Research progress in the development of 3D skin models and their application to in vitro skin irritation testing.

Toxicological assessment of chemicals is crucial for safeguarding human health and the environment. However, traditional animal experiments are associated with ethical, technical, and predictive limitations in assessing the toxicity of chemicals to the skin. With the recent development of bioengineering and tissue engineering, three-dimensional (3D) skin models have been commonly used as an alternative for toxicological studies. The skin consists of the subcutaneous, dermis, and epidermis. All these layers have crucial functions such as physical and biological protection and thermoregulation. The epidermis is the shallowest layer protecting against external substances and media. Because the skin is the first contact point for many substances, this organ is very significant for assessing local toxicity following skin exposure. According to the classification of the United Nations Global Harmonized System, skin irritation is a major potentially hazardous characteristic of chemicals, and this characteristic must be accurately assessed and classified for enhancing chemical safety management and preventing and reducing chemical accidents. This review discusses the research progress of 3D skin models and introduces their application in assessing chemical skin irritation.

Evaluation of alternative methods of tunnel composting (submitted by the European Composting Network) II.

Two alternative methods for producing compost in a tunnel, from certain category (Cat.) 3 animal by-products (ABP) and other non-ABP material, were assessed. The first method proposed a minimum temperature of 55°C for 72 h and the second 60°C for 48 h, both with a maximum particle size of 200 mm. The assessment of the Panel on Biological Hazards (BIOHAZ) exclusively focused on Cat. 3 ABP materials (catering waste and processed foodstuffs of animal origin no longer intended for human consumption). The proposed composting processes were evaluated for their efficacy to achieve a reduction of at least 5 log10 of Enterococcus faecalis and Salmonella Senftenberg (775W, H2S negative) and at least 3 log10 of relevant thermoresistant viruses. The applicant provided a list of biological hazards that may enter the composting process and selected parvoviruses as the indicator of the thermoresistant viruses. The evidence provided by the applicant included: (a) literature data on thermal inactivation of biological hazards; (b) results from validation studies on the reduction of E. faecalis, Salmonella Senftenberg 775W H2S negative and canine parvovirus carried out in composting plants across Europe; (c) and experimental data from direct measurements of reduction of infectivity of murine parvovirus in compost material applying the time/temperature conditions of the two alternative methods. The evidence provided showed the capacity of the proposed alternative methods to reduce E. faecalis and Salmonella Senftenberg 775W H2S negative by at least 5 log10, and parvoviruses by at least 3 log10. The BIOHAZ Panel concluded that the two alternative methods under assessment can be considered to be equivalent to the processing method currently approved in the Commission Regulation (EU) No 142/2011.

Advancing Endocrine Disruptors via In Vitro Evaluation: Recognizing the Significance of the Organization for Economic Co-Operation and Development and United States Environmental Protection Agency Guidelines, Embracing New Assessment Methods, and the Urgent Need for a Comprehensive Battery of Tests.

Efforts are being made globally to improve the evaluation and understanding of endocrine-disrupting chemicals. Recognition of their impact on human health and the environment has stimulated attention and research in this field. Various stakeholders, including scientists, regulatory agencies, policymakers, and industry representatives, are collaborating to develop robust methodologies and guidelines for assessing these disruptors. A key aspect of these efforts is the development of standardized testing protocols and guidelines that aim to provide consistent and reliable methods for identifying and characterizing endocrine disruptors. When evaluating the potential endocrine-disrupting activity of chemicals, no single test is capable of detecting all relevant endocrine-disrupting agents. The test battery approach is designed to reduce the risk of false negative results for compounds with toxic potential. A weight-of-evidence approach is therefore necessary for endocrine disruptor evaluation. This approach considers various types of data from multiple sources, assessing the overall strength, consistency, and reliability of the evidence. OECD guidelines are highly regarded for their scientific rigor, transparency, and consensus-based development process. It is crucial to explore and develop new methodologies that can effectively evaluate the risks associated with potential endocrine disruptors. Integrating these methods into a comprehensive weight-of-evidence framework will enhance risk assessments and facilitate informed decisions regarding the regulation and management of these substances, ensuring the protection of human health and the environment from their adverse effects.

A Cytotoxicity Assay as an Alternative to the Murine Model for the Potency Testing of Bothrops jararaca Venom and Antivenom: An Intralaboratory Pre-validation Study.

Antivenom therapy is the only specific treatment for snakebite envenomation, and antivenom potency determination is key in the efficacy assurance quality control process. Nowadays, this process relies on the in vivo murine model - thus, the development of alternative in vitro methods is imperative. In the current study, the principle of the proposed method is the ability of Bothrops venom to induce cytotoxic effects in Vero cells, and the capacity to evaluate the inhibition of this cytotoxicity by the respective antivenom. After exposure to the venom/antivenom, the relative proportions of adherent (viable) cells were evaluated by direct staining with Coomassie Blue. The optical density (OD) of the lysed cell eluate was directly proportional to the number of adherent cells. This cytotoxicity-based alternative method could represent a potential candidate for validation as a replacement for the current in vivo test. The in vitro-determined cytotoxicity of the Brazilian Bothrops reference venom (expressed as the 50% effective concentration; EC50) was 3.61 µg/ml; the in vitro-determined 50% inhibitory concentration (IC50) of the Brazilian Bothrops reference antivenom was 0.133 µl/ml. From these two values, it was possible to calculate the potency of the reference antivenom. The results from the assays exhibited a good linear response, indicating that the method could be a potential candidate replacement method for use in antivenom quality control prior to lot release, subject to further validation.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Guidelines on Developmental Toxicity Tests: Brief Insights.

Developmental toxicology is a constantly evolving research field which needs to attend to a complex underlying regulatory network. In order to ensure human health and environmental safety, new substances have to be tested for toxic effects on reproduction and development, before being commercialized. Traditional in vivo mammalian models represent the intricacy of human development and provide more adequately an assessment of the interaction of chemical compounds with the reproductive system. However, in the last years, the directives are to reduce the use of vertebrate animals, promoting their use only as a last resort. Consequently, the interest on the development and validation of alternative tests, able to cover the various aspects of the reproductive cycle, has significantly increased. Reproductive toxicity is probably the most difficult endpoint to be replaced by alternative assays, since it should provide information on mechanism interactions essential for female and male fertility and also knowledge on the animal development during the first phases of its life cycle. This complexity explains the slow progress in implementing alternative models for reproductive toxicity safety assays. Alternative test models may be based on in vitro systems and nonmammalian animal models. Many biological processes have been successfully addressed using in vitro models, opening the possibility to study the interference of teratogenic compounds. Their validation and implementation have lagged behind, in part because of difficulties in establishing their predictability. Nevertheless, the advance toward the process of validation is crucial to replace and reduce the use of living animals. Based on the present state of the art, it is not probable that such testing strategies will completely replace the need to assess reproductive toxicity in vivo in the near future, but they will contribute to reduce animal tests and will provide important information. In this chapter, the approved guidelines for standard methods and alternative methods, according to their regulatory and scientific status, are enumerated and briefly described.

The predatory mite Pronematus ubiquitus curbs Aculops lycopersici damage under greenhouse conditions.

BACKGROUND: The tomato russet mite, Aculops lycopersici, is a major worldwide pest infesting tomato crops for which only few control methods are available. At present, no commercialized beneficial organism has proven to be an effective biological control agent of the pest. As there is a strong need to develop alternatives to synthetic insecticides, we assessed the efficacy of an iolinid mite, Pronematus ubiquitus, as a preventive method against A. lycopersici in comparison with a curative treatment in a replicated experiment in the greenhouse. RESULTS: After pre-establishment of P. ubiquitus supplied with cattail pollen, followed by infestation of A. lycopersici, the predator was able to reduce pest populations by 98% as compared with control plants. Probably due to lack of food and high temperature, the number of P. ubiquitus decreased during the season and so the Eriophyid population rose, along with crop damage. The sulphur treatment could stop the progress of A. lycopersici, but their population levels remained high. CONCLUSION: Pronematus ubiquitus has great potential to prevent the establishment of the tomato russet mite. Even if a curative treatment affects the pest mite, the use of a preventive method is preferable as such insecticides/acaricides are harmful for beneficials and are applied after symptom appearance, when the pest pressure is already high. Despite the need to optimise management of the predator throughout the season, P. ubiquitus proved to be able to establish successfully on tomato plants. © 2023 Society of Chemical Industry.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline.

Zebrafish is a popular toxicology model and provides an ethically acceptable small-scale analysis system with the complexity of a complete organism. Our goal is to further validate this model for its regulatory use for reproductive and developmental defects by testing the compounds indicated in the "Guideline on detection of reproductive and developmental toxicity for human pharmaceuticals" (ICH S5(R3) guideline.) To determine the embryotoxic and developmental risk of the 32 reference compounds listed in the ICH S5(R3) guideline, the presence of morphological alterations in zebrafish embryos was analyzed at two different stages to calculateLC50 and EC50 values for each stage. Teratogenic Indexes were established as the ratio between LC50 and EC50 critical for the proper compound classification as teratogenic when it is ≥ 2. A total of three biological replicates have been conducted to study the reproducibility of the assay. The chemicals' concentration in the medium and internally in the zebrafish embryos was evaluated. In this study, the 3 negative compounds were properly categorized while 23 compounds out of the 29 reference ones (sensitivity of 79.31%) were classified as teratogenic in zebrafish. The 6 that had false-negative results were classified 4 as inconclusive, 1 as not toxic, and 1 compound resulted toxic for zebrafish embryos under testing conditions. After the bioavailability experiments, some of the obtained inconclusive results were refined. The developmental defects assay in zebrafish gives an accuracy of 89.66%, sensitivity of 88.46%, specificity and repeatability of 100% compared to mammals; therefore, this is a well-integrated strategy using New Alternative Methods, to minimize the use of animals in developmental toxicity studies.

Repeated intratracheal instillation of whole-cigarette smoke condensate to assess lung damage in a rat model.

Cigarette smoke induces an inflammatory response in the lungs by recruiting inflammatory cells, leading to lung diseases such as lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis. Existing inhalation exposure methods for assessing the adverse effects of cigarette smoke require expensive equipment and are labor-intensive. Therefore, we attempted to develop a novel method to assess these adverse effects using intratracheal instillation (ITI) of whole cigarette smoke condensate (WCSC). The WCSC (0, 5, 10, or 20 mg/mL) was administered by ITI once daily for 6 or 12 days using an automatic video instillator. Repeated WCSC ITI increased the lung weight, and monocyte chemoattractant protein-1 (MCP-1), neutrophil, and lymphocyte levels within bronchoalveolar lavage fluid compared to the control. In the histopathological analysis of the lung tissue, a mild inflammatory response was observed in the 6 and 12 days 20 mg/mL WCSC exposure groups. The genome-wide RNA-seq expression patterns revealed that inflammatory and immune response-related genes, such as the chemokine signaling pathway, Th1/Th2 cell differentiation, and cytokine-cytokine receptor interaction, were employed following WCSC exposure. In addition, MCP-1 was time-dependent and increased in the 10 mg/mL exposure group compared to the control group. These results suggested that the WCSC might induce the potential pulmonary inflammatory response. Furthermore, we proposed that ITI may be a rapid and effective method of evaluating the adverse effects of WCSC within a short exposure period (less than 2 weeks), and it can be used to evaluate cigarette inhalation toxicity studies as an alternative method.

Managing diabetic complications with alternative therapeutic strategies.

Diabetes is a chronic metabolic disease affecting millions worldwide. It is characterized by a lack of insulin production or impaired insulin function, leading to elevated blood glucose levels. Conventional treatment methods for diabetes management typically include lifestyle changes and medications. However, alternative therapies have gained attention in recent years, including traditional medicine containing bioactive compounds, supplements like vitamin D and Omega-3 fatty acids, aromatherapy, and homeopathy. Diabetic complications are common in patients with uncontrolled diabetes and can lead to serious health problems, including diabetic retinopathy, impaired wound healing, kidney disease, nerve damage, and cardiovascular disease. Alternative remedies, such as traditional medicine containing bioactive compounds, supplements, and aromatherapy, have been studied for their potential benefits in managing these complications. Traditional medicines like bitter melon, cinnamon, and fenugreek have been shown to have anti-diabetic effects due to their bioactive compounds. Similarly, supplements like vitamin D and Omega-3 fatty acids have been found to improve glycemic control in patients with diabetes. Aromatherapy, which involves the use of essential oils, has also been explored for its potential benefits in diabetes management. Homeopathy, which uses highly diluted substances to stimulate the body's natural healing abilities, has been used to treat diabetes-related symptoms like neuropathy and wounds. Personalized care is essential in natural diabetes management because each person's body and health needs are unique. A holistic approach that addresses the individual's physical, emotional, and spiritual well-being is essential. As research in this field continues to expand, a more comprehensive understanding of diabetes management will lead to improved outcomes for those living with this condition.

A low-cost alternative method of generating fibronectin micropatterned lines for cellular applications.

The cellular microenvironment contributes to the architecture, differentiation, polarity, mechanics and functions of the cell [1]. Spatial confinement of cells using micropatterning techniques allows to alter and regulate the cellular microenvironment for a better understanding of cellular mechanisms [2]. However, commercially available micropatterned consumables such as coverslips, dishes, plates etc. are expensive. These methods are complex and based on deep UV patterning [3,4]. In this study, we establish a low-cost method for effective micropatterning using Polydimethylsiloxane (PDMS) chips.•We demonstrate this method by generating fibronectin-coated micropatterned lines (width, 5 µm) on a glass bottom dish.•As a proof of concept, we culture macrophages on these lines. We additionally show that this method allows to determine the cellular polarity by measuring the position of the nucleus within a cell on a micropatterned line.

Development of a reference and proficiency chemical list for human steatosis endpoints in vitro.

The most prevalent liver disease in humans is non-alcoholic fatty liver disease, characterised by excessive hepatic fat accumulation, or steatosis. The western diet and a sedentary lifestyle are considered to be major influences, but chemical exposure may also play a role. Suspected environmental chemicals of concern include pesticides, plasticizers, metals, and perfluorinated compounds. Here we present a detailed literature analysis of chemicals that may (or may not) be implicated in lipid accumulation in the liver, to provide a basis for developing and optimizing human steatosis-relevant in vitro test methods. Independently collated and reviewed reference and proficiency chemicals are needed to assist in the test method development where an assay is intended to ultimately be taken forward for OECD Test Guideline development purposes. The selection criteria and considerations required for acceptance of proficiency chemical selection for OECD Test Guideline development. (i.e., structural diversity, range of activity including negatives, relevant chemical sectors, global restrictions, etc.) is described herein. Of 160 chemicals initially screened for inclusion, 36 were prioritized for detailed review. Based on the selection criteria and a weight-of-evidence basis, 18 chemicals (9 steatosis inducers, 9 negatives), including some environmental chemicals of concern, were ranked as high priority chemicals to assist in vitro human steatosis test method optimisation and proficiency testing, and inform potential subsequent test method (pre-)validation.

Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

Correlation between inhibition of lung surfactant function in vitro and rapid reduction in tidal volume following exposure to plant protection products in mice.

Currently, testing of acute inhalation toxicity in animals is required for regulation of pesticide active ingredients and formulated plant protection products. The main outcome of the regulatory tests is "lethal concentration 50″ (LC50), i.e. the concentration that will kill 50% of the exposed animals. However, ongoing work aims to identify New Approach Methods (NAMs) to replace animal experiments. To this end, we studied 11 plant protection products, sold in the European Union (EU), for their ability to inhibit lung surfactant function in vitro in the constrained drop surfactometer (CDS). In vivo, inhibition of lung surfactant function can lead to alveolar collapse and reduction of tidal volume. Therefore, we also assessed changes in breathing patterns of mice during exposure to the same products. Six of the eleven products inhibited lung surfactant function, and six products reduced tidal volume in mice. In vitro inhibition of lung surfactant function predicted reduction in tidal volume in exposed mice with a sensitivity of 67% and a specificity of 60%. Two products were labelled as "harmful if inhaled", both inhibited surfactant function in vitro and reduced tidal volume in mice. Lung surfactant function inhibition in vitro predicted reduction in tidal volume for plant protection products to a lesser degree than for previously tested substances. This could owe to the requirement for rigorous testing of plant protection products prior to approval that might have selected against substances that could potentially inhibit lung surfactant, e.g. due to severe adverse effects during inhalation.

Understanding repolarization in the intracardiac unipolar electrogram: A long-lasting controversy revisited.

Background: The optimal way to determine repolarization time (RT) from the intracardiac unipolar electrogram (UEG) has been a topic of debate for decades. RT is typically determined by either the Wyatt method or the "alternative method," which both consider UEG T-wave slope, but differently. Objective: To determine the optimal method to measure RT on the UEG. Methods: Seven pig hearts surrounded by an epicardial sock with 100 electrodes were Langendorff-perfused with selective cannulation of the left anterior descending (LAD) coronary artery and submersed in a torso-shaped tank containing 256 electrodes on the torso surface. Repolarization was prolonged in the non-LAD-regions by infusing dofetilide and shortened in the LAD-region using pinacidil. RT was determined by the Wyatt (tWyatt) and alternative (tAlt) methods, in both invasive (recorded with epicardial electrodes) and in non-invasive UEGs (reconstructed with electrocardiographic imaging). tWyatt and tAlt were compared to local effective refractory period (ERP). Results: With contact mapping, mean absolute error (MAE) of tWyatt and tAlt vs. ERP were 21 ms and 71 ms, respectively. Positive T-waves typically had an earlier ERP than negative T-waves, in line with theory. tWyatt -but not tAlt-shortened by local infusion of pinacidil. Similar results were found for the non-invasive UEGs (MAE of tWyatt and tAlt vs. ERP were 30 ms and 92 ms, respectively). Conclusion: The Wyatt method is the most accurate to determine RT from (non) invasive UEGs, based on novel and historical analyses. Using it to determine RT could unify and facilitate repolarization assessment and amplify its role in cardiac electrophysiology.

Frequency of Left Atrial Enlargement According to Different Modes of Indexing in Overweight and Obese Individuals.

Background: Current guidelines recommend indexing left atrial volume (LAV) by body surface area (BSA). However, in overweight and obese individuals this may result in the underestimation of left atrial enlargement (LAE). The aim of our study was to assess whether alternative LAV indexing to height and/or height-squared better identifies individuals with LAE among those who are overweight and/or obese. Methods: LAV was indexed to BSA (LAVI), height (LAVh), and height-squared (LAVh2) in 127 individuals with a mean age of 45.7 years and a mean body mass index (BMI) of 34.9 kg/m2 who underwent outpatient echocardiography at the University clinic of cardiology in Skopje. Results: LAVI, LAVh, and LAVh2 showed a progressive increase of respective values with the extent of BMI showing the most enlarged LA size in individuals with Class III obesity. There was a progressive significant increase in the prevalence of LAEh and LAEh2 in obese groups with the highest prevalence among those with class III obesity (p=0.002, p=0.002, respectively), on the contrary of LAEBSA where we could not find any significance in its distribution among obese classes. The greatest degree of reclassification occurred when indexing for height-squared, having relatively less reclassification when indexing for height (p=0.0001). The degree of reclassification varied depending on BMI with the greatest impact among the Class III obese patients, where as many as 76.5% and 88.2% of individuals were reclassified according to height or height-squared, respectively. Conclusions: The use of height, and especially height-squared, in comparison to BSA-based indexing methods are more successful in identifying the LAE prevalence in each class of obesity. Using allometric indexation leads to the significant reclassification of LA size from normal to dilated, especially in women and those with severe obesity, thereby providing an opportunity to identify more individuals at increased risk of adverse events.

Improved Tool for Predicting Skin Irritation on Reconstructed Human Epidermis Models Based on Electrochemical Impedance Spectroscopy.

The rabbit skin irritation test has been the standard for evaluating the irritation potential of chemicals; however, alternative methods that do not use animal testing are actively encouraged. Reconstructed human epidermis (RhE) models mimic the biochemical and physiological properties of the human epidermis and can be used as an alternative method. On RhE methods, the metabolic activity of RhE models is used to predict skin irritation, with a reduction in metabolic activity indicating a reduced number of viable cells and linking cell death to skin irritation processes. However, new challenges have emerged as the use of RhE models increases, including the need for non-invasive and marker-free methodologies to assess cellular states. Electrochemical impedance spectroscopy (EIS) is one such methodology that can meet these requirements. In this study, our results showed that EIS can differentiate between irritant and non-irritant chemicals, with a significant increase in the capacitance values observed in the irritant samples. A ROC curve analysis showed that the prediction method based on EIS met OECD TG 439 requirements at all time points and had 95% within-laboratory reproducibility. Comparison with the MTT viability assay showed that prediction using EIS achieved higher sensitivity, specificity, and accuracy. These results suggest that EIS could potentially replace animal testing in the evaluation of irritation potential and could be a valuable addition to in vitro testing strategies.

Face-to-face and online teaching experience on experimental animals and alternative methods with nursing students: a research study.

BACKGROUND: Animal models are increasingly used in Nursing science to study care approaches. Despite the scientific relevance and the ethical debate surrounding the use of experimental animals, there is a scarcity of scholarly literature exploring this topic in Nursing Schools. AIM: To evaluate perceptions and attitudes of nursing students enrolled in a Pharmacology course on the use of experimental animals and implementation of alternative methods, by comparing the experience for two academic years. An interdisciplinary collaboration has also been developed. METHODS: A descriptive cross-sectional, quantitative study was developed. Undergraduate nursing students were enrolled in the Pharmacology subject at the University of Leon (Spain). The study was carried out in the Pharmacology facilities. Students followed a two-session practical class regarding experimental animals and alternative methods in the Pharmacology course (Degree in Nursing) in two different academic years (2019-20/2020-21). At the end of the activity, they answered a questionnaire to assess their opinions on the use of experimental animals and alternative methods in Pharmacology and the 3Rs principle. RESULTS: A comparison of the students' perception with and without direct participation in the evaluation of promazine effects in mice was made. A total of 190 students participated in the teaching experience, providing high scores in all items (4-5 out of 5 points) regarding the teaching experience. Students became also aware of the advantages and disadvantages on the use of experimental animals, as well as the ethical considerations to bear in mind for their use and the need for alternative methods. CONCLUSIONS: In the students' opinion, the total replacement of animals by alternative techniques was very difficult, and they preferred to do the practice face-to-face. The alternative method designed was useful for the students to accept the employment of experimental animals in biomedical research and education, and know the legislation applied in the protection of animals.

Study on alternative method for vasopressin impurity test of oxytocin injection from biological extraction / 中国药理学通报

Aim To explore the alternative study on rat blood pressure method and HPLC method for vasopressin impurity test of oxytocin injection from biological extraction. Methods The HPLC method for the vasopressin impurity test in vitro was established and validated. The bio-extrac tion oxytocin injection samples and simulated samples were examined for vasopressin impurity by HPLC and rat blood pressure methods respectively. Results Vasopressin and adjacent impurity peaks were successfully separated by the established method. In the range of 210~13 330 IU•L -1the concentration of vasopressin had a good linear relationship with its peak area with r=0.999 9. The results of HPLC method were consistent with the biological examination method-rat blood pressure method in the current standard. Conclusions The method is proved to be specific, sensitive, and accurate, which can be used as a test method for vasopressin impurity to replace the rat blood pressure method in the current standard.