Category Archives: CURRENT DILEMMAS

Single housing of primates in US laboratories: a growing problem with shrinking transparency

Alka Chandna, Michael Niebo, Stacy Lopresti-Goodman and Justin Goodman

Thirty years ago, the United States took steps to enhance
the psychological well-being of primates in laboratories,
including the introduction of social housing requirements.
Now, in an apparent response to questions about
the effectiveness of these measures, federal authorities
are completely shutting down public access to
information on the implementation of social housing

Historical context

In 1985, in response to high-profile cases documenting the mistreatment of non-human primates used in experiments, 1 the US Congress amended the federal Animal Welfare Act (AWA) to mandate that institutions take steps to promote the psychological wellbeing of primates in laboratories.2 As a result, the US Department of Agriculture (USDA) created regulations pertaining to environment enhancement for primates, including provisions aimed at addressing their social needs.3 In federal regulations and guidelines, social housing of primates is now deemed to be the ‘default’, with exemptions permitted only for veterinary or experimental reasons with appropriate documentation and approval.4 In cases in which an experiment-related exemption was granted, institutions have been required to submit these written and approved justifications to the USDA with their annual reports on animal use. These documents — which report the number of primates singly housed for experimental reasons, and why it was deemed necessary — were then made publicly available.

Animal welfare science

The USDA’s primate social housing requirements are evidence-based, as social housing is universally acknowledged to be a key factor in the welfare of primates in laboratories. Moreover, it is well documented that housing primates alone, or single housing, is detrimental to their development, physical health, and psychological well-being. In rhesus macaques, physical contact with conspecifics is essential to normal development, and the amount of time spent caged alone is a significant predictor of stereotypic and self-injurious behaviour — including repeated pacing, circling, hyper-aggression, depression, hair plucking, or self-biting.5, 6  Psychological distress stemming from being caged alone has also been documented in cynomolgus monkeys,7  pigtailed macaques,8 chimpanzees,9 – 11 and baboons.12, 13 In one modified preference test involving capuchin monkeys, the value of social companionship was so high that the primates chose it in lieu of food.14 Singly housed primates have also been documented as having suffered from physiological abnormalities, including depressed immune function and higher incidence of coronary atherosclerosis.15, 16

Surveys on housing of non-human primates

Despite this evidence of the harms caused by single housing, as well as federal regulations and guidelines promoting social housing, the data indicate that many primates in US laboratories continue to be housed alone. A 2000–2001 USDA survey found that 34.7% of primates in US laboratories were housed individually — although the USDA admitted that this was likely a low estimate, as primates who had been housed only temporarily with other primates for breeding purposes were classified as being socially housed.17 A 2003 survey found that 54% of primates at 22 laboratories were singly housed — although this was also likely to have been a low estimate, as the study ill-advisedly included in its definition of “social housing” instances of “grooming-contact” housing, in which singly housed primates have some limited tactile contact with one another through barred or mesh barriers.18 A survey of primate housing at the Washington National Primate Research Center from 2004 to 2006 found that at least 63% of the monkeys were singly caged.19

An ongoing concern is that laboratories sometimes permit single housing of primates for the sake of convenience rather than necessity. For example, many laboratories singly house primates who have had surgical implants, such as head posts or other equipment, even though it is possible to successfully house them socially.20

Given the government mandate that social housing of primates in laboratories should be the default position, the aforementioned figures are cause for concern, particularly because they indicate that rates of single housing may be increasing.

Preliminary analysis of primate single-housing data

In the interest of conducting a current and more comprehensive evaluation of trends in primate single housing and the justifications provided, we attempted to undertake a new analysis of all single housing exceptions submitted in annual reports by laboratories to the USDA from 2010 to 2013 — the years for which data are currently available online. While the total number of facilities that confined primates (191 in 2010, 188 in 2011, 192 in 2012, and 184 in 2013) stayed relatively flat over the four years, there was a steady increase in the number of facilities that reported single-housing exceptions, from 30 (16%) in 2010 to 53 (29%) in 2013. However, when we attempted to look more closely at these exceptions, in order to determine trends in the numbers of singly housed primates and the  explanations given, we found glaring inadequacies in the data available on the USDA site. This inadequacy stemmed mainly from the failure of laboratories in meeting reporting requirements. From 2010 to 2013, the percentage of laboratories reporting singly
housed primates that failed to specify the number of primates singly housed for experimental reasons and the scientific justification for it — both required by law — increased from 36% to 47%. Worryingly, it also appeared that some, or all, of the required information was improperly redacted from many facilities’ reports. We also observed that several facilities that had provided very detailed exception letters in 2010 produced only very vague information in 2013 or, as noted above, redacted all the information, showing
a growing trend toward secrecy.

Government response

In December 2014, we informed the USDA of the problematic reports and requested the agency’s assistance  in securing the missing data. In February 2015, we received correspondence from the USDA (a personal communication), which stated, “We have had
discussions and are finalising our position.” We heard nothing further, but in March, the USDA sent an email to all its stakeholders announcing changes to its Inspection Guide — used by its inspectors to identify violations of the AWA at the facilities they inspect.
The announcement noted that the revisions included a “more consistent procedure for reporting exceptions and exemptions on the Annual Report.”21 Upon closer examination, it became clear that the revised inspection guide now specified that exemption of a primate from “some or all of the environmental enhancement plan” — which would include social housing — “should not be reported on the Annual Report” [emphasis in the original].22 Prior to the recent revision to the inspection guide, it was standard operating procedure for laboratories to report single housing of primates.

Rather than ensuring that laboratories were properly reporting single housing of primates, the USDA instead — we suspect in consultation with the laboratory community — took the backward step of simply exempting facilities from having to submit this information at all.

Discussion

A 2011 study seeking to assess the effectiveness of the 1985 AWA primate psychological health amendments determined that “the current system of laboratory animal care and record keeping is inadequate to properly assess AWA impacts on primate psychological wellbeing and that more is required to ensure the psychological well-being of primates.”17 It was already difficult to ascertain this information, and now the USDA has made this task virtually impossible. Even with the reporting requirement in place, laboratories were often failing to approve, document, and report the single housing of primates. For instance, a 2011 USDA inspection report for a US contract laboratory cited the company for singly housing 83% of the more than 6,000 primates at the facility  without securing the necessary justifications or reporting the matter to the USDA.23,24 This number amounts to more than 4% of all primates housed in US laboratories.

Any previously reported figures are also underestimations, because institutions have only been required to report the numbers of primates who are singly housed for ‘experimental’, but not veterinary, reasons; this could account for a third more singly housed primates.25

Conclusions

On the 30th anniversary of the amendments to promote the psychological well-being of primates, the evidence that singly housed primates suffer is overwhelming, as is the proof that the US government and laboratories are failing to confront this rapidly growing problem effectively. The USDA’s recent revisions to reporting requirements will limit the availability of data, and consequently will stifle informed debate on the suffering of primates in laboratories and failures of the existing regulatory system. To address this issue meaningfully, we need more transparency and accountability, not less.

Author for correspondence:
Dr Alka Chandna
People for the Ethical Treatment of Animals
501 Front Street
Norfolk, VA 23510
USA
E-mail: AlkaC@peta.org

Michael Niebo
People for the Ethical Treatment of Animals
501 Front Street
Norfolk, VA 23510
USA

Dr Stacy Lopresti-Goodman
Department of Psychology
Marymount University
2807 N. Glebe Road
Arlington, VA 22207
USA

Justin Goodman
People for the Ethical Treatment of Animals
501 Front Street
Norfolk, VA 23510
USA
and
Department of Sociology
Marymount University
2807 N. Glebe Road
Arlington, VA 22207
USA

References

1 Carbone, L. (2004). What Animals Want: Expertise and Advocacy in Laboratory Animal Welfare Policy, 304pp. Oxford, UK: Oxford University Press.
2 US Congress (1985). Public Law 99-198, Food Security Act of 1985, Subtitle F — Animal Welfare. Title XVII. Available at: https://awic.nal.usda.gov/public-law-99-198-food-security-act-1985-subtitle-f-animalwelfare (Accessed 31.05.15).
3 US Department of Agriculture (1991). Final Rules: Animal Welfare; Title 9, CFR (Code of Federal Register) Part 3. Standards. Federal Register 55 (No. 32), 6426–6505. Available at: https://awic.nal.usda. gov/final-rules-animal-welfare-9-cfr-part-3 (Accessed 31.05.15).
4 National Research Council (2011). Guide for the Care and Use of Laboratory Animals: Eighth Edition, 248pp. Washington, DC, USA: The National Academies Press.
5 Baker, K.C., Bloomsmith, M.A., Oettinger, B., Neu, K., Griffis, C., Schoof, V. & Maloney, M. (2012). Benefits of pair housing are consistent across a diverse population of rhesus macaques. Applied Animal Behaviour Science 137, 148–156.
6 Bayne, K. (2005). Macaques. From the booklet series Enrichment for Nonhuman Primates. Washington, DC, USA: Department of Health and Human Services. Available at: http://grants.nih.gov/grants/olaw/ Enrichment_for_Nonhuman_Primates.pdf (Accessed 05.04.15).
7 Shively, C.A., Clarkson, T.B. & Kaplan, J.R. (1989). Social deprivation and coronary artery atherosclerosis in female cynomolgus monkeys. Atherosclerosis 77, 69–76.
8 Bellanca, R.U. & Crockett, C.M. (2002). Factors predicting increased incidence of abnormal behavior in male pigtailed macaques. American Journal of Primatology 58, 57–69.
9 Brent, L., Lee, D.R. & Eichberg, J.W. (1989). The effects of single caging on chimpanzee behavior. Laboratory Animal Science 39, 345–346.
10 Kalcher, E., Franz, C., Crailsheim, K. & Preuschoft, S. (2008). Differential onset of infantile deprivation produces distinctive long‐term effects in adult ex‐laboratory chimpanzees (Pan troglodytes). Developmental Psychobiology 50, 777–788.
11 Nash, L.T., Fritz, J., Alford, P.A. & Brent, L. (1999). Variables influencing the origins of diverse abnormal behaviors in a large sample of captive chimpanzees (Pan troglodytes). American Journal of Primatology 48, 15–29.
12 Coelho, A.M., Carey, K.D., & Shade, R.E. (1991). Assessing the effects of social environment on blood pressure and heart rates of baboons. American Journal of Primatology 23, 257–267.
13 Kessel, A. & Brent, L. (2001). The rehabilitation of captive baboons. Journal of Medical Primatology 30, 71–80.
14 Dettmer, E. & Fragaszy, D. (2000). Determining the value of social companionship to captive tufted capuchin monkeys (Cebus apella). Journal of Applied Animal Welfare Science 3, 293–304.
15 Lilly, A.A., Mehlman, P.T. & Higley, J.D. (1999). Traitlike immunological and hematological measures in female rhesus across varied environmental conditions. American Journal of Primatology 48, 197–223.
16 Doyle, L.A., Baker, K.C. & Cox, L.D. (2008). Physiological and behavioral effects of social introduction on adult male rhesus macaques. American Journal of Primatology 70, 542–550.
17 Balcombe, J., Ferdowsian, H. & Durham, D. (2011). Self-harm in laboratory-housed primates: Where is the evidence that the Animal Welfare Act amendment has worked? Journal of Applied Animal Welfare Science 14, 361–370.
18 Baker, K.C., Weed, J.L., Crockett, C.M. & Bloomsmith, M.A. (2007). Survey of environmental enhancement programs for laboratory primates. American Journal of Primatology 69, 377–394.
19 Thom, J.P. & Crockett, C.M. (2008). Managing environmental enhancement plans for individual research projects at a national primate research center. Journal of the American Association for Laboratory Animal Science 47, 51.
20 DiVincenti, L., Jr & Wyatt, J.D. (2011). Pair housing of macaques in research facilities: A science-based review of benefits and risks. Journal of the American Association for Laboratory Animal Science 50, 856.
21 USDA (2015). USDA Revises Its Inspection Guide to Improve Oversight of Research Facilities. Washington, DC, USA: United States Department of Agriculture. Available at: http://content.govdelivery.com/ accounts/USDAAPHIS/bulletins/f4e94e (Accessed 31. 05.15).
22 USDA (2015). Animal Welfare Inspection Guide, 424pp. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.aphis. uda.gov/animal_welfare/downloads/Animal%20Care %20Inspection%20Guide.pdf (Accessed 31.05.15).
23 USDA (2011). Inspection Report for SNBL USA, Ltd. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.mediapeta. com/peta/PDF/July132011-78percent singly housed.pdf (Accessed 25.06.15).
24 USDA (2011). Annual Report for SNBL USA, Ltd. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.mediapeta. com/peta/PDF/SNBL-AnnualReportfor2011.pdf
(Accessed 25.06.15).
25 Lee, D.R. (2013). Social housing strategies for nonhuman primates [PowerPoint slides]. Available at: http://www.aclam.org/content/files/files/forum2013/aclam_forum_2013_lee_social.pdf (Accessed 05.04. 15).

Download a pdf of the article here: Primate single housing.

In Vitro Methodologies in Ecotoxicological Hazard Assessment: The Case of Bioaccumulation Testing for Fish

Helmut Segner

The concerted research efforts undertaken in recent years have
highlighted the potential of in vitro approaches, as part of an
integrated testing strategy, to replace or reduce in vivo bioaccumulation testing in fish

Worldwide programmes for the regulation of chemicals require an assessment of the risks of chemicals to human and environmental health based on three categories of concern: Persistence, Bioaccumulation and Toxicity (PBT). Among these three categories, bioaccumulation refers to the enrichment of environmental chemicals in organisms. It encompasses the absorption, distribution, metabolism and excretion (ADME) of a chemical inside an organism, and ultimately determines the internal toxic dose. For the aquatic environment, the most widely used parameter to estimate the bioaccumulation potential of a chemical is the so-called Bioconcentration Factor (BCF). The BCF represents the ratio of the steadystate chemical concentration in the organism and the chemical concentration in the respiratory medium, i.e. water. For the experimental determination of the BCF, the test procedure as described in OECD Test Guideline 3051 represents the current ‘gold standard’. In this test, fish are exposed to a chemical for 28 days, to reach an equilibrium of chemical concentration between fish and water, followed by a 28-day depuration period to measure the elimination rate. This test, in addition to being lengthy and costly, requires a high number of animals (> 100 fish per test).

Regulatory programmes require bioaccumulation information for chemicals which are lipophilic (for example, those with a log Kow > 3), and which are produced at a certain tonnage (for example, the European Community REACH Regulation requires BCF information for lipophilic chemicals that are produced at > 100 tonnes per year). Experi mentally determined BCF data are not available for the vast majority of existing compounds. For instance,  in a Canadian investigation of 23,000 existing chemicals, it was found that bioaccumulation data existed for less than 4% of them (cf. Nichols et al.2). If the missing BCF data had to be generated by means of the OECD 305 test, this would entail a drastic increase in animal use.3,4 Therefore, there is an urgent need to develop alternative methods to reduce the number of fish used for in vivo bioaccumulation testing. The bioconcentration of chemicals in fish results from the competing rates of chemical uptake via the gills and skin (k1) and chemical elimination via respiratory exchange (k2), faecal egestion (ke) and metabolic biotransformation (km).5 In addition, dilution as a result of growth (kd) can influence bioconcentration.

With the involvement of these different processes, it is clear that non-animal approaches to bioconcentration assessment cannot be based on one single method, but have to rely on an array of methodologies.2,4,6 An initial non-animal based approximation of the bioconcentration potential of an organic chemical in aquatic organisms can be obtained from an in silico hydrophobicity model, which considers bioconcentration as a passive partitioning process resulting from the competing uptake and elimination processes. In this model, bioconcentration can be predicted from the lipophilicity of a chemical, as estimated from its octanol–water partition coefficient, Kow.5 Also, it can actually be measured by using artificial membranes which simulate the passive diffusion processes across the respiratory epithelia.7

The development of in vitro methods

Diffusion-based methodologies have proven instrumental in the prediction of the BCF values of lipophilic chemicals that undergo no endogenous metabolism in the organism. However, as they are not able to take into account chemical loss due to biotransformation (km), they overestimate the BCF values of metabolisable xenobiotics. To correct for the influence of biotransformation on fish BCF values, a possible approach is the use of metabolically competent in vitro assays that show which chemicals are biotransformed, and at what rates. In mammalian toxicology, in vitro assays for the analysis of xenobiotic metabolism largely rely on liver preparations such as subcellular liver fractions (S9, microsomes) and isolated hepatocytes, as the liver is the organ with the highest metabolic activity. Corresponding technologies are also available for fish, and it has been demonstrated that they are suitable for determining biotransformation parameters (see Segner & Cravedi8 and Fitzimmons et al.9). However, their reproducibility and their capability of predicting in vivo BCF values remain to be demonstrated.

In recent years, intensive efforts have been undertaken — largely coordinated by the Health and Environmental Sciences Institute (HESI) — to advance the development of piscine in vitro assays for regulatory purposes. After an initial phase of reviewing the available knowledge and technologies,2,6 in the next step, standardised protocols for liver S9 preparations and isolated hepatocytes from rainbow trout were established.10,11 A major drawback experienced in these studies, particularly with freshly-isolated hepatocyte suspensions, was the between-isolate variability of metabolic capabilities, which is related to factors such as seasonal oscillation, and the nutritional status, gender or genetic background of the donor fishes. Here, a major step forward was the introduction of a cryopreservation method for fish hepatocytes,12 enabling the year-round provision of uniform batches of metabolically characterised hepatocytes to laboratories worldwide. By using a standardised assay protocol, Fay et al.13 recently performed an international ring study with cryopreserved rainbow trout hepatocytes, and were able to demonstrate the good interlaboratory and intra-laboratory reproducibility of the metabolic rate values obtained with the in vitro hepatocyte assay.

To be able to extrapolate from the metabolic rate values measured in the isolated fish hepatocytes to the metabolic rate value (km) in the intact fish, physiologically-based prediction models were developed. 14,15 These models initially scale from the clearance rate of the isolated liver cells to that of the whole liver, and from there to the metabolic transformation rate of the whole fish. The predicted km values are then used to calculate the in vivo BCF value of the test chemical. Currently, the availability of data on BCF values  predicted from in vitro assays is still limited, and it is still too early to come up with a conclusive statement on the predictability of the in vitro approach, partly also because of the variable quality of the in vivo BCF data; however, the existing results look promising.

Looking to the future

There are lessons to be learned from the recent development of in vitro assays as components of alternative integrated testing strategies for the assessment of bioaccumulation in fish. Although a broad spectrum of in vitro assays and methods have been available in ecotoxicology for a while,16 they have never made their way to regulatory implementation. Partly, this is due to the fact that they were considered to be technically not ready nor sufficiently standardised. In the case of the piscine in vitro metabolism assays, this obstacle has been overcome through targeted and internationally concerted research efforts on the standardisation and harmonisation of the assay protocols. Another constraint to the regulatory acceptance of in vitro assays is that they were considered not to be appropriate for the protection goals of ecotoxicology, which are ecological entities such as populations and communities. However, ecotoxicological hazard assessment largely relies on classical toxicity tests for measuring organism-level endpoints such as lethality (cf. Segner17), and these endpoints may well be predictable by in vitro assays, provided that: a) the in vitro assays are rationally selected to represent the critical toxicological processes; b) the assays are standardised; and c) valid extrapolation models are available. These requirements are fulfilled in the case of bioaccumulation assessment — i.e. the in vitro assays measure biotransformation as the critical toxicokinetic process, they are standardised, and there exist physiologically-based models for the scaling of the in vitro metabolic rate values to the in vivo metabolic rates. As ecotoxicology deals with a huge diversity of species, the interspecies scaling of metabolic rates is another critical issue, but this question is also currently under investigation. In conclusion, the concerted research efforts undertaken in recent years have substantially moved the field ahead, and the results obtained highlight the potential of in vitro approaches, as part of an integrated testing strategy,4 to replace or reduce in vivo bioaccumulation testing in fish.

Acknowledgements

The financial support of Stiftung Forschung 3R, ünsingen (Switzerland) and the Health and Environmental Sciences Institute (HESI) is gratefully acknowledged.

Prof. Dr Helmut Segner
Centre for Fish and Wildlife Health
Department of Infectious Diseases and Pathobiology
Vetsuisse Faculty
University of Bern
PO Box 8466
CH 3012 Bern
Switzerland
E-mail: Helmut.segner@vetsuisse.unibe.ch

References

1 OECD (2011). OECD Guideline for Testing of Chemicals No. 305. Bioaccumulation in Fish: Aqueous and Dietary Exposure, 72pp. Paris, France: Organisation for Economic Co-operation & Development.
2 Nichols, J.S., Erhardt, S., Dyer, M.J., Moore, M., Plotzke, K., Segner, H., Schultz, I., Thomas, K., Vasiluk, J. & Weisbrod, A. (2007). Use of in vitro Absorption, Distribution, Metabolism, and Excretion (ADME) data in bioaccumulation assessments for fish. Human & Ecological Risk Assessment 13, 1164–1191.
3 de Wolf, W., Comber, M., Douben, P., Gimeno, S., Holt, M., Léonard, M., Lillicrap, A., Sijm, D., van Egmond, R., Weisbrod, A., & Whale, G. (2007). Animal use replacement, reduction,  and refinement: Development of an integrated testing strategy for bioconcentration of chemicals in fish. Integrated Environmental Assessment & Management 3, 3–17.
4 Lombardo, A., Roncaglioni, A., Benfenati, E., Nendza, M., Segner, H., Fernández, A., Kühne, R., Franco, A., Pauné, E. & Schüürmann, G. (2014). Integrated testing strategy (ITS) for bioaccumulation assessment under REACH. Environment International 69, 40–50.
5 Arnot, J.A. & Gobas, F. (2006). A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environmental Reviews 14, 257–330.
6 Weisbrod, A.V., Sahi, J., Segner, H., James, M.O., Nichols, J., Schultz, I., Erhardt, S.,  Cowan-Ellsberry, C., Bonnell, M. & Hoeger, B. (2009). The state of   science for use in bioaccumulation assessments for fish. Environmental Toxicology & Chemistry 28, 86–96.
7 Kwon, J.H. & Escher, B.I. (2008). A modified parallel artificial membrane permeability assay for evaluating bioconcentration of highly hydrophobic chemicals in fish. Environmental Science & Technology 42, 1787–1793.
8 Segner, H. & Cravedi, J.P. (2001). Metabolic activity in primary cultures of fish hepatocytes. ATLA 29, 251–257.
9 Fitzsimmons, P.N., Lien, G.J. & Nichols, J.W. (2007). A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions. Comparative Biochemistry & Physiology 145C, 485–506.
10 Han, X., Nabb, D., Mingoia, R. & Yang, C. (2007). Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environmental Science & Technology 41, 3269–3276.
11 Johanning, K., Hancock, G., Escher, B., Adekola, A., Bernhard, M.J., Cowan-Ellsberry, C., Domodoradzki, J., Dyer, S., Eickhoff, C., Embry, M., Erhardt, S., Fitzsimmons, P., Halder, M., Hill, J., Holden, D., Johnson, R., Rutishauser, S., Segner, H., Schultz, I. & Nichols, J. (2012). Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction. Current Protocols in Toxicology 53, 14.10.1–14.10.28.
12 Mingoia, R.T., Glover, K.P., Nabb, D.L., Yang, C.H., Snajdr, S.I. & Han, X. (2010). Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): A validation study to support their application in bioaccumulation assessment. Environmental Science & Technology 44, 3052–3058.
13 Fay, K.A., Mingoia, R.T., Goeritz, I., Nabb, D.L., Hoffman, A.D., Ferell, B.D., Peterson, H.M., Nichols, J.W., Segner, H. & Han, X. (2014). Intra- and inter-laboratory reliability of a cryopreserved trout hepatocyte assay for the prediction of chemical bioaccumulation potential. Environmental Science & Technology 48, 8170–8178.
14 Nichols, J.W., Schultz, R.I. & Fitzsimmons, P.N. (2006). In vitro–in vivo extrapolation of quantitative hepatic biotransformation data for fish. I. A review of methods, and strategies for incorporating intrinsic clearance estimates into chemical kinetic methods. Aquatic Toxicology 78, 74–90.
15 Cowan-Ellsberry, C.S., Dyer, S., Erhardt, S., Bernhard, M.J., Roe, A., Dowty, M. & Weisbrod, A. (2008). Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates. Chemosphere 70, 1804–1817.
16 Castano, A., Bols, N.C., Braunbeck, T., Dierickx, P., Halder, M., Isomaa, B., Kawahara, K., Lee, L.E.J., Mothersill, C., Pärt, P., Repetto, G., Sintes, J.R., Rufli, H., Smith, R., Wood, C. & Segner, H. (2003). The use of fish cells in ecotoxicology. The report and recommendations of ECVAM workshop 47. ATLA 31, 317–351.
17 Segner, H. (2011). Moving beyond a descriptive aquatic toxicology: The value of biological process and trait information. Aquatic Toxicology 105, 50–55.

Download a pdf of this article here: 

Current Dilemmas Segner

Animal Use in Education in India: Confusion Continues

Dinesh K. Badyal

In India, there is an urgent need to get a single directive
on the appropriate and relevant use of animals in education
and research which is equally applicable in all institutions

Assessing and regulating the extent of animal use in education in India continues to be complicated, in that there are multiple regulatory bodies, various institutions governed by different councils, and numerous state universities. In August 2014, the University Grants Commission (UGC) wrote to all colleges in India, requesting a compliance report stating that they are not using animals in either undergraduate (UG) or postgraduate (PG)  education.1  In 2011, the UGC wrote to the same colleges, urging that animals be replaced with alternatives in phased manner.2  The recent notification was to ensure that this phased replacement has been carried out, and to ensure that there is currently no animal use in education, in either UG or PG courses. The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) has asked all institutes that use animals in research to constitute an Institutional Animal Ethics Committee (IAEC) and to perform animal house registration every three years.3

However, the colleges in India are also governed by state regulations, and various colleges follow a course which is accepted by their respective professional councils — for example, medical colleges are affiliated to the Medical Council of India (MCI) and pharmacy colleges to the Pharmacy Council of India (PCI). Based on the UGC letter and efforts by PETA India, the Pharmacy Council has issued a directive to stop animal use in all pharmacy courses.4  The recent MCI directive asks colleges to use alternatives in UG medical education, but is silent about PG courses.5

The result of this is that animal use has almost decreased to nil in UG courses, but not in PG courses. The PG courses also have a research component, in which animals are extensively used in research projects. Certain PG courses, such as those in pharmacology and physiology, are currently heavily dependent on animal use. Hence, to replace animal use, a major shift in the curriculum is needed. A news item6  in the Times of India states that animal use in education and research is now banned in India, and that there can be a heavy fine and/or imprisonment, if these guidelines are not followed — however, there is no authentic government letter on which to base this claim.

Due to a number of guidelines and circulars being issued by the UGC, CPCSEA, MCI, and PCI, as well as the Times of India news item about a ban on animal use, it has become very confusing for staff or students working in the various institutions, and it is very difficult to draw any conclusions from this wide range of information. Hence, unfortunately, confusion about the use of animals in education prevails. Indeed, there are some institutions that are using some animals in education, although it is outlawed, and there are others that do not use animals at all. At present, if a member of staff or a student uses animals in education and research in an institution, he can be questioned by animal welfare organisations and other enforcement agencies about such animals,
since the Government has banned animal use for these purposes in India — in fact, there have been police intervention and cases in court related to the use of animals.

In a recent notification, the MCI has said that there is no need to have an animal house servicing UG medical courses — but instead, has advised that an ‘animal hold area’ will be sufficient.5 However, every college running PG courses cannot do away with an animal house, as the animal house is assessed during inspections by the MCI for running physiology and pharmacology PG courses. Animals cannot be bought for use in research projects or education, as prior permission of IAEC is needed for their purchase. However, as no projects involving animals are being carried out, animals cannot be bought just for the sake of an inspection.3  But on the day of inspection, if there are no animals, then questions can be asked — and so this is a very confusing situation indeed.

The staff and researchers in these institutions also have differing views on animal use. Senior faculty, who have used animals during their initial years of education and training, believe that animal use is essential in learning and research. However, most students and junior faculty do not agree with this view. The extrapolation of results from animals to humans is now being challenged. A number of organisations for the welfare of animals question the logic behind the use of animals in education, and even in research.7 Hence, in the present scenario, there is a view that animals have equal rights and hence we should not subject them to the ill-effects of research. However, there is a feeling that there should not be a blanket ban on the use of animals, but that by keeping in view the ‘4Rs’, there should not only be appropriate and relevant  replacement, reduction and refinement,8  but also the effective rehabilitation of animals used in education and in research experiments. This is a currently neglected area of concern.

We now have commercially-available software for computer-simulated experiments on animals, for use in educational institutions.7 High-end manikins that can mimic various conditions are also now available.9  These methods are initially costly, but are economical in the long run. An institution spends a huge amount of money buying animals and maintaining animal houses. However, in research, there are no better alternatives, except for cell lines and cell cultures, and cell culture techniques are also costly.10

However, in my view, even the cell culture phase should now be over, and we should move ahead through the use of simulations that are closer to real life. If movies like Avatar can push toward extremes of simulation, then why can we not use such simulations in education and research? There is a trend to create animal simulations to replace live animal use for medical courses, as it is perceived that medical students object to being taught about the effects of drugs on blood pressure and heart rate, etc., by using live animals. So, to address this objection, we initially created computer simulations of animal models. But then an idea came to us — why not design human simulations instead of animal simulations? We now use computer-based animations of human models in our objective to teach medical students the effects of drugs in human beings.

Thus, in addition to multiple directives, these differing views of staff and students have added to already existing confusion about the use of animals in teaching and research. Hopefully, as awareness levels increase, there should be a change in the views of regulatory bodies, institutions, faculty, researchers and students. Here in India, the need of the hour is to get a single directive on the appropriate and relevant use of animals in education and research, which is equally applicable in all institutions in India.

Professor Dinesh K. Badyal
Department of Pharmacology
Christian Medical College
Ludhiana 141008
India
E-mail: dineshbadyal@gmail.com

 

 

References
1 UGC (2014). The UGC letter on dissection and animal experimentation in zoology/life sciences and allied disciplines in undergraduate, postgraduate and research programmes. New Delhi, India: University Grants Commission. Available at: http://www.ugc.
ac.in/pdfnews/6819407_ugcletterzoology.pdf (Accessed 02.12.14).
2 UGC (2011). Guidelines for Discontinuation of Dissection and Animal Experimentation in Zoology/ Life Sciences in a Phased Manner, 9pp. New Delhi, India: University Grants Commission. Available at: www.ugc.ac.in/pdfnews/6686154_guideline.pdf (Accessed 02.12.14).
3 Government of India (2014). Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). New Delhi, India: Government of India, Ministry of Environment, Forests & Climate Change. Available at: http://envfor.nic.in/division/
committee-purpose-control-and-supervisionexperiments-animals-cpcsea (Accessed 02.12.14).
4 Pharmacy Council of India (2014). Notification, New Delhi, the 25 August 2014. No.10-1/2012-PCI (Pt-I). In The Gazette of India, Part III, Section 4, No. 19. New Delhi, India: Government of India Press. Available at: http://www.pci.nic.in/Circulars/gazette_animals.pdf
(Accessed 02.12.14).
5 Medical Council of India (2014). Notification, New Delhi, the 18 March 2014. No. MCI-34(41)/2013-Med./64022. In The Gazette of India, Part III, Section 4, No. 19. New Delhi, India: Government of India Press. Available at: http://www.mciindia.org/Rulesand-
Regulation/Gazette%20Notifications%20-%20Amendments/msr-50-100-150-200-250.pdf (Accessed 02.12.14).
6 Bigga, L. (2012). Government bans use of live animals for education, research. Gurgaon, India: The Times of India. Available at: http://timesofindia.indiatimes. com/india/Govt-bans-use-of-live-animals-foreducation-research/articleshow/12696452.cms
(Accessed 02.12.14).
7 Badyal, D.K., Modgill, V. & Kaur, J. (2009). Computer simulation models are implementable as replacements for animal experiments. ATLA 37, 191–195.
8 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
9 Anon. (2013). 2012 Lush Training Prize winner: InterNICHE, UK. ATLA 41, 515–516.
10 Arora, T., Mehta, A.K., Joshi, V., Mehta, K.D., Rathor, N., Mediratta, P.K. & Sharma, K.K. (2011). Substitute of animals in drug research: An approach towards fulfillment of 4R’s. Indian Journal of Pharmaceutical Sciences 73, 1–6.

Download a PDF of this article: CLICK HERE

A Three Rs Perspective on the Mouse Bioassay in Routine Seafood Safety Testing for Algal Biotoxins — 1: Replacement

Ian Stewart and Catherine McLeod

Countries that have discontinued routine mouse bioassay testing
might gain a market advantage in domestic and export seafood
products over countries that continue to use the mouse test

Introduction
The laboratory mouse has been the principal tool for routine testing of seafood — mainly shellfish — for the presence of hazardous concentrations of harmful algal toxins, since regulatory oversight of shellfish fisheries began in the 1920s. If undetected, as often is the case in unregulated fisheries, algal biotoxins can cause serious illness and death in people consuming contaminated shellfish and other seafood. A range of alternative testing techniques that do not rely on  the use of live mice are available to the seafood industry and food safety agencies. Some countries — notably Canada, New Zealand, the UK, and now Australia — have discontinued use of the mouse bioassay (MBA) for routine safety testing of shellfish to detect and measure paralytic shellfish toxins and the so-called lipophilic toxins (which includes diarrhetic shellfish toxins), but some other countries seek to maintain access to the mouse test.

Discussions and deliberations at an international level on the credentialing of alternative testing methods occur through the authority of the Codex Alimentarius Commission. Some are frustrated that  the Codex forum is the only conduit for implementing the protracted process by which the anachronistic and unethical MBA for routine shellfish safety testing can be consigned to history. Here, we present some strategies that seafood industries in countries which have discontinued use of the MBA might be able to apply, in order to secure market advantages over those in countries that seek to maintain the status quo. This, in turn, may put pressure on industries in countries that have as yet failed to adopt alternative replacement testing systems, to endorse and refine modern management systems. In a subsequent issue of PiLAS, we will discuss the MBA for routine testing of algal biotoxins in seafood from the perspectives of the other Three Rs initiatives: reduction and refinement.

Validated Alternative Assays
A wide range of chemical, biological and physical tests and assays have been investigated for the purpose of detecting and quantifying marine biotoxins in shellfish and other seafoods, as potential alternatives  to the MBA.1 Many of these alternative techniques have not progressed beyond the stage of research inquiry, but three liquid chromatographic methods 2-4 and a receptor binding assay (RBA)5 are now approved official methods for determining harmful concentrations of specific classes of marine algal toxins. Stewart and McLeod1 recently outlined some of the challenges and constraints in moving from the use of  the MBA for routine shellfish safety testing toward modern chemical or RBA analysis. Not least of these are requirements for sophisticated analytical instruments and high-level skills in analytical chemistry  needed to run these devices. However, analytical laboratories in many countries now have the capability to provide shellfish safety testing by using chemical methods, either under the aegis of government-managed  food safety programmes or as commercial service providers.

Worldwide Regulatory Standards
Requirements for shellfish biotoxin safety testing are determined by standards, guidelines and performance criteria prescribed by the Codex Alimentarius Commission, under the auspices of the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO); Codex is also charged with overseeing fair and equitable trade conventions across the food industry. Revision and refinement of performance criteria for biotoxin safety testing is effected by the Codex Committee on Fish and Fishery Products (CCFFP). At the most recent CCFFP meeting in Norway earlier this year, draft performance criteria for the determination of marine algal biotoxins in molluscs still allow agencies conducting shellfish safety testing to choose between chemical methods or a functional assay, which includes the MBA. The draft criteria6 state that: “The method selected should be chosen on the basis of practicability, and preference should be given to  methods which have applicability for routine use.” While it may be the case that this statement merely represents the status quo, in practice the ability of alternative chemical and functional tests to formally and globally supplant the MBA will likely rest on the accumulation, over years, of a data set demonstrating that alternative shellfish safety testing is indeed protective of public health to the same broad extent as is the MBA. In this regard, deliberations within the CCFFP continue to advance the ability of seafood industries and food safety agencies to adopt alternative replacement testing methods by developing  guidelines to address challenging technical issues pertinent to the implementation of non-animal based methods, such as toxicity equivalence factors and other relevant performance criteria. 6
This is not to suggest, however, that expert opinion is currently of one voice on the need to end the availability of the MBA for shellfish safety testing. National delegates at CCFFP meetings and working groups are divided on the issue. Stewart and McLeod1 have noted some of the countries that have taken a leadership role in ending use of the MBA for routine shellfish safety testing, particularly Canada, New Zealand and the UK. Ireland was another early adopter of chemical- only testing for routine shellfish monitoring. Dissenting opinion in part centres on the claim that removing access to the MBA would represent an unfair trade advantage in favour of developed countries that have the resources to support the infrastructure and skills required to adopt and run chemical testing regimens. This position in favour of maintaining the status quo was expounded in a submission by the Philippines to the 2012 CCFFP meeting, held in Bali, Indonesia. The Philippines supported the position of the USA and Chile that access to the MBA for routine biotoxin monitoring should continue. The Philippines submission7 notes that: “For almost 30 years now, MBA is continuously protecting millions of Filipinos dependent on shellfish as the cheapest source of protein.” Interestingly, the Philippines complements its use of the MBA for shellfish safety testing with a multi-analogue HPLC technique, although this is only performed at a single centre in Manila. However, the MBA itself is performed at several locations across the archipelago.7

Trade Barriers: Who Benefits?
We suggest here that the claim for continuing access to the MBA as a routine biotoxin test for seafood safety, because of the potential trade barriers presented by the skills and infrastructure requirements  of early 21st century chemical testing, deserves closer scrutiny. We do not dispute the suggestion that the skills and equipment demands of modern chemical testing are not inconsiderable. However, the reciprocal assumption that the MBA represents a lowcost option is not a given. While the disapprobation of some scientists and technicians tasked with conducting MBA shellfish safety testing may have had some influence on expert opinion on this matter — author McLeod being one such scientist, who found the assignment objectionable — it is likely that economic  considerations have been a more significant determinant of change in countries that have or are in the process of discontinuing use of the mouse test. The experience in Australia may serve to illustrate  this point.

Scientific tests on live mammals in Australia — including the MBA for routine shellfish safety testing — must be approved and overseen by a properly constituted ethics committee, and Australian state governments must approve and licence facilities in which  such testing is conducted (see  http://www.animalethics.org.au/legislation). Laboratories that conduct scientific testing and research are required to apply rigorous standards of hygiene and husbandry, typically involving buildings that allow for the control of environmental variables such as temperature,  humidity and lighting. In practice, this usually translates to use of specific pathogen-free animals in facilities staffed by appropriately trained and supervised workers. And the operating costs for such facilities are not insubstantial — costs which are factored into their pricing schedule for service provision. The price per sample for paralytic shellfish toxins by MBA testing at the sole Australian laboratory with ethical clearance to perform the MBA for seafood safety was A$390 (including sample preparation); the charges for qualitative screening (detect/nil detect result provided) and confirmatory testing (quantitative result provided) by liquid chromatography with fluorescence detection methods from the Australian provider are A$85 and A$370, respectively. So the MBA for routine biotoxin monitoring in shellfish is not considered to be a low-cost test in Australia. The  compliance cost to Australia’s shellfish industry of routine biotoxin monitoring has been a major incentive for changing to a chemical testing programme. An additional motivating factor for the shellfish  industry and regulators in Australia was the requirement for a more specific test method that returns results only for the regulated marine toxins and excludes other compounds, such as the cyclic imines, which are not subject to food safety oversight and can cause false-positive results in the MBA. A single commercial analytical laboratory in Australia was awarded the tender, after a committee comprising seafood industry representatives and government food safety agencies considered various options for delivery of this service, including the use of providers in each state, a single national provider, and the use of government, university and private sector laboratories. Proponents of continuing use of the MBA for routine seafood safety testing assert that restricting access to the test would represent an unfair trade advantage in favour of developed countries with resources to support  the skills, equipment and infrastructure requirements of liquid chromatography-based analysis. We suggest that these claims are spurious. We propose, considering the extent that the MBA is a low-cost technique in the hands of agencies in some countries that continue to  use the test, that standards of mouse colony breeding and maintenance, husbandry, disease prevention and ethical oversight, must, wholly or in part, be of a lower standard than that seen in the example outlined above of a tightly-regulated dominion like Australia. Therefore, we suggest that countries that conduct the MBA on the cheap, so to speak, are simultaneously cutting corners on animal welfare and potentially experiencing an unfair trade advantage in so doing.

Promoting the ‘Ethical Choice’ to Consumers
An overwhelming majority of shellfish consumers are likely to be unaware that batch and fishery testing, by any method, for algal biotoxin safety is conducted as a matter of routine. Yet there may be a case for the seafood industry in countries that have relinquished  routine MBA testing for algal toxins (e.g. Australia, Canada, Ireland, New Zealand, the UK and some European nations) to take the initiative and actively promote their transition to an ethically unencumbered chemical testing regimen, in order to secure an international market advantage.

Video footage of a bored technician — peering, with stopwatch or electronic timer in hand, at a suffering and terrified mouse in order to ascertain when it has drawn its final asphyxiated breath — will not play well on prime-time television. If and when animal welfare activists decide to campaign on this topic, considerable  economic disruption may ensue.

The fallout from such a campaign should, of course, be visited disproportionately on seafood products from countries and industries that continue to rely on the MBA, but the  potential for some global industry-wide disruption might be realised in the wake of consumer ignorance about the facts of routine monitoring programmes, and the important differences between the MBA and alternative testing methodologies.

A pre-emptive approach by the seafood industry to educate consumers about food safety monitoring programmes could deliver appreciable benefits both to industry and its customers. The trends are for increasing consumer concern and awareness of food safety, particularly in well-educated and higher income  demographics.8 The message of routine monitoring for marine algal toxins should not, in principle, be a difficult sell for the seafood industry, as there is a long history of product safety and public  health protection from monitored fisheries, attributable to both the MBA — since monitoring began in the 1920s in the USA — and more recently, from fisheries that have adopted replacement alternative testing methods.1 Marketing strategies to better inform seafood consumers about routine monitoring programmes for algal biotoxins could potentially realise the dual benefits of a) embedding the food safety message that products from monitored fisheries have an excellent consumer protection record, and b) emphasising that products from countries that have discontinued the use of the MBA for routine  biotoxin safety testing are not subject to unfavourable animal welfare considerations.

In the event that the routine MBA for detection of  algal toxins in shellfish and other seafood becomes the focus of a future public awareness campaign by animal welfare proponent organisations, a pre-emptive information drive by the seafood industry and food safety agencies in countries that have replaced the MBA with alternative testing regimens should effectively insulate local industries against the economic disruption to be expected as a result of such a campaign. The appropriate targets of an animal welfare operation directed at the seafood industry would be fisheries in countries that continue to use the MBA routinely and/or advocate its continued availability. Countries that have discontinued routine  MBA testing might well gain a market advantage in domestic and export seafood products, at the expense of imports from countries that continue to use the mouse test.

Dr Catherine McLeod
Seafood Safety Assessment Ltd
Hillcrest
Kilmore
Isle of Skye IV44 8RG
UK

Author for correspondence:
Dr Ian Stewart
The University of Queensland
National Research Centre for Environmental
Toxicology
Coopers Plains
QLD 4108
Australia
E-mail: i.stewart@uq.edu.au

References
1 Stewart, I. & McLeod, C. (2014). The laboratory mouse in routine food safety testing for marine algal biotoxins and harmful algal bloom toxin research: Past, present and future. Journal of AOAC International 97, 356–372.
2 AOAC International (2012). Method 2005.06 In Official Methods of Analysis. Gaithersburg, MD, USA: AOAC
International. Available at: http://www.eoma.aoac.org/ (Accessed 25.10.14).
3 AOAC International (2012). Method 2011.02 In Official Methods of Analysis. Gaithersburg, MD, USA: AOAC
International. Available at: http://www.eoma.aoac.org/ (Accessed 25.10.14).
4 EU-RL-MB & AESAN (2011). EU-Harmonised Standard Operating Procedure for Determination of Lipophilic Marine Biotoxins in Molluscs by LC-MS/MS, 31pp. Vigo, Spain: European Union Reference Laboratory for Marine Biotoxins & Agencia Española de Seguridad Alimentaria y Nutrición.
5 AOAC International (2012). Method 2011.27 In Official Methods of Analysis. Gaithersburg, MD, USA: AOAC
International. Available at: http://www.eoma.aoac. org/ (Accessed 25.10.14).
6 Codex Alimentarius Commission (2014). Joint FAO/ WHO Food Standards Programme: Codex Alimentarius
Commission. Report of the Thirty-third Session of the Codex Committee on Fish and Fishery Products,
Bergen, Norway, 17–21 February 2014, vi + 64pp. Rome, Italy: Codex Alimentarius Commission.
7 Codex Alimentarius Commission (2014). Joint FAO/ WHO Food Standards Programme: Codex Committee
on Fish and Fishery Products, 32nd Session, Bali, Indonesia, 8pp. Rome, Italy: Codex Alimentarius
Commission.
8 Dosman, D.M., Adamowicz, W.L. & Hrudey, S.E. (2001). Socioeconomic determinants of health- and food safety-related risk perceptions. Risk Analysis 21, 307–317. P56

Lost in Translation: The Need for Better Tools

Susanna Penco, Elena Venco and Alfredo Lio

Although for most pharmaceutical compounds
the final aim is improving human health,
almost all the methods used to identify and
pursue therapeutic targets and to obtain
new potential drugs have traditionally
focused on animal models

Introduction
Recent studies on attrition rate in pharmacological research show that the pharmaceutical industry finds it difficult to turn new experimental compounds into safe and effective drugs. Although, for most pharmaceutical compounds, the final aim is improving human health, almost all the methods used to identify and pursue therapeutic targets and to obtain new potential drugs have traditionally been centered on animal models. The ability of such methods to predict efficacy and safety in humans needs to be carefully reviewed, in the light of more-predictive and more reliable human-based experimental tools.

The overall cost for the development and the marketing of a new pharmaceutical product ranges between one billion and 1.8 billion US dollars.1 It has been estimated that only one in 10,000 new chemical entities (NCEs), also referred to as new molecular entities (NMEs), discovered in the laboratory succeeds in obtaining marketing approval.2, 3 Recent studies have shown that 95% of experimental drugs fail in the clinical phase.4, 5 The main reasons for these poor results can be ascribed to the lack of therapeutic efficacy and safety.6, 7 Such findings point to the significant inadequacy of the current preclinical tests — mainly in vitro cellular assays and animal based disease models — in screening pharmacological compounds. Many in vitro tests are still performed in a two-dimensional format,8 despite the limitations of this approach,8, 9 and are based on animal cells, which complicates the potential extrapolation of the information they provide to humans.6, 10, 11 In spite of this, such preclinical methods are still considered the ‘gold standard’ in pharmaceutical R&D.

The relevance of animal models
Many animals, including mice, rats, dogs, cats, and non-human primates (NHPs), are used in biomedical and toxicological research as human disease surrogates, so they are defined as ‘animal models’. However, there are a number of important limitations that underlie the lack of successful use of these animal models in furthering the understanding of human disease.

Firstly, there are significant differences among species with regard to their anatomy, metabolism and physiology, which correspond to genetic differences, including differences in regulatory genes. This means that even slight molecular differences can be amplified in the extrapolation process from one species to another. For instance, mice (together with rats, which are the most commonly used species in biomedical research) share with humans slightly more than 90% of their gene sequences. Nevertheless, at least 67 major discrepancies have been found in the immunological functions of mice and humans. This fact is hardly surprising, since these two species separated approximately 65- to 75-million years ago, and have since followed different evolutionary path ways.

About 1% of human genes do not have a homologue in the mouse.14 Biochemistry provides many examples concerning similarities and differences between species. Some of the most significant differences are in the cytochrome P450 enzymes (CYPs), which seem to have evolved from a single ancestral gene over a period of 1.36 billion years. To date, at least 14 families of CYPs genes have been identified in mammals.15 Each member of this gene family has many highly conserved regions in its secondary amino acid structure. However, remarkable differences between species also exist in the primary amino acid sequences. Even small differences in amino acid sequence can imply wide differences in substrate specificity.16 Such variations can explain the divergences in drug response between animal models and humans. The scientific literature provides many examples of therapies that proved successful in animal models, but subsequently failed in clinical trials.17-20

A second important issue surrounding the failure of many animal models is the way in which the disease is induced. Diseases induced ‘artificially’ in animals cannot begin to accurately reproduce the very complex aspects and conditions clinically observed in human patients. This is thought to be one of the most crucial reasons for drug attrition.21, 22

In addition, there are relevant species-specific differences in absorption, distribution, metabolism, excretion and toxicity (ADMET) between animals and humans.23 These processes together make up the important concept of ‘pharmacokinetics’.

Pharmacokinetics is one of the main reasons for candidate compound failure in humans.24 A wide range of species-specific metabolic patterns strongly suggest that data can be hardly (at best) extrapolated from one species to another, both quantitatively and qualitatively — i.e. differences in the amino acid sequence of isozymes may influence both the rate of drug metabolism and the metabolite pattern. 25 An outstanding example of species-specific differences between rats and humans is in coumarin metabolism and toxicity, which appears to be mediated through two major phase I metabolic pathways. The first pathway, involving cytochrome CYP2A enzymes and leading to the conversion of coumarin into the non-toxic metabolite 7-hydroxycoumarin, is very efficient in humans and extremely inefficient in rats. The second pathway involves the detoxification of the epoxide intermediate, coumarin 3,4-epoxide, which spontaneously rearranges to o-hydroxyphenylacetaldehyde and is then oxidised to o-hydroxy – phenylacetic acid. In rats, the rate of conversion to o-hydroxyphenylacetic acid is 50 times lower than in humans. These metabolic discrepancies explain the differences in coumarin-mediated hepatotoxicity between the two species.26

There are many significant examples of drug attrition resulting from the limitations of the animal models used in pharmaceutical R&D:
— More than 150 experimental therapeutics for the treatment of sepsis have been successfully tested in animals. None of them proved useful in humans.27
— A total of 800 new drugs showed promising results in animal models for stroke, but only 97 were approved for the clinical phases. Unfortunately, only two showed some efficacy, with aspirin being one of the two.28, 29
— More than 85 different HIV vaccines have been tested in approximately 200 clinical studies,30 but to date no therapeutic or protective effects on humans have been found. The use of resources has been so extensive that, even if an effective HIV vaccine were found as a result of animal experimentation, animal models could not be considered a suitable predictive experimental method, since the PPV (positive predictive value) would be around 0.01.31

The list of failures gets longer with anti-cancer drugs, and there is also an endless list of failures in relation to neurodegenerative diseases. Indeed, anticancer drugs and treatments for neurological diseases have the highest attrition rate in the development process.32 Awareness of the limits of the predictivity of animal models is rapidly growing.33-39 Even the use of transgenic animals seems to have proved inconclusive in translational medicine.6, 34, 40-42 With regard to neurodegenerative diseases, the results obtained by testing new therapies on animals have been very poor.17, 43, 47

The study of bioavailability is a clear example of the differences in drug response occurring among species, as shown by many studies.48-51 Systematic reviews of the predictive accuracy of animal models in the field of teratogenesis52, 53 and carcinogenesis,54 also showed poor predictive power. In a recent analysis of the registration files of all therapeutic monoclonal antibodies (tmAbs) available in the EU, van Meer et al.55 discovered that the incidence of formation of anti-tmAb antibodies in NHPs and patients was comparable in only 59% of cases. In addition, the type of anti-tmAb antibody response was different in NHPs and humans in the same proportion of cases. The authors concluded that monoclonal antibody immunogenicity in NHPs and humans is significantly different.

In a recent review of the use of the dog model and other animal models in drug toxicology, the authors concluded that its predictive value in current toxicology was very poor.56, 57 The issues associated with extrapolating data from animals to humans are probably due to both inadequate testing procedures and to the failure of models to accurately reproduce human diseases, but evidence is growing that the core of the problem could only be resolved by giving up the use of animals as models.33 Therefore, in the light of controversial predictive value, it is not surprising that some scientists consider preclinical animal studies, “generally scarce, unreliable or nonpredictive”. 58-60

Considering the present stalemate of translational medical science, the development of new, reliable experimental approaches to assure efficiency, convenience and safety in clinical therapies is desperately needed. Long-term Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) studies, comprising a set of in vitro tests based on human biological materials, proved more predictive in testing compounds than did traditional animal-based acute toxicity studies.61
Recently, many important improvements have been made in studying acute toxicity, repeated toxicity62 and reproductive toxicology, as assessed by the ESNATS report.63-64 One particularly promising field is that of organs-on-chips, which are micro-engineered physiological systems aimed at reproducing the physiological properties of human tissues and organs and their interactions. Thanks to these biochips, it has been possible to create a model for acute pulmonary oedema that has permitted the evaluation of new clinical and therapeutic interventions.65 In addition to the lung-on-a-chip, other tools have been successfully developed to mimic the human gut66 and kidney.59 The final aim is clearly to develop a ‘human-on-a-chip’, to fully mimic the functions of and interactions between organs, thus getting closer to the human in vivo situation. Indeed, some already trust this approach as a valid alternative to traditional animal tests.67-70 In addition, the use of human pluripotent stem cells seems to be becoming more widely appreciated in pre-clinical toxicology.71-72

Conclusions
Since the available data show that their predictivity can no longer be assumed, there is an urgent need for reviews and meta-analyses of the animal models currently used in medical research. Moreover, science should focus on the development of more-advanced methods, as a result of the limitations of the current pre-clinical tools, the growing bioethical objections surrounding their use, and the ongoing development
of new in vitro and in silico techniques. These alternative methods should be used ideally in the experimental context of an Integrated Testing Strategy.

References
1 Esserman, L.J. & Woodcock, J. (2011). Accelerating identification and regulatory approval of investigational cancer drugs. JAMA 306, 2608–2609.
2 Li, A.P. (2004). In vitro approaches to evaluate ADMET drug properties. Current Topics in Medicinal Chemistry 4, 701–706.
3 Berlin, J.A., Glasser, S.C. & Ellenberg, S.S. (2008). Adverse event detection in drug development: Recommendations and obligations beyond phase 3.
American Journal of Public Health 98, 1366–1371.
4 Hartung, T. (2013). Look back in anger — What clinical studies tell us about preclinical work. ALTEX 30, 275–291.
5 Arrowsmith, J. (2012). A decade of change. Nature Reviews Drug Discovery 11, 17–18.
6 Engle, S.J. & Puppala, D. (2013). Integrating human pluripotent stem cells into drug development. Cell Stem Cell 12, 669–677.
7 Paul, S.M., Mytelka, D.S., Dunwiddie, C.T., Persinger, C.C., Munos, B.H., Lindborg, S.R. & Schacht, A.L. (2010). How to improve R&D productivity: The pharmaceutical
industry’s grand challenge. Nature Reviews Drug Discovery 9, 203–214.
8 Breslin, S. & O’Driscoll, L. (2013). Three-dimensional cell culture: The missing link in drug discovery. Drug Discovery Today 18, 240–249.
9 Arya, N., Sardana, V., Saxena, M., Rangarajan, A. & Katti, D.S. (2012). Recapitulating tumour microenvironment in chitosan–gelatin three-dimensional scaffolds: An improved in vitro tumour model. Journal of the Royal Society, Interface 9, 3288–3302.
10 Knight, A. (2007). Animal experiments scrutinised: Systematic reviews demonstrate poor human clinical and toxicological utility. ALTEX 24, 320–325.
11 van Vliet, E. (2011). Current standing and future prospects for the technologies proposed to transform toxicity testing in the 21st century. ALTEX 28, 17–44.
12 Mestas, J. & Hughes, C.C. (2004). Of mice and not men: Differences between mouse and human immunology. Journal of Immunology 172, 2731–2738.
13 Brady, C.A. (2008). Of mice and men: The potential of high-resolution human immune cell assays to aid the pre-clinical to clinical transition of drug development projects. Drug Discovery World, Winter 2008/09. London, UK: RJ Communications & Media
Ltd. Available at: http://ddw.net-genie.co.uk/enabling_technologies/261930/of_mice_and_men.
html (Accessed 31.08.14).
14 Mouse Genome Sequencing Consortium (2002). Initial sequencing and comparative analysis of the mouse genome. Nature, London 420, 520–562.
15 Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., Waxman, D.J., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W., Gunsalus, I.C. & Nebert, D.W. (1996). P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42.
16 Lindberg, L.P. & Negishi, M. (1989). Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue. Nature,London 339, 632–634.
17 Schnabel, J. (2008). Neuroscience: Standard model. Nature, London 454, 682–685.
18 Gordon, P.H., Moore, D.H., Miller, R.G., Florence, J.M., Verheijde, J.L., Doorish, C., Hilton, J.F., Spitalny, G.M., MacArthur, R.B., Mitsumoto, H., Neville, H.E., Boylan, K., Mozaffar, T., Belsh, J.M., Ravits, J., Bedlack, R.S., Graves, M.C., McCluskey, L.F., Barohn, R.J., Tandan, R.; Western ALS Study Group (2007). Efficacy of minocycline in patients with amyotrophic lateral sclerosis: A phase III randomised trial. Lancet Neurology 6, 1045–1053.
19 Doody, R.S., Raman, R., Farlow, M., Iwatsubo, T., Vellas, B., Joffe, S., Kieburtz, K., He, F., Sun, X., Thomas, R.G., Aisen, P.S.; Alzheimer’s Disease Cooperative Study Steering Committee, Siemers, E., Sethuraman, G., Mohs, R.; Semagacestat Study Group (2013). A phase 3 trial of semagacestat for treatment of Alzheimer’s disease. New England Journal of Medicine 369, 341–350.
20 Gawrylewsky, A. (2007). The trouble with animal models — Why did human trials fail? The Scientist, July 2007. Midland, Ontario, Canada: LabX Media Group. Available at: http://www.the-scientist.com/?articles.view/articleNo/25184/title/The-Troublewith-Animal-Models/ (Accessed 31.08.14).
21 Perel, P., Roberts, I., Sena, E., Wheble, P., Briscoe, C., Sandercock, P., Macleod, M., Mignini, L.E., Jayaram, P. & Khan, K.S. (2007). Comparison of treatment effects between animal experiments and clinical trials: Systematic review. BMJ 334, 197.
22 Pound, P., Ebrahim, S., Sandercock, P., Bracken, M.B., Roberts, I.; Reviewing Animal Trials Systematically (RATS) Group (2004). Where is the evidence that animal research benefits humans? BMJ 328, 514–517.
23 Greek, R. & Hansen, L. (2013). Questions regarding the predictive value of one evolved complex adaptive system for a second: Exemplified by the SOD1 mouse. Progress in Biophysics & Molecular Biology 113, 231– 253.
24 Seymour, M. (2009). The best model for humans is human — How to accelerate early drug development safely. ATLA 37, Suppl. 1, 61–65.
25 Lin, J.F. & Lu, A.Y.H. (1997). Role of pharmacokinetics and metabolism in drug discovery and development. Pharmacological Reviews 49, 403–449.
26 Sutherland, M.L., Fabre, K.M. & Tagle, D.A. (2013). The National Institutes of Health Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline. Stem Cell Research & Therapy 4, Suppl. 1, I1.
27 Seok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., López, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkins, R.G.; Inflammation and Host Response to Injury, Large Scale Collaborative Research Program (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proceedings of the National Academy of Sciences of the USA 110, 3507–3512.
28 Sena, E., Wheble, P., Sandercock, P. & Macleod, M. (2007). Systematic review and meta-analysis of the efficacy of tirilazad in experimental stroke. Stroke 38, 388–394.
29 van der Worp, H.B., Howells, D.W., Sena, E.S., Porritt, M.J., Rewell, S., O’Collins, V. & Macleod, M.R. (2010). Can animal models of disease reliably inform human studies? PLoS Medicine 7, e1000245.
30 Bailey, J. & Taylor, K. (2009). The SCHER report on non-human primate research — Biased and deeply flawed. ATLA 37, 427–435.
31 Greek, R. & Menache, A. (2013). Systematic reviews of animal models: Methodology versus epistemology. International Journal of Medical Sciences 10, 206–221.
32 Kola, I. & Landis, J. (2004). Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery 3, 711–715.
33 Mak, I.W., Evaniew, N. & Ghert, M. (2014). Lost in translation: Animal models and clinical trials in cancer treatment. American Journal of Translational Research 6, 114–118.
34 Enna, S.J. & Williams, M. (2009). Defining the role of pharmacology in the emerging world of translational research. Advances in Pharmacology 57, 1–30.
35 Lazic, S.E. & Essioux, L. (2013). Improving basic and translational science by accounting for litter-to-litter variation in animal models. BMC Neuroscience 14, 37.
36 Leist, M. & Hartung, T. (2013). Inflammatory findings on species extrapolations: Humans are definitely no 70-kg mice. Archives of Toxicology 87, 563–567.
37 Aggarwal, B.B., Danda, D., Gupta, S. & Gehlot, P. (2009). Models for prevention and treatment of cancer: Problems vs promises. Biochemical Pharmacology 78, 1083–1094.
38 Coleman, R.A. (2011). Human tissue in the evaluation of safety and efficacy of new medicines: A viable alternative to animal models? ISRN Pharmaceutics 2011, 806789.
39 Li, A.P. (2014). Biomarkers and human hepatocytes. Biomarkers in Medicine 8, 173–183.
40 Hyman, S.E. (2012). Revolution stalled. Science Translational Medicine 4, 155cm11.
41 Stingl, L., Völkel, M. & Lindl, T. (2009). 20 years of hypertension research using genetically modified animals: No clinically promising approaches in sight. ALTEX 26, 41–51.
42 Bhogal, N. & Combes, R. (2006). The relevance of genetically altered mouse models of human disease. ATLA 34, 429–454.
43 Anon. (2013). Drug testing should be with human iPS cells. Drug Discovery & Development, 06.12.13. Rockaway, NJ, USA: Advantage Media. Available at: http://www.dddmag.com/news/2013/12/drugtesting- should-be-human-ips-cells (Accessed 31.08.14).
44 Mertens, J., Stüber, K., Wunderlich, P., Ladewig, J., Kesavan, J.C., Vandenberghe, R., Vandenbulcke, M., van Damme, P., Walter, J., Brüstle, O. & Koch, P. (2013). APP processing in human pluripotent stem cell-derived neurons is resistant to NSAID-based γ-secretase modulation. Stem Cell Reports 1, 491–498.
45 Rice, J. (2012). Animal models: Not close enough. Nature, London 484, S9.
46 Friese, M.A., Montalban, X., Willcox, N., Bell, J.I., Martin, R. & Fugger, L. (2006). The value of animal models for drug development in multiple sclerosis. Brain 129, 1940–1952.
47 Beal, M.F. (2010). Parkinson’s disease: A model dilemma. Nature, London 466, S8–S10.
48 Grass, G.M. & Sinko, P.J. (2002). Physiology-based pharmacokinetic simulation modelling. Advanced Drug Delivery Reviews 43, 433–451.
49 Akabane, T., Tabata, K., Kadono, K., Sakuda, S., Terashita, S. & Teramura, T. (2010). A comparison of pharmacokinetics between humans and monkeys. Drug Metabolism & Disposition 38, 308–316.
50 Cao, X., Gibbs, S.T., Fang, L., Miller, H.A., Landowski, C.P., Shin, H.C., Lennernas, H., Zhong, Y., Amidon, G.L., Yu, L.X. & Sun, D. (2006). Why is it challenging to predict intestinal drug absorption and oral bioavailability in human using rat model. Pharmaceutical Research 23, 1675–1686.
51 Chiou, W.L., Jeong, H.Y., Chung, S.M. & Wu, T.C. (2000). Evaluation of using dog as an animal model to study the fraction of oral dose absorbed of 43 drugs in humans. Pharmaceutical Research 17, 135–140.
52 Bailey, J., Knight, A. & Balcombe, J. (2005). The future of teratology research is in vitro. Biogenic Amines 19, 97–145.
53 Bailey, J. (2008). Developmental toxicity testing: Protecting future generations? ATLA 36, 718–721.
54 Basketter, D.A., Clewell, H., Kimber, I., Rossi, A., Blaauboer, B., Burrier, R., Daneshian, M., Eskes, C., Goldberg, A., Hasiwa, N., Hoffmann, S., Jaworska, J., Knudsen, T.B., Landsiedel, R., Leist, M., Locke, P., Maxwell, G., McKim, J., McVey, E.A., Ouédraogo, G., Patlewicz, G., Pelkonen, O., Roggen, E., Rovida, C., Ruhdel, I., Schwarz, M., Schepky, A., Schoeters, G., Skinner, N., Trentz, K., Turner, M., Vanparys, P., Yager, J., Zurlo, J. & Hartung, T. (2012). A roadmap for the development of alternative (non-animal) methods for carcinogenicity testing. ALTEX 29, 41–55.
55 van Meer, P.J., Kooijman, M., Brinks, V., Gispen-de Wied, C.C., Silva-Lima, B., Moors, E.H. & Schellekens, H. (2013). Immunogenicity of mAbs in non-human primates during nonclinical safety assessment. MAbs 5, 810–816.
56 Bailey, J., Thew, M. & Balls, M. (2013). An analysis of the use of dogs in predicting human toxicology and drug safety. ATLA 41, 335–350.
57 Bailey, J., Thew, M. & Balls, M. (2014). An analysis of the use of animal models in predicting human toxicology and drug safety. ATLA 42, 181–199.
58 Wang, S.J., Hung, H.M. & O’Neill, R. (2011). Adaptive design clinical trials and trial logistics models in CNS drug development. European Neuropsychopharmacology 21, 159–166.
59 Jang, K.J., Mehr, A.P., Hamilton, G.A., McPartlin, L.A., Chung, S., Suh, K.Y. & Ingber, D.E. (2013). Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integrative Biology (Cambridge) 5, 1119–1129.
60 Giri, S., Braumann, U.D., Giri, P., Acikgöz, A., Scheibe, P., Nieber, K. & Bader, A. (2013). Nanostructured self-assembling peptides as a defined extracellular matrix for long-term functional maintenance of primary hepatocytes in a bioartificial liver modular device. International Journal of Nanomedicine 8, 1525–1539.
61 Ekwall, B. (1999). Overview of the final MEIC results: II. The in vitro–in vivo evaluation, including the selection of a practical battery of cell tests for prediction of acute lethal blood concentrations in humans. Toxicology in Vitro 13, 665–673.
62 Mueller, D., Krämer, L., Hoffmann, E., Klein, S. & Noor, F. (2014). 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicology in Vitro 28, 104–112.
63 Krug, A.K., Kolde, R., Gaspar, J.A., Rempel, E., Balmer, N.V., Meganathan, K., Vojnits, K., Baquié, M., Waldmann, T., Ensenat-Waser, R., Jagtap, S., Evans, R.M., Julien, S., Peterson, H., Zagoura, D., Kadereit, S., Gerhard, D., Sotiriadou, I., Heke, M., Natarajan, K., Henry, M., Winkler, J., Marchan, R., Stoppini, L., Bosgra, S., Westerhout, J., Verwei, M., Vilo, J., Kortenkamp, A., Hescheler, J., Hothorn, L., Bremer, S., van Thriel, C., Krause, K.H., Hengstler, J.G., Rahnenführer, J., Leist, M. & Sachinidis, A. (2013). Human embryonic stem cell-derived test systems for developmental neurotoxicity: A transcriptomics approach. Archives of Toxicology 87, 123–143.
64 Hengstler, J.A., Leist, M. & Hescheler, J. (2013). Briefing paper: The Novel FP7 ESNATS Test Systems of Developmental Toxicity: State-of-the art and Future Perspectives, 8pp. Paris, France: ARTTIC Project Office. Available at: http://www.esnats.eu/uploads/images/ESNATS_Briefing_paper_13-09-2013.pdf (Accessed 31.08.14).
65 Huh, D., Leslie, D.C., Matthews, B.D., Fraser, J.P., Jurek, S., Hamilton, G.A., Thorneloe, K.S., McAlexander, M.A. & Ingber, D.E. (2012). A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Science Translational Medicine 4, 159ra147.
66 Kim, H.J. & Ingber, D.E. (2013). Gut-on-a-chip microenvironment induces human intestinal cells to undergo villus differentiation. Integrative Biology (Cambridge) 5, 1130–1140.
67 Moraes, C., Mehta, G., Lesher-Perez, S.C. & Takayama, S. (2011). Organs-on-a-chip: A focus on compartmentalized microdevices. Annals of Biomedical Engineering 40, 1211–1227.
68 Yum, K., Hong, S.G., Healy, K.E. & Lee, L.P. (2014). Physiologically relevant organs on chips. Biotechnology Journal 9, 16–27.
69 Huh, D., Hamilton, G.A. & Ingber, D.E. (2011). From 3D cell culture to organs-on-chips. Trends in Cell Biology 21, 745–754.
70 Marx, U., Walles, H., Hoffmann, S., Lindner, G., Horland, R., Sonntag, F., Klotzbach, U., Sakharov, D., Tonevitsky, A. & Lauster, R. (2012). ‘Human-on-a-chip’ developments: A translational cutting-edge alternative to systemic safety assessment and efficiency evaluation of substances in laboratory animals and man? ATLA 40, 235–257.
71 Mandenius, C.F., Andersson, T.B., Alves, P.M., Batzl-Hartmann, C., Björquist, P., Carrondo, M.J., Chesne, C., Coecke, S., Edsbagge, J., Fredriksson, J.M., Gerlach, J.C., Heinzle, E., Ingelman-Sundberg, M., Johansson, I., Küppers-Munther, B., Müller-Vieira, U., Noor, F. & Zeilinger, K. (2011). Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines — A report on the Vitrocellomics EU-project. ATLA 39, 147–171.
72 Cao, L., Tan, L., Jiang, T., Zhu, X.C. & Yu, J.T. (2014). Induced pluripotent stem cells for disease modeling and drug discovery in neurodegenerative diseases. Molecular Neurobiology, E-pub ahead of print [doi: 10.1007/s12035-014-8867-6].

Why Animal Studies are Still Being Used in Drug Development

The lack of success of the implementation
of new methods in drug development can
be studied from an innovation perspective

Introduction

In the European Union, about 3.6 million animals per year (30% of all the animals used) are used for drug development.1 Animal studies are the global standard used to evaluate the safety, efficacy and quality of drugs before these drugs are tested in humans. However, the value of animal studies to predict risks for humans has never been extensively established.2  In fact, many studies indicate that the value of animal studies is often limited. 3–5

Pharmaceutical companies and regulatory authorities, as well as the public and governments, aspire to reduce the number of animal studies carried out, because of the limited value of these studies and the ethical issues they involve. The development of innovative methods to replace animal studies received a boost at the end of the 1970s, as a result of campaigns by animal welfare organisations that resulted in increased public awareness and the implementation of Directive 86/609/EEC in the European Union.6

This Directive requires that innovative methods are used whenever possible, and stimulates the development of innovative methods. Many innovative methods  have been developed since the implementation of the Directive. Nevertheless, these innovative methods only incidentally replace animal studies. In my recent doctoral thesis, I tried to elucidate why animal studies are still being used in drug development. 7 To answer the research question, “Which mechanisms explain the lock-in of animal studies in drug development?”, six studies were conducted.

Theoretical framework

The persistent use of animal studies in drug development is an innovation problem, as innovative methods have not yet substituted animal studies on a large scale. This lack of success of the implementation of new methods in drug development can therefore be studied from an innovation perspective — i.e. innovation is the successful implementation of an invention into practice. The main lesson from early innovation studies is that science and technology are only two of the numerous ingredients required for innovation.8 For turning an idea into an innovation, a network of players is essential, that serves to unite the right knowledge, capabilities, skills and resources at the right time.8 Innovation processes are complex, and are characterised by complicated feedback mechanisms and mutual interactions involving science, technology, production, policy and demand.9, 10  Thus, innovation is not an isolated process, but comes about as a result of the interplay of players in a specific context. This context is labelled the ‘innovation system’. The Technological Innovation System (TIS) approach is used as an analytical tool to study  the innovation process of emerging technologies that can replace animal studies from a system perspective.11

The TIS approach is criticised, because it does not take into account processes and influences from outside the TIS itself, such as established conventions (i.e. institutions). Although the success of emerging technologies depends as much on the generation, maturation and use of emerging technologies as it does on escaping practices embedded in the established institutional context, successful innovation is regarded as a consequence of the functioning of the TIS itself. Due to this inward orientation, the TIS approach does not explicitly analyse the persistency of the established institutions, and the effect of this persistency on the innovation process of emerging technologies. To fully understand why animal studies are still being used in drug development, the persistency or ‘lock-in’ of the use of animal studies in drug development, and how that influences the innovation process, is analysed by using institutional theory.

Following institutional theory, established practices such as animal studies are locked-in, because they are embedded in a well-aligned set of institutions (i.e. regulations, norms and values) that are taken for granted, normatively endorsed, and backed up by regulatory authorities.12, 13 The sets of institutions that guide the behaviour of players in a particular scenario are referred to as institutional logics.12, 14  Different sets of institutions guide daily behaviour in different contexts.

When driving to work, people stick to the traffic rules and the norms and values of driving. At work, the behaviour of the staff is guided by the norms, values and rules prevailing in the company by which they are employed. To describe the set of institutions guiding behaviour, Alford and Friedland introduced the concept of institutional logic,15 which involves a set of institutions that independently contribute to a powerful structure that guides daily behaviour of players in specific contexts.12, 14

A framework combining the TIS approach with an analysis of the institutional logic governing the use of animals in drug development was used, in order to improve our understanding of why animal  studies are still being used in drug development. This combined framework enabled us to elucidate a more complete overview of the different mechanisms that hamper the innovation process toward the implementation of new alternative methods in drug development.

Research design

Animal studies will continue to be used in drug development as long as innovative methods are unavailable or are incapable of overcoming the animal studies lockin. Innovative methods can escape the lock-in of animal studies via two routes. Firstly, they can substitute animal studies in established drug development processes. Examples of innovative methods that have substituted animal studies are the Isolated Chicken Eye (ICE) test and the Bovine Corneal Opacity and Permeability (BCOP) test, both of which have replaced the Draize eye irritation test in rabbits. The bacterial Ames test substituted, to a large extent, animal tests for the assessment of mutagenicity, and the Limulus amoebocyte lysate (LAL) test, which uses aqueous extract of blood cells from the horseshoe crab, replaced animal studies to detect and quantify bacterial endotoxins. Secondly, innovative methods can escape the animal studies lock-in, by being adopted in novel drug development processes for new drug classes. New drug classes provide opportunities for innovative methods, because the drug development processes for these new drug classes have to be established.

These novel drug development processes can be considered to be ‘green fields’, wherein innovative methods can be adopted, without the need for substituting existing animal studies. Analysis of the success, or otherwise, of innovative methods to escape the lockin of animal studies in drug development via these two routes, provides a comprehensive overview of mechanisms that influence the lock-in of animal studies during the process of drug development.

Explaining the ‘lock-in’ of animal studies

Two case studies were conducted to identify the barriers that hamper the replacement of animal studies in the current regulations. Based on these studies, it can be concluded that, although regulatory authorities and pharmaceutical companies have an ambition to reduce the use of animal studies, replacing animal studies by innovative methods is challenging. On the one hand, there is no urgency to replace animal studies. This lack of urgency slows down the innovation process, because it can make it more difficult to obtain resources. On the other hand, replacing animal studies by innovative methods is challenging, because innovative methods do not sit well with the institutional logic of animal testing. The institutional logic of animal studies is the selection environment, because innovative methods have to be adopted in the regulations (established institutions) as replacements for animal studies. To validate innovative methods, it is generally required to show that these methods deliver similar results as animal studies.

However, innovative methods are often based on human data and human mechanisms of action, making them a better predictive model. This makes the validation of innovative methods challenging.

Four studies were conducted to identify why animals are implemented in the guidelines for the development of new classes of drug.5, 16-18 Based on these studies, it can be concluded that animal studies often have only limited value in the development of, for example, monoclonal antibodies. These animal studies are implemented in the guidelines, because the design of the development process of new drug classes is experience-driven, rather than being science-driven.

Escaping the animal studies lock-in

Based on the six studies in my thesis, I formulated five recommendations to reduce animal testing in drug development:

a) Governments should create incentives for the pharmaceutical industry to develop and use methods that can substitute animal studies; incentives could be created by rewarding the use of innovative methods and discouraging the use of animal studies.

b) The acceptance of patented innovative methods in regulation will accelerate the innovation process; the patenting of new methods will enable the costs of the development and validation to be recovered.

c) The revision of the validation process will contribute to the implementation of innovative methods; humane endpoints should be used as the reference for validation.

d) ‘Smart’ regulation, enabling science-driven drug development will contribute to the reduction of animal studies; smart regulation provides the opportunity to deviate from the drug development requirements, and thereby enables the use of innovative methods that are not validated.

e) Research on the predictive value of animal studies will increase the innovation process; if more research shows that the predictive value of animal studies is limited, then the legitimacy to use animals as models for humans will decrease, and this will provide opportunities for the implementation of innovative methods.

Dr Marlous Kooijman
Innovation Studies
Copernicus Institute for Sustainable Development
Utrecht University
Heidelberglaan 2
3584 CS, Utrecht
The Netherlands

E-mail: marlous.kooijman@gmail.com

 

References

1 Anon. (2010). Report from the Commission to the Council and the European Parliament: Sixth Report on the Statistics on the Number Of Animals Used for Experimental and Other Scientific Purposes in the Member States of the European Union, SEC(2010) 1107, 15pp. Brussels, Belgium: European Commission. Available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=COM:2010:0511:REV1:EN:PDF (Accessed 23.07.13).
2 Abbott, A. (2008). More than a cosmetic change. Nature, London 438, 144–146.
3 Igarashi, T.I., Nakane, S. & Kitagawa, T. (1995). Predictability of clinical adverse reactions of drugsby general pharmacology studies. Journal of Toxicological Sciences 20, 77–92.
4 Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., Lilly, P., Sanders, J., Sipes, G.,Bracken, W., Dorato, M., Van Deun, K., Smith, P., Berger, B. & Heller, A. (2000). Concordance of the toxicity of pharmaceuticals in humans and in animals. Regulatory Toxicology & Pharmacology 32, 56–67.
5 Van Meer, P.J.K., Kooijman, M., van der Laan, J.W.,Moors, E.H.M. & Schellekens, H. (2013). The value of non-human primates in the development of monoclonal antibodies. Nature Biotechnology 31, 881–882.
6 Anon. (1986). Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes.Official Journal of the European Union L358, 18.12.1986, 1–28.
7 Kooijman, M. (2013). Why Animals are Still Being Used in Drug Development: An Innovation System Perspective, 208pp. [PhD Thesis, Utrecht University.] Ede, The Netherlands: GVO Drukkers & Vormgevers B.V.
8 Fagerberg, J. (2006). Innovation: A guide to the literature. In The Oxford Handbook of Innovation, 1st edition (ed. J. Fagerberg, D.C. Mowery, & R.R. Nelson), pp. 1–26. Oxford, UK: University Press.
9 Edquist, C. (1997). Systems of Innovation: Technologies, Institutions, and Organizations, 432pp. Abingdon, Oxfordshire, UK: Routledge.
10 Lundvall, B-Å., Johnson, B., Andersen, E.S. & Dalum, B. (2002). National systems of production, innovation and competence building. Research Policy 31, 213–231.
11 Markard, J. & Truffer, B. (2008). Technological innovation systems and the multi-level perspective: Towards an integrated framework. Research Policy 37, 596–615.
12 Scott, W.R. (2008). Institutions and Organizations— Ideas and Interests, Third Edn, 266pp. Thousand Oaks, CA, USA: Sage Publications, Inc.
13 Thornton, P.H., Ocasio, W. & Lounsbury, M. (2012). The Institutional Logics Perspective — A New Approach to Culture, Structure and Process, First Edn, 234pp. Oxford, UK: Oxford University Press.
14 Reay, T. & Hinings, C.R. (2009). Managing the rivalry of competing institutional logics. Organization Studies 30, 629–652.
15 Alford, R.R. & Friedland, R. (1985). Powers of Theory: Capitalism, the State, and Democracy, 502pp. Cambridge, UK: Cambridge University Press.
16 Kooijman, M., van Meer, P.J.K., Moors, E.H.M. & Schellekens, H. (2012). Thirty years of preclinical safety evaluation of biopharmaceuticals: Did scientific progress lead to appropriate regulatory guidance? Expert Opinion on Drug Safety 11, 797–801.
17 Kooijman, M., van Meer, P.J.K., Gispen-de Wied, C.C., Moors, E.H.M., Hekkert, M.P. & Schellekens, H. (2013). Risk-based approach for the nonclinical development of ATMPs — useful strategy to realize science-driven regulatory drug testing? Regulatory Toxicology & Pharmacology 67, 221–225.
18 Van Meer, P.J.K., Kooijman, M., Brinks, V., Gispende Wied, C.C., Silva-Lima, B., Moors, E.H.M. & Schellekens, H. (2013). Immunogenicity of mAbs in non-human primates during nonclinical safety assessment. mAbs 5, 810–816.

The Need to Improve Experimental Design

Good experimental design is a valuable strategy
to address the reduction and refinement
principles of the Three Rs

Derek J. Fry

Among the Three Rs, Reduction seems the one which has least progress to record. This may partly be because some principles of good experimental design were established long ago. Some were codified in 1831, when Marshall Hall1 published his “Principles of Investigation in Physiology”, which emphasise the importance of having clear objectives, of minimising severity, and of using observation to avoid invasive procedures. Advances in experimental design since then are eclipsed by great advances in anaesthesia, analgesia and in understanding of animal structure, function and behaviour. The scope for the control of severity in experiments, for reliable measurements, for more-precise formulation of experimental questions and better detection of experimental effects has enormously improved. In particular over the 50 years since Russell and Burch developed the Three Rs concept2 one can trace its spread to worldwide acceptance and see significant improvements in many areas. As well as advances in anaesthesia and analgesic regimes, and much better understanding of animal behaviour, there has been much progress in recognition of pain and distress, a change in attitude among researchers from animals being merely datatools, and wide acceptance of the need for ethical evaluation. However, the major advances in experimental design developed in the 1920s and 30s have been slow to spread through biomedical studies, and experimental design of animal studies tends to consider only one R — Reduction — and omit consideration of the other two, particularly Refinement.

Figure 1: Basic principles in designing experiments
Independent replication — repeating the ‘treatment’,
i.e. the particular set of experimental conditions,
with a number of independent biological units
Randomisation — arranging that inherent differences
in biological units or the measurement process are
equally likely to occur with any of the experimental groups
Control — including comparisons which allow valid
interpretation of results obtained under different
experimental conditions

Over 50 years ago, writing in the UFAW handbook of 1957, Hume3 commented “techniques for designing experiments on the basis of small-sample theory are available” but “one still sees unjustifiably large samples reported”. He was referring principally to the methods in Fisher’s 1935 work, The Design of Experiments.4 Sadly, one could say the same today, with a number of recent papers citing the quality of studies as a likely major reason for difficulties in reproducing animal studies, and for the lack of correlation between their findings and results in the clinic.5–8 Also, the survey by Kilkenny et al. in 20099 considered that “a large number of the studies assessed did not make the most efficient use of the available resources (including the animals), by using the most appropriate experimental design”.
Although there is some concern that researchers are not clear in the objective of an experiment (this was judged to be the case in 5% of studies in the Kilkenny et al. survey9), a much greater problem seems to be that they are not adhering to the fundamental principles which are necessary to ensure that valid comparisons can be made between groups under different experimental conditions. These can be summarised as independent replication, randomisation and control (see Figure 1). To this list may be added ‘blinding’ (concealing the experimental treatment from those allocating experimental material to it, and also from those assessing the outcome).
‘Blinding’ guards against subjective bias, and is particularly important when effects are small. Without adherence to the fundamental principles, the results of an experiment are unreliable and statistical testing is illusory.
Independent replication is essential because of biological variation. It allows the extent of variability to be estimated, and that estimate is the basis for many statistical tests. It means allocating a number of animals or other experimental comparison units to each set of experimental conditions. But the units do have to be truly independent. Placing all test plates in one incubator and control plates in another risks confounding a difference between the incubators as a treatment effect. Similarly, putting ten mice all undergoing one treatment into two cages and another ten all undergoing a different treatment in another two cages superimposes cage effects on any treatment effects. Here, the “experimental unit” (defined by Festing et al.10 as “the unit of replication that can be assigned at random to a treatment”) is strictly speaking the cage and there are only two replicates for each treatment. Commonly, the ten individuals are taken as independent, without taking account of possible cage effects, so the estimates of power and the derived p values may be erroneous.
Randomisation is of crucial importance as it avoids an inherent difference being confounded with the effect of an experimental treatment. A good way of randomising is to give the animals (or other experimental units) numbers, then to use a computer to put the numbers in random order, and then use the experimental units in that order.
Suitable comparisons or controls are essential for the proper interpretation of results. Experimenters should think what the outcomes of an experiment might be, then think what the possible interpretations could be, and then think what controls are needed to avoid misinterpretation. Discussions with experimenters usually indicate that a negative control is routinely included in their experimental design, but other controls are not considered. Importantly, they forget positive controls, which are essential if there are possible causes for lack of effect in an experimental group other than that the treatment is ineffective — a change in animal susceptibility, for example.
The other major failure in experimental design is in the choice of type of design. Often fully randomised group comparison designs are used when there are actually more-efficient arrangements. Use of these more efficient arrangements would result in the gaining of more data and/or the use of fewer animals. Experimental units can be matched into sets according to a characteristic liable to contribute noticeably to variability, such as animal weight range, age or parentage, cage position in a rack, or the time of starting the experimental procedures. With this ‘blocking’ arrangement, the variability
between the sets can then be separately estimated and distinguished from individual variation or that due to treatment. This can provide a more-precise estimate of the effect of treatment, so the ability to detect a real effect is enhanced, and fewer experimental units are needed overall.
Another under-utilised approach is factorial arrangement, in which each experimental group is composed of experimental units with known but different characteristics — both male and female animals, for example, or animals of different strains or age bands.
In such designs, biological variation is estimated by using all the individual units and the number in each treatment-characteristic sub-group can be small. Variability due to the characteristic can be separately estimated from variation due to treatment, and overall numbers are much reduced when compared to repeating the treatments for each characteristic (sex, age, strain and so on).
So how should we address what seems to be a widespread failure to keep to important principles and use advances in design developed in the 1930s? One approach that shows signs of proving successful is education in experimental design by means of workshops of a particular pattern. That pattern involves a mix of information provision with group problem-solving which uses that information, and with discussion with experts in experimental design, statistical testing and control of severity. The comments here come from experience with one-to four-day workshops of this type, run in several countries since 2008. Indications of the need for such education come from the continuing demand for places, from workshop participant comments (e.g. “general statistics courses are too theoretical and too far away from my situation/experiments”), and from survey responses.11 Workshops can emphasise the important fundamentals and provide opportunities to consider a range of types of design. They can also consider refinement, alerting to the effects the severity of procedures and animal distress may have on the results, and suggest how to design for minimal severity.
Pre test and post test results
test scores

Participants’ comments and external assessments of the workshops rate them as informative, educational and enjoyable. Consistently, well over 90% of the participants agree that the workshops exposed them to new knowledge and practices. The routine testing of the knowledge and understanding of the participants has to date always shown a marked improvement between pre-tests and post-tests. The results from a three-and-a-half-day course in an EU country, shown in Figure 2a, are typical of the changes in particular understandings recorded. The distributions of pre- and post-test scores shown in Figure 2b are from a two-day course in an Asian country.
They illustrate the marked shift to higher scoring found so far in all of the workshops. These tests are more like quizzes than examinations, and some element of the improved scores could be improved understanding of the quiz format. However, the greater confidence with which the concepts become part of the group discussions and questioning of experts later in the workshops also show appreciation of the concepts and improved understanding of when different types of design might be used. The long term influence of these workshops has still to be evaluated, but some evidence of long-term effects was picked up in a survey by Howard et al.11

change in attitude
When ‘change in attitude’ was tested, as it was in one RSPCA-led workshop run in an Asian country, a distinct shift was seen. Figure 3 indicates the shift found with one of the questions asked. It shows the numbers of workshop participants giving different levels of agreement with the statement “One animal per cage should be the routine practice for housing rats and mice undergoing experiments”. At the end of the workshop the level of disagreement with routine single-housing had markedly increased.
These workshops have the advantage of providing some limited evidence of effectiveness, but they are only one of a number of approaches to providing education in experimental design. Experimental design has been included in RSPCA courses that also cover refinement, for example. The taught component of certain biomedical, biological and agricultural postgraduate courses in universities will include it, and it is a required element in UK project licensee training and FELASA training for “persons responsible for directing animal experiments”. These would all be expected to have an impact. The concerns raised in publications such as references 5–9, and meeting publication guidelines such as the Gold Standard Publication Checklist12 or the ARRIVE guidelines,13 should also focus attention on improving the design of animal experiments.
However, feedback from former workshop attendees and comments from researchers in many countries indicate that there is still much to be done. Workshop participants return to supervisors who are unwilling to change their time-honoured approaches, and submitted papers meet referees or editors who expect at least six animals per group, or are unfamiliar with blocking or factorial designs. The level of knowledge and confidence reached during a workshop, an undergraduate or postgraduate module, or licensee or FELASA training may well be insufficient of itself to sustain arguments for designs unfamiliar to the home laboratory or referees.
So there is a challenge here for all involved with animal experiments to be open to possibilities for improving design. It would be very sad if, in another 50 years, the comments of Hume,3 quoted above, still applied.

Dr Derek J. Fry
Faculty of Life Sciences
University of Manchester
Oxford Road
Manchester M13 9PT UK
E-mail: dj@fry39.fsnet.co.uk

References
1 Hall, M. (1831). Of the principles of investigation in physiology. In A Critical and Experimental Essay on the Circulation of the Blood; Especially as Observed in the Minute and Capillary Vessels of the Batrachia and of Fishes, 187pp. London, UK: Seeley & Sons.
2 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
3 Hume, C.W. (1957). The legal protection of laboratory animals. In The UFAW Handbook on the Care and Management of Laboratory Animals, 2nd edn (ed. A.N. Worden & W. Lane-Petter), pp 1–14. London, UK: The Universities Federation for Animal Welfare.
4 Fisher, R.A. (1935). The Design of Experiments, 252pp. Edinburgh, UK: Oliver & Boyd.
5 Perel, P., Roberts, I., Sena, E., Wheble, P., Briscoe, C., Sandercock, P., Macleod, M., Mignini, L.E., Jayaram, P. & Khan, K.S. (2006). Comparison of treatment effects between animal experiments and clinical trials: Systematic review. BMJ 334, 197–204.
6 Macleod, M.R., van der Worp, H.B., Sena, E.S., Howells, D.W., Dirnagl, U. & Donnan, G.A. (2008). Evidence for the efficacy of NXY-059 in experimental focal cerebral ischemia is confounded by study quality. Stroke 39, 2824–2829.
7 Prinz, F., Schlange, T. & Asadullah, K. (2011). Believe it or not: How much can we rely on published data on potential drug targets? Nature Reviews Drug Discovery 10, 712.
8 Begley, C.G. & Ellis, L.M. (2012). Raise standards for preclinical cancer research. Nature, London 483, 531–533.
9 Kilkenny, C., Parsons, N., Kadyszewski, E., Festing, .F.W., Cuthill, I.C., Fry, D., Hutton, J. & Altman, D.G. 2009). Survey of the quality of experimental design, statistical analysis and reporting of research using animals. PLoS One 4, e7824 [doi:10.1371/journal. pone.0007824].
10 Festing, M., Overend, P., Gaines Das, R., Cortina Borja, M. & Berdoy, M. (2002). The Design of Animal Experiments: Reducing the Use of Animals in Research Through Better Experimental Design, 112pp. London,
UK: Royal Society of Medicine Press Ltd.
11 Howard, B., Hudson, M. & Preziosi, R. (2009). More is less: Reducing animal use by raising awareness of the principles of efficient study design and analysis. ATLA
37, 33–42.
12 Hooijmans, C.J., Leenaars, M. & Ritskes-Hoitinga, M. (2010). A gold standard publication checklist to improve the quality of animal studies, to fully integrate
the Three Rs, and to make systematic reviews more feasible. ATLA 38, 167–182.
13 Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M. & Altman, D.G. (2010). Improving bioscience research reporting: The ARRIVE guidelines for reporting animal
research. PLoS Biology 8, e1000412 [doi:10.1371/journal.pbio.1000412].

Implementing the In Vitro Pyrogen Test: One More Step Toward Replacing Animal Experimentation

The MAT-based in vitro pyrogen test
should be GLP-accredited for use in
medical device testing

Ulrike Hennig

A pyrogen (from the Greek: pyros — fire, and genesis — generation) is a substance that induces fever upon blood contact in mammals. Deriving, for instance, from fragments of Gram-negative or Gram-positive bacteria, pyrogens can occur even in sterilised products, and have the capacity to pose a serious health threat to patients receiving parenteral treatments. The presence of such pyrogens can be assessed with an in vivo rabbit pyrogen test,1 which was officially introduced in the 1940s for the systematic risk assessment of infusion solutions. In this test, parenterals to be tested are injected into rabbits, and induce a rise in body temperature if they are contaminated with pyrogens. Another test system, which exclusively detects endotoxins from Gram-negative bacteria, is the LAL (Limulus amoebocyte lysate) test.2 The reaction of the lysate with the endotoxin can be measured chromogenically, or by turbidity assessment. The LAL test was a great innovation in the 1970s, but it does not detect the full range of pyrogens and cannot totally replace animal testing.
The in vitro Monocyte Activation Test (MAT) is a validated testing system that constitutes a complete replacement of the rabbit pyrogen procedure for the quality control of injectables.3 It comprises six variants of the same principle: the fact that cells produce cytokines upon contact with fever-inducing substances, so-called pyrogens.4–8 This reaction is (within a certain range) dose-dependent, and permits a quantitative determination of pyrogenic contents. One prominent feature of the MAT is that, unlike the LAL, it is not restricted to endotoxins. An in vitro pyrogen test, based on the MAT, is commercially available from Merck KGaA (Germany), under the name PyroDetect.
With the inclusion, from April 2010, of the Monocyte Activation Test (MAT) in the European Pharmacopoeia,9 an elementary step was taken toward the reduction of animal testing. Nevertheless, even after almost three years, the number of rabbits used for pyrogen testing still reaches approximately 100,000 animals per year in Europe alone.10 To counter this enormous use of animals, there are a number of qualified laboratories that perform the MAT as a service for customers from the pharmaceutical industry. The most important regulation for the pharmaceutical industry is GMP (Good Manufacturing Practice). Pharmaceutical products have to be manufactured and analysed according to these rules. Importantly, the MAT is applicable under GMP regulations, and the aforementioned commercial services can be offered with the necessary quality standard.
The testing of medical devices, however, is regulated separately (e.g. by ISO guidelines), where GLP (Good Laboratory Practice) rules are applied. Therefore, we are currently working on the introduction of PyroDetect for medical device testing in our existing GLP system. In this developmental work (which is being supported by Animalfree Research, Bern, Switzerland), the testing is performed in several steps. The medical devices are either eluated with pyrogen-free water and subsequently subjected to the whole blood assay (see Figure 1), or the devices are incubated directly with diluted human whole blood. Any pyrogenic content present in the eluate or on the devices leads to the secretion of the cytokine, IL-1ß, by blood monocytes. In a final step, the cytokine secretion is quantified via an Enzymelinked Immunosorbent Assay (ELISA).
Incubation and ELISA procedure
After the accreditation of PyroDetect under GLP, we will be able to test medical devices for pyrogenic contamination according to these high standards. With this knowledge, we will continue to also set standards for other applications, such as the testing of air quality. It is notable that pyrogenic limits for medical devices and/or air quality (for working places) are restricted to endotoxins. With a species specific system, based on human whole blood and for a wide range of pyrogens, more-relevant data are available to calculate health-related risks.

Acknowledgements
We thank Stefanie Schindler, from Animalfree Research, Switzerland, for her help with the manuscript. We also thank BioTek Germany for supporting us with their EON Reader and accompanying analysis software (Gen5 secure software).

Ulrike Hennig
Clinical Research Laboratory
Clinic of Thoracic, Cardiac and Vascular Surgery
University Hospital Tübingen
Calwerstr. 7/1
D-72076 Tubingen, Germany
E-mail: Ulrike.Hennig@klinikum.uni-tuebingen.de

References
1 Hort, E.C. & Penfold, W.J. (1912). Microorganisms and their relation to fever: Preliminary communication. Journal of Hygiene 12, 361–390.
2 Levin, J. & Bang, F.B. (1964). The role of endotoxin in the extracellular coagulation of Limulus blood. Bulletin of the Johns Hopkins Hospital 115, 265–274.
3 Hoffmann, S., Peterbauer, A., Schindler, S., Fennrich, S., Poole, S., Mistry, Y., Montag-Lessing, T., Spreitzer, I., Löschner, B., van Aalderen, M., Bos, R., Gommer, M.,
Nibbeling, R., Werner-Felmayer, G., Loitzl, P., Jungi, T., Brcic, M., Brügger, P., Frey, E., Bowe, G., Casado, J., Coecke, S., de Lange, J., Mogster, B., Naess, L.M.,
Aaberge, I.S., Wendel, A. & Hartung, T. (2005). International validation of novel pyrogen tests based on human monocytoid cells. Journal of Immunological Methods 298, 161–173.
4 Poole, S., Thorpe, R., Meager, A. & Gearing, A.J. (1988). Assay of pyrogenic contamination in pharmaceuticals by cytokine release from monocytes.
Developments in Biological Standardization 69, 121–123.
5 Poole, S., Thorpe, R., Meager, A., Hubbard, A.R. & Gearing, A.J. (1988). Detection of pyrogen by cytokine release. Lancet 1, 130.
6 Werner-Felmayer, G., Baier-Bitterlich, G., Fuchs, D., Hausen, A., Murr, C., Reibnegger, G., Werner, E.R. & Wachter, H. (1995). Detection of bacterial pyrogens on the basis of their effects on gamma interferonmediated formation of neopterin or nitrite in cultured
monocyte cell lines. Clinical & Diagnostic Laboratory Immunology 2, 307–313.
7 Hartung, T. & Wendel, A. (1995). Detection of pyrogens using human whole blood. ALTEX 12, 70–75.
8 Eperon, S. & Jungi, T.W. (1996). The use of human monocytoid lines as indicators of endotoxin. Journal of Immunological Methods 194, 121–129.
9 European Directorate for the Quality of Medicines (2009). Monocyte-activation test. In European Pharmacopoeia 6.7, Chapter 2.6.30. Strasbourg, France: Council of Europe.
10 Daneshian, M., Akbarsha, M.A., Blaauboer, B., Caloni, F., Cosson, P., Curren, R., Godberg, A., Gruber, F., Ohl, F., Pfaller, W., van der Valk, J., Vinardell, P.,
Zurlo, J., Hartung, T. & Leist, M. (2011). A framework program for the teaching of alternative methods (replacement, reduction, refinement) to animal experimentation. ALTEX 28, 341–352.

Balancing Reduction and Refinement

By considering reduction before refinement, we risk allowing some animals to experience greater pain and distress. A variety of approaches can help balance reduction and refinement while promoting the welfare of individual animals

Nicole Fenwick and Gilly Griffin

The classic Russell & Burch 1 sequence of applying the Three Rs first examines how to replace the use of the animal, followed by how to reduce numbers through good experimental design. Then ways to minimise pain and distress (and improve animal welfare) through refinements are considered. If this sequence is followed slavishly, there is a risk that the drive to reduce animal numbers will overshadow the goal of minimising pain and distress for each individual animal.

Conflict between reduction and refinement arises when “procedures can be performed such that they either inflict less harm on more animals or inflict more harm on fewer animals”2 (p. 334), as has been recognised in the more-recent literature.2–5 It has also been addressed in animal use policy. For example, the UK Animals (Scientific Procedures) Act 1986 states “the use of fewer animals in each experiment” must occur “…without compromising animal welfare”.6

Similarly, the International Council for Laboratory Animal Science states that the use of genetically-altered animals “may be a Refinement due to the increased accuracy of the animal model, [but] a significant number of animals are required to generate a new animal line, thus bringing the principles of Refinement and Reduction into conflict” 7 (p.3). In addition, Policy #14 of the United States Animal Welfare Act prohibits the use of an animal “in more than one major survival operative procedure, unless the multiple procedures are required to meet the objective of a single animal study activity”.8 The difficulties of balancing reduction and refinement are routinely faced by animal ethics committees (AECs). A study of Canadian AECs found that, when evaluating studies, members often disagreed about whether animal numbers are an important consideration and whether harm from routine housing and husbandry should be considered.9 The original definition of refinement referred only to experimental procedures, not husbandry. However, refinement is now usually taken to embrace the entire lifetime of the animal.10–12 Conflict between reduction and refinement was also identified as a source of “tension” for AECs in the United States.13

Reduction–refinement conflicts appear before AECs in a variety of forms. One form, related to quality of life or the animals’ welfare from birth to death, encompasses concerns about housing and husbandry (not just experimental procedures). One example might be the maintenance of a colony of immunecompromised mice, where the condition of the mice requires they be housed without bedding (to avoid skin irritation) in ventilated cages (where they are exposed to continuous draughts and noise). The colony is maintained while the research is temporarily suspended, because animals will be needed again in the near future. It can be argued that fewer animals are used in the maintenance of the colony than if the colony were euthanised and rederived from frozen embryos when subsequently needed. However, focusing on minimising animal numbers means that debilitated mice continue to experience a poor quality of life, unable to perform the full repertoire of mouse behaviour, such as nest building14 and subject to distress from noise and draughts.14, 15

Animal re-use, defined as sequential use of the same animal for unrelated experiments,16 highlights a further reduction–refinement conflict. One example involves dogs maintained for pharmacokinetic studies in drug research. They are trained to co-operate with the administration of novel drugs and repeated blood sampling, and are housed in an enriched environment. However, the same dogs are re-used many times in order to keep numbers low, so the procedures are experienced repeatedly by the animals, presenting challenges to AECs regarding how to establish limits.

Determining the value of a study is a key factor for AECs, who must weigh harms and benefits. In some cases, they must evaluate how a reduction–refinement conflict might influence experimental outcomes, and therefore the value of the study.

Consider the example of wildlife research on freeliving animals to understand the species biology and inform conservation, where the use of telemetry devices and recapture often allows for fewer animals to be used. However, these individuals may be subjected to harms from capture, wearing tracking devices, biological sampling, and multiple recapture,17–19 leading to concerns that their welfare is compromised and that they no longer represent the typical individuals of their species.

How can reduction and refinement be successfully balanced? Russell and Burch prioritised the experience of the individual animal over reduction. Therefore, when AECs identify a reduction–refinement conflict, they may choose to address it by applying reduction and refinement in conjunction, rather than independently,4 or by considering refinement before reduction. In addition, they may consider that weighing harms and benefits — a utilitarian framework — may not be the best approach for tackling reduction–
refinement conflicts.

Policymakers might address the reduction–refinement conflict by adopting expanded definitions
for refinement that apply to the lifetime experiences of animals. The recent EU Directive 2010/63/EU places conditions on animal re-use, which focus on assessing cumulative severity. Re-use is dependent on the “actual severity of the previous procedures” and the “lifetime experience of the animal” (Article 16).20 The assessment of cumulative suffering, through the use of extended welfare assessment grids,21 should provide more-objective information on whether refinement for individual animals is being achieved. Policymakers can also draw attention to the reduction–refinement conflict by counting and reporting animal re-use and breeding colony animal numbers.

When reduction is prioritised or given equal importance to refinement, greater harms may occur to individual animals. Conversely, when refinement is prioritised, more animals must be used. Arguably, if it is possible to eliminate any pain and distress, then reduction of animal numbers becomes less important.22 To minimise pain and distress and improve animal welfare, a variety of approaches by AECs and policymakers that focus on experiences of the individual animal, will be needed.

Acknowledgements
Thanks to Dr Andrew Winterborn and the Queens University Animal Care Committee, for discussions on this topic.

References
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 Olsson, I.A.S., Franco, N.H., Weary, D.M. & Sandøe, P. (2012). The 3Rs principle — mind the ethical gap. ALTEX Proceedings 1/12, 333–336.
3 Hansen, A.K., Sandøe, P., Svendsen, O., Forsman, B. & Thomsen, P. (1999). The need to refine the notion of reduction. In Humane Endpoint in Animal Experiments for Biomedical Research, pp. 139–144. London, UK: Laboratory Animals Ltd. Available at: http://
www.lal.org.uk/publications/this-is-a-test-subject/ humane-endpoints/ (Accessed 15.05.13).
4 De Boo, M.J., Rennie, A.E., Buchanan-Smith, H.M. & Hendriksen, C.F.M. (2005). The interplay between replacement, reduction and refinement: Considerations where the Three Rs interact. Animal Welfare 14, 327–332.
5 Olsson, I.A.S., Robinson, P. & Sandøe, P. (2010). Ethics of animal research. In Handbook of Laboratory Science, Vol. 1, Third Edition: Essential Principles and Practices (ed. J. Hau & S.J. Schapiro), pp. 21–38. Boca Raton, FL, USA: CRC Press, Taylor & Francis Group.
6 HMSO (1986). Animals (Scientific Procedures) Act 1986. 1986 Chapter 14: An Act to make new provision for the protection of animals used for experimental or other scientific purposes [20th May 1986]. London, UK: Her Majesty’s Stationery Office. Available at: http://www.legislation.gov.uk/ukpga/1986/14 (Accessed 16.07.13).
7 Rose, M., Everitt J., Hedrich, H., Schofield, J., Dennis, M., Scott, E. & Griffin, G. (2013). ICLAS Working Group on Harmonisation: International guidance
concerning the production care and use of genetically-altered animals. Laboratory Animals 47,
146–152.
8 USDA (2011). Policy #14: Major Survival Surgery Dealers Selling Surgically-Altered Animals to Research. Animal Care Resources Guide. Washington, DC, USA: United States Department of Agriculture. Available at: http://www.aphis.usda.gov/animal_
welfare/policy.php?policy=14 (Accessed 06.06.13).
9 Schuppli, C.A. & Fraser, D. (2005). The interpretation and application of the Three Rs by animal ethics committee members. ATLA 33, 487–500.
10 Balls, M., Goldberg, A.M., Fentem, J.H., Broadhead, C.L., Burch, R.L., Festing, M.F.W. & van Zutphen, B.F.M. (1995). The Three Rs: The way forward. ECVAM Workshop Report 11. ATLA 23, 838–866.
11 CCAC (2013). Three Rs Microsite. Montreal, Canada: Canadian Council on Animal Care Available at: http://3rs.ccac.ca/en/ (Accessed 15.05.13).
12 NC3Rs (2013). What are the 3Rs? London, UK: National Centre for the Replacement, Refinement & Reduction of Animals in Research. Available at: http://www.nc3rs.org.uk/page.asp?id=7 (Accessed 15.05.13).
13 Carbone, L. (2011). Pain in laboratory animals: The ethical and regulatory imperatives. PLoS ONE 6, (9):e21578 [doi:10.1371/journal.pone.0021578].
14 Baumans, V. & Van Loo, P.L.P. (2013). How to improve housing conditions of laboratory animals: The possibilities of environmental refinement. The Veterinary Journal 195, 24–32.
15 Jennings, M., Batchelor, G.R., Brain, P.F., Dick, A., Elliott, H., Francis, R.J., Hubrecht, R.C., Hurst, J.L., Morton, D.B., Peters, A.G., Raymond, R., Sales, G.D.,
Sherwin, C.M. & West, C. (1998). Refining rodent husbandry: The mouse. Report of the Rodent Refinement Working Party. Laboratory Animals 32, 233–259.
16 Fentner van Vlissingen, J.M. (1999). The re-use of animals for research — a humane endpoint? In Humane Endpoints in Animal Experiments for Biomedical Research, pp. 145–147. London, UK: Laboratory Animals Ltd. Available at: http://www.lal.org.uk/
publications/this-is-a-test-subject/humane-endpoints/(Accessed 15.05.13).
17 Beaulieu, M., Ropert-Coudert, Y., Le Maho, Y. & Ancel, A. (2010). Is abdominal implantation of devices a good alternative to external attachment? A comparative
study in Adélie penguins. Journal of Ornithology 151, 579–586.
18 Cattet, M., Boulanger, J., Stenhouse, G., Powell, R.A. & Reynolds-Hogland, M.J. (2008). An evaluation of long-term capture effects in ursids: Implications for wildlife welfare and research. Journal of Mammalogy
89, 973–990.
19 Léchenne, M.S., Arnemo, J.M., Bröjer, C., Andrén, H. & Ågren, E.O. (2012). Mortalities due to constipation and dystocia caused by intraperitoneal radio-transmitters in Eurasian lynx (Lynx lynx). European Journal of Wildlife Research 58, 503–506.
20 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 in the protection of animals used for scientific purposes. Official Journal of the European Union L276, 20.10.2010, 33–79.
21 Honess, P. & Wolfensohn, S. (2010). The extended welfare assessment grid: A matrix for the assessment of welfare and cumulative suffering in experimental animals. ATLA 38, 205–212.
22 Weary, D.M. (2012). A good life for laboratory animals – how far must refinement go? ALTEX Proceedings 1/12, 11–13.

The Ethics of Using Laboratory Animals to Develop Medicines for Lifestyle Diseases

Pink pills

Clive Phillips

Humans advance through life with their accolade
of food and laboratory animals to sustain them,
with the former unfortunately increasing the need for the latter

It may be the ultimate expression of speciesism. People fall victim to the numerous addictions that pervade modern society — sugar, alcohol, tobacco, salt, saturated fats, to name but a few — and then expect medicines that have been developed through the use of other animals to sustain them when their bodies fail to cope. Diabetes, obesity, alcoholism, heart disease, cancer ― all of these non-communicable
diseases (NCDs) are promoted by our modern lifestyle. However, some people, when they get to an advanced state of decrepitude in old age, or in many cases middle age, are opting to forego the use of medicaments and their various side-effects and live out their natural lives naturally. Instead of relying on the better-late-than-never band-aids of medicaments developed with the use of animals, how much more effective would it be if preventative medicine and better education enabled people to live to their geriatric years, without becoming
incapacitated? This would benefit the people themselves, but it might not help in the financial health of the country.

Multinational companies increasingly control the production, processing and retailing of those very food products that are causing us to rely on animals to produce the medicines, and they also have a major control over governments through their importance to national economies. For example, in Australia, the recent merger of the multinational company, Cargill, Inc., with the beef processing giant, Teys Bros, created a company that can manage the entire beef food chain — from grain production to cattle carcase processing — for 1.5
million cattle per year. Globally, Cargill has sales revenues of US $120 billion per year and employs 130,000 people, the size of a small city.

The multinational companies have targeted our foibles, and the very appetites that evolved because of their importance for the maintenance of the human body — i.e. sugar for short-term energy, fat for long-term energy, salt for ionic homeostasis — are now exploited to poison our bodies. Food manufacturers use brain imaging to detect human responses in fine detail, such as the flavour burst produced by salt1 (with Cargill being the world’s largest producer of this commodity), and the prolonged aromatic and sensory flavours from high-fat cheeses are well known and exploited.

The human cost in NCDs arising from lifestyle problems is becoming well recognised, but the animal cost is rarely considered. It includes both food animals and laboratory animals, the latter being used to
produce the medicines to treat the NCDs. Last year alone, over 2000 articles which described the use of mice or rats to address the burgeoning problem of diabetes were published in scientific journals recognised by the Web of Science.2 Even if a conservative
estimate of 50 animals were used per study, and only half of the work that was done ended up being published in Web of Science journals, that still amounts to the use of 200,000 rodents and millions of dollars in research funding in one single year.1 Still more mice were used in the production of suitable genetic models that were susceptible to the disease. Whilst few can doubt that progress is being made in the diagnosis and treatment of this disease, if a fraction of the money that was used for these studies was directed toward educating the public about dietary restraint, then the people’s quality of life would improve and fewer research animals would be needed.

The servitude of our minds to the power of potentially-deficient nutrients means that we must satisfy our desires to the detriment of the survival of society. We may yet be able to think, plan and manage our way out of such a complex problem, but that requires strong and just government, better education for the good of society, and less reliance on animals in laboratories and on farms.

A relatively new and powerful tool in persuading people not to eat foods from animals that have been produced in inhumane conditions is to confront them with the reality of where their food comes from.
Cameras in abattoirs, on live-export ships, and in ports of embarkation and disembarkation from these ships, provide the images that many believe will ultimately change people’s way of thinking in relation to the sourcing of their food. The news media obviously believe that people have a hunger for these graphic images, or a conscience that can easily be aroused. Foods produced from animals that are themselves fed nutrient-rich diets, in intensive housing where opportunities for activity are limited, have high saturated fat content (30–50% of total fat content), as compared with the same animals in the wild.3 Generally, the higher the fat content in an animal’s body, the lower the proportion of fat that is polyunsaturated.4 The consumption of saturated fat in meats appears to be related to an increased risk of the major NCDs: i.e. obesity, diabetes and coronary heart disease,4 and public health nutritionists advocate a reduction of saturated fat in the diet, in order to avoid NCDs such as diabetes and cardiovascular disease.5,6

It is only a matter of time before there is more widespread use of cameras in laboratories, which may demonstrate to people the level of animal suffering that accompanies the production of the drugs to help them cope with their NCDs. Of course, we have the Three Rs7 (or indeed Four Rs, as Catherine Tiplady recently argued for in PiLAS8) to help us provide laboratory animals with a respectable environment in which to live. However, another form of speciesism that pervades the discussions of animal ethics committees is the acceptance of inhumane standards for laboratory animals used for drug testing, because of the perceived benefit to society, or at least to individuals within society. No-one can deny the fact that individually-caged rats, a social species that is highly active in the wild, have an inherently low standard of welfare, even when compared to farm livestock in pens or cages, which at least have the benefit of the close proximity of conspecifics. Yet we tolerate the situation in the case of the rats, solely because they are used for scientific research that is seen as essential for our survival.

It is a sorry fact that, in today’s society, humans advance through life with their accolade of food and laboratory animals to sustain them, with the former unfortunately increasing the need for the latter.

Professor Clive Phillips
Centre for Animal Welfare and Ethics
School of Veterinary Science
University of Queensland
Gatton 4343
Queensland
Australia
E-mail: c.phillips@uq.edu.au

References
1 Seo, H.S., Iannilli, E., Hummel, C., Okazaki, Y., Busch – hüter, D., Gerber, J., Krammer, G.E., van Lengerich, B. & Hummel, T. (2013). A salty-congruent odor enhances saltiness: Functional magnetic resonance
imaging study. Human Brain Mapping 34, 62–76.
2 Thomson Reuters (2013). Web of Science. New York,
NY, USA: Thomson Reuters. Available at: http://thomsonreuters.
com/products_services/science/science_
products/a-z/web_of_science/ (Accessed 08.04.13,
via UQ Library Login).
3 Fine, L.B. & Davidson, B.C. (2008). Comparison of lipid and fatty acid profiles of commercially raised pigs with laboratory pigs and wild-ranging warthogs. South African Journal of Science 104, 314–316.
4 Siri-Tarino, P.W., Sun, Q., Hu, F.B. & Krauss, R.M. (2010). Saturated fatty acids and risk of coronary heart disease: Modulation by replacement nutrients. Current Atherosclerosis Reports 12, 384–390.
5 Browning, L.M. & Jebb, S.A. (2006). Nutritional influences on inflammation and type 2 diabetes risk. Diabetes Technology & Therapeutics 8, 45–54.
6 Everitt, A.V., Hilmer, S.N., Brand-Miller, J.C., Jamieson, H.A., Truswell, A.S., Sharma, A.P., Mason, R.S., Morris, B.J. & Le Couteur, D.G. (2006). Dietary approaches that delay age-related diseases. Clinical Interventions in Aging 1, 11–31.
7 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
8 Tiplady, C. (2012). Animal use in veterinary education
— The need for a Fourth R: Respect. ATLA 40, P5–P6