Developmental and reproductive toxicity (DART) mediated by endocrine disruption (ED) is of critical concern for human and environmental health, yet integrated methods to assess DART–ED interactions are still limited, particularly in non-mammalian systems. Current OECD test guidelines and REACH requirements do not fully capture the mechanistic links between endocrine activity and developmental or reproductive outcomes (Holmer et al., 2025; Knapen et al., 2025). Advancing integrated DART–ED approaches in fish and in vitro models will fill major regulatory gaps, enable cross-species extrapolation, and support the broader adoption of mechanistically based, non-animal methods in chemical risk assessment.

This project will provide mechanistic evidence to support chemical safety assessors and regulators by facilitating the increased use of non-animal test data for DART–ED identification and decision-making. Specifically, the project will -

Deliver ED-sensitive endpoints for addition to fish test guidelines for both non-protected (TG 236) and protected life stages (e.g., TG 210, required under Annex IX of REACH).

This project addresses regulatory needs over multiple time frames. In the short term, work on DART–ED methods in fish will build on and complement the ongoing validation of THSD-sensitive endpoints for inclusion in existing OECD test guidelines (TG 236, 210) under the validation management group for ecotoxicology. Integrating ED-sensitive endpoints into these guidelines and REACH requirements will fill the current gap in environmental ED evaluation.

In the medium term, the development of a NAM to assess placental angiogenesis and endocrine disruption will support the characterization and classification of reproductive EDs and may contribute to the weight of evidence for AOP 151 (OECD Project 1.42, under development).

In the long term, replacing the H295R in vitro steroidogenesis assay with more relevant alternatives is crucial for NAM-based assessments of male reproductive toxicants. The project will deliver key outcomes for the development of such models, thus contributing to validated in vitro NAMs for male steroidogenesis.

Developmental immunotoxicity (DIT) is a growing concern because the developing immune system is highly sensitive, and disruption of the immune system can lead to long-term health effects, such as allergies, autoimmune diseases, and increased susceptibility to infections (Hessel et al., 2015). Current chemical regulations offer limited tools to identify immunotoxicants as functional immune endpoints are underrepresented in standard test guidelines. The T-cell-dependent antibody response (TDAR) in the Extended One-Generation Reproductive Toxicity Study (EOGRTS) is the only functional assay required, and the DIT cohort is rarely included due to the absence of validated triggers. Recognizing these limitations, ECHA has identified DIT as a key regulatory challenge, calling for the identification of critical developmental windows and the development of NAMs. Recent OECD activities and guidance (e.g., DRP No. 360 (OECD, 2022)) and frameworks such as the “Key Characteristics of Immunotoxic Agents” (Germolec et al., 2022) provide a solid foundation for advancing non-animal methods and building a NAM-based DIT testing strategy.

In line with ECHA’s KARC recommendations for DIT, this project will -

Identify critical developmental windows (through physiological mapping and literature review) to support NAM development.

The DIT project is directly relevant to multiple regulatory frameworks—including REACH, CLP, the Biocidal Products Regulation, the Plant Protection Products Regulation, and the Cosmetics Products Regulation (OECD, 2025; Dewhurst et al., 2015]—by providing tools for screening, prioritization, read-across, and grouping proposals. Several existing immunotoxicity assays are well-characterized, and some have been developed into OECD test guidelines (e.g., TG 444 on skin sensitization) (OECD, 2023). These constitute a valuable source of methods to be evaluated for their potential to serve as NAMs addressing specific regulatory needs. Further adaptation of these assays to include cell maturation and migration endpoints will be explored. In the long term, new models based on hematopoietic stem cells and hiPSCs will contribute to the development of non-animal tools for hazard identification and characterization, thereby enhancing the overall assessment of DIT properties.

Given its central role in xenobiotic metabolism, the liver represents a primary target organ for chemical-induced toxicity (Rusyn et al., 2021). Increasing evidence indicates that chemical exposures may cause differential effects in men and women, primarily due to sex-specific variations in hepatic metabolic pathways (Hochmuth et al., 2021; Zuri, 2024). Notably, women account for approximately 70% of acute liver-failure cases, indicating a greater susceptibility to hepatotoxicants (Sayaf et al., 2022). While animal studies may partially address sexual dimorphism by selecting the more sensitive sex, current in vitro liver models generally fail to capture these biological differences, thereby limiting their predictive capacity for sex-specific liver toxicity responses.

This project aims to improve in vitro models for chemical-induced hepatotoxicity by incorporating parameters of sexual dimorphism into hepatic cell systems, including primary human hepatocytes, HepaRG cells, and hepatocytes derived from multipotent stem cells (mSCs) and induced pluripotent stem cells (iPSCs). Specifically, the work will carry out the following steps: -

Establish and characterize sexual dimorphism in in vitro hepatic models.

From a regulatory perspective, this project will, for the first time, enable the integration of sexual dimorphism and sex-specific responses in chemical hazard analysis, ultimately supporting better protection of public health and the environment.

This project represents a proof-of-concept (PoC) effort to evaluate the biological relevance of sexual dimorphism in an in vitro context, including assessments of chemical-specific responses (e.g., endocrine disruptors). The approach will enhance mechanistic understanding, which is a prerequisite for developing or refining in vitro test systems and NAMs for defined regulatory contexts. Should PoC data demonstrate robust biological differences, all NAMs currently—or prospectively—used in regulatory frameworks could be affected. Further qualification and validation of these assays will be needed before formal adoption; however, even without full validation, such NAMs could contribute to weight-of-evidence assessments alongside animal studies, in silico models, and historical data.

Current regulatory frameworks for chemical safety rely predominantly on OECD test guidelines, yet many promising NAMs remain underutilized because their regulatory readiness is insufficient (Schmeisser et al., 2023). Within PARC, several NAMs have been developed or refined under WP5, but further work is needed to align these approaches with regulatory expectations (Marx-Stoelting et al., 2024). Enhancing regulatory readiness is crucial to ensuring that NAMs contribute effectively to hazard assessment, screening, and decision-making, thereby supporting the 3Rs principle and improving the protection of human health and the environment.

This project specifically addresses the need to evaluate, harmonize, and advance the most promising NAMs toward regulatory implementation.

The project will support pre-validation and readiness activities for selected NAMs by -

Expanding the ReadEDTest framework to assess methods for priority endpoints within WP5.

Rather than pursuing full OECD-style validation, this project emphasizes structured evidence generation to support regulatory confidence, including the parallel use of NAMs with established methods, assessment of transferability, and integration of promising assays into AOP- and IATA-based test batteries for complex endpoints.

By systematically improving the regulatory readiness of selected NAMs, this project will facilitate their integration into chemical safety assessments. The results will support hazard characterization, prioritization, and weight-of-evidence evaluations across multiple frameworks, including REACH, CLP, the Biocidal Products Regulation, the Plant Protection Products Regulation, and the Cosmetics Regulation.

Strengthening regulatory applicability will ensure that NAMs contribute to science-based decision-making and the progressive reduction of animal testing, thereby producing tangible benefits for human and environmental protection.