Knudsen - Lush Prize Conference 2014

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Is One R the new Three Rs?
Lisbeth E. Knudsen
Professor, PhD, University of Copenhagen, Department of Public Health
liek@sund.ku.dk
Line Mathiesen
Thit Aarøe Mørck
Pernille Winton Hansen
Maria Helena Guerra Andersen
Sara Højriis
FACULTY OF HEALTH AND MEDICINE SCIENCES
UNIVERSITY OF COPENHAGEN
Lisbeth E. Knudsen LUSH Nov 2014

REACH in a nutshell
picture
© ChemSec

EU Chemical legislation: REACH
(Registration, Evaluation, Authorisation of Chemicals)
Investment into safety
30,000 chemicals > 1 t per year to be assessed
billions of Euro, millions of animals,
decades of testing
Major impact of in silico and in
vitro tests

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Categories of medicines
concerned by animal testing for
Quality Control purposes
Vaccines for human use and for veterinary use
Blood products
Biological and biotechnological products
Antibiotics
Radiopharmaceuticals

The TGN1412 (CD28-
superMAB) disaster
• Monoclonal CD28 agonistic antibody (T cell co-activator) developed as a
drug for leukemia and rheumatoid arthritis
• First-in-man study in London 2006 led to catastrophic systemic multi-organ
failure in six young male volunteers
• Animal studies (macaques, mice and rats) showed no adverse effects at
500x the dose used in the human phase I study
News.bbc.co.uk
Antigen-presenting
cell
CD28
Modified from: Alegre et al. (2001), Nat rev immunol

The TGN1412 (CD28-superMAB) disaster
• Animal >< humans?
• Immunologically naïve animals (raised in sterile conditions)
with few memory T cells
• Species differences (no CD28 receptors on memory cells)
• In healthy young men: massive memory cell activation 
cytokine storm
• Could have been detected by in vitro assays
Stebbings et al. (2007), J immunol. Modified from Eastwood et al. (2010), Br J Pharmacol

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Directive 2010/63/EU on the protection of animals
used for scientific purposes requires, as a legal
obligation, to apply the Three Rs in all aspects of the
care and use of animals. Furthermore, a number of
roles under the Directive have a specific responsibility
in ensuring information on the Three Rs are made
available for those who need it. Equally, several
categories of personnel as well as Animal Welfare
Bodies or National Committees, should take the
opportunities to capture and disseminate new Three
Rs innovations for the benefit of the wider user
community, science and animals.

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PROBLEMs in getting engagement in replacement
TRADITIONS favouring animal studies
Established procedures (SOPS) in the Laboratory,
previous publications, age of professor
PREJUDICES/SCEPTICISM towards introducing
new methods Will not work, no comparative
measures, not scientific
LACK OF RELEVANCE which can be stated as
In our area we have always used animal models

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PROBLEMs in getting engagement in replacement
LACK OF EXAMPLES
The 3R area is still developing and need more
substantiation
Validation is ongoing
Human relevance
Concerted research programs in H2020
LACK OF TOOLS
Databases under development/diverse
No full overview

WHAT ?
European Consensus Platform for Alternatives
•International Not-For-Profit
Organisation in Belgium (Europe)
•Members : National Consensus
Platforms
on Alternatives with 4 parties :
Government
Animal Welfare
Academia
Industry

The purposes of ecopa are to facilitate
the exchange of scientific
information, expertise and
experience between NCPs, industry,
science animal welfare and EU and
government institutions to enhance
the further development and
implementation of refinement,
reduction and replacement (3R-concept)
in animal experimentation
in Europe and worldwide.

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Previously ECOPA participated in EU-research
projects with the role of dissemination and public
awareness. The new board of ECOPA wanted to
stress the importance of local activities within the
3Rs – major emphasis on replacement which was
the original remit of ECOPA.
However with the new directive also covering
research much emphasis has been devoted to
3Rs and ECOPA is half heatedly taking the
refinement and reduction on board as the
Replacement R (1R) is our preference.

The Danish 3R-Center

Background
Political decision to establish 3R-Center
in Denmark, June ‘13
Unique, powerful and important
collaboration
- government
- pharmaceutical industry
- animal welfare organizations
Board elected
- initial board meeting November ‘13

Laboratory animals in
Denmark

Board
- Christine Nellemann
- Jan Lund Ottesen
- Axel Kornerup Hansen
- Lisbeth E. Knudsen
- Erwin L. Roggen
- Peter Bollen
- Adrian Smith
- Peter Bie (observer) Secretariat
- Tom Bengtsen
- Anne Sofie Grove
- Rasmus Normann Nielsen
- Mia Vorslund-Kiaer
Organization
Board

Collaborators
Nationally
- Animal welfare organisations
- Animal welfare bodies
- Pharmaceutical industry
- Universities
- Danish Ministry of the Environment /PARERE
- The Danish Health and Medicines Authority / EMA
Internationally
- 3R Research Foundation (Switzerland)
- NC3R (U.K.)
- ZEBET (Germany)
- EURL-ECVAM (EU)
- CCAC (Canada),
- CAAT (USA)

Mission
- Initiate activities that may lead to the
immediate implementation of the 3Rs
- Provide a forum for collaboration,
discussion, exchange and dissemination of
information on the 3Rs
- Initiate research projects and recommend
funds allocation of resources within the
area

Research
Goals
-Work to decrease the use of and the collective strain on
laboratory animals
- Initiate and support research, development and validation
within 3R
- Develop and promote alternatives to animal experimentation
- Initiate/promote research alliances and opportunities for
further education
Initiatives
- Distribute research funding
- Grant applications in 2014: 18 (4 projects supported)
- Publish information regarding and results from the supported
projects

Supported projects 2014
i. Pathological and immunological
consequences of basic experimental
animal procedures in mice
ii. Reducing the group size in studies of
dermatitis by standardization of the gut
microbiota
iii. Refinement of animal models of pain:
Establishment of strategies to alleviate
avoidable pain in rat models for pain and
inflammation
iv. Establishment of an In vitro model for
diabetic atherosclerosis

International collaboration
Goals
- Keep focus on international alliances
- Collaborate with other 3R Centers internationally
Initiatives
- Participate in international meetings, councils, seminars and networks concerning 3R and
Best Practice
- Organise meetings with other 3R Centers worldwide
- NC3R, ZEBET, 3R Research Foundation, EURL-ECVAM
- Host an international conference
- Maintain good international relations
- Participate in” World Congress on Alternatives and Animal Use in the Life Sciences”
- Oral and poster presentations
- Connect with national parties involved in international 3R collaborations
- PARERE national contact person
- The Danish Health and Medicines Authority contact involved in the 3R work in EMA

My Research

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Adverse effects from fetal exposures
Premature birth
Low birth weight
Fetal programming
cardiovascular diseases
Reproductive damage
Neurodev
Diabetes
etc....
Drugs
Alcohol
Diabetes
Nutrition
Air pollution
Medicine
Prenatal exposure

Biomarkers in environmental/food health research
Environmental/food
concentration
(external dose)
dose-response
relationship
toxic effects
internal
dose
biologically
significant
dose
alterations
in structure/
function
early
biological
effects
biomarkers of
exposure biomarkers of risk
biomarkers of individual susceptibility (genetic or acquired)
Biomarkers of exposure (internal dose or biologically significant dose)
Biomarkers of risk (early biological effects or alterations in structure and
function of cellular macromolecules)

Biomonitering
• Blood
• Umbilical cord blood
• Urine
• Hair
• Placenta tissue
• Breast milk
• House dust
• Indoor air
• Questionnaires

Human exposure
Chemical exposure
Human biomonitoring:
Measurement for total
exposure
Biomarker of exposure
- Internal dose
Biomarker of effect
- Early response
Effects of exposure:
Traditional methods
- Animal studies
- Cell toxicology assays

Thit Aarøe Mørck
Human biomonitoring
• EU project DEMOCOPHES (DEMOnstration of a study to
COordinate and Perform Human biomonitoring on a
European Scale)
• 145 mother-child pairs in Denmark
• Two areas (urban and rural)
• Sample collection (urine, hair and blood)
• Questionnaire (mothers)
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64 biomarkers of exposure and effects
URINE BLOOD
Parabens PCBs
Phthalates
PFASs
PBDEs
Phenols
Paracetamol
Mercury
Cotinine
Cadmium
Dioxin-like activity
Micronuclei frequency
HAIR
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Ex vivo placenta transport
Line Mathiesen
95% N2 +
5% CO2
95% O2 +
5% CO2
Fetal Maternal
Lisbeth E.
3
4
Knudsen LUSH

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BeWo cells
Human choriocarcinoma cell line
Trophoblastic cell functions
Monolayer with tight junctions (b30)
Transport pathways

From mother to child
Frederiksen et al., Int. J. Hyg. Environ. Health, (in press); Frederiksen & Vorkamp et al., Environ. Health.
BDE-28
0 100 200 300 400
300
200
100
0
r = 0.86
p <0.001
Maternelt plasma
(pg/g lw)
Føtalt plasma
(pg/g lw)
Plasma
FM-ratio
Ex vivo
FM-ratio
BDE-28 0.45 -
BDE-47 0.30 0.47
BDE-99 - 0.25
BB-153 0.18 -
BDE-153 0.15 -

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I have come to the conclusion the 3 Rs together are
today not compatible with different scientific disciplines,
not normally working together forced to collaborate.
This is my experience from the discussions in the
new Danish 3R centre – strongly associated with the
Animal Experiments Inspectorate (abbreviated AEI).
I do of course give the 3R work a try in the 3R Centre
and emphasize the task of educating and encouraging
young researchers in seeking information about animal
free research and applying this. For animal free to
become the 1R.

Acknowledgements
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Democophes/Cophes
All European countries
Marike Kollosa-Gehring
Greet Schoeters
Argelia Castano
Reinhard and Anke Joas
http://www.eu-hbm.info/
NewGeneris
Jos Kleinjans et al
Mischeline Kirch-Volders et al
Kirsi Vähänkangas et al
Päivi Myllynen et al
Harrie Besselink et al
Margareta Törnquist et al
Andrew Collins et al
Manolis Kogevinas et al
http://www.newgeneris.org/
NanoTEST
Maria Dusinska et al
Margaret Saunders et al
University of Copenhagen
Åse Marie Hansen
Zorana Jovanovic Andersen
Steffen Loft
University Hospital Copenhagen
Morten Hedegård, Morten Dziengiel,
Leif Kofoed, Lisa Maroun, Lise Grupe
Southern Denmark University
Jesper Bo Nielsen, Philippe Grandjean,
Tina Kold Jensen
Aarhus University/NERI
Eva Bonefeld Jørgensen, Herman Autrup,
Katrin Vorkamp
DTU-Food:
Christine Nellemann
Anne Marie Vinggaard

Knudsen - Lush Prize Conference 2014

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