Animal Experimentation

Scrutinised    

Andrew Knight BSc., BVMS, CertAW, MRCVS, FOCAEAnimal Consultants International www.AnimalConsultants.org

1997

“ What you’ve seen so far is only the tip of the iceberg compared to what you will have to do to animals later in the veterinary course. Perhaps you should re-think your choice of career… ”

Humane teaching methods

• high quality videos• ‘ethically-sourced cadavers’ • preserved specimens• computer simulations• non-invasive self-experimentation • clinical/surgical skills models and simulators• supervised clinical/surgical experiences

Computer simulations: dissections

‘Face’

www.Prodissector.com

Ethically-sourced cadavers

Computer simulations: experiments

Models

Clinical skills training mannequins

‘Alternative’ veterinary surgical

training

1. Knot-tying boards, plastic organs and similar models: basic manual skills such as suturing and instrument handling.

2. Ethically-sourced cadavers: simulated surgery.

3. Real patients: observing, assisting with, and then performing beneficial surgery under close supervision (e.g. shelter animal neutering programs).

Surgical simulators: DASIE

Surgical simulators

Alternative surgical training

Alternative Veterinary Surgical

Program, 2000 External clinical experience in private clinics or animal

shelters assisting with or participating in surgery and anaesthesia.

Sterilisations of real patients, e.g., from animal shelters, at Murdoch.

Attendance at all of the terminal surgical laboratories as observers.

Simulated abdominal surgeries on a “DASIE” (Dog Abdominal Surrogate for Instructional Exercises).

Ethically-sourced cadaver surgery: abdominal and orthopaedic surgeries.

Jointly we did not participate as surgeon or assistant surgeon in a total of at most 13 scheduled surgeries at Murdoch.

We performed or assisted with a total of at least 62 additional surgeries instead, not including the abdominal surgeries I performed on a “DASIE” surgical simulator. Surgeries performed under supervision, mostly in private practice.

Outcomes

Depth: Jointly we sterilised 45 dogs and cats, including 21 spays.

Breadth: We also participated in a range of other surgeries as well, e.g., umbilical hernia repair, cruciate ligament repair, cutaneous polyp and lump excisions, aural haematoma excision, abdominal surgeries (exploratory laparotomy, enterotomy, enterectomy, partial splenectomy, spay), orthopaedic surgeries (trochanteric osteotomy, stifle arthrotomy).

Similar depth and breadth of anaesthetic experience.

The effectiveness of humane teaching

methods

Knight A. The effectiveness of humane teaching methods in veterinary education. ALTEX: Altern Anim Experimentation 2007;24(2):91-109.

www.HumaneLearning.info, ‘Published papers, Comparative.’

Results 12 papers published from 1989 to 2006 described 11 distinct

studies of veterinary students:

9 assessed surgical training—historically the discipline involving greatest harmful animal use.

Humane method

SuperiorSuperior EquivalentEquivalent InferiorInferior

45.5% (5/11)45.5% (5/11) 45.5% (5/11)45.5% (5/11) 9.1% (1/11)9.1% (1/11)

All disciplines

Knight A, Balcombe J & De Boo J, www.HumaneLearning.info, ‘Published papers, comparative.’

At least 33 papers sourced from the biomedical and educational literature, covering all educational levels and disciplines, describe studies that have compared the ability of humane alternatives to impart knowledge or clinical or surgical skills.

Humane method

SuperiorSuperior EquivalentEquivalent InferiorInferior

39.4% (13/33) 39.4% (13/33) 51.5% (17/33) 51.5% (17/33) 9.1% (3/33) 9.1% (3/33)

Conclusions

Well-designed humane alternatives usually perform at least as well as methods that rely upon harmful animal use, in some cases achieving superior learning outcomes.

Their financial and time savings, repeatability, increased flexibility of use, and potential to increase active learning and computer literacy, all provide other important advantages when compared to traditional methods reliant upon harmful animal use.

From Guinea Pig to Computer Mouse: Alternative Methods for a Progressive, Humane Education. www.InterNICHE.org

www.vetmed.ucdavis.edu/Animal_Alternatives www.clive.ed.ac.uk

Alternatives libraries, free on-line computer simulations, comprehensive alternatives databases, academic reviews of leading alternatives, and hundreds of educational studies of alternatives organized by discipline:

www.HumaneLearning.info www.EURCA.org

Alternatives sources

Other major student successes

2000 University of Sydney: all terminal veterinary surgical labs all terminal veterinary surgical labs stopped, alternatives introduced, conscientious objection policy passedstopped, alternatives introduced, conscientious objection policy passed

2000, 2004 University of Illinois: all physiology vivisection labs all physiology vivisection labs stopped, conscientious objection policies passedstopped, conscientious objection policies passed

2000 Massey University, New Zealand: most veterinary most veterinary physiology vivisection labs stopped, conscientious objection policy physiology vivisection labs stopped, conscientious objection policy passedpassed

2004 University of Queensland: alternatives to terminal veterinary alternatives to terminal veterinary surgical laboratories introducedsurgical laboratories introduced

2004 University of Melbourne: alternatives to terminal veterinary alternatives to terminal veterinary surgical laboratories introducedsurgical laboratories introduced

Educating students

After vet school…

Scientific resistance to alternatives

Non-compliance of US researchers with the alternatives regulations of the Animal Welfare Act:

Most common: inadequate consideration of alternatives (600 - 800 research facilities).

Fourth most common: unnecessary experimental duplication (~ 250 facilities).

Others: inadequate justification for animal numbers, alleged uncertainty of research personnel about signs indicative of pain and/or distress (USDA-APHIS-AC, 2000).

Supporting animal experimentation

Animal experimentation is vital for preventing, curing or alleviating human diseases (e.g. Brom 2002, Festing 2004).

The greatest achievements of medicine have been possible only due to the use of animals (e.g. Pawlik 1998).

The complexity of humans requires nothing less than the complexity of laboratory animals to effectively model during biomedical investigations (e.g. Kjellmer 2002).

Medical progress would be “severely maimed by prohibition or severe curtailing of animal experiments,” and “catastrophic consequences would ensue” (Osswald 1992).

Concordance or discordance?

Drugs causing serious side effects or death in animalsthat are harmless to humans:

Penicillin Morphine Aspirin …

Drugs released onto the market after passing rigorous Drugs released onto the market after passing rigorous testing in animals, and very limited testing in testing in animals, and very limited testing in humans, that have caused serious human side effects: humans, that have caused serious human side effects:

TGN1412 (UK, 2006)TGN1412 (UK, 2006) VioxxVioxx Thalidomide, Eraldin, Chloramphenid, Ibufenac, Flosint, Zipeprol, Thalidomide, Eraldin, Chloramphenid, Ibufenac, Flosint, Zipeprol,

Zomax, Accutane, Benedectin, PhenforminZomax, Accutane, Benedectin, Phenformin Many, many more… Many, many more…

Such adverse drug reactions are the Such adverse drug reactions are the 4-6th leading cause of death in US hospitals, and kill over 10,000 people annually in and kill over 10,000 people annually in the UK.the UK.

Calls for systematic reviews

Pound and colleagues (British Medical Journal 2004):

Clinicians and the public often consider it axiomatic that animal research has contributed to human clinical knowledge, on the basis of anecdotal evidence or unsupported claims.

These constitute an inadequate form of evidence for such a controversial area of research, particularly given increasing competition for scarce research resources.

Hence, formal evaluation of existing and future animal research is urgently required, e.g., via systematic reviews of existing animal experiments.

Systematic reviews: ‘gold standard’ evidence

Critically examine human clinical or toxicological utility of animal experiments

Examine large numbers of experiments

Experiments selected without bias, via randomisation or similarly methodical and impartial means

Studies published in peer-reviewed biomedical journals

Literature survey 2007

27 systematic reviews of the utility of animal experiments in advancing human clinical outcomes (20), or in deriving human toxicity classifications (7)

Three different approaches sought to determine the maximum clinical utility that may be achieved by animal experiments …

1. Experiments expected to lead to medical advances

Lindl Lindl et al.et al. (2005 & 2006) examined animal experiments (2005 & 2006) examined animal experiments conducted at three German universities between 1991 and conducted at three German universities between 1991 and 1993, that had been approved by animal ethics committees at 1993, that had been approved by animal ethics committees at least partly on the basis of claims by researchers that the least partly on the basis of claims by researchers that the experiments might lead to concrete advances towards the cure experiments might lead to concrete advances towards the cure of human diseases. of human diseases.

For 17 experiments meeting the inclusion criteria, citations For 17 experiments meeting the inclusion criteria, citations were analysed for at least 12 years. 1,183 citations were were analysed for at least 12 years. 1,183 citations were evident. evident.

However …However …

Only 8.2% of all citations (97) were in clinical publications.Only 8.2% of all citations (97) were in clinical publications.

Of these, only 0.3% of all citations (4 publications) Of these, only 0.3% of all citations (4 publications) demonstrated a direct correlation between the results of animal demonstrated a direct correlation between the results of animal experiments and human outcomes. experiments and human outcomes.

However, even in these four cases the hypotheses that had However, even in these four cases the hypotheses that had been successfully verified in animals failed completely when been successfully verified in animals failed completely when applied to humans. applied to humans.

None of these 17 experiments led to any new therapies, or, None of these 17 experiments led to any new therapies, or, indeed, any beneficial clinical impact during the period indeed, any beneficial clinical impact during the period studied!studied!

2. Clinical utility of highly cited animal

experiments

Highly cited animal experiments are most likely to be subsequently tested in clinical trials. Hence, Hackam & Redelmeier (2006) searched for experiments with more than 500 citations, published in the 7 leading scientific journals when ranked by journal impact factor.

76 animal studies were located with a median citation count of 889 (range: 639-2,233).

However…

Only 36.8% (28/76) were replicated in human randomized trials. 18.4% (14/76) were contradicted by randomized trials, and 44.7% (34/76) had not translated to clinical trials.

Ultimately, only 10.5% (8/76) of these medical interventions were subsequently approved for use in patients.

Translation rates of most animal experiments are

even lower

Most experiments are neither highly cited nor published in leading journals. Many experiments are not published at all.

The selective focusing on positive animal data while ignoring negative results (optimism bias) is one of several factors identified that may have increased the likelihood of translation beyond that scientifically merited.

Rigorous meta-analysis of all relevant animal experimental data would probably significantly decrease the translation rate to clinical trials (Hackam, 2007).

Additionally, only 48.7% (37/76) of these highly cited animal studies published in leading journals were of good methodological quality.

Few included random allocation of animals, adjustment for multiple hypothesis testing, or blinded assessment of outcomes.

Accordingly, Hackam & Redelmeier cautioned patients and physicians about extrapolating the findings of even highly cited animal research to the care of human disease.

Poor methodological quality

3. Chimpanzee experimentation

Prominent advocates of invasive research on captive chimpanzees such as Vandeberg et al. (Nature, 2005) have called passionately for the funding of such research to be increased.

They have stated that such research has been of great importance during our struggles against major human diseases such as AIDS, hepatitis and cancer, and that the genetic similarities between humans and chimpanzees — our closest living relatives — makes them ideal biomedical research models.

Contributions to biomedical knowledge

749 studies of captive chimpanzees or chimpanzee tissues, conducted from 1995-2004:

Figure 1: Chimpanzee experiments 1995-2004 (total 749)

48%

42%

3%

3%

2%

2%

Biology (363)

Diseases: virology (311)

Therapeuticinvestigations (26)Diseases: parasitology(23)Miscellaneous (14)

Diseases: other (12)

Figure 2: Biology experiments (363 of 749)

37%

21%

10%

9%

7%

7%

6%

2%

1%Cognition/Neuroanatomy/Neurology (133)

Behavior/Communication (75)

Immunology (37)

Biochemistry (34)

Reproduction/Endocrinology (27)

Genetics (25)

Anatomy/Histology (20)

Physiology (9)

Microbiology (3)

21 others: Six: FV. Four: HAV. Two each: GBV – B, HIV & HV, IV, PIV, Noroviuses. One each: Bacteriophages, Dengue v., Ebola v., HCMV, HGV, HMPV, H/S TLV, LCV, Papillomaviruses, RV2, Rhinovirus, VZV, WMHBV, Unspecified.

Figure 3: Virology experiments (311 of 749)

31%

31%

9%

4%

4%

3%

3%

2%

2%

11%

HCV (97)

HIV (97)

HBV (29)

RSV (12)

HEV (11)

STLV (9)

HIV & SIV (8)

SIV (7)

TTV (7)

21 others (34)

HCV = hepatitis C v., HIV = human immunodeficiency v., HBV = hepatitis B v., RSV = respiratory syncytial v., HEV = hepatitis E v., STLV = simian T-cell lymphotropic v., SIV = simian immunodeficiency v., TTV = transfusion-transmitted v., FV = foamy v (human and simian FV), HAV = hepatitis A v., GBV-B = GB virus B, HV = herpes v., IV = influenza v., PIV = parainfluenza v., HCMV = human cytomegalovirus, HGV = hepatitis G v., HMPV = human metapneumovirus, H/S TLV = human/simian T-cell leukemia v., LCV = lymphocryptoviruses, RV2 = rhadinovirus (or gamma-2-herpesvirus) genogroup 2, VZV = varicella-zoster v., WMHBV = woolly monkey hepatitis B v.

Therapeutic investigations

3.5% (26/749) of all chimpanzee experiments.

61.5% (16/26) of these investigated the pharmacological properties of various compounds.

Others included: the testing of surgical techniques or prostheses, anaesthesiology and toxicology experiments.

Implications?

On the face of it, research on captive chimpanzees or chimpanzee tissue appears to have contributed towards a large array of biomedical disciplines.

However, …

Not all knowledge has significant value, nor is worth the animal welfare-related, bioethical and financial costs that may be incurred!

Statistically-significant subset of 95 chimpanzee studies:

49.5% (47/95) were not cited by any future papers.

Given that much research of lesser value is not published…

True conclusion:

The majority of chimpanzee experiments generate data of questionable value, which makes little obvious contribution toward the advancement of biomedical knowledge.

Efficacy in combating human diseases

38.5% (34/95) were cited only by 116 subsequent papers that clearly did not describe well developed methods for combating human diseases.

Instead, papers focused primarily on:

non-human species ranging from bacteria to elephants, including a large variety of primates;

human subjects in relation to a variety of biological disciplines other than pathology;

examinations of the aetiological or other aspects of human diseases.

Only 14.7% (14/95) of chimpanzee studies were cited by a total of 27 papers that appeared to describe prophylactic, diagnostic or therapeutic methods with sound potential for combating human diseases.

Figure 4: Citations of 95 randomly selected

published chimpanzee studies

47

34

14

0

10

20

30

40

50

Not subsequentlycited

Cited by otherpaper

Cited by medicalpaper

Contributions of chimpanzee studies

63.0% (17/27) of these medical papers: wide-ranging reviews of 26-300 (median 104) references, to which the cited chimpanzee study made a relatively small contribution.

Research methodologies contributing most: in vitro studies human clinical and epidemiological studies molecular assays and methods genomic studies

12 cases: the cited chimpanzee studies appeared redundant, as humans or human sera were studied concurrently, or because they only served to confirm previous human-based observations.

7 cases: the methods explored in the chimpanzee study were not developed further, sometimes because later clinical trials in humans failed to demonstrate safety or efficacy, contrary to positive chimpanzee results.

5 cases: the chimpanzee study examined a disease or method peripheral to the prophylactic, diagnostic or therapeutic method described.

Remainder: the chimpanzee study yielded results inconsistent with other human or primate data, or merely illustrated historical findings, or was only cited in order to discuss concurrent human outcomes within the cited chimpanzee study.

No chimpanzee study demonstrated an essential contribution, or ― in a clear majority of cases ― a significant contribution of any kind, towards papers describing well-developed prophylactic, diagnostic or therapeutic methods for combating human diseases!

27 systematic reviews:overall results

The authors concluded that the animal models were useful in advancing human clinical outcomes or substantially consistent with human outcomes in only 2 of 20 studies, and the conclusion in 1 case was contentious.

7 reviews failed to clearly demonstrate utility in predicting human toxicological outcomes such as carcinogenicity and teratogenicity.

Consequently, animal data may not generally be assumed to be substantially useful for these purposes.

Scientific limitations of animal models

Differences between species and genders — with subsequent effects on toxico- and pharmacokinetics (the study of bodily distribution), or pharmacodynamics (the study of mechanisms of action, and drug effects).

Loss of biological variability or predictivity resulting from the use of in-bred strains, young animals, restriction to single genders, and inadequate group sizes.

Lack of co-morbidities (concurrent illnesses) or other human risk factors.

Physiological or immunological distortions resulting from stressful environments and procedures.

1. Interspecies differences

Altered susceptibility to, causes and progression of diseases

Differing absorption, tissue distribution, metabolism, and excretion of pharmaceutical agents and toxins

Differences in the toxicity and efficacy of pharmaceuticals

2. False positive results of chronic high dose rodent

studies

Differences in rodent physiology when compared to humans, for example, increased metabolic and decreased DNA excision repair rates

Overwhelming of natural physiological defences such as epithelial shedding, inducible enzymes, DNA and tissue repair mechanisms, which effectively protect against many naturally occurring toxins at environmentally relevant levels

Unnatural elevation of cell division rates during ad libitum feeding studies

Variable, yet substantial, stresses caused by handling and restraint, and frequently stressful routes of administration, and subsequent effects on hormonal regulation, immune status and disease predisposition

3. Poor methodological quality of animal

experiments At least 11 systematic reviews demonstrated the poor

methodological quality of many of the animal experiments examined

None demonstrated good methodological quality of a majority of experiments

Common deficiencies: lack of sample size calculations, sufficient sample sizes, randomised treatment allocation, blinded drug administration, blinded induction of ischaemia in the case of stroke models, blinded outcome assessment and conflict of interest statements.

Balls et al. (2004):

“…surveys of published papers as well as more anecdotal information suggest that more than half of the published papers in biomedical research have statistical mistakes, many seem to use excessive numbers of animals, and a proportion are poorly designed”.

Conclusions

The historical and contemporary paradigm that animal models are generally fairly predictive of human outcomes provides the basis for their continuing widespread use in toxicity testing and biomedical research aimed at developing cures for human diseases.

However, their use persists for historical and cultural reasons,

rather than because they have been demonstrated to be scientifically valid. E.g., many regulatory officials “feel more comfortable” with animal data (O’Connor 1997), and some even believe animal tests are inherently valid, simply because they are conducted in animals (Balls 2004).

However, most systematic reviews have demonstrated that animal models are insufficiently predictive of human outcomes to offer substantial benefit in advancing clinical outcomes, or in deriving human toxicity assessments.

Consequently, animal data may not generally be assumed to

be of substantial use for these purposes.

The poor human clinical and toxicological utility of most animal models for which data exists, in conjunction with their generally substantial animal welfare and economic costs, justify a ban on animal models lacking scientific data clearly establishing their human predictivity or utility.

Animal experiments may sometimes be followed by a concordant human outcome. However, they are a highly inefficient means of advancing human health.

Additionally, they accord insufficient ethical weighting to the interests of other species.

3Rs alternatives

Replacement Reduction Refinement

(Recycling?) (Rehabilitation)

Replacement alternatives

Mechanisms to enhance sharing and assessment of existing data, prior to conducting further studies.

Physicochemical evaluation and computerized modelling, including the use of structure-activity relationships (predict biological activity such as toxicity on the basis of structure), and expert systems (seek to mimic the judgment of expert toxicologists, by using known rules about factors affecting toxicity, in combination with physicochemical or other information about a specific compound).

These allow predictions about toxicity and related biological outcomes, such as metabolic fate.

Minimally-sentient animals from lower phylogenetic orders, or early developmental vertebral stages, as well as microorganisms and higher plants.

A variety of tissue cultures, including immortalised cell lines, embryonic and adult stem cells, and organotypic cultures.

In vitro assays (tests) utilising bacterial, yeast, protozoal, mammalian or human cell cultures exist for a wide range of toxic and other endpoints. These may be static, or perfused, and used individually, or combined within test batteries.

Human hepatocyte (liver cell) cultures and metabolic activation systems offer potential assessment of metabolite (product of metabolism, usually by the liver) activity — a very important consideration when assessing toxicity.

cDNA microarrays (‘gene chips’) allow assessment of large numbers of genes simultaneously. This may allow genetic expression profiling (detection of up- or down-regulation of genes, caused by exposure to test compounds). This can increase the speed of toxin detection, well prior to more invasive endpoints.

The safety profile and predictivity for diverse human patient populations of clinical trials should be improved using microdosing, biomarkers, staggered dosing, more representative test populations, and longer exposure periods.

Surrogate human tissues and advanced imaging modalities.

Human epidemiological, psychological and sociological studies.

Particularly when human tissues are used, non-animal models may generate faster, cheaper results, more reliably predictive for humans, yielding greater insights into human biochemical processes.

Reduction alternatives

Improvements in experimental design and statistical analysis; particularly, adequate sample sizes.

Minimising animal numbers without unacceptably compromising statistical power, through decreasing data variability:

Environmental enrichment, aimed at decreasing physiological, psychological or behavioural variation resulting from barren laboratory housing and stressful procedures.

Choosing, where possible, to measure variables with low inherent variability.

Genetically homogeneous (isogenic or inbred) or specified pathogen-free animal strains.

Screening raw data for obvious errors or outliers.

Meta-analysis (aggregation and statistical analysis of suitable data from multiple experiments). For some purposes, treatment and control groups can be combined, permitting group numbers to be minimised.

Refinement alternatives

Analgesics and anaesthetics. (Around 60% of UK procedures are conducted without anaesthetics). While such drugs undoubtedly alter normal physiology, claims that such alterations are sufficiently important to hypotheses under investigation, to warrant their exclusion, require careful scrutiny.

Non-invasive imaging modalities. Telemetric devices to obtain information remotely. Faecal analysis (e.g. faecal cortisol monitoring). Training animals (especially primates) to participate (e.g.

presenting arms for blood-sampling), rather than using physical or chemical restraint.

Environmental enrichment. Socialisation opportunities.

Increasing 3Rs compliance

Technology reproducibility and transfer: increased methodology description, e.g., via publicly-accessible databases, linked to scientific articles.

Redirection of public funds from animal models to alternatives development/implementation.

Prerequisite for research funding, ethics committee approval, and publication of results. Would require education and cooperation of funding agencies, ethics committees and journal editors about the limitations of animal models, and the potential of alternatives.

National centre for the development of alternative methods.

Scientific recognition: awards, career options.

Greater selection of test models that are truly predictive of human outcomes

Increased safety of humans exposed to chemicals that have passed toxicity tests

Increased efficiency during the development of human pharmaceuticals and other therapeutic interventions

Decreased wastage of animal, personnel and financial resources.

Likely benefits

The scientific and logistical limitations incurred by the use of animal models of humans within biomedical research and toxicity testing are substantial, and increasingly recognized.

So is social concern about, and consequent regulatory restriction of, laboratory animal use.

In defiance of these factors, such use remains enormous. The use of GM animals, and the implementation of large-scale chemical testing programs, are increasing laboratory animal use internationally.

Conclusions

These trends clearly demonstrate the need for considerably greater awareness of, and compliance with, the principles of the 3Rs.

These principles are universally recognized as essential to good laboratory animal practice, for animal welfare-related and ethical reasons, and also, to increase the quality of the research, and the robustness of subsequent results.

Published papers: www.animalexperiments.info www.aknight.info, ‘publications’