On the 1st of June 2021, the Swedish 3Rs Center held an hour long webinar on the Identification of Endocrine Disruptors. This webinar included a short introduction to endocrine disruptors, a presentation on the history and current works of PEPPER, an interactive breakout session in which the audience got to share their work on endocrine disruptors to identify studies that could be useful for the PEPPER platform, and of course a Q&A at the end.
The first to present was Gregory Moore of both the Swedish 3Rs Center and the Swedish board of Agriculture. He was given 3 questions to cover; What is an endocrine disruptor? Why are we concerned with them? and Why is it important to develop and validate test methods?
He explained that endocrine disruption is a form of toxicity where the endogenous hormone system is affected by these disruptors. The endocrine disruptors (EDs) are naturally occurring in the human body and are important in teenage development as well as the environment. But they can also be synthetic, such as BPA, and cause adverse effects to health, namely developmental, fertility, immune and other issues 1. Regulations saw the need for a definition and for this he directed our attention to the WHO definition which can be found here.
The reason we are concerned with EDs is because of the adverse health effects but also due to the fact that EDs can have many mechanisms of action making them harder to predict/target. They imitate the normal hormones in the body altering the ability for synthesis of the natural hormones. These alterations can be in the form of agonistic or antagonistic effects or by disrupting the endocrine system balance. A very small concentration of EDs can cause a big effect on the body. The cost of inaction was also mentioned as a reason for concern alongside the lack of harmonisation in the EU of ED regulations (this is being worked on). On the subject of the EU, only CA 15 known EDs have been identified to date and are accessible here. The endpoint methods and tests for these disruptors are both time consuming and expensive to develop and at present leave a lot to be desired. So they are looking towards new approaches for these.
The importance of development and validation was also emphasised. Information is needed for regulatory assessment and the information should be reliable and reproducible. A few different organisations are working on systems to produce this data. The OECD has devised a Mutual Acceptance of Data (MAD) system. An Endocrine Disruptors Testing and Assessment Advisory Group (EDTA AG) has been established for 20 years and has been working on both in vitro and in vivo validation along with new approach methods (NAMs). EURL ECVAM have been working on in vitro validation implementing the 3R approach to animal use in their established network of laboratories. The PEPPER project is endeavouring to speed up the validation process and EURION has a collection of projects in the pipeline for the production of new screening and testing methods funded by the EU’s Horizon 2020 program.
The main aim of this webinar was the introduction of the PEPPER project. This was delivered by Phillipe Hubert the Director of PEPPER. The project is a public-private platform for pre-validation of ED characterization methods and is part of the French National Strategy on EDs. It’s a not for profit organisation and their aim, as mentioned by Mr. Moore, is to speed up the validation process. Its main source of funding comes from the private sector but also from members. They aim to increase resources both public and private and see the european dimension as the way forward. They help with organisation and funding in labs developing pre-validation methods. This funding goes a long way in encouraging the development of new methods and compiling validation evidence for the governing bodies. This can be used for internationally recognised validation as a step towards quickening the process. But validation is very slow, in Sweden they put funding towards validation but the lack of this elsewhere is contributing to the lag. There is no systematic way to prioritise validation.
He sees an increasing concern and decreasing confidence in the science and the authorities over the science. ED related diseases cost the EU €157 billion and the US $340 billion. This brings up the importance of species relevant assays especially due to the increase in environmental affairs caused by biodiversity. There is an ongoing divide between academic experiments and regulatory science. This often leads to disunited interpretation of experimental results, which in turn weakens the commercial activities, environmental preservation, and confidence of the general public in science. The current timeframe for validation is an average of 7 years but can be as high as 20 years emphasising the need for a shortened process.
Since the initial proposal of a pre-validation platform in 2013, PEPPER has established a key role in the governance of validation. A shared concern across the EU is the need for more validated methods for EDs. PEPPER plays an integral part of the pre-validation process, there are 5 steps for the development of new methods and PEPPER is involved at all stages helping with identifying the methods, the pre-validation operations and providing assistance for the application. The relevance and scientific committees of PEPPER are involved in the other two steps. PEPPER is also working on including anonymised test subjects into these steps.
Moving on to more recent work, in 2020, they set their sights on identification and quantification methods as well as building contacts with developer labs. Through this they have answered the call from institutions and are filling the gaps in pre-validation. An example of an ongoing project, hPLACENTOX-PE, is looking at the link from endocrine function to female infertility and placental function. The pre-validation of this method includes 5 steps for both mammalian and non-mammalian in vivo toxicology and PEPPER is working to shorten this process to 3 steps.
Throughout this there have been some challenges. These were in the form of identifying relevant methods that also had potential, striking a balance between promising acceptance and actual acceptance by the regulatory bodies, and making academics aware of the constraints of these processes. They had some difficulty in the collection of historical data and some confusion with the validation in EC programmes. But moving forward they hope to provide a toolbox for ED characterisation which, in turn, will give the OECD more mature submissions more likely to be validated, therefore speeding up the validation process. Another aim of theirs is to open up communication on the need for more validated methods via working groups on organoids, MPS etc. and webinars. More on PEPPER can be found here.
We were then passed over to his colleague Kristina Fant who gave a brief overview of what the relevance committee does within PEPPER. They work with test method validation including all the steps from defining the method to the actual validation. They test the biological and chemical safety of all new methods in a large-scale, GMP compliant way.
The webinar finished with a Q&A session that mainly consisted of specific questions about the PEPPER platform and funding. One interesting thing mentioned was that along with updates to the pre-criteria and criteria for application to PEPPER, criteria for new methods like in silico will soon be added to the website increasing the categories of new methods they deal with. The webinar was well run and, aside from the sound difficulties, was easy to understand and follow. It presented encouraging developments for the 3R method of animal use with more funding and time being put into new methods.
3Rs – Reduce, Refine and Replace animal use in testing
BPA – Bisphenol A
EC – European Commission
ED – Endocrine Disruptors
EDTA AG – Endocrine Disruptors Testing and Assessment Advisory Group
EURL ECVAM – EU Reference Lab for alternatives to animal testing
GMP – Good Manufacturing Practices
MAD – Mutual Acceptance of Data system
MPS – Microphysiological Systems
NAMs – New Approach Methods
OECD – Organisation for Economic Co-operation and Development
WHO – World Health Organisation
1. Endocrine Disruptors, https://www.niehs.nih.gov/health/topics/agents/endocrine/index.cfm (accessed 2 June 2021).
Written by Saoirse Foley.