3R Alternatives

1
3R Alternatives
Scientists' Competence Course 2009 - Category C -
University of Kuopio

• Matti Viluksela
• National Institute for Health and Welfare (THL)
• (formerly KTL)
• Department of Environmental Health
• P.O. Box 95, FI-70701 Kuopio

2
The 3 Rs

• William M.S. Russell Rex Burch
• The Principles of Humane Experimental Technique
• 1959
• http//altweb.jhsph.edu/publications/humane_exp/he
  t-toc.htm
• Reduction
• Refinement
• Replacement

3
What are the alternatives?

• Reduction - fewer animals
• Refinement - less painful procedures
• Replacement - alternative techniques

4
The 3 Rs

• Reduction any decrease in the numbers of
  animals used to obtain information of a
  given amount and precision
• Refinement any decrease in the incidence or
  severity of procedures applied to animals
  necessarily used
• Replacement the substitution of conscious
  living vertebrates by non-sentient material

5
Legislation

• EU Directive 86/609/EEC
• An experiment shall not be performed on an
  animal, if another scientifically satisfactory
  method of obtaining the result sought, is
  reasonably and practicably available.
• The European Commission and the EU Member States
  must actively encourage and support the
  development, validation and acceptance of methods
  which could reduce, refine or replace the use of
  laboratory animals.

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The 4th R
The 4th R

• Responsibility

7
Reduction alternatives

• Good planning of studies
• Rational and efficient use of animals
• no wasting
• pilot studies
• screening tests
• Proper statistical design
• Use of inbred strains (for some study types)

8
Refinement alternatives

• Minimized potential for pain or distress
• Enhanced animal well-being
• Improved housing conditions and experimental
  techniques

9
Replacement alternatives (1)

• Efficient use of existing information
• In silico methods (computer simulations,
  mathematical models, QSAR)
• In vitro methods isolated organs
• tissue slices
• tissue cultures
• cell cultures
• subcellular fractions
• Lower organisms
• Early stages of development

10
Replacement alternatives (2)

• In vitro methods are not primarily
  replacements of in vivo methods. Typically
  these methods have different roles in research,
  and they are complementary for each others.
• Depending on the objective of the study in vitro
  methods may be the most appropriate methods for
  certain areas of interest, because they can more
  accurately provide the information required (e.g.
  cellular and molecular events).

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Replacement alternatives (3)

• Absolute replacement no need to use animals
  (cell lines, human or invertebrate cells and
  tissues)
• no need to test for skin irritation if pH lt2.0 or
  gt11.5
• no need to test for eye irritation if the
  chemical is a skin irritant
• Relative replacement humane killing of animals
  to provide cells or tissues for in vitro studies
• Partial replacement e.g. use of non-animal
  methods as prescreens in toxicity testing

12
Replacement alternatives (4)

• Direct replacement e.g. human or guinea pig skin
  is used in vitro to replace guinea pig tests in
  vivo
• Indirect replacement e.g. Limulus amoebocyte
  lysate (LAL) test or a test based on whole human
  blood is used to replace rabbit pyrogen tests

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Limulus amoebocyte lysate (LAL) test
Horseshoe grab (Limulus polyphemus)
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MTT cytotoxicity test in cell culture
http//www.ib.amwaw.edu.pl/home/dslado/video/mtt.h
tml
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Current use of replacement alternatives

• Safety testing of chemicals
• Local toxicity
• Genotoxicity
• Screening tests
• Quality control and potency testing of vaccines
• Production of monoclonal antibodies

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Skin corrosion and skin irritation tests

• In vitro
• Skin corrosion artificial human skin cultures

• In vivo
• Corrosivity test on rabbit skin

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Eye irritation tests

• In vitro
• Eye irritation
• HET-CAM (hens egg chorio-allantoic membrane)
  test
• BCOP (bovine corneal opacity) test
• ICE (isolated chicken eye) test

• In vivo
• Draize test in rabbits eye

HET-CAM hens egg chorio-allantoic membrane
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Production of monoclonal antibodies

• In vivo
• Mouse ascites method

• In vitro
• Cell culture

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Pyrogen tests

• In vivo
• Rabbit pyrogen test

• In vitro
• Limulus test
• Human whole blood test

Limulus Amebocyte Lysate Test Kit
Horseshoe grab (Limulus polyphemus)
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Analysis of biologically active compounds

• In vitro
• Pregnancy test (immune assay)
• Hormones, vitamins (HPLC, immune assays)
• Insulin determination

• In vivo
• Frog pregnancy bioassay
• Bioassays in chicken, rats and mice
• Convulsion test (mouse), blood glucose
  determination (mouse, rabbit)

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Advantages of in vitro tests

• Controlled testing conditions
• Lack of systemic effects
• Reduction of variability between experiments
• Testing is fast (and cheap)
• Small amount of test material is required
• Limited amount of toxic waste is produced
• Human cells and tissues can be used
• Transgenic cells carrying human genes can be used
• Reduction of testing in animals

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Limitations of in vitro tests

• General toxicity profile of a chemical cannot be
  assessed
• In vivo dose-responses cannot be obtained (for
  human risk assessment)
• Systemic effects cannot be evaluated
• Interactions between tissues and organs cannot be
  tested
• Pharmacokinetics cannot be evaluated
• Specific organ sensitivity cannot be assessed
• Chronic effects cannot be tested

23
Safety testing of chemicals

• Replacement alternatives in use for testing
• Genotoxicity mutagenicity
• chromosomal effects
• Local tolerance phototoxicity
• skin corrosivity
• skin irritation
• eye irritation
• Toxicological screening general cytotoxicity
• Percutaneous absorption

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A Typical Safety Evaluation Program
Chemical Identification and Characterization
Literature Review, Structure-Activity
Relationships
Local Toxicity
Acute Toxicity
Sensitization
Pharmacokinetics Toxicokinetics
Genotoxicity
Subchronic Toxicity
Reproductive and Developmental Toxicity
Chronic Toxicity
Special Studies
Carcinogenicity
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No relevant replacement alternatives

• Pharmacokinetics / toxicokinetics
• Systemic toxicity
• Organ systems toxicity (CNS, respiratory,
  cardiovascular, gastrointestinal etc.)
• Immunotoxicity
• Male and female reproduction toxicity (fertility
  tests, developmental toxicity tests, peri- and
  postnatal toxicity tests)
• Subchronic and chronic toxicity
• Carcinogenicity tests

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Stepwise in vitro and in vivo testing

• Skin / eye irritation and corrosivity testing
  (OECD 404, 405)
• Existing data on human and laboratory animals
• Toxicity based on physico-chemical properties
  QSAR
• Toxicity in vitro
• positive result ? classification
• negative or equivocal result ? in vivo testing
• Toxicity in vivo
• first one or few animals
• positive ? classification
• negative ? more animals ? classification -

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European Centre for the Validation of Alternative
Methods

• Established in 1991 (Directive 86/609/EEC )
• Located at the Joint Research Centre, Ispra,
  Italy
• Part of the Institute for Health and Consumer
  Protection (IHCP)
• http//ecvam.jrc.it/

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Responsibilities of ECVAM

• Coordinates the validation of alternative methods
  at the European Community level
• Acts as a focal point for the exchange of
  information on the development of alternative
  methods
• Maintains and manages a data base on alternative
  procedures
• Promotes dialogue among legislators, industries,
  biomedical scientists, consumer organizations and
  animal welfare groups, with a view to the
  development, validation and international
  recognition of alternative methods

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ECVAM Database Service
http//ecvam-dbalm.jrc.cec.eu.int/

• Database on alternative methods (method
  descriptions)
• Database on Test Methods (INVITTOX) provides
  step-by-step descriptions for the method
  performance
• Bibliographic database
• (Q)SAR (quantitative structure-activity
  relationship) model search

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Stages in test method development
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Predictive ability of a toxicity test

• Specificity
• The percentage of negative chemicals correctly
  identified.
• Sensitivity
• The percentage of positive chemicals correctly
  identified.
• Predictivity
• The percentage of predictions for a particular
  classification, which were correct.
• Accuracy
• The overall percentage of correct
  classifications.

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What is validation?

• The process by which the reliability and
  relevance of a procedure are established for a
  particular purpose.
• In a general sense authentication,
  confirmation, establishing credibility,
  ratification, substantiation.

33
Validation study

• A formal interlaboratory blind trial (4
  laboratories / test)
• Two phases
• preliminary phase (training set of
  chemicals)
• definitive phase
• Main stages study design
• selection of tests
• selection of laboratories
• data collection and analysis
• assessment of outcome

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Methods with accepted validation

• Chemicals testing
• Phototoxicity test in vitro (1998)
• Skin corrosivity test in vitro (4 methods 1998,
  2000)
• Skin sensitisation test (local lymph node assay
  2000) - REFINEMENT, REDUCTION
• Percutaneous absorption in vitro (OECD, 2002)
• Embryotoxicity test (embryonic stem cell test,
  rat whole embryo culture test, micromass test
  2002) REDUCTION (only for in vitro-screening
  for moderate to strong teratogens)
• The colony forming unit-granulocyte/macrophage
  (CFU-GM) assay for predicting acute neutropenia
  in humans (2006) requires also in vivo data
• Skin irritation test in vitro (2 methods 2007
  2 new methods 2008)
• Severe eye irritation test (2 methods 2007)

35
Methods with accepted validation

• Biologicals testing
• In vitro production of monoclonal antibodies
  (1998)
• Batch testing of tetanus vaccines (2 methods,
  ELISA, ToBI, 2000)
• Batch potency testing of erysipelas vaccines
  (ELISA, 2002)
• Batch potency testing of erythropoietin
  concentrated solution (2002) REFINEMENT
• Five in vitro pyrogen tests (2006) only for
  Gram-negative endotoxins

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Genotoxicity tests in OECD Guidelines

• 471 Bacterial Reverse Mutation Test, 1997
  (originally 1982)
• 473 In vitro Mammalian Chromosomal Aberration
  Test, 1997
• 476 In vitro Mammalian Cell Gene Mutation Test,
  1997
• 477 Sex-Linked Recessive Lethal Test in
  Drosophila melanogaster, 1984
• 479 In vitro Sister Chromatide Exchange Assay in
  Mammalian Cells, 1986
• 480 Saccharomyces cerevisiae, Gene Mutation
  Assay, 1986
• 481 Saccharomyces cerevisiae, Mitotic
  Recombination Assay, 1986
• 482 DNA Damage and Repair, Unscheduled DNA
  Synthesis in Mammalian Cells in vitro, 1986

37
Principle of the 3T3 NRU phototoxicity test

• Mouse fibroblast 3T3 cell line
• Comparison of cytotoxicity of the test chemical
  in the presence and in the absence of exposure to
  a noncytotoxic dose of UVA/vis light
• Cytotoxicity is evaluated using the Neutral Red
  Uptake (NRU) assay

38
Artificial human skin (EPISKINTM )
39
Principles of EPISKINTM and EPIDERMTM skin
corrisivity tests (2)

• EPISKINTM or EPIDERMTM commercial kits
• Reconstructed human epidermis stratified
  differentiated epidermis from human keratinocytes
  on a human collagen matrix
• Test material is applied to the epidermal surface
• Viability of epidermis is evaluated using the MTT
  test (mitochondrial activity)

40
Principles of the TER skin corrosivity test

• TER transcutaneous electrical resistance
• Skin disks from humanely killed young rats
• Test material is applied to the epidermal
  surfaces of skin disks
• Corrosivity is measured as a reduction in the
  inherent TER below the threshold level (5 k?)

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Development of in vitro skin corrosivity tests
Timetable

• 1993 - 94 Prevalidation study involving three
  methods
• 1994 ECVAM workshop on outcome of the
  prevalidation study
• 1996 - 97 Formal validation study involving four
  methods
• 1998 Successful outcome of formal validation
  study endorsed by ESAC for two methods (the
  rat skin TER method and the EPISKINTM
  reconstituted human skin method)
• 2000 Regulatory acceptance (EU, Annex V)
• 2002 Regulatory acceptance (OECD Guidelines for
  Testing of Chemicals)

42
In vitro methods in risk assessment

• Suitable for
• hazard identification (local effects,
  genotoxicity)
• screening tests, stepwise testing strategies
• mechanistic studies
• Cannot be used for
• characterization of the toxicity profile of a new
  chemical
• dose extrapolation to humans
• studies for systemic effects
• studies for long-term effects
• Highly useful as complementary studies together
  with in vivo methods

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Replacement alternatives in perspective

• Replacement methods are good alternatives for
  animal experiments in certain limited areas of
  research (e.g. testing for local toxicity and
  genotoxicity, screening tests, quality control of
  biologicals, production of monoclonal antibodies)
• Reliability and relevance of validated methods is
  high
• The role of in vitro and in vivo methods in
  research is often complementary
• Majority of current knowledge in life sciences
  and biomedical sciences is based on animal
  experiments
• Future development of biomedical sciences is
  likely to be highly dependent on animal
  experiments

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Challenges with alternative methods

• Scientists
• lack of interest to develop candidate methods
• Regulators
• general distrust of alternative methods
• Animal welfare interests (general public,
  politicians)
• unrealistic expectations

45
Cosmetics testing in the EU (1)

• Cosmetics legislation
• No hazard for human health
• Safety evaluation based on assessment of
  ingredients
• Cosmetics Directive (76/768/EEC) phasing out of
  animal testing
• Consumers are exposed to cosmetics daily
  throughout their life.
• Currently 0.025 of laboratory animals are used
  for cosmetics testing in the EU (2002).
• A conflicting situation high degree of safety is
  required, but the use of necessary tools for
  safety assessment (animals) is prohibited.

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Cosmetics testing in the EU (2)
7th Amendment to the Cosmetics Directive
(76/768/EEC)