One important tool within Next Generation Risk Assessment (NGRA) for closing data gaps and avoiding animal testing is read-across. This involves transferring data from a well-studied chemical to a similar, less well-studied chemical, providing their structure, properties and mechanisms of action are sufficiently comparable.

Next Generation Risk Assessment (NGRA) combines modern in vitro methods, computer-based models and other new approach methodologies (NAMs) to provide scientifically sound assessments of chemicals and pharmaceuticals while progressively reducing the use of animal testing.

For many years, the European Union has pursued a policy aimed at the replacement, reduction and refinement of animal testing (the 3Rs principle). To put the ethical principle of the 3Rs into practice, the European Commission has now published a roadmap for the gradual phase-out of animal testing in the safety assessment of chemicals. The ASPA (Alternative Safety Profiling Algorithm) workflow is a central, structured tool for fully animal-free, risk-based safety assessment. It is one of the most specific scientific concepts of modern Next Generation Risk Assessment (NGRA), a field in which new methods and software solutions have been developed to put the roadmap into practice. The workflow was developed by the ASPIS cluster, a collaboration of the EU-funded projects ONTOX, PrecisionTox and RISK-HUNT3R. Researchers at Fraunhofer ITEM have played a key role in developing the ASPA workflow within the RISK-HUNT3R project.

“The ASPA workflow is a transparent, reproducible and modular approach to animal-free Next Generation Risk Assessment of chemicals. It enables us to assess chemical risks using animal-free in silico and in vitro methods,” says Sylvia Escher, a research scientist at Fraunhofer ITEM. “With ASPA, we transparently integrate biological activity data from a range of new approach methodologies (NAMs), information on human uptake and exposure, and toxicological mechanisms. The aim is to enable scientifically sound safety assessments that can be readily used by regulatory authorities and to reproducibly assess the risks posed by chemicals on the basis of three main pillars: toxicokinetics (absorption, distribution, metabolism and excretion), hazard and exposure.”

NAMs are regarded as key technologies for the future safety assessment of chemicals. These include human-relevant cellular test systems, such as in vitro and ex vivo models, as well as computer-based models. Together, they form the foundation of NGRA. The ASPA workflow supports safety assessments. It comprises around 55 assessment elements, providing a multi-stage, modular strategy for risk analysis.

The aim of ASPA is to make safety assessments fully transparent for the recipient—such as a regulatory authority—by showing which steps and decisions led to the final outcome. ASPA requires precise justification of interim decisions, which in turn necessitates the continuous assessment of uncertainties. This complex process is supported by the ASPA-assist software, which guides users step by step through ASPA’s 55 assessment modules and facilitates the reporting process in a reproducible and standardized manner.

Fraunhofer ITEM has advanced several aspects of the ASPA workflow, including the development of toxicokinetic PBK (physiologically based kinetic) models. These mathematical computer models from pharmacology and toxicology represent the human body in order to calculate how chemicals are absorbed, distributed, metabolized and excreted (ADME: absorption, distribution, metabolism and excretion). In effect, kinetic models translate these processes into mathematical equations based on anatomical and physiological data as well as substance-specific ADME parameters.

The Fraunhofer PBK model specifically focuses on inhalable substances, including gases and aerosols consisting of liquid droplets or solid particles. Existing PBK models for inhalation absorption are designed either for gases and vapors or for airborne particles and represent the different regions of the lung only in simplified form. Working together with other teams at Fraunhofer ITEM, Sylvia Escher can also experimentally determine the specific ADME parameters needed to assess absorption, metabolism and excretion following exposure to airborne substances. “For example, we investigate what causes the toxicity and what type of toxicity it is. We determine this using in vitro models that allow us to measure biological processes. With PBK models, we can translate results from in vitro test systems more effectively to real-world human exposure scenarios. We convert doses measured in the Petri dish into realistic doses in the human body,” explains Escher.

Case studies are essential when it comes to testing ASPA and the in vitro and in silico models in practice and identifying their uncertainties. For an OECD IATA case study combining different methods and data sources within an integrated assessment approach, researchers at Fraunhofer ITEM combined high-throughput in vitro assays with biokinetic modeling and machine learning methods to prioritize substances with potential systemic toxicity. The aim was to identify substances with high systemic toxicity at an early stage and prioritize them for further assessment. For the risk assessment, the researchers used extensive omics data to characterize the biological activity profiles of the substances under investigation. This high-dimensional data made it possible to systematically evaluate changes at the cellular and molecular levels and identify the most predictive features for detecting potentially toxic substances. The OECD IATA case study demonstrates how modern data-driven approaches can be applied to regulatory decision-making.

The goal is not an immediate ban on all animal testing but a coordinated regulatory transition. The European Commission is formally incorporating methods and concepts developed primarily through research projects such as RISK-HUNT3R. “For the research community, regulatory acceptance is a crucial step. Many NAMs have existed for a long time and are already scientifically well advanced. The real challenge is to continue validating these models so that regulatory authorities accept them and integrate them into regulatory processes,” says the researcher. The new EU roadmap underscores Fraunhofer ITEM’s strategic focus within NGRA. For many years, the institute has been working on human-relevant models, inhalation toxicology, PBK models and data-integrated approaches to regulatory risk assessment.