Final Opinion on The need for non-human primates in biomedical

Non-human primates testing (update 2017)

Final Opinion

Scientific Committee on Health Environmental and Emerging Risks

SCHEER

Final Opinion on

The need for non-human primates in biomedical

research, production and testing of products and devices

(update 2017)

The SCHEER adopted this final Opinion at its plenary meeting on 18 May 2017

Non-human primates testing (update 2017)

Final Opinion

ABSTRACT

Following a request from the European Commission, the Scientific Committee on Health,

Environmental and Emerging Risks (SCHEER) reviewed recent evidence to update the

2009 Opinion of the Scientific Committee on Health and Environmental Risks (SCHEER)

on ‘The need for non-human primates in biomedical research, production and testing of

products and devices’.

This Opinion responds to six main issues in the mandate and highlights the many

scientific approaches that could significantly contribute to the replacement, reduction

and refinement (3Rs) of Non-Human Primates (NHP) studies and tests. However, there

are significant issues that go beyond scientific rationale that prevent widespread

adoption and development of alternatives for NHP laboratory use and these are

discussed with suggestions of the opportunities to overcome them.

Although the current state of knowledge does not permit to propose a timetable for

phasing-out the use of NHP in Europe, the Opinion provides recommendations on how to

advance 3Rs for NHP use, such as through alternative methods, training, improvement

of techniques and protocols, sharing of knowledge and removal of barriers. Finally,

research needs are given.

Keywords:

SCHEER, scientific opinion, non-human primates, biomedical research, toxicity testing,

neuroscience, vaccines, infectious diseases, ophthalmology, (xeno)transplantation.

Opinion to be cited as:

SCHEER (Scientific Committee on Health, Environmental and Emerging Risks), Final

Opinion on ‘The need for non-human primates in biomedical research, production and

testing of products and devices (update 2017)’, 18 May 2017.

Non-human primates testing (update 2017)

Final Opinion

ACKNOWLEDGMENTS

Members of the Working Group (WG) are acknowledged for their valuable contribution to

this opinion. The members of the WG are:

The SCHEER members:

Peter Hoet

Renate Krätke

Emanuela Testai

Theo Vermeire (Chair)

External experts:

Romina Aron Badin, Atomic Energy Commission, France

Michelle Epstein (Rapporteur), Medical University of Vienna, Austria

Paul Andrew Flecknell, Newcastle University, UK

Michelle Hudson-Shore, Fund for the Replacement of Animals in Medical Experiments

(FRAME), UK

David Jones, Safety Working Party, European Medicines Agency (EMA), UK

Jan Langermans, Foundation Biomedical Primate Research Centre, The Netherlands

Mark Prescott, National Centre for the Replacement, Refinement and Reduction of

Animals in Research (NC3Rs), UK

Alain Simonnard, Institut National de Recherche et Sécurité, France

All Declarations of Working Group members and supporting experts are available at the

following webpage:

http://ec.europa.eu/health/scientific_committees/experts/declarations/scheer_wg_en

Non-human primates testing (update 2017)

Final Opinion

About the Scientific Committees (2016-2021)

Two independent non-food Scientific Committees provide the Commission with the

scientific advice it needs when preparing policy and proposals relating to consumer

safety, public health and the environment. The Committees also draw the Commission's

attention to the new or emerging problems which may pose an actual or potential threat.

They are: the Scientific Committee on Consumer Safety (SCCS) and the Scientific

Committee on Health, Environmental and Emerging Risks (SCHEER). The Scientific

Committees review and evaluate relevant scientific data and assess potential risks. Each

Committee has top independent scientists from all over the world who are committed to

work in the public interest.

In addition, the Commission relies upon the work of other Union bodies, such as the

European Food Safety Authority (EFSA), the European Medicines Agency (EMA), the

European Centre for Disease prevention and Control (ECDC) and the European Chemicals

Agency (ECHA).

SCHEER

This Committee, on request of Commission services, provides Opinions on questions

concerning health, environmental and emerging risks. The Committees addresses

questions on:

- health and environmental risks related to pollutants in the environmental media and

other biological and physical factors in relation to air quality, water, waste and soils.

- complex or multidisciplinary issues requiring a comprehensive assessment of risks to

consumer safety or public health, for example antimicrobial resistance,

nanotechnologies, medical devices and physical hazards such as noise and

electromagnetic fields.

SCHEER members

Roberto Bertollini, Teresa Borges, Wim de Jong, Pim de Voogt, Raquel Duarte-Davidson,

Peter Hoet, Rodica Mariana Ion, Renate Kraetke, Demosthenes Panagiotakos, Ana

Proykova, Theo Samaras, Marian Scott , Remy Slama, Emanuela Testai, Theo Vermeire,

Marco Vighi, Sergej Zacharov

Contact:

European Commission

DG Health and Food Safety

Directorate C: Public Health, Country Knowledge, Crisis management

Unit C2 – Country Knowledge and Scientific Committees

Office: HTC 03/073 L-2920 Luxembourg

SANTE-C2-SCHEER@ec.europa.eu

ISSN 2467-4559

doi:10.2875/337906

ISBN 978-92-79-80131-0

EW-CA-18-004-EN-N

The Opinions of the Scientific Committees present the views of the independent

scientists who are members of the committees. They do not necessarily reflect the views

of the European Commission. The Opinions are published by the European Commission

in their original language only.

http://ec.europa.eu/health/scientific_committees/policy/index_en.htm

Non-human primates testing (update 2017)

Final Opinion

TABLE OF CONTENTS

ABSTRACT .......................................................................................................... 2

ACKNOWLEDGMENTS ......................................................................................... 3

1 SUMMARY ..................................................................................................... 7

2 MANDATE .................................................................................................... 11

2.1 BACKGROUND .......................................................................................... 11

2.2 TERMS OF REFERENCE ............................................................................. 13

3 OPINION .................................................................................................... 14

3.1 Overview ................................................................................................. 14

3.2 The areas of research (fundamental, translational and applied) and testing

of products and devices in which non-human primates continue to be used

today ................................................................................................................ 15

3.3 The currently available possibilities by type of research or testing to

replace their use either with methods not entailing the use of animals or by

using other species of animals including those genetically altered .................. 15

3.4 The opportunities for the reduction and refinement of their use in areas

where no replacement can be foreseen in medium or long term as per the

principles of the Three Rs ................................................................................. 17

3.5 Identification of specific research areas where effort should be made to

advance replacement, reduction and refinement of the use of non-human

primates in scientific procedures ..................................................................... 19

3.6 The scientific viewpoint on when their use would no longer be necessary,

considering the type of research and areas of testing with a view to the

establishment of a specific phasing-out time-table where possible ................. 20

3.7 Potential implications for biomedical research (e.g., immune based

diseases, neurodegenerative disorders, infectious diseases and serious

diseases) should the use of non-human primates be banned in the EU ............ 21

4 MINORITY OPINION ................................................................................... 23

5 DATA AND METHODOLOGY.......................................................................... 24

6 SCIENTIFIC RATIONALE ............................................................................. 24

6.1 Introduction ............................................................................................ 24

6.2 Ethical issues ........................................................................................... 24

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Final Opinion

6.3 Housing and husbandry ........................................................................... 26

6.4 Animal welfare standards outside the EU ................................................. 27

6.5 Experimental design and staff training .................................................... 28

6.6 Areas of research (fundamental, translational and applied) and testing of

products and devices ....................................................................................... 30

6.6.1 Overview on the use of NHPs in research and testing ................................. 30

6.6.2 Development and safety testing of pharmaceuticals and medical devices ...... 33

6.6.3 Treatment and prevention of infectious diseases ....................................... 44

6.6.4 Neuroscience ....................................................................................... 48

6.6.5 Other uses .......................................................................................... 57

7 RECOMMENDATIONS FOR FURTHER WORK ................................................. 63

7.1 Advancing 3Rs ......................................................................................... 63

7.2 How to overcome barriers? ...................................................................... 65

8 CONSIDERATION OF RESPONSES RECEIVED IN PUBLIC CONSULTATION ... 67

9 ABBREVIATIONS AND GLOSSARY OF TERMS ............................................... 67

10 REFERENCES ............................................................................................ 69

ANNEXES .......................................................................................................... 91

Annex I- Definitions and examples of replacement, reduction and refinement

......................................................................................................................... 91

Annex II - Publically available information concerning the publication of

statistical data under Article 54(2) of Directive 2010/63/EU........................... 92

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1 SUMMARY

In 2009, the SCHER adopted its Opinion on ‘The need for non-human primates in

biomedical research, production and testing of products and devices’. The SCHER

recognised that animals should only be used in medical research when it is unavoidable

and validated alternative methods are not available. The SCHER also considered NHPs

essential for scientific progress in important areas of disease, biology, research and

safety testing. The SCHER requested a regular review of this position that was reflected

in Directive 2010/63/EU. Therefore, the European Commission has requested SCHEER to

issue in 2017 an update of the 2009 Scientific Opinion.

NHPs are generally considered the best available animal models for addressing particular

research questions because of the close phylogenetic relationship with humans.

However, research in NHPs represents a serious ethical dilemma which gives rise to a

high level of concern from European Union (EU) citizens. Therefore, human interest in

potential benefits for mankind must be balanced against avoiding harm to NHPs and

adopting ethical limits or boundaries on NHP use. Opinion polls show that the general

public are more accepting of animal research where animal use and suffering are

minimised, in line with the 3Rs principle (Replacement, Reduction, Refinement) and with

the acceptance of replacement of NHPs in experiments as an achievable goal. This would

also include clinical trials and the use of ex vivo organs. Directive 2010/63/EU requires

implementation of the 3Rs during the design and conduct of animal studies. To fully

apply the 3Rs and maximise the benefits, there is a need to ensure that as new

knowledge, technologies and approaches emerge there is timely assessment and

evolution of research strategies, study designs, scientific procedures and husbandry,

throughout the lifetime of research projects. Applying the 3Rs has both scientific and

economic merit.

If NHPs are considered necessary for certain uses, it is essential to adopt the highest

standards of NHP housing and husbandry and to follow best practice in the conduct and

refinement of scientific procedures. In addition, experimental design, analysis and

reporting are key means of maximising the knowledge gained from animal experiments

and avoiding wastage of animals. Appropriate training for all those working with NHPs is

essential to ensure compliance with legislation, excellence in science, animal welfare and

full implementation of the 3Rs.

It is recognised that tightening of the existing strict EU regulations for NHP use may lead

NHP research to transfer to other countries to the detriment of animal welfare. This can

be avoided by international cooperation that engages as many stakeholders and

organisations as possible to promote the international development of high standards for

research and animal welfare and ethical use.

This Opinion responds to six main issues in the mandate and highlights the many

scientific approaches that could significantly contribute to the replacement, reduction

and refinement of NHP studies and tests. However, there are significant issues that go

beyond scientific rationale that prevent widespread adoption and development of

alternatives for NHP laboratory use and these are discussed with suggestions of the

opportunities to overcome them. Lastly, the SCHEER provides recommendations on how

to advance 3Rs for NHP use, specifically on training, improvement of techniques and

protocols, sharing of knowledge, removal of barriers and research needs.

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Final Opinion

1- The areas of research (fundamental, translational and applied) and

testing of products and devices in which NHPs continue to be used today

In 2014, 8898 procedures on NHPs were reported in the EU, including first use and reuse

of animals. Currently, NHPs are predominantly used in the following areas a)

development and safety testing of pharmaceuticals and medical devices, b) treatment

and prevention of infectious diseases, c) neuroscience, d) ophthalmology and e)

(xeno)transplantation. The first three categories comprise the majority of NHPs used.

While NHPs are often the species of choice, their use for development and safety testing

of pharmaceuticals and medical devices also meets regulatory requirements.

2- The currently available possibilities by type of research or testing to

replace their use either with methods not entailing the use of animals or

by using other species of animals including those genetically altered

Progress has been made in identifying opportunities to avoid NHP use where, 1) they are

not the relevant species, 2) alternative species are available and 3) other methods can

be used. With regard to the study of disease, there is consensus in the scientific

community that one model can never fully recapitulate all aspects of human diseases.

This implies that a variety of models, animal and non-animal, should be used. If a one-

to-one replacement of a test may not be achievable, an integrated testing strategy

should should be used in which in silico, in vitro, ex vivo and in vivo experiments, and

clinical research are used in combination with a weight-of-evidence approach. Recent

developments in biomedical research will potentially improve the selection of the most

promising candidates for new therapies before further assessment in vivo.

Examples of currently available possibilities for replacement are:

• Case-by-case approach for the choice of a second non-rodent species and greater

emphasis in regulatory guidelines on the use of alternative methods in the safety

assessment of pharmaceuticals.

• Replacement strategies for treatment and prevention of infectious diseases with the

development and use of controlled human challenge models for typhoid, Plasmodium

falciparum malaria and transmission studies with specific influenza strains.

• The potential to use new, high spatial and temporal resolution imaging techniques in

humans to replace some cognitive neuroscience experiments performed in NHPs.

• NHPs are no longer considered acceptable organ donors for practical and ethical

reasons.

3- The opportunities for the reduction and refinement of NHP use in areas

where no replacement can be foreseen in medium- or long-term, as per

the principles of the 3 Rs

Researchers are encouraged to increase the yield of data per animal and experimental

session and to share data and tissues with other researchers and to publish negative/null

results. Greater efforts are needed to assess the degree of pain and distress experienced

by NHPs, so that refinements can be implemented effectively. For studies examining the

effects of lesions or other interventions within or between groups of animals, factors

such as effect size should be considered to reduce sample sizes. Examples of currently

available possibilities for reduction and refinement are:

 Safety assessment of chemicals and drugs is being enriched by increasing knowledge

and substantial experience of non-animal techniques, which potentially greatly

Non-human primates testing (update 2017)

Final Opinion

reduces the number of NHPs used. Additionally, there are experimental clinical pain

protocols developed for providing early clinical proof of concept within the boundaries

of a phase I clinical trial design (analgesic drugs).

 Increasing costs of drug development of investigational medicinal products favour

limited human exposure in exploratory clinical trials, including microdosing.

 Patient-derived induced pluripotent stem cells (iPSCs) have the potential to reduce

the number of animal-based preclinical tests and even replace some of them.

 Application of novel sophisticated imaging techniques to study the development of

infectious diseases may result in more information from fewer animals.

 Technological developments in neuroscience have enabled refinement of surgical and

other procedures e.g., refining and de-sizing the devices used in invasive

experiments, improving the anaesthetics and analgesics with faster recovery used in

imaging experiments and surgery, non-invasive imaging methods help reduce and

refine invasive techniques such as surgery, refinement of food and fluid control

protocols and wireless technology have a positive impact on NHP welfare.

4- Identification of specific research areas where effort should be made to

advance replacement, reduction and refinement of the use of non-human

primates in scientific procedures

There is an urgent need to conduct systematic reviews and meta-analysis of all areas of

NHP use. This would potentially significantly reduce the number of NHPs used and the

resources required by identifying where they are unsuitable models or where they have

contributed very little to current knowledge. Additionally, it may provide evidence for

more targeted use of NHPs, which is important for ethics committees

and funders of

research. Emphasis should also be given to ensuring proper reporting of NHP studies and

effective knowledge transfer, focusing NHP research in centres of excellence and the

development of suitable, harmonised training courses. Continued work is necessary to

develop improved means of assessing pain, suffering and distress in NHPs, including the

psychological impact of their use in research. Scientific knowledge about the welfare

impact of husbandry and procedures, even after refinement measures have been

applied, needs to be factored into harm-benefit assessments.

Examples of efforts needed in specific research areas are:

 To progress towards complete replacement of NHPs in drug safety testing, it will be

necessary to gain new insights into molecular biology, including a better

understanding of signalling pathways, modelling and bioinformatics and further

research into integrated testing strategies, including the identification of Adverse

Outcome Pathways (AOPs) leading to human diseases. Additional work is needed on

new models for investigating abuse potential, assessment of reproductive toxicity

and characterisation of the safety of biopharmaceuticals.

 Further research is necessary into 1) the relevance and limitations of the use of NHP

models for infectious diseases and therapies, 2) the optimisation of novel non-NHP

animal models, such as humanised mice and 3) development of new techniques such

as organoïds and –omics technology.

 In neuroscience, progress is required in refinement of awake, behaving

The role of the ethics committees is to do comprehensive NHP project evaluations which address all aspects

of NHP programs including ethical considerations

Non-human primates testing (update 2017)

Final Opinion

electrophysiology studies. There should also be a focus on significantly improving the

spatial and temporal resolution of non-invasive imaging technologies to enable the

replacement of electrophysiological studies in NHPs.

5- The scientific viewpoint on when their use would no longer be necessary,

considering the type of research and areas of testing with a view to the

establishment of a specific phasing-out time-table where possible

A number of factors determine the timetable for complete replacement of NHP use,

among others:

 Availability of funding and resources for developing alternatives to NHP models and

ensuring they are fit for purpose.

 Progress in the formal validation of alternative test methods within the regulatory

arena and in reducing the timescale and bureaucracy associated with this process;

 Lack of regulatory and guideline harmonisation between countries and the condition

that is often included that an alternative method must be formally validated and

accepted by regulatory authorities before it can be used.

 New demands for NHP use in science, such as emergence and re-emergence of

infectious diseases where NHPs are the only relevant model.

 While NHPs have been advocated by WHO as better models for Biosimilars because

they have a higher tolerance for human proteins, EMA and FDA in April 2015

adopted guidelines proposing a tiered approach suggesting that only in vitro studies

are acceptable.

 The risk aversive nature of society makes it difficult to move away from familiar

methods to new alternative methods where there is less historical data to fall back

on.

This wide spectrum of positive and negative incentives for NHP use makes it difficult to

predict a timetable for complete replacement for each of the research areas.

6- Potential implications for biomedical research (e.g., immune based

diseases, neurodegenerative disorders, infectious diseases and serious

diseases) should the use of non-human primates be banned in the EU.

Recognising the high levels of public concern about NHP research, regulatory authorities

in some world regions have also adopted ethical limits or boundaries on NHP use.

However, the close phylogenetic relationship of NHPs with humans makes them the best

available animal models for addressing particular research questions.

Therefore, there is consensus within certain sections of the scientific community that,

where alternatives do not exist, appropriate use of NHPs remains essential in some areas

of biomedical and biological research and for the safety assessment of pharmaceuticals.

As long as sufficiently validated alternatives are not available, a total ban would make

further progress in such research and some safety studies impossible, at least in Europe.

Since animal welfare standards for laboratory NHPs are on average higher in many

European countries than in other parts of the world, it follows that if NHP research is

forced outside of Europe then there would likely be a net decrease in animal welfare.

This could also have an impact on the quality of the research, on public health and

accessibility of treatments developed under different standards and on local economy.

As long as a total ban is not feasible, when communicating about NHP use with the

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Final Opinion

public, the scientific community should provide an accurate description of the benefits,

harms to animals and limitations of such research, and be realistic about the potential

outputs and impacts.

2 MANDATE

2.1 BACKGROUND

Directive 2010/63/EU on the protection of animals used for scientific purposes

, revising

and replacing Directive 86/609/EEC, was adopted in 2010. It provides for controls of the

use of live animals for scientific purposes including a systematic project evaluation and

authorisation, sets binding standards for housing and care as well as for the education,

training and competence of personnel both handling animals and supervising the

experiments.

Due to their genetic proximity to humans and highly developed social skills, the use of

non-human primates in scientific procedures raises specific ethical questions and

practical problems in terms of meeting their behavioural, environmental and social needs

in a laboratory environment. Furthermore, the use of non-human primates for scientific

purposes is of the highest concern to the citizens. As a result, the use of non-human

primates attracted significant attention during the review of the Directive.

In this context, it is useful to recall the 2002 Scientific Steering Committee, SSC, report

highlighting the continuing need to use non-human primates in biomedical research. This

was followed by the European Parliament declaration adopted in 2007 urging the

Commission to

1. Make an end to the use of great apes and wild-caught monkeys in scientific

experiments;

2. Establish a timetable for replacing with alternatives the use of all primates in

scientific experiments.

The Commission stated in its response

to the European Parliament (EP) that with the

current scientific knowledge at the time a timetable with a fixed deadline to phase out all

use of non-human primates in the area of biomedical research was not possible.

However, the Commission also recognised that science is evolving rapidly in this field

and novel technologies, such as genomics and computer modelling, were gradually

emerging.

As a result of these events, DG ENV requested the Scientific Committee on Health and

Environmental Risks (SCHER) to issue an opinion on the status of alternatives for the

use of non-human primates to facilitate an informed debate during the negotiations of

the European Parliament and the Council on the revision of the Directive.

SCHER adopted its opinion in January 2009

. The SCHER opinion concurred with the

Commission's view seeing no scientific reasons to support a discontinuation of the use of

non-human primates in basic and applied research, or in the development and testing of

OJ L 276, 20.10.2010, p. 33–79

The Scientific Steering Committee: “The need for non-human primates in biomedical research”, statement

adopted 4-5 April 2002: http://europa.eu.int/comm/food/fs/sc/ssc/out253_en.pdf

http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm

http://ec.europa.eu/environment/chemicals/lab_animals/pdf/scher_o_110.pdf

Non-human primates testing (update 2017)

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new drugs. The opinion assessed the different areas in which non-human primates were

being used. It examined areas for which partial or full replacement could be foreseen,

and discussed the opportunities for the reduction and refinement of the use of non-

human primates in areas where no replacement could be expected in medium or long

term.

Use of non-human primates in scientific procedures today

There has been a decrease in the use of NHPs compared to the situation in 2008.

According to the latest available statistics of the EU from 2011, around 11 million

animals were used in scientific procedures in the EU. Of these, approximately 6,000 were

NHPs, compared to almost 10,000 in 2008.

From a scientific point of view, the use of NHPs, at the time of the SCHER report, was

considered essential for scientific progress in a number of important areas of disease

biology, research and in safety testing, including:

− Development of pharmaceuticals, in particular safety testing, to assess potential

toxicity in animals to identify unacceptable adverse reactions in humans;

− Understanding the pathophysiology of infectious diseases such as HIV/AIDS, where

the NHP was considered the only susceptible species and therefore the only useful

animal model to study the disease, and to develop safe and effective vaccines and

therapies;

− Learning how complex brains of primates, humans included, are structured and

function. Again, NHPs were considered the best model due to their close similarity to

humans with regard to brain complexity and function;

− Developing and testing xenotransplantation methodologies.

This is reflected in the EU statistics which show that 56% of the non-human primates

were used for the purposes of toxicological and other safety evaluations, 10% in the

area of basic research and 23% in applied research, development and quality control of

products and devices for human medicine and dentistry and for veterinary medicine.

Need for an update

A number of recommendations put forward by SCHER are reflected in the Directive. The

Directive restricts the purposes for which non-human primates can be used, requires

scientific justification that no other species can be used and requires more stringent

inspections of establishment keeping or using non-human primates. An individual history

file must be kept for each non-human primate and projects using non-human primates

cannot to be authorised using simplified administrative procedures.

In addition, any work involving the use of non-human primates is subject to a

retrospective assessment at the end of the project. The use of wild-caught non-human

primates for scientific purposes is prohibited and specific measures are required to move

towards using only second (or higher) generation purpose-bred non-human primates.

Finally, SCHER recommended that their position should be regularly reviewed in the light

of alternative approaches that are constantly being developed.

This is reflected in Recital 49 of the Directive that provides that "Technical and scientific

advancements in biomedical research can be rapid, as can the increase in knowledge of

factors influencing animal welfare. It is therefore necessary to provide for a review of

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this Directive. Such review should examine the possible replacement of the use of

animals, and in particular non-human primates, as a matter of priority where it is

possible, taking into account the advancement of science…."

The legal obligation for the review is embedded in the first paragraph of Article 58:

"The Commission shall review this Directive by 10 November 2017, taking into account

advancements in the development of alternative methods not entailing the use of

animals, in particular of non-human primates, and shall propose amendments, where

appropriate."

2.2 TERMS OF REFERENCE

In view of the above, the Commission asks the SCHEER to issue a scientific opinion,

updating the SCHER opinion of 13 January 2009, "The need for non-human primates in

biomedical research, production and testing of products and devices", on:

• The areas of research (fundamental, translational and applied) and testing of

products and devices in which non-human primates continue to be used today;

• The currently available possibilities by type of research or testing to replace their use

either with methods not entailing the use of animals or by using other species of

animals including those genetically altered;

• The scientific viewpoint on when their use would no longer be necessary, considering

the type of research and areas of testing with a view to the establishment of a

specific phasing-out time-table where possible;

• The opportunities for the reduction and refinement of their use in areas where no

replacement can be foreseen in medium or long term as per the principles of the

Three Rs;

• Identification of specific research areas where effort should be made to advance

replacement, reduction and refinement of the use of non-human primates in scientific

procedures.

• Potential implications for biomedical research (e.g., immune based diseases,

neurodegenerative disorders, infectious diseases and serious diseases) should the

use of non-human primates be banned in the EU.

SCHEER's opinion would be appreciated by the end of December 2016 to contribute to

the preparation of Commission Review of the Directive as provided by Article 58 of the

Directive with a deadline of 10 November 2017.

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3 OPINION

3.1 Overview

In 2009, SCHER adopted its first Opinion on ‘The need for NHPs in biomedical research,

production and testing of products and devices’. The SCHER acknowledges that animals

should only be used in medical research when it is unavoidable and validated alternative

methods are not available. The SCHER also considered NHPs essential for scientific

progress in important areas of disease, biology, research and safety testing. The SC

requested a regular review of this position that was reflected in Directive 2010/63/EU.

Therefore, the European Commission requested SCHEER to issue an update of the 13

January 2009 Scientific Opinion. This current Opinion addresses six areas from the

Commission's mandate on “the need for non-human primates in biomedical research,

production and testing of products and devices" (Section 3.2).

NHPs are generally considered the best available animal models for addressing particular

research questions because of the close phylogenetic relationship with humans.

However, this similarity renders the acceptance of inducing pain or distress as a

consequence of their use in scientific procedures even more debatable/challenging than

for other species of animals. Hence, research in NHPs represents a serious ethical

dilemma which gives rise to a high level of concern from EU citizens. Therefore, human

interest in potential benefits for mankind arising from NHP research must be balanced

against avoiding harm to NHPs and adopting ethical limits or boundaries on NHP use.

Opinion polls show that the general public is more accepting of animal research where

animal use and suffering are minimised, in line with the 3Rs principle. If NHPs are

considered necessary for certain uses, it is essential to adopt the highest standards of

NHP housing and husbandry

and to follow best practice in the conduct and refinement

of scientific procedures. In addition, experimental design, analysis and reporting are key

means of maximising the knowledge gained from animal experiments and unnecessary

use. Appropriate training for all those working with NHPs is essential to ensure

compliance with legislation, excellence in science, animal welfare and full implementation

of the 3Rs.

It is recognised that tightening of the existing strict EU regulations for NHP use may lead

NHP research to transfer to other countries to the detriment of animal welfare.

Decreased animal welfare may also lead to less reliable results. This can be avoided by

international cooperation that engages as many stakeholders and organisations as

possible to promote the international development of high standards for research and

animal welfare and ethical use.

In this section, SCHEER will address each of the six mandate issues beginning with a

general section that lists topics relevant for all research areas, followed by research

area-specific topics. The scientific rationale, including supporting references, for this new

opinion is given in Section 6. Recommendations are given in Section 7.

Upgraded legal obligations concerning space allowances for NHPs were taken effect 1.1.2017 Dir 2010/63/EU,

Annex III

Non-human primates testing (update 2017)

Final Opinion

3.2 The areas of research (fundamental, translational and applied)

and testing of products and devices in which non-human

primates continue to be used today

Currently, NHPs are predominantly used in the following areas a) development and

safety testing of pharmaceuticals and medical devices, b) treatment and prevention of

infectious diseases, c) neuroscience, d) ophthalmology and e) (xeno)transplantation. The

first three categories comprise the majority of NHPs used. While NHPs are often the

species of choice, their use for development and safety testing of pharmaceuticals and

medical devices also meets regulatory requirements.

3.3 The currently available possibilities by type of research or

testing to replace their use either with methods not entailing

the use of animals or by using other species of animals including

those genetically altered

General issues

Significant progress has been made in identifying opportunities to avoid NHP use where,

1) they are not the relevant species, 2) alternative species are available and 3) other

methods can be used. Where NHP use is scientifically justified, progress has been made

in identifying models in which the human target molecule is present in NHPs but not in

other animals and in improving study designs to use fewer animals. Greater progress

would be made in the replacement of NHP experiments, if this was accepted as an

achievable goal by the scientific community. Importantly, decisions about the need for

NHP use should be made case-by-case based on harm-benefit assessment taking into

account ethics and scientific rationale.

With regard to the study of disease, there is consensus in the scientific community that a

single model can never fully recapitulate all aspects of human diseases. The type of

scientific question asked and the methodology used will determine how useful and how

predictive the results obtained are. This implies that a variety of models, animal and

non-animal, should be used to address different aspects of the same disease. If one-to-

one replacement of a test may not be achievable, an integrated testing strategy in which

in silico, in vitro, ex vivo and in vivo experiments and clinical research are combined in a

weight-of-evidence approach, could fulfil the task. Moreover, recent developments in

biomedical research, e.g., cell culture techniques, in silico modelling, –omics, organoids

and novel fully artificial whole-body models will potentially improve the selection of the

most promising candidates for new therapies before further assessment in vivo.

Notably, substitution of NHPs with rodents or other laboratory animal species is not

replacement as defined by Russell and Burch (1959). However, this may be ethically

desirable if the available evidence indicates that the non-primate species is likely to

suffer less harm.

Development and safety testing of pharmaceuticals and medical devices

Replacing NHP models in new medicine development and safety testing has the potential

to significantly reduce the number of NHPs used in European laboratories, as regulatory

use and routine production are consistently the areas of greatest NHP use. Examples are

in vitro and in silico models for studying liver injury and testing for idiosyncratic adverse

drug reactions, a new mouse model for understanding human-specific menstrual and

pregnancy associated disease, and swine models as second species next to rodents.

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Although international regulations specify that non-rodent species should be used for

safety assessment of pharmaceuticals prior to their being used for the first time in man

and that one of these species can be an NHP species, a significant change in emphasis is

that the need to use NHPs should now be considered on a case-by-case basis. ICH

guideline S6 (R1) on preclinical safety evaluation of biotechnology-derived

pharmaceuticals, in force since December 2011, emphasises the use of in vitro

alternative methods for safety evaluation and opening the way to the replacement of

current standard methods. NHPs may be substituted by transgenic animals (usually

mice) or by the use of homologous proteins in rodents. The EU Guideline on similar

biological medicinal products containing monoclonal antibodies – non-clinical and clinical

issues, which came into effect on 1 December 2012, states “the conduct of toxicological

studies in NHPs is usually not recommended”.

Arguments against phasing out animals in safety testing of pharmaceuticals are:

incomplete knowledge in new models of integrated body systems and pathophysiology,

poor representation of pharmacokinetics by isolated in vitro systems (so far only partially

overcome by using PB-PK modelling) and difficulties in extrapolating from in vitro data to

benchmark doses, which is vital for human risk assessment. Human models are

progressing in translational medicine; however, they are not yet a substitute for well-

designed animal models. Important progress is still required to develop and validate new

alternative methods and to validate them, before obtaining scientific acceptance and

regulatory recognition.

Treatment and prevention of infectious diseases

Infectious disease models in NHPs, because of the similarity of NHP and human immune

systems, resemble human disease more closely than in other animal models. However,

NHP infectious disease models are used when the course of disease resembles human

disease with a similar host range of cells, organs or tissues involved and reflect the host

response in humans. Progress has been made in replacement strategies for treatment

and prevention of infectious diseases with the development and use of controlled human

challenge models for typhoid, Plasmodium falciparum malaria and transmission studies

with specific influenza strains. Other examples in development, which might offer new

possibilities for vaccine research, include a human attenuated Tuberculosis (TB)

challenge model. These studies are particularly important because of the many ethical

and safety barriers that prevent human challenge studies with virulent pathogens. It is

not always possible to study fundamental aspects of the disease and to perform in depth

analysis of host-pathogen interactions in human volunteers or patients. In some cases,

non-NHP animal models, for example, new genetically modified mouse models may

substitute for NHPs. Importantly, it is unlikely that new technologies will negate the need

for infectious NHP models in the near future due to emerging and re-emerging

pathogens.

Neuroscience

In behavioural neuroscience studies, many of the procedures used to monitor and

manipulate brain elements are invasive and therefore, only rarely is it considered ethical

to carry out these procedures in healthy humans. Opportunities for minimal risk

microelectrode recording should be utilised with the patient’s informed consent and

ethics committee approval. However, data from an injured human brain should be

carefully interpreted and cannot replace in vivo controlled studies in animals.

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There is a potential to use imaging studies in humans to replace some cognitive

neuroscience experiments currently being done on NHPs. However, fMRI measures can

only indirectly reflect neuronal activity and imaging techniques require further

development to completely replace studies in NHPs and other animals.

In vitro disease modelling possibilities have expanded and have potential for

replacement, e.g., microfluidic chambers/brain chips, mixed cell cultures, patient-

derived-induced pluripotent stem cells (iPS cells), “mini-brains” or organoids. The

absence of a blood-brain-barrier, a vascular system and an immune system, however,

are major limitations for complex pathophysiological investigations.

Other uses: Ophthalmology

NHPs represent a more relevant model for the discovery and development of new

therapeutic strategies as compared with other species. No currently available rodent, in

vitro or in silico model appears to recapitulate the architectural features of the primate

eye (i.e., the macula) or the complex structural and functional interactions of retinal

cells. New, more pertinent models of ocular pathology are currently being developed in

NHPs and new gene therapy strategies are being tested in NHPs before their translation

to the clinic. However, good progress is being made developing suitable in vitro models

of retinal defects for screening and efficacy testing of new therapies. The potential for

replacement is encouraging, especially through the use of human stem cells in

developing human cell-based models of visual pathways, particularly the retina, and

models of macular degeneration (Singh et al., 2012).

Other uses: (Xeno)transplantation

NHPs are no longer considered acceptable organ donors for practical and ethical reasons.

Pigs have the potential to be the prime candidates for organ donation. However, due to

their anatomic and immunologic similarity to humans, NHPs have been used as

recipients to investigate the fundamental aspects of organ transplantation and may be

required in the future to evaluate new immunosuppressive therapies and new methods

to prevent organ rejection. The scientific justification of the use of NHPs as recipients

should still be examined on a case-by-case basis and should take into account the in

silico, in vitro and in vivo data acquired in a non-primate species.

3.4 The opportunities for the reduction and refinement of their use

in areas where no replacement can be foreseen in medium or

long term as per the principles of the Three Rs

General issues

Researchers are encouraged to increase the yield of data per animal and experimental

session, to share data and tissues with other researchers and to publish negative/null

results. Greater efforts are needed to assess the degree of pain and distress experienced

by NHPs, so that refinements can be implemented effectively. For studies examining the

effects of lesions or other interventions within or between groups of animals, factors

such as effect size should be considered to reduce sample sizes.

Development and safety testing of pharmaceuticals and medical devices

Safety assessment of drugs is being enriched by increasing knowledge and substantial

experience of non-animal techniques, which greatly reduces the number of NHPs used.

Additionally, there are experimental clinical pain protocols developed for providing early

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clinical proof of concept, within the boundaries of a phase I clinical trial design (analgesic

drugs).

Exploratory Clinical Trials, including microdosing, were established by regulatory

authorities to reduce the nonclinical testing prior to First in Human (FIH) clinical trials

(ICH M3 (R2)). These studies allow an early decision based on clinical data with

associated time and cost savings, and reduced drug manufacturing requirements (Burt

et al., 2016).

New applications of human microdosing studies to areas beyond exploratory

pharmacokinetic data could significantly reduce the number of unsuccessful candidate

drugs from progressing into the full developmental process and as a consequence reduce

the number of pre-clinical animal tests. This includes using microdosing to investigate

drug-drug interactions, site of action, pharmacokinetics in vulnerable populations,

intravenous data and metabolic profiling. The implementation of human microdosing

studies prior to full Phase I clinical trials can eliminate unsuccessful candidates from

progressing through the full developmental pathway. As microdosing studies are allowed

based on a reduced safety package compared to a full Phase I trial, then both the cost

and the number of required animal-based (including NHPs) preclinical test are reduced.

Patient-derived induced pluripotent stem cells (iPSCs) have the potential to reduce the

number of animal-based preclinical tests and even replace some of them. They can be

used to assess the toxicity and clinical efficacy of drug candidates before they are

progressed into market.

Cross-company and cross-sector data sharing has identified efficient study designs for

regulatory toxicology studies, helping to reduce NHP use per study/test compound (e.g.,

fewer dose groups, animals per group, and recovery animals.

Treatment and prevention of infectious diseases

New imaging techniques may enable the study of the development of certain infections,

including early effects of infection, and/or therapy over time in the same individual. The

application of novel sophisticated imaging techniques may result in more information

from fewer animals.

Identifying correlates of protection to avoid the need for progression to severe clinical

signs would help to refine infectious disease studies.

Neuroscience

Technological developments have enabled refinement of surgical and other procedures

within the neurosciences, e.g.:

 Refining and de-sizing the devices used in invasive experiments,

 Using wireless techniques to record neuronal activity,

 Using positive training techniques to reduce stress in housing management and

procedures.

 Improving the anaesthetics and analgesics with faster recovery used in imaging

experiments and surgery,

 Using non-invasive imaging methods that help reduce and refine invasive

techniques such as surgery,

 Refining food and fluid control protocols to minimise the impact on NHP welfare.

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3.5 Identification of specific research areas where effort should be

made to advance replacement, reduction and refinement of the

use of non-human primates in scientific procedures

General issues

There is an urgent need to conduct systematic reviews and meta-analysis of all areas of

NHP use and to build up public databases so that no duplication is allowed. This could

potentially significantly reduce the number of NHPs used and the resources required by

identifying where they are unsuitable models or where they have contributed very little

to current knowledge. Additionally, research funders should ensure that NHP proposals

are only funded where there is no suitable alternative approach, and where there is a

high likelihood of scientific, medical or social benefit. Emphasis should also be given to

ensuring proper reporting of NHP studies and effective knowledge transfer, to maximise

the value of funded research.

Importantly, it is advisable to ensure the harmonisation and implementation of

regulations leading to improvements of replacement, reduction and refinement. It is

essential to stimulate changes in both scientific and societal attitudes and in scientific

practice by improving and expanding the 4Cs (Commitment, Communication,

Cooperation and Coordination). This may require improvements to peer review processes

for project proposals, (e.g., appointment of panel members with expertise in alternatives

to NHPs, statisticians and clinicians; amendments to application forms, guidance and

training, to ensure provision of quality information about the justification for NHP use,

animal numbers and experimental design, with robust scrutiny of this information).

It is recommended that consideration be given to focusing NHP research in centres of

excellence, defined by performance of excellent research in combination with the

provision of optimal care and use of NHP, and improving existing networks for

information sharing. Moreover, researchers and animal care staff must ensure that they

keep abreast of the latest techniques that enable reduction in animal numbers and of the

refinement of existing methods and techniques to reduce suffering, and put this evidence

base into practice. Consideration should be given to continuous development of an

accredited training course for those working with NHPs, harmonised across Member

States, and improved access to training in experimental design. Work needs to continue

to develop improved means of assessing pain, suffering and distress in NHPs, including

the psychological impact of their use in research. Scientific knowledge about the welfare

impact of husbandry and procedures, even after refinement measures have been

applied, needs to be assessed and factored into harm-benefit assessments. A high

quality social and physical environment for NHPs must always be assured, beyond the

scope of a particular study.

Development and safety testing of pharmaceuticals and medical devices

Progress has been made in identifying opportunities to avoid NHP use where they are not

a relevant species or alternative species can be used and in identifying efficient study

designs using fewer animals where NHP use is scientifically justified. In addition,

progress has been made in identifying situations where nonclinical testing can be

conducted in a single relevant species. To progress towards complete replacement of

NHPs in safety testing, it will be necessary to gain further insight into molecular biology,

including a better understanding of signalling pathways, modelling and bioinformatics

and additional research into integrated testing strategies.

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More work is needed on new models for investigating abuse potential, the assessment of

reproductive toxicity and the characterisation of the safety of biopharmaceuticals.

Treatment and prevention of infectious diseases

Research is necessary for

 Determining the relevance and limitations of the use of NHP models for infectious

diseases and therapies,

 The improvement of techniques and NHP characterisation to further refine models

and reduce the number of NHPs required,

 The optimisation of novel non-NHP animal models, such as humanised mice, and

 Development of new techniques such as organoids and –omics technology to

further reduce the current need to use NHPs to study important infections.

Neuroscience

There has been progress with refinement of awake, behaving electrophysiology studies,

but further progress is required, given the high impact on animal welfare. The basic

paradigm for study of brain activity at the single cell level involves repeated electrode

penetration, prolonged restraint, fluid or food control, and surgical procedures to implant

head fixation and recording or other devices. There needs to be a focus on significantly

improving the spatial and temporal resolution of non-invasive imaging technologies in

order to refine and ultimately replace this use of NHPs.

Other uses: Ophthalmology

For advanced replacement techniques in vision research and to learn more about the

structure and function of the visual cortex, new technology is necessary for whole organ

eye-culturing, in vitro and in silico models and simulations.

3.6 The scientific viewpoint on when their use would no longer be

necessary, considering the type of research and areas of testing

with a view to the establishment of a specific phasing-out time-

table where possible

General Issues

A number of factors determine the timetable for complete replacement of NHP use:

 The availability of funding and resources for developing alternatives to NHP models

and ensuring they are fit for purpose. Criteria for prioritisation of areas of further

research could be used, e.g., the absolute number of the NHP used and the severity

of procedures.

 The progress in the formal validation of alternative test methods within the

regulatory arena and in reducing the timescale and bureaucracy associated with this

process.

 Lack of regulatory harmonisation both within and across sectors and the condition

that is often included that an alternative method must be formally validated and

accepted by the scientific community and regulatory authorities before it can be used

 The progress to validate new non-animal models against existing animal models.

 A continued prominent role and investment of the EU in promoting and implementing

alternative methods.

 The extent to which justification for the use of alternative models is laid down in

internationally accepted guidelines.

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 The risk aversive nature of society makes it difficult to move away from familiar

methods to new alternative methods where there is less historical data to fall back

on.

 Factors related to scientific practice and career progression where dynamics such as

competition, the reputation and track record of researchers (in terms of grants and

publications), and entrenchment discourage switching from NHPs to alternative

(animal and non-animal) models.

 New demands for NHP use in science, such as emergence and re-emergence of

infectious diseases where NHPs are the only relevant model, and biosimilars for

which NHPs have been advocated by WHO as better models because they have a

higher tolerance for human proteins. It is, however, noted that EMA and FDA in April

2015 adopted guidelines proposing a tiered approach and suggesting that only in

vitro studies are acceptable (Chapman et al., 2016).

This wide spectrum of positive and negative incentives for NHP use makes it difficult to

predict a timetable for complete replacement for each of the research areas.

3.7 Potential implications for biomedical research (e.g., immune

based diseases, neurodegenerative disorders, infectious

diseases and serious diseases) should the use of non-human

primates be banned in the EU

Recognising the high levels of public concern about NHP research, regulatory authorities

in some world regions have also adopted ethical limits or boundaries on NHP use.

However, the close phylogenetic relationship of NHPs with humans makes them the best

available animal models for addressing particular research questions.

Therefore, there is consensus within certain sections of the scientific community that,

where alternatives do not exist, appropriate use of NHPs remains essential in some areas

of biomedical and biological research and for the safety assessment of pharmaceuticals.

For example: NHPs can play a key role in drug development due to their similarity to

humans with regard to sensory organs, hormonal systems, reproduction, immune

system etc., to evaluate efficacy and safety, especially for biopharmaceutical

compounds. There has been progress in a move toward a non-animal predictive

mechanism-based approach, e.g., for testing of drugs for reproductive toxicity, but there

are still obstacles to overcome in terms of regulatory acceptance and scientific validity.

In spite of this, NHPs should only be used when it is scientifically demonstrated that

none of the other non-rodent species commonly used in safety testing is appropriate for

the purpose of the study.

As long as sufficiently validated alternatives are not available, a total ban would make

further progress in such research and some safety studies impossible, at least in Europe.

On the other hand, a ban may lead to acceleration of investment into the development

of alternative methods as seen for cosmetics (European Commission, 2016).

However, a ban in Europe would likely force NHP research outside of Europe and since

animal welfare standards for laboratory NHPs are on average higher in many European

countries than in other parts of the world, it follows that there would likely be a net

decrease in animal welfare. Though some companies have developed global animal

welfare policies, there are concerns that European scientists are transferring their

research programmes outside the EU to countries where welfare/scientific standards

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would not be judged ethically acceptable in Europe. This could have an impact also on

the quality of the research, on public health and accessibility of treatments developed

under different standards and on local economy

As long as a total ban is not feasible, when communicating about NHP use with the

public, the scientific community should provide an accurate description of the benefits,

harms to animals and limitations of such research, and be realistic about the potential

outputs and impacts.

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4 MINORITY OPINION

None.

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Final Opinion

5 DATA AND METHODOLOGY

An initial literature research was undertaken by the SCHEER in July 2016 to identify key

publications for this Opinion. The aim of the literature review was to collect publications

from January 2009 to July 2016 for this Opinion. The following terms were used for the

literature review in the title, abstract, key word fields: "non-human primates" AND

(("human+health" AND "relevance") OR (ethics OR "alternative+methods" OR

"research+needs")) resulting in 26 hits for publications. The following documents were

included: peer-reviewed articles, journal entries, reviews, and public polls. The SCHEER

WG included additional relevant publications in areas of their expertise.

A call for information

was published on 1 January 2009 for collecting papers on new

technologies used to replace, reduce and refine the use of non-human primates in

biomedical research, production and testing of products and devices. The Call for

information was opened between 8 June and 3 July 2016. There were 19 submissions

containing more than 100 papers from individual scientists, research organisations,

professional societies, pharmaceutical industry and animal protection organisations.

Submitted information was considered for the Opinion, if it was relevant for the scope of

the Opinion.

6 SCIENTIFIC RATIONALE

6.1 Introduction

This Opinion made use of the relevant scientific literature on the use of NHPs and

alternatives in biomedical research worldwide. However, in the interpretation of studies it

is taken into account that different jurisdictions may be involved and that the regulations

with regard to NHPs can be different from those in the EU.

6.2 Ethical issues

The close phylogenetic relationship of NHPs with humans makes them the best available

animal models for addressing particular research questions. Therefore, there is

consensus within certain sections of the scientific community that, where alternatives do

not exist, appropriate use of NHPs remains essential in some areas of biomedical and

biological research and for the safety assessment of pharmaceuticals (Weatherall et al.,

2006). However, this similarity renders the acceptance of inducing pain or distress as a

consequence of their use in scientific procedures even more debatable/challenging than

for other species of animals (The Boyd Group, 2002; Weatherall et al., 2006).

Furthermore, animals cannot consent to their participation in research and generally will

not benefit from it. Hence, their use in research represents a serious ethical dilemma,

and there is a variety of views on whether NHP experiments should be permitted

(Prescott, 2010). Opinion polls of the European public repeatedly show low levels of

acceptance of the use of NHPs in research (European Commission, 2006; Crettaz von

Roten, 2012; Clemence and Leaman, 2016).

The approach to this ethical dilemma most often adopted in regulatory frameworks on

the use of animals for scientific purposes is a pragmatic, utilitarian one, which is

important for protecting human safety. Utilitarianism is a branch of ethics in which the

http://ec.europa.eu/health/scientific_committees/consultations/calls/scheer_call_info_01_en

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Final Opinion

moral worth of an action is determined by its outcome. It requires us to strike the most

favourable balance of benefits and costs for all the sentient individuals affected by what

is proposed to be done. In the case of NHP research, the human interest in potentially

obtaining some benefit for mankind must be balanced against the interests of the NHPs

in avoiding harm (Quigley, 2007). This case-by-case approach, referred to as “harm-

benefit assessment”, forms a key part of the authorisation process for projects involving

animals under Directive 2010/63/EU. The EC and others have published guidance on

how to perform robust harm-benefit assessments (European Commission, 2013; Home

Office, 2015; Brønstad et al., 2016; Laber et al., 2016). However, there is some

question whether the Utilitarian approach is the most appropriate one for NHP research

(Rossi, 2009; Walker, 2006; Quigley, 2007).

Further recognising the high levels of public concern about NHP research, regulatory

authorities in some world regions have also adopted ethical limits or boundaries on NHP

use, giving rise to legislation, which is a hybrid of utilitarian and deontological ethics (the

latter being based on duties and adherence to rules, rather than the consequences of

actions). For example, Directive 2010/63/EU places an effective ban on the use of Great

Apes in procedures, save for exceptional circumstances (subject to the use of a

safeguard clause; see Article 55). For a summary of the provisions relating to NHPs in

international legislation on animal use in science, see Chapman et al., 2015 and Bayne

and Morris, 2012.

Opinion polls of the general public show greater acceptance of animal research where

animal use and suffering are minimised (e.g., Ipsos MORI, 2014) in line with the 3Rs

principle, which can be summarised as:

 Replacement of animals with non-animal methods,

 Reduction of the number of animals used to obtain information of a given amount

and precision,

 Refinement of scientific procedures and husbandry to minimise harm (e.g., pain,

suffering, distress) and improve animal welfare.

Definitions of the 3Rs have evolved since their original inception by Russell and Burch

(1959). For example, refinement is now generally accepted to apply to all aspects of the

animal’s lifetime experience and to encompass promotion of positive welfare states as

well as minimisation of suffering (Buchanan-Smith et al., 2005). More detailed and

contemporary definitions are provided in Annex 1.

Directive 2010/63/EU requires implementation of the 3Rs during the design and conduct

of animal studies. However, their successful implementation relies on the proactive and

coordinated engagement of multiple stakeholders, including researchers, veterinarians

and animal care staff, research funders and regulatory bodies requiring animal test data.

To fully apply the 3Rs and maximise the benefits, there is a need to ensure that as new

knowledge, technologies and approaches emerge there is timely assessment and

evolution of research strategies, study designs, scientific procedures and husbandry,

throughout the lifetime of research projects.

In addition to its value as a practical ethical framework for more humane in vivo

research, thereby helping to address societal concerns, the 3Rs concept also has

considerable scientific merit (Graham and Prescott, 2015). This is because replacement

is about accelerating the development and use of human-relevant tools, based on the

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Final Opinion

latest technologies, which may be more predictive of responses in man, more cost

effective, and allow a higher throughput of studies (Sewell et al., 2016). Approaches to

reduction often focus on principles of good experimental design, and better

interpretation and reporting of studies, helping to improve the quality and reproducibility

of animal experiments (Parker and Browne, 2014). Animals free from unnecessary pain

and distress yield better quality and more reproducible data (Poole, 1997). Hence, there

are strong scientific and business drivers for the 3Rs, which increasingly are leading to

changes in practice in both industry and academia (Burden et al., 2015).

There is interplay between the 3Rs and conflicts can arise, such as when procedures that

enable a reduction in animal numbers lead to cumulative distress for the fewer animals

that are used (as might occur in a longitudinal study using imaging, for example). This

conflict is usually resolved by carefully weighing the harms and benefits to the animals

involved, or else by prioritising the experience of the animals (i.e., refinement) over

reduction. The re-use of NHPs can decrease the number of animals used overall,

however there are ethical considerations against as well as for re-use. Recital 25 of

Directive 2010/63/EU states: “The number of animals used in procedures could be

reduced by performing procedures on animals more than once, where this does not

detract from the scientific objective or result in poor animal welfare. However, the

benefit of reusing animals should be balanced against any adverse effects on their

welfare, taking into account the lifetime experience of the individual animal. As a result

of this potential conflict, the reuse of animals should be considered on a case-by-case

basis.” (Recital 25 of the Directive 2010/63/EU).

In the majority of cases, NHPs used in research and testing are euthanised as an integral

part of the experiment for analysis of tissues post mortem. In situations where this is not

required, or in the case of former breeding or surplus stock animals, it may be possible

to rehome the animals as an alternative, and this is permitted under Directive

2010/63/EU. However, the rehoming process can entail welfare costs to the animals

involved. NHPs should thus only be rehomed if it is clear that the process will be truly in

the best interest of the individual animals, that it will not harm their welfare and that the

new home offers a good quality of life (Kerwin, 2006; Prescott, 2006, NCad 2016 report:

file:///C:/Users/mprescott/Downloads/ncad-opinion-rehoming-former-lab-animals.pdf).

6.3 Housing and husbandry

Europe has implemented major improvements in the housing and husbandry of

laboratory NHPs over the last decade. Under Directive 63/2010/EU, it is now mandatory

to house these highly social animals in socially stimulating environments, unless there

are exemptions for scientific, animal welfare or animal health reasons (Article 33(3)). In

addition, environmental enrichment must be provided, to allow performance of a wide

range of normal behaviour. Enriched housing conditions can have a positive impact on

both the psychological wellbeing of the animals and the research outcomes (Gottlieb et

al., 2015; Hannibal et al., 2017). Improvements also include the development and

implementation of training protocols designed to encourage animals to co-operative

voluntarily with husbandry and scientific procedures (e.g., Prescott and Buchanan-Smith,

2003, 2007; Prescott et al., 2005; Graham et al., 2012; Coleman and Maier, 2010). In

the past EUPRIM-Net has developed such training protocols and has disseminated them

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extensively across European NHP facilities. A new EU COST Action (PRIMTRAIN

) is in

place to develop and exchange training protocols, building on the earlier work of the

EUPRIM-Net consortium. It aims to implement a minimum European standard for animal

training for NHP facilities.

The early life experience of NHPs is important for their welfare later in life. Care should

be taken to source animals that are weaned at an appropriate age (Prescott et al., 2012)

and that are well habituated to humans at the breeding centre, as this will mean that

they are better able to cope with experimental use in the laboratory, especially for

studies that involve frequent/close human-animal interaction (Tardiff et al., 2006;

Jennings and Prescott, 2009). There is the potential for breeding centres to prepare

animals for their future use by training them using positive reinforcement methods to

perform behaviours such as targeting shifting, and entering a transport cage. There is

evidence that voluntary exposure to simple operant tasks at the breeding centre, such as

pressing a button for automated delivery of food or fluid reward, can expedite learning of

more complex tasks in the neuroscience laboratory, and hence provide a basis for

selecting those animals likely to perform well on-study (Tulip et al., 2017). The breeding

unit provides a natural complexity of environment that lends itself to studies of primate

welfare and behaviour.

6.4 Animal welfare standards outside the EU

Animal welfare standards for laboratory NHPs are on average higher in many European

countries than in other parts of the world. There are a number of reasons for this,

including differences in legislation, practice, awareness of the welfare needs of NHPs and

animal health status. For example, the minimum enclosure sizes and space allowances

permissible for macaques under Annex III to Directive 2010/63/EU are, depending on

the age of the animals, six- to ten-times greater than the minimum permissible under

the USA ILAR Guide (European Union 2010; National Research Council 2011). NHPs in

Europe are more often housed in pens or customised rooms rather than small stainless

steel cages with gridded floors (though some laboratories outside of Europe offer EU-

style housing) (see the NC3Rs Macaque Website for examples:

). Housing NHPs in pairs

and groups is more standard in Europe than the USA and Asia (e.g., Couch et al., 2015),

where Institutional Animal Care and Use Committees (IACUCs) may grant exemptions

from social housing for reasons that would not generally be acceptable in most European

countries. Accreditation schemes, such as the one administered by the Association for

Assessment and Accreditation of Laboratory Animal Care (AAALAC), are valuable but do

not require EU standards outside of Europe.

It follows that if NHP research is forced outside of Europe then there would likely be a

net decrease in animal welfare. A decline in welfare standards need not be axiomatic in

all cases, however. For example, the major UK public funders of NHP research require

standards equivalent to the UK Animals (Scientific Procedures) Act and the NC3Rs

guidelines ‘Primate accommodation, care and use’, wherever in the world the research is

conducted (NC3Rs 2006; NC3Rs/AMRC/BBSRC/Defra/MRC/NERC/Wellcome Trust 2015).

Compliance is assessed case-by-case for each grant application by the NC3Rs. In recent

years, the European pharmaceutical industry has opened animal facilities in China and

http://www.cost.eu/COST_Actions/ca/CA15131

http://www.nc3rs.org.uk/macaques/captive-management/housing/

Non-human primates testing (update 2017)

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other growing economies to take advantage of emerging markets. Outsourcing of studies

to the expanding contract research base in China has also increased (Xia and Gautam

2015). To maintain appropriate standards, some companies have developed global

animal welfare policies that apply at all of their facilities, and most consider animal

welfare issues when choosing and auditing contractors and suppliers (Underwood, 2007).

These are good examples on how the commitment to actions related to international

harmonisation of regulations and welfare standards, including engaging with other

countries through the International Council on Harmonisation of Technical Requirements

for Registration of Pharmaceuticals for Human Use (ICH) and the Organisation for

Economic Co-operation and Development (OECD), can improve conditions abroad to

bring research practices in line with European standards.

However, there are also concerns that European scientists are transferring their research

programmes outside the EU to countries where welfare/scientific standards would not be

judged ethically acceptable in Europe (Cryanoski, 2016; Anon, 2016). Decreased animal

welfare may lead to more stressed animals and this could have an impact on the quality

of the research. This may also affect public health and accessibility of treatments

developed under different standards and on local economy.

6.5 Experimental design and staff training

Good experimental design, analysis and reporting are key means of maximising the

knowledge gained from animal experiments and avoiding wastage of animals. With NHPs

being such precious resources, it is crucial for scientific, ethical and economic reasons

that their use is optimised. However, recent years have seen growing concern about the

reliability and reproducibility of animal studies, and poor experimental design and

reporting have been implicated as major contributing factors (see Begley and Ellis, 2012,

Collins and Tabak, 2014, and other articles in the Nature specials archive: Challenges in

irreproducible research; AMS/BBSRC/MRC/Wellcome Trust 2015, 2016). There is no

reason to suspect that NHP research is any better in this regard than other fields of in

vivo research.

For example, Kilkenny and colleagues (2009) conducted a systematic survey of the

quality of reporting, experimental design and statistical analysis in 271 papers reporting

research on live rats, mice and NHPs carried out in UK and US publicly funded research

establishments. Only 59% of the studies stated the hypothesis or objective of the study

and the number and characteristics of the animals used. Most of the papers surveyed did

not report the use randomisation (87%) or blinding (86%), to reduce bias in animal

selection and outcome assessment. Only 70% of the publications that used statistics

described the statistical tests used and presented the results with a measure of precision

or variability. These findings are by no means isolated. Poor reporting is consistently

highlighted in systematic reviews of animal research (de Vries et al., 2014). These are

important factors in the design of studies and have serious implications for animals,

science and society.

Good experimental design is one of the most effective and immediate ways to reduce

and refine animal procedures, but researchers are not getting sufficient training in this

essential area for completing ethical, rigorous and efficient research (Howard et al.,

2009). Therefore, it is essential that training in experimental design is improved and

made more widely available. There have been a number of important

initiatives/resources launched to aid researchers to improve the design and reporting of

Non-human primates testing (update 2017)

Final Opinion

research using animals. The ARRIVE Guidelines of the NC3Rs

(Kilkenny et al., 2010)

lay out the criteria that should be met in reporting animal studies in order that their

results and conclusions can be properly evaluated by readers. These criteria address a

range of issues relating to transparency and validity of experimental design, the

avoidance or minimisation of bias, and the adequacy of statistical aspects of the study,

such as power and analysis methods. The Guidelines have been endorsed by over 1,000

journals internationally, including major titles like Nature, Cell and PLoS, and by UK

biomedical research funders, who have revised and update their peer review processes

in response. The NC3Rs has also launched the Experimental Design Assistant (EDA)

(https://eda.nc3rs.org.uk/) a free online resource to support researchers, particularly

those in the early-stage of their career, in the planning of animal experiments. The EDA

consists of a website with comprehensive guidance on experimental design and a web

application which uses computer-based reasoning to provide tailored feedback and

advice on individual experimental plans. The system also includes dedicated support for

randomisation, blinding and sample size calculation, helping to ensuring robust study

design and reliable and reproducible findings.

Appropriate training for staff working with NHPs is essential to ensure compliance with

legislation, good quality science, good animal welfare and full implementation of the 3Rs.

Special knowledge and practical skills are required in order to be able to address the

complex behavioural, social and psychological needs of NHPs and the ethical

considerations involved in their use in research (Jennings and Prescott, 2009).

Guidance to Member States on the minimum training requirements for those involved in

the care and use of animals under Directive 2010/63/EU includes some species-specific

learning outcomes (Anon, 2014). NHP-specific material has been provided as part of this

initial, mandatory training in some Members States for many years, with some courses

being accredited by the competent authority or accrediting bodies on their behalf.

However, a number of issues with the current training provision in the EU need to be

addressed to deliver the best science and animal welfare and to avoid perpetuation of

out-dated practices and unwanted variation between laboratories.

First, NHP-specific training provided to prospective licensees is often very short (one day

or less) and can only provide a very brief overview of the required information. A very

short period of initial training also means that there is a particular need for Continuing

Professional Development (CPD) to expand and maintain the knowledge and skills of

those working with NHPs. Many institutions do not provide this on-going training, so

there is a strong case for increasing the scope of both initial training courses and

introduction of mandatory CPD (Weatherall et al., 2006).

Second, there is variation in the syllabus content and delivery for initial training courses

in Member States and no common agreed approach. A survey at the 2010 NC3Rs

Primate Welfare Meeting, a key forum for CPD and networking within the EU NHP

research community, found that 79% of delegates considered there was a need for an

accredited course on NHP use, care and welfare. Specific gaps in existing training

provision identified at the meeting were

 NHP behaviour

 Animal training

http://www.nc3rs.org.uk/ARRIVE

Non-human primates testing (update 2017)

Final Opinion

 Surgery

 Anaesthesia and analgesia

 Welfare assessment

 Humane endpoints

In response to this, the NC3Rs organised workshops on animal training and refinement

of chronic implants, and also a network for animal welfare officers working in NHP units.

The training courses run from 2007 to 2015 as part of the EC-funded projects EUPRIM-

Net I and II have also helped to support training in a range of areas. They take a

blended learning approach and cover aspects of experimental design, imaging

techniques, housing and husbandry, colony management and animal behaviour

management.

Training in practical skills is another key issue. The Primate Welfare Meeting survey

found only 15% of delegates believed there are sufficient practical training opportunities

for those working with NHPs. This may be related to the way in which some Member

States have interpreted Article 8 of the Directive, which prevents the use of NHPs for

“higher education, or training for the acquisition, maintenance or improvement of

vocational skills”. However, this article should not prevent staff with the appropriate

licenses gaining practical skills by undertaking, under supervision, authorised procedures

required as a necessary part of on-going research projects. The problem may also be

related to financial and time constraints preventing researchers from visiting other

laboratories to observe and learn new techniques, for example approaches for surgical

implantation of new recording, dosing or sampling devices. Although the concept of “see

one, do one, teach one” is still considered valid within medical surgical training (Kotsis

and Chung, 2013), those performing procedures in NHPs will only rarely have undergone

the extensive training in surgical skills which underpins the assimilation of more complex

procedure by surgeons.

New opportunities to implement the 3Rs arise continually and it is important that

researchers and animal care staff keep abreast of the latest developments in NHP use,

care and welfare. Published literature and scientific events provide opportunities to

expand knowledge and skills. Increasingly, these traditional routes of information

exchange are being complemented by dedicated online resources aimed at sharing best

practice and enhancing the training and CPD of staff working with NHPs (Prescott, 2016).

These include The Macaque Website

, Common Marmoset Care website

, Chronic

Implants Wiki

and Experimental Design Assistant of the NC3Rs.

See Section 7 for recommendations.

6.6 Areas of research (fundamental, translational and applied) and

testing of products and devices

6.6.1 Overview on the use of NHPs in research and testing

The SCHEER has used publicly available information from Member States for 2014 to

provide an overview on uses of NHPs in Europe (for the sources see Annex 1).

For 2014, a total of 8898 uses (uses includes reuse of the same NHP, does not refer to

http://www.nc3rs.org.uk/macaques/

http://www.marmosetcare.com

https://www.ciwiki.net//

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the total number of animals) of NHPs were reported by European Member States (Table

1, refers to the whole EU). Species most frequently used were cynomolgus monkeys

(7098, 79.7%), marmoset and tamarins (743, 8.4%) and rhesus monkeys (612, 6.9%).

Importantly, these numbers are based on experimental procedures and include first use

as well as any subsequent reuse of the NHPs

. The number of animals used in

procedures could be reduced by performing procedures on animals more than once,

where this does not detract from the scientific objective or result in poor animal welfare.

However, the benefit of reusing animals should be balanced against any adverse effects

on their welfare, taking into account the lifetime experience of the individual animal. As

a result of this potential conflict, the reuse of animals should be considered on a case-

by-case basis. The Directive 2010/63/EU on the protection of animals used for scientific

purposes lays down basic criteria for the reuse of animals. In accordance with

veterinarian advice, an animal may be reused if the previous procedure was of “mild” or

“moderate” severity, the animal's state of health and well-being has been fully restored

and the subsequent procedure is classified as “mild”, “moderate” or “non-recovery” (Art.

16).

Table 1: Experimental procedures reported for NHP species by all European

Member States in 2014

Prosimians

203

Cercopithecoidea (OW)

Cynomolgus monkey

7098

Rhesus monkey

612

Vervets, Chlorocebus spp.

Baboons

183

Other species of OW monkeys

Ceboidea (NW)

Marmosets and tamarins

743

Squirrel monkey

Other species of NW monkeys

Total uses of NHPs

8898

NW: New World species (primates of the superfamily Ceboidea, of Central and South America),

OW: Old World species (primates of the superfamily Cercopithecoidea, originating from Africa, the

Arabian Peninsula, and Asia).

The EU distinguishes different categories of research areas were animal experiments

may be conducted, i.e., 1) basic research, 2) translational and applied research, 3)

regulatory use and routine production and 4) higher education or training for the

acquisition, maintenance or improvement of vocational skills. These categories are

included in the forms for Member States to communicate data on use of experimental

animals. Table 2 illustrates the distribution of NHP testing in relation to these categories

Revised requirements for the reporting of statistical data on the use of animals in the EU in Annex II of

Commission Implementing Decision 2012/707/EU http://eur-lex.europa.eu/legal-

content/EN/TXT/?uri=CELEX:02012D0707-20140115

Non-human primates testing (update 2017)

Final Opinion

which are reported by most Member States. Regulatory use and routine production is the

category with the most NHPs used. With 74.5% of all NHP uses (6196) this category is of

high importance regarding the application of the 3Rs.

Table 2: Distribution of NHP testing in relation to the different purposes (2014)

as published by the majority of Member States having used NHPs

Basic research

Translational and

applied research

Regulatory use and

Routine production

Higher education or

training for the

acquisition,

maintenance or

improvement of

vocational skills

Total

(1)

(2)

(3)

(4)

Prosimians

201

203

Cercopithe

coidea

(OW)

Cynomolgus

monkey

155

744

5713

6640

Rhesus monkey

233

182

101

480

Vervets,

Chlorocebus spp.

Baboons

134

151

Other species of

OW monkeys

Ceboidea

(NW)

Marmosets and

tamarins

314

368

743

Squirrel monkey

Other species of

NW monkeys

Total

1074

1012

6196

8314

Additional data as well as statistical analysis on the use of NHPs like breakdowns e.g. by

primary purposes, specific research area or severity of treatment vary among the

Member State reports. In addition, 2014 was the first year for reporting under a new

format. Therefore no further statistics could be carried out by the SCHEER to provide

reliable further information or trends regarding the use of NHPs. A collation of the

Member State reports on NHP use including comments on comprehensiveness of the

reporting and the quality of the statistics that were published by Taylor and Rego

(2016).

Uses were part of projects that were authorised before 1.1.2013; 18 uses were incorrectly reported (correct

category should have been regulatory use as these concerned training of the animals in regulatory procedures)

Non-human primates testing (update 2017)

Final Opinion

For the sake of transparency and monitoring progress in the application of the 3Rs, it

would be desirable that all Member States report the same type of information and that

included categories would include the level of severity of experimental procedures,

specific research area, origin of animals, generation and first time use (see e.g., Home

Office, Annual Statistics of Scientific Procedures on Living Animals – Great Britain, 2014).

6.6.2 Development and safety testing of pharmaceuticals and medical

devices

6.6.2.1 Introduction

The focus in this section is on pharmaceuticals and medical devices because for all other

products testing on NHPs is generally not permitted. In addition, the use of NHPs is only

permitted in those biomedical areas essential for the benefit of human beings, for which

no other alternative replacement methods are yet available. A report commissioned by

several major UK funding agencies argued that evaluation of any proposed primate

research project should actually consider four factors: quality and importance of the

science, likelihood of medical or other public benefit, likelihood of animal suffering, and

availability of alternatives (Bateson, 2011).

In 2012, the EC adopted a package of measures on innovation in health that included

proposals to revise existing legislation on general medical devices and in vitro diagnostic

medical devices. The revisions affected all kinds of medical devices. Any animal model

selected should be generally accepted for the study of the device type and there should

be a reasonable amount of scientific evidence that the animal model has utility for the

intended study of the device type.

Currently, animal research is viewed as an essential part of the drug development

process. In its report, “Working to reduce the use of animals in scientific research” (UK

Government, 2014), the UK Government states: “over the past twenty years, the

development of monoclonal antibody therapies has completely transformed the ability to

treat diseases, including cancers, rheumatoid arthritis and multiple sclerosis, and the

development of this technology would not have been possible without the use of

animals, including nonhuman primates (NHPs)”.

The development of a pharmaceutical is a stepwise process involving an evaluation of

animal and human efficacy and safety information. The nonclinical safety testing of new

medicines is conducted according to international regulatory testing requirements,

frequently in accordance with guidelines agreed in meetings under the auspices of the

International Council for Harmonisation (ICH), formerly known as the International

Conference on Harmonisation. The ICH’s mission is to make recommendations towards

achieving greater harmonisation in the interpretation and application of technical

guidelines and requirements for pharmaceutical product registration, thereby reducing or

obviating duplication of testing carried out during the research and development of new

human medicines. The goals of the nonclinical safety evaluation generally include a

characterisation of toxic effects with respect to target organs, dose dependence,

relationship to exposure, and, when appropriate, potential reversibility. Inclusion of a

non-rodent species is required in the safety assessment of pharmaceuticals, see ICH

guidelines ICH M3 (R2), 2009; ICH S9, 2009 and ICH S6 (R1), 2011.

Testing the safety of medical devices, such as orthopaedic joint replacement, in animals

Non-human primates testing (update 2017)

Final Opinion

such as NHPs before they are introduced in human clinical trials is required for both

ethical and legal reasons. Nonclinical safety testing, however, also includes many

improvements in technique, in vitro screening of devices, and investigator understanding

of study model selection. These factors all enhance the ability to predict the safety and

performance of medical devices in humans, as well as reducing the number of animals

required for testing. It should be noted, however, that very few medical devices, e.g.,

orthopaedic devices, need to be tested in NHPs.

6.6.2.2 Progress Made In the Last 10 Years

Several alternatives for animal model testing are proposed every year (see Doke and

Dhawale, 2015 for a review), including the increasing use of human tissues (Holmes,

Bonner and Jones, 2015; Strauss and Blinova, 2017).

Bowes et al. (2012) pointed out that in vitro pharmacological profiling is increasingly

being used earlier in the drug discovery process to identify undesirable off-target activity

profiles that could hinder or halt the development of candidate drugs or even lead to

market withdrawal if discovered after a drug is approved. In addition, the utility of

animal models of disease for assessing the safety of novel therapeutic modalities has

become an increasingly important topic of discussion as research and development

efforts focus on improving the predictive value of animal studies to support accelerated

clinical development (Graham and Prescott, 2015; Cavagnaro and Silva Lima, 2016).

Scientists have also been conducting extensive reviews to challenge the existing testing

paradigms outlined in regulatory guidelines (Galijatovic-Idrizbegovic et al., 2016).

It is also recognised that in some cases earlier access to human data can provide

improved insight into human physiology/pharmacology, knowledge of drug candidate

characteristics and therapeutic target relevance to disease. Streamlined early

exploratory approaches can accomplish this end and involve limited human exposure,

with no therapeutic intent, see ICH M3 (R2). Such studies can be used to investigate a

variety of parameters such as PK, PD and other biomarkers often identified by means of

in vitro studies, which could include PET receptor binding and displacement or other

diagnostic measures. Although still needing nonclinical studies to be conducted before

initiating such clinical trials, use of any of these approaches can reduce overall animal

use in drug development.

There has also been considerable progress made over the last few years, both in terms

of reducing the numbers of NHPs required for the safety assessment of medicinal

products and in the husbandry practices (Baldrick, 2011, Beaumont et al., 2011; Buckley

et al., 2011; Chapman et al., 2012; Chapman et al., 2013; van Meer and Schellekens,

2015; Backes et al., 2016 and Chapman et al., 2016).

The value of a second species in regulatory toxicology studies is beginning to be

questioned more generally. For example, the NC3Rs and ABPI has launched an initiative

to investigate whether data from one species could be sufficient for the progression of a

potential new drug in human clinical trials (Monticello, 2015; Mangipudy et al., 2014).

The addendum to the ICH S6 (R1) guideline, which specifically deals with biotechnology

derived medicines where use of NHP is widespread, was written with an aim to reduce

the use of animals in accordance with the 3Rs principles. The guideline also emphasises

“rodent species should be considered unless there is a scientific rationale for using non-

rodents”. The guideline also states that when no relevant species can be identified

Non-human primates testing (update 2017)

Final Opinion

because the biopharmaceutical does not interact with the orthologous target in any

species, use of homologous molecules or transgenic models can be considered. In

addition, it is considered to be justified to use one species for all general toxicity studies

when the clinical candidate is pharmacologically active in only one species. In these

cases, studies in a second species with a homologous product are not considered to add

further value for risk assessment and are not recommended. This revision to the

guideline is expected to lead to a decrease in NHP numbers used in this area.

The EU Guideline on similar biological medicinal products containing monoclonal

antibodies – non-clinical and clinical issues (CHMP 2010), which came into effect on 1

December 2012, states “the conduct of toxicological studies in NHPs is usually not

recommended”. The background and consequence of this paradigm shift was discussed

by van Aerts et al. (2014). A further call for the waiving of animal studies on a global

basis was made in a paper by Chapman et al. (2016).

Over the past few decades, many improvements have been implemented in animal

studies on medical devices. These improvements have effectively reduced the number of

animals used, while enhancing the effectiveness of the overall assessment of nonclinical

safety. The development of established and reproducible animal models to mimic the

human condition has resulted in the site-specific implantation of devices in the

anatomical positions in which the device would likely be used. Improved understanding

of these models, relative to animal versus device interactions, has resulted in a more

accurate assessment of the device.

6.6.2.3 Justification for NHP Use

Arguments against phasing out NHPs in safety testing of pharmaceuticals are therefore

similar in many ways to those regarding using rodents for toxicity testing, i.e.

incomplete knowledge of integrated body systems and pathophysiology, poor

representation of pharmacokinetics by in vitro systems (SCHER, 2005).

Safety testing of pharmaceuticals is intended to safeguard human subjects used in

clinical trials through risk assessment based on the results of all nonclinical studies,

including those from animal experiments. The Declaration of Helsinki

is a set of ethical

principles developed by the World Medical Association (WMA) for the medical community

regarding human experimentation. It states that the wellbeing of the human subject

should take precedence over the interests of science and society.

The ICH safety guidelines are written to ensure that duplication of studies is not required

for various regions in the world. These guidelines also indicate that the nonclinical

studies should be performed in “relevant species”, and that pivotal studies for risk

assessment of pharmaceuticals, such as the repeated dose toxicity testing, usually have

to be performed in two species, one of which must be a non-rodent.

1 Adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, and amended by the:

29th WMA General Assembly, Tokyo, Japan, October 1975

35th WMA General Assembly, Venice, Italy, October 1983

41st WMA General Assembly, Hong Kong, September 1989

48th WMA General Assembly, Somerset West, Republic of South Africa, October 1996

52nd WMA General Assembly, Edinburgh, Scotland, October 2000

53th WMA General Assembly, Washington 2002 (Note of Clarification on paragraph 29 added)

55th WMA General Assembly, Tokyo 2004 (Note of Clarification on Paragraph 30 added)

59th WMA General Assembly, Seoul, October 2008

Non-human primates testing (update 2017)

Final Opinion

The use of a non-rodent species for the characterisation of new medicinal products aims

at limiting the uncertainty in the extrapolation process from in vitro and animal toxicity

data to the human situation. In Europe, dogs are most frequently used as the non-

rodent species, and NHPs are only used when no other species showed the same primary

pharmacodynamic response. NHPs make up less than 0.1% of the animals used in

research. However, they can play a key role in drug development due to their similarity

to humans with regard to sensory organs, hormonal systems, reproduction, immune

system etc., to evaluate efficacy and safety, especially for biopharmaceutical compounds

(Brennan et al., 2015). Species specificity can be crucial for some new medicines.

Nevertheless, the species for toxicity testing should be selected based on their

similarities to humans with regard to pharmacology and pharmacokinetics, including

biotransformation and in certain cases also where anatomical similarities are essential. A

scientific justification for the choice of non-rodent species used is a regulatory

requirement.

There still exist many “pharmaceutical gaps” for various diseases, diagnostics, and

conditions, as outlined in the 2013 Report “Priority Medicines for Europe and the World”

(Kaplan et al., 2013). This is an update to the original 2004 report and provides a public-

health-based medicines development agenda, taking into account increasing life

expectancy and related population aging. Research is considered essential for medical

progress, and the report outlines various key findings and recommendations. The EU is

pushing “for the translation of basic research into therapies”, a transition that often

requires the testing of new medicines in primates, as the most relevant nonclinical

species. However, restrictions on primate experiments could hinder the development of

new medicines (Abbott, 2014).

For some disease indications, specific NHP models were developed which successfully

mimic the disease in humans, e.g., for Graves’ disease (Wang et al., 2013).

The European Committee for Medicinal Products for Human Use (CHMP) has defined

criteria on the demonstration of relevance of an animal species to predict human safety

in their Guideline on Strategies to Identify and Mitigate Risks for First-in Human Clinical

Trials with Investigational Medicinal Products (EMEA/CHMP/SWP/ 28367/07), which is

currently being revised. The guideline states that scientific requirements specific to the

substance include:

 Presence of the required pharmacodynamic (PD) binding site and response

 Similarity to human toxicity or pharmacokinetic (PK) profile based on in vitro data or

prior experience with related compound(s) of the same class

 Similarity to human in aspects of anatomy or physiology of specific organ systems

 Indication for the need of an additional species to investigate a toxic effect or the

effects of a significant metabolite in humans which is not produced in the original

non-rodent species

The Association of British Pharmaceutical Industry (ABPI) and the UK’s Home Office

(2002) also gave the following specific recommendations on the selection and

justification of the relevance of an animal species for safety testing:

 Use of a well characterised species may be quicker and require fewer animals

 Unknown and contradictory neurophysiological sensitivity (meant to reflect

differences in suffering, harm, etc.) of the species (e.g., dog vs. pig)

 Public perception (e.g., dogs and other pets)

Non-human primates testing (update 2017)

Final Opinion

 Availability of new pharmaceuticals in limited quantities in the early stages of

development, requiring initial assessments in animals of small body size

According to all these recommendations, NHPs should only be used when it is

scientifically demonstrated that none of the other non-rodent species commonly used in

safety testing is appropriate for the purpose of the study. It is also important to consider

the limitations of the NHP when choosing which species to use in drug safety test. For

example, a recent study raises significant concern about the scientific validity to humans

of drug safety studies undertaken in NHPs. Specifically, the cynomolgus monkey has

been found to be remarkably resistant to liver injury caused by paracetamol (Yu et al.,

2014). This finding is important for numerous reasons, including the fact that

cynomolgus monkeys are the NHPs used most commonly to assess drug safety.

There are in vitro systems in development that could negate the need for NHPs and

indeed other animals (see Replacement Possibilities section below).

The following examples illustrate areas of research, where therapeutic development and

safety testing in the NHP may be preferred over that in other mammalian species and

also reflect on the limitations of the model in some cases.

Menstrual Cycle: Due to the similar menstrual cycle and the anatomy and physiology of

the mammary gland of NHP females and human females, NHPs (cynomolgus monkeys)

are the more pertinent species in terms of the prediction of relevant reproductive effects

(Buse et al., 2003; Cline, 2007; Luetjens et al., 2005) and are, therefore, often chosen

as the non-rodent species for classes of compounds that are expected to provoke effects

on the female genital organs. Recently, Buse et al. (2014) have provided a good review

of available in vitro and animal models of the placenta for toxicological purposes. This

includes highlighting the limitations of the cynomolgus monkey model as well as its

suitability. It gives details of some NHP species, which should not be used. It also

suggests other models that could and may be preferable to use in place of NHPs in given

circumstances such as rodents, pigs, bovine and horses. In addition, a new study raises

the possibility that a new rodent model, the spiny mouse (Nowogrodzki, 2016), could be

used as a future replacement for NHP menstrual-related study (see replacement

possibilities section below).

Vomiting and Nausea: NHPs are less susceptible to vomiting than dogs. Thus,

pharmaceuticals with an emetic effect in the dog may be tested in the monkey (Weber,

2005). Vomiting does not only limit exposure of the pharmaceutical administered, but is

also a major hurdle to accurately characterise early effects on behaviour and on the

cardiovascular system. However, a review by Holmes et al. (2009) cautions that it

should not be assumed that an NHP is necessarily the best predictor for emetic liability in

humans as it can depend on the emetic challenge. The paper discusses opportunities for

the replacement of animals in the study of nausea and vomiting and includes

suggestions that are relevant to replacing and refining NHP studies in this area.

Blood Coagulation: The coagulation system of NHP is more similar to humans than that

of any other species (Abildgaard et al., 1971; Lewis, 1996) and thus, NHP are often the

most suited model for humans to assess potential toxicity of coagulation factors and

anti-coagulation agents.

Biopharmaceuticals: NHPs are the most appropriate animals to characterise safety of

many biotechnology-derived pharmaceuticals, especially monoclonal antibodies (mAbs),

Non-human primates testing (update 2017)

Final Opinion

since the most relevant species for testing is selected based on species-specific aspects

of the immune system. Monoclonal antibodies are highly specific to their targets and

accurate prediction of ‘on-target’ effects requires testing in a species that shows cross-

reactivity, thus frequently requiring testing in NHPs as the only species cross-reacting

with humanised monoclonal antibodies (APBI-NC3Rs, 2006; van Meer et al., 2015).

However, an Expert Working Group convened by the National Centre for Replacement,

Refinement and Reduction (NC3Rs) in the UK cautioned that:

‘The use of the NHP plays an important role in assessing the safety of mAbs.

Nevertheless, the close relatedness of the NHP to man does not necessarily guarantee

that they are the most appropriate species for the development of mAbs as illustrated

by the TGN1412 clinical trial in 2006 where the NHP had limited utility in detecting the

cytokine storm subsequently observed in the phase 1 clinical trials. Species selection

should be based on the biology of the mAb and in particular its pharmacological

activity. The NHP should not be used as ‘default’ species, as a screen or because NHP

studies have previously been conducted.’ (Chapman et al., 2007, Chapman et al.,

2016).

The report of the NC3Rs Working Group suggests strategies for the scientific and

regulatory community to implement to minimise NHP use and increase the efficiency of

mAb development (See Reduction and Refinement Possibilities section below).

An ongoing challenge in the development of biopharmaceuticals is preventing

immunogenicity, which can lead to side effects and more commonly an immune

response that causes loss of efficacy as the body develops neutralising or clearing

antidrug antibodies (van Meer et al., 2015). Although NHPs are often the only relevant

animal model for the development of mAbs, van Meer and colleagues’ study (van Meer

et al., 2015) suggests that the immunogenic response in NHPs is poorly predictive of the

response in humans. This finding strengthens the need to develop innovative alternative

models to ensure progress in this important therapeutic research area.

Central nervous system (CNS) pharmaceuticals: Assessing novel CNS

pharmaceuticals may further increase the need for testing in NHPs (Vuillemenot et al.,

2016). While the rat is in principle acceptable for self-administration studies in the EU,

NHPs are preferred in Japan.

Reproductive Toxicity: Historically, non-primate species have been used for

reproductive toxicity studies, generally mice, rats and rabbits. However, rodents and

rabbits are not necessarily the most accurate predictor of teratogenicity or reproductive

toxicity in humans due to differences in placental anatomy and number of fetuses. In

addition, they are not suitable models for all aspects of human reproductive toxicity,

specifically for the investigation of agents suspected or known to interfere with the

menstrual cycle. In such cases, NHPs may be more predictive for human toxicity. The

male cynomolgus is also a good model of male fertility in specific cases (Ehmcke et al.,

2006; Millar et al., 2000). Rodents can also not be used to assess the safety of novel

hormonal intrauterine devices or cognitive dysfunction associated with the menopause

(Schlatt et al., 2008; Wistuba and Schlatt, 2002). However, there is a strong impetus

within the scientific community to move away from animal-based reproductive and

developmental toxicity testing (Stallman Brown et al., 2012). This has resulted in

progress in the development of non-animal alternatives to move toward a predictive

mechanism-based approach.

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Other challenges for the development of new medicines are the novelty of the molecules

that are currently under development, the unique delivery systems, use of alternative

routes of administration, long half-life and mismatch between pharmacokinetics and

pharmacodynamics.

6.6.2.4 Replacement possibilities

Replacing NHP models in new medicine development and safety testing has the potential

to significantly reduce the number of NHPs used in European laboratories, as regulatory

use and routine production are consistently the areas of greatest NHP use, as discussed

above. Substantial progress has been made in the development of new in vitro and in

silico methods and integrated testing strategies have been proposed by OECD

and

ECVAM

which have the potential to reduce, refine and ideally replace the need for NHPs

and indeed other animal studies in the near future, based on the identification of AOPs

(Adverse Outcome Pathways) through the integration of Mode of Action, kinetics and

dynamics. The examples given include substituting NHPs for other species, which is not

without ethical considerations, but can be viewed in the context of progressing toward

the ideal position of complete replacement with non-animal alternatives.

Liver Injury and Testing for Idiosyncratic Adverse Drug Reactions (IADRs) in

Humans: Thompson et al., (2012) evaluated an in vitro approach, which explored both

cellular effects and covalent binding to assess IADR risks for drug candidates using 36

drugs which caused different patterns and severities of IADRs in humans. They propose

that this integrated in vitro panel of five assays approach has the potential to enable

selection of drug candidates with reduced propensity to cause IADRs in humans. Shoda

et al., (2014) describe the DILIsym® software, a mechanistic model of drug-induced

liver injury (DILI) and how its development will improve the rationale design of new

drugs. DILIsym® simulates the mechanistic interactions and events from compound

administration through the progression of liver injury and regeneration. Modelling

mitochondrial toxicity illustrates the type and use of in vitro data to represent biological

interactions, as well as insights on key differences between in vitro and in vivo

conditions.

Menstrual Cycle and Associated Disorders: Advances in research relating to

menstruation and associated disorders such as endometriosis and pre-menstrual

syndrome, have been hampered by the lack of an appropriate model. The ideal model

would be a small species with a short reproductive cycle and similar placental form, but

traditionally the species most resembling humans has been cynomolgus monkeys (Buse

et al., 2014). However, very recently evidence that the spiny mouse (Acomys cahirinus)

is the first rodent species known to menstruate indicates that it could provide an

unprecedented natural non-NHP model to study the mechanisms of menstrual shredding

and repair and may be useful in further understanding human-specific menstrual and

pregnancy associated disease (Bellofiore et al., 2016). This is new research and further

studies will be required to check the validity of this rodent as a model for the human

condition, but it does hold great promise as a future alternative to NHPs.

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clanguage=en

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ategy.pdf

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Swine Models: There is an expanding body of research that supports the use of swine

models (especially mini-pig strains) as an alternative to the use of dogs and NHPs as the

choice of non-rodent species in nonclinical toxicology testing of pharmaceuticals. Swine

models share similarities in anatomical and physiological characteristics with humans

such as the cardiovascular, urinary, integumentary and digestive systems (Swindle et

al., 2012). The pig has been particularly useful in treatment of atherosclerosis and

myocardial studies. The pig and mini-pig are increasingly being used as toxicological

models in safety assessment for reproductive toxicology, development of paediatric

pharmaceuticals and routine toxicology (Swindle et al., 2012).

In addition, Forster et al., (2010) concluded that the mini-pig is well placed to meet the

challenges of emerging technologies and the toxicology of the future due to the

sequence homology between pigs and humans, the potential to perform toxicogenomic

studies, and because the mini-pig is the only non-rodent toxicology model where

transgenic animals can be readily generated and where reproductive technologies are

well developed. Ellegaard et al., 2010 concluded that it is easier to keep mini-pigs to a

good standard of welfare under laboratory conditions than it is for NHPs or dogs.

6.6.2.5 Reduction and refinement possibilities

Where replacement of NHPs is not yet possible, there are several opportunities to reduce

the numbers required in this area of research and to refine the procedures involved,

which are outlined here.

Biopharmaceuticals: To investigate evidence-based opportunities to minimise the use

of NHPs in the development of therapeutic monoclonal antibodies, an expert working

group was convened by the NC3Rs. The report of the working group (Chapman et al.,

2007) and subsequent papers (e.g., Chapman et al., 2012, 2016) provide detailed

strategies and approaches that can be adopted to ensure that NHP studies are minimised

and when they are necessary are efficient and effective, such as using homologous

proteins and appropriate genetically altered rodents, and designing the programme of

work to optimise the study duration, dose regimes and number of groups. The efficient

study designs identified by the NC3Rs-industry data sharing enable the number of NHPs

per mAb in development to be reduced by half. Other opportunities for reduction and

refinement identified by the NC3Rs working in collaboration with industry and regulatory

bodies internationally include: avoiding the use of NHPs for candidate selection (Lave et

al., 2009, Beaumont et al., 2011), reducing the number of recovery animals on

toxicology studies (Sewell et al., 2014), group housing during cardiovascular telemetry

recordings on toxicology and safety pharmacology studies (Prior et al., 2016), reducing

the upper limits of body weight loss in short term toxicology studies (Chapman et al.,

2013), and use of micro-samples to assess drug exposure in blood, plasma and/or

serum (Chapman et al., 2014a,b).

Microdosing: New applications of human microdosing studies to areas beyond

exploratory pharmacodynamics and biomarkers, which could include PET receptor

binding and displacement or other diagnostic measures data, could potentially reduce

the number of unsuccessful candidate drugs from progressing into the full developmental

process and as a consequence reduce the number of nonclinical animal tests. Lappin et

al., 2013 and Burt et al., 2016 describe how the microdosing technique has been

extended to assess the magnitude of potential drug-drug interactions and early

indications of metabolic profiling of a drug. The latter could lead to improved prediction

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Final Opinion

of drug clearance in humans. Therefore, there is greater potential for the so far limited

application of microdosing to be improved and its utility widely expanded to the benefit

of animal models, which could include NHPs.

Patient derived induced pluripotent stem cells (iPSCs): Recently, Giri and Bader

(2015) argued that iPSCs can be used as an in vitro clinical trial and as a consequence

can provide an opportunity within the drug development process to assess human

disease pathology without the need for animal models. Giri and Bader (2015) discuss the

utility of iPSC methods, the technical challenges and how they can be further developed

including comments on the infrastructure needed to ensure widespread use. iPSC

technology enables exploration of disease mechanisms of normal healthy and diseased

cells in vitro. This provides clues to novel therapeutic molecular targets that are directly

relevant to humans. Once the targets have been identified, iPSCs can be used to screen

potential drug candidates to predict their effectiveness in terms of toxicity, dosage and

individual human susceptibility. Giri and Bader (2015) also note that such technology

could be used to modify the conventional drug discovery process to ensure improved

relevance to human disease and to produce high-quality efficacious drugs. Therefore,

iPSCs have the potential to reduce the number of animal-based preclinical tests and

even replace some of them.

6.6.2.6 Identification of specific research areas

This section provides information on areas of research that have the potential to require

increased NHP use in the future or where there is great potential to develop alternative

models and methods to replace current NHP use.

Debilitating Disease: There are a number of examples for major new treatment

options for debilitating diseases where NHPs have been used in the safety assessment as

the best available model for humans due to close similarities in physiology and anatomy

that can be found in the European Public Assessment Reports (EPARs) on the European

Medicines Agency (EMA) website

. This is probably a reflection of an increased number

of biotechnology-derived medicines being developed for indications such as rheumatoid

arthritis and cancer. Biotechnology derived medicines have tended to use NHPs as a

default non-rodent species, although this is being questioned (Baumann et al., 2014;

Backes et al., 2016).

Cognitive Processes: Brain disorders are among the largest cause of disease burden

globally, affecting millions of people and imposing enormous societal and economic

costs. Many of these disorders are chronic and incurable conditions for which existing

treatment options are inadequate and in some cases almost completely ineffective. One

specific challenge in modelling brain disorders is that many of the clinical symptoms

involve higher cognitive functions that are controlled by the prefrontal cortex (PFC),

which is much less developed in rodents than in primates

(Kaas, 2013) and thus, NHPs

are generally considered the most suitable experimental animals for research on higher-

level cognitive and behavioural processes (Giarola, 2015).

Abuse Potential: New drugs must be evaluated for “actual or relative potential for

abuse” (CHMP, 2006; FDA 2010). Abuse potential can be determined in multiple ways,

most frequently using drug discrimination and drug self-administration. Animals,

http://www.ema.europa.eu

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Final Opinion

primarily rats and monkeys, self-administer most of the drugs humans abuse and, in

many cases, the use of rodent and monkey models provides similar information. While

the rat is, in principle, acceptable for self-administration studies in the EU, NHPs are

preferred in Japan. Working primarily with Pfizer, the NC3Rs built a case to use the rat,

supporting a change in ICH M3 to accept rodent instead of NHP data (O’Connor et al.,

2011).

There are cases, however, when species differences emerge and it is advantageous to

use NHPs for these assessments. There are differences in brain anatomy between

rodents and primates (including NHPs) that make NHPs more suitable for translational

longitudinal neuroimaging studies (Howell and Murnane, 2011; Murnane and Howell,

2011; Nader and Banks, 2014). As well as potential for abuse of new drugs, the

regulatory uncertainty in industry about the testing and safety of e-cigarettes could lead

to the use of NHPs in inhalation and addiction studies, but Combes and Balls, (2015a;

2015b) present an extensive assessment of how these new tobacco-related products

could be tested using mainly cutting edge in vitro techniques and human clinical studies.

Prenatal Drug Exposure: Another advantage of the use of NHPs in new medicine

development includes the study of prenatal drug exposure (Gill et al., 2012; Soto et al.,

2012). Macaques develop at a similar rate to humans during the embryonic period, but

at an accelerated rate thereafter. Skeletal growth and maturation is a continuous

process that occurs during the pre and postnatal period through to adult. Therefore,

disruptions in this process at any time during development could potentially affect

growth. However, disruptions in the prenatal and early postnatal periods are likely to

have the most notable effects since this is when the skeletal structures are being formed

and the ossification centres appear. The choice of species is, however, driven by the

same considerations as for general safety assessment in that only relevant species

should be used, meaning that NHPs are used most often for biotechnology derived

medicines.

Paediatric Medicine: Guidance documents on the safety assessment of medicines for a

paediatric population are available (FDA 2006, CHMP 2008). A harmonised ICH guideline

on the Nonclinical Safety Testing in Support of Development of Paediatric Medicines (ICH

S11) is currently in development. There is agreement that juvenile toxicity testing

should be considered if the available nonclinical and clinical data are not sufficiently

comprehensive to support the intended paediatric clinical trials. This has led, in some

instances, for the conduct of safety assessment in juvenile animals across several

species. Mostly these studies are performed using rodent models (rats and mice), but in

a number of cases, NHP models have also been used. For those organs that continue to

develop post-birth, the most rapid maturation period is during the first 3 to 6 months in

macaques and this corresponds developmentally to the first 2 to 8 years in humans. The

choice of species is basically driven by the same considerations as for general safety

assessment in that only relevant species should be used (Baldrick 2013, Barrow et al.,

2013).

The timing of the various postnatal phases is obviously different across species (rodents,

dog, mini-pig, NHPs) and these differences should be considered for study planning

and/if the test item targets a particular organ system (Baldrick, 2013). For NHPs, there

is a lack of accepted and consistent definition of the various postnatal development

phases. However, efforts have been made to provide this information for the cynomolgus

monkey (Morford et al., 2011; Weinbauer et al., 2011).

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Final Opinion

Vomiting and Nausea: Holmes et al. (2009) note that the use of animal models,

including NHPs, for establishing emetic liability is an issue for discussion as robust data

on their predictive value are lacking. They argue that there are scientific and ethical

drivers for applying 3Rs to this area. They suggest that alternatives to animal models in

nausea and vomiting research have not been fully explored due to the complexity and

multisystem reflexes involved. The paper calls for experts to come together and examine

opportunities for replacement and to inspire innovation in 3Rs implementation in this

field of research. Some of the key avenues of research that they suggest should be

explored are;

 Invertebrates and other lower organisms – have the potential to act as screening to

identify emetic potential early in the drug development process. Assessing the utility

of these models is potentially quick and validating them against existing clinical and

pre-clinical data will be essential.

 Pharmacogenomics – microarray based gene expression profiling could offer

researchers the opportunity to identify emetic liability of novel compounds in vitro.

 In vitro approaches – tissue models of enteroendocrine cells from the gastrointestinal

tract developed for investigating neurotransmitter responses to mechanical forces

and dietary components could potentially be applied to nausea and vomiting. Further,

techniques such as isolated abdominal vagal grease gap preparation could be used to

assess emetic liability.

Holmes et al., (2009) also provide a useful stepwise strategy incorporating these

approaches that will help to reduce and refine animal use, including NHPs, in this area.

Reproductive Toxicity: There has been progress in a move toward a non-animal

predictive mechanism-based approach but there are still obstacles to overcome in terms

of regulatory acceptance and scientific validity. This is an area where resources and

collaborative effort should be focused as there is significant scope for replacing NHPs and

other animals. A report (Stallman Brown et al., 2012) of a 2012 FDA Workshop to

discuss emerging in vitro tools for predicting reproductive and developmental toxicity

focuses on the ability of in vitro test methods to predict in vivo outcomes and the

potential to incorporate these new methods into regulatory decision making. The report

highlighted emerging methods such as whole embryo culture and embryonic stem cell

tests, zebrafish and metabolomics. It also highlights several collaborative efforts to

develop and validate in vitro assays and what strategies are need to apply such assays

to regulatory decision making.

In addition, when considering toxicity testing more generally the recent findings of Bailey

et al. (2015) indicate that tests results inferring an absence of toxicity provide no

evidential weight of an absence of toxicity in another species even when data from NHPs

and humans are compared. The paper strongly argues that animal tests including those

involving NHPs have very little value in predicting human toxicity and that human-

focused alternatives need to be adopted.

Transgenic Techniques: Transgenic macaques were first reported 15 years ago (Chan

et al., 2001) and an overexpression model of Huntington's disease was described in

2008 (Yang et al., 2008). Despite these advances, however, the widespread adoption of

transgenic primate models appeared impractical until recently, given the paucity of

methods for making precise genetic changes in primate embryos. Recent developments

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Final Opinion

in transgenic techniques such as CRISPR/Cas9 and TALENS have now led to calls for the

creation of lines of transgenic NHPs and arguments for their necessity to biomedical

science (Jennings et al., 2016; Scaduto, 2016).

Basic and translational neuroscience has made, and will continue to make, great

progress by studying mice and other simpler organisms. Much can also be learned by

studying humans directly, and technological advances in areas such as neuroimaging,

genomics and induced pluripotent stem (iPSC) cells are allowing human disease

researchers to address questions that were previously restricted to experimental

animals. NHP research will in no way replace these approaches, and, for both ethical and

practical reasons, primate genetic models should only be considered where other

alternatives are not available.

In addition, this is an area of research where some argue that the moral and welfare

arguments vastly outweigh the potential benefits of any such use (Coors et al., 2010;

Combes and Balls, 2014; Bateson and Ragan, 2014). It is therefore important to

carefully consider when, why and how such projects should be attempted.

When considering these ethical issues, it is useful to distinguish between different types

of transgenic studies. Many questions about brain function can be addressed in mouse

transgenic knock-in lines expressing genetically encoded reporters or effectors under the

control of an endogenous promoter, allowing monitoring and manipulation of activity in

specific subsets of cells. The development of primate genetic models, if deemed

essential, will be an international effort, involving many countries with different cultural

traditions and public attitudes toward animal research. It will be important to establish

shared standards and regulations and to assure all stakeholders that work is performed

to the highest standards of animal welfare regardless of where it is conducted.

Finally, there is an ethical obligation to use animal resources wisely, minimising

unnecessary duplication of effort and maximising the benefit obtained from each animal

by sharing data and (where applicable) cell and tissue samples. This applies to all animal

research, but it is especially so for NHP research given the high costs, the long lead

times and the need to minimise the numbers of animals used. Achieving this will require

coordination at national and international levels.

6.6.3 Treatment and prevention of infectious diseases

6.6.3.1 Introduction

Despite the development of various advanced in vitro, in vivo and ex vivo techniques,

the pathophysiological aspects responsible for morbidity and mortality of many infectious

diseases are still largely unknown. Therefore, the use of live models to study biological

aspects of infectious diseases and to develop new therapies and vaccines remains

indispensable. By definition, the most predictive model to study a disease is in the target

species itself. However, safety and ethical barriers to study infectious diseases in

humans limit the use of most human challenge models and/or patients. In general,

clinicians only see patients when the disease is in an advanced stage and treatment is

required. Although important data can be obtained from patient studies, both practical

and ethical issues limit the in depth analysis of host-pathogen interactions. Animal

studies are, therefore, important to study certain aspects of infectious diseases in depth.

Furthermore, it is important that these models mimic the various responses in humans

as much as possible including immunological and pathological responses. Such models

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Final Opinion

allow studies on safety, immunogenicity and efficacy of new vaccines and/or drugs and

also provide possibilities for relevant research on host-pathogen interactions and the

interplay with the host immune system. In some cases, NHPs provide the best, or even

only, models to study these aspects in infectious disease research. Over the last

decades, NHPs have been instrumental in gaining an understanding of the pathogenesis

of various infectious diseases and have provided relevant models to develop new

therapies, e.g., development of vaccines against polio, yellow fever, Hepatitis B and

Ebola, identification of the causative agents of infectious diseases such as SARS, typhoid

fever and mumps, and more in-depth understanding of infections such as HIV (e.g.,

Cong et al., 2016; Curtis et al., 2015; Fouchier et al., 2003; Garcia-Tellez et al., 2016;

Kaushal et al., 2012; Martinez et al., 2015; Mason et al., 1973; Prince and Brotman,

2001; Racaniello, 2006; Rivera-Hernandez et al., 2014; Shedlock et al., 2009, Pena and

Ho, 2015; Scanga and Flynn, 2014).

6.6.3.2 Justification for the use of NHP in Infectious Disease

Research

The selection of an animal model in infectious disease research depends on the specific

scientific questions. Macaques, Syrian gold hamsters and mice may be used to study

yellow fever and are associated advantages and disadvantages (Julander, 2016). In

contrast to the other models, however, macaque models have a disease pattern similar

to humans and clinical isolates can be used. Therefore, NHP models are used to study

the whole array of disease manifestations and optimal protection, which is the case for a

variety of other infectious diseases. To select NHPs as an animal model, the course of

the disease should: 1) resemble human disease with a comparable host range of cells,

organs or tissues involved and 2) reflect the host response in humans as much as

possible. Additional reasons for selecting NHPs include 1) the target of a potential drug

or vaccine is only present in NHPs and humans (‘t Hart, 2015) or 2) NHPs are natural

hosts for the pathogen (Ploquin et al., 2016).

6.6.3.3 Progress made in the last 10 years

During the past 10 years, there have been many developments in the infectious disease

and vaccine research field. There have been striking improvements in in-depth analysis

of various aspects during infections with the aim to select biomarkers and/or vaccine

candidates. Development of –omics technologies such as genomics and proteomics, and

new cell culture techniques and development of organoïds have helped the

downselection of vaccine candidates for preclinical studies. Rapidly developing imaging

techniques and telemetry in infectious disease research provide more data from animal

studies and thus, reduce animal numbers in experiments. Human challenge models and

clinical data are additional developments in infectious disease and therapy research as

well as the development of humanised mice and non-mammal animal models. Taken

together, new approaches lead to reduction, refinement and replacement of NHP models.

One major advance has been the full replacement of NHPs for testing of the

neurovirulence of polio vaccines (Dragunsky et al., 2003).

Replacement

For replacement, controlled human challenge models were developed and implemented

for typhoid, Plasmodium falciparum malaria (Pollard. et al., 2012) and transmission

studies with specific influenza strains (Killingley et al., 2012) as well as the human

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Final Opinion

attenuated TB challenge model, which is currently in development for vaccine

development (Kaufmann et al., 2016). However, with ethical and safety barriers

preventing human challenge studies with virulent pathogens e.g., HIV, TB and Zika

virus, NHP models remain in use. Moreover, because many aspects of certain infections

are mimicked in NHPs, NHPs remain suitable research models (Mothé, 2015; Capuano,

2003; Garcia-Tellez et al., 2016; Dudley et al., 2016). Furthermore, validated correlates

of protection against most of these pathogens have not been identified, which prevents

the assessment of new vaccine efficacy in humans without challenge studies.

Replacement and Reduction

In vitro modelling: Recent developments in biomedical research have improved the

selection of ‘most promising candidates’ for new therapies in vitro before further

assessment in vivo. A recent example is a cell culture technique to study drugs against

dormant stages of specific malaria species that substantially reduced the number of NHP

experiments (Dembele, 2014; Zeeman, 2016). In vitro/ex vivo models, -omics

technologies and systems biology (in silico modelling) provide important new

opportunities for understanding host response to pathogenic organisms, e.g., growth

inhibition assays for M. tuberculosis (Zelmer et al., 2016) and can provide information on

important components of pathogens for the selection of best vaccine candidates.

Promising new technology, e.g., growing organoïds from specific stem cells and

improved 3D cell culture techniques and techniques such as MIMIC may also provide

new insights into infectious diseases and/or development of new therapies (Dauner et

al., 2017). Collectively, new methods may downselect new vaccine candidates and drug

targets. However, these alternative methods may only provide partial information on the

complex interaction between host and pathogen, though there is hope for the near

future, with the development of fully artificial whole-body models.

Non-NHP animal models: The development of non-NHP animal models, such as

transgenic and humanised mice or zebrafish may eventually reduce NHP use (Holz et al.,

2016; Arainga et al., 2016; Neely, 2017, Ibeh et al., 2016). Unfortunately, data may be

difficult to interpret due to variations in immune systems between certain non-NHP hosts

and humans (Garcia-Tellez, 2016). Additionally, there may be the need to adapt the

pathogen to the host, e.g., influenza or Dengue virus in mice, which is less problematic

with NHPs. Currently, there are very few non-NHP models available that have the

capacity to fully replace NHP models in the near future.

Refinement and Reduction

Imaging techniques: Imaging techniques, such as PET-CT scanning or fluorescent

imaging within infectious disease may enable the study of the development of certain

infections, including the early effects of infection, and/or therapy over time in the same

individual (Lin et al., 2016). Novel sophisticated imaging techniques including new

tracers and labelled mAbs will enable detailed analysis of the development of infections

in a single animal, which will increase the amount of information from fewer animals

(Bocan et al., 2015).

6.6.3.4 Identification of specific research areas

The (re)emergence of infectious diseases over the past years demonstrates the need for

research and new therapies. A recent example is the re-emergence of whooping cough

caused by Bordetella pertussis for which small animal models are not reliable for vaccine

Non-human primates testing (update 2017)

Final Opinion

development. However, a new baboon model was developed that mimics whooping

cough in patients (Warfel et al., 2013). Important diseases such as TB and simian

immunodeficiency virus (primate analogue of HIV) are more human-like in NHPs than in

other animal models. Human challenge models are currently not possible because of the

potential chronic nature of the infection and absence of relevant non-NHP models for the

specific research question. Plasmodium vivax, one of the most prevalent malaria

infections in the world, can only be partially studied in humanised mice (Mikolajczak et

al., 2015). Additionally, Macaques infected with a macaque-specific analogous parasite

may only provide limited information on Plasmodium vivax infection (Joyner et al.,

2015). Although NHPs are important the study of malaria infection and therapy, NHP

models also have their limitations (Martinelli, 2016). Depending on the question, malaria

liver stage research may be studied in mouse models (Holz et al., 2016). There are

several studies stating that there is a lack of available vaccines, even after many years

of research in NHP models for HIV research (Akhtar, 2015; Bailey, 2014). However,

NHPs have been essential for improving our understanding of HIV and in the

development of new interventions that resulted in a longer survival of HIV patients.

Recently, a specific NHP HIV model was found to have many similarities with disease

development in HIV-infected children with non-progressive disease (Muenchhoff et al.,

2016). NHP models are thus, indispensable for the study of the fundamental aspects of

HIV and development of prophylactic and therapeutic therapies.

Changes in spread, infectivity and pathogenicity of infectious agents are realistic

dangers. Examples are recent Ebola and Zika epidemics as well as new pandemic

influenza strains. Because of many years of NHP research on Ebola, vaccines were

available relatively quickly and could be tested in endemic areas. Zika was originally

discovered in NHPs in the 1940s and currently, Zika infections are studied in NHPs.

Dengue virus, the most prevalent arbo-virus with potential severe morbidity, does not

naturally infect non-human species. However, in contrast to all other immune-competent

animal species, Dengue infects and replicates in NHPs and stimulates relevant immune

responses (Clark et al., 2013). Thus, NHPs provide essential models for understanding

and combatting (re)emerging infectious pathogens.

Infectious disease models are better in NHPs than in other animals when the disease

resembles that in humans compared with other animal models. A major advantage is the

similarity of NHP and human immune systems. Most reagents for analysis of human

immune responses can be reliably used in NHPs. Additionally, increasing knowledge of

the NHP major histocompatibility complex (MHC) genes has substantially improved the

understanding of HIV and will have an impact on other diseases, such as emerging viral

infections, TB and malaria, which will also be essential for elucidating mechanisms

underlying host immune control of pathogens. Because genetics between humans and

other species, including NHPs are not identical, small differences may influence the

outcome of an infectious disease or therapy (Bailey, 2014). Differences between humans

and NHP in their innate response to LPS have been reported (Barreiro, 2010), which can

be used both as weaknesses or strengths for NHP models in this field.

Due to their phylogenetic relationship, the MHC of NHPs and humans are more closely

related than that of other animal species and as a result often offer the best fit-for-

purpose models. Small differences in genetics between different species (e.g., rhesus

versus cynomolgus macaques) or even within species derived from different

geographical origins in combination with different disease outcomes (e.g., HIV and TB)

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Final Opinion

provide the opportunity to study fundamental mechanisms underlying pathogenesis and

host response (‘t Hart et al., 2015; Sharpe et al., 2016).

Future Research

Further optimisation and characterisation of non-NHP animals, such as humanised or

transgenic mice and zebrafish, can reduce the use of NHP models for specific questions.

Further development of new techniques such as organoïds and -omics technology, 3-

dimensional cell culture techniques and systems biology for vaccine development will

reduce the current necessity to use NHP to study important infections

. Development of

human challenge models should be encouraged when possible. Platforms for

collaboration and sharing of data from population studies, genome wide association

studies and clinical studies should be strengthened to further advance knowledge and

thus development of new therapies and vaccines. The use of imaging techniques in both

preclinical and clinical studies are important to reduce or replace NHP models and will be

helpful to compare NHP and patient data. Improvement and development of new

imaging techniques, tracers (e.g., to allow PET imaging) and labelled antibodies are

necessary to follow infections in vivo in individual animals. Research leading to the

identification of predictive biomarkers for vaccine efficacy in animal studies, including

NHP, and patients will result in the possibility to perform better predictive vaccine

efficacy studies in humans without requirement of a pathogen challenge. Reduction and

refinement of NHP models in infectious disease research can also be achieved by further

and better characterisation of NHP. Examples include the in-depth characterisation of the

genetic background, which will improve the understanding of disease, therapy, improved

animal selection (Haus et al., 2014; Vierboom et al., 2016) and characterisation of the

microbiome. Additional refinement will be reached by continuously improving animal

training protocols to reduce potential stress responses that can influence the outcome of

the research. Sharing of biological material obtained from NHPs, e.g., through biobanks,

should be encouraged to further reduce the number of animals used. In some cases, the

use of NHPs is mandatory to test a new vaccine master or working seed lots, such as for

yellow fever (WHO Technical Report Series, No 872, 1998). Replacement of live vaccines

that have a risk for neurological effects and were master or working seed lots are now

tested in NHPs by subunit vaccines. Currently, it is not possible to provide a fixed time-

schedule for reducing the number of NHPs used in infectious disease research. It is

unlikely that the new technologies will fully replace NHPs in the near future, especially

with the (re)emergence of pathogens. Moreover, this will also depend on potential

outbreaks with novel pathogens in which NHPs are the most relevant or possibly, the

only reliable model species.

6.6.4 Neuroscience

6.6.4.1 Introduction

Historically, NHPs are used as models of the human brain in fundamental research to

elucidate how brain circuits contribute to memory, attention, decision making, reward,

emotion, visual and auditory perception and motor control (Bystron et al., 2006; Dehay

and Kennedy, 2007; Garcia-Cabezas et al., 2008; Letinic et al., 2002; Meyer et al.,

2000; Georgopoulos, 2000; Sanchez-Gonzalez et al., 2005; Smart et al., 2002; Schultz,

http://www.iprove-roadmap.eu

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2002; Fabbri-Destro and Rizzolatti, 2008; Rizzolatti and Fabbri-Destro, 2008). Although

the number of NHPs used per study is typically small, many neuroscience research

groups within Europe are engaged in this type of academic research. The continuum

between such fundamental primate research and applied neuroscience research in

patients remains the key to advances in the field.

In addition to advancing scientific knowledge about the brain, information gained from

fundamental research on NHPs has helped to understand brain dysfunction and has

sometimes contributed to the identification of treatments against disease and injury in

patients. For example, behavioural techniques to study the consequences of brain injury

in NHPs have been translated to allow assessment of human patients. Changes in

learning after lesions of different regions within the frontal lobe (Kennerley et al., 2006,

Rudebeck et al., 2008) were used by another research team to elucidate the difficulties

of human patients with frontal lobe lesions (Camille et al., 2011). Understanding the

consequences of brain lesions in NHPs had a direct bearing on understanding the

impairment of patients with lesions that included parts of the same brain areas. The

clinical work could not have been done without the initial animal studies. Fundamental

neuroscience research has also helped, along with data from rodents and man, construct

new experimental in silico and in vitro models and to develop new computational

technologies to simulate how the brain works. An ongoing example of this endeavour is

the Human Brain Project

. These replacement techniques partially rely on

electrophysiological data acquired in animals. Substantial work in the field of imaging

techniques has been achieved due to the work with NHPs in fundamental neuroscience

research (Logothetis, 2001). Electrophysiology studies in the awake, behaving state are

generally assessed as imposing a high welfare impact due to the number of procedures

involved (e.g., surgeries under anaesthesia, chronic restraint and penetration of

microelectrodes, food or fluid control), their likely effects on the monkeys, their

repetition over many months or years, and long-term housing in the laboratory

environment (Bateson et al., 2011) (also see Severity Classification below).

Translational neuroscience research aims at understanding the causes of, and developing

new therapies against, a range of brain disorders like Parkinson’s disease, Alzheimer’s

disease or multiple sclerosis. These disorders remain poorly understood and have an

important impact on society in terms of number of patients (510 million in the EU in

2015) and their affected relatives, as well as on public health cost (WHO NeuroAtlas,

EBC). Multiple strategies should be deployed to advance knowledge in this area. Whilst

rodents are used for this research, applied and translational research in NHPs can be key

in some steps of the process because of their greater similarity to man. Both

fundamental and applied studies can show discrepancies between primates and non-

primate species, thus avoiding the pursuit of certain research directions that could fail if

translated to the clinic.

Modelling neurodegenerative diseases in NHPs can cause a high degree of suffering to

the animals. For example, the MPTP model of Parkinson’s disease can result in severe

impairment of the well-being and condition of the animals, necessitating daily

examination, warming, weighing, hand feeding and grooming until they regain autonomy

(Jackson and Jenner, 2012). In the chronic phase, motor symptoms of Parkinson’s

disease closely resemble the pathology in patients, which includes abnormalities in gait,

https://www.humanbrainproject.eu/2016-overview

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balance and posture, slowness of movement, freezing, rigidity and levodopa-induced

dyskinesias. The caging environment needs to be adapted to these symptoms (reduced

height of elevated platforms in the cage, use of grids instead of bars, plastic instead of

metal grids to avoid contact with cold surfaces on platforms and cage flooring, adjusting

the height of food and beverage receptacles to grant ease of access, etc.) and to

minimise risk of injury. Alternatively, other NHP models of neurodegenerative disease

like 3NP systemic intoxication or quinolinic acid intra-striatal lesions for Huntington’s

Disease, overexpression of mutant proteins like huntingtin (for Huntington’s Disease),

alpha-synuclein (for Parkinson’s Disease), amyloid or Tau (for Alzheimer’s Disease), are

mostly asymptomatic and currently classified as moderate since they only require one

surgical intervention that does not induce long lasting suffering throughout the lifespan

of the animal.

However, given the potential for serious adverse effects of some models and techniques

and the long-lasting nature of some of these studies, it is particularly important that

funders and ethics committees establish robust peer review procedures to ensure that

only those projects with a very high likelihood of producing scientific, medical or social

benefit go ahead, and that there is full application of the available opportunities to

implement all three Rs.

6.6.4.2 Severity classification

Directive 2010/63/EU requires that severity classification take into account, among other

things, the lifetime experience of the animals, the duration, frequency and multiplicity of

harmful techniques, the potential for cumulative suffering within a procedure, and the

application of refinement techniques (see Annex VIII). Prospective severity classification

for the purposes of project evaluation is done for the entire group of animals undergoing

the procedure/s, and is based on the most severe effects likely to be experienced by any

individual animal after applying all appropriate refinement techniques. On the other

hand, retrospective reporting of actual severity (e.g. for annual statistical reporting) is

based on the actual experience of each individual animal. Guidance on severity

assessment from national competent authorities

states “The actual severity to be

reported for the individual animal should be the highest level experienced during the

course of the procedure and not based on the severity at the end of the procedure. Nor

should the evaluation be considered a simple additive process, e.g., a number of mild

procedures = moderate severity. It should be based on an overall assessment of the

animal's experience from the start of the procedure to the end.” Permission to re-use is

dependent on actual severities of the previous procedure/s in combination with

prospective classification of proposed re-use. Reuse permission is not based on

prospective classification of the previous procedure.

Annex VIII of the Directive gives examples of procedures under each severity category.

Surgery under general anaesthesia with appropriate analgesia is a ‘moderate’ procedure,

whereas use of metabolism cages involving severe restriction of movement over a

prolonged period (e.g., up to 5 days) is a ‘severe’ procedure. Most non-human primates

on long-term electrophysiology studies will experience a number of surgical procedures

during their lifetimes (Pickard et al., 2013), and restraint in a restraint chair for up to 6

http://ec.europa.eu/environment/chemicals/lab_animals/pdf/guidance/severity/en.pdf

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hours per day most days per week (McMillan et al., 2017). The Pickard Committee

analysed data extracted from laboratory records on the number of adverse effects and

complications experienced by macaques and marmosets used in neuroscience studies

predominantly in the UK (Pickard et al., 2013). Infection around implants occurred in

23% of macaques with short-term implants, rising to 39% of macaques with long-term

implants. Bone infection occurred in 6% of animals, brain infection in 1%, and cerebral

haemorrhage in 1%. The incidence of seizures in studies involving lesions to the central

nervous system was 9% of cases. Non-elective euthanasia to end suffering was reported

in 26/234 cases, of which 42% were procedure-related. The Pickard data indicates that

underestimation of severity can occur (e.g., see Table 9, page 49 for four case studies

where the animals failed to cope and premature euthanasia was performed on welfare

grounds – identified as clear examples of ‘severe’ by the UK competent authority but

classified by the researcher/s involved as ‘moderate’).

Prospective severity classification should be made on case-by-case basis and in line with

the highest severity that may be experienced by an individual animal. It is important to

remember that reuse conditions are based on actual reported severity.

6.6.4.3 Justification for continuing to use NHP in the specific area

A variety of animal species are used in neuroscience. NHPs are generally used where

other species such as rodents lack the brain structures of interest, the fine visual and

motor abilities under investigation, the use of neurotransmitter systems, the functional

connectivity, or the ability to perform complex cognitive and behavioural tasks similar to

those used in humans (Jennings, 2016).

There is consensus in the scientific community that one animal model can never fully

recapitulate all aspects of human brain diseases. The type of scientific question asked

and the methodology used to explore will determine how useful and how predictive the

results will be. This implies that a variety of models, animal and non-animal, must be

used to address different aspects of the same disease. As an example, mouse transgenic

models of Huntington’s disease, bearing the same genetic defect as humans, do not

exhibit all the major aspects of the disease, whereas non-transgenic NHPs models do

(Palfi et al., 2007; Palfi et al., 1996; Pouladi et al., 2013).

The similarity of follow-up techniques used in pre-clinical studies and the appropriate

scaling up of the treatment delivered is a very important factor in the predictability of

the results for clinical safety and efficacy trials. For gene therapy strategies, assessing

adverse events from either off-target infection or deleterious motor consequences such

as dyskinesias (abnormal movements) can only be explored in a brain that is as complex

as the human brain (e.g., in terms of number of cortical layers) and that presents a

similar motor phenotype (which implies a similar connectivity across specific brain

regions). For cell therapy, the number of cells needed, the rate of their differentiation

(much more rapid in rodents than in NHPs) and the length of the axonal connections are

key factors for circuitry reconnection that cannot always be appropriately assessed in

small brains before initiating clinical trials (Lindvall, 2016). Perhaps more importantly,

the potential adverse effects of these cells (migration/implantation to undesired targets

over long distances, overgrowth) can be monitored non-invasively using magnetic

resonance imaging (MRI) techniques. The availability of peripheral fluids, in particular

cerebrospinal fluid, to follow up adverse events such as rejection and immunogenicity of

grafted cells longitudinally using specific immune biomarkers can be challenging in small

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animals compared with NHPs and may not predict the clinical scenario (de Lange, 2013).

Imaging techniques like fMRI and PET pharmacology have high translational potential

and are increasingly used to identify cell populations involved in disease and these

approaches may then be modelled in silico or in less complex systems where relevant

(Herrmann et al., 2015). The use of NHPs in certain experiments can be justified by the

sub-anatomical rearrangement of neuronal populations and their connectivity across

brain structures that are not always present or partially present in rodent species like the

neocortex, the prefrontal cortex, and visual areas as well as attentional and oculomotor

networks (Hutchison and Everling, 2012). Importantly, the connectivity across

hemispheres, the presence of homologous structures in both hemispheres (redundancy),

or the lateralisation and intra-hemisphere connections are distinctive features in human

and NHPs (Passingham, 2009; Stout, 2012; Izpisua Belmonte et al., 2015; Procyk,

2016).

6.6.4.4 Progress made in the last 10 years

There has been extraordinary progress in recent years in the field of Brain-Machine

Interfaces (BMIs) that use neuronal action potentials (spikes) recorded by implanted

electrode arrays to restore function after paralysis. NHPs have and continue to play an

important role, particularly in the development of upper-limb BMIs, since the motor

systems controlling the human arm and hand are shared only by other primates (Borton

et al., 2013). Key advances made first with NHPs and subsequently translated to

paralysed human patients include: the control of computer interfaces in monkeys (Taylor

et al., 2002) and humans (Hochberg et al., 2006), the control of robotic prostheses in

monkeys (Velliste et al., 2008) and humans (Hochberg et al., 2012), the control of

functional electrical stimulation of muscles in monkeys (Ethier et al., 2012) and humans

(Bouton et al., 2016) and artificial sensory stimulation in monkeys (Tabot et al., 2013)

and humans (Flesher et al., 2016). In each case, NHP research has driven translation to

patients (Bensmaia and Miller, 2014). The next generation of BMIs may act as artificial

(neuroprosthetic) connections between disconnected parts of the nervous system

(Hampson et al., 2012; Jackson et al., 2006); Hampson et al., 2012; Jackson et al.,

2006). For example, work is underway to develop methods of reconnecting the brain to

the spinal cord (Zimmermann and Jackson, 2014), requiring new spinal cord stimulating

technologies which (due to the different neuroanatomy of quadrupeds) must be tested

first in NHPs. In addition, improved longevity and stability of BMIs may require new

cortical electrodes and signal processing techniques (Hall et al., 2014) which must first

be developed and tested in NHPs before they can safely be translated to humans.

Applied studies conducted in animal models, and in particular in NHPs, have also had an

important role in the advancement of gene therapy for neurodegenerative diseases

(Hocquemiller et al., 2016). Taking Parkinson’s disease as an example a gene therapy

vector that produces dopamine in the brain has been shown to be safe and efficacious in

pre-clinical studies in rodents and NHPs and in the first generation of patients treated in

Europe (Jarraya et al., 2009; Palfi et al., 2014).

Finally, immunotherapies and silencing strategies in aggregation diseases like

Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis and

Frontotemporal Dementia aim at neutralising or eliminating aggregated proteins believed

to be pathological and result in neuronal death (Arevalo-Villalobos et al., 2016;

Crunkhorn, 2016; Merienne et al., 2015; Nielsen and Nielsen, 2013; Valera and Masliah,

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2013). These proteins are naturally present in the brain, have one or more physiological

functions that are not completely understood and are present in many different

conformations in the brain. While the refinement of these strategies can be tested in

vitro by making gene silencing more specific or improving the specificity of antibodies

targeted against pathological protein epitopes, an assessment of the systemic effects,

both beneficial and adverse, of reducing protein load in the whole body has been

assessed in NHPs and proven to be safe before testing them clinically (McBride et al.,

2011; Grondin et al., 2012; Stiles, Zhang et al., 2012; Grondin et al., 2015;

Kordasiewicz et al., 2012; Keiser et al., 2016).

Despite this progress, a 2011 review by an independent panel convened by the major

public UK funders of NHP research found little direct evidence of actual medical benefit in

the form of changes in clinical practice or new treatments arising from the NHP

neuroscience portfolio funded over a ten year period between 1997 and 2006 inclusive

(Bateson et al., 2011). There were a number of reasons for this including lack of a

genuine link to medicine, the time that had elapsed between the research and the review

and a lack of awareness on the part of the researchers about the pathway to medical

benefit and how to move discovery to practical application. The panel also noted,

however, that the neuroscience was good quality and highly cited. It is important that

the justifications offered for individual NHP research projects are soundly based and

realistic and any potential health benefits are demonstrable. In addition, there should be

effective knowledge transfer and dissemination to ensure maximum benefit.

6.6.4.5 Replacement possibilities

Most behavioural neuroscience studies combine measurements of sensory, cognitive,

motor or other psychological functions with some method of monitoring and

manipulating the activity of individual neurons, circuits or systems to establish a causal

relationship between the brain and behaviour or cognition. Because many of the

procedures used to monitor and manipulate brain elements are invasive (such as

electrophysiological recording or stimulation of neurons, and production of temporary or

permanent lesions via surgical, chemical or physical means), only rarely is it considered

ethical for them to be carried out in humans. It has been proposed that many more

studies involving recording from single neurons could be made directly in people (Bailey

and Taylor, 2016). However, when these studies are conducted, the electrode placement

sites are driven exclusively by clinical need (e.g., implanting DBS electrodes) and often

the brain tissue under study is compromised by a disease process like epilepsy,

Parkinson’s disease or traumatic brain injury (Jacobs et al., 2013; Priori et al., 2013;

Worrell et al., 2012). Moreover, the recording duration of this type of studies is limited

compared to the longitudinal follow-up available in the experimental setting. Hence, their

potential for replacing NHP studies is limited. Nonetheless, where opportunities for

microelectrode recording exist, with the patient’s informed consent, minimal risk and

ethics committee approval, they should be utilised (Mamelak, 2014).

The potential to use functional MRI studies in humans to replace some cognitive

neuroscience experiments performed in NHPs has been repeatedly proposed. However,

there are limitations in the interpretation of the blood oxygenation BOLD signal, that

does not reflect neuronal activity at the single cell or small population level, that prevent

this technique from explaining the neural correlates of cognitive neuroscience in the

healthy and pathological brain (Logothetis et al., 2008; Schridde et al., 2008; Buzsaki et

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al., 2012; Rigotti et al., 2013; Buckner et al., 2013). fMRI is a powerful non-invasive

neuroimaging method to extrapolate fundamental neuronal mechanisms that can be

revealed and interpreted by electrophysiology. Both methods are complementary and

many laboratories use approaches in NHPs and humans to advance our understanding of

the human brain while reducing the numbers of NHPs used in the laboratory (Procyk et

al., 2016; Passingham, 2009).

Moreover, recent studies have stressed several critical factors concerning statistical

approaches used in fMRI studies (Eklund et al., 2016), concluding that the widespread

use of this technique has to some extent slowed down and mislead current neuroscience

research.

Regarding other non-invasive imaging techniques proposed to be able to replace

neurophysiological studies in NHP, their limitations have been previously reviewed

(Bateson et al., 2011):

“despite the opportunities offered by imaging techniques, they are a long way from being

able to replace completely studies in NHPs and other animals. For example, information

on the direction of an anatomical connection (anterograde versus retrograde) is

unavailable from MRI-based techniques, and it is impossible to infer fine grained

connectivities (at the level of individual cells or groups of cells) using these approaches.

Functional mapping techniques such as fMRI are limited in recording a haemodynamic

signal, rather than the neuronal activity itself. This makes it impossible, for example, to

make inferences about the relative timing of events at a fine temporal scale. Techniques

such as magnetoencephalography (MEG) can offer greater insights into neuronal timing

and do directly reflect the electrical activity of a region, but the MEG signal reflects

synchronised activity across populations of cells, rather than the single cell level

information that is available from electrophysiological studies in animals. Non-invasive,

reversible interference with localised human brain functions using TMS allows mapping of

brain structure-behaviour relationships, providing a powerful alternative to NHP lesion

studies. However, TMS can only be reliably targeted to structures on the cortical surface;

deep brain structures, or medial cortical areas, are inaccessible to this technique yet are

important to study because of their role in many neuropsychiatric disorders.”

Acknowledging the limitations of existing imaging techniques is important to determine

the scientific hypotheses that can be tested through them and what experiments can be

directly performed in humans (Perry and Singh, 2014; Ruff et al., 2008; Schmid et al.,

2010). Significantly improving the spatial and temporal resolution of non-invasive

imaging technologies should be a high priority given the potential, after appropriate

validation, to advance the 3Rs in of NHP research (Boto et al., 2016). Transcranial

focused ultrasound (tFUS) / focused ultrasound neuromodulation (FUN) is an emerging

form of non-surgical neuromodulation that confers advantages over existing electro and

electromagnetic technologies by providing a superior spatial resolution on the millimeter

scale as well as the capability to target sub-cortical structures non-invasively in NHPs

and human (Deffieux et al., 2013; Lee et al., 2016; Leo et al., 2016).

The use of microfluidic chambers/brain chips and mixed cell cultures constitutes

replacement strategies are important for understanding cellular processes such as cell-

to-cell communication, protein trafficking or the role of glial cells, in health and disease.

Advances in stem cell biology have significantly contributed to biomedical research in the

last 10 years. The use of patient-derived iPS (induced pluripotent stem) cells has

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considerably changed the ethical dilemma of using embryonic stem cells to understand

differentiation or as a therapeutic option (Santostefano et al., 2015). In vitro disease

modelling possibilities have expanded thanks to this technology, in particular when

monogenetic diseases rather than sporadic diseases are concerned (Avior et al., 2016).

These cells can recapitulate some aspects of the disease, just like animal models can,

and allow the replacement animals in the study of cellular mechanisms of disease by

assessing neuron-to-neuron or neuron-to-glial cell communication in microfluidic

chambers for example. Moreover, it allows the replacement of animals in novel drug

screening where the molecular target is known and toxicity assays. The challenges

remaining are, for example, the use of viruses for the reprogramming of patients cells

derived from skin (fibroblasts) or blood (PBMCs), residual somatic memory that can

affect differentiation “purity”, and the time that neurons and non-neuronal cells can be

kept alive after differentiation in order to study their activity (Korecka et al., 2016). The

same holds true for “mini-brains” or organoids where neuronal and non-neuronal cells

can form networks, but the degree of connectivity and axonal outgrowth remains to be

determined. Moreover, given the differentiation states of cells achieved at present, these

“brains in a dish” seem more suitable to study neurodevelopment and developmental

disorders of the brain than processes involving aged neurons (Bae and Walsh, 2013).

The absence of a blood-brain-barrier, a vascular system and an immune system

constitute major limitations when investigating complex pathological processes in vitro.

6.6.4.6 Reduction and refinement possibilities

Generally within basic neuroscience the number of NHPs used per study is small. For

studies where the experimental unit is the neuron, the standard for publication is two

animals per condition; if the results obtained from the second monkey confirming the

results can be replicated in another animal. Efforts are made to optimise the yield of

data per animal and experimental session, for example via the use of multi-electrode

arrays, and in some cases to share data and tissues with other researchers. For studies

examining the effects of lesions or other interventions within or between groups of

animals, sample sizes may be around four per group, depending of factors such as effect

size.

Over the last decade, advances in brain imaging technologies and non-invasive

electrophysiological methods have furthered efforts to refine and replace NHP

investigations in cognitive neuroscience and pharmacology. For example, integration of

structural and functional MRI with transient inactivation of targeted brain regions allows

studies to be performed without longer-lasting impairments or disability in the animals

(Schmid et al., 2010). Transcranial magnetic stimulation (TMS) provides a way of

establishing the causal role of particular brain cortical areas in cognitive function, without

causing the permanent tissue destruction that has long been the traditional method.

Structural MRI is being used to more accurately target recording, stimulation, lesioning

and transplantation procedures, yielding better quality data (t’Hart et al., 2006). MRI

scans are also being used to produce head restraint devices custom-fitted to the animal’s

skull, improving integration and stability and reducing the likelihood of infection, bone

necrosis and loosening of the device (Mulliken et al., 2015). Functional MRI in NHPs is

developing rapidly, offering a powerful tool for directly refining and potentially replacing

some electrophysiological studies (Vanduffel and Farivar, 2014, Ortiz-Rios et al., 2015).

Technological developments have also enabled refinement of surgical and other

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procedures within the neurosciences. For example, infrared reflection and video-based

systems for tracking eye position/movement have replaced the use of surgically

implanted scleral eye coils (Kimmel et al., 2012). Screw-mounted headposts and

recording chambers machined from single pieces of titanium have in some cases

replaced traditional devices using dental acrylic, which create greater defects in the

scalp, are less biocompatible and must be maintained assiduously to prevent infection

(Adams et al., 2007; www.ciwiki.net). In some studies, non-invasive means of head

restraint can replace surgically implanted devices (Hadj-Bouziane et al., 2014, Slater et

al., 2016). Use of antimitotic compounds (e.g., 5-flurouracil) have reduced, and in some

cases removed, the need for dural scrapes (Spinks et al., 2003). Modern anaesthetics,

from which the animals recuperate more rapidly (Bertrand et al., 2017), have enabled

more rapid reintroduction of NHPs to the social group, reducing the likelihood of

aggression due to disturbance of the group hierarchy (Jennings and Prescott, 2009).

Food and fluid control protocols, used to motivate reliable performance on cognitive and

behavioural tasks, have been refined to reduce the impact on animal welfare (Prescott et

al., 2010; Hage et al., 2014; Gray et al., 2016). Technological advances have resulted in

smaller devices for multi-electrode array recordings and the possibility to obtain in vivo

electrophysiological data wirelessly (Eliades and Wang, 2008; Fernandez-Leon et al.,

2015; O'Shea et al., 2017; Yin et al., 2014).

6.6.4.7 Identification of specific research areas

Many large and small animal species have been used to identify anatomical structures

and pathways that are relevant to vision, sensation, hearing motor control and cognition.

Traditionally, electrophysiological recordings have been performed in vivo and brain

slices. An alternative that is heavily investigated is the use of optogenetic techniques in

which a virus can infect neurons and render them sensitive to light to modulate their

activity. Optogenetics has contributed immensely to identifying networks of connected

cell populations, functional pathways and circuitry in vitro and in the rodent brain both in

health and disease in the last decade (Adamantidis et al., 2015; Deisseroth, 2015). An

important refinement is that optogenetic experiments in rodents could better inform

deep brain stimulation (DBS) treatments to modulate neural activity in pathways that

are anatomically relevant to a given disease and combine it with pharmacological

modulation for a symptomatic treatment (Luscher and Pollak, 2016). However, there are

several technological aspects (light penetration, stable opsin protein expression without

cell dysfunction/inflammation, single neuron versus neuronal population modulation,

etc.) that currently prevent this technique from being translated to large brains and

replacing standard electrophysiology are under investigation in NHPs.

An important factor in the progress of gene therapy, antibodies, PET radiotracers,

contrast imaging molecules and drug delivery strategies for neurodegenerative disease is

the crossing of the blood-brain-barrier. The efficient passage of these imaging or

therapeutic agents into the brain will determine the success of these approaches in the

clinic. Focus ultrasound (FUS) uses ultrasound and a systemic injection of microbubbles

in the vasculature in combination with anatomical MRI imaging to transiently and focally

open the barrier. Given the size of the brain, the potential adverse events (like tissue

heating, lesions, and duration of the opening) and the specificity of the cell populations

that need to be targeted, animal models, in particular NHPs, might still be required to

validate such new approaches (San Sebastian et al., 2013).

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More recently, some research groups have been working on direct reprogramming in the

rodent brain, using the proliferation of astrocytes in neurodegenerative diseases to

reconvert these cells into neurons, thus avoiding an invasive surgical procedure for cell

transplantation (Chen et al., 2015). Because the exact role (beneficial versus

detrimental) of astrocytic/microglial proliferation during inflammatory processes that

may be different in rodents and primates, some work to confirm the mechanisms in

NHPs may be a requirement before clinical trials in patients.

6.6.5 Other uses

It is not possible to report on all NHP models in this Opinion. Primates are used in

behavioural research and in various other areas of biomedical research in addition to the

areas focused on in the Opinion. NHP models are used to study inflammatory disorders,

aging (Mattison and Vaughan, 2016), developing and evaluating new tracers for

neurodegenerative diseases (Golla et al., 2015) and other diseases, developing and

evaluating new drugs and therapies against a variety of disorders (Haanstra et al., 2016)

and developing gene therapies (Tadin-Strapps et al., 2015). NHPs will mostly likely be

required to study the effects and safety issues for stem cell therapy and organoïd

transplants.

6.6.5.1 Ophthalmology

6.6.5.1.1 Introduction

Blindness is a debilitation condition affecting more than a million people in Europe alone.

The major causes of sight loss are retinal dystrophies, age-related macular

degeneration, glaucoma, and diabetic retinopathy. Given the heterogeneity of inherited

eye diseases and the impact on public health, efforts have been made at the EU level to

create networks for rapid translation of therapies to the clinic (Cuna-Vaz and Zrenner,

2011).

The retina of NHP and man show some unique features (e.g., both NHPs and humans

have a macula lutea/fovea) not found in other mammals (Eichenbaum et al., 2014) and

therefore NHPs represent a more relevant model for specific ophthalmology research.

Combes and Shah (2016) provide a summary of the scientific advantages of using NHP

models such as forward facing eyes, binocular vision and ability to train them to perform

complex tasks. In conjunction, they discuss the associated welfare concerns and

limitations of using NHPs including the highly invasive nature of some of the techniques

and re-use of animals. The paper gives a comprehensive overview of the in vivo, ex vivo,

in vitro, computational models and volunteer studies that can be used in vision research.

For example, they describe the restricted possibility that species such as fruit fly, zebra

fish and amphibia could be used in some circumstances and outline the pros and cons of

subcellular, tissue culture, retinal cell cultures, stem cells and brain slices. However, they

argue that the potential for relative replacement is more encouraging especially through

the use of human stem cells in developing human cell-based models of visual pathways,

the availability of suitable in vitro models for efficacy testing of new therapies

particularly in the retina (Krishnamoorthy et al., 2016; cited in Combes and Shah,

2016). They provide evidence that the rodent visual system displays similarities with the

visual pathway of humans and NHPs and that transgenesis has opened up new avenues

of research with rodents. Furthermore, they argue that;

Non-human primates testing (update 2017)

Final Opinion

‘There is an urgent need to improve the methodology for whole organ eye-culturing, and

to discover more information about the structure and function of the visual cortex, in

order to expedite the development of improved in vitro and in silico models and

simulations as a way of increasing implementation of advanced replacement techniques

in vision research’ (Combes and Shah, 2016).

6.6.5.1.2 Pertinence of NHP in the specific research area

No currently available in vitro or in silico model appears to recapitulate the architectural

complexity of retinal cells or their complex structural and functional interactions and

many animal models have been attempted to mimic ophthalmic diseases like macular

degeneration or glaucoma (Chen et al., 2014; Burgoyne, 2015). Many differences in eye

anatomy and function exist between rodents and NHP including colour perception, the

presence of forward-looking eyes, a binocular processing that gives rise to the

perception of depth, the presence of a fovea, saccadic foveation and a divergent

specialisation of the spatial visual attention system; the presence of a macula, central

part of the retina, that is responsible for visual acuity and colour vision in human and

NHPs (Meier and Reinagel, 2013).

6.6.5.1.3 Progress and future research directions

For some of these models, NHP might be indispensable, such as Usher type I syndrome,

an inherited disorder causing deafness and blindness. Many transgenic mouse models

have been generated since the discovery of the genetic mutations leading to the disease.

Although these mouse strains are deaf, none reproduces the retinal degeneration

observed in humans. A recent publication argues that unlike mice, NHPs have the five

USH1 proteins colocalising at membrane–membrane connection sites between the outer

segment of the photoreceptors and surrounding subcellular compartments called the

“calyceal processes” (Sahly et al., 2012). A NHP model of this disease is under

development by specifically targeting the proteins in these regions through a shRNA

silencing strategy delivered by AAV (Institute of Vision, France).

A major axis of research is the restoration of visual perception in patients suffering from

degenerative retinopathies who eventually become blind. One example of these

conditions is the genetically inherited retinitis pigmentosa (Dalkara and Sahel, 2014).

«Artificial retinas» have been shown to functionally replace photoreceptors in patients

suffering from retinal degeneration and new devices are currently being developed and

tested in NHPs to improve the number of photodiodes, the biomaterials and the optimal

morphology of the implant (Stingl et al., 2016; project SIGH AGAIN

Alternatively, gene therapy and optogenetic light-mediated activation of remaining

transfected retinal neurons are ideal for specifically stimulating the cells that have not

degenerated and thus, restore electrical stimulation of the optic-brain circuitry (Duebel

et al., 2015).

Gene therapy for a different inherited disease, Leber’s Congenital Amaurosis, that causes

impaired vision from birth and blindness within a decade, has given promising results in

patients but seems to be less efficacious than in a dog model and a new viral vector is

under investigation in NHPs to improve the clinical results (Bainbridge et al., 2008;

http://www.rpfightingblindness.org.uk/newsevent.php?newseventid=419&tln=newsevents

Non-human primates testing (update 2017)

Final Opinion

Kostic et al., 2010). Finally, cell therapy using embryonic stem cells or induced

pluripotent stem cells has given promising results in rodent and primate models of

retinitis pigmentosa and researchers have now developed cell lines that might be less

immunogenic after transplantation by matching the MHC molecules in the cell donor and

the recipient (Shirai et al., 2016; Sugita et al., 2016a; Sugita et al., 2016b). Because of

the uniqueness of the visual system in primates, it is certain that some of these and

novel therapeutic approaches will be tested in NHPs as well as rodents.

6.6.5.2 Transplantation

NHPs have been used as recipients for investigating the fundamental aspects of organ

transplantation (Kean et al., 2006). They have been essential for developing various

aspects of organ transplantation and prevention of rejection. Insights gained from NHPs

in organ transplantation are used in clinical medicine and clinical research. This has

reduced the need for NHPs in transplantation research, though NHPs may still be

required to evaluate new immunosuppressive therapies and methods to prevent organ

rejection (Zeiser and Blazar, 2016).

The success of organ transplantation in patients with organ failure has increased the

demand for human cells, tissues and organs in the treatment of human disease and the

shortage of organ donors for transplantation is considered to be a major social problem.

Only a minority of patients who may benefit from a transplant will be able to receive one

and 10 to 20% of patients on the waiting list for organ transplants will die before a donor

organ becomes available (UK National Health Service, 2015). In addition, waiting times

are growing and are typically 2 to 5 years. Furthermore, as the transplants themselves

may also need replacing, this will exacerbate the situation.

In addition to treatment of the terminal failure of organs such as kidney, lung, liver and

heart, transplantation is also being seen as a therapy for other diseases such as cystic

fibrosis and for patients affected by diabetes and Parkinson disease.

The concept of “xenotransplantation”, i.e. organs from animals, was pioneered a century

ago, when transplanting human organs was considered ethically controversial. Grafts

were, however, quickly rejected, however, because of unknown forces later identified as

immune responses.

The unmet and growing demand for human cells, tissues, and organs coupled with

recent advances in the science of immunology and molecular biology (e.g., potent

immunosuppressive drugs, transgenic techniques) have stimulated a renewed interest in

the transplantation of animal cells, tissues, and organs instead of human cells and

organs.

The subject of ethics in relation to xenotransplantation has been widely explored (Sykes,

2003). Various animal rights activists are opposed to the idea of xenotransplantation

because they maintain that humans do not have right to breed and use other animals for

their own needs. While these issues require considerable debate, the accepted opinion is

that animals used for research or clinical xenotransplantation must be treated

respectfully and humanely, and they must not be used without regulatory approval.

Xenotransplantation does, however, also raise a major public health dilemma. There is a

need to balance the potential promise of this technology to alleviate the shortage of

human cells, tissues, and organs currently available for transplantation with the risk of

potential transmission of infectious agents to the patient, their close contacts, and the

Non-human primates testing (update 2017)

Final Opinion

public at large. Experience with human-to-human transplantation has demonstrated the

transmissibility of infectious agents from donor to recipient (e.g., Human

immunodeficiency virus (HIV), Creutzfeldt-Jacob disease, Hepatitis B virus, and Hepatitis

C virus).

There are anatomic and immunologic similarities between NHPs and humans and these

similarities can reduce the immunological and other barriers to the survival and adequate

functioning of a xenograft in a human host. For these reasons, some investigators have

preferred the use of NHPs as potential sources of cells, tissues, and organs for

xenotransplantation. However, the structural and functional similarities may also

facilitate the transmission of certain infectious agents.

In light of the lack of supply of human organs for transplantation, several alternatives

have been investigated and debated. Implantable mechanical devices have been

explored in the field of cardiac transplantation. Recently, research has increased in the

area of transplanting embryonic cells across species and growing kidneys and endocrine

pancreas cells in situ (Mohiuddin, 2014) Organs from pigs have been the focus of much

of the research in xenotransplantation, in part because of the public acceptance of using

pigs and the physiological similarities between pigs and human and NHPs.

Xenografts have been proposed as appropriate for infants who are physically too small to

accommodate organs retrieved from adult or paediatric donors. Additionally, organs from

animal sources could be transplanted into patients currently excluded from the human

organ transplantation list.

NHPs are not acceptable organ donors for both practical and ethical reasons. In addition

to being uncomfortably close to humans on the evolutionary ladder, they also produce

few offspring, are slow to mature, and would be difficult to rear under the sterile

conditions required to minimise contamination by shared pathogens.

For xenotransplantation research pigs are the optimal candidates for organ donation

(Cooper et al., 2016).

6.6.5.3 Barriers against the implementation of alternatives and

opportunities to progress

The spirit of EU Directive 2010/63/EU, which lays down the rules on animals used for

scientific purposes, is to facilitate and promote the advancement of alternative

approaches and to ensure a high level of protection for animals needed for animal

testing. To achieve this, it requires Member States to promote the use of 3Rs-principles

in scientific research and wherever possible a scientifically satisfactory method or testing

strategy, not entailing the use of live animals, shall be used instead of a procedure (on

animals). This Opinion highlights that in the case of NHPs there are many scientific

approaches that could significantly contribute to the replacement, reduction and

refinement of NHP studies and tests. However, there are also significant issues that go

beyond the scientific rationale, which are preventing the widespread adoption and

development of alternatives to NHP laboratory use. This section describes these barriers

to the implementation of alternatives and provides some suggestions of the

opportunities to overcome them and make progress toward achieving the ethos of the

Directive in Europe.

Schiffelers et al. (2014) provide a framework for understanding the barriers and drivers

associated with the regulatory acceptance and use of 3Rs models in pharmaceutical and

Non-human primates testing (update 2017)

Final Opinion

chemical testing more generally, which can be effectively applied to the case of NHP use

in science more specifically. They discuss the issues in terms of three levels that have to

align in order for an alternative approach to be accepted and implemented;

micro level: consists of the niche in which innovations such as new test methods are

developed and tested. Here, drivers and barriers are found relating to the development

and validation of 3R models;

meso level: entails a mix of existing rules and regulations, expertise, practices and

institutions that strongly influence the acceptance of innovations like 3R models;

macro level: where broader societal features, like the existing material infrastructure,

the political culture and coalitions, broad social values, world views, the macro-economy,

demography and the natural environment, can be found (Schiffelers et al., 2014, p.42).

In terms of this Opinion, the barriers to NHP alternatives are applicable to animal use

more generally but are amplified due to the strong ethical and social concerns

surrounding NHP experimentation. In this context, the micro-level barriers are the

scientific limitations of alternative methods that have been described in this Opinion as

well as the uncertainty of how to translate the findings from such models and build up

the necessary knowledge base to refer to. The main meso-level barriers are legislative,

in particular, the lack of regulatory harmonisation both within and across sectors and the

condition that is often included that that an alternative method must be scientifically

valid, justified and accepted (Schiffelers et al., 2014). A second meso level problem is a

lack of resources for developing alternatives to NHP models. At the macro level, the

potential to replace primates is not just about scientific data but is strongly

related/reliant on factors related to scientific practice where dynamics such as

competition, the reputation of the researchers and entrenchment and policy create

polarisation (Hudson-Shore, 2015; Innovative UK, 2015) and add to the problems at the

micro and meso levels. Another major macro-level barrier is the risk aversive nature of

society which makes it difficult to move away from familiar methods to new alternative

methods where there is less historical data to fall back on.

When considering these barriers and how to overcome them, this Opinion illustrates that

there is a great deal of work being conducted at the micro-level with many examples of

alternatives to NHP use that have been or are being developed. It also provides the

impetus for encouraging innovation and further scientific progress in terms of model and

technique development. There have also been a small number of funded European

initiatives to further the implementation of 3Rs principles in the field of NHP research

including EUPRIMNet

, ANIM.AL.SEE

and PRIMTRAIN

. There is an urgent need to

conduct systematic reviews and meta-analysis of all areas of primate use. While this will

be time consuming in the short-term, in the medium- and long-term, it could result in

significantly reducing the number of NHPs used by identifying where they are unsuitable

models or where they have contributed very little to current knowledge. This will save

resources and animals in conducting further unnecessary studies. It will also provide

evidence for much more targeted use of NHPs which could lead to reduction and

refinement and will ensure that the scientific justification for their use is much more

http://www.euprim-net.eu/

http://www.inemm.cnr.it/animalsee/index.html

http://www.cost.eu/COST_Actions/ca/CA15131

Non-human primates testing (update 2017)

Final Opinion

specific and robust. For advice and guidance on systematic reviews and meta-analyses of

animal studies, researchers may wish to consult the NC3Rs/CAMARADES Systematic

Review Facility (http://syrf.org.uk/), which includes a free app to help researchers to

utilise these methodologies.

At the meso-level, there is still much to be done in terms of ensuring regulations are

harmonised and fully implemented to prevent opportunities to replace primates being

missed. In particular, it is important that the problems with validation are addressed so

that the paradox of in vitro models being expected to meet criteria that were never met

by most animal tests is resolved. In recent years, there has been some improvement in

the resources and funding available for developing alternatives and implementing 3Rs

initiatives. However, this is still small in comparison to the figures involved in animal

based-research. In addition, Research Councils and Funding Bodies should conduct

regular reviews of the outcomes of their NHP projects to ensure that the work has

resulted in a significant enough outcome to justify the use of NHPs and that funding is

being effectively distributed. While not perfect, the Bateson Report (2011) provides an

example of the kind of review, which can be conducted.

The macro-level barriers are perhaps the most difficult to address as they require

changes in attitude both scientific and societal and in scientific practice. Schiffelers et al.

(2014), Taylor (2014) acknowledge these difficulties, but they provide a core set of tools

to enhance the process of aligning the levels and moving the process forward which is

very applicable to NHP use. They advocate the 4Cs;

Commitment: This includes an international commitment to 3Rs models for scientific

and ethical reasons as well as the practical commitment of allocation of resources for the

development, validation and implementation of 3Rs models.

Communication: Finally, NHP researchers should have access to training opportunities

to help them communicate the aims and impacts of their research to the general public.

The Concordat on The Concordat on Openness initiative in the UK is a welcome

development, which is being replicated in other EU countries (e.g. Tierversuche

Verstehen Initiative in Germany and GIRCOR in France). Organisations signing the

Concordat agree to be more open and transparent about their animal research, to

provide accurate descriptions of the benefits, harms and limitations, be realistic about

the potential outputs of such research, and be open about its impact on animal welfare

and the ethical considerations involved. The Bateson Committee (Bateson et al., 2011)

also commented on public engagement, cautioning against overstating and generalising

the medical benefit of NHP research given that much of it is funded primarily for its

scientific value.

Cooperation: Cooperation or collaboration is arguably the most important means by

which to progress 3Rs initiatives in NHP use. Schiffelers et al. (2014) summarise it as

the process where two or more parties interact to create shared understanding. It is not

only about exchanging information but also about education and the mutual use of

information. It is important at all stages in the 3Rs process. This opinion includes several

examples where such cooperation has been very effective such as the NC3Rs working

groups on NHP use in mAb development and vomiting and nausea, and the EUPRIM-Net

initiative, which facilitated the implementation of the 3Rs among NHP researchers from

different lines of research. Cooperation needs to be inter and intra-disciplinary and needs

to cut across different stakeholder sectors such as regulators, industry and animal

Non-human primates testing (update 2017)

Final Opinion

protection groups to drive innovation and change.

Coordination: This is needed to guide alternatives to NHP models through the chain

from models development to the ultimate implementation in regulatory guidelines and/or

everyday scientific practice. This will involve the cooperation detailed above on an

international as well as national scale and the creation of clear roadmaps to follow.

Communication will be key to successfully and efficiently completing each part of the

chain.

7 RECOMMENDATIONS FOR FURTHER WORK

Some of the recommendations given below are already requirements of EU Directive

2010/63, however they are incorporated into the committees series of recommendations

to emphasise their particular importance in relation to the use of non-human primates,

and to encourage their full and rapid implementation in all member states.

7.1 Advancing 3Rs

R01 Decisions about the need for NHPs in research projects or regulatory testing should

be made case-by-case based on strong scientific rationale and the availability of

alternative approaches. For instance, it may be possible that data from one species

is sufficient for progression of a potential new drug into human clinical trials.

Although international regulations for drug safety testing specify that non-rodent

species should be used and that one of these species can be NHPs, a significant

change is consideration of the need to use NHPs on a case-by-case basis. The choice

of species for a research project should, in each case, be based on scientific data on

the most appropriate model. There should also be harm-benefit assessment taking

into account the importance of the biological or medical question and the anticipated

benefits, the quality of experimental design and likelihood of successfully answering

the question, and ethical considerations, including the number of animals involved

and the harm caused to them. The EC and others have published guidance on how

to perform robust harm-benefit assessments (European Commission, 2013; Home

Office, 2015; Brønstad et al., 2016; Laber et al., 2016).

R02 In contrast to current areas of research with great potential to replace current NHP

use such as in safety testing of pharmaceuticals, other areas of research, partly

new, may require increased NHP use in the future, e.g., emerging infectious

diseases. A solid harm-benefit assessment is needed here, too. The majority of NHP

use in the EU is for safety assessment studies, performed to meet regulatory

requirements. NHPs should only be used when there are no alternatives and it is

scientifically demonstrated that none of the other non-rodent species commonly

used in safety testing is appropriate for the purpose of the study. In addition NHP

use can be avoided when in vitro preliminary studies demonstrate NHP are not a

suitable animal model. It is indeed important to consider the limitations of the NHP

when choosing which species to use in drug safety test: the use of an appropriate

species or combination of species/models is essential to obtaining the most reliable

and translatable information.

R03 Progress has been made in identifying opportunities to avoid NHP use where they

are not a relevant species or alternative species can be used, and in identifying

efficient study designs using fewer animals where NHP use is scientifically justified.

This progress has largely been led by the NC3Rs working in collaboration with

industry companies and regulatory bodies internationally, acting as a honest broker

Non-human primates testing (update 2017)

Final Opinion

for pre-competitive data sharing and providing an open forum for dialogue between

the parties. The evidence base and recommendations are being put into practice,

but there is scope for wider uptake across the industry to further advance the 3Rs.

R04 Research funders and ethics committees should ensure that research is conducted

on NHP only where there is no suitable alternative approach, and where there is a

high likelihood of producing scientific, medical or social benefit. This may require

improvements to existing peer review processes to make them more robust. It is

recommended that an international working group develops a clear listing of the

elements that should be scrutinised by project evaluators for their authorisation

and/or funding including experimental design and systematic inclusion of a

publication plan (including for negative results) for non-regulatory projects.

Researchers should report their NHP studies in compliance with the ARRIVE

Guidelines in order to maximise the information published and avoid unnecessary

studies.

R05 With regard to transgenic techniques (e.g., CRISPR) in NHPs, the SCHEER

recommends that the European Commission form a working group to assess the

scientific and ethical implications of such research to determine if it should be

allowed in the EU and, if so, within what constraints.

R06 Conduct systematic reviews in all areas of NHP research, where possible, to conclude

on its value, translational relevance and necessity in the context of alternative

approaches. Cross-company and cross-sector data sharing would be a path forward

for this type of retrospective assessment of NHP studies.

R07 Where NHP research is necessary and justified, it should be performed to genuinely

high standards of experimental design and technical practice, ethics and animal

welfare. Experiments should be performed by appropriately trained and skilled staff

with the necessary knowledge, resources and infrastructure to fully implement the

3Rs, as required by legislation and expected by general public. To this end, we

recommend that consideration be given to focusing NHP research in centres of

excellence and improving existing networks for information sharing.

R08 Researchers, ethics committee members, animal welfare committee members and

animal care staff must ensure that they keep abreast of the latest techniques that

enable reduction in animal numbers and the refinement of methods and techniques

to reduce suffering, and put this evidence base into practice. Ideally, this is a

continuous process of improvement and CPD. Institutional constraints on

implementation of the 3Rs should be recognised and addressed. Training should be

included in non-animal methods to improve skills in multidisciplinary science and

technology can help drive the development of non-animal technologies by training

researchers in new skills for continuing their research in their field, without animal

use.

R09 CPD should be seen as an essential part of the training process for research workers

using NHPs. Researchers, veterinarians and animal technicians should make full use

of the CPD opportunities available, and this should be supported by their research

organisation. CPD should form part of training records and assessment of

competency.

R10 Available training opportunities are fragmented and not always aligned with best

practice. Consideration should be given to development of an accredited,

harmonised training course for those involved in NHP research to provide a solid

foundation in NHP behaviour and best practice in their care and use.

Non-human primates testing (update 2017)

Final Opinion

R11 Funding should also be provided for exchange visits and practical workshops for

those directly involved in the care and use of NHPs to facilitate sharing of best

practice.

R12 Whilst there has been progress with refinement of neuroscience studies involving

NHPs, funders and researchers should focus on significantly refining devices and

methods as well as improving the spatial and temporal of existing and new non-

invasive imaging technologies to refine, validate and ultimately replace the use of

highly invasive techniques in NHP. Experiments should make full use of modern

imaging, biotelemetry, virtual models and other technologies, and sharing of data

and resources (animals, tissues and equipment) between researchers and

institutions, to reduce and refine NHP use.

R13 There needs to be improved means of assessing pain and distress in NHPs, including

the psychological impact of their use in research. Scientific knowledge about the

welfare impact of husbandry and procedures, even after refinement measures have

been applied, needs to be factored into harm-benefit assessments.

R14 Reduction in NHP use would be assisted by greater use of more efficient

experimental designs (e.g., factorial designs). Careful consideration should also be

given to sample size calculations for NHP studies. The NC3Rs Experimental Design

Assistant (https://eda.nc3rs.org.uk/) is one source of tailored advice for those

lacking institutional access to expert statistical support.

R15 Breeding facilities should contribute to improvements in both animal welfare and

quality of science by ensuring that animals are well habituated to humans and better

able to cope with scientific and husbandry procedures.

R16 Literature searches, mainstream scientific and specialist conferences and resources

from institutions like the NC3Rs are all sources of information and advice on

opportunities to apply the 3Rs and the benefits of this. Researchers and associated

animal care staff should ensure that they continually review and adapt their working

practices accordingly.

R17 For the sake of transparency and monitoring progress in the application of the 3Rs,

it would be desirable that all Member States report on the level of severity of

experimental procedures, origin of animals, generation and first time use.

7.2 How to overcome barriers?

R18 To overcome the wider social, non-technological barriers to the implementation of

alternatives it will be necessary to stimulate changes in both scientific and societal

attitudes and in scientific practice by improving and expanding the 4Cs

(Commitment, Communication, Cooperation and Coordination). This will influence

the risk aversive nature of society that makes it difficult to move away from familiar

methods to new alternative methods, where there is less historical data to fall back

on. The 4Cs will also help to reduce problems related to competition, the reputation

of researchers and entrenchment and policy create problems.

R19 When communicating about NHP use with the public, the scientific community

should provide an accurate description of the benefits, harms to animals and

limitations of such research, and be realistic about the potential outputs and

impacts. For safety testing, regulatory requirements and scientific consideration, it is

possible to use NHPs, with proof that NHPs are most representative of humans,

regarding pharmacodynamics and pharmacokinetics.

R20 Researchers should report their experimental methods and results comprehensively,

Non-human primates testing (update 2017)

Final Opinion

accurately and transparently. They should transparently evaluate and report on the

progress in the development of alternative methods and their validation, but also on

scientific limitations of alternative methods as well as the uncertainty of how to

translate the findings from such models and build up the necessary knowledge base

to refer to.

R21 It is also necessary to reduce the timescale and bureaucracy associated with the

process of formal validation and to overcome the lack of regulatory harmonisation

both within and across sectors.

R22 The EC should instigate strategic research funding initiatives to support the scientific

and technological development required to achieve NHP replacement, or at least

considerable progress towards it. This would also help the scientific community meet

the policy objectives of the Commission.

R23 To progress towards complete replacement of NHPs in safety testing, it will be

necessary to gain advance molecular biology techniques, including, for example, a

better understanding of signalling pathways, mode-of-action information, modelling

and bioinformatics. By integrating these data and the results of in vitro testing, —

omics technologies applied to in vitro systems and physiologically-based

pharmacokinetic modelling, non-animal models will together more closely represent

what happens in a human body.

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Final Opinion

8 CONSIDERATION OF RESPONSES RECEIVED IN PUBLIC

CONSULTATION

A public consultation on this Opinion was opened on the website of the Scientific

Committees from 10 February to 26 March 2017. Information about the public

consultation was broadly communicated to national authorities, international

organisations and other stakeholders.

The public consultation involved 190 contributors from Academia, researchers, Member

States, Non-Governmental Organisations and industry, providing a total of 318

comments, each of them addressing several issues. Each submission was carefully

considered by the SCHEER and the Scientific Opinion and biblography was revised

accordingly. In addition, a public hearing took place on 14 March 2017

. 19

organisations participated in the public hearing.

The comments from the public consultation and SCHEER responses are available at:

https://ec.europa.eu/health/scientific_committees/consultations/public_consultations/sc

heer_consultation_03_en

9 ABBREVIATIONS AND GLOSSARY OF TERMS

3Rs

Replacement, Reduction, Refinement

4Cs

Commitment, Communication, Cooperation and Coordination

AAALAC

Association for Assessment and Accreditation of Laboratory Animal

Care

ABPI

Association of British Pharmaceutical Industry

BMIs

Brain-Machine Interfaces

CHMP

European Committee for Medicinal Products for Human Use

CNS

Central Nervous System

CPD

Continuing Professional Development

DG ENV

Directorate-General for Environment in the European Commission

DBS

Deep Brain Stimulation

DILI

Drug-Induced Liver Injury

EDA

Experimental Design Assistant

EFSA

European Food Safety Authority

EMA

European Medicines Agency

https://ec.europa.eu/health/scientific_committees/events/ev_20170314_en

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Final Opinion

EPAR

European Public Assessment Report

European Commission

European Union

FDA

US Food and Drug Administration

FRAME

UK Fund for the Replacement of Animals in Medical Experiments

FUS

Focus ultrasound

IACUCs

Institutional Animal Care and Use Committees

IADRs

Idiosyncratic Adverse Drug Reactions

ICH

International Council for Harmonisation

iPSCs

Induced Pluripotent Stem Cells

mAbs

Monoclonal Antibodies

NC3Rs

UK National Centre for the Replacement, Refinement and Reduction

of Animals in Research

NHPs

Non-human primates

PFC

Prefrontal Cortex

PRIMTRAIN

European Network Behavioural Management and Training of

Laboratory non-human Primates and Large Laboratory Animals

Scientific Committee

SCENIHR

Scientific Committee on Emerging and Newly Identified Health Risks

SCHEER

Scientific Committee on Health, Environmental and Emerging Risks

SCHER

Scientific Committee on Health and Environmental Risks

SCCS

Scientific Committee on Consumer Safety

Tuberculosis

TMS

Transcranial magnetic stimulation

WMA

World Medical Association

Working Group

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Final Opinion

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Final Opinion

ANNEXES

Annex I- Definitions and examples of replacement, reduction and refinement

‘R’

Definition

Examples

Replacement

Methods that avoid or replace the

use of animals in areas where they

would have otherwise been used.

In some cases, relative

replacement (i.e. replacing the use

of live ‘protected’ vertebrates with

vertebrate cells or tissues, early

life-stages or non-vertebrates) has

been implemented as a first step to

absolute replacement.

Human volunteers, tissues and

cells; mathematical and computer

models; established animal cell

lines, or cells and tissues taken

from animals killed solely for this

purpose (i.e. not having been

subject to a regulated procedure);

non-protected immature forms

vertebrates; invertebrates, such

as Drosophila and nematode

worms

Reduction

Methods that minimize the number

of animals used per experiment or

test, either by enabling researchers

to obtain comparable levels of

information (of a given amount and

precision) from fewer animals, or

to obtain more information from

the same number of animals

(thereby avoiding further animal

use).

Improved experimental design and

statistical analysis; sharing of data

and resources (e.g., animals and

equipment) between research

groups and organizations; use of

technologies, such as imaging,

that enable longitudinal studies in

the same animals.

Refinement

Methods that minimize any pain,

suffering, distress or lasting harm

that may be experienced by the

animals, and improve animal

welfare.

Refinement applies to all aspects of

animal use, from the housing and

husbandry used to the scientific

procedures performed upon them.

Use of appropriate anaesthetics

and analgesics regimens; avoiding

stress by training animals to

cooperate with procedures such as

blood sampling; providing animals

with appropriate housing and

environmental enrichment which

allows the expression of species-

specific behaviours.

Reproduced from Graham ML, Prescott MJ (2015) The multifactorial role of the 3Rs in

shifting the harm-benefit analysis in animal models of disease. European Journal of

Pharmacology 759, 19-29.

In the European Union, non-protected immature forms are embryonic and fetal

mammals, birds and reptiles up to the last third of their gestation or incubation period,

larval forms of amphibians and fish until they can feed independently, and cephalopods

until the point at which they hatch.

Non-human primates testing (update 2017)

Final Opinion

Annex II - Publically available information concerning the publication of

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etre_animal_NL

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2014

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http://vmvt.lt/node/607

http://vmvt.lt/sites/default/files/2014_m_bandomuju_gyvunu_statistine_ataskaita.pdf

http://www.pvd.gov.lv/lat/kreis_izvlne/veterinr_uzraudzba/majas_un_izmeginajumu_dzivn

iek/izmeginajumu_projektu_netehnis/publiskie_parskati

http://www.ma.public.lu/ministere/rapport/index.html

No animals were used in Malta in 2014

https://www.rijksoverheid.nl/documenten/jaarverslagen/2016/03/01/zo-doende-2014

Non-human primates testing (update 2017)

Final Opinion

http://www.bip.nauka.gov.pl/sprawozdania_zwierzeta/

http://www.dgv.min-

agricultura.pt/portal/page/portal/DGV/genericos?generico=1149097&cboui=1149097

http://www.ansvsa.ro/documente/admin/Statistica%202014_49571ro.pdf

Not yet published.

http://www.uvhvvr.gov.si/si/delovna_podrocja/dobrobit_zivali/zascita_zivali_v_poskusih/

http://www.svps.sk/dokumenty/zvierata/VPHU_1_2015.pdf

UK: https://www.gov.uk/government/statistics/statistics-of-scientific-procedures-on-living-

animals-great-britain-2014

Northern Ireland: https://www.dhsspsni.gov.uk/sites/default/files/publications/dhssps/asp-

statistics-of-scientific-procedures-on-living-animals-ni-2014.pdf