Considerations for the development of guidance on dose level selection for developmental and reproductive toxicity studies.

In 2022, the European Chemicals Agency issued advice on the selection of high dose levels for developmental and reproductive toxicity (DART) studies indicating that the highest dose tested should aim to induce clear evidence of reproductive toxicity without excessive toxicity and severe suffering in parental animals. In addition, a recent publication advocated that a 10% decrease in body weight gain should be replaced with a 10% decrease in bodyweight as a criterion for dose adequacy. Experts from the European Centre for Ecotoxicology and Toxicology of Chemicals evaluated these recent developments and their potential impact on study outcomes and interpretation and identified that the advice was not aligned with OECD test guidelines or with humane endpoints guidance. Furthermore, data analysis from DART studies indicated that a 10% decrease in maternal body weight during gestation equates to a 25% decrease in body weight gain, which differs from the consensus of experts at a 2010 ILSI/HESI workshop. Dose selection should be based on a biological approach that considers a range of other factors. Excessive dose levels that cause frank toxicity and overwhelm homeostasis should be avoided as they can give rise to effects that are not relevant to human health assessments.

Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

A retrospective analysis of in vivo eye irritation, skin irritation and skin sensitisation studies with agrochemical formulations: Setting the scene for development of alternative strategies.

Analysis of the prevalence of health effects in large scale databases is key in defining testing strategies within the context of Integrated Approaches on Testing and Assessment (IATA), and is relevant to drive policy changes in existing regulatory toxicology frameworks towards non-animal approaches. A retrospective analysis of existing results from in vivo skin irritation, eye irritation, and skin sensitisation studies on a database of 223 agrochemical formulations is herein published. For skin or eye effects, high prevalence of mild to non-irritant formulations (i.e. per GHS, CLP or EPA classification) would generally suggest a bottom-up approach. Severity of erythema or corneal opacity, for skinor eye effects respectively, were the key drivers for classification, consistent with existing literature. The reciprocal predictivity of skin versus eye irritation and the good negative predictivity of the GHS additivity calculation approach (>85%) provided valuable non-testing evidence for irritation endpoints. For dermal sensitisation, concordance on data from three different methods confirmed the high false negative rate for the Buehler method in this product class. These results have been reviewed together with existing literature on the use of in vitro alternatives for agrochemical formulations, to propose improvements to current regulatory strategies and to identify further research needs.

GHS additivity formula: A true replacement method for acute systemic toxicity testing of agrochemical formulations.

Acute systemic (oral, dermal, inhalation) toxicity testing of agrochemical formulations (end-use products) is mainly needed for Classification and Labelling (C&L) and definition of personal protection equipment (PPE). A retrospective analysis of 225 formulations with available in vivo data showed that: A) LD50/LC50 values were above limit doses in <20.2% via oral route but only in <1% and <2.4% of cases via dermal and inhalation route, respectively; B) for each formulation the acute oral toxicity is always equal or greater than the Acute Toxicity Estimate (ATE) via the other two routes; C) the GHS (Global Harmonised System) computational method based on ATE, currently of limited acceptance, has very high accuracy and specificity for prediction of agrochemical mixture toxicity according to the internationally established classification thresholds. By integrating this evidence, an exposure- and data-based waiving strategy is proposed to determine classification and adequate PPE and to ensure only triggered animal testing is used. Safety characterisation above 2000 mg/kg body weight or 1.0 mg/L air should not be recommended, based on the agrochemical exposure scenarios. The global implementation of these tools would allow a remarkable reduction (up to 95%) in in vivo testing, often inducing lethality and/or severe toxicity, for agrochemical formulations.

Physiological and pathological roles of Apaf1 and the apoptosome.

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer.