Animal Experiments

and Alternatives

    

ANDREW KNIGHT ANDREW KNIGHT

DipECAWBM (WSEL), PhD, MRCVS, FOCAEDipECAWBM (WSEL), PhD, MRCVS, FOCAE

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 test plans often failed to follow minimal EPA guidance about 3Rs alternatives:

failed to use existing published data failed to avoid duplicative or otherwise unnecessary animal testing proposed irrelevant or unnecessary tests (such as acute fish toxicity

tests on water-insoluble chemicals) Ignored opportunities to use non-animal tests failed to utilise opportunities to combine protocols, sometimes

doubling the number of animals killed

In its responses to test plan proposals, the EPA frequently failed to encourage companies to follow basic animal welfare principles (Sandusky et al., 2006).

High Production Volume (HPV) test program

Scientific 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 some laboratory animal species that are harmless to humans:

Penicillin Morphine Aspirin …

Drugs released onto the market after passing more 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 have been recorded as the 4-6th leading cause of death in US hospitals, and kill over 10,000 people annually in the UK.

Calls for systematic reviews

Clinicians and the public often consider it axiomatic that animal research has contributed to human clinical knowledge, on the basis of anecdotes 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.

- Pound et al. Brit Med J, 2004.

Systematic reviews: ‘gold standard’ evidence

Critically examine human clinical or toxicological utility of animal experiments

Examine large numbers of experiments, usually sourced via multiple bibliographic databases

Any subsets of experiments must be selected without bias, via randomisation or similarly methodical and impartial means

Studies published in peer-reviewed biomedical journals

- Altern Lab Anim, 2007.- Rev Recent Clin Trials, 2008.

- Palgrave Macmillan, 2011.

27 systematic reviews of the utility of animal studies 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 studies…

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 partly on the basis of researcher claims 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 when applied to humans.

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

2. Clinical utility of highly cited animal

experiments

Animal studies with > 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

- Hackam & Redelmeier. J Am Med Assoc, 2006.

Even in these cases human benefit cannot be assumed,

because adverse reactions to approved interventions are the 4th - 6th leading cause of death in US hospitals

- Lazarou & Pomeranz. J Am Med Assoc, 1998.

Translation rates of most animal experiments are

much 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 warranted.

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

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

Common deficiences:

lack of random allocation of animalsblinded assessment of outcomes

- Hackam & Redelmeier. J Am Med Assoc, 2006.

Poor methodological quality

3. Invasive chimpanzee research

Passionate calls for increased funding of such research, e.g. VandeBerg et al. (Nature 2005):

Such research has been of critical importance during struggles against major human diseases such as AIDS, hepatitis and cancer

The genetic similarities between humans and chimpanzees — our closest living relatives — makes them ideal biomedical research models

J Appl Amim Welf Sci, 2007 Philos, Ethics, Humanit Med, 2008

Contributions to biomedical knowledge

749 studies of captive chimpanzees or chimpanzee tissues, 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.

Remaining chimpanzee studies

pharmacological and toxicological studies of various compounds

testing of surgical techniques or prostheses, and anaesthesiology experiments

investigations of laboratory/husbandry techniques

radiation studies

various disease studies, including endotoxaemia and eight parasitic species

Implications?

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 costs that may be incurred.

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

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 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 demonstrate reliable predictivity of human toxicological outcomes such as carcinogenicity and teratogenicity

- Knight. - Knight. Altern Lab Anim, Altern Lab Anim, 2007.2007.

- Knight. - Knight. Rev Recent Clin Trials, Rev Recent Clin Trials, 2008.2008.

Causes: 1. Interspecies differences

Altered susceptibility to 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 comorbidities (concurrent illnesses) or other human risk factors.

Physiological or immunological distortions resulting from stressful environments and procedures.

2. Stressful environments and protocols

Most laboratory animals spend most of their lives in small, relatively barren cages. A review of 110 studies from the biomedical literature revealed the outcomes:

- Balcombe - Balcombe et al. Lab Anim et al. Lab Anim 20062006

Impacts of laboratory housing

Deleterious neuroanatomical, psychological (eg, stereotypical behaviour) and physiological effects

Distortion of many subsequent scientific results

Even so-called ‘enriched’ environments fail to ameliorate most of these deficits

- Balcombe - Balcombe et al. Lab Anim et al. Lab Anim 20062006

Impacts of common procedures

All common laboratory species suffer marked stress, fear and possibly distress (indicated by the distortion of a broad range of physiological parameters) when subjected to:

Handling Blood sampling Gavaging (insertion of an esophageal tube for

the oral administration of test compounds — a common procedure in toxicity studies)

- Balcombe - Balcombe et al. Contemporary Topics Lab Anim Sci et al. Contemporary Topics Lab Anim Sci 20042004

Animals do not readily habituate to these procedures over time.

This stressful alteration of normal physiological parameters also predisposes to a range of pathologies and distorts scientific results.

- Balcombe - Balcombe et al. Contemporary Topics Lab Anim Sci et al. Contemporary Topics Lab Anim Sci 20042004

3. False positive results of chronic high dose rodent

studies

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

Differences in rodent physiology when compared to humans, e.g.: increased metabolic and decreased DNA excision repair rates

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

4. 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 deficiences

Lack of:

sample size calculationssufficient sample sizesrandomised treatment allocationblinded drug administrationblinded induction of injury (ischaemia in the case of stroke models)blinded outcome assessmentconflict 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 assessment.

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

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, and expert systems.

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 (a 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 modelling to alternatives development/implementation.

Increased 3Rs compliance should be necessary 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 centres for the development of alternative methods.

Scientific recognition: awards, career options.

Greater selection of test models more predictive of human outcomes

Increased safety of people 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. Increased 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.

Policy reforms:1. Animals protected

Regulatory protection should be based on current scientific knowledge about:

neuroanatomical architecturecognitive, psychological, and social characteristicsconsequent capacity for suffering in laboratory environments and protocols.

Sufficient scientific evidence exists to warrant the protection of:

all living vertebratesadvanced larval forms and foetal developmental stagescertain invertebrates such as cephalopods

Similar protection is warranted for:

animals used to develop or maintain GM strainsbred for organ or tissue harvestingbred or intended for laboratory use, including those killed when surplus to requirements

2. Species and procedures associated with high welfare risks

Primate sourcing and useTerminal or surgical proceduresMajor physiological challengesThe production of GM animalsProcedures resulting in pain, suffering, or distress likely to be severe or long-lasting

3. Scrutiny of animal use

Independent scientific and public scrutiny of proposed protocolsIndependent ethical review

Directive 2010/63/EU on the protection of animals

used for scientific purposes

‘It is essential, both on moral and scientific grounds, to ensure that each use of an animal is carefully evaluated as to the scientific or educational validity, usefulness and relevance of the expected result of that use.’

‘The likely harm to the animal should be balanced against the expected benefits of the project.’

Thorough searches for replacement, reduction, and refinement methodologies Where scientifically suitable alternatives are identified, they should be used

4. Retrospective evaluation

To assess the degree to which experimental objectives were successfully metThe extent to which animals sufferedTo inform future research strategyFuture experimental licensing decisionsMinimise unwarranted experimental duplication

Cited studies:www.AnimalExperiments.info

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