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4/25/2014
The Future of Animal Research in the Context of the 3Rs (Replacement, Reduction & Refinement)
Joanne Zurlo, PhD
Johns Hopkins Center for Alternatives to Animal Testing
A Short History Lesson
A few key names:
• Rene Descartes – 17th century (animals are automata)
• Jeremy Bentham – 18th century (“The question is not, can they reason? Nor can they talk? But, can they suffer?)
• Claude Bernard – 19th century (dubbed by Pasteur as “Physiology itself)
Darwin Quotes on Animals
"You ask about my opinion on vivisection. I quite agree that it is justifiable for real investigations on physiology; but not for mere damnable and detestable curiosity. It is a subject which makes me sick with horror, so I will not say another word about it, else I shall not sleep to‐night.”
Lecture Outline
• Brief history of animals in research • Laboratory animal protection and legislation in the US and the Three Rs
• Advances in replacement and reduction –
toxicity testing and drug development
• Advances in refinement
• Final thoughts
Darwin and Animal Welfare
• Both animal rightists and scientists embraced Darwin as their own in a period of “spirited discussion” regarding the use of animals in research leading up to the passage of the 1876 Cruelty to Animals Act in England
• In truth, Darwin was very much a centrist, but certainly conflicted as evidenced by some of his quotes
“From all that I have heard, however, I fear that in some parts of Europe little regard is paid to the sufferings of animals, and if this be the case, I should be glad to hear of legislation against inhumanity in any such country.”
‐ Letter to Prof. Frithiof Holmgren (1881)
‐ Letter to Professor R. Lankester (1871)
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“On the other hand, I know that physiology cannot possibly progress except by means of experiments on living animals, and I feel the deepest conviction that he who retards the progress of physiology commits a crime against mankind.”
‐ Letter to Prof. Frithiof Holmgren (1881)
Since Darwin’s time, advances made in ethology have shown that some animals are capable of:
– Self‐awareness – Facial recognition
– Empathy and grief
– Tool making and usage
– Cooperativity
– Altruism
– Language
– Aggression
U.S. ANIMAL PROTECTION LEGISLATION
• 1966 Laboratory Animal Welfare Act ‐focused on dealers to prevent pet theft (included dogs, cats, non‐
human primates, guinea pigs & rabbits)
• 1970 amendment to LAWA ‐expanded to include all warm‐blooded animals used in research (except farm animals)
• 1971 ‐USDA excluded rats, mice and birds from LAWA
“The difference in mind between man and the higher animals, great as it is, certainly is one of degree and not of kind.”
‐ The Descent of Man (1871)
In his book The Expressions of the Emotions in Man and Animals (1872), Darwin asserted that facial and bodily expressions reveal inner feelings, e.g. pain, fear, pleasure, in both humans and animals
Laboratory Animal Protection and Legislation in the US
U.S. ANIMAL PROTECTION LEGISLATION (cont’d)
• Animal Welfare Act (1985) ‐ amendment of LAWA:
– Created Institutional Animal Care and Use Committees (IACUCs)
– Mandated minimization of pain and distress
– Specified exercise requirements for dogs
– Established guidelines to maintain psychological well‐being of nonhuman primates
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Public Health Service Policy on Humane Care and Use of Laboratory Animals
• Applies to all institutions with PHS funding
• Protects ALL vertebrate animals
• Institutions must comply with guidelines set forth in the Guide for the Care and Use of Laboratory Animals (NRC)
• Each assured facility must have an IACUC and report whether they are accredited by AAALAC International
Recent Changes to NIH Policy
• Random source dogs and cats for use in NIH‐
funded research may no longer be obtained from Class B dealers (based on recommendations of the ILAR report)
• Most use of chimpanzees for biomedical research is unnecessary and any proposed future research must meet strict criteria (based on recommendations from IOM report)
Association for Assessment and Accreditation of Laboratory Animal Care International
• Voluntary accreditation for laboratory animal care programs (www.aaalac.org)
• Uses the Guide for the Care and Use of Laboratory Animals (the Guide) as the basis for accreditation (along with other reference documents)
• Conducts announced site visits every three years
Guiding principles for the use of animals in research – the 3Rs
Replacement Reduction
Refinement
WMS Russell and R Burch (1959) Principles of Humane Experimental Technique
Replacement considerations
“…by now it is widely recognized that [the most humane] possible treatment of experimental animals, far from being an obstacle, is actually a prerequisite for successful animal experiments.”
W.M.S. Russell and R. Burch
The Principles of Humane Experimental
Technique, 1959
• Living systems, e.g., organ, tissue, or cell culture techniques (in vitro), invertebrate models, microorganisms, and if necessary less sentient animal species
• Non‐living systems (chemical, physical)
• Computer models or simulations (in silico)
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Reduction considerations:
• Rational selection of group size including pilot studies and power analysis
• Careful experimental design and appropriate statistical analysis
• Maximizing use of animals
• Correct choice of model
• Optimal animal husbandry (monitoring for infections, avoiding overbreeding)
Refinement Considerations
• Pain ‐ Recognition and alleviation
• Distress ‐ Minimization (prevention), recognition and alleviation
• Humane Endpoints
• Environmental enrichment
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Toxicology
Advances in Replacement and Reduction – Toxicity Testing
Toxicity Testing
• Systematic approach to assessing adverse effects of a drug, chemical or other agent on human health
• Historically done in animals to measure a variety of endpoints
• Why test?
• Informally ‐ The science of poisons • Paracelsus – “the dose makes the poison”
• More formally ‐ Toxicology is the study of the adverse effects of chemical, physical, or biological agents on people, animals, and the environment. A Brief History of Toxicity Testing
• Federal Food, Drug and Cosmetic Act (FDCA) –
1938
• Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) – 1947 • Toxic Substances Control Act (TSCA) – 1976 • Endocrine Disruptor Screening Program ‐ 2009
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Toxic Substances Control Act (TSCA) – 1976 • Control of new and existing industrial chemicals not regulated by other legislation
• No specific toxicity testing was required by EPA
• Chemicals in commerce at the time TSCA was passed (60,000) could remain in commerce unless EPA could show that a chemical posed a hazard • For new chemicals, companies must bring data to EPA to help EPA assess potential adverse effects
• Many thousands of chemicals remain untested (e.g.,4‐methylcyclohexanemethanol spill in WV
Traditional Toxicity Testing Paradigm
• Relies on administering high doses of chemicals and drugs to animals, then results are extrapolated for low dose exposures
• Animals are typically inbred strains, and the results are extrapolated to heterogeneous human populations
• Results from animal studies do not always reflect results in humans
Vision of the Committee
A 2007 report by the National Research
Council of the National Academies requested by the EPA
Options for Future Toxicity Testing Strategies
Option I
In Vivo
Option II
Tiered In Vivo
Option III
In Vitro/In Vivo
Option IV
In vitro
Animal biology
Animal biology
Primarily human
biology
Primarily human
biology
High doses
High doses
Broad range of
doses
Broad range of
doses
Low throughput
Improved throughput
High and medium
throughput
High throughput
Expensive
Less expensive
Less expensive
Less expensive
Time consuming
Less time consuming Less time
consuming
Less time
consuming
Relative large
number of animals
Fewer animals
Substantially fewer
animals
Virtually no animals
Apical endpoints
Apical endpoints
Perturbations of
toxicity pathways
Perturbations of
toxicity pathways
In silico screens
possible
In silico screens
Some in silico and
in vitro screens
To effect a paradigm shift in the approach to toxicity testing that would:
• Provide broad coverage of chemicals, chemical mixtures, outcomes and life stages
• Reduce the cost and time of testing
• Use fewer animals and cause minimal suffering in the animals used
• Develop a more robust scientific basis for assessing health effects of environmental agents
A New Paradigm: Activation of Toxicity Pathways
Exposure
Tissue Dose
Biologic Interaction
Low Dose
Higher Dose
Higher yet
Perturbation
Normal
Biologic
Function
Biologic
Inputs
Early Cellular
Changes
Adaptive Stress
Responses
(Courtesy of Mel Andersen)
Cell
Injury
Morbidity
and
Mortality
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Toxicity Pathways
Progress in Implementation
• NTP Vision and Roadmap • Memorandum of Understanding Toxicity Pathway: A cellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect. – US EPA (National Center for Computational Toxicology)
– NIH/NIEHS (National Toxicology Program)
– NIH (National Center for Advancing Translational Sciences)
– US FDA Center for Drug Evaluation & Regulation
ToxCast Program (EPA)
Agency Commitment
“With an advanced field of
An atmosphere
of departure in
regulatory science, new
toxicology
tools, including functional
genomics, proteomics,
New technologies
from
metabolomics,
highthroughput
biotech and
(bio-screening, and
systems
biology, we can
)informatics
revolution
replace current toxicology assays with tests
that
incorporate
mechanisticof
Mapping
ofthe
pathways
underpinnings of disease and of underlying
toxicity (PoT)
toxic side effects.” M.A. Hamburg, FDA 2011
“We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments” F. Collins, NIH, 2008
Uses automated chemical screening technologies,
called “high throughput screening assays”, to expose
living cells or isolated proteins to chemicals.
• Phase‐I: 309 data‐rich chemicals (primarily pesticides) having over 30 years of traditional animal studies valued at $2B; 500 assays • Phase‐II: screened over 2000 chemicals from a broad range of sources (e.g., industrial and consumer products, food additives, failed drugs) using 700 assays for over 300 toxicity pathways • Now being used for Endocrine Disruptor Screening
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ToxCast Program (EPA)
Emerging Technologies
• “Omics” technologies – genomics, transcriptomics, metabolomics, proteomics
• “Organ/Human on a Chip” – building a whole human, organ by organ
– Project supported by FDA, NIH, DARPA and DTRA
– Desire to achieve better predictability of new drugs for humans (cf. FDA animal rule for medical countermeasures)
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Other Organs on Chips
Lung‐on‐a‐Chip (Wyss Institute for Biologically Inspired Engineering at Harvard University)
•
•
•
•
•
•
Published by AAAS
Liver
Heart
Kidney
Bone Marrow
GI
Brain
Published by AAAS
Future of Testing
HUMAN on a chip????? – achieve integration of all organ systems to create a virtual human
Some Advances
• In vitro methods used early in drug development to identify potentially toxic agents leads to significant reduction in animal use
• In vitro assays to identify biomarkers for toxicity reduces numbers of animals needed
• Use of chemical structure analysis and information in databases to help select potential new drugs with less toxicity
Scientific and Animal Welfare Innovations in Drug Development and Safety Assessment
Advances (cont’d)
• Use of stem cell cultures to assess cardiotoxicity, and less so for hepatotoxicity and neurotoxicity
• Use of human and animal hepatocyte co‐
cultures to assess metabolism and liver toxicity
• Mechanism based‐investigative toxicology to identify critical organ systems and potential intervention systems
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Advances in Refinement
Specific Refinement Issues for Laboratory Animals
• Meeting biological needs
• Confinement in cages – little freedom of movement
• Few guidelines for enrichment – are laboratory animals “normal?”
• Caregivers not proficient in animal behavior of laboratory animals
Abstract
“Mus musculus enjoys pride of place at the center of contemporary biomedical research. Despite being the current model system of choice for in vivo mechanistic analysis, mice have clear limitations. The literature is littered with examples of therapeutic approaches that showed promise in mouse models but failed in clinical trials. More generally, mice often provide poor mimics of the human diseases being modeled. Available data suggest that the cold stress to which laboratory mice are ubiquitously subjected profoundly affects mouse physiology in ways that impair the modeling of human homeostasis and disease. Experimental attention to this key, albeit largely ignored, environmental variable is likely to have a broad transformative effect on biomedical research.”
From Medical Unit blog: ABCnews.com
Environmental Enrichment
Providing novel or complex stimuli to encourage species‐specific behaviors in a laboratory setting, and/or to avoid distress and stereotypical behaviors resulting from boredom or fear
From C.L. Karp (2012) Unstressing intemperate models: How cold stress undermines
mouse modeling. J Exp Med 209, 1069‐1074
Enrichment – group housing considerations
• Is the species social or solitary in the wild?
• Normal complex socialization in the wild, e.g. rhesus macaques – different for males and females
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Enrichment – group housing benefits and costs
•
•
Benefits include support of species specific behavior, social buffering, increased resistance to disease, increased immune response
Costs include increased aggression and wounding, food competition, infant mortality, increased variability (dominance rank in NHP groups), separation effects, e.g., depression
Other Types of Enrichment –
Nonhuman Primates
• Arboreal component in cage
• Visual barriers
• Food enrichment
– Novel foods
– Puzzle feeders
– Foraging opportunities
• Manipulanda (toys, tools)
• Visual (TV, computer games)
• Audio (music)
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Other Types of Enrichment – Dogs
Considerations for Rodents
• Cage conditions
• Human interaction***
– Rats prefer opaque cages when humans are present (Cloutier & Newberry, 2010) and like hiding shelters
– Mice like to burrow and build nests (nest building also helps mice to maintain body temperature
– Handling
– grooming
•
•
•
•
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Visual access to conspecifics
Toys Audio (music)
Access to outdoors
• Lighting
• Noise
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Positive Reinforcement Training (PRT)
The Future of Refinement
• Used to habituate laboratory animals to routine procedures
• Much in the literature on nonhuman primates
• Other animals can be trained as well
• Provides a positive experience + reward for the animals
• Results in significantly less stress
• Can eliminate stereotypic behavior
• Linked to advances in animal behavior research
• Necessary to understand normal behavior to meet animals’ needs
• Further incorporation of positive reinforcement training to enhance animal welfare
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Final Thoughts
Thank you!
• Will there be an end to animal use for invasive research? – Not “if” but “when” • When is difficult to predict: depends on scientific advances, funding for research, commitment of agencies
• Until that time, we must employ the principles of the 3Rs
Questions?
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