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In vivo developmental neurotoxicity (DNT) testing is resource intensive and lacks information on cellular processes affected by chemicals. To address this, DNT new approach methodologies (NAMs) are being evaluated, including: the microelectrode array neuronal network formation assay; and high-content imaging to evaluate proliferation, apoptosis, neurite outgrowth, and synaptogenesis. This work addresses 3 hypotheses: (1) a broad screening battery provides a sensitive marker of DNT bioactivity; (2) selective bioactivity (occurring at noncytotoxic concentrations) may indicate functional processes disrupted; and, (3) a subset of endpoints may optimally classify chemicals with in vivo evidence for DNT. The dataset was comprised of 92 chemicals screened in all 57 assay endpoints sourced from publicly available data, including a set of DNT NAM evaluation chemicals with putative positives (53) and negatives (13). The DNT NAM battery provides a sensitive marker of DNT bioactivity, particularly in cytotoxicity and network connectivity parameters. Hierarchical clustering suggested potency (including cytotoxicity) was important for classifying positive chemicals with high sensitivity (93%) but failed to distinguish patterns of disrupted functional processes. In contrast, clustering of selective values revealed informative patterns of differential activity but demonstrated lower sensitivity (74%). The false negatives were associated with several limitations, such as the maximal concentration tested or gaps in the biology captured by the current battery. This work demonstrates that this multi-dimensional assay suite provides a sensitive biomarker for DNT bioactivity, with selective activity providing possible insight into specific functional processes affected by chemical exposure and a basis for further research., (Published by Oxford University Press on behalf of the Society of Toxicology 2022. This work is written by US Government employees and is in the public domain in the US.)

Recently, we showed that the addition of physiological concentrations of ascorbic acid, a tear antioxidant, to the OptiSafe™ macromolecular eye irritation test reduced the false-positive (FP) rate for chemicals that had reactive chemistries, leading to the formation of reactive oxygen species (ROS) and molecular crosslinking. The purpose of the current study was to 1) increase the number of chemicals tested to comprehensibly determine whether the antioxidant-associated reduction in OD is specific to FP chemicals associated with ROS chemistries and 2) determine whether the addition of antioxidants interferes with the detection of true positive (TP) and true negative (TN) ocular irritants. We report that when ascorbic acid is added to the test reagents, retesting of FP chemicals with reactive chemistries show significantly reduced OD values (P < 0.05). Importantly, ascorbic acid had no significant effect on the OD values of TP or TN chemicals regardless of chemical reactivity. These findings suggest that supplementation of ascorbic acid in alternative ocular irritation tests may help improve the detection of TN for those commonly misclassified reactive chemicals., (Copyright © 2022. Published by Elsevier Ltd.)

The present study evaluates the in vitro developmental toxicity of 4- and 5-ring polycyclic aromatic hydrocarbons (PAHs) including benz[a]anthracene (BaA) and benzo[a]pyrene (BaP) and six of their monomethylated congeners, and dibenz[a,h]anthracene (DB[a,h]A) using the zebrafish embryotoxicity test (ZET). In general, the tested PAHs induced various developmental effects in the zebrafish embryos including unhatched embryos, no movement and circulation, yolk sac and pericardial edemas, deformed body shape, and cumulative mortality at 96 h post fertilization (hpf). The methyl substituent on different positions of the aromatic ring of the PAHs appeared to change their in vitro developmental toxicity. Comparison to a previously reported molecular docking study showed that the methyl substituents may affect the interaction of the PAHs with the aryl hydrocarbon receptor (AhR) which is known to play a role in the developmental toxicity of some PAHs. Taken together, our results show that methylation can either increase or decrease the developmental toxicity of PAHs, and suggest this may in part relate to effects on the molecular dimensions and resulting consequences for interactions with the AhR., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study describes the within- and between-laboratory reproducibility (WLR and BLR) of a Time-to-Toxicity (TTT) approach for chemicals based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). The WLR and BLR was assessed with three participating laboratories. Each laboratory tested 40 coded chemicals in three independent runs. The predictive capacity of the method was assessed on a larger set of 150 chemicals (70 liquids and 80 solids) by combining the results of this study with the results of the test method developer. The WLR for the 20 liquids ranged from 85% to 95% with a BLR of 90%. For the 20 solids, a WLR and BLR of 100% was obtained. The test method developer obtained a WLR of 80% and 95%, based on 50 liquids and 48 solids tested in three independent runs, respectively. Regarding the predictive capacity, the SkinEthic™ HCE TTT test method identified 80.8% Cat. 1, 69.2% Cat. 2, and 74.9% No Cat. correctly. An independent peer review panel concluded that based on all available data, the relevance and reliability of the SkinEthic™ HCE TTT has been demonstrated for discriminating the three UN GHS eye hazard categories., (Copyright © 2022. Published by Elsevier Ltd.)

The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity rely mainly on animal studies, which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Here, human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized a fetal bovine serum (FBS)-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as beating frequency and area of beating cardiomyocytes by video analyses are measured. The embryotoxic positive and non-embryotoxic negative controls, 5-Fluorouracil (5-FU) and Penicillin G (PenG), respectively, were correctly assessed in the hiPS Test. More compounds need to be screened in the future for defining the assay's applicability domain, which will inform us of the suitability of the hiPS Test for detecting adverse effects of substances on embryonic development.

The U-SENS™ assay was developed to address the third key event of the skin sensitization adverse outcome pathway (AOP) and is described in OECD test guideline 442E, Annex II. A dataset of 68 fragrance ingredients comprised of 7 non-sensitizers and 61 sensitizers was tested in the U-SENS™ assay. The potential for fragrance ingredients to activate dendritic cells, measured by U-SENS™, was compared to the sensitization potential determined by weight of evidence (WoE) from historical data. Of the non-sensitizers, 4 induced CD86 cell surface marker ≥1.5-fold while 3 did not. Of the sensitizers, 50 were predicted to be positive in U-SENS™, while the remaining 11 were negative. Positive and negative predictive values (PPV and NPV) of U-SENS™ were 93% and 21%, respectively. No specific chemical property evaluated could account for misclassified ingredients. Assessment of parent and metabolite protein binding alerts in silico suggests that parent chemical metabolism may play a role in CD86 activation in U-SENS™. Combining the U-SENS™ assay in a "2 out of 3" defined approach with the direct peptide reactivity assay (DPRA) and KeratinoSens™ predicted sensitization hazard with PPV and NPV of 97% and 24%, respectively. Combining complementary in silico and in vitro methods to the U-SENS™ assay should be integrated to define the hazard classification of fragrance ingredients, since a single NAM cannot replace animal-based methods., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Animals have been indispensable in testing chemicals that can pose a risk to human health, including those delivered by inhalation. In recent years, the combination of societal debate on the use of animals in research and testing, the drive to continually enhance testing methodologies, and technology advancements have prompted a range of initiatives to develop non-animal alternative approaches for toxicity testing. In this review, we discuss emerging in vitro techniques being developed for the testing of inhaled compounds. Advanced tissue models that are able to recreate the human response to toxic exposures alongside examples of their ability to complement in vivo techniques are described. Furthermore, technology being developed that can provide multi-organ toxicity assessments are discussed., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This report introduces a novel method, rabbit whole embryo culture (WEC) combined with toxicokinetics (TK), for toxicity testing. Rodent WEC has been extensively used for in vitro screening of developmental toxicity. To improve the reliability of in vitro data, it is important to consider TK and species specificity. To test the utility and effectiveness of this method, we investigated the toxic effect of thalidomide on rabbit embryos and its behavior in test systems both in vitro and in vivo under the same experimental condition. The data showed that thalidomide induced embryo malformations such as embryonic brain hypoplasia, short limb buds, and declined embryonic growth both in vitro and in vivo. The toxic effect increased with the increasing exposure of the embryo to thalidomide. In addition, we observed similar toxic effects and exposure-effect relationships in vivo and in vitro. Therefore, we preliminarily conclude that this new method can effectively predict and explain thalidomide toxicity. Furthermore, we investigated the behavior of test compounds in the WEC system for the first time, and this method is expected to be an important technique for in vitro toxicity study after extensive verification., (© 2021 John Wiley & Sons, Ltd.)

The safety assessment of fragrance materials for photoirritation utilized by The Research Institute for Fragrance Materials has recently been modified and is described in detail. Materials demonstrating significant absorbance in the ultraviolet and visible light (UV/VIS) range (290-700 nm) may present a concern for photoirritation and require further investigation. If there are no photoirritation data or data are insufficient, then data on read-across materials are considered before a tiered approach for testing begins. The hazard-based 3T3-Neutral Red Uptake (NRU) Phototoxicity Test (OECD TG 432) is used as a first-tier assay; if it predicts photoirritation, it is followed by the reconstructed human epidermis (RhE) phototoxicity assay (OECD TG 498). The RhE phototoxicity assay is used to determine a No Observed Effect Level (NOEL) for photoirritation that is used in a confirmatory human photoirritation test. Data are presented on 108 fragrance materials exhibiting significant UV/VIS absorbance and evaluated in the 3T3-NRU Phototoxicity Assay. Twenty-one materials were predicted to be phototoxic; twenty were evaluated in the RhE Phototoxicity Assay to establish a NOEL. Fourteen materials were then evaluated in a confirmatory human phototoxicity test. The tiered testing approach presented represents a scientifically pragmatic method to minimize the likelihood of photoirritation from fragrance materials., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acute inhalation toxicity testing for chemical classification and labeling has been performed using animal models, however, these models have limited predictibility of the toxicity on the respiratory system. Thus, non-animal models have been emerging as alternatives for preclinical assessment of respiratory toxicity of chemicals, comprising in chemico, in vitro, ex vivo, and in silico approaches. In this study, we characterized and evaluated the applicability of a new ex vivo bovine bronchial model for addressing key aspects of pulmonary toxicity. Standardized bronchial fragments were cultured at an air-liquid interface for seven days showing cell viability, morphology, and function during the ex vivo time of cultivation. Different exposure ways, liquid or aerosol exposure, were also studied using paraformaldehyde (PFA) as a positive control. In a concentration-dependent manner, a decrease in tissue viability was observed for aerosols instead of direct liquid exposure upon tissue surface. Moreover, PFA exposure allowed the addressment of several damage biomarkers, including epithelium thickness, mitochondrial activity, ROS production, and caspase-3 activation. Besides, the bronquial tissue was exposed to chemicals from different UN GHS inhalation toxicity categories and presented a concentration-dependent response for most of the evaluated materials. The proposed airway ex vivo model represents a low-cost and reproducible tool applicable for pulmonary toxicity assessment of chemicals., (Copyright © 2022. Published by Elsevier Ltd.)

Assessment of aerosols effects on liver CYP function generally involves aqueous fractions (AF). Although easy and efficient, this method has not been optimized recently or comparatively assessed against other aerosol exposure methods. Here, we comparatively evaluated the effects of the AFs of cigarette smoke (CS) and Tobacco Heating System (THS) aerosols on CYP activity in liver spheroids. We then used these data to develop a physiological aerosol exposure system combining a multi-organs-on-a-chip, 3D lung tissues, liver spheroids, and a direct aerosol exposure system. Liver spheroids incubated with CS AF showed a dose-dependent increase in CYP1A1/1B1, CYP1A2, and CYP2B6 activity and a dose-dependent decrease in CYP2C9, CYP2D6, and CYP3A4 activity relative to untreated tissues. In our physiological exposure system, repeated CS exposure of the bronchial tissues also caused CYP1A1/1B1 and CYP1A2 induction in the bronchial tissues and liver spheroids; but the spheroids showed an increase in CYP3A4 activity and no effect on CYP2C9 or CYP2D6 activity relative to air-exposed tissues, which resembles the results reported in smokers. THS aerosol did not affect CYP activity in bronchial or liver tissues, even at 4 times higher concentrations than CS. In conclusion, our system allows us to physiologically test the effects of CS or other aerosols on lung and liver tissues cultured in the same chip circuit, thus delivering more in vivo like data., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach. The outputs from each tier are classification categories, safe doses, and risk assessments, and progress through the tiers depends on the output from previous tiers. We have exemplified the use of the framework with three examples. The outputs were the same or more conservative than parallel assessments based on conventional studies. The framework allows a transparent and phased introduction of NAMs in chemical safety assessment and enables science-based safety decisions which provide the same level of public health protection using fewer animals, taking less time, and using less financial and expert resource. Furthermore, it would also allow new methods to be incorporated as they develop through continuous selective evolution rather than periodic revolution., (© 2022. The Author(s).)

In vitro microelectrode array (MEA) assessment using human induced pluripotent stem cell (iPSC)-derived neurons holds promise as a method of seizure and toxicity evaluation. However, there are still issues surrounding the analysis methods used to predict seizure and toxicity liability as well as drug mechanisms of action. In the present study, we developed an artificial intelligence (AI) capable of predicting the seizure liability of drugs and identifying drugs using deep learning based on raster plots of neural network activity. The seizure liability prediction AI had a prediction accuracy of 98.4% for the drugs used to train it, classifying them correctly based on their responses as either seizure-causing compounds or seizure-free compounds. The AI also made concentration-dependent judgments of the seizure liability of drugs that it was not trained on. In addition, the drug identification AI implemented using the leave-one-sample-out scheme could distinguish among 13 seizure-causing compounds as well as seizure-free compound responses, with a mean accuracy of 99.9 ± 0.1% for all drugs. These AI prediction models are able to identify seizure liability concentration-dependence, rank the level of seizure liability based on the seizure liability probability, and identify the mechanism of the action of compounds. This holds promise for the future of in vitro MEA assessment as a powerful, high-accuracy new seizure liability prediction method., (© 2022. The Author(s).)

The development of a universal, label-free, and reliable in vitro toxicity testing method for nanoparticles is urgent because most nanoparticles can interfere with toxicity assays. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable in vitro toxicity assay for testing nanoparticles without such interference. Recently, the Organisation for Economic Co-operation and Development (OECD) developed a 60 × 15 mm Petri dish-based CFE assay for testing nanoparticles in MDCK-1 cells. However, further investigations are needed, including testing with other cell types, at a smaller scale for greater efficiency, and the application of the co-culture technique. In this study, we selected TiO 2 , CuO, CeO 2 , and SiO 2 as test nanoparticles and successfully developed a 6-well plate-based CFE assay using HepG2 and A549 cells and a co-culture assay for combinations of HepG2 cells and THP-1 macrophages or A549 cells and THP-1 monocytes. The results suggest that the 6-wellplate-based CFE assay for HepG2 and A549 cells can be applied to nanoparticles, but the co-culture CFE assay has limitations in that it is not different from the single culture study, and it inhibits colony-formation by A549 cells in the presence of macrophages; this warrant further study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The database of practical examples where toxicokinetic (TK) data has benefitted all stages of the human health risk assessment process are increasingly being published and accepted. This review aimed to highlight and summarise notable examples and to describe the "state of the art" in this field. The overall recommendation is that for any in vivo animal study conducted, measurements of TK should be very carefully considered for inclusion as the numerous benefits this brings continues to grow, particularly during the current march towards animal free toxicology testing and ambitions to eventually conduct human health risk assessments entirely based upon non-animal methods., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ciguatoxins (CTXs) which are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa and share a ladder-shaped polyether structure, are causative compounds of one of the most frequent foodborne illness disease known as ciguatera fish poisoning (CFP). CFP was initially found in tropical and subtropical areas but nowadays the dinoflagellates producers of ciguatoxins had spread to European coasts. Therefore, this raises the need of establishing toxicity equivalency factors for the different compounds that can contribute to ciguatera fish poisoning, since biological methods have been replaced by analytical techniques. Thus, in this work, the effects of six compounds causative of ciguatera, on their main target, the human voltage-gated sodium channels have been analyzed for the first time. The results presented here led to the conclusion that the order of potency was CTX1B, CTX3B, CTX4A, gambierol, gambierone and MTX3. Furthermore, the data indicate that the activation voltage of sodium channels is more sensitive to detect ciguatoxins than their effect on the peak sodium current amplitude., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Formaldehyde mainly emitted from wood adhesives, finishing materials, paint for furniture represents, together with wood dust, a potential carcinogenic risk for wood workers. Aims of this multidisciplinary study are to investigate the possibility of replacing urea-formaldehyde (UF) adhesives in the wood industry with organic and/or inorganic-based glues to obtain a final less toxic product and to evaluate the potential toxicity of wood glued with such new adhesives. For this purpose we selected poplar wood to test an organic new adhesive HBP (Hemp Based Protein), a mixture of hemp flour and cross-linker PAE (polyaminoamide epichlorohydrin), and spruce wood to test an inorganic adhesive geopolymer K-PSS (potassium-polysiloxosialate) plus polyvinyl acetate. For the poplar wood, we also used a commercial panel glued with UF for comparison. We reproduced occupational inhalation exposure during sawing activities of mentioned woods, collected and characterized the wood dusts emitted during sawing and evaluated in vitro their potential cyto-genotoxic and inflammatory effects. We used human lung cells (A549) exposed for 24 h to 20 and 100 μg/mL of collected PM 2.5 wood dust. We found that both the new adhesives wood dusts induced a slightly higher apoptotic effect than untreated natural wood dusts particularly in spruce wood. Only geopolymer K-PSS wood dust induced membrane damage at the highest concentration and direct and oxidative DNA damage that could be explained by the different chemical composition and the lower particle sizes in respect to organic HBP adhesive wood dust. We found slight induction of IL-6 release, not influenced by K-PSS treatment, at the highest concentration in spruce wood. For poplar wood, IL-6 and IL-8 induction was found particularly for untreated and UF-treated wood at the highest concentration, where hemp adhesive treatment induced lower inflammation while at lower concentration similar slight cytokine induction was found for all tested wood dusts. This preliminary study shows that natural adhesives used to replace UF adhesives represent an interesting alternative, particularly the organic hemp-based adhesive showing very low toxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds (1) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), (2) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), (3) classified as non-embryotoxic by ECVAM (Penicillin G), and (4) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine)., (© 2022. The Author(s).)

Ethnopharmacological Relevance: Traditional Chinese medicine (TCM) has been applied for more than 2000 years. However, modern basic research on the safety of TCMs is limited. Establishing safety evaluation technology in line with the characteristics of TCM and conducting large-scale basic toxicity research are keys to comprehensively understand the toxicity of TCMs. In recent years, zebrafish has been used as a model organism for toxicity assessment and is increasingly utilized for toxicity research of TCMs. Yet, a comprehensive review in using zebrafish as a toxicological model for TCMs is lacked., Aim of the Study: We aim to summarize the progress and limitation in toxicity evaluation of TCMs using zebrafish and put forward the future research ideas., Materials and Methods: The scientific databases, including Springer, Science Direct, Wiley, Pubmed and China Knowledge Resource Integrated (CNKI) were searched using the key words of zebrafish, toxicology, traditional Chinese medicine, acute toxicity, liver injury, cardiotoxicity, kidney toxicity, developmental toxicity, neurotoxicity, gastrointestinal irritation, immunotoxicity, ototoxicity, and osteotoxicity., Results: Zebrafish assays are low experimental cost and short cycle, easily achieving high-throughput toxicity screening, and exemption from ethical legislation up to 5 dpf. It has been widely used to evaluate the acute toxicity, liver toxicity, cardiotoxicity, nephrotoxicity, developmental toxicity, neurotoxicity, gastrointestinal irritation, immunotoxicity, and ototoxicity caused by TCMs, although some physiological difference limited its application., Conclusions: Zebrafish is a powerful model for TCMs toxicity evaluation, but it is not flawless. The toxicity testing criterion and high throughput assays are urgent to be established. This review provides references for future studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Cuscuta reflexa Roxb. (C. reflexa) is a well-known traditional herbal plant, with numerous inherent therapeutic potentials including anticancer, antitumor, antibacterial, analgesic, anthelmintic, laxative and others. Moreover, the anticancer and antitumor potentials of this herb are ongoing with several trails, thus an attempt was made to assess the anticancer and hepatoprotective potentials of traditional C. reflexa herbs., Method: The dried ethanolic extract of C. reflexa was tested for acute oral toxicity in the treated animals subsequently their behavioral, neurological, and autonomic profiles changes were observed. The preliminary anti-cancer effects of extracts against 1, 2- Dimethyl hydrazine (DMH) induced animals were assessed through barium enema X-ray, colonoscopy, and Aberrant crypt foci (ACF) studies. The blood samples of the animals (treated and untreated) were collected and their in-vitro histological parameters were evaluated by the experienced technician., Results: It was observed that C. reflexa significantly reduced Disease activity indexing (DAI) level and ACF counting, as well as demonstrated similar activity as of the standard drug 5-Fluorouracil (5-FU). Histopathological results revealed that the apoptotic bodies decreased in the DMH-induced group (group II) during cancer progression while in 5-FU treated (group III) and C. reflexa treated (group IV and V) animals the apoptotic bodies were increased. Inversely, the mitotic bodies increased in group II animals and reduced in group III, IV, and V animals. In the colonic section, DMH-induced cancer assay exhibited significant effects on the levels of hemoglobin, Packed cell volume (PCV), Red blood cell (RBC) counts, Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), and Mean cell hemoglobin (MCH), and was found to be less in group II animals whereas administration of C. reflexa efficiently recovered back the loss probably by healing the colon damage/depletion of cancer progression. Moreover, compared to the group II animals, the neutrophil count was within the normal range in C. reflexa administered group., Conclusions: In the present study, the major hematological parameters significantly increased within DMH treated animals and exhibited extensive damage in the hepatic regions. Moreover, the histopathological findings demonstrated that the C. reflexa extracts potentially reduced the cell proliferation, with no toxicity. The C. reflexa extracts exhibited impending anti-cancer activity as well as protected the hepatic cells and thus could be potentially used in the management of colon or colorectal cancer and hepatic impairments., (Copyright © 2021 Elsevier B.V. All rights reserved.)

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (C max ) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC 10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Mansoa hirsuta is a medicinal plant native to the Brazilian semi-arid region. This approach aimed to investigate the in vitro and in vivo toxicity and anti-inflammatory and analgesic actions of the M. hirsuta fraction (MHF). In vitro cell viability was assessed in 3T3 cells. In vivo, the acute toxicity test, a single dose of the MHF was administered. For the subchronic toxicity test, three doses of were administered for 30 days. Locomotion and motor coordination were assessed using open field and rota-rod. The anti-inflammatory activity was evaluated in carrageenan-induced paw edema and zymosan-induced air-pouch models. Myeloperoxidase (MPO) and total proteins were also measured. The antinociceptive activity MHF was determined using acid acetic-induced abdominal writhing and formalin models. In the cytotoxicity assay, MHF showed no significative impairment of cell viability and in the acute toxicity study, did not cause mortality or signs of toxicity. Repeated exposure to MHF did not cause relevant toxicological changes. The evaluation in the open field test showed that the MHF did not alter the locomotor activity and there was no change in motor coordination and balance of animals. MHF significantly reduced edema, MPO production, the migration of leukocytes and protein leakage. In addition, MHF reduced abdominal writhing and significantly inhibited the first and second stage of the formalin test. The results of this study indicated that MHF has an anti-inflammatory and analgesic potential without causing acute or subchronic toxic effects and it can be a promising natural source to be explored., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

There is a pressing need to understand the impact of contaminants on Arctic ecosystems; however, most toxicity tests are based on temperate species, and there are issues with reliability and relevance of bioassays in general. Together this may result in an underestimation of harm to Arctic organisms and contribute to significant uncertainty in risk assessments. To help address these concerns, a critical review to assess reported effects for these species, quantify methodological and endpoint relevance gaps, and identify future research needs for testing was performed. We developed uniform criteria to score each study, allowing an objective comparison across experiments to quantify their reliability and relevance. We scored a total of 48 individual studies, capturing 39 tested compounds, 73 unique Arctic test species, and 95 distinct endpoints published from 1975 to 2021. Our analysis shows that of 253 test substance and species combinations scored (i.e., a unique toxicity test), 207 (82%) failed to meet at least one critical study criterion that contributes to data reliability for use in risk assessment. Arctic-focused toxicity testing needs to ensure that exposures can be analytically confirmed, include environmentally realistic exposure scenarios, and report test methods more thoroughly. Significant data gaps were identified as related to standardized toxicity testing with Arctic species, diversity of compounds tested with these organisms, and the inclusion of ecologically relevant sublethal and chronic endpoints assessed in Arctic toxicity testing. Overall, there needs to be ongoing improvement in test conduction and reporting in the scientific literature to support effective risk assessments in an Arctic context. Environ Toxicol Chem 2022;41:46-72. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for higher throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of ENMs using NAMs. In this study, member agencies within the Interagency Coordinating Committee on the Validation of Alternative Methods were queried about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENM toxicity testing. To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed.

In compliance to animal welfare 3Rs principle there is a great demand for refined tests alternative to classical mammal teratogenicity tests. We propose a refined alternative amphibian method (R-FETAX) to evaluate chemical induced embryotoxicity. The human foetal valproate spectrum disorder (FVSD) characteristics are morphological defects (including cranio-facial, neural tube defects) and behavioural alterations due to valproate (VPA) exposure in pregnancy. Vertebrate assays to evaluate FVSD include classical and alternative mammal (implying adult sacrifice), and non-mammal developmental models (zebrafish, amphibians, chick). Among these latter only zebrafish assays report in the same test both morphological and behavioural examinations. Compared to zebrafish, the amphibian Xenopus laevis excels having a more comparable organ development and morphology to mammalian systems. We used X. laevis embryos exposed during developmental specific windows to VPA therapeutic concentrations. Different VPA effects were observed depending on the exposure window: concentration-related embryo-lethal and teratogenic effects (neural tube, facial, tail defects) were observed in groups exposed at the organogenetic phylotypic stages. Neurobehavioral deficits were described using a functional swimming test at the highest VPA concentration exposure during the phylotypic stages and at any concentration during neurocognitive competent stages. Malformations were compared to those obtained in a mammalian assay (the rat post-implantation whole embryo culture method, WEC), that we used in the past to evaluate VPA teratogenicity. R-FETAX and WEC data were modelled and their relative sensitivity was calculated. We suggest the amphibian R-FETAX as a refined windowed alternative test for the evaluation of chemicals inducing both morphological and behavioural anomalies, including VPA., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inter-species differences in toxicodynamics are often a critical source of uncertainty in safety evaluations and typically dealt with using default adjustment factors. In vitro studies that use cells from different species demonstrated some success for estimating the relationships between life span and/or body weight and sensitivity to cytotoxicity; however, no apparent investigation evaluated the utility of these models for risk assessment. It was hypothesized that an in vitro model using dermal fibroblasts derived from diverse species and individuals might be utilized to inform the extent of inter-species and inter-individual variability in toxicodynamics. To test this hypothesis and characterize both inter-species and inter-individual variability in cytotoxicity, concentration-response cytotoxicity screening of 40 chemicals in primary dermal fibroblasts from 68 individuals of 54 diverse species was conducted. Chemicals examined included drugs, environmental pollutants, and food/flavor/fragrance agents; most of these were previously assessed either in vivo or in vitro for inter-species or inter-individual variation. Species included humans, the typical preclinical species and representatives from other orders of mammals and birds. Data demonstrated that both inter-species and inter-individual components of variability contribute to the observed differences in sensitivity to cell death. Further, it was found that the magnitude of the observed inter-species and inter-individual differences was chemical-dependent. This study contributes to the paradigm shift in risk assessment from reliance on in vivo toxicity testing to higher-throughput in vitro or alternative approaches, extending the strategy to replace use of default adjustment factors with experimental characterization of toxicodynamic inter-individual variability and to also address toxicodynamic inter-species variability.

Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish ( Danio rerio ), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.

Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results., (© 2021 John Wiley & Sons, Ltd.)

The generation of degradation products (DPs) like ions and organo-metallic particles from corroding metallic implants is an important healthcare concern. These DPs generate local and systemic toxicity. The impact on local toxicity is well documented, however, little is known about systemic toxicity. This is mainly due to the limited scope of the current microtiter plate-based (static) toxicity assay techniques. These methods do not mimic the systemic (dynamic) conditions. In this study, it is hypothesized that DPs incubated with cells in static conditions might provide improper systemic toxicity results, as there is no movement mimicking the blood circulation around cells. This study reports the development of a three-chambered prototype microfluidic system connected to the operational hip implant simulator to test the cellular response induced by the DPs. This setup is called a dynamic microfluidic bioreactor-hip simulator system. We hypothesize that a dynamic microfluidic system will provide a realistic toxicology response induced by DPs than a static cell culture plate. To prove the hypothesis, Neuro2a (N2a) cells were used as representative cells to study systemic neurotoxicity by the implant DPs. The microfluidic bioreactor system was validated by comparing the cell toxicity against the traditional static system and using COMSOL modeling for media flow with DPs. The hip implant simulator used in this study was a state-of-the-art sliding hip simulator developed in our lab. The results suggested that static toxicity was significantly more compared to dynamic microfluidic-based toxicity. The newly developed DMBH system tested for in situ systemic toxicity on N2a cells and demonstrated very minimum toxicity level (5.23%) compared to static systems (31.16%). Thus, the new DMBH system is an efficient tool for in situ implant metal systemic toxicity testing., (© 2021 Wiley Periodicals LLC.)

Ascorbate has many biological activities that involve fundamental cellular functions such as gene expression, differentiation, and redox homeostasis. Biochemically, it serves as a cofactor for a large family of dioxygenases (> 60 members) which control transcription, formation of extracellular matrix, and epigenetic processes of histone and DNA demethylation. Ascorbate is also a major antioxidant acting as a very effective scavenger of primary reactive oxygen species. Reduction of Fe(III) by ascorbate is important for cellular uptake of iron via DMT1. Cell culture models are extensively used in toxicology and pharmacology for mechanistic studies of nutrients, drugs and other xenobiotics. High-throughput screens in vitro, such as a large-scale Tox21 program in the US, offers opportunities to assess hazardous properties of a vast and growing number of industrial chemicals. However, cells in typical cultures are severely deficient in ascorbate, raising concerns about their ability to accurately recapitulate toxic and other responses in vivo. Scarcity of ascorbate and a frequently unrecognized use of media with its thiol substitute alters stress sensitivity of cells in different directions. Remediation of ascorbate deficiency in tissue culture restores the physiological state of many cellular processes and it should improve a currently limited toxicity predictability of in vitro bioassays., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Regulatory toxicology seeks to ensure that exposures to chemicals encountered in the environment, in the workplace, or in products pose no significant hazards and produce no harm to humans or other organisms, i.e., that chemicals are used safely. The most practical and direct means of ensuring that hazards and harms are avoided is to identify the doses and conditions under which chemical toxicity does not occur so that chemical concentrations and exposures can be appropriately limited. Modern advancements in pharmacology and toxicology have revealed that the rates and mechanisms by which organisms absorb, distribute, metabolize and eliminate chemicals-i.e., the field of kinetics-often determine the doses and conditions under which hazard, and harm, are absent, i.e., the safe dose range. Since kinetics, like chemical hazard and toxicity, are extensive properties that depend on the amount of the chemical encountered, it is possible to identify the maximum dose under which organisms can efficiently metabolize and eliminate the chemicals to which they are exposed, a dose that has been referred to as the kinetic maximum dose, or KMD. This review explains the rationale that compels regulatory toxicology to embrace the advancements made possible by kinetics, why understanding the kinetic relationship between the blood level produced and the administered dose of a chemical is essential for identifying the safe dose range, and why dose-setting in regulatory toxicology studies should be informed by estimates of the KMD rather than rely on the flawed concept of maximum-tolerated toxic dose, or MTD., (© 2021. The Author(s).)

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The growth in drug development over the past years reflects significant advancements in basic sciences and a greater understanding of molecular pathways of disease. Benchmarking industry practices has been important to enable a critical reflection on the path to evolve pharmaceutical testing, and the outcome of past industry surveys has had some impact on best practices in testing. A survey was provided to members of SPS, ACT, and STP. The survey consisted of 37 questions and was provided to 2550 participants with a response rate of 24%. Most respondents (∼75%) came from the US and Europe. The survey encompassed multiple topics encountered in nonclinical testing of pharmaceuticals. The most frequent target indications were oncology (69%), inflammation (55%), neurology/psychiatry/pain (46%), cardiovascular (44%), and metabolic diseases (39%). The most frequent drug-induced toxicology issues confronted were hepatic, hematopoietic, and gastrointestinal. Toxicological effects that impacted the no observed adverse effect level (NOAEL) were most frequently based on histopathology findings. The survey comprised topics encountered in the use of biomarkers in nonclinical safety assessment, most commonly those used to assess inflammation, cardiac/vascular, renal, and hepatic toxicity as well as common practices related to the assessment of endocrine effects, carcinogenicity, genotoxicity, juvenile and male-mediated developmental and female reproductive toxicity. The survey explored the impact of regulatory meetings on program design, application of the 3 Rs, and reasons for program delays. Overall, the survey results provide a broad perspective of current practices based on the experience of the scientific community engaged in nonclinical safety assessment.

Reproductive toxicity chemical safety assessment involves extensive use of vertebrate animals for regulatory testing purposes. Although alternative methods such as the zebrafish embryo teratogenicity assay (identified in the present manuscript by the acronym ZETA) are promising for replacing tests with mammals, challenges to regulatory application involve lack of standardization and incomplete validation. To identify key protocol aspects and ultimately support improving this situation, a comprehensive review of the literature on the level of harmonization/standardization and validation status of the ZETA has been conducted. The gaps and needed advances of the available ZETA protocols were evaluated and discussed with respect to its applicability as an alternative approach for teratogenicity assessment. Based on the review outcomes, a set of minimum reporting standards for the experimental protocol is proposed. Together with other initiatives towards implementation of alternative approaches at the screening and regulatory levels, the application of minimum reporting requirements is anticipated to support future method standardization and validation, as well as identifying potential improvement aspects. Present findings are expected to ultimately support advancing the ongoing validation initiatives towards the regulatory acceptance of the ZETA., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60°C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37°C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60°C or 37°C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60°C and purified via TCA (12 ± 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37°C and purified using UF (6 ± 1 MNi/1000 binucleated cells) after 9 days (P < 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60°C relative to 37°C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transgenic zebrafish models have been successfully used in biomonitoring and risk assessment studies of environmental pollutants, including xenoestrogens, pesticides, and heavy metals. We employed zebrafish larva (transgenic SR4G line) with a cortisol-inducible green fluorescence protein reporter (eGFP) as a model to detect stress responses upon exposure to compounds with environmental impact, including bisphenol A (BPA), vinclozolin (VIN), and fluoxetine (FLX). Cortisol, fluorescence signal, and mRNA levels of eGFP and 11 targeted genes were measured in a homogenized pool of zebrafish larvae, with six experimental replicates for each endpoint. Eleven targeted genes were selected according to their association with stress-axis and immediate early response class of genes. Hydrocortisone (CORT)and dexamethasone (DEX) were used as positive and negative controls, respectively. All measurements were done in two unstressed and stressed condition using standardized net handling as the stressor. A significant positive linear correlation between cortisol levels and eGFP mRNA levels was observed (r> 0.9). Based on eGFP mRNA levels in unstressed and stressed larvae two predictive models were trained (Random Forest and Logistic Regression). Both these models could correctly predict the blunted stress response upon exposure to BPA, VIN, FLX and the negative control, DEX. The negative predictive value (NPV) of these models were 100%. Similar NPV was observed when the predictive models trained based on the mRNA levels of the eleven assessed genes. Measurement of whole-body fluorescence intensity signal was not significant to detect blunted stress response. Our findings support the use of SR4G transgenic larvae as an in vivo biomonitoring model to screen chemicals for their stress-disrupting potentials. This is important because there is increasing evidence that brief exposures to environmental pollutants modify the stress response and critical coping behaviors for several generations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nozari, Do and Trudeau.)

Advanced in vitro systems often combine a mechanical-physical instrument with a biological component e.g. cell culture models. For testing of aerosols, it is of advantage to consider aerosol behavior, particle deposition and lung region specific cell lines. Although there are many good reviews on the selection of cell cultures, articles on instruments are rare. This article focuses on the development of in vitro instruments targeting the exposure of aerosols on cell cultures. In this context, guidelines for toxicity investigation are taken into account as the aim of new methods must be the prediction of human relevant data and the replacement of existing animal experiments. We provide an overview on development history of research-based instruments from a pharmaceutical point of view. The standardized commercial devices resulting from the research-based instruments are presented and the future perspectives on pulmonary in vitro devices are discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Previous embryonic fish data have shown caffeine to induce potential teratogenic and long-term neurodevelopmental outcomes through oxidative stress-mediated apoptosis. In this context, antioxidants may have the potential to counteract the caffeine-induced effects. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proven antioxidant properties, against caffeine-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 0.5 mM caffeine, co-exposed to 24-EPI (0.01, 0.1 and 1 μM) and to 24-EPI alone (1 μM) for 96 h. During exposure, lethal and sublethal developmental parameters were evaluated. At the end of the exposure, biochemical evaluations were made, and 24 h after, different behavioural paradigms were assessed. An increased number of animals showing oedema and malformations were observed after caffeine exposure, while these were reduced after co-exposure to 24-EPI concentration, namely the tail curvature. The results showed oxidative stress and related parameters similar among treatments. Yet, caffeine exposure resulted in locomotor deficits (decreased speed and distance) and disrupted anxiety-like and avoidance responses. The co-exposure to caffeine and to the highest 24-EPI concentrations resulted in less pronounced behavioural deficits. Overall, there was an absence of effects in the embryo/larvae exposed solely to 24-EPI, while caffeine caused developmental and neurotoxic effects. Although further studies are needed, the results showed promising protective effects of the highest 24-EPI concentration tested against the toxicity induced by caffeine in zebrafish., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

Essential oils (EOs) are increasingly consumed as food supplements. The few published recommended doses available generally lack details both on the methodology used and concentration limits for substances of concern, including genotoxic carcinogens. We propose a tiered approach based on the toxicological evaluation of maximized concentrations of each constituent present in the EO investigated. The genotoxic potential of each constituent is assessed using literature data or QSAR analyses. Genotoxic constituents are evaluated according to the methodology provided in the ICHM7 guideline. A Toxicological Reference Value (TRV) is associated to each non-genotoxic constituent, using one of the following methodologies (decision-tree successive steps): extraction from recognized databases or clinical studies, application of adequate safety factors to NOAELs established in animal studies, read-across analyses and when none was possible, TTC of Cramer classes. An EO recommended dose is considered safe when the safety margin (ratio between TRV and systemic exposure) for all constituents is all at least equal to 1. In conclusion, this methodology has proven to be robust to establish safe recommended doses for EOs used as food supplements, consistent with those publicly available, and avoiding unnecessary dedicated new animal testing., (Copyright © 2021 Pierre Fabre Research Institute: Institut de recherche Pierre Fabre SAS Centre de Recherche et Developpement. Published by Elsevier Ltd.. All rights reserved.)

Given the increasing importance of establishing better risk assessments for mycotoxins, novel in vitro tools for the evaluation of their toxicity are mandatory. In this study, an in vitro 3D spheroid model from SH-SY5Y cells, a human neuroblastoma cell line, was developed, optimized and characterized to test the cytotoxic effects caused by the mycotoxin sterigmatocystin (STE). STE induced a concentration- and time-dependent cell viability decrease in spheroids. Spheroids displayed cell disaggregation after STE exposure, increasing in a dose-dependent manner and over time. STE also induced apoptosis as confirmed by immunofluorescence staining and Western blot. Following the decreased proliferation and increased apoptosis, STE cytostasis effects were observed by migration assays both in 2D and 3D cell culture. Increased ROS generation, as well as DNA damage were also observed. Taken together, these data highlight the cytotoxic properties of STE and suggest that cell culture models play a pivotal role in the toxicological risk assessment of mycotoxins. The evaluation of cytotoxicity in spheroids (3D) rather than monolayer cultures (2D) is expected to more accurately reflect in vivo-like cell behaviour., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

When considering test species for soil ecotoxicity, the development of new model organisms is often suggested to increase the reliability of ecological risk assessments. Ubiquitous soil algae could offer potential test species for assessing various soil pollution levels. Currently, there are few reviews offering comprehensive perspectives on stressors-based toxicological studies using microalgae in soil media, with the majority of scholarly attention paid to the toxicological effects of freshwater algae or marine algae in aquatic ecosystems. In this review, we focus on current toxicological studies of microalgae assessed in soil-related media and suggest considerations for using microalgae in soil toxicity tests based on 22 publications (1998-2021). In addition, we analyzed characteristics of soil algae based on criteria for selecting test species and suggest that future research should be directed toward the standardization of soil algal toxicity test methods. This review discusses a promising method using soil algae as new test species for soil toxicity assessment as cost-effective and environmentally sound soil quality bioindicators. The review also addresses the lack of understanding behind how soil algae can serve as important test species for soil ecotoxicity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Acetaminophen (APAP), one of the most widely used antipyretic and analgesic drugs, principally contributes to drug-induced liver injury when taken at a high dose. APAP-induced liver injury (AILI) results in extensive necrosis of hepatocytes along with the occurrence of multiple intracellular events such as metabolic activation, cell injury, and signaling pathway activation. However, the specific role of the immune response in AILI remains controversial for its complicated regulatory mechanisms. A variety of inflammasomes, immune cells, inflammatory mediators, and signaling transduction pathways are activated in AILI. These immune components play antagonistic roles in aggravating the liver injury or promoting regeneration. Recent experimental studies indicated that natural products showed remarkable therapeutic effects against APAP hepatotoxicity due to their favorable efficacy. Therefore, this study aimed to review the present understanding of the immune response in AILI and attempted to establish ties among a series of inflammatory cascade reactions. Also, the immune molecular mechanisms of natural products in the treatment of AILI were extensively reviewed, thus providing a fundamental basis for exploring the potential pharmacological targets associated with immune interventions., Competing Interests: Declaration of Competing Interest All the authors read and approved the final manuscript. They declared that they have no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nanotoxicological studies using existing models of normal cells and animals often encounter a paradox: retention of nanoparticles in intracellular compartments for a long time is not accompanied by any significant toxicological effects. Can we expect that the revealed changes will be not harmful after translation to practice, outside of a sterile laboratory and ideally healthy organisms? Age-associated and pathological processes can affect target organs, metabolism, and detoxification in the mononuclear phagocyte system organs and change biodistribution routes, thus making the use of nanomaterial not safe. The potential solution to this issue can be testing the toxic properties of nanoparticles in animal models with chronic diseases. However, current studies of nanotoxicity in animal models with a brain, cardiovascular system, liver, digestive tract, reproductive system, and skin diseases are unsystematic. Even though these studies demonstrate the emergence of new toxic effects that are not present in healthy animals. In this regard, we set the goal of this review as the formulation of the requirements for an animal model capable of assessing the potential toxicity of nanoparticles based on the nanosafety approach., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Developmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro-in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration-response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose-response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED 50 ) amounted to 67 and 45 mg/kg bw being comparable to the ED 50 derived from the in vivo dose-response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro-in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing., (© 2021. The Author(s).)

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80-400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 10 5 Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays., (© 2021. The Author(s).)

The safety assessment of cosmetic products is based on the safety of the ingredients, which requires information on chemical structures, toxicological profiles, and exposure data. Approximately 6% of the population is sensitized to cosmetic ingredients, especially preservatives and fragrances. In this context, the aim of this study was to perform a hazard assessment and risk characterization of benzalkonium chloride (BAC), benzyl alcohol (BA), caprylyl glycol (CG), ethylhexylglycerin (EG), chlorphenesin (CP), dehydroacetic acid (DHA), sodium dehydroacetate (SDH), iodopropynyl butylcarbamate (IPBC), methylchloroisothiazolinone and methylisothiazolinone (MCI/MIT), methylisothiazolinone (MIT), phenoxyethanol (PE), potassium sorbate (PS), and sodium benzoate (SB). Considering the integrated approaches to testing and assessment (IATA) and weight of evidence (WoE) as a decision tree, based on published safety reports. The hazard assessment was composed of a toxicological matrix correlating the toxicity level, defined as low (L), moderate (M), or high (H) and local or systemic exposure, considering the endpoints of skin sensitization, skin irritation, eye irritation, phototoxicity, acute oral toxicity, carcinogenicity, mutagenicity/genotoxicity, and endocrine activity. In a risk assessment approach, most preservatives had a margin of safety (MoS) above 100, except for DHA, SDH, and EG, considering the worst-case scenario (100% dermal absorption). However, isolated data do not set up a safety assessment. It is necessary to carry out a rational risk characterization considering hazard and exposure assessment to estimate the level of risk of an adverse health outcome, based on the concentration in a product, frequency of use, type of product, route of exposure, body surface location, and target population., (© 2021 John Wiley & Sons, Ltd.)