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Testing Times

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Title: Testing Times


1
Testing Times
(or, Dragging governments environmental groups
kicking screaming into the 21st century)
  • Troy Seidle
  • Science Policy Advisor
  • Animal Alliance of Canada
  • People for the Ethical Treatment of Animals
  • TroyS_at_peta.org

VITAL Symposium 27 March 2004 Ottawa,
Ontario
2
Presentation Overview
  • Status Quo
  • Government data requirements
  • Test method case studies
  • Animal testing statistics
  • Alternatives
  • History
  • Validated/accepted methods
  • Methods undergoing pre/validation
  • Factors affecting progress
  • Emerging Challenges
  • New Ongoing Campaigns

VITAL Symposium 27 March 2004 Ottawa,
Ontario
3
Regulatory Testing Requirements
  • Under the Pest Control Products Act of 2002,
    Health Canadas Pest Management Regulatory Agency
    (PMRA) is responsible for regulating the sale,
    importation and safety review of all
  • Conventional (chemical) pesticides
  • Insecticides, fungicides, rodenticides
    antimicrobial pesticides
  • Bio-pesticides
  • Pheremones, floral attractants, microbials,
    plant incorporated protectants
  • The Health Protection Branch of Health Canada is
    empowered by the Food and Drug Act and the
    Canadian Environmental Protection Act to review
    the safety of existing and new
  • Industrial chemicals polymers
  • Drugs vaccines
  • Food additives
  • Cosmetics

VITAL Symposium 27 March 2004 Ottawa,
Ontario
4
Regulatory Testing Requirements
  • Pesticides
  • Human health ecological effects of active
    inert ingredients
  • Acute toxicity7-20 rats x 3 exposure routes
    (oral inhalation skin)
  • Eye skin corrosion/irritation1-3 rabbits/test
  • Skin sensitization30 guinea pigs or 16 mice
  • Subacute (28-day repeated-dose) toxicity40 rats
    x 2 exposure routes
  • Subchronic (90-day repeated-dose) toxicity32
    dogs 120 rats/exposure route
  • Chronic toxicity32 dogs 160 rats
  • Reproductive toxicity in 2 generations2,500
    rats
  • Developmental toxicity900 rabbits 1,300 rats
  • Genetic toxicity80 hamsters/mice x 2-5 separate
    tests
  • Carcinogenicity400 rats 400 mice
  • Nervous system toxicity80 hens 80-2,500 rats
    x 3-5 separate tests
  • Acute chronic effects on fish120-360 fish x
    1-3 species (fresh /or salt water)
  • Acute, dietary reproductive effects on
    birds60-1,450 birds x 1-2 species

VITAL Symposium 27 March 2004 Ottawa,
Ontario
5
Regulatory Testing Requirements
  • New Chemicals
  • Substances marketed post-1986 products of
    biotechnology
  • Acute toxicity7-20 rats x 2 exposure routes
    (oral /or inhalation /or skin)
  • Subacute (28-day repeated-dose) toxicity40 rats
    via most relevant exposure route
  • Skin irritation0-3 rabbits/test
  • Skin sensitization0-30 guinea pigs or 0-16 mice
  • Genetic toxicity0-80 hamsters/mice x 1-5
    separate tests
  • Acute effects on fish120 fish x 1-3 species
    (fresh /or salt water)
  • Existing Chemicals
  • Substances that have been on the market as of
    1986 or earlier
  • Acute toxicity7-20 rats x 2 exposure routes
    (oral /or inhalation /or skin)
  • Subacute (28-day repeated-dose) toxicity40 rats
    via most relevant exposure route
  • Reproductive toxicity in 1 generation1,300
    rats
  • Developmental toxicity900 rabbits 1,300 rats
  • Genetic toxicity80 hamsters/mice x 1-5 separate
    tests
  • Acute effects on fish120 fish x 1-3 species
    (fresh /or salt water)
  • Acute effects on birds60 birds x 1-2 species

6
Regulatory Testing Requirements
  • Drugs
  • Safety efficacy of both active ingredients
    vehicles
  • Acute toxicity7-20 rats dogs or primates
  • Subchronic (14- to 180-days) toxicity120 rats
    32 dogs or primates
  • Chronic toxicity160 rats 32 dogs or primates
  • Carcinogenicity400 rats 400 mice
  • Reproductive toxicity
  • Segment I Reproductive toxicity in 2
    generations2,500 rats
  • Segment II Developmental toxicity900 rabbits
    1,300 rats
  • Segment III Peri-postnatal effects??? rats
  • Metabolism pharmacological interaction of
    active ingredients
  • Specialty studies
  • Genetic toxicity80 hamsters/mice x 2-5 separate
    tests
  • Immunotoxicity32 rats
  • Skin/eye/mucosal irritation3 rabbits/test
  • Combination of 2 or more drugs considered new
  • Must be subjected to same assessments required
    for a new drug

VITAL Symposium 27 March 2004 Ottawa,
Ontario
7
Regulatory Testing Requirements
  • Food Additives
  • Extensive testing to determine allowable daily
    intake (ADI)
  • Acute toxicity7-20 rats x 3 exposure routes
    (oral inhalation skin)
  • Subacute (28-day repeated-dose) toxicity40 rats
    x 2 exposure routes
  • Subchronic (90-day repeated-tose) toxicity32
    dogs 120 rats/exposure route
  • Chronic toxicity32 dogs 160 rats
  • Reproductive toxicity in 2 generations2,500
    rats
  • Developmental toxicity900 rabbits 1,300 rats
  • Genetic toxicity80 hamsters/mice x 2-5 separate
    tests
  • Carcinogenicity400 rats 400 mice

VITAL Symposium 27 March 2004 Ottawa,
Ontario
8
Regulatory Testing Requirements
  • Cosmetics
  • No specific animal testing requirements
  • Numerous companies market non-animal tested
    cosmetics in Canada
  • Section 16 of Food Drug Act
  • Prohibits the sale of any cosmetic that may cause
    injury to the user when applied according to
    directions on label
  • Responsibility rests with cosmetic manufacturer
    to ensure that ingredients substances are
    tested according to acceptable protocols
  • i.e., Organization for Economic Cooperation
    Development (OECD)Test Guidelines (TGs)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
9
Case Study Acute Toxicity
  • OECD TGs 420, 423 425
  • Issues of concern
  • Mortality or moribundity as the endpoint
  • Exposure via 3 routes is unnecessary redundant
  • 5-20 animals killed, depending on protocol
    exposure route
  • Reliability relevance to humans (scientific
    validity) has never been established
  • Poor inter-lab reproducibility concordance
    among rodent species between rodents humans
  • Rat LD50 prediction of mouse LD50 (R2 0.61)
  • Rodent LD50 prediction of human lethal doses (R2
    0.65)
  • (Ekwall et al. 1999)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
10
Case Study Eye Irritation
  • OECD TG 405
  • Issues of concern
  • Potential to inflict severe pain distress
  • Reliability relevance to humans (scientific
    validity) has never been established
  • Poor inter-lab reproducibility concordance
    between rabbits humans
  • An extensive interlaboratory study documented
    very high variability among 24 labs (CV
    42-117) (Weil Scala 1971)
  • A comparison of 281 cases of accidental human eye
    exposure to various household products to Draize
    test data for these products found little or no
    predictive value to the rabbit test (R2 0.48)
    (Freeberg et al. 1984)
  • FDA investigators found no clear relationship
    between rabbit human eye responses concluded
    that the Draize test is plagued by lack of
    reproducibility (Koch et al. 1989)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
11
Case Study Skin Irritation
  • OECD TG 404
  • Issues of concern
  • Potential to inflict severe pain distress
  • Reliability relevance to humans (scientific
    validity) has never been established
  • Poor inter-lab reproducibility concordance
    between rabbits humans
  • An extensive interlaboratory study documented
    very high variability among 24 labs (CV
    42-117) (Weil Scala 1971)
  • A comparison of data from Draize rabbit tests and
    4-hr human skin patch tests for 65 substances
    found that 45 of existing classifications of
    chemical irritation potential are incorrect
    (Robinson et al. 2002)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
12
Case Study Carcinogenicity
  • OECD TGs 451 453
  • Issues of concern
  • 800 animals (400 rats 400 mice) killed per test
  • Animals may endure severe pain distress
  • Reliability relevance to humans (scientific
    validity) has never been established
  • Poor inter-lab reproducibility concordance
    among rodent species between rodents humans
  • Of 19 known human oral carcinogens, rodent cancer
    studies have identified only 37 (Salsburg 1983)
  • 46 of substances were found to have been
    carcinogenic in rats but not in mice, vice
    versa (Di Carlo 1984)
  • A very recent analysis of duplicate rodent
    carcinogenicity data showed that there was only a
    57 agreement between results for 121 chemicals,
    each of which had been tested on two occasions
    (Gottmann et al. 2001)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
13
Canadian Animal Testing Statistics
  • Canadian Council on Animal Care statistics for
    2000
  • 259,272 animals used for regulatory testing
    purposes
  • 58 were subjected to D or E-level procedures
  • 82 of all E-level procedures were undertaken
    for regulatory testing purposes

Experiments which cause moderate to severe
stress or discomfort to procedures which cause
pain near, at, or above the pain tolerance
threshold of unanesthetized, conscious animals
Figure 1 Proportion of animals per species used
in government-mandated toxicity testing
VITAL Symposium 27 March 2004 Ottawa,
Ontario
14
European Animal Testing Statistics
  • European Commission statistics for 1999
  • 800,788 animals used for regulatory testing
    purposes
  • 33 biologicals/vaccines
  • 21 drugs/pharmaceuticals
  • 11 industrial chemicals
  • 8 agricultural pesticides
  • 7 enviro. contaminants
  • 3 food additives
  • 1 cosmetics toiletries
  • 16 other evaluations

Figure 2 Distribution of animal use by toxicity
endpoint
VITAL Symposium 27 March 2004 Ottawa,
Ontario
15
History of Alternatives
  • 1959
  • Russell Burch publish Principles of Humane
    Experimental Technique
  • 1969
  • Fund for the Replacement of Animals in Medical
    Experiments (FRAME) founded
  • 1989
  • German Center for Documentation Evaluation of
    Alternatives (ZEBET) founded
  • 1993
  • NIH Revitalization Act calls for the development
    of alternative methods
  • 1994
  • European Centre for the Validation of Alternative
    Methods (ECVAM) founded
  • Netherlands Centre for Alternatives founded
  • 1996
  • 1st Organization for Economic Cooperation
    Development (OECD) Validation Conference, Solna,
    Sweden

VITAL Symposium 27 March 2004 Ottawa,
Ontario
16
History of Alternatives
  • 1997
  • U.S. Interagency Coordinating Committee on the
    Validation of Alternative Methods (ICCVAM)
    established as an ad hoc standing committee
  • 2000
  • ICCVAM Authorization Act establishes ICCVAM as a
    permanent entity
  • 2001
  • First ever congressional appropriation in the
    U.S. specific to non-animal test method
    developmentEPA directed to spend 4 million on
    this effort
  • 2002
  • 2nd OECD Validation Conference, Stockholm, Sweden
  • OECD National Coordinators endorse 3 in vitro
    test guidelines
  • OECD Task Force on Endocrine Disruptor Testing
    Assessment establishes a dedicated subgroup for
    non-animal methods

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Validated /or Accepted Methods
  • Embryotoxicity
  • Rodent embryonic stem cell test (Genschow et al.
    2002)
  • ECVAM-validated but not yet widely accepted by
    regulators
  • Eye Irritation
  • Bovine Corneal Opacity Permeability Test
    (Gautheron et al. 1994)
  • Accepted in UK, Germany Belgium for detection
    of severe irritants
  • Chicken Enucliated Eye Test (Prinsen Koëter
    1993)
  • Accepted in the UK, Germany Netherlands for
    detection of severe irritants
  • Genetic Toxicity (Garner 1991)
  • Bacterial Reverse Mutation/Ames Test
  • In Vitro Chromosomal Aberration Test
  • In Vitro Sister Chromatid Exchange Test
  • In Vitro Cell Gene Mutation Test
  • All four methods accepted internationally as
    OECD Test Guidelines (TGs)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Validated /or Accepted Methods
  • Phototoxicity
  • 3T3 Neutral Red Uptake (NRU) Phototoxicity Test
    (Spielmann et al. 1998)
  • ECVAM validated accepted as an OECD TG
  • Pyrogenicity
  • Human Whole Blood Pyrogen Test (Spreitzer et al.
    2002)
  • ECVAM validated but not yet widely accepted by
    regulators
  • Limulus Amebocyte Lysate (LAL) Test (Hartung et
    al. 2001)
  • ECVAM validated widely accepted by regulators
  • Skin Absorption (Diembeck et al. 1999)
  • Use of excised skin to measure the rate of
    chemical absorption
  • Extensively peer reviewed accepted as an OECD
    TG
  • Skin Corrosion
  • EpiSkin EpiDerm assays (Fentem Botham 2002)
  • ECVAM-validated accepted as an OECD TG

VITAL Symposium 27 March 2004 Ottawa,
Ontario
19
Validated /or Accepted Methods
  • Skin Corrosion
  • CORROSITEX (ICCVAM 1999)
  • Validation review by U.S. ICCVAM accepted for
    certain chemical classes
  • Skin Irritation
  • Human clinical skin-patch test (Robinson et al.
    2001)
  • Accepted as a replacement by Health Canada
  • Vaccine Potency Testing (Hendriksen et al. 1994)
  • ELISA ToBi tests for tetanus vaccines
  • MAPREC test for neurovirulence of polio vaccine
  • All tests have been validated by ECVAM
    recommended for acceptance by the European
    Pharmacopoeia
  • Acute Toxicity to Fish
  • DarT Fish Egg Test (Schulte Nagel 1994)
  • Validated accepted in Germany as a replacement
    for fish in wastewater effluent testing
    recommended for development into an OECD TG

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Methods Undergoing Pre/Validation
  • Acute Toxicity (mammalian)
  • Basal cytotoxicity assays using normal human
    keratinocytes (NHK) or mouse fibroblast (Balb/c
    3T3) cell lines
  • Battery of 3 human cell line tests found to be
    highly predictive (R2 0.79) of peak human
    lethal blood concentrations (Clemendson et al.
    1988)
  • Prediction increased (R2 0.83) when
    supplemented with biokinetic modeling
    (blood-brain barrier algorithm)
  • Joint ECVAM-ICCVAM validation study presently
    underway
  • Acute Toxicity (fish)
  • TETRATOX (Sinks Schultz 2001)
  • Uses the protozoan Tetrahymena pyriformi as a
    biomarker for lethality in fish
  • Results of a preliminary ring test found assay
    results to be highly concordant with fish LC50
    data
  • Further validation efforts underway through the
    German EPA

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Methods Undergoing Pre/Validation
  • Carcinogenicity
  • Cell transformation assays (IARC/NCI/EPA 1985)
  • Cell transformation highly correlated to
    carcinogenesis
  • Systems include immortalized cell lines (e.g.
    BALB/c 3T3 C3H/10T1/2) primary cells (e.g.
    Syrian hamster embryos)
  • Skin Sensitization
  • Step-wise testing strategy
  • Assessment of physio-chemical properties
    (molecular weight, etc.)
  • Human skin equivalent tests to verify lack of
    corrosivity
  • Knowledge-based computer models (e.g. DEREK,
    TOPKAT CASE) to identify the presence/absence
    of molecular structural alerts for skin
    sensitization
  • Assessment of permeability/partition parameters
    for chemicals exhibiting structural
    alerts/potential reactivity

VITAL Symposium 27 March 2004 Ottawa,
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Factors Affecting Progress
  • Provision of adequate resources for test method
    development validation
  • Regulatory bodies in North America have not yet
    committed to a strategy to develop, validate
    utilize non-animal test methods in risk
    assessments
  • Where dedicated funding has been made available
    (e.g. Congressionally mandated reallocation of 4
    million from the U.S. Environmental Protection
    Agencys 2001 budget), they have not been spent
    on focused, endpoint-specific non-animal test
    method development or pre/validation

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Factors Affecting Progress
  • International coordination of RD efforts
  • Europe Japan leading the way with in vitro
    method development, while North America is a
    stronger proponent of (Q)SAR and other in silico
    systems
  • Until ECVAM published its May 2002 report on
    Alternative Methods for Chemicals Testing,
    there was no comprehensive and globally-recognized
    overview of the current status future
    prospects for non-animal test methods
  • International coordination through ECVAM has
    traditionally been Euro-centric, while
    OECD-coordinated RD activities have focused
    almost exclusively on animal tests (e.g.
    endocrine disruptors)

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Factors Affecting Progress
  • Availability of trained personnel experienced
    laboratories to participate in validation studies
  • A critical observation from validation studies
    carried out to date
  • Tendency among laboratories to deviate from
    standardized protocols

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Factors Affecting Progress
  • Availability of high quality human reference data
    for validation
  • Data from non-validated animal tests should not
    be considered the gold standard to be met by
    non-animal methods
  • Recognized limitations of animal studies for
    acute toxicity, skin/eye irritancy other
    endpoints (Ekwall et al. 1999 Weil Scala 1971)
  • Highly variable in vivo data has created
    difficulties in validating non-animal replacement
    methods (Balls et al. 1995)
  • OECD Stockholm conference recommended that an
    expert meeting be held to organize creation of a
    human health effects database
  • Occupational exposure/effects data
  • Product use/hazard data

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Factors Affecting Progress
  • Regulatory acceptance of scientifically valid
    non-animal test methods strategies
  • Current processes are duplicative much too slow
  • Driven as much by politics as by science
  • Non-animal methods consistently held to a more
    rigorous standard of scientific validation
  • Even non-animal methods that have undergone
    successful scientific validation have been
    accepted in one jurisdiction may not be fully
    accepted in others, e.g. skin corrosion tests
  • ECVAM Scientific Advisory Committee statement of
    validity issued in 2000
  • Accepted as stand-alone methods under EU
    Directive 67/548/EEC
  • Recommended for acceptance as OECD TGs 430/431 in
    May 2002
  • U.S. ICCVAM insists that negative results be
    confirmed using a non-validated in vivo skin
    corrosion study

VITAL Symposium 27 March 2004 Ottawa,
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Current Challenge
  • Total replacement of animal tests required by
    drug pesticide regulators
  • Acute toxicity7-20 rats x 3 exposure routes
    (oral inhalation skin)
  • Eye skin corrosion/irritation1-3 rabbits/test
  • Skin sensitization30 guinea pigs or 16 mice
  • Subacute (28-day repeated-dose) toxicity40 rats
    x 2 exposure routes
  • Subchronic (90-day repeated-dose) toxicity32
    dogs 120 rats/exposure route
  • Chronic toxicity32 dogs 160 rats
  • Reproductive toxicity in 2 generations2,500
    rats
  • Developmental toxicity900 rabbits 1,300 rats
  • Genetic toxicity80 hamsters/mice x 2-5 separate
    tests
  • Carcinogenicity400 rats 400 mice
  • Nervous system toxicity80 hens 80-2,500 rats
    x 3-5 separate tests
  • Metabolism pharmacological interaction of
    active ingredients
  • Acute chronic effects on fish120-360 fish x
    1-3 species (fresh /or salt water)
  • Acute, dietary reproductive effects on
    birds60-1,450 birds x 1-2 species

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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Emerging Challenges
  • Pressure from environmental groups others to
    reassess pesticides for effects on children
    sensitive subpopulations, endocrine disruption,
    etc.
  • Trend toward perpetual new flavour-of-the-month
    animal tests
  • Developmental neurotoxicity
  • Developmental immunotoxicity
  • Endocrine disruption
  • Acute reference dose
  • (?)
  • Current trend seriously undermines the trend
    towards reducing reliance on animal testing,
    which government agencies claim to support

VITAL Symposium 27 March 2004 Ottawa,
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Going After the Mean Greenies
  • Most of the animal testing battles we are facing
    today have emerged as a direct result of lobbying
    by environmental groups
  • 3 groups have consistently emerged as the worst
    culprits
  • World Wildlife Fund (WWF)
  • Chief proponent of U.S. international
    endocrine disruptor testing programs, as well
    as a European program to (re)test some 30,000
    common chemicals
  • Natural Resources Defense Council (NRDC)
  • Major proponent of developmental neurotoxicity,
    endocrine disruptor cancer tests on animals
  • Environmental Defense
  • Chief proponent of U.S. program to test 2,000
    high production volume chemicals
  • After years of attempts persuade these groups to
    kick their animal testing habit, PETA took its
    case to the public by launching the following
    websites
  • MeanGreenies.com
  • WickedWildlifeFund.com

VITAL Symposium 27 March 2004 Ottawa,
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Give the Animals 5
  • PETA campaign to educate lawmakers,teachers
    the public by highlighting 5non-animal test
    method success stories
  • Skin corrosion, irritation, absorption,
    phototoxicity pyrogenicity
  • Multi-faceted campaign
  • Pressures U.S. federal agencies to permanently
    do away with5 outdated animal tests todevelop
    more alternatives
  • Urges teachers to incorporatemore material on in
    vitro toxico-pharmacology into their curriculum
  • More at StopAnimalTests.com

VITAL Symposium 27 March 2004 Ottawa,
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Global Problem Global Campaign
  • International Council on Animal Protection in
    OECD Programs (ICAPO)
  • Representing 30 million supporters in Europe,
    North America Asia
  • Organizational members
  • Animal Alliance of Canada British
    Union for the Abolition of Vivisection
  • Doris Day Animal League Eurogroup for
    Animal Welfare
  • Humane Society of the U.S. Japan
    Anti-Vivisection Association
  • People for the Ethical Treatment of Animals
  • Physicians Committee for Responsible Medicine
  • ICAPO has invited expert status at high-level
    OECD meetings
  • National Coordinators of the OECD Test Guidelines
    Program
  • Nearly 100 OECD TGs on the books, of which
    nearly 1/2 are animal tests
  • OECD Task Force on Endocrine Disrupters Testing
    Assessment
  • Developing strategies methods to test chemicals
    for hormonal effects
  • OECD Task Force on Existing Chemicals
  • Part of an international effort to test 2,000
    high production volume chemicals

VITAL Symposium 27 March 2004 Ottawa,
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On-line Resources
  • Animal Alliance of Canada
  • CruelScience.ca AnimalAlliance.ca
  • European Centre for the Validation of Alternative
    Methods
  • ECVAM.jrc.it
  • Fund for the Replacement of Animals in Medical
    Experiments
  • FRAME.org.uk
  • Organization for Economic Cooperation
    Development
  • OECD.org/oecd/pages/home/displaygeneral/0,3380,EN-
    documentation-524-nodirectorate-no-no-no-8,FF.html
  • People for the Ethical Treatment of Animals
  • PETA.org StopAnimalTests.com
    StopEUChemicalTests.com MeanGreenies.com
    WickedWildlifeFund.com

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Literature Cited
  • Balls et al. (1999) Alt. Lab. Anim. 27, 53-77
  • Basketter et al. (1997) Food Chem. Toxicol. 35,
    845-852
  • CCAC (2002) Animal Use Survey2000, www.ccac.ca
  • Clemendson et al. (2000) Alt. Lab. Anim. 28
    (Suppl 1), 159-234
  • Di Carlo FJ (1984) Drug Metabol. Rev. 15, 409-13
  • Diembeck et al. (1999) Food Chem. Toxicol. 37,
    191-205
  • Ekwall et al. (1999) Toxicol. In Vitro 13,
    665-673
  • European Commission (2003) 3rd Commission Report
    on the Statistics on the Number of Animals Used
    for Experimental and Other Scientific Purposes in
    the EU
  • Fentem Botham (2002) Alt. Lab. Anim. 30 (Suppl
    2), 61-67
  • Freeberg et al. (1986) J. Toxicol. Cut. Ocular
    Toxicol. 5, 115-123
  • Garner (1991) In Animals Alternatives in
    Toxicology, Nottingham, UK FRAME
  • Gautheron et al. (1994) Toxicol. In Vitro 8,
    381-392
  • Genschow et al. (2002) Alt. Lab. Anim. 30,
    151-176
  • Gottmann et al. (2001) Environ. Hlth. Perspect.
    109, 509-51

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Literature Cited
  • Hartung et al. (2001) Alt. Lab. Anim. 29, 99-123
  • Hendriksen et al. (1994) Alt. Lab. Anim. 22,
    420-434
  • IARC/NCI/EPA Working Group (1985) Cancer Res. 45,
    2395-2399
  • ICCVAM (1999) NIH Publication No. 99-4495,
    Research Triangle Park, NC NIEHS
  • Koch et al. (1989) J. Toxicol Cut. Ocular
    Toxicol. 8, 17-22
  • Prinsen Koëter (1993) Food Chem. Toxicol. 31,
    69-76
  • Robinson et al. (2001) Contact Derm. 45, 1-12
  • Robinson et al. (2002) Food Chem Toxicol.
  • Salsburg D (1983) Fundam. Appl. Toxicol. 3, 63-67
  • Schulte Nagel (1994) Alt. Lab. Anim. 22, 12-19
  • Sinks Schultz (2001) Environ. Toxicol. Chem.
    20, 917-921
  • Spielmann et al. (1998) Toxicol. In Vitro 12,
    305-327
  • Spreitzer et al. (2002) ALTEX 19 (Suppl 1), 73-75
  • Weil Scala (1971) Toxicol. Appl. Pharmacol. 17,
    276-360
  • Worth et al. (1998) Alt. Lab Anim. 26, 709-720

VITAL Symposium 27 March 2004 Ottawa,
Ontario
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