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
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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
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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
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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
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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

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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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,
Ontario
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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

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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

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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