Integration of QSAR and NAM in the Read-Across Process for an Effective and Relevant Toxicological Assessment.

Read-Across (RAx) serves as a strategy to fill a data gap in the toxicological profile of a substance (target) using existing information on similar source substances. The principle is applied also to a category of substances for which similarity may follow a regular trend. Demonstration of similarity is not trivial and requires the analysis of different steps, starting from the precise analytical characterization of both target and source substances and including the analysis of the impact that each minor difference can have on the final outcome. Application of QSARs and performing new experimental tests within the new approach methodologies (NAMs) is necessary to increase confidence in the final prediction and reduce the uncertainty.

In Vitro Cell-Based MTT and Crystal Violet Assays for Drug Toxicity Screening.

The development of novel drug candidates is a current challenge in pharmacology where therapeutic benefits must exceed side effects. Toxicology testing is therefore a fundamental step in drug discovery research. Herein, we describe the first line of toxicology testing program, consisting in cell-based high-throughput screening assays, which have the advantage of being easy, rapid, cheap, and reproducible while providing quantitative information. We illustrate MTT and Crystal Violet assays, two common colorimetric tests able to assess both cytostatic and cytotoxic effects, respectively, of a drug candidate. MTT assay allows evaluation of cellular metabolic activity, by which cell viability can be inferred; Crystal Violet staining is directly correlated with attached viable cells, thus allowing direct assessment of cell survival and death. Therefore, combination of the two methodologies represents a useful tool for predicting drug sensitivity and efficacy, the first milestones in pre-clinical toxicology workflow.

TIRESIA and TISBE: Explainable Artificial Intelligence Based Web Platforms for the Transparent Assessment of the Developmental Toxicity of Chemicals and Drugs.

Developmental toxicity is key human health endpoint, especially relevant for safeguarding maternal and child well-being. It is an object of increasing attention from international regulatory bodies such as the US EPA (US Environmental Protection Agency) and ECHA (European CHemicals Agency). In this challenging scenario, non-test methods employing explainable artificial intelligence based techniques can provide a significant help to derive transparent predictive models whose results can be easily interpreted to assess the developmental toxicity of new chemicals at very early stages. To accomplish this task, we have developed web platforms such as TIRESIA and TISBE.Based on a benchmark dataset, TIRESIA employs an explainable artificial intelligence approach combined with SHAP analysis to unveil the molecular features responsible for calculating the developmental toxicity. Descending from TIRESIA, TISBE employs a larger dataset, an explainable artificial intelligence framework based on a fragment-based fingerprint encoding, a consensus classifier, and a new double top-down applicability domain. We report here some practical examples for getting started with TIRESIA and TISBE.

Faster ciguatoxin extraction methods for toxicity screening.

Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.

Systematization of a toxicity screening method based on a combination of chemical analysis and the delayed fluorescence algal growth inhibition test for use in emergency environmental surveys.

In recent years, heavy rainfall disasters linked to climate change have become more frequent, raising concerns about the release of chemicals stored in factories. Assessing chemical contamination during such emergencies therefore necessitates the development of a quick and easy method for evaluating hazardous contaminants in combination with toxicity testing. This study proposes a "toxicity screening" method that combines biological response testing and chemical analysis to systematically evaluate hazardous contaminants in emergency situations. The toxicity screening method evaluates the water quality in three steps, including water quality measurements and a delayed fluorescence (DF) assay, metal content measurements and a DF assay, and targeted screening analysis and a DF assay. The efficacy of this method was tested using industrial wastewater from 14 locations. Seven of the samples were non-toxic, while the other seven samples were toxic, displaying no observed effect concentration (NOEC) values ranging from 0.625 to 20%. Two toxic samples in the first phase possessed high total chlorine concentrations (0.4 mg L-1) and conductivities (2200 mS m-1), indicating that the main sources of toxicity were residual chlorine and a high salt concentration. In the second phase, metal content analysis identified metals as the toxicity cause in four samples. In the third phase, the organic contaminants were analyzed, and tri-n-octyl phosphate (TNOP) was detected at a concentration of 0.00027 mg L-1. The results of solid-phase extraction experiments and exposure tests with TNOP alone indicated that the contribution of TNOP to the toxicity was negligible and that chemicals not adsorbed on the solid-phase extraction cartridges were the cause of toxicity. The proposed method can therefore be considered effective for disaster-related water quality assessment, delivering results within 12 days.

Priority screening of contaminants of emerging concern (CECs) in surface water: Comparing cell-based bioassays and exposure-activity ratios (EARs).

In this study, we compared a wide range of cell-based bioassays to the use of chemical analysis followed by exposure-activity ratio (EAR) and Toxicological Prioritization index (ToxPi) for prioritizing chemicals, sites, and hazard concerns in water samples. Surface water samples were collected from nine sites in three Central Pennsylvania streams and analyzed for a forty-six contaminants of emerging concern (CECs), including pesticides, personal care products, and pharmaceuticals. Cell-based reporter assays evaluated human and zebrafish molecular initiating events (MIEs) in endocrine and metabolic disruption, altered lipid metabolism, and oxidative stress. Bioassays showed that 12 out of 40 assays had at least one site with activity over the effect-based trigger (EBT) values. The receptors that exhibited the highest number of samples above the EBT that would be expected to cause toxicity were Aryl hydrocarbon receptor (AhR, human and zebrafish), Pregnane X Receptor (PXR), Estrogen Receptor-beta (ERB), and Androgen Receptor (AR). Characterizing the collection sites by their bioactivity aligned closely with the stream in which samples were collected. The sum of all EARs for each chemical indicated that the pharmaceutical Carbamazepine and the pesticides Carbaryl and Atrazine posed the greatest concern. However, predicted activity and site prioritization based on individual chemical analysis and calculated EAR were different than those measured by bioassay, indicating that biologically active chemicals are present in the samples that were not included in the targeted analytes. Taken together, these data show that chemical analysis and EAR analysis are beneficial for prioritization of chemicals, whereas mechanism-based bioassays are more inclusive of known as well as unknown chemical contaminants and thus of more use for overall water quality analysis and site prioritization.

Ecotoxicological assessment of sanitary sewer overflows and rainfall dynamics offers insights into conditions for potential adverse ecological outcomes.

Sewer overflows are an environmental concern due to their potential to introduce contaminants that can adversely affect downstream aquatic ecosystems. As these overflows can occur during rainfall events, the influence of rainwater ingress from inflow and infiltration on raw untreated wastewater (influent) within the sewer is a critical factor influencing the dilution and toxicity of the contaminants. The Vineyard sewer carrier in the greater city of Sydney, Australia, was selected for an ecotoxicological investigation of a sanitary (separate from stormwater) sewerage system and a wet-weather overflow (WWO). Three influent samples were collected representing dry-weather (DW), intermediate wet-weather (IWW) and wet-weather (WW). In addition, a receiving water sample was also collected downstream in Vineyard Creek (WW-DS) coinciding with a WWO. We employed direct toxicity assessment (DTA) and toxicity identification evaluation (TIE) approaches to gain comprehensive insights into the nature and magnitude of the impact on influent from rainwater ingress into the sewer. Three standard ecotoxicological model species, a microalga, Chlorella vulgaris, the water flea, Ceriodaphnia dubia and the midge larva, Chironomus tepperi were used for both acute and chronic tests. The study revealed variable toxicity responses, with the sample of influent collected in wet-weather displaying lower toxicity compared to the dry-weather sample of influent. Ammonia, and metals, were identified in dry weather as contributors to the observed toxicity, however, this risk was alleviated through rainwater ingress in wet-weather with further dilution within the receiving water. Based on toxicity data, dilutions of influent to minimise effects on C. vulgaris and C. dubia ranged from 1 in 12 in DW to 1 in 2.8 in WW, and further diminished in the receiving water to 1 in 1.8. The successful application of ecotoxicological approaches enabled the assessment of cumulative effects of contaminants in influent, offering valuable insights into the sanitary sewer system under rainwater ingress.

Ecotoxicological study of seven pharmaceutically active compounds: Mixture effects and environmental risk assessment.

Pharmaceutically active compounds (PhACs) have been detected in several aquatic compartments, which has been of environmental concern since PhACs can cause adverse effects on the aquatic ecosystem at low concentrations. Despite the variety of PhACs detected in surface water, ecotoxicological studies are non-existent for many of them, mainly regarding their mixture. In addition, water bodies can continuously receive the discharge of raw or treated wastewater with micropollutants. Thus, PhACs are subject to mixture and interactions, potentiating or reducing their toxicity. Therefore, the present study evaluated the toxicity on Aliivibrio fischeri of seven PhACs, which still needs to be explored in the literature. The effects were evaluated for the PhACs individually and for their binary and tertiary mixture. Also, the experimental effects were compared with the concentration addition (CA) and independent action (IA) models. Finally, an environmental risk assessment was carried out. Fenofibrate (FEN), loratadine (LOR), and ketoprofen (KET) were the most toxic, with EC50 of 0.32 mg L-1, 6.15 mg L-1 and 36.8 mg L-1, respectively. Synergistic effects were observed for FEN + LOR, KET + LOR, and KET + FEN + LOR, showing that the CA and IA may underestimate the toxicity. Environmental risks for KET concerning algae, and LOR e 17α-ethynylestradiol (EE2) for crustaceans and fish were high for several locations. Besides, high removals by wastewater treatment technologies are required to achieve the concentrations necessary for reducing KET and LOR risk quotients. Thus, this study contributed to a better understanding of the toxic interactions and environmental risks of PhACs.

Non-conventional endpoints show higher sulfoxaflor toxicity to Chironomus riparius than conventional endpoints in a multistress environment.

Evidence grows that standard toxicity testing might underestimate the environmental risk of neurotoxic insecticides. Behavioural endpoints such as locomotion and mobility have been suggested as sensitive and ecologically relevant additions to the standard tested endpoints. Possible interactive effects of chemicals and additional stressors are typically overlooked in standardised testing. Therefore, we aimed to investigate how concurrent exposure to environmental stressors (increased temperature and predation cues) and a nicotinic acetylcholine receptor (nAChR)-modulating insecticide ('sulfoxaflor') impact Chironomus riparius across a range of conventional and non-conventional endpoints. We used a multifactorial experimental design encompassing three stressors, sulfoxaflor (2.0-110 µg/L), predation risk (presence/absence of predatory cues), and elevated temperature (20 °C and 23 °C), yielding a total of 24 distinct treatment conditions. Additional stressors did not change the sensitivity of C. riparius to sulfoxaflor. To assess potential additive effects, we applied an Independent Action (IA) model to predict the impact on eight endpoints, including conventional endpoints (growth, survival, total emergence, and emergence time) and less conventional endpoints (the size of the adults, swimming abilities and exploration behaviour). For the conventional endpoints, observed effects were either lower than expected or well-predicted by the IA model. In contrast, we found greater than predicted effects of predation cues and temperature in combination with sulfoxaflor on adult size, larval exploration, and swimming behaviour. However, in contrast to the non-conventional endpoints, no conventional endpoints detected interactive effects of the neurotoxic insecticide and the environmental stressors. Acknowledging these interactions, increasing ecological context of ecotoxicological test systems may, therefore, advance environmental risk analysis and interpretation as the safe environmental concentrations of neurotoxic insecticides depend on the context of both the test organism and its environment.

Construction of a pumpless gravity-driven vascularized Skin-on-a-Chip for the study of hepatocytotoxicity in percutaneous exposure to exogenous chemicals.

The utilization of existing Skin-on-a-Chip (SoC) is constrained by the complex structures, the multiplicity of auxiliary devices, and the inability to evaluate exogenous chemicals that are hepatotoxic after percutaneous metabolism. In this study, a gravity-driven SoC without any auxiliary devices was constructed for the hepatocytotoxicity study of exogenous chemicals. The SoC possesses 3 layers of culture chambers, from top to bottom, for human skin equivalent (HSE), Human Umbilical Vein Endothelial Cells (HUVEC) and hepatocytes (HepG2), and the maintenance and expression capacity of the corresponding cells on the SoC were verified by specificity parameters. The reactivity of the SoC to exogenous chemicals was verified by 2-aminofluorene (2-AF). The SoC can realistically simulate the in vivo exposure process of exogenous chemicals that are percutaneously exposed and metabolized into the bloodstream and then to the liver to produce toxicity, and it can achieve the same effects on transcriptome as those of animal tests at lower exposure levels while examining multiple toxicological targets of the skin, vascular endothelial cells, and hepatocytes. Both in terms of species similarity, the principles of reduction, replacement and refinement (3R), or the level of exposure suggest that the present SoC has a degree of replacement for animal models in assessing exogenous chemicals, especially those that are hepatotoxic after percutaneous metabolism.

Transcriptomic Point of Departure (tPOD) of androstenedione in zebrafish embryos as a potential surrogate for chronic endpoints.

The transcriptomic Point of Departure (tPOD) is increasingly used in ecotoxicology to derive quantitative endpoints from RNA sequencing studies. Utilizing transcriptomic data in zebrafish embryos as a New Approach Methodology (NAM) is beneficial due to its acknowledgment as an alternative to animal testing under EU Directive 2010/63/EU. Transcriptomic profiles are available in zebrafish for various modes of action (MoA). The limited literature available suggest that tPOD values from Fish Embryo Toxicity (FET) tests align with, but are generally lower than, No Observed Effect Concentrations (NOEC) from long-term chronic fish toxicity tests. In studies with the androgenic hormone androstenedione in a Fish Sexual Development Test (FSDT), a significant shift in the sex ratio towards males was noted at all test concentrations, making it impossible to determine a NOEC (NOEC <4.34 µg/L). To avoid additional animal testing in a repetition of the FSDT and adhere to the 3Rs principle (replacement, reduction, and refinement), a modified zebrafish FET (zFET) was conducted aiming to determine a regulatory acceptable effect threshold. This involved lower concentration ranges (20 to 6105 ng/L), overlapping with the masculinization-observed concentrations in the FSDT. The tPOD analysis in zFET showed consistent results with previous FSDT findings, observing strong expression changes in androgen-dependent genes at higher concentrations but not at lower ones, demonstrating a concentration-response relationship. The tPOD values for androstenedione were determined as 24 ng/L (10th percentile), 60 ng/L (20th gene), and 69 ng/L (1st peak). The 10th percentile tPOD value in zFET was 200 times lower than the lowest concentration in the FSDT. Comparing the tPOD values to literature suggests their potential to inform on the NOEC range in FSDT tests.

[Development of a Novel Male Reproductive Toxicity Assessment Method to Predict Male-mediated Effects on the Next Generation].

Less than 10% of the candidate drug compounds are associated with male reproductive toxicity. Genetic and/or epigenetic information on sperm may be crucial for fetal development. Therefore, developmental toxicity, such as paternally transmitted birth defects, is possible if genetic abnormalities in the male germ line persist and accumulate in the sperm during spermatogenesis. First, this study provides an overview of chemical and male reproductive toxicity, which may lead to developmental toxicity from the perspective of male reproduction. Second, we demonstrate methods for evaluating male reproductive toxicity to anticipate male-mediated developmental toxicity. We developed a novel staining technique for evaluating sperm quality, as well as a noninvasive imaging analysis of male reproductive toxicity. The former is a mammalian male germ cell-specific staining method using reactive blue 2 dye (RB2), as previously confirmed in human sperm, and a method for detecting the early-stage DNA fragmentation in a single nucleus from mouse spermatozoa using single-cell pulsed-field gel electrophoresis. The latter is a new, ready-to-use, and compact magnetic resonance imaging (MRI) platform utilizing a high-field permanent magnet to evaluate male reproductive toxicity. The histopathological analysis supported the suitability of the MRI platform. The present study, for the first time, revealed a rapid, noninvasive evaluation of male reproductive toxicity in vivo using compact MRI. These novel toxicity assessments can help predict male-mediated developmental toxicity, contributing to accelerated drug discovery and drug repositioning.

Inhalation Toxicity Screening of Consumer Products Chemicals using OECD Test Guideline Data-based Machine Learning Models.

This study aimed to screen the inhalation toxicity of chemicals found in consumer products such as air fresheners, fragrances, and anti-fogging agents submitted to K-REACH using machine learning models. We manually curated inhalation toxicity data based on OECD test guideline 403 (Acute inhalation), 412 (Sub-acute inhalation), and 413 (Sub-chronic inhalation) for 1709 chemicals from the OECD eChemPortal database. Machine learning models were trained using ten algorithms, along with four molecular fingerprints (MACCS, Morgan, Topo, RDKit) and molecular descriptors, achieving F1 scores ranging from 51 % to 91 % in test dataset. Leveraging the high-performing models, we conducted a virtual screening of chemicals, initially applying them to data-rich chemicals generally used in occupational settings to determine the prediction uncertainty. Results showed high sensitivity (75 %) but low specificity (23 %), suggesting that our models can contribute to conservative screening of chemicals. Subsequently, we applied the models to consumer product chemicals, identifying 79 as of high concern. Most of the prioritized chemicals lacked GHS classifications related to inhalation toxicity, even though they were predicted to be used in many consumer products. This study highlights a potential regulatory blind spot concerning the inhalation risk of consumer product chemicals while also indicating the potential of artificial intelligence (AI) models to aid in prioritizing chemicals at the screening level.

Evaluation of rat and rabbit embryofetal development studies with pharmaceuticals: the added value of a second species.

Embryofetal development (EFD) studies are performed to characterize risk of drugs in pregnant women and on embryofetal development. In line with the ICH S5(R3) guideline, these studies are generally conducted in one rodent and one non-rodent species, commonly rats and rabbits. However, the added value of conducting EFD studies in two species to risk assessment is debatable. In this study, rat and rabbit EFD studies were evaluated to analyze the added value of a second species. Information on rat and rabbit EFD studies conducted for human pharmaceuticals submitted for marketing authorization to the European Medicines Agency between 2004 and 2022 was collected from the database of the Dutch Medicines Evaluation Board, along with EFD studies conducted for known human teratogens. In total, 369 compounds were included in the database. For 55.6% of the compounds similar effects were observed in rat and rabbit EFD studies. Discordance was observed for 44.6% of compounds. Discordance could often be explained based on occurrence of maternal toxicity or the compound's mechanism of action. For other compounds, discordance was considered of limited clinical relevance due to high exposure margins or less concerning EFD toxicity. For 6.2%, discordance could not be explained and was considered clinically relevant. Furthermore, for specific therapeutic classes, concordance between rat and rabbit could vary. In conclusion, in many cases the added value of conducting EFD studies in two species is limited. These data could help identify scenarios in which (additional) EFD studies could be waived or create a weight-of-evidence model to determine the need for (additional) EFD studies.

Use of the threshold of toxicological concern (TTC) approach as an alternative tool for regulatory purposes: A case study with an inert ingredient used in pesticide products.

This study investigates the potential of the Threshold of Toxicological Concern (TTC) as an alternative to traditional animal testing in pesticide regulatory risk assessments. The TTC is a principle that establishes exposure threshold values for chemicals with certain structural features, below which there is no appreciable risk to human health. A case study was conducted with α-terpineol, an inert ingredient proposed to be used at low concentrations in pesticide products, to compare a conventional risk assessment using animal data with one using the TTC method. For the conventional risk assessment, animal data showed that there was no toxicity endpoint of concern, which resulted in a qualitative assessment and no risks of concern identified. For the risk assessment using the TTC method, a 5th percentile no-observed-effect level (NOEL) selected based on α-terpineol's Cramer classification was used as a point of departure (POD) for a quantitative risk assessment that resulted in no risks of concern identified. Therefore, the same conclusion was reached with both approaches and α-terpineol is considered safe for use in pesticide products at low concentrations. A comparative analysis was also performed to determine the applicability of the TTC method in calculating potential dietary risk from common pesticide use patterns for chemicals that fall within different Cramer classes. Results showed that use of the TTC method may be feasible for inert ingredient risk assessments when chemicals are used in a pesticide product at concentrations below 1%. This research underscores the TTC as a valuable and robust tool for assessing the potential hazards from inert ingredient use in pesticide formulations, considering factors such as chemical properties and the concentrations at which a chemical may be used in pesticide products. These findings contribute to the ongoing efforts by the United States Environmental Protection Agency (US EPA) to reduce animal testing in chemical safety assessments. The TTC method presents a viable alternative for risk evaluations of chemicals used at low concentrations, with anticipated low exposure, and with a predicted low toxicity potential.

Encapsulation and release of tetrabromobisphenol A in microplastics: Trade-off in their individual toxicity to Xenopus tropicalis tadpoles.

The toxicity of microplastics (MPs) to aquatic animals is closely related to the presence and release kinetics of contained additives, as most plastic products contain various additives. However, the relationship between the occurrence and release of additives from MPs, and their individual or combined toxicity remains unclear. In this study, the nanoscale distribution and release of tetrabromobisphenol A (TBBPA, a common flame retardant with endocrine-disrupting effect) in polystyrene (PS) MPs, and the long-term (60 days) toxicity of TBBPA and MPs containing TBBPA (at doses of 0 %, 1 %, 10 %, w/w) to Xenopus tropicalis tadpoles were investigated. Exposure to 10 µg/L TBBPA alone was the most toxics, while the encapsulation of TBBPA in MPs significantly delayed its lethal toxicity to tadpoles by inhibiting the rapid and extensive release of TBBPA. PS MPs alone and MPs containing 10 % TBBPA exhibited delayed survival toxicity compared to TBBPA alone, whereas PS MPs containing 1 % TBBPA did not show this effect but inhibited growth. These findings suggest that chronic toxicity assessments should be based on long-term (months or even years) exposure experiments due to the encapsulation-controlled slow release of toxic additives.

Development of Sensitive In Vitro Protocols for the Biocompatibility Testing of Medical Devices and Pharmaceuticals Intended for Contact with the Eyes: Acute Irritation and Phototoxicity Assessment.

This study introduces a novel in vitro methodology that employs the 3-D reconstructed tissue model, EpiOcular, to assess the irritation and phototoxicity potential of medical devices and drugs in contact with the eye. Our study evaluated diverse test materials, including medical devices, ophthalmological solutions and an experimental drug (cemtirestat), for their potential to cause eye irritation and phototoxicity. The protocols used in this study with the EpiOcular tissue model were akin to those used in the ultra-mildness testing of cosmetic formulations, which is challenging to predict with standard in vivo rabbit tests. To design these protocols, we leveraged experience gained from the validation project on the EpiDerm skin irritation test for medical devices (ISO 10993-23:2021) and the OECD TG 498 method for photo-irritation testing. The predictions were based on the tissue viability and inflammatory response, as determined by IL-1α release. By developing and evaluating these protocols for medical devices, we aimed to expand the applicability domain of the tests referred to in ISO 10993-23. This will contribute to the standardisation and cost-effective safety evaluation of ophthalmic products, while reducing reliance on animal testing in this field. The findings obtained from the EpiOcular model in the photo-irritation test could support its implementation in the testing strategies outlined in OECD TG 498.

Generation, characterization, and toxicological assessment of reference ultrafine soot particles with different organic content for inhalation toxicological studies.

Ultrafine particles (UFP) are the smallest atmospheric particulate matter linked to air pollution-related diseases. The extent to which UFP's physical and chemical properties contribute to its toxicity remains unclear. It is hypothesized that UFP act as carriers for chemicals that drive biological responses. This study explores robust methods for generating reference UFP to understand these mechanisms and perform toxicological tests. Two types of combustion-related UFP with similar elemental carbon cores and physical properties but different organic loads were generated and characterized. Human alveolar epithelial cells were exposed to these UFP at the air-liquid interface, and several toxicological endpoints were measured. UFP were generated using a miniCAST under fuel-rich conditions and immediately diluted to minimize agglomeration. A catalytic stripper and charcoal denuder removed volatile gases and semi-volatile particles from the surface. By adjusting the temperature of the catalytic stripper, UFP with high and low organic content was produced. These reference particles exhibited fractal structures with high reproducibility and stability over a year, maintaining similar mass and number concentrations (100 µg/m3, 2.0·105 #/cm3) and a mean particle diameter of about 40 nm. High organic content UFP had significant PAH levels, with benzo[a]pyrene at 0.2 % (m/m). Toxicological evaluations revealed that both UFP types similarly affected cytotoxicity and cell viability, regardless of organic load. Higher xenobiotic metabolism was noted for PAH-rich UFP, while reactive oxidation markers increased when semi-volatiles were stripped off. Both UFP types caused DNA strand breaks, but only the high organic content UFP induced DNA oxidation. This methodology allows modification of UFP's chemical properties while maintaining comparable physical properties, linking these variations to biological responses.

Advancing ecotoxicological studies: Utilizing new approach methodologies to enable cross-species extrapolation and reduce avian testing.

Ecological risk assessments of agrochemicals have traditionally depended on in vivo guideline tests using northern bobwhite and mallard to provide relevant endpoints for avian species. However, these studies have limitations, including animal welfare concerns, the time and cost involved, limited potential for extrapolation to more realistic exposure conditions, and the lack of mechanistic understanding. The proof-of-concept work presented a case study for thiamethoxam in three avian species, demonstrating the potential of physiologically based kinetic (PBK) modeling to enable dosimetry extrapolations that inform hazard characterization in risk assessment, and reduce the use of avian testing. The model structure for northern bobwhite and mallard contained ten compartments, while an additional ovulation model was included for chicken in the physiological state of egg-laying. The model was first parameterized and evaluated for chicken and northern bobwhite using in vitro kinetic measurements and in vivo toxicokinetic (TK) data. The chicken model was then extrapolated to mallard based on allometric scaling. The models were then used to map the TK profiles across species by simulating internal dose metrics in different avian toxicology studies. These metrics, including peak blood concentrations (Cmax) and area under the curve (AUC) for blood concentration, were determined for acute, subacute, or chronic toxicity endpoints for mallard and northern bobwhite, enabling a quantitative cross-species and cross-route comparison of dosimetry. The results suggested that the chronic toxicological response of birds exposed to thiamethoxam is highly dependent on internal exposure, while mallard appeared to be more dynamically sensitive to thiamethoxam on an acute oral exposure basis. The case study increases the confidence in using new approach methodologies (NAMs) for interpreting avian toxicity studies and facilitating in vitro-in silico-based ecological risk assessments of agrochemicals.

New approach methodologies to confirm developmental toxicity of pharmaceuticals based on weight of evidence.

The aim of embryo-fetal developmental toxicity assessments for pharmaceuticals is to inform potential risk of adverse pregnancy outcome, which has traditionally relied on studies in pregnant animals. Recent updates to international safety guidelines (ICH S5R3) have incorporated information on how to use weight of evidence and alternative assays to reduce animal use while still informing risk of fetal harm. Uptake of these alternative approaches has been slow due to limitations in understanding how alternative assays translate to in vivo effects and then relevance to human exposure. To understand the predictivity of new approach methodologies for developmental toxicity (DevTox NAMs), we used two pharmaceutical examples (glasdegib and lorlatinib) to illustrate the value of DevTox NAMs to complement weight of evidence (WoE) assessments while considering the relationship of concentration-effect levels in NAMs to in vivo studies. The in vitro results generated in a battery of assays (mEST, rWEC, zebrafish, and human based stem cells) confirmed the WoE based on literature and further confirmed by preliminary embryo-fetal development data. The data generated for these two compounds supports integrating DevTox NAMs into the developmental toxicity assessment for advanced cancer indications.