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ADVANCING SAFETY SCIENCE AND HEALTH RESEARCH WITH INNOVATIVE, NON-ANIMAL TOOLS

An Expert Report by Humane Society International/Europe

Written by Dr Gill Langley M.A., Ph.D. (Cantab)

About HSI EXECUTIVE SUMMARY

Humane Society International/Europe5 Underwood StreetLondon N1 7LYUnited Kingdom

hsi.org/endanimaltesting hsieurope.org

This publication is supported by a grant from the World Society for the Protection of Animals

computational biology, and high-speed robot automation of cell-based (in vitro) screening systems, to name a few, has sparked a quiet revolution in biology.

Together, these innovations have produced new tools and ways of thinking that can help uncover exactly how chemicals and drugs disrupt normal processes in the human body at the level of cells and molecules. From there, scientists can use computers to interpret and integrate this information with data from human and population-level studies. The resulting predictions regarding human safety and risk are potentially more relevant to people in the real world than animal tests.

Innovative Health Research for a Sustainable Future

Toxicology for the 21st century. Tox21. Next-generation safety testing. Whatever term is used, it refers to an exciting and fundamental change in the way chemical and product testing and risk assessments are carried out. The National Research Council in the United States has expressed its vision of “a not-so-distant future in which virtually all routine toxicity testing would be conducted

in human cells or cell lines”, and science leaders across the EU and globally have echoed this view.

The ultimate goal is to assess safety:

> Of a much larger number of substances than is currently possible

> Of combinations of chemicals so that effects of real-life exposure can be better assessed

> More rapidly, efficiently, and cost-effectively than at present

> In systems that are likely to be more relevant to toxicity in humans, as well as capable of identifying the cellular mechanisms at the root of toxicity and disease

> Using fewer or no animals.

The European Commission and Member States have already begun to invest in this research area, and collaborative agreements have been struck between EU and global research teams to maximise co-ordination, data sharing and potential synergies. However, the 7th Framework Programme (FP7)-funded co-ordination project AXLR8 (axlr8.eu) estimates that a further combined

EXECUTIVE SUMMARY 3

Humane Society International (HSI) and its partner organisations together constitute one of the world’s largest animal protection organisations— backed by 11 million people. For nearly 20 years, HSI has been working for the protection of all animals through the use of science, advocacy, education and hands-on programmes. HSI’s European Policy Office is a leading force for animal protection in the EU, with active public policy initiatives to reduce and replace animal use in scientific research and regulatory testing, to combat the slaughter of marine life, and to improve conditions for animals farmed for food, skins and fur.

Robotic “high throughput” cell culture systems can screen thousands of chemicals in a single day.

Chemicals, including those used in consumer goods, industrial processes and medicinal products, are essential to modern life, yet we lack innovative, efficient and human-relevant testing tools to inform regulatory safety decisions. New approaches are needed to promote economic growth, protect against adverse health and environmental impacts of chemicals, replace animal use, and support greener chemistries and safer products.

But hope is on the horizon: Europe’s new Framework Programme for research and innovation—Horizon 2020—has the potential to revolutionise not only our approach to safety testing, but the field of human health research as a whole.

Advancing Health Research and Safety Testing: A Societal Challenge

For nearly a century, drug and chemical safety assessments have been based on laboratory testing involving rodents, rabbits, dogs, and other animals. Aside from the ethical issues they pose, animal tests are time- and resource-intensive, restrictive in the number of substances that can be tested, provide little understanding of how chemicals behave in the body, and in many cases do not correctly predict real-world human reactions.

Current drug attrition rates see nine out of every 10 candidate medicines that appear safe and effective in animal studies fail when given to humans—a waste of billions of €, millions of animal lives, and all the while failing to effectively address pressing human health needs. Similarly, scientists are increasingly questioning the relevance of research aimed at ‘modelling’ human diseases in the laboratory by artificially creating symptoms in other animal species. In contrast, unprecedented scientific and technological advances are being made which have true ‘game changer’ potential. The sequencing of the human genome and birth of functional genomics, the explosive growth of computer power and

Asthma rates are on the increase, yet most treatments that perform well in “animal models” fail in humans due to lack of safety and efficacy.

ContentsAbout HSI ............................................................................................................... 2

Executive Summary ............................................................................................... 3

Specific recommendations for Horizon 2020 ...................................................... 5

Introduction .......................................................................................................... 6

Changing regulatory needs ................................................................................ 7

The new science in toxicology and health research ............................................. 8

Why this report ................................................................................................. 8

Chapter 1: Conventional safety testing—time to change .................................... 9

Scientific and technical limitations of animal testing ............................................ 9

Economic and resource limitations of animal testing ........................................... 13

Regulatory limitations of animal testing .............................................................. 14

Chapter 2: 21st century testing—safer for us all ................................................. 18

Safety testing: state-of-the-art tools ................................................................... 19

Exposure science and risk assessment ................................................................. 25

The benefits of advanced safety testing .............................................................. 27

Chapter 3: Advancing the science of health research .......................................... 28

Outdated animal ‘models’ of disease .................................................................. 28

The 21st century approach .................................................................................. 31

Advanced techniques: supporting safe medical innovation ................................... 31

Strategy and investment .................................................................................... 32

Chapter 4: Supporting the next steps in 21st century safety testing and health research .............................................................................................. 33

Strategic direction and visionary funding ............................................................ 33

Regulatory support and change ......................................................................... 34

2 About HSI Executive Summary 3

4 Executive Summary

investment by the EU and industry of the order of €325 million, together with equivalent funding commitments in other regions (ideally through formal public-public partnerships with the EU and public-private partnerships with industry), may be needed to fully address the scientific challenges that lie ahead in the safety testing area.

But that’s just the beginning. The wider field of human health research could benefit from a similar shift in paradigm. Many disease areas have seen little or no progress despite decades of animal research. Some 300 million people currently suffer from asthma, yet only two types of treatment have become available in the last 50 years. More than a thousand potential drugs for stroke have been tested in animals, but only one of these has proved effective in patients. And it’s the same story with many other major human illnesses.

A large-scale re-investment in human-based (not mouse or dog or monkey) research aimed at understanding how disruptions of normal human biological functions at the levels of genes, proteins and cell and tissue interactions lead to illness in our species

could advance the effective treatment or prevention of many key health-related societal challenges of our time.

EU Policy Drivers

The Treaty on the Functioning of the European Union acknowledges animals’ sentience and requires the Union and Member States to “pay full regard to the welfare requirements of animals” in formulating and implementing EU policies. Decision No. 1982/2006/EC of the European Parliament and of the Council concerning FP7 aims, among other things, to reduce and ultimately replace the use of animals in research and testing. Directive 2010/63/EU for the protection of animals used for scientific purposes provides a further overarching mandate for “coordinating and

Two high-throughput testing robots at the US National Human Genome Research Institute.

Specific Recommendations for Horizon 2020

European Commission legislative proposals for the establishment of Horizon 2020, its specific work programmes and associated regulatory instruments, provide an initial point of departure for a paradigm shift in EU health research and safety testing, and the ongoing political process provides EU Institutions the opportunity to refine and augment this basic framework and demonstrate their political commitment to the revolutionary change that is needed. To this end, we recommend the following:

> Establishment of a high-level public-public partnership between EU Directorates General for Research & Innovation and Joint Research Centre and regulatory/research agencies in the United States and other countries that are actively working to map human disease and toxicity pathways and to develop efficient, human biology-based experimental and computational tools for their study, to share the workload in a co-ordinated manner, maximising synergies and avoiding duplication.

> A funding commitment of at least €325 million under Horizon 2020 to a scientific work programme as outlined above and by the FP7 project AXLR8 (axlr8.eu). Achievement of this funding level through one or more targeted public-private partnerships with industry is encouraged.

> A strategic ‘top-down’ and tightly coordinated framework for this research effort using non-traditional funding models (e.g., longer-term, larger budgets, fewer partners, etc.). This should include centrally co-ordinated mechanisms to identify relevant research in Member States and internationally and ensure its integration with EU-funded projects.

> Increased the proportion of health research funding dedicated to human biology-based in vitro, -omics, computational, and other innovative, non-animal tools and technologies. Applying these to better define human disease pathways is a basis for developing more targeted and effective medical interventions. This can also be regarded as providing a financial incentive for adherence to the requirements of Decision No 1982/2006/EC and Directive 2010/63/EU.

> Development of health discipline-specific (e.g., cancer, immunology, etc.) research and development roadmaps to expand the strategic application of advanced human biology-based advanced tools and technologies in EU health research strategies.

> Establishment of more rigorous and transparent processes for ethical and scientific merit review of new projects involving animal use, including substantiation of claims regarding the relevance of animal models to the human health outcomes, and a requirement for retrospective assessment and systematic analysis of all vertebrate animal experiments expected to cause more than ‘moderate’ suffering, regardless of species.

Specific recommendations for Horizon 2020 5

promoting the development and use of alternatives to [animal] procedures including in the areas of basic and applied research and regulatory testing”.

An investment in the science needed to replace the use of animals in testing and research is more than a political objective driven ethical considerations; it is an opportunity to improve our fundamental understanding of human biology as a means of achieving optimal health and being able to effectively treat—and ideally, prevent—disease. This remains a pressing societal challenge, which can only be met by moving beyond the research paradigm from last century and fully embracing the science of the 21st century.

Disease pathways based on gene, protein and cell networks can be researched using human cells and tissues in the test tube.

Virtually every manufactured product used in our homes, gardens, fields and industries contains chemicals, either synthetic or natural. Chemicals are in most of our medicines and are key ingredients in cosmetics. Chemicals are added to our foods as colourings, flavourings and preservatives. They can also contaminate foods when used in packaging, or as leftover residues of plant protection products (pesticides) applied to combat fungi, weeds and insects in our fields. In our homes they are used as disinfecting agents (biocides) and are found in numerous products such as solvents, paints, furniture, plastics and air fresheners.

As a result of decades of widespread use, synthetic chemicals are found in the air, food, water, dust and soil—even in our bodies. In 2003, blood samples were taken from volunteers from 17 European countries, including Members of the European Parliament (MEPs).1 The samples were analysed for a variety of persistent, bioaccumulative and toxic (PBT) substances such as DDT and other organochlorine pesticides, PCBs, phthalates, brominated fire retardants, and perfluorinated chemicals.

Every volunteer tested was contaminated by a cocktail of potentially hazardous chemicals. Children, as they develop physically and mentally, are particularly at risk, and they are also contaminated. The typical daily diet of French children was tested for chemical residues in 2010. The foods contained 81 different synthetic chemical substances, including 36 pesticides and 47 suspected carcinogens.2

The problem is worldwide: A recent report from the US Centers for Disease Control and Prevention (CDC) records the outcome of the most comprehensive assessment of the exposure of the American population to chemicals in the environment. The CDC looked for and found 212 chemicals in people’s blood or urine, including toxic substances such as arsenic, acrylamide and bisphenol A.3

For much of the 20th century, chemicals were not comprehensively tested for their

potential toxicity to humans, animals or the environment. As problems developed and were recognised, safety-testing legislation was introduced in a piecemeal way in the different industry sectors. For example, medicines began to undergo substantial animal testing during the 1960s (after the Thalidomide disaster); pesticides during the 1970s; but prior to 1981 safety testing was not comprehensive in the European chemical industry. Even after 1981, the 100,000 then existing chemicals were exempted from testing requirements.4

Meanwhile, the medical research which underpins our health has suffered a roadblock for many years. Major illnesses such as strokes, motor neuron disease, Alzheimer’s and Parkinson’s diseases are still not sufficiently understood, and efforts to develop effective therapies have had limited success.

Legislation and regulations are being introduced or amended at all levels and in all

Changing Regulatory Needs

Introduction 7

Hazard labelling continues to be a major driver for “tick-box” animal testing, including immensely cruel “acute lethality” tests.

INTRODUCTION

product sectors to improve the safety testing and risk assessment of new chemicals and to hasten the evaluation of the untested, pre-1981 chemicals.5 At the same time, updated EU legislation controlling animal experimentation and emphasising the need to replace, reduce and refine animal use applies across all safety testing and health research areas.

Most of the changes in EU regulatory requirements reflect a greatly increased effort to assess the safety of chemicals—but how effective will that effort be using existing methodologies?

The traditional testing framework was established in the 1960s and is widely recognised as inappropriate for modern demands, even with incremental changes over the years that have been intended to update some of the animal test methods. As the US Environmental Protection Agency (EPA) stated in its 2009

strategic plan6 for evaluating the toxicity of chemicals:

“This approach has led over time to a continual increase in the number of tests, cost of testing, use of laboratory animals, and time to develop and review the resulting data. Moreover, the application of current toxicity testing and risk assessment approaches to meet existing, and evolving, regulatory needs has encountered challenges in obtaining data on the tens of thousands of chemicals to which people are potentially exposed and in accommodating increasingly complex issues (e.g., lifestage susceptibility, mixtures, varying exposure scenarios, cumulative risk, understanding mechanisms of toxicity and their implications in assessing dose-response, and characterization of uncertainty)”.

The limitations of conventional toxicology also include difficulties in translating the results of tests in different species and breeds of animals to human populations; and problems in understanding the human health significance of tests using unrealistically high chemical doses (see Chapter 1).

Emerging safety concerns about particular effects, such as endocrine disruption,7 learning disabilities, effects on fertility,

1 WWF (2004). Chemical Check Up—An analysis of chemicals in the blood of Members of the European Parliament. WWF/Co-operative Bank. assets.panda.org/downloads/checkupmain.pdf

2 Générations Futures and the Health and Environment Alliance published the results of tests on typical children’s meals in France in December 2010. menustoxiques.fr/

3 CDC (2009). Fourth National Report on Human Exposure to Environmental Chemicals. cdc.gov/exposurereport/pdf/FourthReport.pdf

4 Commission of the European Communities (2001). White Paper: Strategy for a Future Chemicals Policy. COM(2001) 88 final.

developmental damage to vulnerable groups such as unborn fetuses and children, and effects of chemical mixtures, highlight the need for improved test regimes because existing methods can be inconclusive when required to address subtle and complex human health and environmental impacts. The introduction of novel substances, some with previously unknown effects, has further exposed the inadequacy of traditional safety testing.

Simultaneously, there are legislative imperatives to replace and reduce animal use in testing and research. The new Directive 2010/63/EU on the protection of animals used for scientific purposes (like its predecessor Directive 86/609/EEC) calls for the replacement of animal tests to be advanced, as does the seventh amendment of the Cosmetics Directive 76/768/EEC, which introduced progressive bans on animal testing. Similarly, the ‘REACH’ chemicals regulation calls for non-animal testing to be progressed so as to produce safety data relevant to humans, and to replace current animal studies. These legislative drivers are underpinned by the Treaty on the Functioning of the European Union8, which acknowledges animals’ sentience and requires the Union and Member States to “pay full regard to the welfare requirements of animals” in formulating and implementing EU policies.

5 European Chemicals Agency (2009). Press Release 28 August 2009. echa.europa.eu/doc/press/pr_09_11_animal_testing_20090828.pdf

6 EPA (2009). The US Environmental Protection Agency’s Strategic Plan for Evaluating the Toxicity of Chemicals. EPA 100/K-09/001. epa.gov/osa

7 An endocrine disrupter is a substance or mixture that alters the body’s hormonal (endocrine) system and consequently causes adverse health effects.

8 Treaty on the Functioning of the European Union (2008). eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:C:2008:115:0047:0199:en:PDF

6 Introduction

In 2007, toxicology began a massive paradigm shift with the publication of the US National Research Council (NRC) report Toxicity Testing in the 21st Century: A Vision and a Strategy.9 This truly visionary report was a pivotal document which explained why toxicology must incorporate the revolutions in biology and computer science and move away from animal testing. The driving force behind the NRC report was not to replace animal tests only for ethical reasons, but to develop an approach that would better protect people, wildlife and the environment.

The NRC vision has helped to re-energise non-animal testing initiatives globally, such as the chemical safety programme at the Organisation for Economic Co-operation and Development (OECD), and the US Food and Drug Administration’s (FDA) Critical Path to New Medical Products initiative. In the EU, it is stimulating a transition in toxicology that is reflected in the plans for the next Research and Innovation Framework Programme, Horizon 2020.

Reflecting the new paradigm in safety testing, fundamental health research and drug discovery also need to be re-focused primarily on human disease pathways, away from efforts to mirror human illnesses in other animals.

The purpose of this Humane Society International/Europe report is to explain why the 21st century approach is the essential framework for making chemicals safer and health research more effective, while also minimising animal use and suffering; and to recommend the next steps for the EU to support and accelerate this exciting scientific and technological revolution through Horizon 2020.

Unless the new research and testing technologies are used within completely fresh paradigms for effective health research and safety testing, there is little chance

of radically improving the prevention and treatment of human diseases, or of ensuring that the chemical environment in which we live is a safe one.

This challenge—to improve safety testing and health research by replacing outdated animal studies with modern techniques—is universal. It is not limited to one or a few industries, countries or regions, but is relevant worldwide. The changes will not be easy to achieve, but they are essential. The development and implementation of advanced approaches must continue to be prioritised in the EU and worldwide.

The New Science in Toxicology and Health Research

Introduction 9 8 Introduction

9 Committee on Toxicity Testing and Assessment of Environmental Agents, National Research Council (2007). Toxicity Testing in the 21st Century: A Vision and a Strategy. ISBN: 978-0-309-10988-8.

Why this Report?

Animal tests failed to detect the cancer-causing effects of asbestos, benzene, cigarette smoke, and other substances, delaying consumer warnings by many years.

CHAPTER 1 CONVENTIONAL SAFETY TESTING—TIME TO CHANGE

Animal-based safety testing of chemicals, medicines and other products dates back to the early 1900s, as do some of the most widely used test methods even today. Regulatory toxicology has become virtually frozen in time. This is most unfortunate, given that toxicity testing on animals suffers from scientific, practical and ethical limitations that cannot be overcome and can no longer be ignored.10

Scientific and Technical Limitations of Animal Testing

Species Variations

Different species—and even different breeds or genders of the same animal species (mainly rats, mice, rabbits, dogs and primates)—often vary in their susceptibility to chemicals and reactions to medicines. This is partly due to differences in the way they absorb chemicals, how the substances are distributed through the body, how organs are affected by them, how they are metabolised and the rate at which they are excreted. Each of these processes directly affects the safety of chemicals and medicines.

For example, regarding the testing of nanomaterials, the EU Scientific Committee on Emerging and Newly-Identified Health Risks (SCENIHR) has commented11:

“There may be large species differences in deposition in the respiratory tract between humans and rodents …existing animal tests may not be sensitive enough to detect all possible adverse effects of nanoparticles.”

Species variations are due to evolutionary differences in anatomy, physiology, pharmacology, biochemistry and metabolism, and are a major cause of discrepancies in outcomes between test animals and humans. This is hardly surprising: in evolutionary terms, humans diverged from rodents about 80 million years ago.

Regulatory systems in all sectors are confronted with challenges that cannot be adequately addressed with animal testing. Whether the aim is to protect human health (workers and consumers) or the environment, it cannot be assumed that extensive animal testing can underpin robust and responsive regulatory decision-making.


11 SCENIHR (2007). Opinion on the Appropriateness of the Risk Assessment Methodology in Accordance with the Technical Guidance Documents for New and Existing Substances for Assessing the Risks of Nanomaterials. ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_010.pdf.

Using the results of animal tests to predict human health effects involves many uncertainties, not only about species variations, but also whether high doses in animals predict effects at the lower doses to which humans may be exposed, etc. Consequently, a number of ‘uncertainty factors’ are applied by regulators.12 This means dividing the maximum safe dose in animals by several ten-fold uncertainty factors (e.g., a ‘safe’ dose level in animal tests are often divided by 10 x 10 x 10 or more to try to compensate for the many uncertainties that exist). These uncertainty factors are simply ‘guesstimates’ with little basis in science.

Chapter 1 11 10 Chapter 1

12 Uncertainty factors are used to (i) extrapolate animal data to humans, (ii) extrapolate from short-term test results to lifetime exposure, (iii) extrapolate from animal data when the database is incomplete, (iv) extrapolate from a ‘lowest-’ to a ‘no-observed adverse effect level’ and (v) account for variations in sensitivity in the human population, including between children and adults. NRC (2000). Scientific Frontiers in Developmental Toxicology and Risk Assessment. Washington, DC: National Academies Press.

13 DiGangi J, Strakova J (2011). A survey of PBDEs in recycled carpet padding. Report by the International POPs Elimination Network (IPEN). ipen.org/cop5/wp-content/uploads/2011/04/POPs-in-recycled-carpet-padding-23-April-20111.pdf

14 Suvorov A, Takser L (2008). Facing the challenge of data transfer from animal models to humans: The case of persistent organohalogens. Environmental Health 7, 58-75.

15 Johnson FM (2002). How many food additives are rodent carcinogens? Environmental & Molecular Mutagenesis 39, 69-80.

16 One example is the artificial sweetener saccharin, which was considered a carcinogen on the basis of test results in male rats. It was subsequently delisted when it was realised that humans are not susceptible to the carcinogenic effect seen in male (but not female) rats. European Commission Directorate-General III Industry Scientific Committee for Food (1997). Annex III To Document III/5157/97 CS/ADD/EDUL/148-FINAL: Opinion on Saccharin and its Sodium, Potassium and Calcium Salts.

17 FDA (2004). Innovation or Stagnation: Challenge and opportunity on the critical path to new medical products. fda.gov/ScienceResearch/SpecialTopics/CriticalPathInitiative/ucm076689.htm

18 Mahmood I (2010). Theoretical versus empirical allometry: Facts behind theories and application to pharmacokinetics. Journal of Pharmaceutical Sciences 99, 2927-2933.

The PBDEs (polybrominated diphenyl ethers) are flame-retardant chemicals found in building materials, computer casings, electronics, furniture, plastics, foams and textiles. PBDE production began in 1965, and these chemicals were first found in the environment in 1979. Levels of PBDEs in wildlife (including fish, whales, dolphins, birds and seals) have doubled every three to five years. In humans, PBDEs have increased exponentially for three decades, doubling every five years.

Intensive testing of PBDEs began in the 1980s, but regulatory action was delayed because of uncertainties regarding the relevance of animal test results. The chemicals were banned in the EU in 2008, after they had been contaminating human populations and the environment for decades. Use of deca-BDEs is permitted in the US at least until 2013; additionally, banned PBDEs are present in recycled foam being used for carpet padding, putting workers and consumers at continuing risk in the EU.13 In other regions of the world, PBDEs are still manufactured and used.

Most testing has been conducted on rats and mice, who metabolise these chemicals differently from humans. Tests are also affected by other species differences.

More than 40 years passed between the first manufacture of PBDEs and significant regulatory control. There are many reasons for this delay, but the flaws in conventional testing were central, as shown in Box 1.

Case Study: Brominated Flame Retardants and Species Variations

Box 1 Animal Test Limitations that Delayed the Regulation of PBDE Flame Retardants14

> The animal tests were slow to deliver results

> Regulation was delayed because the interpretation of results was hindered by species differences in pregnancy, fetal development and in metabolism and excretion

> Tests used chemical doses orders of magnitude higher than actual human exposures; so the results were challenged for their relevance

> The animal test endpoints selected were insensitive and failed to reveal toxicity which occurs at low doses, leading to a false sense of security

The example of the PBDEs is not unique. Animal testing uncertainties caused long delays in the regulation of many toxic substances (e.g., PCBs, benzene, asbestos, tobacco and in food additives15), and continuing controversy about the relevance of animal tests to chemicals suspected of human toxicity (e.g., bisphenol A). Citizens have continued to be exposed to unsafe chemicals while seemingly endless re-testing is carried out. Sometimes, though, the marketing of substances has been unnecessarily restricted because of difficult-to-interpret animal tests, until final proof of their safety for humans was established.16

The same is true for medicines development. While nine out of every 10 novel medicines that pass animal tests are later found to be unsafe or ineffective for human patients,17 equally many potentially safe and effective drugs are lost because they are screened out by faulty animal tests.

Size Matters

Size differences between humans and rodents influence the effects of substances in the body and make data interpretation difficult, requiring an approach called ‘scaling’. Scaling is intended to compensate for size-related differences in the rate at which drugs or chemicals circulate to the body’s organs, and are metabolised and excreted by the body. Factors based on body weight, fractions of body weight, body surface area, metabolic rate and lifespan have all been proposed, but without any consensus as to which is the most predictive (Box 2). According to the US Food and Drug Administration, the conventional notion of a fixed or universal scaling factor has “no evidence to support it.”18

Box 2 Size Differences Impact Toxic Effects—But a Universal Scaling Factor is Misleading

> The volume of a person’s blood is 2,000-times that of a mouse; but the human heart pumps about 300-times more blood per minute than the mouse heart

> Larger animals (e.g., humans) have more cells susceptible to toxic effects than small animals

> European humans live about 39-times longer than laboratory mice (77 years versus 2 years)

> The average body weight of a European human (70 kg) is 2,333-times greater than that of a mouse (30 g)

> The average surface area of the human body is about 231-times that of a mouse and 65-times that of a rat*

> Rates of cell division in mice and rats are roughly double those in humans; this affects immune reactions and the frequency of mutations (and cancers) in response to toxic chemicals and drugs

* US FDA (2005). Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers.

fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances ucm078932.pdf

Megadosing

Rodents have short lifespans, so the duration of a chemical test is limited. It takes time for some toxic effects, such as cancer, to develop. Consequently, rodent tests cannot be made sufficiently sensitive to detect toxic effects at the low, prolonged doses of chemicals to which humans are usually exposed.

Conventional cancer (and other) tests are conducted using unrealistically high doses in an effort to maximise sensitivity. However, people and wildlife are very seldom exposed to toxic chemicals at such megadoses. Estimating the effects of low chemical exposures in humans in the real world on the basis of megadose results

To consume the level of the pesticide Alar that was fed to rats and mice in one toxicity test would require a human to eat 12,700kg of apples daily for 10 years.


in rats and mice in the laboratory involves large margins of error and uncertainty.19 For example, brominated flame retardants (described above) have been tested on animals at doses many orders of magnitude higher than human exposures, “preventing the direct transfer of animal results to human studies” according to Canadian experts.20

Each cancer test for a chemical or medicine requires about five years to design, conduct and analyse, and uses at least 800 rats and mice, at a cost of up to €3 million. Clearly such an inefficient test method is unfit for assessing the cancer risk of thousands of chemicals, pesticides and medicines.

Regulatory scientists from all these sectors have reported difficulties in interpreting the data from these unrealistic ‘megadosing’ tests.21,22 Consequently regulatory agencies must resort to vague and non-committal classifications (such as “unclassifiable” or “possible human carcinogen”) for many

tested chemicals. These designations are virtually useless from a public health point of view; and from an animal welfare perspective, in 2008 these tests subjected 20,807 animals to prolonged and extensive suffering in the EU.23 Chemical Mixtures

Humans and wildlife are exposed to complex chemical cocktails, but risk assessment is based almost entirely on tests of single substances, mainly in animals, even though some chemical combinations can have excessively toxic effects.

According to a 2009 report produced for the European Commission 24 :

“There is a consensus in the field of mixture toxicology that the customary chemical-by-chemical approach to risk assessment might be too simplistic. It is in danger of underestimating the risk of chemicals to human health and to the environment”.

21st century toxicology utilises advanced, high-throughput tests that offer a rapid, inexpensive approach to screening large numbers of chemical and mixtures for human health and environmental safety.

19 For example, at chemical megadoses, enzymes function differently, chemicals may be metabolised to different forms, detoxification mechanisms may fail, and organ systems may be overwhelmed.

20 Suvorov A, Takser L (2008). Facing the challenge of data transfer from animal models to humans: The case of persistent organohalogens. Environmental Health 7, 58-75.

21 Pastoor T, Stevens J (2005). Historical perspective of the cancer bioassay. Scand J Work Environ Health 31 (suppl 1),129-140.

22 Gaylor DW (2005). Are tumour incidence rates from chronic bioassays telling us what we need to knowabout carcinogens? Regulatory Toxicology and Pharmacology 41,128-133.

A mouse suffering from a chemically-induced tumour in a two-year cancer study.

Economic and Resource Limitations of Animal Testing

All industry sectors need to reduce the escalating costs of developing new products and ingredients and establishing the safety of existing ones. Here we look at examples from the medicines, chemicals and pesticides sectors to illustrate the demands made by animal testing for results that still do not provide the data needed for effective regulation.

Medicines

In 2004, the average cost of developing and testing one new medicine was US $900 million (€681 million);25 in 2008 a low estimate was US $1,394 million (€1,042 million)26. The time taken for a successful new medicine to progress from development to the market is 10 to 13 years. Yet 92% of novel medicines that pass animal tests fail to reach the market, mainly because of unpredicted toxicity or lack of efficacy in humans.27

This means that most of the lengthy and costly medical safety tests that currently cause suffering to around half a million animals each year in the EU28 do not identify toxic effects later found in humans during clinical trials. This failure rate massively increases overall costs and also risks the health of patients in clinical trials. Rates of successful drug innovation are no higher now than they were 50 years ago.

In Germany, the number of animals used in laboratories each year decreased from 2.7 million in 1989 to 1.6 million in 1999. The decrease was seen mainly in medicines development, where animal use fell by 50% due to the introduction of techniques of molecular biology and genetics, including cell and tissue culture models allowing very rapid screening of thousands of new drug candidates.29

Chemicals

The ‘chemical data gap’ describes the lack of adequate safety information for tens of thousands of chemicals in the environment to which people and wildlife are exposed. It was this worrying backlog of unevaluated chemicals that helped to trigger the introduction of the REACH regulation in the EU. As explained by Dr Richard Judson and colleagues from the US EPA:30

“This data gap is due largely to the high cost and length of time required to conduct animal testing in rodents and other species. A complete set of regulatory tests for a single chemical (including those for carcinogenicity and for chronic, reproductive, and development toxicity)

uses thousands of animals and costs millions of dollars. In addition, traditional animal tests often yield limited information on mechanism of action, and hence on the cellular pathways that could lead to toxicity in humans”.

In 2008, 82,434 animals were used in the EU to test the safety of industrial chemicals. This figure will rise massively as REACH is implemented and the backlog of insufficiently tested pre-1981 chemicals is tackled.

The European Chemicals Agency says that under the REACH regulation, about nine million animals will be used in the laboratory tests required and the costs of testing would be €1.3 billion.31 Other estimates are much higher: using conventional testing, REACH may directly cost industry €3.08 billion (US $4.2 billion) and use more than 45 million animals over the next 15 years.32 In 2011, BASF, the world’s largest chemical company, predicted that its total REACH costs over 11 years would be €500 to 550 million, the bulk of which is the cost of animal tests.33

Ten of the world’s top 20 chemical companies are based in the EU. It is essential that safety testing is modernised to maintain the competitiveness and reputation of the industry.

For REACH chemicals, modern, non-animal testing tools could go a long way toward filling the data gap.

23 European Commission (2010). Commission Staff Working Document (corrected version): Accompanying document to the 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. ec.europa.eu/environment/chemicals/lab_animals/pdf/sec_2010_1107.pdf

24 Kortenkamp A, Backhaus T, Faust M (2009). State of the Art Report on Mixture Toxicity: Final Report. ec.europa.eu/environment/chemicals/pdf/report_Mixture%20toxicity.pdf

25 Kola I, Landis J (2004). Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery 3, 711-715.

26 Munos B (2009). Lessons from 60 years of pharmaceutical innovation. Nature Reviews Drug Discovery 8, 959-968.

Drug failures due to lack of efficacy and safety demonstrate the need for the development of human-relevant models to accurately predict real-world human reactions.

12 Chapter 1 Chapter 1 13


27 US FDA (2004). Innovation or Stagnation: Challenge and Opportunity on the Critical Path to New Medical Products. fda.gov/ScienceResearch/SpecialTopics/CriticalPathInitiative/ucm076689.htm

28 529,497 animals were used to “test products/substances or devices for human medicine and dentistry and for veterinary medicine” according to the EU statistics of animal use for 2008. ec.europa.eu/environment/chemicals/lab_animals/pdf/sec_2010_1107.pdf

29 Spielmann, H (2002). Animal use in the safety evaluation of chemicals: harmonization and emerging needs. ILAR Journal 43(suppl.), S11-S17.

30 Judson RS, Houck KA, Kavlock RJ, et al. (2010). In vitro screening of environmental chemicals for targeted testing prioritization: The ToxCast project. Environmental Health Perspectives 118, 485-492.

31 European Chemicals Agency (2009). Press Release 28 August 2009. echa.europa.eu/doc/press/pr_09_11_animal_testing_20090828.pdf

32 Rotroff DM, Wetmore BA, Dix DJ, et al. (2010). Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening. Toxicological Sciences 117, 348-358.

33 The Society of Chemical Industry.soci.org/Chemistry-and-Industry/CnI-Data/2011/2/REACHing-for-a-price-tag

Plant Protection Products

Plant protection products (herbicides, insecticides and fungicides) are designed to be toxic to agricultural pests, but ideally not to humans or to non-target animal species. They are applied in the wider environment, contaminate waterways, and may remain as residues in food and animal feed.

The number of authorised pesticides has decreased by about half in the last decade due to changes in EU regulation requiring safety data gaps to be filled to contemporary standards. Upwards of 10,000 animals may be used to test each new chemical active ingredient in a product according to EU regulations, and in 2008 in the EU the industry used 74,000 animals in tests that can be lengthy, expensive and difficult to interpret for human safety.

For these reasons the development of novel, safer pesticides is likely to be impaired unless advanced testing techniques and data analysis are implemented.

EU regulatory agencies and expert committees have the responsibility of making judgements about the safety of chemicals and products. Their judgements involve risk assessments based on safety testing data and the likely levels of exposure of people (workers, consumers, bystanders) and wildlife to the products in question. Chemicals and other products must be classified and labelled for specific hazards, safe uses must be specified, and unsafe applications controlled.

In recent decades there has been an explosion in manufactured products, especially those containing synthetic chemicals. At the same time, citizens have rightly demanded higher standards of safety. New types of toxicity (such as endocrine disruption and effects on neurodevelopment) have been recognised and entirely novel chemical and physical forms have been synthesised, such as nanomaterials. In medicine, new therapies include protein-based drugs (e.g., antibodies) which are highly species-specific, causing difficulties in testing their safety and efficacy in other animal species (see Chapter 3). All these products offer exciting possible benefits, but issues of safety assessment remain problematic.

Conventional toxicology has struggled to respond to the new regulatory requirements, because animal tests are slow and resource-intensive and subject to many scientific problems. Tests with inconclusive results are often repeated, sometimes many times for high-value substances, leading to a stockpile of frequently conflicting data.34 In the case of the solvent trichloroethylene, there have been 29 risk assessments of the same cancer test data. Four contradictory classifications of the chemical as a cancer risk have resulted, in part due to disagreements about the human relevance of long-term cancer assays in rodents.35

Animal testing does not support precautionary regulation or offer adequate protection to citizens and the environment. In fact, in many cases—such as bisphenol A, asbestos, water chlorination by-products and brominated flame retardants—the never-ending search for conclusive animal test data has hindered risk assessment and regulatory control and exposed people unnecessarily to toxic harms.

Regulatory Limitations of Animal Testing

Dozens of different animal tests are required to bring a new pesticide to market, yet in most cases only one study is used in the final risk assessment.

33 For instance, at least eleven rodent cancer tests have been conducted for benzene. EPA (1998). Carcinogenic Effects of Benzene: An Update. EPA/600/P-97/001F. 34 Rudén C (2001). Interpretations of primary carcinogenicity data in 29 trichloroethylene risk assessments. Toxicology 169, 209-225. 35 Nanomaterials are substances less than 100 nanometres in size in at least one dimension (one nanometre is one thousand millionths of a metre. 36 Choi JY, Ramachandran G, Kandlikar M (2009). The impact of toxicity testing costs on nanomaterial regulation. Environmental Science and Technology 43, 3030-3034.

Nanomaterials

Because of the microscopic size of nanomaterials,36 their properties—physical, chemical and biological—can be significantly different from those of the same substance in its ordinary ‘bulk’ size. These differences vary with each type of nanomaterial and are strongly influenced by the physical context in which they are used. These novel and distinct properties may offer medical and economic benefits, but will also have unpredictable and little-understood effects on human health and environmental safety.

Currently, nanomaterials are only controlled in a general way in the EU under existing regulatory frameworks (e.g., chemicals, pharmaceuticals, cosmetics). There are no validated test methods specifically for nanomaterials. To test just the nanomaterials already in use by conventional animal methods, cost estimates range from US $249 million to 1.18 billion, and the time needed is likely to be 34 to 53 years.37

The European Commission has established a Competent Authorities Sub-Group to explore exactly how the REACH chemicals regulation can be properly applied to manufactured nanomaterials. Until specific test guidelines for nanoscale substances emerge, safety testing will be carried out according to existing guidelines, and “...manufacturers, importers and downstream users must ensure that their nanomaterials do not adversely affect human health or the environment”.38

According to the 2006 opinion of the EU Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR):39

“...there is insufficient knowledge and data concerning nanoparticle characterisation, their detection and measurement, the fate (and especially the persistence) of nanoparticles in humans and in the environment, and all aspects of toxicology

and environmental toxicology related to nanoparticles, to allow for satisfactory risk assessments for populations and ecosystems to be performed”.

The results from animal tests so far show potentially dangerous properties of nanomaterials (see Box 3); but, as ever, the relevance of the animal data for humans is unknown.

Box 3 Potentially Dangerous Properties of Nanomaterials

In animal tests, nanomaterials have been shown to:

> Pass through the lining of the nose into the brain

> Move from the lungs (after inhalation) into the liver, spleen and lymph nodes

> Cause gene mutations that can lead to cancer

> Cause inflammation of the lungs

> Change the shape and behaviour of important proteins in the blood and brain

> Bioaccumulate in some wildlife, with potential to concentrate up the food chain

> Have toxic and even lethal effects on some micro-organisms, crustaceans and fish

A model of the inside of a carbon nanotube – sheets of carbon atoms in a hexagonal arrangement and curved into tubes.

36 Nanomaterials are substances less than 100 nanometres in size in at least one dimension (one nanometre is one thousand millionths of a metre.

37 Choi JY, Ramachandran G, Kandlikar M (2009). The impact of toxicity testing costs on nanomaterial regulation. Environmental Science and Technology 43, 3030-3034.

38 European Commission (2008). Follow-up to the 6th Meeting of the REACH Competent Authorities for the implementation of Regulation (EC) 1907/2006 (REACH). Concerns: Nanomaterials in REACH Doc. CA/59/2008 rev. 1.

39 SCENIHR (2006). Modified Opinion: The Appropriateness of Existing Methodologies to Assess the Potential Risks Associated with Engineered and Adventitious Products of Nanotechnologies. SCENIHR/002/05.

34 For instance, at least eleven rodent cancer tests have been conducted for benzene. EPA (1998). Carcinogenic Effects of Benzene: An Update. EPA/600/P-97/001F.

35 Rudén C (2001). Interpretations of primary carcinogenicity data in 29 trichloroethylene risk assessments. Toxicology 169, 209-225.

14 Chapter 1

Economic and Resource Limitations of Animal Testing

According to the Organisation for Economic Co-operation and Development (OECD), 13 nanomaterials are already, or are about to be, marketed.40 Over 600 products containing nanomaterials are currently available in the global marketplace. Some of these products are intended to come into contact with our food and drink; some may migrate into food with potentially toxic effects; others are applied to our skin. Examples include polyethylene beer bottles with a nano-clay gas barrier; polypropylene food containers with antimicrobial nano-silver; nano-zinc oxide-containing sunscreens and films for food wrapping.41

At the moment the situation is circular: nanomaterials must be shown to be safe for humans and the environment; existing test methods and frameworks are inadequate; the properties and behaviour of nanomaterials are not sufficiently understood; yet nanomaterials are already on the market and manufacturers are planning many more.

The EU is in an impossible position and must introduce a moratorium on the use of nanomaterials in products until appropriate testing and risk assessment methods are available.

Endocrine-Disrupting Chemicals: Bisphenol A

In the early 1990s, unusual developmental changes in birds, fish and other wildlife populations were a new cause of concern. These included reduced reproductive ability,

altered behaviour and thyroid problems, and were particularly noticed in the offspring of adult animals exposed to certain chemicals. These chemicals were termed ‘endocrine disrupters’, because their major toxic effects were mediated through hormone (endocrine) systems.

Bisphenol A is one of the highest-volume chemicals produced worldwide, and is widely used in the linings of tin cans, baby bottles (not in the EU) and other plastics,

in food-wrapping films and in many other applications. More than 100 tons of bisphenol A are released into the atmosphere each year during production. Over 90% of people are exposed to bisphenol A on a long-term basis.42

Since 2000, more than 1,000 animal studies of bisphenol A have been conducted, with controversial results. Toxicologists assumed that results from high-dose testing could be extrapolated to low doses, with a

safe threshold below which there would be no toxic effects. But they found no safe low dose. They also expected that an increased dose would produce a normal increase in toxic effects, but sometimes the reverse occurred.43

Different species and breeds of animals reacted with varying sensitivities to the chemical. For example, bisphenol A can cause enlargement of the womb and vagina in rodents, as well as abnormal cell proliferation

and mucous secretion. However, Sprague-Dawley rats are highly resistant while Fisher-344 rats are highly sensitive to these bisphenol A effects.44 No one knows which breed or species, if any, predicts effects in people.

Developing fetuses and newborn animals are often more susceptible to endocrine-disrupting effects, but classic animal studies conducted over several generations are costly and time-consuming—and give no clear

answers to questions of human safety.45

The controversies are complicated by the lack of detailed information about human exposures.46 New sources of exposure are still being discovered, such as thermal (carbonless) shop receipts, the leaching of bisphenol A from children’s books, and its presence in cigarette filters.

Reviews of bisphenol A in 2008 by the US Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) concluded that current human exposure levels were safe. Those conclusions, however, were based on data from a small number of animal studies which some scientists have criticised. Further, the few test results chosen by the FDA and EFSA as definitive were contradicted by the results of hundreds of studies published in the open literature.47

In 2010, EFSA reported that the established daily intake for humans was safe and that there was no convincing evidence that bisphenol A causes neurobehavioural toxicity.48 But in 2012, EFSA started a full re-evaluation of the human risks, in the light of newly discovered exposure routes and the questionable relevance of rodent low-dose test data. In the EU, the use of bisphenol A in the manufacture of infant feeding bottles is prohibited but it is still permitted in food contact materials in the EU, USA and Japan. The endless controversies and re-analyses are likely to continue unless and until the safety assessment of endocrine-disrupter and other chemicals is modernised.

New animal tests are being created to detect endocrine disrupting chemicals, but will additional testing really lead to better regulation?

40 OECD (2010). Series on the Safety of Manufactured Nanomaterials No. 27. List of Manufactured Nanomaterials and List of Endpoints for Phase One of the Sponsorship Programme for the Testing of Manufactured Nanomaterials: Revision. ENV/JM/MONO(2010)46.

41 European Food Safety Authority (2009). Scientific Opinion on the Potential Risks Arising from Nanoscience and Nanotechnologies on Food and Feed Safety. The EFSA Journal 958,1-39.

42 Vandenberg LN, Chauhoud I, Heindel JJ, et al. (2010). Urinary, circulating and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environmental Health Perspectives 118, 1055-1070.

43 Vandenberg LN, Maffini MV, Sonnenschein C, et al. (2009). Bisphenol A and the great divide: A review of controversies in the field of endocrine disruption. Endocrine Reviews 30, 75-95.

44 Spearow JL, Barkley M (2001). Reassessment of models used to test xenobiotics for oestrogenic potency is overdue: Opinion. Human Reproduction 16, 1027-1029.

45 Vandenberg LN, Maffini MV, Sonnenschein C, et al. (2009). Bisphenol A and the great divide: A review of controversies in the field of endocrine disruption. Endocrine Reviews 30, 75-95.

46 Bucher JR (2009). Bisphenol A: Where to now? Environmental Health Perspectives 117, A96.

47 Hunt PA, Susiarjo M, Rubio C, et al. (2009). The bisphenol A experience: A primer for the analysis of environmental effects on mammalian reproduction. Biology of Reproduction 81, 807-813.

48 EFSA (2010). Scientific Opinion on Bisphenol A: Evaluation of a Study Investigating its Neurodevelopmental Toxicity, Review of Recent Scientific Literature on its Toxicity and Advice on the Danish Risk Assessment of Bisphenol A. EFSA Journal 8, 1829-1939.


Regulatory Limitations of Animal Testing

CHAPTER 2 21ST CENTURY TESTING— SAFER FOR US ALL

The publication of the US National Research Council’s (NRC’s) pivotal 2007 report49 has prompted a new approach to safety testing and risk assessment. As the NRC explained, the conventional strategy of dosing animals with chemicals and evaluating the resulting signs of poisoning “is so time-consuming and resource-intensive, it has had difficulty in meeting many challenges encountered today”.

Instead of poisoning animals, the NRC report developed a science-based and human-relevant approach with four core elements:

1. Chemical characterisation: knowledge of the chemical and physical properties of substances and how these relate to potential hazards

2. Determining the effects of test substances on important biochemical pathways within and between human cells, assessed with high-throughput, state- of-the-art human cell-based methods

3. In-depth (targeted) testing where necessary, to clarify uncertainties and investigate possible toxic metabolites

4. Computer modelling of relationships between chemical doses and effects, to allow risk assessment decisions to be made.

The function of the key biochemical pathways is to maintain normal cell activities, control communication between cells and allow cells to adapt to changes. The pathways are ‘perturbed’ by toxic chemicals,

leading to adverse effects in the whole organism. An advantage of assessing toxicity at molecular and cellular levels is the ability to study cells and sub-cellular components from humans, rather than using surrogate animals, eliminating problems of species differences.

The NRC scientists understood clearly that recent revolutions in biology and biotechnology have not been properly integrated into regulatory safety testing. They proposed that basing the new toxicology on computational and cell-based (in vitro) methods, automated where possible, would allow the development of a truly science-based toxicology, while also enabling a dramatic increase in the number of chemicals that can be tested in a cost-effective manner. For instance, automated (high-throughput) screening tests now enable pharmaceutical companies to scan or ‘screen’ more than 100,000 compounds in a single day,50 and these techniques can also be applied to safety testing.

Other components of the NRC’s vision were to reduce the duration of testing programmes and increase efficiency and flexibility, expediting regulatory decision-making; and eventually to eliminate animal tests, meanwhile using far fewer animals and minimising suffering to animals who continue to be used for the time being.

Identifying and characterising the hazards of substances demand relevant and reliable safety tests. Risk assessors also need to know the sources, routes and levels of exposure


50 Andersen ME, Al-Zoughool M, Croteau M, et al. (2010). The future of toxicity testing. Journal of Toxicology and Environmental Health, B 13, 163-196.

51 Ankley GT, Bennett RS, Erickson RJ, et al. (2010). Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment. Environmental Toxicology and Chemistry 29, 730-741.

which can be provided by human- and population-based studies. The interplay between hazard identification, dose-response relationships and exposure is the cornerstone of risk assessment.

Finally, regulatory decisions are made about whether and how substances should

Safety Testing: State-of-the-Art Tools

be authorised or controlled, taking into account the costs and benefits of authorising the use of a substance. Some side effects would be tolerated in a very important and effective medicine, but harm caused by a chemical ingredient in a cosmetic cannot be acceptable.

What are the revolutionary developments in molecular biology, chemistry and computer science which the NRC proposes that will underpin the new science of safety testing? The key ‘tools’ and their applications are explained in Table 1.

1. Chemical Characterisation

Computational models of quantitative structure/activity relationships (QSARs) for chemicals offer a first, rapid screen, relating basic molecular properties (e.g., cell-membrane solubility, electric charge) with likely toxicity to people and the environment. For example, there are several useful QSAR models for predicting short-term toxicity

to fish and other aquatic life. The Organisation for Economic Co-operation and Development (OECD) has developed computer software and guidance to assist governments and industries to apply QSARs to related groups of chemicals, allowing identification of relevant structural characteristics and potential modes of action of a target chemical. The EpiSuite model includes a QSAR that predicts the general (i.e., non-specific) toxicity of chemicals to fish. It is included in the OECD application toolbox; has been used to screen large numbers of chemicals, such as in the Canadian Domestic Substances List; and is also utilised by the US EPA.51

Structure-activity relationship modelling can predict a chemical’s biological properties based on its molecular structure, as similar molecules often have similar activities.


2. Effects on Biochemical Pathways

In the next stage, high-throughput biological screening tests are applied. These are often robot-automated techniques using sub-cellular components (such as receptors, enzymes and other functional proteins) and human cells in test tube culture. These tests can screen thousands of chemicals at dozens of doses per day, providing comprehensive data on perturbations of biochemical pathways likely to cause health problems. High-throughput tests are already being used to assess substances for potential to harm the immune system and to cause mutations and cancer.52

52 Andersen ME, Al-Zoughool M, Croteau M, et al. (2010). The future of toxicity testing. Journal of Toxicology and Environmental Health B 13, 163-196.

Table 1. State-of-the-art in vitro and computational safety testing tools

Scientific tools

Quantitative structure/activity relationships (QSARs) and similar computational methods

Molecular and cell-level techniques e.g., biochemical assays; fluorescence techniques using laser cytometry; high-content cell imaging; electronic sensing of cell activities; receptor binding assays and reporter gene assays, etc.

Toxicogenomic tools for transcriptomics (e.g., DNA microarray techniques), proteomics and metabolomics (using mass spectrometry and magnetic resonance spectrometry)

Additional test-tube models, such as enzymes and liver cells to study in vitro metabolism; three-dimensional tissue cultures

Systems biology

Physiologically based pharmacokinetic (PBPK) and similar computational models

What they are

Computational models and databases for predicting toxicity, based on structure and properties of chemical molecules and their ability to produce reactions at cell-level.

Numerous assays measuring many cell pathways (e.g., endocrine signalling via estrogen receptors) and cell health endpoints (cell stress and structure changes, cell toxicity, cell proliferation, etc.)

Biological tools that measure toxic chemical effects on whole-cell patterns of gene activities, protein production and metabolism. Provide a ‘fingerprint’ of chemical effects

In vitro techniques to study toxic effects in detail. This may also involve animal tests in the short term. 3D tissue spheroids, e.g., heart tissue, are more sophisticated cell cultures

Computational methods to integrate and interpret data from molecular and cell pathways and chemical effects on them

Computer models that predict how a substance will be absorbed, distributed, metabolised or excreted, using human parameters and cell data

How they can be used

Very rapid and cost-effective sorting method to characterise chemicals and prioritise them for testing, both for human health and environmental effects

Rapid-throughput assays using human enzymes, receptors, ion channels and cells in the test tube. Identify chemicals that perturb biochemical pathways causing toxicity e.g., embryotoxicity or cancer

Screen chemicals for potential hazards. Understand cell pathways and mechanisms of toxicity. Identify biomarkers of toxicity and exposure

In-depth or targeted testing, used if necessary to clarify uncertainties or provide data on how substances are metabolised

Understand chemical or drug dose-response relationships based on cell data. Interpret screening assays. Translate molecular and cell data into physiological outcomes

Predict absorption, distribution, metabolism and excretion. Dose/ response relationships. Predict individual and life-stage variations

Likely sequence of application

DNA microarrays examine how chemical exposures affect the activity and interactions of human genes, proteins and metabolites.

53 Toxicogenomics integrates modern genetic techniques into toxicology, studying the effects of chemicals on all the genes in cells.

Toxicogenomic tools53 can help identify pathways of toxicity at the cell level, revealing changing patterns of gene and protein activities caused by chemical exposure.

Computer techniques of ‘systems biology’ combine cell and molecular information to better understand how more complex biological systems function.

The computational tools are used for interpreting the new test data, linking chemicals to cellular gene and protein changes, and helping to associate chemical exposures with diseases in humans or wildlife.

Many cell pathways are already mapped and important data are being produced now, as illustrated by the US ToxCast programme.



54 Judson RS, Houck KA, Kavlock RJ, et al. (2010). In vitro screening of environmental chemicals for targeted testing prioritization: The ToxCast project. Environmental Health Perspectives 118, 485-492.

55 Reif DM, Martin MT, Tan SW, et al. (2010). Endocrine profiling and prioritization of environmental chemicals using ToxCast data. Environmental Health Perspectives 118, 1714-1720.

56 Judson RS, Martin MT, Reif DM, et al. (2010). Analysis of eight oil spill dispersants using rapid, in vitro tests for endocrine and other biological activity. Environmental Science and Technology 44, 5979-5985.

57 MacDonald JS, Robertson RT (2009). Toxicity testing in the 21st century: A view from the pharmaceutical industry. Toxicological Sciences 110, 40-46.

58 Raschi E, Ceccarini L, De Ponti F, et al. (2009). hERG-related drug toxicity and models for predicting hERG liability and QT prolongation. Expert Opinion in Drug Metabolism and Toxicology 5, 1005-1021.

The US EPA’s ToxCast programme is developing a cost-effective screening approach to prioritise the testing of many thousands of chemicals in the environment.54

Phase I of ToxCast profiled more than 300 chemicals, mostly food-use pesticides. Computational and in vitro screening tests, with nearly 500 biological parameters, have provided activity profiles or ‘signatures’ for each chemical.

In 2010, Phase II of the programme began screening 700 additional chemicals used in industrial and consumer products, food additives and drugs.

ToxCast is also developing statistical and computational models to interpret and integrate chemical toxicity data, formulating a numerical scoring system. The score for each chemical, plus the likelihood of humans being exposed, will be used to prioritise each substance for further in-depth testing.

Scientists have defined endocrine-disrupter signatures for the 300 chemicals, and ranked them according to scores for these activities.55 An index called the Toxicological Priority Index (ToxPi) combines multiple test results and provides a visualisation and ranking of each chemical’s potential endocrine activity.

Visually, ToxPi represents chemical hazards as slices of a circle, with each slice representing one piece (or similar pieces) of information about a chemical. Each slice is coloured to represent grouped data from related in vitro tests (green), chemical properties (orange) or biological pathways (blue). The distance from the centre of the circle represents the ‘strength’ or potency of each chemical activity. For example, in Figure 1, a light-green slice representing female hormone (estrogen) activity results for bisphenol A extends farther from the circle’s centre than the corresponding data slice for tebuthiuron. This illustrates that bisphenol A is more likely to have estrogen-related toxic effects than is tebuthiuron.

The practical value of ToxCast has already been demonstrated by its application to a real-world scenario: the disastrous oil-spill in the Gulf of Mexico after the explosion on the Deepwater Horizon oil platform. Efforts to break up the oil-slicks involved more than a million gallons of dispersant chemicals, mainly surfactants and solvents, for which little safety information was available.

ToxCast high-throughput in vitro tools allowed these complex chemical mixtures to be tested and ranked according to their likely endocrine activities and toxicity to cells, with results published within a month. No animals were used and, indeed, animal tests are incapable of providing such quick, cost-effective results, especially for chemical mixtures.56

Case Study: The ToxCast Initiative for Chemical Screening

Figure 1. ToxPi diagrams allow the potential toxic properties of chemicals to be visualised(Reproduced with permission from R Kavlock (2010). ToxCast & Tox21: Providing high-throughput decision support tools for chemical risk management. In: AXLR8 Consortium—Alternative Testing Strategies—Progress Report 2010. Replacing,

reducing and refining use of animals in research. Eds. Seidle T, Spielmann H, Kral V, Schoeters G, Rowan A, McIvor E).

3. Targeted Testing

The NRC envisaged that with the new paradigm, knowledge of the biochemical pathways affected by a toxic substance would be complemented where needed by more ‘targeted testing’. Targeted tests could clarify uncertainties about toxicity pathways, or find out if a substance is metabolised to toxic forms.

Prompted by high levels drug failures, the pharmaceutical industry has begun implementing advanced testing methods into its drug development programmes. Considerable effort has gone into developing ‘early warning’ tests which can screen out novel drugs that would otherwise go through years of development and fail at a later, more costly stage—or even have to be withdrawn after marketing.

Guidelines provide advice on testing new drug molecules for a wide range of potential toxicities, and a major example is cardiovascular safety (does the drug damage the heart or blood circulation?). In the past, cardiovascular effects were not assessed until a later stage in the development of a drug, using rats and dogs and sometimes non-human primates or pigs. However, in the last decade, advanced technologies have been introduced to screen drug molecules for cardiovascular harm caused by changes in the electrical properties of the heart, as explained in the Case Study, below.

Use of human cells eliminates uncertainties associated with species differences, and testing at environmentally relevant dose levels eliminates uncertainties encountered in high-dose animal experiments.

Between 1998 and 2008, several drugs such as Hismanal and Seldane (anti-histamines), Posicar (a drug for high blood pressure) and Propulsid (for heartburn), were withdrawn after having fatal effects on the heartbeat.57

Underlying this fatal side effect is a disruption in the electrical activity of the heart muscles, caused by the drugs blocking the channels in cell membranes that control the movement of potassium into and out of the heart muscle cells. A gene called hERG regulates these potassium channels.

A method which measures ion channel activities in cells was developed to assess the toxic effects of novel drug molecules on hERG activity, using cell cultures. The technique is applied early in the discovery process so that dangerous drug molecules can be discarded before wasting resources and lives.

In 2004, the new in vitro technique was accepted into formal guidelines, and by 2008 most pharmaceutical companies were using it. Already the method is being improved so it can be applied in a high-throughput format and computational QSAR systems will give an even earlier warning about this toxic effect.58

The test saves animals, time and money as well as the health and lives of volunteers and patients.

Case Study: Advanced Testing of Medicines for Heart Safety

Chapter 2 23


22 Chapter 2

59 Bois FY, Jamei M, Clewell HJ (2010). PBPK modelling of inter-individual variability in the pharmacokinetics of environmental chemicals. Toxicology 278, 256-267.

60 Jamei M , Marciniak S, Feng K (2009). The Simcyp® population-based ADME simulator. Expert Opinion in Drug Metabolism and Toxicology 5, 211-223.

4. Dose-Response Modelling

An essential aspect of safety testing is understanding the effects of test substances at different doses. Animal testing is usually insensitive to low doses, necessitating cumbersome and often misleading interpretations of results.

Computational systems biology tools are used to interpret the new testing data. They link chemical doses to cell pathway changes, describing changes in toxic effects at different doses and providing important thresholds of toxicity.

Physiologically-based pharmacokinetic (PBPK) models are computational systems now commonly used in drug development and increasingly in regulatory toxicology.59 They predict the absorption, distribution, metabolism and excretion (ADME) of substances in the body, at different doses.

PBPK models based on human rather than animal characteristics avoid the problem of species differences. They can also help predict variations in susceptibility between individuals and at different developmental life stages,

which commonly occur but cannot be properly addressed by conventional animal testing.

Functional PBPK models have developed alongside the rapid advances made in the use of human in vitro systems and in understanding gene function. It is now possible to predict ADME outcomes in ‘virtual humans’ with increasing confidence.60

Exposure Science and Risk Assessment

As the data from test-tube and computational assays accumulate, the links between genes, proteins, toxic pathways and harm to humans and wildlife will be increasingly well understood. The NRC report recommended the generation and use of population-based and individual exposure data for interpreting laboratory test results.

Conventional safety testing has neglected the importance of ‘exposure science’. Assessing complex human health risks, in particular, requires the characterisation of chemical hazard, susceptibility and exposure. State-of-the-art exposure science is essential to assess potential for risk to individuals and populations and to inform public health decisions.61 To protect people and the environment, approaches are needed to monitor and characterise chemical emissions, model chemical dispersion, and understand the impact of people’s physical surroundings and other relevant factors.

With the new approach to safety testing, exposure data will be used to characterise which exposures to chemicals have significance to real-world health outcomes. For example, if environmental chemicals disrupt important biochemical cellular pathways at concentrations similar to those detected from human biomonitoring, those chemicals should be prioritised for regulatory control.

As exposure information becomes available for more chemicals, it will allow a more realistic assessment of the likelihood of adverse effects to human health and the

Computational systems biology modelling can simulate the human body to describe and predict complex interactions among cells, tissues, organs and organ systems.



61 Hubal EA (2009). Biologically relevant exposure science for 21st century toxicity testing. Toxicological Sciences 111, 226-232.

64 Website epa.gov/risk/nexgen/docs/NexGen-Program-Synopsis.pdf

The Benefits of Advanced Safety Testing

As the landscape of safety testing is transformed, so too must the risk assessment framework undergo re-development. Expanded testing programmes such as REACH will produce volumes of new test data for regulatory assessment.

Advancing the Next Generation of Risk Assessment (NexGen) is a US federal agency initiative to develop a more cost-effective, faster and more robust system of chemical risk assessment incorporating the new knowledge from advanced safety testing and exposure data. The collaborative programme aims to discover how best to use advanced safety testing data.64

NexGen is developing prototype risk assessments for three human-health disorders caused by chemical exposures:

> Lung injury and related respiratory disorders caused by chlorine and ozone

> Reproductive damage and developmental neurotoxicity in fetuses and children, caused by endocrine-disrupter effects of bisphenol A and perchlorate

> Cancers resulting from exposure to polycyclic aromatic hydrocarbons

In these three areas, NexGen will assess the validity of the new safety data, based on molecular and cellular tests, for these chemicals with known public-health risks and existing animal test data.

The planned outcome is to develop a novel health assessment paradigm based on three different levels or tiers. Tier 1 assessments will address data for thousands of chemicals mainly from high-throughput tests and from quantitative structure-activity relationships (QSARs). In tier 2, additional data from more targeted testing will be considered, probably for hundreds of chemicals. In-depth, tier 3 assessments would be reserved for a much smaller number of chemicals of high concern: those with the highest hazard and wider public exposure.

Case Study: NexGen—Transforming Risk Assessment

Modern safety testing tools provide numerous advantages compared with conventional animal testing, as summarised in Box 4. Achieving these benefits will depend on multi-sector and multi-institute investments and collaborations throughout Europe and globally.

Box 4 Summary of the Benefits of 21st Century Safety Testing

> Advanced safety testing focuses on how chemicals and medicines affect human, rather than animal, systems—eliminating problems of species and breed differences

> Novel, rapid and cost-effective tools can tackle the backlog of inadequately tested chemicals

> High-throughput tests allow quick prioritisation of the most toxic chemicals, including endocrine disrupters

> They enable a side-by-side comparison of similar chemicals of different toxicities, to select the least toxic

> Chemical mixtures can be tested

> Advanced tests better mimic real-world exposures including repetitive and low- dose scenarios

> They can address individual and life-stage differences in susceptibilities, for better predictions of toxicity to fetuses and to children

> Advanced test-tube methods offer scope to test the safety of nanomaterials

> Animal pain and distress caused by safety testing will be massively reduced, and eliminated in the longer term

> Risk assessment will become more evidence-based and less dependent on uncertainty factors

62 Scientific Committee on Consumer Products (SCCP)—Scientific Committee on Health and Environmental Risks (SCHER)—Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) (2008). Preliminary report on the Use of the Threshold of Toxicological Concern (TTC) Approach for the Safety Assessment of Chemical Substances. ec.europa.eu/health/ph_risk/committees/documents/sc_o_001.pdf

63 Fry RC, Navasumrit P, Valiathan C, et al. (2007). Activation of inflammation/NF-kappaB signaling in infants born to arsenic-exposed mothers. PLoS Genetics 3, e207.plosgenetics.org/article/info%3Adoi%2F10. 1371%2Fjournal.pgen.0030207

environment. One such decision-making approach being discussed and developed in the US and in Europe at the moment is the Threshold of Toxicological Concern (TTC).62 The TTC is an exposure level below which the likelihood of damage to human or environmental health is negligible.

The TTC concept is applied to families of structurally related chemicals so that when reliable toxicity data are available for a chemical, the toxicity of a structurally similar substance can be deduced. Then the presence of the chemical in the environment or people’s exposure to it directly (e.g., in a medicine) is evaluated, and if it is below the TTC then the risk is minimal. The TTC concept is already used for evaluating the safety of food-contact materials, flavouring chemicals and contaminants in medicines. With care, its wider application would reduce the need for extensive safety testing and allow a greater focus on high-risk chemicals.

These developments require advances in exposure science, so that state-of-the-art techniques can be applied to the rapid and cost-effective assessment of exposure to chemicals. As with safety testing, these tools include advanced computational and biological techniques.

In exposure science, computational tools are used to: > collect and analyse new data, making connections between exposure, hazard and illness

> extract new information from existing data

> link exposure data to safety testing results to help validate new tests

> identify gaps in exposure information

> model human-environment interactions

> identify levels of human exposure likely to affect toxicity pathways in molecular and cell tests

> predict how chemicals disperse and are transformed in the environment.

Biological techniques will be used to routinely monitor people, wildlife and the environment—air, water and soil—for levels of potentially toxic chemicals, and to understand their effects on human health. Research is identifying ‘biomarkers’—easily

measurable biological signals that indicate exposure or toxic effects—to recognise when people, plants and animals have been exposed to chemicals, and to spot health changes that result. For humans, common biomarkers include chemical metabolites in urine, blood or breath; changes in gene activity; and DNA damage.

An example of this approach is a study of human infants born to mothers who had had different exposures to arsenic during pregnancy.63 Using umbilical cord blood, advanced gene techniques combined with computational models demonstrated that prenatal arsenic exposure led to changed patterns of gene activity in babies. These gene activity patterns were found to be highly reliable biomarkers for early exposure to arsenic.


Exposure Science and Risk Assessment

Human biomonitoring detects toxic chemicals in human blood, urine or other tissues that can be used to identify markers of exposure and toxicity.

CHAPTER 3 ADVANCING THE SCIENCE OF HEALTH RESEARCH

Health research aims to discover the causes human diseases and to prevent or treat them, but it relies heavily on animal ‘models’ of human illnesses, usually artificially induced by chemical treatment or genetic modification. However, fewer new drugs became available in the first decade of this century than at any time in the last 25 years65. The average investment needed to develop and test a single drug has spiralled to US $1,394 million (€1,042 million)66, and it now takes 10 to 13 years to bring a new drug to market.

Even so, 92% of novel drugs fail in clinical trials because of unpredicted toxicity or insufficient efficacy in humans67. Apart from the economic and other resources wasted by these failures, patients’ chances of improved health seem endlessly postponed.

As with safety testing, disease research and drug development have failed to keep pace with the exciting advances achieved in basic science, still relying on the tools and concepts of the last century. A paradigm shift is also needed in health research. It would involve moving the strategic focus away from animal

65 Kaitin KI, DiMasi JA (2011). Pharmaceutical innovation in the 21st century: new drug approvals in the first decade, 2000-2009. Clinical Pharmacology & Therapeutics 89, 183-188.

66 Munos B (2009). Lessons from 60 years of pharmaceutical innovation. Nature Reviews Drug Discovery 8, 959-968.

67 Food and Drug Administration (2004). Innovation and Stagnation: Challenge and Opportunity on the Critical Path to New Medical Products. fda.gov/ScienceResearch/SpecialTopics/CriticalPathInitiative/ucm076689.htm

Outdated Animal ‘Models’ of Disease

Trying to mirror human diseases by artificially creating symptoms in animals such as mice, rats, rabbits and monkeys has severe scientific limitations, reflecting an early 20th-century approach to health research.

As in the area of safety testing, there is the fundamental problem of species differences, e.g. between humans and rodents, both in disease susceptibilities and in the range of symptoms displayed. For example, research into multiple sclerosis uses animals injected with toxic proteins, creating a condition known as experimental autoimmune

encephalomyelitis. Yet experimental autoimmune encephalomyelitis is a weak model of multiple sclerosis: its symptoms vary between different animal species, and it provides very limited insights into the cellular processes of multiple sclerosis itself. Consequently, 99% of treatments effective in animals have failed to help humans68.

It is probably impossible to create a reliable disease model in other animals when the interacting causes of the human illness are still unknown. Mice have been genetically modified to develop a limited array of

symptoms of Alzheimer’s disease. Alzheimer’s disease arises through some combination of genetic and environmental influences, yet animal research focuses solely on genetic causes. The four commonly used Alzheimer’s drugs only stabilise the condition, temporarily, in about half of patients. For 10 years no effective new treatment has emerged, although many were successful in tests on mice.

According to the European Union Joint Programme on Neurodegenerative Disease Research69, the poor predictability of animal

experiments and towards understanding human disease pathways, using advanced human biology-based in vitro and computational tools. Disease pathways, like toxicity pathways, are disruptions of normal pathways at the levels of genes, proteins, cells and tissues. They provide the key to unlocking why and how human illness occurs. A strategic change of focus could dramatically help advance fundamental health research, as well as drug discovery and development.

‘models’ and the uncertain relevance of the behavioural tests applied to animals are important reasons for failing to discover effective treatments. More emphasis on understanding the causes and underlying characteristics of the human disease would surely be more effective than the current focus on mice.

Another unavoidable outcome of using animals in health research is that their symptoms and responses to potential treatments are frequently dissimilar to those of patients. 300 million people currently suffer from asthma, but only two types of treatment have become available in the last 50 years. Experiments on rodents have dominated asthma research, yet the animal ‘models’ do not duplicate human asthma70. There is a real need for better translation of results from the laboratory bench to the clinic.

It is the same story with many other major human illnesses. More than 1,000 potential drugs for stroke have been tested in animals, but only one of these has proved effective in patients71. Animal research into motor neuron disease (a fatal paralysis affecting one in 500 people) has produced many treatments which were promising in mice, but none has benefited humans72.

Failures in research because of an over-reliance on animal ‘models’ of human diseases not only delay medical progress, but also consume limited resources and risk the health and safety of volunteers in clinical trials.

68 Sriram S, Steiner I (2005). Experimental allergic encephalomyelitis: A misleading model of Multiple Sclerosis. Annals of Neurology 58, 939-945.

69 European Union Joint Programme on Neurodegenerative Disease Research (2011). Basic Research Workshop, Madrid, Final Report. neurodegenerationresearch.eu

70 Buckland GL (2011). Harnessing opportunities in non-animal asthma research for a 21st-century science, Drug Discovery Today 16, 914-927.

71 O’Collins VE, Macleod MR, Donnan GA, et al. (2006) 1,026 experimental treatments in acute stroke. Annals of Neurology 59, 467-477.

72 Benatar M (2007). Lost in translation: treatment trials in the SOD1 mouse and in human ALS. Neurobiology of Disease 26, 1-13.

Human cell-based tests can be used to model critical “circuits” in human toxicity pathways and test for chemically-induced perturbations.


Systems biology, which draws on recent developments in computer power, can interpret and integrate complex data to provide knowledge at the physiological and whole-organism levels.

Advanced Techniques: Supporting Safe Medical Innovation

73 Brennand KJ, Simone A, Tran N, et al. (2012). Modeling psychiatric disorders at the cellular and network levels. Molecular Psychiatry. Apr 3. doi: 10.1038/mp.2012.20. 74 Chapman K, Pullen N, Coney L, et al. (2009). Preclinical development of monoclonal antibodies: considerations for the use of non-human primates. MAbs 1, 505-516.

Reflecting the new conceptual approach in safety testing, fundamental health research and drug development need to be based primarily on human disease pathways—not on misplaced efforts to mirror human illnesses in mice and rats. And as safety testing is moving away from simply causing recognisable toxic effects in animals without understanding the toxic pathways involved, so too should research shift its focus from creating artificial ‘symptoms’ in animals and towards understanding underlying disease pathways in humans.

Disease pathways based on gene, protein and cell networks can be researched using human molecules, cells and tissues in the test tube, and will help explain the causes and consequences of illnesses, as well as revealing new therapeutic opportunities. For example, researchers are already studying schizophrenia using human neuron cells in culture, identifying completely new pathways and potential drug targets that had not been found in animal studies73. The discovery of useful molecular signs or ‘biomarkers’ will facilitate early diagnosis as well as the study new drugs more safely in clinical trials.

Systems biology, which draws on recent developments in computer power, can interpret and integrate complex data to provide knowledge at the physiological and whole-organism levels. This will be aided

by data from safe clinical studies of patients, using modern tools including imaging and genetic analysis. Other computational modelling techniques will contribute key information about drug metabolism and likely effects in patients, as well as ‘virtual’ clinical trials.

Pharmaceutical companies are hoping to move into a modern era of treatments that are tailored or personalised to individual patients, to increase therapeutic benefits and minimise side effects. How can research to develop personalised medicine be progressed by using standard strains and species of animals? There is an urgent need to change the conceptual framework and tools of health research, so that it is based on human—not animal—biology and on cellular disease pathways. Only then can we realistically hope to have 21st century advances in the diagnosis, prevention and treatment of the human illnesses which burden society.

The new vision for research and safety testing must be pursued if Europe is to benefit from novel products and processes without risking the health of consumers, workers and the environment. The revolutions must happen because the traditional systems are too cumbersome, costly and scientifically unreliable to be adapted for developing and testing novel products and technologies.

Animal testing threatens to hold back innovation, especially in the field of biopharmaceuticals. Biopharmaceuticals are medicines produced from biological materials such as genetically modified cells or bacteria. They offer potential treatments for serious illnesses including cancer, diabetes, multiple sclerosis, rheumatoid arthritis and Alzheimer’s disease. By 2009, the global biopharmaceuticals market was US $106 billion, and is expected to grow to US $220 billion by 2016. Advanced research and testing is likely to play a key role in realising this potential, by offering cost-effective computational and in vitro methods relevant to humans. These advances would also solve the ethical problems raised by the industry’s growing use of primates in testing these products.74

Assessing the efficacy and safety of biopharmaceuticals like monoclonal antibodies is very challenging using animal tests. These special proteins are highly specific to each species: a monoclonal antibody medicine that is safe and effective in animals may not be in humans, and vice versa. Rodents are unsuitable, being


The 21st Century Approach

too dissimilar to humans. The use of non-human primates is increasing, but does not eliminate the danger posed to participants in clinical trials and also brings serious ethical dilemmas.

TGN1412 was a monoclonal antibody drug evaluated in a clinical trial in England in 2006, after extensive animal safety tests, including administering 500-times the normal dose to cynomolgus monkeys. In humans, TGN1412 had disastrous effects, causing six volunteers to suffer severe adverse reactions including multi-organ failure. Modern scientific techniques need to be applied to the development and testing of biopharmaceuticals75.

Strategy and Investment

A new strategy for health research will require an investment in the science needed to replace the outdated use of animals as research ‘models’. This is much more than a political objective driven by animal welfare considerations: it is also an opportunity to improve our understanding of human biology and so to gain better ways of preventing, diagnosing and treating human diseases, through the use of modern tools and technologies that are directly relevant to our species.

As with safety testing, there is a need for European leaders in each discipline (e.g., immunology, neurodegeneration, cancer, etc.) to work together to identify opportunities to increase the prominence of molecular, cellular, genomic, computational and other non-animal methods in EU research strategies. EU research funding should be made available to support the development and implementation of these discipline-specific roadmaps, through well co-ordinated programmes and intramural and extramural research.

CHAPTER 4 SUPPORTING THE NEXT STEPS IN 21ST CENTURY SAFETY TESTING AND HEALTH RESEARCH

There has never been such strong international momentum for a change of paradigm in safety testing, uniting toxicologists, regulatory agencies, politicians and non-governmental organisations in a unique endeavour to improve the safety of chemicals and medicines. A similar paradigm shift is needed in health research, moving the strategic focus away from animal experiments and towards understanding human disease pathways, using advanced research techniques. These will provide the key to unlocking why and how human illness occurs and could overcome the long-standing roadblocks in drug development worldwide.

This pivotal moment has arisen from a convergence of events: a new generation of scientific tools and analytical approaches; a realisation that traditional safety testing and risk assessment are impractical for the challenge facing them; the ongoing failure to understand human diseases and find effective treatments; a deep concern for the tens of millions of animals used in conventional tests and research; and a visionary concept for how recent technical developments could be applied to benefit human health and the environment.

It is essential that the existing momentum is not lost, yet there are significant challenges. These initiatives will require strategic direction for achieving their goal, visionary funding decisions (pan-European and international public-public and public-private partnerships),

regulatory support and expertise, and targeted scientific developments based on multi-sector (medicines, food additives, pesticides, biocides, chemicals and cosmetics) and multi-disciplinary co-operation (e.g., between toxicologists, medical scientists, chemists, biologists, biochemists, bioinformatics experts and computer scientists). To capitalise on its investment, the EU needs to increase its efforts in terms of funding and overall strategy, co-ordination and co-operation through Horizon 2020 and beyond.

Strategic Direction and Visionary Funding

Much of the research to replace animal testing funded under the 6th and 7th Framework Programmes was aimed narrowly at specific safety issues, such as allergy testing or reproductive toxicity. What is needed under Horizon 2020 is an over-arching strategy for achieving the necessary step-change to a new toxicology: a roadmap which incorporates short- and longer-term milestones, sets out how the goal will be reached and how the transition to a 21st century approach will be managed.

An investment in human-relevant tools for safety assessment and health research promises a better tomorrow for our children.


Advanced Techniques: Supporting Safe Medical Innovation

75 Langley G, Farnaud S (2010). Microdosing: safer clinical trials and fewer animal tests. Bioanalysis 2, 393-395.

Bio-engineers at Harvard’s Wyss Institute have pioneered the lung-on-a-chip, which mimics the complicated mechanical and biochemical behaviours of human lungs.

In health research, a new roadmap would shift the focus away from animal models of human diseases and towards human biology-based studies seeking to understand the biochemical pathways related to different human diseases. The advanced research tools need further development, and a strategy is also required in this arena to achieve the necessary transformation.

The next Framework Programme, Horizon 2020, runs from 2014 to 2020, and the European Commission has acknowledged the critical importance of accelerating the development and use of novel in vitro and computational tools in safety testing and health research.76

EU research has traditionally been driven by policy decisions, to replace animal experiments for example, and research proposals have sometimes been considered without sufficient regard to a wider strategy. A more effective approach, adopted in other regions, may be to drive research programmes more by excellent science and with a ‘top-down’ strategy, to maximise co-ordinated outcomes.

With a projected budget of €80 billion, Horizon 2020 should include significant strands supporting the development of advanced safety testing and health research. The FP7-funded co-ordination project AXLR8 suggests that an investment, from the EU and industry, of at least €325 million should be committed to research for advancing and implementing a 21st century safety testing programme.77

Regulatory Support and Change

The regulatory community’s collaboration and input will be very important to the safety testing transition. Risk assessors will need to refocus their perspective away from evaluating toxic effects in animal surrogates at high doses, towards new approaches to integrating data from human cellular pathways and human exposure studies. More scientific, reliable and predictive information will be a powerful driver to acceptance by regulators of the new safety testing paradigm.

Several components of risk assessment are important in achieving harmonisation, including transparency, terminology, weight of evidence, flexibility, and communication. Europe-wide and international-level activities to encourage regulatory support should be starting now.

There are substantial challenges to implementing the new visions for toxicology and health research but, equally, the need is great. This pivotal moment sees a strong, shared desire to move decisively away from the limitations of animal studies. For the first time, 21st century scientific and analytical tools are available to support innovation and achieve improved health for EU citizens and their environment.

34 Chapter 4

Strategic Direction and Visionary Funding

A “lab on a chip” may some day help doctors rapidly diagnose a wide range of conditions in the clinic. Credit: Maggie Bartlett, US National Human Genome Research Institute.

76 European Commission (2011). Proposal for a Council Decision establishing the Specific Programme Implementing Horizon 2020 - The Framework Programme for Research and Innovation (2014-2020). Brussels, 30.11.2011. COM(2011) 811 final. 2011/0402 (CNS).

77 AXLR8 Consortium (2011). Alternative Testing Strategies: Progress Report 2011 & AXLR8-2 Workshop Report on a ‘Roadmap to Innovative Toxicity Testing’. axlr8.eu/publications/

Chapter 4 35

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