Alternatives to animal experimentation

Dr Maaike van Zijverden Netherlands Knowledge Centre on Alternatives to animal use (Nationaal KennisCentrum Alternatieven voor dierproeven) www.NKCA.nl 1

Content

-Biomedical research -Animal use -3R: legislation and motives -Replacement -Reduction -Refinement -Validation, barriers and drivers, info

2

3

Case: skin allergy

Source: van der Jagt et al 2004

Case: skin allergy

Research model ● Study a research hypothesis in a standardized environment under standardized conditions, generally by using surrogate models: analytical, in silico, in vitro, ex vivo and in vivo models ● NB: differs from the target organism ● Animal model: historical use, widespread

6

History The rise of the animal model in the 19th century: ● Availability of anesthetics ● Understanding of genetics (inbreeding) ● Breeding colonies of rats and mice ● Understanding of microbiology and disease processes

7

Characteristics of the ideal research model ● ● ● ● ● ●

8

Relevant Reproducible, robust Mechanistically based Inexpensive, not laborious or time consuming Ethically acceptable …

Strenghts and weaknesses animal models

9

Strenghts and weaknesses of animal models

10

Motives to develop alternatives ● Ethics ● Public Opinion ● Economics ● Better Science ● Regulation/legislation

11

Laboratory animal use I: numbers&trends NL: total number of laboratory animal experiments (2011): 589.853 (581.776 animals, mostly mouse, rat, chicken and fish) Same number killed in stock (75% genetically modified). Number reduced to 37% of number in 1978 (start registration): a.o.more info per animal, but recently also higher demands for safety EU total number (2008) 12 million Worldwide use: 100-200 million/year

Laboratory animal use I: numbers&trends Trends: Possible causes of decline: • Better experimental set up and statistics • Critical evaluation of existing animal models • Shifts in research strategy and paradigm Possible causes of increase: • New safety legislation • New safety issues and other new challenges • Lifestyle-related diseases • Risk acceptance

Laboratory animal use II: Purposes (NL, 2011)

56%: scientific research

14

33%: development, control, production or biological standardisation of vaccines, medicines or veterinary products

Laboratory animal use II: purposes (NL, 2011)

6%: safety of chemicals 15

Laboratory animal use II: purposes (NL, 2011) 2%: diagnostics

3%: education and training ● Bron: Zo doende 2011, NVWA 16

Laboratory animal use III: regulatory purposes (NL, 2011) Laboratory animal use for regulatory purposes: 22%

Toxicology: approx 90% is required by regulations, i.e. routine testing. Introduction of alternatives will have great effect on animal use. 17

The 3 R’s: alternative? ● Russell and Burch (1959) ● Replacement ● Reduction ● Refinement ● Alternative: implicates a choice between (two) possibilities

18

Legislation and regulation Declaration of Helsinki, ethical principles for medical research involving human subjects, from 1964 onward

Medical research involving human subjects must conform to generally accepted scientific principles, be based on a thorough knowledge of the scientific literature, other relevant sources of information, and adequate laboratory and where appropriate animal experimentation.

19

Legislation and regulation ● Wet op Dierproeven (WoD, Dutch law on experimental animal use) section 10 quotes: No animal experiment shall be conducted for a purpose which may also be achieved: ● By means other than an animal experiment, or ● By means of an experiment using fewer animals, or ● Entailing less distress than the experiment in question. ● 3R = Red line in the new EU regulation EU/2010/63 ● Part of European Science Foundation, and adopted by regulatory bodies such as OECD, European Pharmacopoeia, WHO, etc 20

Principle

● No, unless

21

3R in practice The researcher: mindset, IQ, check existing literature Data sharing: ● Scientific literature of high quality (e.g. Animal Research Reporting In Vivo Experiments ARRIVE guidelines, NC3R 2010/Gold Standard Publication Checklist Hooijmans et al 2011) ● Publish “meaningful negative data” ● Appreciate meta-analyses and validations ● Central databases

Replacement: overview ● Physico-chemical and immunochemical methods (I) ● In vitro (tissue cultures) (II) ● Technological innovation: in silico (modelling and audiovisual), omics, TIM (III) ● Invertebrate organisms ● Human material (IV)

23

Replacement I: Physico-chemical methods •Pyrogenicity test: from rabbit to horseshoe crab to monocyte

Replacement II: in vitro/tissue cultures ● ● ● ● ● ● ● ● ●

Cell organelles Primary cell cultures Cell lines Organs Slices Slaughterhouse material Human material Tissue engineering Stemcells

25

Replacement II: pros and cons “classic” in vitro - no ADME (NB bio-kinetics and transformation: modelling)* - extrapolation* - cell culture not 100% animal free, FCS/BSE needed

* Except for technological innovative methods e.g. human-on-a-chip

Wyss Institute, Harvard University, USA

Replacement II: pros and cons in vitro ● Primary cells: + cell differentiation, also human material, … - limited life span (de-differentiation and cell death), source needed, … ● Cell lines: + immortal, robust, standardisation - no differentiation, aspecific characteristics ● Organ culture/slices: + complexity, intercellular communication, various cell types - limited life span, complexity, no systemic interactions

Replacement II: Slaughterhouse

Replacement II: human material ● From donor: for transplantation research only ● Surgical waste: NB possible pathology ● From patient/volunteer: for research ● Post mortem Pros: no animals, no interspecies extrapolation

Replacement II: tissue engineering •Growing of different cell types on a structure (scaffold) to form a complex structure •NB the right stimuli

Replacement II: Stem cells

Replacement III: technological innovation •TIM (TNO) •Omics

Pathways of Toxicology and Adverse outcome (disease)

Chemical

Molecular initiating event

Cellular response

Organ response

Toxicity pathway

Mode of action pathway

Adverse Outcome pathway

Organism response

Replacement III: technological innovation Audiovisual

Replacement IV: human volunteers

non-invasive techniques, micro-dosing: 1/100 of the pharmacologically active dose, or 100 microgram

Case: skin allergy •1) bioavailability

•2) (reactive) metabolite •3) reaction with proteins •4) formation complete antigen, at 2nd contact: spec memory Tcell response •5) inflammation •6) A) immune recognition B) responses •7) dendritic cell migration •8) T cell reaction = key event

Source: AOP skin sensitization part 1 dec 2011, OECD

Case: skin allergy •2nd or further contact: memory T cell response •Reactions in skin and local lymph nodes •After sensitization follows elicitation •Visible effect: inflammation •Disease: allergic contact dermatitis

Source: AOP skin sensitization part 1 dec 2011, OECD

Reduction I ● ● ● ● ●

38

See replacement: technological innovation such as -omics Experimental set up (standardisation, statistics) Choice animal model (literature/systematic review) Ethical verification Cryopreservation (cancer research: tumor material)

Reduction II ●Longitudinal measurements (e.g. physico-chem, immunochem, imaging, wires) ●Cooperation ●Education/training

Reduction III Integrated testing strategies: ● Gather all experimental data (vitro, vivo, human, modelling), share data, search literature ● Determine underlying mechanism/pathway * ● Determine exposure * ● Perform non-animal testing ● Perform (reduced) animal testing ● Probabilistic* instead of deterministic risk assessment * ITS vs tiered testing

Source: OSIRIS

Reduction III

KP-6 project

Reduction in practice • • • • •

Pay attention to statistics, perform a pilot if needed Optimise standardisation of the experiment Use real time monitoring Re-use animals Use quantitative instead of qualitative data

Standardisation: example i.p. injection Animal technician Pigment localisation A

B

C

Injectional canal

6

12

7

Subcutis

17

9

18

peritoneal cavity

10

23

14

Stomach

9

1

Small intestine

6

1

Caecum

11

2

Colon

9

1

Case skin allergy: from guinea pig to mouse

20-30

8-20

Tiered strategy, starting with 8 mice (reduced mouse test)

Refinement Reduce suffering and enhance welbeing ● ● ● ● ●

Animal care Skilled personell Analgesia, anaesthesia, euthanesia Non-invasive procedures Animal behaviour

Info a.o: www.humane-endpoints.info

46

Case skin allergy: from guinea pig to mouse

Read out: from late (actual disease) to early (read out of onset of immune response in Local Lymph Node Assay LLNA, in mice)

Roadmap for 3R in toxicology (CAAT 2012) and biomedical research In vivo database, share data Mutual acceptance of data ITS, flexible Pathways of Toxicity In vitro methods In silico methods Biokinetics Optimization existing tests: better science Abolition useless tests

Carcinogenicity in vivo 18-24 months 600 animals 53% of tested substances = positive In human approx 5-20% Known human carcinogens test negative Reproducibility low: 57% Interspecies correlation low (mouse to rat, to human) Alternative: fixed ITS Accept negative repeated dose tox data Should be: flexible, adaptive, including non-genotoxic mechanisms

Consistency approach vaccines •

Current paradigm: every batch is unique

•

New paradigm: consistency approach for batch release testing = monitor every production step with phys/chem, immuno/chem methods, apply GLP/GMP

•

Scientific, economic and animal welfare benefit (Coenraad Hendriksen et al)

The chain from research to implementation Review current method Research

Is there need for new method Investigate mechanisms, biomarkers

Development

Incorporate biomarkers into test method

Prevalidation

Optimize transferable SOP

Validation

Determine relevance and reliability

Peer review

Independent scientific evaluation

Acceptance

Determine acceptability for regulatory assessment

Implementation

Effective use by regulators and users

Case skin allergy: barriers? + mechanistic knowledge (pathways), different alternatives for the different steps available, ITS possible…. - Complex endpoint (no stand alone, validation ITS), lack of experience, test infrastructure, diverse regulations, trust, risk aversion, …

Barriers and drivers in the actual use (implementation) of 3R methods + variability animal model, extrapolation gap, costs animal testing, education, better science, harmonisation, concern animal welfare

(a.o.Schiffelers et al, ALTEX 24, 2007)

- validation process, lack of experience, lack of understanding of biol. mechanism, costs alternative test (battery), no stand alone (complex endpoints), gold standard, information asymmetry, diverse regulations, risk aversion, culture of litigation

ITS skin sensitization scientifically validated Mode of action Test 1: Protein reactivity

Test 2: Keratinocyte activation

Both negative: non sensitizer

Test 3: Dendritic cell activation

positive: sensitizer

Contradicting? Majority: 2 out of 3 determine classification

C Bauch et al 2012

Validation is the scientific process determining reliability and relevance of a new procedure for a specific goal

Validation: modular approach P r e v a l .

V a l i d a t i o n

Test definition Within-lab. variability Transferability

Reproducibility

Between-lab.variability Predictive capacity Relevance Applicability domain Minimum performance standards

Data interpretation model Prediction model

Disadvantages formal validation - Takes (a lot of) time - Takes (a lot of) money - Validation = comparison with “golden standard” (in vivo test) - In regulatory testing: does the test answer the right question? NB retrospective validation might be sufficient! NB scientific validity should prevail!

3R and NL government Cabinet view 2008 Establishment NKCA 2010 Trend scientific and societal analyses on use of animals and 3Rs 2010 Action plan 2011 ZonMw funds “More knowledge with less animals” programme

NKCA activities Mission: Stimulate information exchange and application of 3R ⋅ ⋅ ⋅ ⋅ ⋅

Sharing knowledge with and between professionals Communication, a.o www.nkca.nl and newsletter Policy advise Education Focusing Dutch international contributions

The chain from research to implementation

websites NKCA NC3Rs (UK) Altweb ECVAM DBALM Go3Rs -

www.nkca.nl www.nc3rs.org.uk/ altweb.jhsph.edu/ (portal) ecvam.jrc.it/index.htm ecvam-dbalm.jrc.ec.europa.eu/ www.Go3R.org (3R search engine)

Contact information www.nkca.nl [email protected] [email protected]

3R, better science, ...