1

What are "alternative test methods " ?

The term "alternative" is generally associated with the Principles of the 3Rs, - Replacement, Reduction and Refinement - of animal testing. In this context an alternative method serves to fully replace an animal test, to reduce the number of animals needed in a test, or to refine an animal testing procedure in order to reduce pain and suffering. Alternative test methods that are developed to reduce or replace animal experiments are typically based on either in vitro systems or on computerbased models. In vitro test methods use (reconstructed) tissues, whole cells or parts of cells. Recent advances in cell-based research include the development of two-dimensional and three-dimensional cell (co)-cultures which mimic very closely cells and tissues in the human body. The growing use of 'omics' technologies (e.g. transcriptomics, proteomics and metabonomics) in combination with in vitro test systems allows a comprehensive analysis of the impact of a chemical at the molecular level and can indicate potential toxicity pathways that may lead to adverse health effects. Computer-based approaches (often termed in silico or non-testing methods) are becoming increasingly powerful and can be used effectively to predict the toxicity of a chemical from its basic properties. Computer models are also an important tool for efficiently integrating toxicological information derived from complimentary in vitro and in silico methods. A non-testing approach frequently used in the safety assessment of industrial chemicals, for example, is called 'read-across' technique where toxicological effects for one chemical are predicted using data for the same toxicological effect from another chemical, which is considered to be similar in terms of chemical structural, physico-chemical properties, or bioactivity.

2

What is the historical context for efforts within EU legislation to reduce testing on animals?

As far back as the 1980s, the EU made a commitment to pursue efforts to replace animals used in experiments and to improve the welfare of those still being used. This led in 1986 to the adoption of a Directive (86/609/EEC) on the protection of animals used for experimental and other scientific purposes. Besides measures related to the use of experimental animals, such as their housing and care, requirements for the authorisation of persons and establishments and the minimisation of pain, suffering and distress, the Directive also requested the use of alternative (non-animal) methods when reasonably and practicably available, and in case animals need to be used, requires methods that use the minimum numbers and inflict minimum pain, suffering, distress and lasting harm on animals. It also called for encouragement of research into the development and validation of alternative methods. The text, however, did not explicitly mention the principles of the 3Rs. In 2008 the Commission adopted a proposal to revise the Directive 86/609/EEC. Following this, in November 2010, a new, significantly more detailed and comprehensive Directive (2010/63/EU) was adopted, which replaced the previous Directive from 1 January 2013. The revised Directive spells out the principles of the 3Rs, removes ambiguities as to the legal requirements for using alternatives and requires the Commission and Member States to contribute to the development and validation of alternative methods. It also stipulates the legal basis for the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and introduces further measures to facilitate the acceptance and promotion of alternative approaches. Furthermore, the REACH Regulation constitutes a step towards the protection of experimental animals. One of the objectives of this Regulation

1

states that testing on vertebrate animals for the purposes of the Regulation shall be undertaken only as a last resort and that duplication of tests should be limited. In addition, the Regulation aims at the promotion of the development of alternative methods for the assessment of hazards of substances. Other relevant legislation includes the Cosmetics Directive with its amendments, Directive 91/414/EEC concerning plant protection products, the Plant Protection Regulation 1107/2009, the Biocides Directive 98/8/EC, the future Biocides Regulation, and Directive 2004/10/EC on principles of good laboratory practice. 3

What are the 3Rs?

"3Rs" stands for "Replacement, Reduction and Refinement" in animal testing. Replacement refers to methods which avoid or replace the use of animals in areas where animals would have otherwise been used. Reduction refers to methods which minimise animal use and enable researchers to obtain comparable levels of information from fewer animals or to obtain more information from the same number of animals, thereby reducing future use of animals. Refinement refers to improvements which minimise actual or potential pain, suffering, distress or lasting harm, or which improve animal welfare in situations where the use of animals is unavoidable.

4

What is the mandate of EURL ECVAM?

The European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission's Joint Research Centre was originally founded in 1991. Recently, however, its role has been broadened and explicitly captured within legislation, namely the Directive 2010/63/EU on the protection of animals used for scientific purposes. In this context ECVAM has been nominated as the European Union Reference Laboratory for Alternatives to Animal Testing, with the new acronym EURL ECVAM. According to the Directive, the main tasks of the EURL ECVAM are (1) to promote the development and use of alternatives in the area of regulatory testing but also in biomedical research; (2) to coordinate at the European level the validation of alternative methods, also by involving the newly established EU Network of Validation laboratories (EU-NETVAL) located in Member States, and to participate with its own laboratories in the evaluation and validation of test methods; (3) to disseminate information on alternative test methods through databases (DB-ALM http://ecvamdbalm.jrc.ec.europa.eu/) and other media; (4) to act as a focal point for information exchange on development, use and acceptance of methods and to promote dialogue between all relevant players in the field. To this end, EURL ECVAM is fostering dialogue with EU Member States' competent authorities and EU agencies (e.g. ECHA, EFSA and EMA) through its consultation body for the regulatory relevance of proposed methods (so-called PARERE) and is maintaining close dialogue with its stakeholders through the ECVAM Stakeholder Forum (ESTAF) that brings together European industry associations, non-governmental organisations (e.g. animal welfare groups) and academic research associations.

5

Which alternative methods has EURL ECVAM already validated?

EURL ECVAM has validated full replacement methods in the toxicological areas of skin irritation, skin corrosion, skin absorption/penetration as well as phototoxicity. EURL ECVAM has also validated methods for partial replacement and reduction of animal testing in the areas of eye irritation, eye corrosion, skin sensitisation, genotoxicity, carcinogenicity, reproductive toxicity (embryotoxicity and endocrine disruption) and acute oral toxicity. Many of these validated methods, besides being used in research, have been taken up into EU law and other forms of internationally accepted test

2

methods, such as the Test Guidelines of the OECD (Organisation for Economic Cooperation and Development) and the European Pharmacopoeia. 6

Which are the main steps in the validation of an alternative method?

EURL ECVAM's validation process encompasses 4 major steps, each involving extensive interaction with stakeholders and international partners: (1) Typically the process starts with the submission of a test method and its scientific and technical evaluation by ECVAM scientists. If necessary, ECVAM scientists may request the advice of ECVAM's Scientific Advisory Committee (ESAC) on scientific/technical issues, and interacts with its regulatory and stakeholder bodies (PARERE and ESTAF) with regard to relevance of the submitted method for application in particular contexts (e.g. for safety assessment in different regulatory sectors such as cosmetics, plant protection products or industrial chemicals). Moreover, ECVAM involves other international validation organisations collaborating under an umbrella cooperation agreement called "International Collaboration on Alternative Test Methods (ICATM)". Consultation with ICATM partners is a key element to gauge the potential global usability of an EU-validated test method i.e. to judge to which extent will the test method, once validated, be usable by EU companies to generate data on their substances in agreement with, for instance, laws in the US or Japan. (2) Once the test method has been fully defined, the next step is to design and execute a validation study including a ring trial in view of addressing the defined purpose of the assay. At this step ECVAM will decide on the involvement of laboratories in EU Member States (e.g. EU-NETVAL labs) or elsewhere. (3) Once the study has been completed, it is peer reviewed by the ESAC which provides ECVAM with an independent scientific opinion on the quality of study and the potential use of the method; (4) Taking ESAC's opinion into account, EURL ECVAM develops its Recommendation summarising the outcome of the validation study including a description of the overall performance of the method, its applicability, its limitations and its potential use in different contexts. Before publishing this recommendation, ECVAM consults with its regulatory and stakeholder bodies, the ICATM, and in addition invites comments by the test method developer ("right to be heard") and from the general public.

7

Why does validation often take a long time?

Although validation is an intrinsic part of the scientific process, it is also an essential prerequisite for regulatory acceptance. Validation has to be rigorous and impartial in order to convince regulators and end-users that an alternative method can provide the same or an enhanced level of protection of human health and the environment when compared to traditional methods. Methods undergoing validation are typically assessed with respect to their reliability and relevance. In terms of reliability, a method must first be stable and reproducible within one laboratory and then shown to be transferable and reproducible in at least two other laboratories that have not performed the method before. Such "ring trials" can be difficult and time consuming to set up and execute due to their technical and administrative complexity. The selection, sourcing and distribution of test chemicals and the training of lab personnel can often pose considerable challenges. In addition, pilot "pre-validation" trials are often necessary to optimise and refine the test protocol. On completion of the validation study the results are then subject to independent scientific peer review. Nevertheless, EURL ECVAM is continually looking for opportunities to streamline its validation process to maximise efficiency while retaining high levels of quality and rigour.

3

8

Besides validating alternative methods, what else has EURL ECVAM accomplished?

Besides coordinating alternative methods validation in the EU, the tasks entrusted to the JRC and EURL ECVAM include the European Commission's contribution to the Test Guidelines Programme at the OECD, research and development of both in vitro and computational methods, operation of a GLP compliant in vitro test facility, dissemination and promotion of methods, training in alternative methods, provision of direct support to other Commission Services and Agencies, organisation of expert workshops, contribution to projects of the European Partnership for Alternative Approaches to Animal Testing (EPAA, http://ec.europa.eu/enterprise/epaa/), and participation in the International Cooperation on Alternative Testing Methods (ICATM, http://iccvam.niehs.nih.gov/about/icatm.htm). EURL ECVAM also contributed significantly to international standards related to the validation of alternative test methods, now enshrined in the globally applicable OECD guidance document No. 34 on validation and international acceptance of test methods. The JRC and EURL ECVAM has also been involved in the implementation of the European chemicals legislation (REACH Regulation), participating in the development of user guidance to describe the test methods and strategies, often involving nonanimal tests that can be used to generate information for REACH dossiers. Finally, scientists working for the EURL ECVAM participated and still participate in key EU research projects aiming at the development of safety assessment approaches and alternative methods.

9

What is the EU doing to promote research and validation on alternative methods?

The European Commission is doing everything it can to guide and support the development, validation and promotion of non-animal methods for use in regulatory safety assessment and as tools in biomedical research. Public-private partnerships such as SEURAT-1 (http://www.seurat-1.eu/) and the EPAA (http://ec.europa.eu/enterprise/epaa/) have a very important role to combine efforts across industry, academia, governmental and nongovernmental organisations so that everybody is pulling in the same direction. The Joint Research Centre (JRC) and its EURL ECVAM will continue to look for innovative ways to translate the results of scientific research into useful and valid tools that can significantly reduce society's reliance on animal testing while ensuring the highest level of protection for human health and the environment and maintaining industrial and economic competitiveness of the EU.

10

What is the EPAA?

The aim of the European Partnership for Alternative Approaches to Animal Testing (EPAA) (http://ec.europa.eu/enterprise/epaa/) is to promote the development of new ‘3R’ methods - replacement, reduction, refinement - as alternative approaches to the use of animals in safety assessment.

11

Are there cultural barriers preventing a larger diffusion of alternative methods?

In the past the regulatory community has been typically cautious when considering the use of alternative methods for safety assessment. Today, however, there is overwhelming consensus in the toxicology community that alternative test methods are indeed powerful tools capable of delivering relevant information useful in many regulatory contexts – especially if validated by international and independent bodies, such as ECVAM.

12

Which scientific challenges are still limiting a reduction in animal testing?

The main challenge concerning the reduction of animal testing is to sufficiently advance many areas of toxicological and biomedical sciences to deliver complete and effective non-animal solutions that can detect all the possible adverse effects that a chemical could possibly cause to an organism. For instance, human reproduction involves multiple organs, tissues and cells together with hormonal systems acting on the level of the entire body. Chemicals that are potentially toxic to reproduction could therefore act in a plethora of ways through many different mechanisms. This level of complexity most likely can not be modelled in one in vitro test but needs to be recapitulated through the use of multiple complimentary non-animal

4

tests and computational models. 13

What scientific advantages do alternative methods have when compared to animal tests?

The animal tests that are standard nowadays in toxicology have been developed decades ago and thus are not based on modern toxicological science or the latest biomedical technologies. Although there is considerable experience worldwide with using animals testing for protecting human health and the environment, animal tests suffer from a number of drawbacks. For one, animal and human physiology can be very different in some aspects, which makes extrapolation of results from animal studies to the human context difficult. Moreover, most animal tests are based on simply the observation of adverse effects (e.g. lesions or tumours) caused by a chemical rather than looking at the underlying cause. Thus, conducting animal testing in the traditional manner does not contribute to our overall understanding of toxicity or any possibility to predict it. Many animal tests take a long time to conduct (e.g. years in some cases) and are expensive. In vitro methods very often use human cells which are more representative of the human response to toxins than the animal; once developed and validated, they are for the most part inexpensive and practical to implement.

14

Why do we look for validation of replacements – the animal tests were themselves never validated?

In the past, testing on animals has been the best available and most reliable source to carry out risk assessment in many industrial sectors. While the animal testing methods have not undergone the same type of validation which has been set up for alternatives, long-standing experience with their use was attained and they have been included in the European Test Method Regulation. In many circumstances, therefore, they are required by law. There are of course limitations with animal testing and thus the development and validation of non-animal methods is not only important from an ethical standpoint, but it is also important to ultimately provide a better safety assessment framework for protecting human health and the environment.

15

Is animal testing for cosmetics not already prohibited in the EU?

Yes, there can be no testing for cosmetic purposes in the EU. Animal testing for finished cosmetic products has been banned in the EU since 2004, and animal testing for cosmetic ingredients has been banned since 11 March 2009. As from March 2009, it is also prohibited to market in the EU cosmetic products containing ingredients which have been tested on animals regardless of their origin. This applies to a series of aspects to be tested in order to demonstrate the safety of cosmetic products, such as skin and eye corrosion/irritation, acute photo toxicity, skin penetration, genotoxicity and acute systemic toxicity. For the three most complex aspects (repeated-dose toxicity, including skin sensitisation and carcinogenicity, reproductive toxicity and toxicokinetics) the marketing ban th came into force on the 11 of March 2013.

16

Is it true that alternative methods are not yet available for some adverse health effects?

It is correct to say that alternative test methods are not yet sufficiently developed to fully replace the standard animal tests for the following adverse health effects or "toxicological endpoints": 

repeated-dose toxicity (problems related to long term repeated exposure to a chemical)



skin sensitisation (the toxicological impact associated with chemicals that have the intrinsic ability to cause skin allergy)



carcinogenicity (the ability of substances to cause cancer)



reproductive toxicity (refers to a wide variety of adverse effects that may occur in different phases within the reproductive cycle, as a consequence of one or more exposures to a toxic substance, including effects on fertility, sexual behaviour, embryo implantation)



toxicokinetics (the penetration into, fate within and elimination from the body of a toxic substance, including its absorption,

5

distribution, metabolism and excretion). 17

How did the Commission establish the availability of alternatives to animal testing?

On 23 July 2010, the Directorate-General for Health and Consumers launched a public consultation on a draft "Report on alternative (nonanimal) methods for cosmetic testing: current status and future prospects – 2010". The report was drawn up by working groups of experts nominated by stakeholders and by the ECVAM. Experts were appointed in their personal expert capacity, not as stakeholder representatives. The findings of the expert group were published in a peer reviewed scientific journal. (http://ec.europa.eu/consumers/sectors/cosmetics/files/pdf/animal_testing/fi nal_report_at_en.pdf

18

Which validated alternative methods are available today?

Validated alternative methods are available for the identification of corrosive substances, skin irritants and severe eye irritants, skin phototoxicity and skin penetration as well as to assess genotoxicity. In addition, significant advances have been made in reducing the number of animals used in tests, for example in relation to acute toxicity. However, certain toxicological effects based on more complex processes are not yet or not fully covered by alternative methods. These relate to very complex biological processes for which the scientific basis to fully replace animal testing is still not fully established. They mainly relate to: 

repeated-dose toxicity (problems related to long term repeated exposure to a chemical)



skin sensitisation (the toxicological impact associated chemicals that have the intrinsic ability to cause skin allergy)



carcinogenicity (the ability of substances to cause cancer)



reproductive toxicity (refers to a wide variety of adverse effects that may occur in different phases within the reproductive cycle, as a consequence of one or more exposures to a toxic substance, including effects on fertility, sexual behaviour, embryo implantation)



toxicokinetics (the penetration into and fate within the body of a toxic substance, including its absorption, distribution, metabolism).

with

While full replacement is not yet accomplished for those endpoints there is a potential for partial replacement strategies, to reduce the number of animals used. For more information please refer to a report "Alternative (non-animal) methods for cosmetics testing: current status and future prospects—2010" at

http://ec.europa.eu/consumers/sectors/cosmetics/files/pdf/animal_test ing/final_report_at_en.pdf 19

Which scientific improvements are still needed to address the most complex cases?

We still lack basic scientific understanding of how exactly chemicals lead to toxicological effects. One common challenge for assessing a chemical against complex systemic health effects is to simply predict where the chemical ends up in the body and for how long it stays there. Moreover, chemicals are typically transformed and broken down in the body and sometimes the transformation products (metabolites) are actually the source of toxicity. Understanding the fate of a chemical in the body, i.e. how it is absorbed, distributed, metabolised (transformed) and excreted, and its so-called toxicological "mode of action" is essential. The identification of key events in toxicity pathways would enable the targeted development of specialised test methods to produce the information needed to decide if a chemical would trigger an adverse outcome, and also, hopefully, the intensity of this effect in the organism. In this context, there will not be one alternative method to replace one animal test method. Combinations of tests and computer models need to

6

be used in an integrated way to predict systemic toxicity effects. 20

If testing on animals for cosmetics is forbidden, what kinds of tests are still being done on animals in the EU?

No animal testing for cosmetic purposes can be carried out in the EU since March 2009. However, animal testing can be still carried out for several other purposes. According to the sixth Report from the Commission to the Council and the European Parliament on the Statistics on the number of animals used for experimental and other scientific purposes in the member states of the European Union, about 12 million animals were used in the EU annually in 2008 for experiments. 8.7% (about 1 million) were used for toxicological testing and other safety evaluations, about 15% for production and quality control in human and veterinary medicine, while the vast majority being used in science (biological and medical research and development). Reductions in the number of animals used in these experiments since 2009 would be difficult to quantify. In relation to the overall number of animals used in the EU for testing in all fields, those used for cosmetics testing was very low even prior to the 2009 ban: 5500 in 2005, 1818 in 2007, 1510 in 2008.

21

Is animal testing for cosmetics done in countries beyond Europe?

Europe upholds the strictest position worldwide with regard to animal testing for cosmetics. No other country prohibits testing for cosmetics and a number of countries, such as for example China, explicitly requires animal testing data in order to bring cosmetics ingredients on the market.

22

Which animals are used in testing for cosmetics?

Animals used for cosmetics testing are rabbits, rats and guinea pigs.

23

How can I know that a cosmetic product I buy is not tested on animals, if it comes from outside the EU?

Consumers can be sure that no finished cosmetic products have been subject to animal testing in the EU since 2004. Consumers can also be sure that no cosmetic ingredients have been tested for cosmetic purposes in the EU since 2009. This so called 'testing ban' was reinforced by a 'marketing ban' on all cosmetic ingredients or products tested outside the EU after the same date. The only exception related to animal testing for the more complex toxicological effects (endpoints), such as repeated dose toxicity, reproductive toxicity and toxicokinetics, for which the deadline was set to 11 March 2013, Thus, from this date the consumer can be sure that also imported cosmetic products are not tested on animals. It is however important to realize that many ingredients used in cosmetics are used for other purposes as well – such as in pharmaceuticals, detergents, food, paints etc., so they may be subject to animal testing requirements under the legislation which covers these products.

24

What is the meaning of animal testing-free labels on cosmetics?

Animal testing-free labels usually mean that animal testing has not been conducted on any of the ingredients in the cosmetic product after a certain (cut-off) date. However, this does not necessarily mean that all the ingredients were never tested on animals, since in some cases the producing company may still rely on old animal test data generated before the cut-off date.

25

Does the EU promote alternative methods at international level?

At international level, the European Commission promotes cooperation to facilitate the development and the validation of alternative methods and their recognition by our main trading partners. A framework of cooperation has been established between the US, Japan, Canada and the Republic of Korea in a regulatory dialogue called "International Cooperation on Cosmetic Regulation" (ICCR), which led to the International Cooperation on Alternative Test Methods (ICATM).

26

Can substances tested on animals under REACH be used as cosmetics ingredients?

The majority of cosmetics ingredients will fall also under the scope of REACH, which means that they might have to be the object of animal testing in order to fulfil the REACH requirements. It is, however, important to note that testing requirements are not the same for all legislative domains. For example REACH does not require carrying

7

out a two-generation reprotoxicity test or repeated dose toxicity tests for an industrial chemical produced in low tonnage (1 to 10 tonnes). Performing such tests on a substance and annexing them to a REACH registration for the sole purpose of allowing then the use of this substance as a cosmetics ingredient would be probably subject to legal challenges. 27

Are alternative methods accepted under the EU regulation on chemicals, REACH?

In general, REACH supports using any existing information to establish the toxicological profile of a substance. This includes "non-standard" information produced using, for example, in vitro methods, structure-activity relationships, read-across and chemical categories (comparing a given chemical with other ones for which toxicological information is available). Thus in reality in many cases information requirements of REACH can be satisfied by a "weight of evidence" approach which combines various pieces of evidence coming from alternative sources, thereby avoiding prospective animal testing.

28

Did EURL ECVAM ever validate a method based on human embryonic stem cells as an alternative method to animal testing?

To date EURL ECVAM has not received any submission of a test based on human embryonic stem cells (hESC), nor has it conducted any validation activities on in vitro methods based on hESC. Moreover, it is the current policy of EURL ECVAM not to enter into the validation of methods that rely on hESC. EURL ECVAM is focusing on the validation of alternative methods that are of relevance for regulatory safety testing. Validation is a prerequisite for the acceptance of toxicological test methods for chemical hazard assessment both within Europe and worldwide, and as such the validation process contributes to the global mutual acceptance of toxicological data. With respect to test methods based on hESC that might be proposed for regulatory safety assessment, it is unlikely that such methods would be accepted by all EU member states because of differences in views on the use of hESC. As a consequence, a mutual acceptance of hESC-derived toxicological data would not be attainable. A way out of this dilemma was the discovery of induced pluripotent stem cells (iPSC) that share similar characteristics of hESCs. These cells are generated by genetic manipulation of human cells, e.g. derived from the skin, and make the destruction of any human embryo superfluous. iPSC hold great potential for in vitro methods, both for identifying therapeutic targets and for early safety assessment. In particular, iPSC can be a powerful tool for understanding and considering specific side-effects of drugs due to slight differences in the metabolising capacity (polymorphism) of the human population. However, iPSC technology is still rather new and in a rapid phase of development, supported both by industry and by the European Commission through its research Framework Programme. To date, ECVAM has not received any submission of an in vitro method based on iPSC, but we are aware of numerous research initiatives involved in their development.

29

How does ECVAM's work relate to the new vision of toxicity st testing in the 21 Century?

The paradigm shift in toxicological hazard assessment, which is for example at the heart of the so-called Tox21 initiative in the United States (http://www.ncats.nih.gov/research/reengineering/tox21/tox21.html), is a move from the traditional data-driven approach based on observations of effects derived from animal studies towards a knowledge-based framework, where we use our understanding of toxicological modes-of-action to rationally design integrated assessment and testing systems that are fit for a particular regulatory purpose/endpoint. The push towards the use of mode-of-action based knowledge and information within a regulatory context has been led in particular by the WHO International Programme on Chemical Safety, and is also spearheaded by the OECD, who recently announced a new Programme on "Adverse Outcome Pathways" (AOP), which will officially start in January 2013.

8

The JRC as a whole and EURL ECVAM in particular are engaging proactively in this important endeavour, including co-chairing of the OECD Advisory Group on Molecular Screening and Toxicogenomics, which is responsible for the AOP Programme. The first concrete indication of how we can use AOPs as the knowledge base for developing integrated test systems (ITS) is in the area of skin sensitisation. The OECD has already published a comprehensive AOP related to this endpoint and EURL ECVAM has proposed to use this to guide the optimal combination of in vitro test methods and in silico tools (e.g. QSAR – Quantitative structure activity relationship models) to both identify and characterise skin sensitisation hazard in support of various pieces of chemicals legislation. A number of the relevant in vitro methods to be included in such ITS are currently undergoing validation by ECVAM in order to assess their individual performance. 30

Is it possible to contemplate a future where animal testing is not necessary?

A lot of progress has been made during the last 20 to 30 years with regard to the development of alternative methods, especially in the field of routine toxicity and safety testing of chemicals and biologicals. It is conceivable that in the mid-term most safety testing can be done through the intelligent combination of non-animal testing methods, computer models and integrative approaches. But also in the area of basic research, non-animal models can be used to advance our knowledge. However, there are limits to the use of reductionist systems. It is for instance difficult to imagine how we could truly understand the brain function without studying the entire organ as well. However, also here, there is great potential for non-invasive approaches such as advanced imaging techniques.

9