Project platform

Replacement of animal-based potency tests for human rabies vaccines

Project aim

The aim of this project is to replace the current in vivo potency test for the release of human rabies vaccines (NIH, mice intracranial challenge test) with an in vitro antigen (G glycoprotein) quantification assay using ELISA technology.

Background

The problematic NIH test involves the use of large numbers of animals, of which half develop distressful rabies symptoms. Furthermore, the variation of the NIH test is high and the test, therefore, may have to be repeated to meet regulatory requirements. Thus, its replacement will have a substantial impact on animal use.

3Rs added value and EPAA actions

EPAA’s contributions have been indispensable for this project by supporting previous workshops and facilitating manufacturers, regulatory and scientific bodies to elaborate a clear strategy for the replacement of the human rabies vaccine NIH method. Since 2012, the EPAA has enabled the creation of an international working group to coordinate a more harmonised approach of the alternative assay development through the acquisition and distribution of a common set of rabies vaccines. The EPAA supports the BSP 148 project which is required for full validation of the in vitro method.

This activity requires input from various stakeholders (control authorities, manufacturers, and academia) and cannot be carried out by a single manufacturer. The 2 European manufacturers of human rabies vaccines (Sanofi Pasteur and GlaxoSmithKline) are members of EPAA and provide additional in-kind contribution by participating in the study. The project continues the work started within the EPAA Vaccines consistency approach project and if successful it will contribute to the promotion of 3Rs and overall harmonisation of vaccine quality control via dissemination workshops.

The results of the collaborative study have been presented under the name of EPAA in various world congresses on alternatives and other international conferences.

Stakeholders involved

In addition to EDQM that coordinates the BSP 148 collaborative study, a number of laboratories and international experts from control authorities, manufacturers, academia and EURL ECVAM contribute to the project.

Milestones

• A pre-collaborative study involving 5 laboratories evaluating 3 ELISA methods was run in 2013 to identify a suitable replacement method. Compliant sub-potent samples of vaccines prepared from different virus strains were obtained from European and non-European sources with the support of WHO, EDQM and industry.
• Based on the results of this pre-collaborative study, a candidate method was selected.
• The detailed study report has been published in Vaccine (Morgeaux S., et al., 2017.) Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA – Results of an international collaborative study.
• A major achievement in 2016 has been the endorsement of the proposal for a wide collaborative study to validate the transferability and robustness of the selected ELISA.
• This collaborative study is being run since 2017 under the biological standardisation programme of the Council of Europe and the EU Commission, coordinated by the EDQM (study coded BSP148) with the support of EPAA.
• First experiments confirmed that the selected ELISA is applicable to most virus strains used worldwide for human rabies vaccines.
• In 2018 the results were presented to the developing countries vaccine manufacturers network (DCVMN: India, May 2018), the European society for alternatives to animal testing (EUSAAT: Austria, September 2018), and the joint meeting of national toxicology program interagency center for evaluation of alternative toxicological methods (NICEATM).
• In 2023, Phase 2 of the collaborative study has been completed; the study participants discussed the results and learnings during an expert meeting jointly organised by EDQM and EPAA in June. This meeting also discussed the planning of Phase 3 of the study, which includes interlaboratory testing in production, assessing the specificity and reproducibility of the ELISA-based assay.

Next steps

It is expected that the data generated throughout the ongoing study will support the revision of the Ph. Eur. monograph on human rabies vaccines as well as global acceptance of the replacement method. The project has prompted considerable interest from international regulators and NGOs.

Identifying clinical signs that are predictive of mortality

Project aim

This project aims to identify opportunities to waive the acute toxicity animal testing requirements completely or, where this is not possible, to refine the decision-making steps or assessment strategies so as to minimise the suffering of animals. The ultimate goal is to develop an animal-free decision framework for acute systemic toxicity testing.

Background

Whilst acute toxicity testing is no longer needed in the pharmaceutical sector and in vivo acute toxicity testing is no longer possible in the cosmetics sector, evaluation of acute toxicity remains a requirement for chemicals and agrochemicals in order to establish their overall hazard profile and to meet classification, labelling and packaging (CLP) requirements that are relevant to human safety, for example, in emergency situations. The REACH standard information requirements for the endpoint of acute toxicity (REACH Annex VIII, point 8.5.3.) were revised in May 2016 allowing a waiving of acute toxicity testing via the dermal route under certain circumstances.

In previous years, the EPAA has provided scientific justification in support of such a ‘weight of evidence’ approach. Acute toxicity by the oral route is the most common testing requirement and therefore this route has been prioritised by EPAA.

3Rs added value and EPAA actions

Because the EPAA brings together a wide range of industry sectors, it can offer a unique overview of the regulatory and scientific issues in this field, and can recommend 3Rs approaches that could be adopted widely across different sectors.

Stakeholders involved

The project is based on collaboration with industry partners, experts from EURL ECVAM, the European Commission, UK National Centre for the 3Rs (NC3Rs) and the UK Chemicals Regulation Directorate (CRD).

Milestones

• The project was initiated in 2008 and reviewed the scientific and regulatory drivers for acute toxicity testing in order to promote the use of 3Rs approaches in this area.
• In 2010, a workshop was organised and a paper was published on ‘cross-sector review of drivers and available 3Rs approaches for acute systemic toxicity testing’ (T. Seidle et al, 2010, Toxicological Sciences). The workshop identified opportunities to waive acute animal testing requirements completely or, where this is not possible, to refine the decision-making steps or strategies to minimise suffering of test animals. Recommendations on a 3Rs-based classification & labelling decision framework were prepared including replacement of death as an endpoint.
• Since 2014, additional evidence in support of this decision framework is being developed through data mining of acute oral toxicity studies in collaboration with the NC3Rs and the UK Chemicals Regulation Directorate.
• Data from previously filed acute toxicity studies are collected and will be analysed to confirm that clinical signs (evident toxicity) are an appropriate alternative to death as an endpoint.
• The findings will be considered in the overall decision framework document.
• Due to delays in selection and accessibility of the data, the project has made slow progress in the last years. In 2016, more than 450 previously filed, acute toxicity studies were screened and in 2018 data from more than 70 studies were collected and evaluated by the NC3Rs.
• Following the statistical analysis of the results, a webinar was co-organised with the NC3Rs in 2022, during which the preliminary results were presented and the recommendations were discussed with the data contributing companies.
• In 2023, a manuscript with the results of the analysis and recommendations to support wider use of TG 420 has been completed and submitted for publication to a peer reviewed journal. The results will also be presented at relevant scientific conferences, e.g. WC12 in Canada and EUROTOX.

Next steps

The project dissemination plan includes, in addition to the peer-reviewed publication, presentation to international conferences, discussions with regulatory authorities and the OECD. 

Supporting the deletion of international regulatory requirements for in vivo safety tests

Project aim

In the production of biological products, manufacturers are required to confirm potency and safety of each batch of product. This may involve the use of laboratory animals. Directive 2010/63/EU prohibits manufacturers in the EU from using an animal test method, if an alternative, non-animal method is recognised by the European pharmacopoeia.

However, if alternatives are not internationally harmonised and accepted in other regions, then excessive or unnecessary animal testing may be undertaken by manufacturers, in order to gain access to other, non-EU markets.

This project aims to achieve global harmonisation in batch testing requirements for human and veterinary vaccines, as well as other biologicals. It is hoped the project will lead to better incorporation of the 3Rs in potency and safety testing strategies. Project deliverables include mapping of regulatory bodies responsible for establishing potency and safety testing requirements, identifying key differences in QC testing requirements between pharmacopoeias or equivalents assessing test methods with related benefits and risks.

Stakeholders involved

European Medicines Agency, European Commission, EURL ECVAM, pharmaceutical industry, animal health industry, Council of Europe - European Directorate for the Quality of Medicines and Healthcare (EDQM).

Milestones

• 2013: Project launch
• 2013: Mapping of regulatory bodies responsible for potency and safety testing requirements
• 2015: EPAA workshop on international harmonisation of biologicals, Egmond aan Zee, the Netherlands. The mapping results, case studies and recommendations were discussed with regulatory authority representatives at this workshop, with a view to progressing to full international harmonisation.
• 2015: Publication of the September 2015 workshop report in a peer-reviewed journal
• 2016-2018: The project team submitted formal requests to WHO, OIE and Ph. Eur. to encourage deletion of specific tests (GST / ATT and TABST) from their recommendations or requirements.
• 2018: The Ph. Eur. Commission endorsed the complete suppression of the test for abnormal toxicity (ATT) from 49 monographs in the Ph. Eur. (implementation from 1 January 2019).
• 2022-2023: Supporting the replacement of the rabbit pyrogenicity testing (RPT) by in vitro methods.Pyrogenicity testing is relevant to a wide range of products including vaccines, chemicals and blood products. The Ph. Eur. monographs encourage replacement ofpyrogen testing in rabbits by suitable alternative methods, however, more than 50 Ph. Eur. product-specific monographs mention the rabbit pyrogen test (RPT) and not the alternatives. As such, the rabbit test continues to be used widely. Moreover, in vitro tests require product-specific validation and are often not accepted outside EU. Stimulated by the EPAA project team, EDQM defined a strategy in 2021 to amend Ph. Eur. monographs (removal of RPT) in the years to 2026.  New chapters on Monocyte Activation Test (MAT) for vaccines and on pyrogenicity were published for comments in July 2022. Revision of various chapters started in 2023. In addition, a new chapter of the Ph. Eur. was introduced in July 2020 for the bacterial endotoxin test (BET) using a recombinant Factor C assay as a potential replacement of the existing test based on horseshoe crab blood extract.
• A joint EDQM-EPAA Workshop was organised in February 2023 to discuss the deletion of RPT together with a training in MAT. The event was open to all stakeholders impacted by RPT deletion (e.g., health authorities, industry users, service providers) and invited international perspectives to support global alignment in promoting alternative assays such as MAT and BET. A paper reporting the main discussions from the February workshop was published in Biologicals in August 2023.

Next steps

As a follow-up to the joint EDQM-EPAA Pyrogenicity Workshop in February 2023, the team continues activities to support inter-pharmacopoeias dialogue towards the acceptance of animal-free pyrogenicity testing also outside Europe.

An assessment is ongoing by the project team to identify and work upon additional priority areas where harmonisation discussions are needed.

Optimal duration of non-clinical studies to assess safety of monoclonal antibodies (mAbs)

Project aim

The project aims to re-evaluate regulatory practices from a non-clinical perspective focusing on monoclonal antibodies, building on previous research experience at the Dutch Medicines Evaluation Board (MEB) with support from EPAA, several pharmaceutical companies and the NC3Rs.The results demonstrate ongoing commitment of both regulatory agencies and industry to strive towards initiating fewer animal studies without compromising the ability to assess benefit and risk.

Background

In order to evaluate the safety and efficacy of new drugs or indications, it is often necessary to conduct animal studies. These animal studies have evolved over time and are embedded in (inter-) national guidance and legislation. However, the translational and predictive value of animal studies is increasingly being debated and questioned in the public, scientific and regulatory community. With technological advances being made, new opportunities are emerging to further implement 3R principles (refinement, reduction and replacement) in drug development. In addition, re-evaluation of regulatory guidance can further provide opportunities to restrict the use of animals in safety and efficacy studies to those which provide meaningful information that is relevant to humans.

3Rs added value and EPAA actions

This project provides a scientific basis to further reduce non-clinical studies for specific biotechnology products (monoclonal antibodies) on a case-by-case basis. While the gains could be seen as modest at face value, they are an important basis to demonstrate the ongoing commitment of both regulatory agencies and industry to strive towards initiating fewer animal studies without compromising the ability to assess benefit and risk.

Stakeholders involved

In addition to the Medicines Evaluation Board (MEB), experts from the European Commission, pharmaceutical companies and the NC3Rs contribute to the project.

Milestones

• The project started in October 2020. Criteria were established on the basis of which decisions could be made on the need and duration of non-clinical safety studies for monoclonal antibodies based on approved and non-approved drug development programmes
• A workshop was organised in April 2021 to discuss and disseminate the results, offering a potential starting point for formal discussions between agencies and companies at international level.
• The findings of the project were published in two papers, one detailing the outcome of the survey data analysis and the development of a Weight of Evidence model and the second led by the NC3Rs on the use of recovery animals; both accepted for publication by the journal of Regulatory Toxicology and Pharmacology. In addition, the project was widely presented and discussed in meetings and symposia, with further active dissemination of the results planned.

Links to the two publications from the project can be found here:

• Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach
• The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain
• A joint NC3Rs/EPAA webinar was organised on 17 April 2023 to provide an overview of the two publications from the project, discuss key results and recommendations. In particular, a proposed weight-of-evidence model was highlighted, with case-studies explaining the potential decision process. Additionally, 3Rs opportunities identified within the dataset, including group sizes and use of recovery animals, were presented. A recording of this webinar is available.

Next steps

The results of the project could provide a scientific basis to further reduce non-clinical studies for specific biotechnology products (monoclonal antibodies) on a case-by-case basis.

Waiving of 2-year carcinogenicity studies

Project aim

The objectives of this project are to identify opportunities for improving the science supporting the regulatory testing of agrochemicals, and to achieve a reduction in the use of animals when assessing the potential for carcinogenicity.

Background

The project was launched in 2017 as a follow-up of a previous, successful EPAA project on the prediction of carcinogenicity of pharmaceuticals which provided evidence that in many cases a 2-year carcinogenicity study in rats could be waived without compromising human safety. The waiver could be applied based on prior knowledge of the pharmacological properties of the compounds in question, integrated with histopathological findings from 3-6 month repeat dose toxicity studies and together with evidence for lack of genotoxic potential and lack of hormonal perturbation. The conclusions were based on data analysis of 289 pharmaceutical compounds and demonstrated a prediction rate of 92% and 98% for noncarcinogens and for carcinogens, respectively.

Two-year carcinogenicity studies are part of the regulatory requirements for pharmaceuticals, food additives, chemicals and agrochemicals. Such studies entail the use of large numbers of animals. Currently, to assess the potential for a non-genotoxic compound (i.e. not inducing DNA damage) to increase the risk of cancer in humans, 2-year carcinogenicity studies in rats or mice are performed. The relevance to the human safety of data from rodent carcinogenicity studies has often been questioned but still this type of study remains the default requirement. Regulatory requirements also include repeated dose toxicity studies of 3-6 months duration for compounds intended for long-term administration.

3Rs added value and EPAA actions

The expected impact to the 3Rs is substantial. Based on the results obtained in the previous EPAA-supported project for pharmaceuticals (Van der Laan et al. 2016), the number of agrochemicals requiring a carcinogenicity study may be reduced by 40-60%. Since the proposed approach makes use of sub-chronic (3-month) in vivo toxicity tests that are performed anyway, each agrochemical for which a carcinogenicity study may be waived would save a large number of animals from being tested for a long period of time. If successful, this project could be possibly extended to other types of chemicals in other industrial sectors.

Stakeholders involved

The project is supported and coordinated by EPAA and is being conducted by RIVM (National Institute for Public Health and the Environment, The Netherlands). The project team includes members from industry and regulatory bodies, EURL ECVAM, as well as researchers from the previous pharmaceutical-focused project.

Milestones

• 2017: An initial 2-year project was launched as a follow-up of the previous, successful EPAA project on the prediction of carcinogenicity of pharmaceuticals. Data was collected for >400 agrochemicals. Of these, 170 are considered to be non-genotoxic carcinogens and thus relevant to the objective of providing an overview of modes of action (MOA) and key events in carcinogenicity. Analysis of the data identified the most relevant MOAs and target organs involved in agrochemical carcinogenesis, and determined potential parameters and assays for detecting those MOA, non-genotoxic compounds, and target organs.
• 2019: From the MOAs identified in this first agrochemical project, a subset was discussed in an EPAA expert workshop in June 2019, in Brussels, with participants including toxicologists, regulators, industry and NGOs. The main outcome of the workshop was that the MOA-driven approach was strongly supported as the way forward, complementing other relevant international activities, e.g. by the OECD and US-EPA.
• Although the project identified a selection of 10 MOAs or MOA networks underlying non-genotoxic carcinogenic potential of agrochemical compounds, some crucial data gaps were also identified. These include the observation of treatment-related tumours for which no MOA information could be identified (“known unknowns”) as well as assessment of the human relevance of each of the MOAs. For the majority of the MOAs, an alternative approach (i.e. without the need for a 2-year carcinogenicity assay) remains to be developed.
• The project generated two peer-reviewed publications: one manuscript on all the work completed in the project and another reporting from the 2019 workshop.

         - Heusinkveld H. et al. (2020) Towards a mechanism-based approach for the prediction of nongenotoxic carcinogenic potential of agrochemicals. Critical Reviews in Toxicology 50 

         - Luijten M. et al (2020) A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals. Regulatory Toxicology and Pharmacology 118 

• A second agrochemicals-focused project started in March 2020 with the objectives of

        (a) identifying “known unknowns” and consolidation of MOAs

        (b) developing a weight of evidence approach to predict carcinogenic potential of agrochemicals without the need for two-year rodent studies.

• An approach for identifying “known unknowns” has been established. This primarily includes filtering irrelevant findings. For example, those related to high dose and excessive toxicity. Based on a database of 115 tumors in various organs, involving 72 substances, criteria for filtering high-dose findings have been agreed. These were applied to the set of 115 tumour cases for which the MOA was unknown. This resulted in the definition of “known unknowns”, together with a consolidated list of MOAs.

Next steps

The project will next focus on defining a WoE approach for predicting carcinogenic potential, together with disseminating the results for “known unknowns” via presentations at international conferences and a peer-reviewed publication that is expected to be completed by early 2024.

A user forum sharing knowledge and experience on the use of NAMs for skin sensitisation decision-making.

Project aim

This project aims to promote training and knowledge-sharing regarding the application of non-animal strategies for assessing skin sensitisation.

Background

Sensitisation of human skin to chemicals is a potential danger to human health and therefore reliable hazard and risk assessments need to be performed to ensure these ingredients can be used safely. The current legislation in Europe for the safety evaluation of chemicals (REACH: 1907/2006) and cosmetics (EU Cosmetics Regulation: 1223/2009) includes the requirement to assess the skin sensitisation potential of a substance or formulation.

The focus of intensive previous work of many stakeholders has been the assessment without the use of animals, and as a result a number of validated non-animal tests or NAMs are accepted as OECD Test Guidelines (TGs).

These and other approaches are being increasingly used as part of Defined Approaches (DAs) to inform Integrated Approaches to Testing and Assessment (IATA) for skin sensitisation.The project has focused on training and peer-to-peer knowledge-sharing workshops that EPAA organised in collaboration with Cefic LRI, Cosmetics Europe, IFRA and other stakeholders in 2013, 2015 and 2019.

These workshops were hosted by ECHA, in Helsinki, and targeted experts from industry and regulatory authorities.

The concept of the user forum as a mechanism for sharing knowledge and experience among members emerged from the EPAA Partners Forum (PF) on “Skin Sensitisation New Approach Methodologies (NAMs)” that was held in Brussels in October 2019.

3Rs added value and EPAA actions

The knowledge-sharing envisaged through this project is expected to improve communication about the scientific and regulatory realities. It will indicate to companies, regulators and validation authorities the advantages and potential limitations of the non-animal approaches that are currently the only possibility in the cosmetics sector, the default methods in REACH regulation and strongly encouraged in other pieces of EU legislation.

This also has the potential to enhance coordination of the various research initiatives in this field, and make progress towards replacing animal testing for the sensitisation endpoint.

Stakeholders involved

The project team includes experts from all EPAA industry sectors, EURL ECVAM and ECHA. Experts from national regulatory authorities and NGOs are invited to the training workshops.

Milestones

• February 2019:EPAA/Cefic-LRI/IFRA Europe cross sector workshop, ECHA Helsinki, 7 and 8 February2019. See the flash report, publication and documents and watch the video
• 2020-2023: Building on the recommendations of the previous workshops and the 2019 Partners Forum, activities have progressed through

       (a) an exchange of ideas in a “User Forum” including practical experience for regulatory decision-making and

       (b) EPAA-sponsored training sessions including an online training successfully completed during the 11^th World Congress (Maastricht, 2021) in collaboration with Altertox academy. Presentations were given by NICEATM and Industry members of EPAA.

• The user forum as a mechanism to build confidence in the use of NAMs was established by the EPAA Skin sensitisation team in 2020. Several successful User Forum sessions have taken place since then; each focused on a case study presentation followed by Q&A with 10+ organizations (EPAA members) participating.
• A case study from Cosmetics Europe has been accepted as an OECD IATA. To maximise the impact of the user forum, the team is inviting other interested parties to share knowledge and to involve a wider audience.
• In 2023, a new round of user forum sessions is in preparation to gain confidence in NAMs with some complex case studies and involvement of a wider audience.
• The dentification of sector-specific needs and gaps is ongoing. User forum sessions on agrochemical and pharmaceutical assessments are planned next.The potential to share case studies from the medical devices area is also being explored.

Next steps

Continue to share knowledge and experience via a User Forum on the use of NAMs for skin sensitisation decision-making.

Project aim

The project aims to provide a cross Industry/EC environment for appraisal of the current use of NAMs for decision-making and to define the needs to increase the confidence to use NAMs more routinely for Chemicals Registration in the EU. Discussions focus on regulatory safety decision-making using information from NAMs.

The main goal is arguably the design of novel strategies that will allow characterization of the potential of chemicals to cause systemic adverse health effects, the ultimate objective being an alternative to repeat dose toxicity studies in animals.

Background

The EPAA has a long history of exploring how new science can contribute to the 3Rs in the area of safety assessment. A series of expert Workshops have been organised over the years by EPAA focusing on systemic toxicity research by combining computational chemistry, systems biology and toxicology, e.g., the “New Perspectives on Safety” workshop in 2008;

“Harnessing the Chemistry of Life: Revolutionising Toxicology” in 2010; and the

“Blue Sky” workshop in 2019.

– Kimber et al (2011) J Appl. Toxicol., 31, 206-209

– Mahony et al (2020) Regul. Toxicol. Pharmacol.,114, 104668

3Rs added value and EPAA actions

The project focuses on and benefits from the experience of EPAA partners in use of NAMs / non-animal science for decision-making and exchange of this between Industry sectors and Commission partners in EPAA. The topic is very relevant to reduction of animal usage in REACH, C&L, and CSS.

Stakeholders involved

The two working groups (WG1 and WG2, see below)  include experts from all EPAA industry sectors, EURL ECVAM / JRC , ECHA and EFSA. Experts from national regulatory authorities, academia, NGOs and CROs are invited to the expert workshops based on recommendations of the organising team.

Milestones

• The project began with a ‘Deep-dive’ workshop on 23-24 November 2021 to share information from groups evaluating NAMs in regulatory decisions for chemical safety. A workshop flash report is available. A full report of the workshop is available in RTP. 
• The workshop aimed to share information including case studies from groups currently using NAMs for various regulatory purposes. It explored circumstances where NAMs could be used, how NAMs could provide alternative DNELs and potential to contribute to CSS. Scientific exchange focused on programmes particularly relevant to EPAA partners and discussions aimed to identify major challenges faced by policy makers and NAM users.
• A poster summarising the workshop conclusions was presented at the EU conference ‘ONE – health, environment, society’ (Brussels, 21-24 June 2022). An OpEd article in Euractiv was also published in 2022.

• The Deep-dive workshop identified keyareas for further development of NAMs

1. Building trust through defining criteria for robust, reliable and reproducible use of NAMs and level of acceptable variability (Scientific)
2. The existing regulation could be revised to further explore tiered schemes that include exposure and NAMs without seeing animal studies as the gold standard (Regulatory)
3. Increasing opportunities to use NAMs that are fit for regulatory needs (e.g. Annexes of REACH) such as sharpening the text to better facilitate the use of NAMs (Regulatory)
4. Industry and regulators will find ways to explore more NAM assessments in regulatory submissions. This is to increase confidence in use of NTheAMs in regulatory discussions (Education/training/exchange of knowledge)

These recommendations formed the basis of creating two EPAA NAMs Working Groups in 2022

• WG1 to work on NAMs for Classification of chemicals, building on the ECETOC Framework for chemical safety assessment incorporating NAMs within REACH (Ball et al 2022)
• WG2 to establish a “NAMs User Forum”, where NAM-based safety assessments addressing EU regulatory requirements can be discussed with industry and regulatory NAM end users.
• WG1: A F2F meeting of a sub-team (Ispra, 5-6 December 2022) and  follow-up sessions of full WG1 have taken place in 2023 to explore further future use of NAMs for chemical classification using systemic toxicity as a case study (employing NAMs for both bioactivity and bioavailability). This challenge will be run as a ‘Designathon’ Challenge for human systemic toxicity asking for solutions from the global scientific community and NAM developers.
• The Designathon challenge was presented and launched at the ECHA NAMs Workshop on 31 May 2023 in Helsinki (more information on this)
• WG is making progress on implementing the NAMs Users’ Forum for sharing experiences and dialogue on applying NAMs for regulatory decisions to chemical safety. Case studies are being sought from the European Commission as well as industry.

Next steps

• WG1: An EPAA Workshop will be organized in 1Q 2024 to discuss proposed solutions arising from the Designathon.
• WG2: EPAA NAM User Forum is planned for 7-8 December 2023 (Helsinki) hosted by ECHA to discuss case studies on the use of NAMs to address priority regulatory testing requirements for chemicals.
• The project is also seeking to actively collaborate / coordinate with other projects (e.g. EPAA Skin Sensitisation User Forum, ASPIS, PARC, etc).

Project aim

The aim of this project is to identify opportunities for improving the science behind the regulatory testing of medicines and chemicals through the application of the 3Rs. Among the seven sectors involved primarily in this project are those concerned with the development of human medicines, veterinary medicines, and crop protection products.

• The main goal is to examine how each sector approaches the issue of regulatory toxicology and to identify opportunities for cross-sector alignment on best practice and on the introduction of new methodologies advancing the 3Rs.
• Alignment has 2 aspects: industry alignment on testing methodologies, and regulatory harmonisation across sectors and global regions.
• Thus the project aims not only at identifying possibilities but also how these might be translated into regulatory practice

Sectors involved

• Pharmaceuticals, animal health, crop protection

Development and priorities

The project team initially conducted a survey of regulatory requirements in the various sectors and then sent a questionnaire to the relevant EPAA member associations (EFPIA, IFAH-Europe and ECPA).

Respondents were asked for more detailed information on

• the scope for variation in study design within the existing guidelines
• regional variations in regulatory requirements
• individual company practice
• the development of alternatives that could advance the 3Rs

The questionnaire was distributed to the member companies of the aforementioned organisations and the results were collated and analysed by the project team.

It was clear that there is a considerable divergence in practice within sectors, between sectors and between geographical areas, despite the existence of international harmonisation bodies such as ICH and VICH.

Moreover, the differing practice was as likely to be a result of tradition than the application of science.

Following these discussions, the Project Team selected the area of carcinogenicity testing as one that offered great potential for the project. Therefore, all sectors need to consider the carcinogenic potential of their products. Sector practice is quite divergent, the scientific value of some study designs is currently being questioned and the introduction of in vitro methods offers the possibility of a more targeted and progressive approach to animal testing.

Milestones

• 28 February 2013: EPAA workshop on carcinogenicity testing: scope for harmonisation and advancing the 3rs flash report (87 kB)

Next steps

• Ongoing discussions on MTD and human effects database

Project aim

Since late 2008, the EPAA has been exploring the opportunities that stem cells could play in developing novel approaches for the potential hazard characterisation of chemicals and drugs

• Much research on stem cells is currently ongoing within the EU and beyond, and this is considered valuable in assessing how such work may fit into an overall strategy for evaluating safety without animal testing
• This EPAA project aims to direct the stem cells research towards the industry need for safety assessment
• In the opinion of the EPAA platform on science, the current research on the use of stem cells lacks clear guidance to develop novel testing strategies for safety assessment
• The identification of gaps in testing strategies is relevant in order to select the cell types of interest, as well as the most appropriate readout to identify modes of action. This is important for multiple sectors and therefore a good opportunity for the EPAA to coordinate this activity.

Workgroups

• Workgroup 1 on fundamental research on stem cells
• Workgroup 2 on communication: stem cells forum

Project added value on 3Rs and priorities

• establishing a permanent stem cell network, which brings together experts from both sides (those working on alternatives and those working on basic research)
• setting up a central unit monitoring the quality of the stem cells
• focusing on inducible pluripotent stem cells (iPSC)
• focusing on pathways, instead of organs or cell types is preferred
• noting that first steps in analysing the signal cascades of human iPSC should be performed with well-known compounds that address a well-defined pathway

Milestones

• 28-29 April 2008: EPAA new perspectives on safety workshop, flash report (480 KB)
• October 2009: Expert meeting on the future directions of stem cells research for safety testing
• 4-5 October 2011: Stem cells and their derivatives in toxicological research programmes and as a possible regulatory tool – A gap analysis, flash report (710 KB)
• 25 April 2013: EPAA stem cells Forum kickoff meeting (WG3), flash report (360 KB)
• 28-29 August 2013: Stem cell-derived organ-like models for analysis mid-term and long-term dosing dynamics, flash report (135 KB)
• August 2014: Joint EPAA-Liverpool University CDSS stem cells forum international meeting

Project aim and rationale

The risk assessment of chemicals and pharmaceuticals continues to rely on the use of in vivo assessments, although there is an international drive towards the replacement of animal testing with mechanistic, in vitro systems.

This new approach includes the in vitro measurement of concentration-response relationships to identify where prolonged or excessive perturbations of biochemical pathways are likely to cause adverse health effects.

• This approach requires a framework, into which a wide range of in vitro, in silico and in chemico generated data, may be integrated and used to predict the consequences of exposure in humans.
• One of the major drawbacks of most in vitro methods compared to intact organisms is indeed their inability or low competence to metabolise xenobiotics. To address this issue, the EPAA launched the absorption, distribution, excretion & metabolism (ADME) project with the following goal: to develop a new, innovative bioinformatics tool to predict the ADME or human exposure based on inputs from in vitro data (and vice versa).

The tool will be web based on a Peters’ model and freely available for downloading and independent use on desktops PC to ensure confidentiality of data.

Project status

A prototype was already developed in partnership with the British Health and Safety Laboratory (HSL). This project aims to develop a tool that is more tailored to industry needs. The first tasks to be undertaken included the building of the model and interface. These were completed as planned, within the allocate 6-month period.

Stakeholders involved

UK Health and Safety Laboratory, GlaxoSmithKline, European Council of the Chemical Industry - Long-range initiative (CEFIC LRI), European Union Reference Laboratory - European Centre for Validation of Alternative Methods (EURL ECVAM), European Centre for Ecotoxicology and Toxicology of Chemicals (ECETC)

Milestones

• November 2008: EPAA In vitro absorption, distribution, excretion & metabolism (ADME) in safety testing workshop to evaluate jointly with regulators, what information was concerning metabolism is needed to permit safety assessment of substances.
• October 2011: EPAA –ECVAM joint ADME workshop on the potential for further integration of PBTK modelling into the prediction of in vivo dose-response curves without animal experiments
• June 2014: Launch of the latest project workstream on user-friendly, web-based tools for toxicokinetics in exposure assessments

References

• Bessems et al., Reg. Tox. Pharmacol., 68 (2014) 119-139): 'PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment. Recommendations from a joint EPAA – EURL ECVAM ADME workshop'
• Peters, S.A., 'Evaluation of a generic physiologically based pharmacokinetic model for lineshape analysis.' Clin. Pharmacokinet., 2008. 47(4): p. 261-75.

This project was supported by EPAA during 2006-2012.

Project aims and background

Transferring successful approaches from one sector to another is a deliberate strategy of EPAA. In 2006-2008, the EPAA devoted attention to whether a valuable alternative test developed for agrochemicals by the ILSI/HESI project on agricultural chemical safety assessment (ACSA) could also be applied to other sectors such as industrial chemicals. The feasibility of the ACSA extended one-generation study protocol was evaluated.

The ACSA testing proposal was originally designed as an intelligent testing strategy for the evaluation of agrochemicals. It aimed to address inefficient development of data, much of which is not used in the final risk assessment. This provided a scientifically robust approach to reduce and refine the studies required for registration of agrochemicals. One of the core elements was the extended one-generation reproductive toxicity study proposed to replace the classical two-generation study (OECD 416).

Potentially saving a substantial number of animals, the EOGRTS design was considered as an alternative to the two-generation test (OECD TG 416) with potential applicability and impact on chemical safety assessment across many industry sectors. The OECD states that the EOGRTS can provide all the information regulators need to make a proper safety assessment of a substance without automatically requiring the use of a second generation of animals, thus potentially saving substantial numbers of animals per test.

Stakeholders involved

Experts from companies (chemical, crop protection, soaps & detergents), EURL ECVAM, ECHA, national and international regulators

Milestones

A 2006 EPAA workshop concluded that the extended one-generation study could, in principle, be applicable to safety testing under REACH. However, it was also agreed that the complex ACSA protocol would have to be modified to meet the requirements for industrial chemical safety testing. The EOGRTS addresses far more endpoints than currently required for chemical risk assessment. Therefore, the ACSA test protocol for use under REACH needed modification to design reliable triggering and/or waiving criteria for the components of the protocol as modules.
In 2008, a task force of the European Centre for Ecotoxicology and Toxicology of Chemicals developed these criteria (ECETOC Doc. No. 45, 2008). A subsequent April 2008 workshop by the ECETOC and EURL ECVAM discussed the criteria, their relevance, and possible validation needs with invited representatives of industry, academia and the regulatory community.

The OECD held a number of expert discussions in 2010 to advance the approach.
Studies on model compounds, based on the ACSA protocol, were conducted by industry partners of EPAA (BASF, Bayer CropScience, Dow Agroscience, and Syngenta). The results from these feasibility studies have been published in a peer reviewed journal1.
[1Fegert et al. Feasibility of the extended one-generation reproductive toxicity study (OECD 443). Reprod Toxicol. 2012 Nov; 34(3):331-9]

In July 2011 the OECD endorsed the extended one-generation as test guideline 443.
Following the adoption of the test method by the OECD, an expert sub-group under CARACAL (competent authorities for REACH and CLP) was tasked with considering all the options to apply it and to explore how it could be used in the context of REACH. Through the EPAA, industry was invited to contribute to this discussion with information on costs and practical issues related to applying the test guideline. Cefic coordinated the industry response.

Practical questions related to the use of EOGRTS were discussed during further CARACAL meetings. The Commission included EOGRTS in the test method regulation and has modified the corresponding REACH annexes. The design of EOGRTS under REACH regulation was modified further by a set of triggers for the use of F2 generation and DIT, DNT cohorts to underline further the 3Rs aspects.

In 2018, the European Commission published the communication 'Towards a comprehensive European Union framework on endocrine disruptors in the different legislative frameworks to improve identification of endocrine disruptors'. It highlights a number of initiatives which are also focused on the EOGRTS test. These can provide useful information about ED properties, as far as the study is considered in its full scope, (i.e. inclusion of the use of F2 generation and DIT, DNT cohorts). This will most probably have an impact on the current chemical legislation.

Project aim

The overall goal of this platform is to synchronise initiatives for 3R methods in safety and potency testing of vaccines in Europe.

This project aims to create a technical platform for human and veterinary vaccines. The platform

• includes general strategies and policies to introduce the consistency approach in routine release activities
• prioritises the replacement of compendial in vivo tests with validated alternative in vitro tests
• defines minimal acceptance criteria for alternative in vitro tests
• reviews work plans and reports provided by human and veterinary vaccine task forces or groups working on test development and validation

Background

Vaccines require batch-specific quality control to ensure their quality, including safety and efficacy. Part of quality control, particularly for the final product, is based on animal tests required by legislation. Out of about 100 million animals that are used each year in laboratories throughout the world, 10 to 15 million animals are still being used for vaccine batch testing.

The vaccines consistency approach (VCA) for batch release is based on a thorough characterisation of the vaccine during manufacture, including formulation, using non-animal testing. The quality of subsequent batches is guaranteed by the strict application of quality systems and of consistent production of batches that are comparable to reference lots of known potency and safety.

The VCA is already used for recently registered vaccines, whereas many vaccines developed several decades ago, continue to rely on animal tests for confirming the quality of each batch.

Due to the potential of the VCA to significantly reduce the number of animal tests used in vaccines quality control, the EPAA initiated this project with the aim to provide a framework for resolving remaining scientific and technical issues and for fostering the regulatory adoption of VCA as a non-animal approach for quality control of established (conventional) vaccines.

Stakeholders involved

The European Commission, industry (European vaccines manufacturers), European Medicines Agency (EMA), national regulators, OMCLs (Official Medicines Control Laboratory), EDQM (European Directorate for the Quality of Medicines and HealthCare), international regulators (observers from the US, Canada and India) and academia.

Project organisation

The project activities were organised by a project committee (PC) chaired by the coordinator (Ian Ragan, consultant to EPAA) in consultation with a technical committee (TC) composed of experts from vaccine manufacturers, EDQM, EURL ECVAM, OMCLs and regulatory authorities for both human and veterinary vaccines. The TC was chaired by Coenraad Hendriksen, academic member of the PC.

At the technical committee meeting on 30 September 2011, 4 priority vaccines were identified and progressed further by expert working groups: DTaP, human rabies, veterinary rabies, and clostridial vaccines.

Milestones

January 2010: Workshop jointly organised by EPAA and EURL ECVAM in Brussels discussed the VCA and its potential to reduce the number of animal tests used in quality control of human and veterinary vaccines (De Mattia et al 2011, The consistency approach for quality control of vaccines: A strategy to improve quality control and implement 3Rs, Biologicals 39, p 59-65). As a follow-up, in late 2010 the EPAA agreed to initiate a VCA project.

7 April 2011: Kick-off meeting - Application of the 3Rs and the consistency approach for improved vaccine quality control, flash report (265 KB)

Milestones of the specific working groups

1. DTaP

• Workshop, 30-31 August 2012,flash report (409 KB)
• No activities started under EPAA umbrella since several activities were ongoing elsewhere (to avoid duplication)
• In 2016: activities supported by IMI 2 funding programme (VAC2VAC – IMI 2 Call 3)

2. Human rabies

This workstream aims at replacing the current in vivo immunisation challenge test for batch release (the NIH test) by in vitro tests based on antigen quantification (ELISA).

• Workshop, 8-9 October 2012,flash report (264 KB)
• Workshop, 9-11 May 2015 Joint veterinary & human rabies vaccines workshop (report in preparation, to be published soon). Agreed that in vitro ELISA is acceptable to replace current challenge/serology tests
• Collaborative study to identify most suitable ELISA was finalised in 2015
• Global agreement reached regarding the way forward
• In 2016: in cooperation with EDQM (Council of Europe), the preparation of a formal validation study supported by EPAA

3. Veterinary rabies

• Workshop, 5-6 November 2012,flash report (403 KB)
• Activities ongoing at manufacturer level
• Cross-fertilisation from the learnings of the Human rabies project
• In 2016: supported by the IMI-2 funding programme (VAC2VAC – IMI 2 Call 3)

4. Clostridial vaccines

This work stream aims at replacing in-process controls which use testing on animals, with cell-culture-based assays that have been developed by MSD animal health with support by the NC3Rs. The application of the consistency approach is feasible for clostridial vaccines but in vitro tests remain to be developed for some important strains.

• Workshop, 19 March 2013 (co-organised with EDQM, Council of Europe), flash report (138 KB)
• Workshop, 11 September 2013 (co-organised with EDQM, Council of Europe), flash report (426 KB). This workshop helped prepare the clostridium septicum vaccine collaborative study (BSP130). The EDQM BSP130 study was successfully completed in 2015 and demonstrated concordance of in vivo and in vitro methods
• Workshop, 15-16 September 2015 (co-organised with EDQM, Council of Europe, in Egmond aan Zee, the Netherlands). This workshop discussed the results of the BSP130 study as a proof of concept and start of validation for clostridium septicum vaccine in process control methods
• In 2016: a phase III study (formal in vitro test full validation) will be progressed as part of BSP130 and with the support of EPAA

The final aim is to introduce alternative methods into the Ph. Eur. relevant monograph(s).

Project achievements

• One peer-reviewed publication: De Mattia et al 2015, The vaccines consistency approach project: an EPAA initiative, Pharmeuropa Bio& SN, May 2015, p 30-56

The technical committee, where the Commission provided visibility and all parties committed to alternatives shared scientific know-how, has

• raised awareness in all stakeholders (industry, regulators, civil society) on the vaccines consistency approach
• facilitated cross-sharing between human and veterinary vaccines helping to promote new testing paradigms
• offered coordination, identified scientific gaps and enabled early collaboration helping to address them
• prepared the ground for the IMI-2 Vaccines consistency project to work towards ultimately replacing animal testing in the batch release of vaccines. This project has been accepted and will start in March 2016. With funding of approximately €8 million, this project will help develop new methods, work towards their validation and ensure regulatory engagement with an ultimate aim to lead to acceptance by regulators, implementation and use by industry

EPAA contributions

The EPAA has

• provided funding and organisational support for the regular expert meetings, 7 workshops and 2 peer-reviewed publications (De Mattia et al. 2011 and 2015).
• provided a neutral, scientifically based platform for regular discussions and exchanges between the various stakeholders facilitating the development, validation and acceptance of the approach
• supported specific collaborative studies (e.g. by funding shipment of testing materials for the clostridials collaborative study BSP130, the European Commission (via EURL ECVAM) funded data analysis within the human rabies vaccines project)
• promoted the VCA through presentations at EPAA annual conferences and other events (e.g. briefings to MEPs / intergroup for animal welfare)

Current status

• December 2015: Activities under the general VCA project are completed.

• 2016: Human rabies and Clostridial vaccines continue as stand-alone EPAA projects (see above).

Acknowledgements

The EPAA partners would like to thank the members of the technical committee and project committee for their valuable contributions to the project.

Comparison of 3D skin models for the assessment of ‘difficult to test substances’

Project duration: 2016 - 2019

Project aim

The aim of this EPAA project is to evaluate the 4 most advanced 3D skin model-based methods for their reliability in predicting skin sensitisation.

Background

Sensitisation of human skin to chemicals is a potential danger to human health and requires reliable hazard and risk assessments in order to provide appropriate risk management. The current legislation in Europe for the safety evaluation of chemicals (REACH: EU Regulation No.1907/2006) and cosmetics (EU Regulation No. 1223/2009) includes the requirement to assess the skin sensitisation potential of a substance or formulation.

Assessment without the use of animals has been the focus of intensive previous work of many stakeholders and, as a result, a number of validated non-animal tests are accepted as OECD test guidelines (TGs). These and other approaches are being increasingly used as part of optimised testing strategies based on integrated approaches to testing and assessment (IATA) of safety.

Furthermore, validated nonanimal test results are now the default information requirement for assessment of skin sensitisation potential of chemicals in REACH. 3 in vitro methods, 2 of which are based on cell culture in aqueous media (KeratinoSens®, human cell line activation test (h-CLAT)) and the in chemico direct peptide reactivity assay (DPRA) are now adopted OECD TGs.

However, these methods have certain limitations, for example when testing substances with a very low water solubility (highly hydrophobic substances) or pH instability. Therefore, 3D skin tissue models were developed and relevant markers were identified to detect sensitisers and to differentiate from non-sensitisers.

These 3D skin models are able to better mimic the skin structure and organisation, and offer other advantages such as the possibility to directly apply the substances to the model skin. However, the utility of these models for the evaluation of hydrophobic and other ‘difficult to test substances’ was unclear.

3Rs added value and EPAA actions

Establishing the complementarity of the 3D skin model-based methods with the existing methods is very important. The study addresses a critical challenge for industry, as testing of ‘unusual chemicals’ (unusual in the sense of physicochemical properties as compared to chemicals used in validation studies) is not simple. In addition, regulatory bodies need this type of information to understand the use of these models for regulatory classifications.

The project should therefore help to better understand the applicability of the models, give guidance on which methods to further sponsor and promote at regulatory level, and to facilitate the acceptance, use, and expansion of the non-animal test methods available for sensitisation testing.

Stakeholders involved

This project brings together stakeholders from European and national regulators and a wide range of industry stakeholders from various sectors (chemicals, cosmetics, fragrances, etc.). The EPAA has been collaborating with Cefic LRI and Cosmetics Europe to ensure optimal cross-fertilisation across sectors.

Milestones

• The project started in Q1 2016
• A small set of reference chemicals (N=12) for 3D-assays was selected from the list of ‘difficult substances’ assessed at the time by Cosmetics Europe in a variety of in vitro test methods
• 2017-2018: Test data on these 12 blind-coded chemicals were generated by testing them in four 3D-assays
• The results were presented at the Eurotox 2018 conference in Brussels
• In 2019 the results were described in a peer-reviewed Open Access publication

Next steps

The EPAA welcomes opportunities for further sharing of non-animal approaches and experience in testing challenging substances and mixtures in the context of the Skin sensitisation User Forum.

Novel in vitro methods to replace animal-based in-process controls

Project aim

This pilot project on clostridium septicum antigen aims at validating in vitro assays for toxicity and antigenicity and at proposing their inclusion in the European Pharmacopoeia (Ph. Eur.) to replace the current animal-based tests.

Background

Vaccines for protection against diseases caused by clostridial species in animals are widely used. Many of these vaccines currently require that the toxoid in the bulk preparation used to produce the final vaccine batches is evaluated by animal-based tests, such as the minimum lethal dose (MLD) for toxicity and the total combining power (TCP) for antigenicity. New in vitro methods to replace these animal-based tests are highly desirable and, because of higher sensitivity and accuracy, they offer the potential for improved assurance of quality and safety of the vaccines.

As the vast majority of veterinary clostridial vaccines are based on detoxified cytotoxic antigens, it is expected that these assays could be adapted to all cytotoxin-based clostridial antigens with the potential to greatly reduce the total animal usage in in-process control testing of veterinary vaccines.

3Rs added value and EPAA actions

The number of animals used for clostridial vaccines quality control tests, including the tests for toxicity and antigenicity, represents about 50% of all the animals used for quality control tests of veterinary vaccines. It is thus a high priority to identify alternative tests. Further to recommendations of the original EPAA vaccine consistency project, the EPAA has been supporting since 2013 the collaborative study coordinated by EDQM (Council of Europe) as part of the biological standardisation programme (BSP 130).

Stakeholders involved

This large EDQM project, to which EPAA is contributing, involves 14 vaccine manufacturers and official control laboratories in Europe, USA, Morocco and Mexico.

Milestones

• A collaborative study organised by the European Directorate for the Quality of Medicines & Healthcare (EDQM, Council of Europe) co-sponsored by the EU started in 2013 and was successfully completed in 2015 with the support of the EPAA.
• The results demonstrated that the non-animal, cell line-based assays for in-process toxicity and antigenicity testing of Cl. septicum vaccines outperform the animal-based methods. Based on the successful completion of the project, the in vitro assays have been incorporated into the relevant Ph. Eur. monographs.
• Proceedings from the 2015 EPAA workshop, where the results of the validation of the tissue culture sero-neutralisation methods were discussed, have been published in Pharmeuropa Bio.

Sinitskaya, N., Redhead, K., Daas, A., Bruckner, L., Behr-Gross, M-E. (2015). Validation of alternative/3Rs methods for the in-process quality control of Clostridium septicum vaccines BSP130 participants workshop report 15-16 September 2015. Egmond aan Zee, Netherlands.

• After a further optimisation, the validation of the in vitro assays was completed in  2018 through an extension of the collaborative study.

Behr-Gross M-E, Siklodi B, Le Tallec D, Halder M, Manou I, Sinitskaya N, Bruckner L, Dalmadi B, Kiss L, Redhead K. (2021) Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 2: Optimisation of cell line assays. Pharmeuropa Bio Sci Notes (2021) 101-156

• In light of the results of this project, the European Pharmacopoeia (Ph. Eur.) Group of Experts 15V revised the monographs for veterinary vaccines against cytotoxic Clostridia to introduce in vitro methods.
• Subsequent to a Ph. Eur. public inquiry, the revised monographs were adopted by the Ph. Eur. Commission in June 2021, published in Ph. Eur. 10.8 and implemented on 1^st July 2022. Importantly, the revisions allow not only replacement of in vivo by in vitro tests but will also require only residual toxicity testing of antigens rather than of the final product.

Next steps

The project has stimulated considerable interest, including the potential for application of the optimised protocol developed in this project to other, in vitro replacement, assay validation research work (e.g. Vac2Vac).

Project duration: 2018-2019

Project aim

Under this a multi-stakeholder project, a workshop was organised in 2019 to generate new ideas for non-animal approaches to predict repeated-dose systemic toxicity.

Background

The problem of predicting repeated-dose systemic toxicity with non-animal methods remains unsolved. Although there have been excellent results in predicting acute, site-of-contact effects, progress on repeated-dose, systemic toxicity has been limited. There are many reasons for this: the numerous modes of action that can cause toxicity, many of which are still undefined, the interplay among different cell types, temporal aspects of toxicity, including the relationship between repeated lesions and repair, and pharmacokinetic and metabolic considerations that are only imperfectly recapitulated in vitro.

The complexity of this problem raises it to the level of one of the grand challenges of modern science. Recognising the complexity of this problem, and the urgency in solving it, in 2008 the EPAA convened a group of thought leaders from basic life sciences, chemistry and medicine to brainstorm possible ways forward. Most of what emerged from that ‘New perspectives on safety’ Workshop was a list of possible technologies that might be helpful in constructing predictive models. This became the framework for SEURAT-1, the large, multi-centre programme funded later by Cosmetics Europe and the European Commission. While SEURAT-1 created many useful tools, it has become clear that this programme was not sufficient to deliver alternatives for systemic toxicity.

3Rs added value and EPAA actions

The recommendations of the workshop and the publication reporting from it will be used to inform/advise future research strategies of the EPAA partners and other stakeholders, thereby increasing the chances to accelerate the development of future non-animal integrated approaches/strategies for assessing repeated dose toxicity.

Stakeholders involved

The project team includes experts from different industry sectors, the E. Commission and EURL ECVAM. The workshop will be organised with international experts from different scientific disciplines coming from industry, academia and regulatory bodies.

Milestones

• The workshop took place in October 2019, in Brussels
• The workshop summary with recommendations was published in a peer-reviewed journal

Next steps

The workshop conclusions and recommendation have been addressed through further EPAA projects related to New Approach Methodologies (NAMs) for assessing repeated dose toxicity.

Project duration:2019-2022

Project aim

The objective of this project was to test the effectiveness of a computational algorithm developed to convert in vitro concentration-response data to in vivo dose-response data (known as QIVIVE) and its applicability to a range of chemical structures. More specifically, the project aims to
1. Increase confidence in the approach by demonstrating applicability with diverse chemical structures
2. Demonstrate with representative compounds a robust evidence of applicability on a broader landscape and across different industrial sectors
3. Provide evidence of a tool that may be incorporated within R-Vis, specifically designed for QIVIVE, which will be unique for modelling software platforms
4. Compare the predicted in vivo BMD to existing experimental BMD values used in chemical safety assessment by a regulatory agency

Background

The overall context of this project is the development of a reliable non-animal in vitro bioassay-based testing strategy for human safety testing of chemicals. Specifically, the aim is to test the effectiveness of a computational algorithm that was developed earlier to convert in vitro concentration-response data to in vivo dose-response data (known as quantitative in vitro to in vivo extrapolation or QIVIVE) and its applicability to a range of chemical structures.

The algorithm could be incorporated as a tool in a future third phase of development of the earlier Cefic-LRI and EPAA funded user-friendly, freely available modelling platform called R-Vis. This algorithm was not a deliverable of the R-Vis project but was developed separately to address limitations in current approaches to QIVIVE which use the term ‘reverse dosimetry’. It would therefore expand the existing reverse dosimetry capability of R-Vis.

3Rs added value and EPAA actions

An automated computational algorithm that accepts in vitro concentration response data as an input and returns in vivo dose response data as an output would represent a significant milestone in the development of tools that could contribute to the 3Rs.

The outputs from this work, which include characterising specific uncertainties associated with the computational tools and in vitro data used, will be presented according to the OECD PBK template currently under development. The suitability, utility and concordance of in vitro assay systems could be readily and reasonably examined. This would further encourage and facilitate uptake of alternative to animal methods.

Stakeholders involved

The project is supported by EPAA and is conducted by HSE (Health Safety Executive, UK). In addition, experts from the European Commission, the Joint Research Centre, EURL ECVAM, EFSA and interested companies contribute to the project.

Milestones

• This 2-year project started in 2019 and was completed in Q1 2022.
• Building of PBPK model, perform sensitivity analysis, and identify most sensitive parameters
• Translating in vitro concentration responses to in vivo
• Determining in vivo BMD values
• The following papers were published in the peer-reviewed literature

References

Loizou G, McNally K, Dorne J-LCM, Hogg A. 2021. Derivation of a human in vivo benchmark dose for perfluorooctanoic acid from toxcast in vitro concentration–response data using a computational workflow for probabilistic quantitative in vitro to in vivo extrapolation. Frontiers in Pharmacology. 12 (570).

Loizou G, McNally K, Paini A, Hogg A. 2021. Frontiers | Derivation of a Human In Vivo Benchmark Dose for Bisphenol A from ToxCast In Vitro Concentration Response Data Using a Computational Workflow for Probabilistic Quantitative In Vitro to In Vivo Extrapolation | Pharmacology (frontiersin.org)

Loizou G, McNally K, Paini A, Hogg A. 2022. Derivation of a Human In Vivo Benchmark Dose for Chlorpyrifos from ToxCast In Vitro Concentration Response Data using RVis, an Open-Source PBK Modelling Platform and a Computational Workflow for Probabilistic Quantitative In Vitro to In Vivo Extrapolation (submitted to Frontiers in Pharmacology – Predictive Toxicology

Next steps

The EPAA partners will consider how to best built on the outcomes of this project.

Project duration: 2019-2022

Project aim

This project aims to review the progress in PBK, assess the chemical space coverage of existing PBK models and determine methods to identify ‘similar’ chemicals. More specific project aims are to

1. undertake and publish a complete systematic review and collation of existing, published PBK models in rats and humans in order to provide a readily updatable resource for PBK model developers and users.
2. assess the chemical space coverage of existing PBK models in relation to the chemical space of food additives, drugs, cosmetics, pesticides and industrial chemicals. This will help to identify where gaps exist in current knowledge and which regions of chemical space are associated with higher confidence in potential read-across.
3. investigate a range of similarity assessment metrics (e.g. chemical fingerprints) to determine which individual or consensus method leads to most appropriate selection of source chemicals to predict behaviour/properties of target chemicals and serve as templates in PBK model development.
4. develop a freely available software tool to assist the identification of appropriate chemicals to use as templates via an automated workflow.

Background

Increasing confidence in the use of non-animal alternatives for safety assessment is an important goal for many sectors. Read-across is increasingly being used as an alternative, whereby data from one or more source chemicals is used to predict the effect of a target chemical of interest. For any chemical (food additive, drug, cosmetic, pesticide etc) to have an effect, the chemical (or its transformation product) must not only possess intrinsic activity, but must also reach the relevant site of action at sufficient concentration.

Hence, for more reliable risk assessment consideration must be given to both intrinsic activity and internal exposure. physiologically-based kinetic (PBK) models are used to predict the overall time-concentration curves for chemicals in blood/organs; they are increasingly being used by industry, academia and regulators. The models can be used in conjunction with pharmacological or toxicological information in order to determine the true potential of a chemical to elicit an effect – desirable or undesirable.

Also, the 2017 EPAA partners forum on ‘toxicokinetics and read across’ reiterated the importance of in silico toxicokinetics in this context.

3Rs added value and EPAA actions

A systematic review of existing PBK models that may serve as templates for the development and evaluation of new PBK models will be published, providing a valuable data resource for academia, industry and regulators. Using existing models and data as templates will help to reduce the number of new animal tests for PBK model development. Detailed methodology for the review would also be provided ensuring that the review could be readily updated as more information becomes available, ensuring sustainable impact.

Read-across is increasingly recognised as an important non-animal alternative in safety assessment; however, barriers to its acceptance and uptake remain due to lack of confidence in the predictions. Incorporating information from PBK models into the read-across prediction provides additional information and support of the prediction; this may help to promote the acceptability of read-across as an alternative to animal testing.

Stakeholders involved

The project is supported by EPAA and is conducted by the Liverpool John Moores University (LJMU). Furthermore, experts from EURL ECVAM, the US Environment Protection Agency, HSE and industry contribute to the project.

Milestones

The project started in the autumn of 2019 and was completed at the end of 2022.

All agreed milestones, deliverables and tasks proposed were completed as anticipated. The only exception was that the submission of the systematic review was slightly delayed because it was decided to collate models for all species (not just human and rat as initially proposed) and to publish the chemical space analysis as part of this review. In addition, the following two key outputs were delivered.

• Output 1 – PBK Model Dataset has been developed and published. A published systematic review which incorporated the chemical space analysis is available from Thompson et al 2021
• Output 2 – a software tool for analogue selection, in the form of a KNIME workflow, was launched during the ICT conference in September 2022. Information on the tool was made available via a poster presentation during the conference, with a follow-up webinar on use of both the tools derived from the projects on 29^th September 2022.
• US EPA shared an Excel spreadsheet of results from their non-systematic review of PBK models on PubChem.
• A protocol for the formal systematic review had been previously accepted and published

Presentations made to ASCCT virtual conference (October 2020), QSAR 2021 (June 2021), WC11 (Aug 2021), andSOT (San Diego, Mar 2022). Two presentations on the final tools were given at ICT / Eurotox (Maastricht, Sept 2022). A webinar on 29 September 2022 provided a practical demonstration of the tools.

References

1. Thompson C, Penson P, Madden J. (2020) PROSPERO: Research Protocol Registration: Systematic review to determine the chemical space of existing physiologically-based kinetic (PBK) models

2. Sayre R, Grulke C, Tan Y-M, Madden J, Thompson C, Webb S, Penson, P (2019) Existing physiologically-based kinetic (PBK) models, identified via PubMed searches (with associated EndNote library) Figshare

3. Thompson CV, Firman JW, Goldsmith MR, Grulke CM, Tan Y-M, Paini A, Penson PE, Sayre RA, Webb S and Madden JC (2021) A Systematic Review of Published Physiologically-based Kinetic Models and an Assessment of their Chemical Space Coverage. Alternatives to Laboratory Animals 49(5):197-208

4. Thomspon PhD Thesis entitled “Development of Tools to Support the Application of Physiologically-Based Kinetic Modelling in Safety Assessment”, LJMU, 2022  

Next steps

The strength of the PMD is the collation of existing PBK models, that facilitate ready search of models that have been capture. Expanding this dataset to include all recently published models (as well as any earlier models inadvertently excluded from the systematic review) would greatly enhance the utility of the PMD.

In this study, fingerprints and key physico-chemical properties were used to assess similarity of analogues. Selecting analogues based on similarity in ADME properties may produce more suitable analogues and this possibility warrants further investigation.

This study was shown to be successful for two chemicals that were used in the case studies but further investigation with additional chemical types, covering a wider range of properties is recommended to test the overall applicability of the approach.