consensus  paper  containing  concepts 
and  suggestions  for  a  road-map  for  future  re-
search.

Stakeholders  (among  them  European  pharma-
ceutical  industries)  have  identified  bottlenecks 
in  drug  development  and  in  the  integration  of 
in  vitro  methods.  Early  identification  of  wrong 
candidates  for  further  development  and  avoid-
ing efforts for under-performing candidates are 
essential  for  the  competitiveness  of  European 
industry. Identification of bottlenecks in the im-
plementation  of  the  3Rs  in  drug  research  and 
development  should  help  identify  the  best  in 
vitro and in vivo systems, as well as speed up the 
drug  development  process.  Existing  hurdles  at 
the  scientific,  technological,  ethical,  regulatory 
and political levels play a substantial role and are 
rate-limiting in developing new drugs, including 
biological entities. 

The  project  is  structured  around  three  work-
shops,  preceded  by  two  expert  meetings  rede-
fining and prioritising current bottlenecks in the 
3Rs  methodology,  drug  discovery  and  develop-
ment. The limitations and gaps of each phase will 
be addressed, e.g. many cell systems do not have 
the  required  stability  for  genomics,  proteomics 
or metabonomics and many current cell systems 
lack  crucial  bioactivation  capability.  Thus,  the 
status  of  satisfactory  ‘predictive’  pharmacology 
and toxicology in vitro has not been reached yet. 
The final goal is a consensus document in which 
a road map for implementing the strategy for a 
better integration of 3Rs in the EU drug discovery 
and development strategy will be proposed.

Coordinator

Vera Maria Rogiers
Vrije Universiteit Brussel
103 Larbeeklaan
1090 Brussels, Belgium
E-mail: vrogiers@vub.ac.be 

Partner

Tamara Vanhaecke
Vrije Universiteit Brussel
Brussels, Belgium

92

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ALTERNATIVE TESTING STRATEGIES – FP7

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

93

INDEX OF PROJECTS

ALTERNATIVE TESTING STRATEGIES

ACuteTox 
ARTEMIS  
carcinoGENOMICS  
COMICS  
EXERA  
Invitroheart  
LIINTOP  
MEMTRANS  
PREDICTOMICS  
ReProTect  
Sens-it-iv  
TOXDROP  
VITROCELLOMICS  
BBMO  
CONAM  
ForInViTox  
InViToPharma  
NHR DevTox  
RAINBOW  
RETHINK  
SCARLET  

FP7 Projects

ESNATS  
NANOTEST  
OPENTOX  
PREDICT-IV  
START-UP  

11
17
19
22
25
29
33
37
39
44
49
53
58
61
64
67
69
71
73
76
78

83
86
88
90
92

94

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

ALTERNATIVE TESTING STRATEGIES

INDEX OF
COORDINATORS

 75
80
38
 84
43
 68
24
90
28
87
77
89
21
18
63

Benfenati, Emilio (RAINBOW) 
Benfenati, Emilio (SCARLET)  
Bermejo, Marival (MEMTRANS)  
Bremer, Susanne (ESNATS) 
Castell, José V. (PREDICTOMICS)  
Clemedson, Cecilia (ForInViTox) 
Collins, Andrew (COMICS)  
Dekant, Wolfgang (PREDICT-IV)  
Di Lorenzo, Diego (EXERA)  
Dusinska, Maria (NANOTEST)  
Forster, Roy (RETHINK)  
Hardy, Barry (OPENTOX)  
Kleinjans, Jos (carcinoGENOMICS)  
Lenas, Petros (ARTEMIS)  
Lindholm-Sethson, Britta (BBMO)  
Mandenius, Carl-Fredrik
(Invitroheart, scientific coordinator)  
32 
Mandenius, Carl-Fredrik (VITROCELLOMICS)   60
72
Penman, Mike (NHR DevTox)  
14
Risteli, Leila (ACuteTox)  
 51 
Roggen, Erwin L. (Sens-it-iv) 
 66
Rogiers, Vera (CONAM) 
 92
Rogiers, Vera Maria (START-UP) 
57
Schaack, Béatrice (TOXDROP)  
47
Schwarz, Michael (ReProTect)  
70
Toft, Erica (InViToPharma)  
36
Zucco, Flavia (LIINTOP)  

Alternative Testing Strategies: Replacing, reducing and refining use of animals in research

95

Directorate F – Health

Unit F5 – Health Biotechnology

Head of Unit  Arnd HOEVELER (arnd.hoeveler@ec.europa.eu)

  Secretary: Andrea JAEGER (andrea.jaeger@ec.europa.eu)

 Alternative Testing Strategies 

Juergen Buesing (juergen.buesing@ec.europa.eu)

  Secretary: Isabel CANUETO MORO (isabel.canueto-moro@ec.europa.eu)

Communications  Charles Kessler (charles.kessler@ec.europa.eu) 

  Secretary: Caroline RICHMOND (caroline.richmond@ec.europa.eu)

 
 
 
 
                       
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Alternative Testing Strategies
Replace  the  need  for  animal  experiments,  reduce  the  number  of  animals  required  and 
refine  techniques  are  the  three  concepts  underlying  the  development  of  alternative 
testing  strategies  used  in  pharmaceutical  discovery  and  development,  and  in  safety 
assessment of chemicals. Research into alternative testing strategies formed part of the 
EU’s  Sixth  Framework  Programme  (2002-2006)  and  the  purpose  of  this  catalogue  is  to 
demonstrate the activities initiated over the duration of the programme and the initial 
results generated. A particular focus of the work is the development of in vitro methods 
and on methods which can be validated to international standards, achieve approval of 
regulatory authorities and be adopted by industry.