Information about development and validation of methods
The Swedish 3Rs Center has made an inventory of online resources for development, evaluation and formal validation of in vitro* methods. Many of the tools are generally useful to ensure method robustness, and some are more specific for methods that are meant to be used in regulatory risk assessment.
* In vitro is Latin for “within the glass”. In vitro methods are performed outside of a living organism and often refer to different types of cell cultures.
Infographics on development and validation of in vitro methods
The Swedish 3Rs Center has produced infographics to visualize and explain the process of development, evaluation and formal validation of in vitro methods. These documents are a good starting point to get an overview of the process. They also include links to other useful resources.
Tools for development of in vitro methods
We have gathered guidance documents, articles and online courses that are useful in development of robust in vitro methods.
Good Cell Culture Practice (GCCP)
Good Cell Culture Practice (GCCP) is a set of six principles intended to support best practice and define minimum standards for cell culture methods. The principles were originally published in 2005 and an updated version, GCCP 2.0, was published in 2022 to include more complex cell culture systems. Both versions are useful in development of cell culture methods.
Good In Vitro Method Practices (GIVIMP)
Good In Vitro Method Practices (GIVIMP) is a guidance document from the Organization for Economic Co-operation and Development (OECD). GIVIMP combines scientific advances with principles of quality assurance, and is a good tool both for test method developers striving for regulatory usefulness, and for the general scientist wanting to generate robust and reliable data.
The EU education and training platform ETPLAS has created an online education module (EU-60) based on the GIVIMP guidance document. The module is free of charge but you have to create an account and login to access the contents.
Recommendations and templates for reporting of detailed protocols
To ensure that other scientists can implement and use the methods that you have developed, they need to be described in sufficient detail in your publication.
Promoting Reusable and Open Methods and Protocols (PRO-MaP) and Reporting In Vitro Experiments Responsibly (RIVER), are two sets of recommendations aiming to increase and improve the reporting of detailed protocols for methods used in the life sciences. These recommendations outline actions that can be taken by each of four stakeholder groups; researchers, research institutions and departments, publishers and editors, and research funding bodies, in order to produce clear step-by-step protocols for research methods.
For regulators working with risk assessment to be able to use data from methods that are not part of an established test guideline, the methods need to be clearly described. The Template for the Description of Cell-Based Toxicological Test Methods (ToxTemp), has been developed to help researchers meet the documentation requirements from regulators.
Formal validation
Methods that are to be used in regulatory risk assessment need to be formally validated. Formal validation is a rigorous, science-based evaluation process. This process is used to assess the method’s reliability and its relevance for the intended use.
The EU Reference Laboratory for alternatives to animal testing (EURL ECVAM), is responsible for formal validation within the EU. There are also European initiatives that prepare methods for formal validation. One example is the PEPPER platform for pre-validation of methods to characterize endocrine disruptors.
The Organization for Economic Co-operation and Development (OECD) and the International Organization for Standardization (ISO) work globally to standardize and harmonize guidelines and ensure reliable data. Validated test methods can be taken up into the OECD test guideline program or into a standard developed by the ISO, and thus gain regulatory acceptance.
The OECD also provides guidelines and instructive webinars on how validation studies should be performed.
EURL ECVAM
The EU reference laboratory EURL ECVAM is tasked to promote and facilitate the use of non-animal methods in research and safety testing. EURL ECVAM is also responsible for formal validation within the EU. EURL ECVAM thus assess and priorities methods that are submitted for validation based on development, optimization, eventual pre-validation as well as potential relevance for the 3Rs. If a method is found relevant and mature enough for validation, EURL ECVAM decides on the type of study needed and coordinates the study. The hands-on laboratory work is conducted in multi-laboratory validation teams, often by laboratories in the EURL ECVAM network of specialized laboratories (EU-NETVAL), where Sweden is represented by RISE Research Institutes of Sweden.
At the EURL ECVAM website you can find information about validation, the method submission process, validated test methods, computational methods, the EU-NETVAL network of specialized laboratories and more. You can also find the form for submission of new test methods.
PEPPER
PEPPER is a public-private platform that acts to speed up validation of methods specifically for evaluation of endocrine disruptors. PEPPER coordinates and helps to fund pre-validation studies to promote further validation and regulatory acceptance.
The PEPPER platform has produced a Self Assessment Questionnaire (SAQ) to assess the readiness of your method for characterization of endocrine disruptors with respect to validation. This is called the ReadEDTest and is formed like a checklist for in vitro methods.
OECD
The OECD is an international organization that works to build better policies for better lives in many different areas.
In the area of risk assessment of chemicals, the OECD works globally to standardize and harmonize guidelines to ensure reliable data.
The OECD has described the validation process in guidance document number 34 (GD34). This document is currently being updated and a new version will soon be available.
A validation study should preferably be performed in a quality assured work flow, ensuring data integrity and the possibility to reconstruct the study. The OECD also provides guidance on the quality system Good Laboratory Practice (GLP) which can be used for this purpose.
More guidance documents that can be relevant for the validation process are found below.
- Guidance Document for the Development of OECD Guidelines for the Testing of Chemicals (GD1) (oecd.org)
- Revised Guidance Document on Developing and Assessing Adverse Outcome Pathways (GD184) (oecd.org)
- Guidance Document for Describing Non-Guideline in vitro test methods (GD211) (oecd.org)
- Guidance Document for the Use of Adverse Outcome Pathways in Developing Integrated Approaches to Testing and Assessment (IATA) (GD260) (oecd.org)
A complete list of available OECD publications of test guidelines, can be found here:
The OECD also provides webinars with information on validation. Some of the webinars are directed towards researchers with only little or no previous knowledge on method validation. Other webinars are directed towards regulators.