Animal Experiments and Alternatives

Animal Experiments: Human Utility and Alternative Methodologies Andrew Knight BSc., BVMS, CertAW, MRCVS, FOCAE Animal Consultants International www.AnimalConsultants.org

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).

 Chemical companies submitting High Production Volume (HPV) test plans to the US EPA have often failed to follow even minimal EPA guidance about 3Rs alternatives. They have:  failed to use existing published data,  failed to avoid duplicative or otherwise unnecessary animal testing,  proposed irrelevant tests (such as acute fish toxicity tests on water- insoluble chemicals), or tests clearly unnecessary to meet HPV requirements.  Opportunities to use non-animal tests have been ignored,  companies have failed to take advantage of opportunities to combine protocols, sometimes doubling the number of animals killed.  In its responses to such test plan proposals, the EPA frequently failed to encourage companies to follow basic animal welfare principles (Sandusky et al., 2006).

Support for 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 animals that are harmless to humans:  Penicillin  Morphine  Aspirin  …

Drugs released onto the market after passing rigorous testing in animals, and very limited testing in humans, that have caused serious human side effects:  TGN1412 (UK, 2006)  Vioxx  Thalidomide, Eraldin, Chloramphenid, Ibufenac, Flosint, Zipeprol, Zomax, Accutane, Benedectin, Phenformin  Many, many more… Such adverse drug reactions are the 4-6th leading cause of death in US hospitals, and kill over 10,000 people annually in 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 et al. (2005 & 2006) examined animal experiments conducted at three German universities between 1991 and 1993, that had been approved by animal ethics committees at least partly on the basis of claims by researchers that the experiments might lead to concrete advances towards the cure of human diseases. For 17 experiments meeting the inclusion criteria, citations were analysed for at least 12 years. 1,183 citations were evident. However …

Only 8.2% of all citations (97) were in clinical publications. Of these, only 0.3% of all citations (4 publications) demonstrated a direct correlation between the results of animal experiments and human outcomes. However, even in these four cases the hypotheses that had been successfully verified in animals failed completely when applied to humans. None of these 17 experiments led to any new therapies, or, indeed, any beneficial clinical impact during the period 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 randomised trials. 18.4% (14/76) were contradicted by randomised 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. Even in these cases human benefit cannot be assumed, because adverse reactions to approved interventions are sufficiently common that they are the 4 -6th leading cause of death in US hospitals (Lazarou & Pomeranz 1998).

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).

Poor methodological quality 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.

3. Chimpanzee experimentation Prominent advocates of biomedical 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 critical 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.

Figure 1: Citations of 95 randomly-selected published chimpanzee studies 50 47 45 40 34 35 30 25 20 14 15 10 5 0 Not Cited by other Cited by human subsequently paper medical paper cited

Citations by medical papers 63% (17/27) of these medical papers were wide-ranging reviews of 26 - 300 (median 104) references, to which the cited chimpanzee study made only a small contribution. No chimpanzee study made an essential contribution, or, in a majority of cases, a significant contribution of any kind, towards papers describing well-developed 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 useful for these purposes.

Causes: 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

 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.

2. False positive results of chronic high dose rodent studies  Differences in rodent physiology when compared to humans, e.g.: 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 (‘at will’) 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.

Conclusions  Historical and contemporary paradigm:  Animal models are fairly predictive of human outcomes.  Provides the basis for their 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).  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 during the development of clinical interventions, or during human toxicity assessments.  Consequently, animal data may not generally be assumed to be useful 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.

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.

Likely benefits  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.

Conclusions 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.

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.

References/published papers www.AnimalExperiments.info Thanks for your attention!

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Animal Experiments and Alternatives

by Andrew Knight on May 11, 2010

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