Focus on the 3Rs: Validation of new methods

Focus on the 3Rs

March 2022

Validation of new methods

Solid evaluations of a new test method are crucial to gain trust and acceptance from those who will use it. Methods intended for regulatory risk assessment also has to undergo a formal validation. We try to sort these concepts out and we highlight the need for collaboration to speed things up concerning a transition to a more human-based research and to reduce the use of animals for scientific purposes.

We will give you an insight into the system for development, evaluation and validation of in vitro methods.

What is method validation?

Method validation is a process of determining how well a method performs for a given purpose, to ensure that it is based on sound science and is useful from a regulatory perspective. Validation is a rigorous scientific process where a large number of test items with a known effect on the endpoint of interest are being evaluated numerous times in numerous laboratories.

Ideally, the laboratory work should be performed under a quality system, preferably Good Laboratory Practice (GLP) to ensure reliability and traceability of the data. All the data from a validation study must be available for an independent review.

Formal validation is the key to regulatory utility of a test method. To be accepted by authorities in all OECD member countries, data needs to be generated:

by a test method, that has been validated successfully and described in an OECD guideline
by the quality system of Good Laboratory Practice.

This means that experiments for safety testing of the same product do not need to be repeated for different markets.

Guidance for the validation studies

OECD has published a guidance document on the validation process: OECD GD 34. It gives advice on how to design and conduct validation studies. Validation can be prospective, i.e. generate new data, or retrospective, i.e assessing data that already exists or comparing with an existing guideline (catch-up validation). OECD GD 34 describes the validation steps in a modular way and some of the modules can be performed together. The modules are answering different questions regarding the performance and characteristics of the test method:

Module 1: Test method definition

What endpoint is a method intended to predict, and what is the scientific rationale for why the method can predict this endpoint? Why is the method needed? How is the method to be performed? What are the decision criteria for the classification of data? What are the limitations of the method?

Module 2: Intra-laboratory repeatability and reproducibility

How do results from different runs compare in the same laboratory?

Module 3: Inter-laboratory transferability

Can the method be performed in another laboratory than the one where it was developed?

Module 4: Inter-laboratory reproducibility

How do results from different laboratories compare?

Module 5: Predictive capacity

How well does the method predict the biological endpoint it is designed to evaluate?

Module 6: Applicability domain

What kind of samples is the method suitable for?

Module 7: Performance standards

What should a similar method or biological test system be able to achieve to be judge dequivalent to the one that has been validated?

OECD Guidance Document 34: Validation and International Acceptance of New or Updated Internationally Acceptable Test Methods for Hazard Assessment (oecd.org)

Validation institutions in the European Union

The European Union Reference Laboratory for alternatives to animal testing, EURL ECVAM,is responsible for coordinating and undertaking validation studies for alternative methods for safety testing of chemicals in the EU.

The EURL ECVAM Scientific Advisory Committee (ESAC) performs independent peer-review of performed validation studies independent of whether they have been coordinated by EURL ECVAM or performed by other actors. ESAC can issue recommendations that a test method should be adopted as an internationally accepted test guideline.

Who can submit a method for validation?

Any scientist can submit a test method to EURL ECVAM for uptake in their validation program. EURL ECVAM will assess the regulatory potential and the efforts required for finalising the method, and undertakes validation studies depending on available resources. EURL ECVAM hosts a network of qualified laboratories, EU-NETVAL, that assist in data generation and also supports other tasks related to promoting the 3Rs.

Adoption of validated methods

A method that has been successfully validated can be submitted for adoption either as an OECD test guideline or as an international standard via ISO.

This brings about a process: first, drafting of a guidance document or a standard, than review by international experts and finally, voting on a finalized document by the members of the organisation that issues it.

The OECD's work with test methods takes place mostly in various working groups with experts from authorities, academia, research institutes, industry, environmental organizations and animal welfare organizations. In Sweden, the Swedish Chemicals Agency is responsible for nominating experts to these expert groups.

In ISO, the drafting is based on consensus between members of the technical committee responsible for the area. Members in the committees are experts organised by country. In Sweden, the national partner is the Swedish Institute for Standards.

Webinars and other resources on validation

In the fall of 2021, the Swedish 3Rs Center organised a mini-series of webinars with the focus on validation. We have covered several parts of the path from development of a novel in vitro method to a validation study and the subsequent peer-review.

During the first webinar, we invited Dr. Raffaella Corvi to introduce a submission process and prioritization of methods for validation at EURL ECVAM and Dr. Emma Pedersen to bring attention to the OECD Guidance Document No. 286 on Good In Vitro Method Practices (GIVIMP). In the subsequent webinar, we had Dr. Kristina Fant talk about hands-on laboratory-based validation work according to the OECD GD No. 34 and Prof. Ian Cotgreave introduce, the scientific peer-review from the ESAC member perspective.

The series were well received by the participants, the majority of which use or develop in vitro methods. Participants expressed a strong interest to receive further information on method validation. We had a Q&A session after each webinar, where participants could ask questions and receive answers and comments from our guest lecturers. Questions with regard to timeline, use of animal-free ingredients and funding were of particular interest to the audience and we would like to outline them in more detail below.

Timeline of the validation process

Validation is a multi-step process and several factors influence its timeline, like an overall development level of the method on submission and eventual need for supplemental information or clarification. However, a process can be further optimised to shorter the timeline by:

employment of integrated testing strategies e.g “cluster validation” instead of current “onetest-one validation” approach
dedicated funding to make it possible for more laboratories to join in and for more methods to be validated faster.

Use of the animal-free ingredients

Those who submit in vitro methods to ECVAM for validation are strongly encouraged to use non-animal derived reagents. Besides contribution to improved reproducibility of science, the use of animal-free reagents may help with prioritization, in case two similar methods are submitted at the same time. Therefore, efforts should be made to replace ingredients like serum, matrigel, trypsin and antibodies with non-animal derived alternatives.

Peer review, on the other hand, it is solely based on the evaluation of scientific evidence. However, in its opinion ESAC can give recommendations e.g. to replace animal-derived components. For in vitro methods that are at advanced stages of validation (ring-trial), a “catch-up” validation with performance standards to replace animal-derived components can be made.

Funding opportunities

Funding opportunities for validation studies are currently rather limited and difficult to find. Opportunities vary in each member state, but generally funding is available through national funding, 3Rs Centres or the EU funding programs (e.g Horizon 2020 or the newHorizon Europe). Furthermore, industry, NGOs and private foundations might have an interest to fund validation.

Joint Research Centre cannot push for increased national funding for validation, but stakeholders in respective countries can.

The Swedish Research Council is funding validation studies with 2 million SEK annually, when it comes to projects that have been prioritized by the European network PARERE. You can learn more about PARERE in another article in this focus letter.

Infographics and upcoming webinar

During the spring of 2022, the 3Rs Center will release infographics describing the validation process, both for scientific and regulatory purposes. We will publish the infographics in social media and on our website. We will also distribute it to our networks and stakeholders.

We further aim to organise a new webinar on method validation to meet the active interest from stakeholders.

Join the Replace network

In 2019, we instated a national Replace network. Keep in touch with our work by becoming a member of the network. To register, send an e-mail to us at: 3Rcenter at jordbruksverket.se.

Join the LinkedIn group

Find contacts and collaboration between different stakeholders with an interest in non-animal methods and Replace. Join the group and discuss Replace-related topics!

Swedish 3Rs Network for Replace on LinkedIn (linkedin.com)

New national contact point for the PARERE network

PARERE – EURL ECVAM’s Network for Preliminary Assessment of Regulatory Relevance – is a network of national regulators. The network shall provide opinions on regulatory relevance of methods or approaches submitted to EURL ECVAM for validation and peer review.

It is of great importance that nationalregulators are involved early in the process of method validation in order to speed up theregulatory acceptance of alternative methods.

PARERE has its mandate in the EU Directive on the protection of animals used for scientificpurposes (2010/63/EU) and each member state shall nominate a national contact point forthe network. The contact point shall provide advice on the regulatory relevance and suitability of alternative approaches proposed for validation. The network also includesrelevant Commission services and the EU regulatory agencies EFSA, ECHA and EMA.

The 3Rs Center is the new national contact point

PARERE held its first meeting in 2011. Sweden has previously been represented by theSwedish Center for Animal Welfare (SCAW) but as of November 2021, the Swedish 3RsCenter’s administrative officer Kaisa Askevik is assigned as national contact point forPARERE:

– We are currently working on establishing a formal structure for the work, but we willutilize our close contact with Swedish agencies and our networks of experts in order toprovide relevant feedback to EURL ECVAM, says Kaisa.

February meeting

The Swedish 3Rs Center attended its first meeting on February 17 and the topics discussed at this meeting was “Extended REACH information requirements and opportunities tointroduce NAMs” and “Roadmap for respiratory sensitization testing”.

If you want to have more information regarding the PARERE network and the ongoing work,please contact the Swedish 3Rs Center.

EURL ECVAM Network for Preliminary Assessment of Regulatory Relevance (ec.europa.eu)

Sofia Kamlund: an engineer with expertise in nanotechnology and intellectual property rights

One of the recent members in the Swedish National Committee is Sofia Kamlund, engineer and former researcher, working with intellectual property rights. Sofia is dedicated to the development of alternative research methods.

Sofia is an engineer trained in nanotechnology at Lund University. While doing her specialisation in bionanomedicine, she met Professor Stina Oredsson in the lab on a toxicology course. Under her influence, Sofia decided to continue working with cell cultures and non-animal methods. After graduation, she started doctoral studies in Oredsson’s research group as an industrial PhD.

A new microscope technique

During her PhD studies, Sofia developed a microscope technique that combines holography with fluorescence. The technique makes it possible to study movement and division of cancer cells, where as fluorescence helps identify different biomarkers.

– I did much laboratory work and data analysis as a PhD student, says Sofia. My engineering skills helped me understand how to tweak the microscope settings to get maximum data. I collaborated with a company on holographic imaging to improve the microscopy and its’ potential.

To work with the patents is to work with the future

After her dissertation in 2019, Sofia started a trainee program at AWA, an intellectual property firm that has offices all over the world. She wanted to leave the academic world and use her engineering skills in a more hands-on approach. Working with patents, is working with the future; Sofia says and refers to the daily insight in innovative products and methods. Today she is a regular employee at AWA and works with all sorts of patents.

Her motivation comes from helping people to protect their inventions – making sure to find and describe the uniqueness. She wants to encourage Swedish researchers to patent their methods:

– To patent does not mean that you lose control or will have to give it up to industry, says Sofia. It is you, the inventor, that decides how to use the patent. You can sell it to whomever you want, and make it easier for others to use, if that is what you want.

Economic incentives are crucial in a transition

To patent new research methods would also reinforce the economic incentives to develop new alternative methods. Sofia means that this is crucial to speed up the transition to non-animal research.

– Both financing and publishing is depending on people’s mind-sets, says Sofia. We need to change attitudes and frames of reference. I do not talk about the methods developed ten years ago; I mean new methods – better methods – to predict human health and physiology. A transition to human-based methods will reduce the number of animals used for scientific purposes, but above all, it will improve science.

The National Committee is a strong knowledge base

Sofia brings a broad competence into the National Committee, where she has been analternate member for a year now. In her own words, she contributes with her skills in technology and engineering, and her experiences in individual property rights and how to make business out of the alternative methods.

Sofia is also committed to animal rights. She is vice chairperson in the Swedish association Djurens Rätt (Animals Right) and she emphasises the need for a humble dialogue about the3Rs.

– The National Committee can do this – we can offer arenas for good and productive talks, where all perspectives are represented. Finally, it is all about political decisions and that is why politicians and others must know where to find objective information and facts. To offer a strong knowledge base for all parties is the Committee’s most important task, I think.

Horizon Europe with the 3Rs in focus

Horizon Europe is the EU's ninth framework programme for research and innovation, which runs from 2021 to 2027. The budget is €95.5 billion, which includes €5.4 billion from EU recovery programme from the covid-19 crisis.

The aim of the programme is to strengthen the EU's scientific and technological base, support research and innovation from idea to the market and complement national and regional funding. The programme also contributes to achieving UN’s sustainable development goals and promotes competitiveness and growth.

Through participation in the programme, one can receive funding to develop, validate or use animal-free methods. Furthermore, one has an opportunity to contribute to different objectives in the programme by using alternative methods to replace, reduce and refine animal experiments.

The Swedish 3Rs Center has listed all projects with relevance for the 3Rs with information on subject, deadline and national contact points.

Educational material for upper secondary schools

The Swedish 3Rs Center has produced a basic 3Rs educational material for upper secondary schools. Additional material and instructions for teachers and pupils are included.

The basic education has the form of three e-learning modules, free of charge, on subjects such as science, scientific methods, animal experiments, the 3Rs, ethics and animal-free methods. Every module consists of texts, films, graphics, additional material, discussion tasks and knowledge tests. One can choose to go through all three modules coherent or each module separately.

We will publish the material on a Swedish website for teachers (gratisiskolan.se) and at the 3Rs Center’s website.

Estimated release is in April.

Short films about replacement and non-animal methods

The 3Rs Center will produce several films about replacement and new research methods based on human data. Two films will describe cell models more in-depth with examples from todays’ research. Swedish researchers will tell about different methods and about how they have replaced animal models in their own research.

The other films will discuss replacement in terms that are more general and what it takes to speed up the transition to research without the use of animals.

The films will be launched continuously during 2022.

Swedish 3Rs Center │ Swedish Board of Agriculture

3Rcenter@jordbruksverket.se │ jordbruksverket.se/3R │ Twitter: @Swedish3Rs

The Swedish 3Rs Center is the executive body of the National Committee for the Protection of Animals used for Scientific Purposes and part of the Swedish Board of Agriculture.

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