A modular non-dietary exposure assessment approach for pesticides (PPPs) is proposed, emphasizing a dose-based prediction of dermal absorption. The Modular Dermal Exposure Model (MODEXMO) defines new nomenclature: exposure, dermal absorption and surface distribution prediction models, respectively abbreviated as EXPREMOs, DAPREMOs, SUDIPREMOs. Approach and nomenclature are generic and can be used for probabilistic models or simulation-based approaches. The modularity aids the integration of refined exposure data and enhances the adaptability of risk assessments to better reflect dynamic real-world scenarios. DAPREMOs could replace relative dermal absorption values (DAVs) by directly predicting internal exposure from external doses with a statistical model. It is well-known that absolute dermal penetration depends primarily on applied dose. EXPREMOs can predict relatively low area doses, since exposure estimates are averaged over large body surfaces, regardless of the actual residue surface distribution. While this has no consequence in the current assessment approach using DAVs, it ignores the exposure scenario-dependent spatial distributions of residue on skin, e.g., due to individual splash events during mixing and loading operations. SUDIPREMOs can refine EXPREMO-assumed default surface areas to provide DAPREMOs with more accurate inputs for different area doses in multiple exposure sub-streams.

Non-animal methods for skin sensitization assessment have been developed and adopted as OECD test guidelines. However, no single new approach methodology (NAM) can fully replace animal-based methods, leading to the development of defined approaches like OECD GL497. This study advances quantitative risk assessment (QRA) for skin sensitization using Artificial Neural Network (ANN) models to predict LLNA EC3 values. As a case study, six substances were evaluated using ANN models based on the Direct Peptide Reactivity Assay (DPRA) and the Amino acid Derivative Reactivity Assay (ADRA). These substances included four with known structures (Metol, Dibenzyl Ether, Safranal, and Lyral) and two with unknown structures (Verbena Oil and Oakmoss Extract). Most predicted EC3 values were within a 10-fold range of observed values, demonstrating model reliability. Incorporating ADRA molar and gravimetric method data, ANN models successfully predicted EC3 values for both substances with known and unknown structure, showing their applicability to natural complex substances like botanical extracts. A new skin sensitization risk assessment flow incorporating ANN models is proposed, contributing to the 3Rs by providing a reliable, non-animal method for determining Points of Departure (PoD) and advancing Next Generation Risk Assessment (NGRA) for cosmetic ingredients.

During development, potential adverse effects of pharmaceuticals on male and female fertility are evaluated. Although non-human primates (NHP) can be an appropriate model for some pharmaceuticals, there are scientific, ethical and practical limitations. We performed a retrospective analysis of the use of NHP for fertility assessment. A database was created, consisting of pharmaceuticals submitted to the European Medicines Agency (2011-2022). Pharmaceuticals with repeat-dose toxicity studies or dedicated studies to evaluate surrogate fertility endpoints in NHP were included. Publicly available data on product characteristics, study design, observations on standard surrogate fertility endpoints (changes in reproductive organ weight and histopathology), additional surrogate fertility endpoints (changes in reproductive hormone levels, menstrual cycle, and sperm parameters), and labeling were collected. In total, 263 pharmaceuticals were included. For 22 pharmaceuticals, adverse effects on surrogate fertility endpoints in NHP were reflected in the label. In all 22 cases, effects were predictable based on mechanism of action or rodent studies provided sufficient information on fertility. Hence, no clear scientific value of fertility assessment in sexually mature NHP was determined. In conclusion, using NHP to evaluate effects on surrogate fertility endpoints of pharmaceuticals did not have a significant impact on fertility labeling.

The United States Environmental Protection Agency (USEPA) has long championed the use of the most modern toxicity testing approaches to meet its pesticide registration mandates, often collaborating with stakeholders to develop and implement innovative approaches that strengthen environmental and human health protections. Despite these efforts, the adoption of 21st-century testing approaches in pesticide registration has been slowed by various factors, including limited resources, inefficient processes for establishing confidence in new methods, and the retention of outdated data requirements codified in the Code of Federal Regulations. This paper provides a brief overview of the current USEPA legislative landscape for pesticide toxicity testing, describes the progress and remaining challenges in using new testing approaches to fulfill regulatory requirements, and highlights opportunities to address these challenges and enhance protection of human health and the environment.

Fluoxapiprolin is a new fungicide developed for the control of oomycetes that damage crops such as tomato, potato, grapes and leafy vegetables. Human safety studies have shown no toxicity clearly attributable to fluoxapiprolin, highlighting its favourable toxicological profile compared to many other fungicides. It meets all human health risk assessment criteria without difficulty. However, fluoxapiprolin was tested up to a Kinetically-Derived Maximum Dose (KMD) rather than the conventional limit dose, making it necessary to justify this approach in the context of regulatory hazard assessment. Official guidance documents encourage the application of KMD approaches under specific conditions. In the case of fluoxapiprolin, oral absorption was shown to become saturated as doses increased, supporting the selection of the high dose in long-term studies based on a KMD rather than using a fixed limit dose. Even if a limit dose had been used, systemic exposure would have been only about twice as high as that observed at the KMD. Given the absence of treatment-related toxicity in any study and the relatively small difference in systemic exposure, the toxicological database is considered sufficient to identify relevant hazards and to support a robust human health risk assessment.

Silver nanoparticles (AgNPs) are widely used in nanotechnology products. However, the health risks associated with co-exposure to these emerging contaminants and environmental pollutants, such as non-essential metals, are poorly understood. The present study aimed to investigate the cytotoxicity and toxicological interaction of AgNPs (0.36 and 3.6 μg mL) + lead (Pb, 25 and 250 μM) and AgNPs + mercury (Hg, 15 and 150 μM) using the macrophage cell line RAW 264.7 as a model. Effects were observed after a few hours (4 h) on NO levels, phagocytic activity, and DNA damage. Cell viability (24 h-exposure) was affected mainly by the higher concentrations of the contaminants and their mixtures, preceded by increases in NO levels and DNA damage, but without effects on ROS levels. Co-exposure potentiated some effects (ROS and NO levels and DNA damage), indicating toxicological interaction. These important findings must be further investigated, since the interaction of Pb and Hg with AgNPs from nanoproducts may impair the function of macrophages and represent a health risk for humans.

Error-corrected sequencing (ECS) is a transformative method for in vivo mutagenicity assessment, enabling direct, highly sensitive measurement of mutation frequency and spectrum. ECS addresses key limitations of the transgenic rodent (TGR) assay, including lack of integration into standard toxicity studies, restricted model availability, and limited alignment with the 3R principles. To support regulatory acceptance, an expert workgroup of the International Workshops on Genotoxicity Testing (IWGT) reviewed ECS technologies and developed consensus recommendations for its inclusion into Organisation for Economic Co-operation and Development (OECD) test guidelines. The working group agreed that ECS: produces results that are concordant with validated TGR assays; can be incorporated into standard ≥28-day repeat-dose toxicity studies; and, data interpretation should be based on overall mutation frequency compared with concurrent vehicle controls. The working group emphasized harmonized data reporting aligned with OECD principles and endorsed study designs that enable quantitative risk assessment. Overall, the working group agreed that ECS offers a significant advancement over current mutagenicity assays by enabling the use of diverse models beyond conventional TGR systems described in OECD test guideline 488. The working group fully supports the application of ECS to generate in vivo mutagenicity data for regulatory submissions and recommends its inclusion in future OECD test guidelines.

The impact of firsthand and secondhand smoking on the development of various types of cancer is well established. However, the association of thirdhand smoke (THS) with malignant diseases has only recently been recognized, with limited research investigating the underlying mechanisms. THS refers to the dynamic mixture of residual tobacco smoke constituents that persist in air, dust, and on surfaces, and can occur through dermal absorption, inhalation of re-emitted gases and particles, and ingestion - particularly via dust intake. Tobacco smoke contains hundreds of toxic compounds, 60 of which are classified as carcinogens. These include aromatic amines, nicotine, nitrosamines, nitric oxide and polycyclic aromatic hydrocarbons. These substances can react with environmental chemicals, forming highly toxic secondary pollutants that can be released from surfaces over months and be absorbed through the skin or mucosa. The metabolization of tobacco smoke components can increase the incidence of cancer by altering key molecular pathways. Main mechanisms of carcinogenesis include DNA adduct formation and DNA structural changes, interference with tumor suppressor genes, and chronic inflammation. This review examines the risks of THS exposure, the potential mechanisms of carcinogenesis associated with THS and the role of THS in the development of different cancers linked to tobacco use.

Serial blood collection is a key requirement for pharmacological and toxicological studies in rodents, but existing approaches such as tail vein, retro-orbital, or catheter-based sampling are constrained by limited sample volume, variability, and welfare concerns. We developed a percutaneous technique for serial blood sampling from the external jugular vein in male Wistar rats under isoflurane anaesthesia. The method was evaluated over 14 days by monitoring body weight, behaviour, haematological and clinical chemistry parameters in a preclinical safety study at a No-Observable-Adverse-Effect-Level dose setting. Rats tolerated repeated sampling without distress, skin lesions, or behavioural abnormalities. Isoflurane induction was rapid and uneventful, and blood samples (0.5-1 ml) were consistently obtained within 2 min. Body weight changes were minimal across sessions (1.2 % after the first to 0.2 % after the sixth), indicating procedural adaptation. Haematological values remained within reference ranges, with only a significant reduction in RDW-CV, while biochemical parameters including AST, CK, and lipids showed no significant alterations; minor, non-adverse trends included reduced ALT (p < 0.041) and non-significant cholesterol and minimal non-significant increases in plasma creatinine. This study establishes repeated percutaneous jugular vein puncture as a minimally invasive, welfare-compliant, and reliable method for longitudinal blood sampling in anaesthetised rats without catheterisation, supporting robust preclinical research.

This position paper presents a science-based, holistic assessment of the toxicological risks posed by organic impurities in drug-linkers (D-L) used as intermediates in the production of Antibody-Drug Conjugates (ADCs) for oncology indications. The analysis outlined in this manuscript demonstrates that organic impurities present at levels at or below 1.0 % w/w in D-L intermediates are unlikely to result in adverse toxicological effects upon administration of the final ADC drug product. Due to the extremely low exposure (molar and weight) associated with 1.0 % w/w impurity level in the D-Ls, the authors propose to maintain the ICH Q3A criteria of not more than 1 mg/day limit however increase the qualification limit criteria for D-L related impurities from 0.15 % to 1.0 % w/w. Additionally, a methodology is introduced to assess the level of concern for non-conjugatable organic impurities, utilizing process-specific and ADC-specific factors to justify specifications for these impurities in D-L intermediates. Based on the minimal risk associated with D-L impurities at or below the discussed levels, the paper proposes a workflow for quality risk management of such impurities in D-Ls.

Phase 1 clinical trial participants could potentially be exposed to significant health risks. Findings from a standard battery of genetic toxicology tests typically are the only data available to inform about cancer hazards at the initiation of clinical trials. Although uncommon, a question occasionally arises that is not clearly defined in current guidance: how many doses of an Ames-positive (potentially DNA-reactive) drug can be administered safely to healthy adult subjects during Phase 1 clinical trials? A literature survey was undertaken to identify information on carcinogenic risks from short-term exposures to Ames-positive agents, which might inform about administering an Ames-positive drug as a single dose or over a period of up to 14 days in healthy adult subjects. Limited information was identified on risk predictions for short-term exposures from modeling applications and from human studies, with more extensive data available using animal models. Relevant information on cancer outcomes following short-term exposures to Ames-positive agents suggest there is an increased cancer risk for administering even a single dose of an Ames-positive drug to healthy adult subjects. These findings indicate that Phase 1 studies with Ames-positive drug candidates should be exceedingly rare, and that additional mutagenicity testing should be performed before drug administration.

Key lime (Citrus aurantiifolia) peel, a major by-product of juice processing, is rich in flavonoids and other bioactives. To support its safe use as a food-derived ingredient, we chemically characterized key lime peel ethanol extract (KLPE) and conducted a full toxicological evaluation under OECD Test Guidelines (No. 420, 408, 471, 473, 474) and in compliance with GLP. The program included acute oral toxicity, a 90-day repeated-dose study with a 28-day recovery, and in vitro and in vivo genotoxicity assays. KLPE caused no mortality or clinical signs, and body weight, food intake, hematology, and organ weights were comparable to controls. No histopathological lesions or genotoxic effects were observed. Both the LD and the No Observed Adverse Effect Level (NOAEL) exceeded 2000 mg/kg BW/day, the highest dose tested, indicating a wide safety margin. These results provide regulatory-grade evidence confirming the safety of KLPE for human consumption and support its application as a functional food and nutraceutical ingredient in global markets.

Novel waxes require safety documentation, which traditionally involves using a battery of assays in experimental animals. For a class of well-studied substances (e.g., waxes) generally recognized to be of low or no toxicity, bridging methods that leverage safety information to fill data gaps for new substances may be considered. The aim of this assessment was to use a bridging assessment procedure to fill data gaps to demonstrate safety for two novel plant waxes (rice bran wax and sunflower wax). Using the European Chemicals Agency (ECHA) framework methodology, compositional similarity was established between the novel waxes and four well-evaluated plant waxes. Using the evidence bases for the evaluated plant waxes, the bridging assessment predicted that rice bran and sunflower waxes would have limited absorption. The bridging assessments also predicted that rice bran and sunflower waxes had limited repeat-dose oral toxicity, and were not reproductive or developmental toxicants or carcinogenic. Predicted lack of acute and mutagenic activity by rice bran wax was supported by experimental data. In conclusion, this case study shows that the bridging approach can fill toxicity data gaps, supporting a waiver of additional in vivo tests, thus ensuring safety, as well as reducing the use of animal testing.

The use of pesticides is an inseparable and undeniable part of modern agriculture to ensure sufficient food supply for a still-growing population but, due to their intrinsic toxic nature, rigorous testing is needed before making them available on the market. In the European Union, in vitro metabolism studies with the active ingredients became mandatory in 2013. Unfortunately, in contrast to most other toxicological endpoints, a harmonised test guideline for their conduct is not yet available. More than 10 years after its introduction, we aimed to analyse the practical experience with that data requirement. For this purpose, 70 comparative in vitro metabolism studies were reviewed. Significant methodological differences were noticed among the studies, including species selection, test systems, tested concentrations, and incubation times. These differences and the deviations from existing but not legally binding recommendations limit the use that could be made of these studies, in particular beyond a simple comparison of human and rat metabolism. There is an urgent need for a harmonised guidance how to perform the in vitro biotransformation studies. Its implementation would not only increase the likelihood to identify unique or disproportionate human metabolites but might also support next-generation risk assessment.

Policymakers in Europe utilize biomonitoring to regulate exposure to arsenic and protect public health, but potentially high exposure levels among ancestral subgroups remain understudied. We assessed arsenic exposure in a random population sample of Amsterdam, the Netherlands, stratified by European-Dutch vs Asian and African-Dutch ancestry (n = 60, 30 women, aged 40-63 years). We analyzed total spot urinary arsenic with inductively coupled plasma mass spectrometry and used food frequency questionnaires to calculate the exposure to inorganic arsenic (reference point <0.8 nmol/kg body mass/day) from rice, fish, and water consumption. Urinary arsenic (40-4828 nmol/L, mean 532 (SE 116); respectively 312 (60) in European-Dutch vs. 642 (169) in Asian and African-Dutch), was independently associated with rice consumption (343 nmol/L (95 % CI, 112-575)/100 g rice). Inorganic arsenic intake (87-976 nmol/day, mean 397, SE 32), respectively 175 (14) in European-Dutch (2.4 (0.2) nmol/kg body mass/day) vs 508 (37) in Asian and African-Dutch (6.7 (0.5) nmol/kg body mass/day), was independently associated with systolic blood pressure (3.1 (1.5-4.8) mm Hg/100 nmol iAs). The high dietary arsenic exposure among certain ancestry subgroups living in Europe is concerning. Health policies should be developed to monitor these subgroups and reduce their inorganic arsenic intake to the unavoidable minimum.

This study evaluates the potential of using historical control data (HCD) in rat fertility studies to replace/reduce concurrent control animal use and improve statistical analysis. This analysis was conducted using data sourced from a single test facility using consistent animal attributes and study procedures and consisted of 13 datasets from 12 rat fertility studies conducted between 2018 and 2023. For required fertility endpoints, we have evaluated full and hybrid Virtual Control Group (VCG) and Bayesian statistical approaches compared with standard statistics used for concurrent control groups (CCG). Our findings demonstrate that the sensitivity and specificity of most required fertility endpoints were generally similar between CCG and full or hybrid VCGs of sufficient sample size. Bayesian analyses leveraging all available HCD offered similar or superior sensitivity and specificity to CCGs and VCGs (full or hybrid) for all required fertility endpoints, with some exceptions. A retrospective case study implementing these approaches with HCD illustrated similar statistical performance across all approaches in addition to the benefit of reduced animal use. Overall, this effort illustrates the potential to improve sensitivity and reduce animal use with VCG (full or hybrid) or Bayesian approaches for required fertility endpoints in the rat.

The Globally Harmonized System standardizes chemical hazard communication. Within this system, germ cell mutagenicity holds unique importance due to the potential of heritable mutations. Yet, such testing is rarely performed, requiring alternative approaches to assess germ cell mutagenic potential. One strategy involves using physiological parameters to predict if somatic cell mutagens reach the gonads and induce mutations in germ cells. Therefore, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee examined whether blood concentrations of somatic cell mutagens can predict genotoxic effects in germ cells. We analyzed 11 substances with dominant lethal test data and 30 with transgenic rodent gene mutation data in male germ cells, together with blood concentrations. While significant associations were found between genotoxic outcomes in somatic tissues (e.g., micronuclei, mutation induction) and outcomes for germ cell mutagenicity, considerable variability was noted in genotoxic responses. This variability could not be explained by blood concentrations alone. Notably, blood levels of substances positive in both somatic and germ cells were similar to those that were positive for somatic cell genotoxicity and negative in germ cells. We conclude that the concentration of a somatic cell genotoxic substance in blood is insufficient to predict germ cell mutagenicity.

In ICH Q3C five residual solvents are classified as "should be avoided" and are designated as Class 1 solvents. The solvents in question are: benzene, carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethene and 1,1,1-trichloroethane. Although multiple revisions to ICH Q3C have been made, PDE (permitted daily exposure) limits for Class 1 solvents remain unchanged from those originally proposed in 1997. Since that time, new toxicological data have become available, and additional expert assessments have been published. A detailed review of information currently available indicates that there is a case for a change to limits for all Class 1 solvents except benzene. Two of the solvents can be classified as mutagenic carcinogens making them eligible for determination of AI (acceptable intake) limits as described in ICH M7(R2). In addition, the concept of expressing limits as concentrations, based on the assumption of a daily drug-substance dose of 10 g, is challenged.