Animals In Safety Testing Research Articles

Effect of oncolytic Newcastle disease virus harboring PTEN gene on pancreatic adenocarcinoma growth through inhibition of PI3K/mTOR signaling and promotion of apoptosis.

49 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and intractable cancer, warranting the need for more effective therapies. Cell proliferation, progression, and severity in PDAC are highly activated owing to KRAS mutations and low PTEN expression. Methods: A recombinant Newcastle disease virus (rNDV) harboring PTEN (rNDV-PTEN) was constructed to infect PDAC cells to artificially induce PTEN expression. The cancer killing effect of the rNDV-PTEN virus was observed in an in vitro assay, and 107.0 pfu/dose(0.1 ml) of the rNDV-PTEN virus was inoculated through intravenous injection into a PANC-1 cell transplanted mouse to prove the tumor growth inhibition effect. Apoptotic signaling-related proteins were analyzed with Western blotting and qPCR assays based on the mRNA. During the treatment, biomarkers were also analyzed. Results: Factitious PTEN expression in KRAS-mutated PDAC cells following rNDV-PTEN infection lowered PI3K/AKT/mTOR signaling, induced PDAC cell death, and suppressed tumor growth. PTEN overexpression promoted apoptotic and autophagic signaling pathways in PANC-1 cells and orthotopic xenograft mice. rNDV-PTEN has substantial cancer cell-killing effects and inhibits tumor growth. rNDV-PTEN injection into animals did not elicit an immune response against rNDV and improved blood parameters, such as glucose, triglyceride, and total cholesterol levels. Conclusions: rNDV-PTEN showed a strong tumor-suppressive effect on PDAC, which was caused by an abnormally activated PI3K/AKT/mTOR signaling pathway due to KRAS mutations and PTEN loss. We also showed that rNDV-PTEN is relatively safe for use in cancer therapy. Further studies using patient-derived PDAC cells with the same genetic background, as well as more extensive animal safety tests, are required to demonstrate rNDV-PTEN as a PDAC therapeutic. However, PTEN gene-containing rNDV may be a very promising therapeutic candidate for pancreatic cancer treatment.

Development and evaluation of high permeability liposomes for improving anti-melanogenic activity of taxus chinensis fruit polysaccharide

Taxus chinensis var. mairei fruit polysaccharide (TMFP) is a natural resource with effective hypoglycemic and anti-tumor properties. However, its impact on melanogenesis remains largely unexploited. This study explores the anti-melanogenic activity of TMFP using in vitro and in vivo models. It was found that TMFP can safely and effectively eliminate melanin and inhibit tyrosinase activity without cytotoxicity. More specifically, the inhibitory effect of TMFP on melanin synthesis was verified in zebrafish embryos. In addition, nine formulations of TMFP-Liposomes were prepared to improve drug transdermal absorption, and TMFP-Liposome E was determined as the optimal formulation after characterization and transdermal comparison experiments. Most importantly, in vivo skin whitening experiments gave that TMFP-Liposome E has better skin whitening activity than control, model, blank liposomes, TMFP aqueous, and arbutin groups. The cell viability and animal safety tests confirmed the good biosafety of TMFP-Liposome E. Overall, this research suggests that TMFP may serve as a potential therapeutic agent for skin diseases and a potent whitening agent for cosmetic use. TMFP-Liposome E may enhance the therapeutic effect of melanin through improved transdermal absorption.

Synbiotic Yogurt Fortified with Bawang Dayak (Eleutherine palmifolia) Extract

Aims: The purpose of this research was to study the quality of yogurt symbiotic fortified with Bawang Dayak (Eleutherine palmifolia) extract.
 Study Design: This research used Completely Randomized Design (CRD) experimental method and consist of 4 treatments.
 Place and Duration of Study: Sample: Animal Product Technology Laboratory Faculty of Animal Science Universitas Brawijaya, Food Quality and Safety Testing Laboratory Department of Agricultural Product Technology, Faculty of Agricultural Technology Universitas Brawijaya, between June 2022 – December 2022.
 Methodology: The material used are skim milk, fresh milk, yogurt starter bacteria (Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophillus), and bawang dayak extract. Bawang Dayak were extracted using Microwave-Asisted Extraction (MAE) with temperature 60-700C. This research which consist of 4 treatments and 3 replications with various concentration of Bawang Dayak extract of T0 (0%), T1 (1%), T2 (4%), and T3 (6%), respectively. The samples of synbiotic yogurt with fortification of Bawang Dayak extract were analyzed for their total lactic acid bacteria, antioxidant activity, viscosity and color analysis.
 Results: Fortification of Bawang dayak extract in synbiotic yogurt results in highly significant different effects on the quality parameters of antioxidant and color (lightness, redness and yellowness) (P<0.01), but had no significant effect (P>0.05) on the total quality of lactic acid bacteria. Fortification of 6% Bawang dayak extract (T3) in yogurt produced the best quality in terms of antioxidant content and color (lightness, redness, and yellowness).
 Conclusion: The best treatment of synbiotic yogurt was achieved in T3 with the 6% of Bawang Dayak extract.

Nonopioid analgesics discovery and the Valley of Death: EMA401 from concept to clinical trial.

Translation of promising preclinical efficacy data for investigational analgesics to positive clinical trial outcomes is limited, despite the large collective effort to date. However, one target with positive proof-of-concept clinical trial data is the angiotensin II type 2 (AT2) receptor. This review addresses the obstacles impeding successful preclinical to clinical research translation in the novel analgesics field, and it also provides an overview of the discovery and development of EMA401, a peripherally restricted, highly selective, orally active, small-molecule AT2 receptor antagonist for relief of neuropathic pain. Multiple AT2 receptor antagonists evoked dose-dependent antiallodynia in the chronic constriction injury of the sciatic nerve rat model of neuropathic pain. In AT2 receptor knockout chronic constriction injury mice, antiallodynia was abolished, affirming the AT2 receptor as the target. Subsequently, AT2 receptor antagonists were shown to evoke pain relief in multiple rodent chronic pain models. EMA401 (sodium salt) was selected as the drug candidate based on its >10,000-fold binding selectivity c.f. the angiotensin II type 1 receptor, good potency, and favourable pharmacokinetics. Animal toxicology and safety testing along with phase 1 clinical trials in healthy volunteers showed that oral EMA401 was safe and well-tolerated. Based on these data, a proof-of-concept clinical trial of oral EMA401 was undertaken in patients with postherpetic neuralgia. This 4-week trial showed that EMA401 evoked superior relief of postherpetic neuralgia relative to placebo and there were no serious adverse events in the EMA401 group.

The role of ‘big data’ and ‘in silico’ New Approach Methodologies (NAMs) in ending animal use – A commentary on progress

In silico (computational) methods continue to evolve as part of a robust 21st century public health strategy in risk assessment, relevant to all sectors of chemical safety including preclinical drug discovery, industrial chemicals testing, food and cosmetics. Alongside in vitro methods as components of intelligent testing and pathway driven strategies, in silico models provide the potential for more human relevant solutions to the use of animals in safety testing and biomedical research. These are often termed ‘New Approach Methodologies’ (NAMs). Some NAMs incorporate the use of ‘big data’, for example the information provided from high throughput or high content in vitro screening assays or ‘omics’ technologies. Big data has increasing relevance to predictive toxicology but must be appropriately defined, particularly with regard to ‘quality vs quantity’. The purpose of this article is to provide a commentary on the progress of in silico human-based research methods within the context of NAMs, as well as discussion of the emerging use of big data with relevance to safety assessment. The current status of in silico methods is discussed, with input from researchers in the field. Scientific and legislative drivers for change are also considered, along with next steps to address challenges in funding and recognition, to achieve regulatory acceptance and uptake within the research community. To provide some wider context, the use of in silico methods alongside other relevant approaches (e.g., human-based in vitro) is also discussed.

Recombinant Myeloperoxidase as a New Class of Antimicrobial Agents.

ABSTRACTHeme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. The difficulty of producing MPO at a large scale hinders its study and utilization. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study. rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities. In this study, rMPO was secreted into culture medium as a monomer. rMPO revealed enzymatic activity similar to that of native MPO. rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. In animal safety tests, the administration of 100 nM rMPO via tail vein did not result in any sign of toxic effects. Taken together, the data suggest that rMPO purified from a stably expressing human cell line is a new class of antimicrobial agents with the ability to kill a broad spectrum of pathogens, including bacteria and fungi with or without drug resistance.IMPORTANCE Over the past 2 decades, more than 20 new infectious diseases have emerged. Unfortunately, novel antimicrobial therapeutics are discovered at much lower rates. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death, and high health care costs. Currently, this is best seen with the lack of a cure for coronavirus disease 2019 (COVID-19). To combat such untreatable microorganisms, there is an urgent need to discover new classes of antimicrobial agents. Myeloperoxidase (MPO) plays an important role in host defense. The difficulty of producing MPO on a large scale hinders its study and utilization. We have produced recombinant MPO at a large scale and have characterized its antimicrobial activities. Most importantly, recombinant MPO significantly reduced the morbidity and mortality of murine pneumonia induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. Our data suggest that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.

Evaluation of the stability of Yamakagashi (Rhabdophis tigrinus) Equine Antivenom after 20 years storage.

In 2000, an equine Yamakagashi (Rhabdophis tigrinus) antivenom (Lot 0001) was testmanufactured as an unapproved drug for treatment of Yamakagashi bites. It was stocked on the premise of super-legal use from the viewpoint of emergency health crisis management. The antivenom showed a strong neutralizing ability against the hemorrhagic and coagulation activity of the Yamakagashi venom in its potency test. One vial of the antivenom can effectively neutralize at least about 4 mg of Yamakagashi venom. Its efficacy has also been confirmed in patients with severe cases of R. tigrinus bite that has been used in emergency. In 2020, this antivenom (Lot 0001) has reached 20 years after its production. To evaluate the integrity and potency of the antivenom, quality control, safety and potency tests had been conducted almost every year since 2012. Physical and chemical tests (property test, moisture content test, insoluble foreign matter test, osmotic pressure ratio test, pH test, protein content test, endotoxin test, sterility test) of the antivenom, showed no significant changes throughout the years, when compared to the results immediately after its production in 2000. All the parameters measured were also within the standard values. In animal safety tests (test for absence of toxicity and pyrogen), there was no change in the test results during the storage period and no abnormalities were observed. The potency test (anti-coagulant activity) after 20 years of the product, showed the same potency as those recorded immediately after production. Therefore, in all of the stability monitoring tests conducted so far, the product did not show any significant change compared to the results immediately after production. This confirms the stability of the product during the stockpiling period to the present, that is, 20 years after production.

Exploring in vitro to in vivo extrapolation for exposure and health impacts of e-cigarette flavor mixtures

In vitro to in vivo extrapolation (IVIVE) leverages in vitro biological activities to predict corresponding in vivo exposures, therefore potentially reducing the need for animal safety testing that are traditionally performed to support the hazard and risk assessment. Interpretation of IVIVE predictions are affected by various factors including the model type, exposure route and kinetic assumptions for the test article, and choice of in vitro assay(s) that are relevant to clinical outcomes. Exposure scenarios are further complicated for mixtures where the in vitro activity may stem from one or more components in the mixture. In this study, we used electronic cigarette (EC) aerosols, a complex mixture, to explore impacts of these factors on the use of IVIVE in hazard identification, using open-source pharmacokinetic models of varying complexity and publicly available data. Results suggest in vitro assay selection has a greater impact on exposure estimates than modeling approaches. Using cytotoxicity assays, high exposure estimates (>1000 EC cartridges (pods) or > 700 mL EC liquid per day) would be needed to obtain the in vivo plasma levels that are corresponding to in vitro assay data, suggesting acute toxicity would be unlikely in typical usage scenarios. When mechanistic (Tox21) assays were used, the exposure estimates were much lower for the low end, but the range of exposure estimate became wider across modeling approaches. These proof-of-concept results highlight challenges and complexities in IVIVE for mixtures.

Evaluation of the Neurobehavioural Toxic Effects of Taurine, Glucuronolactone, and Gluconolactone Used in Energy Drinks in Young Rats.

The neurotoxic effects of food additives used in energy drinks have been investigated since the 1900s but safety concerns are rising and reassurance via safety testing in animals is demanded by the public. Rigorous safety testing is performed for dose optimisation and duration of treatment and to detect the methods to assess changes in mood and behaviour. Hence, we studied the neurobehavioral effects of selected food additives used in energy drinks and their combination in rats when consumed in high doses. Young Sprague Dawley rats were divided into six groups. Group 1 was treated with the vehicle, group 2 was treated with 25 mg/kg p.o. caffeine, group 3 was treated with 5 mg/kg p.o. glucuronolactone, group 4 was treated with 8 mg/kg p.o. taurine, group 5 was treated with 84 mg/kg p.o. gluconolactone, and group 6 was treated with a combination of the three food additives. Neurobehavioral changes were evaluated on days 7, 14, and 21 using behavioural parameters. Neurobehavioral scoring and neurotransmitter estimation in rat brain tissue was performed on day 21. Significant changes were observed in the neurobehavioral parameters and neurobehavioural scoring in group 4 and group 6, compared with the control group (p<0.001). Furthermore, the significant decreases in neurotransmitter levels in the brains of rats that were treated with food additives indicated the neurotoxic effects of these substances. This study elaborated the neurobehavioral effects of selected food additives, namely glucuronolactone, taurine, and gluconolactone, when administered orally for 21 days in young rats. The highest toxic effects, including alterations in neurotransmitter levels, were observed in animals treated with a combination of food additives at high doses.

A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay.

The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, β-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.

Predicting toxicity of chemicals: software beats animal testing.

We created earlier a large machine‐readable database of 10,000 chemicals and 800,000 associated studies by natural language processing of the public parts of Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) registrations until December 2014. This database was used to assess the reproducibility of the six most frequently used Organisation for Economic Co‐operation and Development (OECD) guideline tests. These tests consume 55% of all animals in safety testing in Europe, i.e. about 600,000 animals. With 350–750 chemicals with multiple results per test, reproducibility (balanced accuracy) was 81% and 69% of toxic substances were found again in a repeat experiment (sensitivity 69%). Inspired by the increasingly used read‐across approach, we created a new type of QSAR, which is based on similarity of chemicals and not on chemical descriptors. A landscape of the chemical universe using 10 million structures was calculated, when based on Tanimoto indices similar chemicals are close and dissimilar chemicals far from each other. This allows placing any chemical of interest into the map and evaluating the information available for surrounding chemicals. In a data fusion approach, in which 74 different properties were taken into consideration, machine learning (random forest) allowed a fivefold cross‐validation for 190,000 (non‐) hazard labels of chemicals for which nine hazards were predicted. The balanced accuracy of this approach was 87% with a sensitivity of 89%. Each prediction comes with a certainty measure based on the homogeneity of data and distance of neighbours. Ongoing developments and future opportunities are discussed.

Target animal safety testing of an oral salicylanilide suspension, oxyclozanide, for the treatment of fascioliasis in bovine in China

The aim of this study was to determine the potential toxicity risk of an oxyclozanide suspension to the target animal, bovine. In this experiment, 32 Simmental beef cattle were fattened and fed a full-price diet without antimicrobial agents. The test cattle were divided into 4 groups, which were treated with 0, 1, 3, and 5 times the recommended dosage through continuous intermittent oral administration at intervals of 2 days. The body weight of the cattle was recorded before and after the experiment, and the weight changes were calculated. The safety of the drugs was evaluated by weight gain, observation of clinical toxicity, haematology, clinical chemistry and histopathology. The results showed that the cattle had different degrees of diarrhoea, loss of appetite and depression after administration. The results of clinicopathology had no significant effect. The results of pathological examination showed that there was a certain degree of damage in the 5 times recommended dose group. The recommended dose was safe to use. Thus, the recommended dose should be given by a single oral administration to ensure the safe use of this drug in the clinic.

Guideline on safety evaluation of cell-based medicinal products for animal use.

With the increased use of cell therapy in the veterinary sector, there is a growing demand for the development of cell-based medicinal products and the determination of their safety. Currently, the Korean Animal and Plant Quarantine Agency has established a guideline for evaluating the safety of cell-based medicinal products for animal use. The guideline includes items related to definition, classification, management, manufacturing procedure and quality control (standard and test method), stability testing, toxicity testing, pharmacological testing, and performance of clinical trials. In addition, testing protocols related to safety assessment of animal cell-based products such as chromosome karyotyping, tumorigenicity testing, confirmatory testing of biodistribution and kinetics, and target animal safety testing are described in detail. Moreover, because cell-based medicinal products are novel therapies, deviations from traditional designs may be justified in order to obtain relevant safety information on the treatment. Additionally, this guideline can be amended on the basis of new scientific findings.

Refinement, Reduction, and Replacement (3R) Strategies in Preclinical Testing of Medical Devices.

The U.S. Food and Drug Administration Center for Devices and Radiological Health (FDA/CDRH) has recently published several in vivo test guidance documents that mention refinements, reductions, or replacement animal testing strategies to facilitate the leveraging of data from large animal safety tests for conventional rodent testing. In response to the recently enacted Food and Drug Administration Safety and Innovation Act Section 907, which facilitates expedited access to novel therapies commonly described as Breakthrough Therapy Designation, FDA/CDRH has discussed efficient regulatory strategies for first-in-human investigation, including early feasibility study guidance. Large gains in humane care and translational research could also be attained by examples in FDA's Guidance for the Use of International Organization for Standardization 10993-1, which states that large animal safety studies may be considered as replacement rodent tests if the scientific principles, methods, and end points (SPME) are considered and applied. This article discusses SPME for the replacement of conventional rodent testing by the inclusion and integration of clinical, diagnostic, and pathologic data obtained from well-designed large animal studies. The recommendations include consideration for study designs that utilize methods for an overall more comprehensive interrogation of animal systems.

Gene expression profiling toward the next generation safety control of influenza vaccines and adjuvants in Japan

Influenza becomes epidemic worldwide every year, and many individuals receive vaccination annually. Quality control relating to safety and potency of influenza vaccines is important to maintain public confidence. The safety of influenza vaccines has been assessed by clinical trials, and animal safety tests are performed to monitor the consistent quality between vaccines used for clinical trials and marketing; the biological responses in vaccinated animals are evaluated, including changes in body weight and white blood cell count. Animal safety tests have been contributing to the quality relating to the safety of influenza vaccines for decades, but improvements are needed. Although precise mechanisms involving biological changes in animal safety tests have not been fully elucidated, the application of cDNA microarray technology make it possible to reliably identify genes related to biological responses in vaccinated animals. From analysis of the expression profile of >10,000 genes of lung in animals treated with an inactivated whole virion influenza vaccine, we identified 17 marker genes whose expression patterns correlated well to changes in body weight and leukocyte count in vaccinated animals. In influenza HA vaccine-treated animals exhibiting subtle changes in biological responses, a robust expression pattern of marker genes was found. Furthermore, these marker genes could also be used in the evaluation of adjuvanted influenza vaccines. The expression profile of marker genes is expected to be an alternative indicator for safety control of various influenza vaccines conferring high sensitivity and short turnaround time. Thus, gene expression profiling may be a powerful tool for safety control of vaccines in the future.

Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions.

High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.

Establishment of a New Quality Control and Vaccine Safety Test for Influenza Vaccines and Adjuvants Using Gene Expression Profiling

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.

"BINACLE" assay for in vitro detection of active tetanus neurotoxin in toxoids.

Tetanus neurotoxin (TeNT) consists of two protein chains connected by a disulfide linkage: The heavy chain mediates the toxin binding and uptake by neurons, whereas the light chain cleaves synaptobrevin and thus blocks neurotransmitter release.Chemically inactivated TeNT (tetanus toxoid) is utilized for the production of tetanus vaccines. For safety reasons, each toxoid bulk has to be tested for the "absence of toxin and irreversibility of toxoid". To date, these mandatory tests are performed as toxicity tests in guinea pigs. A replacement by an animal-free method for the detection of TeNT would be desirable. The BINACLE (BINding And CLEavage) assay takes into account the receptor-binding as well as the proteolytic characteristics of TeNT: The toxin is bound to immobilized receptor molecules, the light chains are then released by reduction and transferred to a microplate containing synaptobrevin, and the fragment resulting from TeNT-induced cleavage is finally detected. This assay offers a higher specificity for discriminating between toxic TeNT and inactivated toxoid molecules than other published assays. Validation studies have shown that the BINACLE assay allows the sensitive and robust detection of TeNT in toxoids, and thus may indeed represent a suitable alternative to the prescribed animal safety tests for toxoids from several European vaccine manufacturers. Product-specific validations (and possibly adaptations) of the assay protocol will be required. A European collaborative study is currently being initiated to further examine the applicability of the method for toxoid testing. The final aim is the inclusion of the method into the European Pharmacopoeia.

Toxicity testing in the 21st century beyond environmental chemicals.

After the publication of the report titled Toxicity Testing in the 21st Century - A Vision and a Strategy, many initiatives started to foster a major paradigm shift for toxicity testing - from apical endpoints in animal-based tests to mechanistic endpoints through delineation of pathways of toxicity (PoT) in human cell based systems. The US EPA has funded an important project to develop new high throughput technologies based on human cell based in vitro technologies. These methods are currently being incorporated into the chemical risk assessment process. In the pharmaceutical industry, the efficacy and toxicity of new drugs are evaluated during preclinical investigations that include drug metabolism, pharmacokinetics, pharmacodynamics and safety toxicology studies. The results of these studies are analyzed and extrapolated to predict efficacy and potential adverse effects in humans. However, due to the high failure rate of drugs during the clinical phases, a new approach for a more predictive assessment of drugs both in terms of efficacy and adverse effects is getting urgent. The food industry faces the challenge of assessing novel foods and food ingredients for the general population, while using animal safety testing for extrapolation purposes is often of limited relevance. The question is whether the latest paradigm shift proposed by the Tox21c report for chemicals may provide a useful tool to improve the risk assessment approach also for drugs and food ingredients.

Gluten and wheat intolerance today: are modern wheat strains involved?

Celiac disease is a food-induced enteropathy resulting from exposure to gluten in genetically predisposed individuals. The non-celiac gluten sensitivity (NCGS) is a less known syndrome whose prevalence is under-estimated. The last decades have seen changes in the clinical presentation of both diseases. One possible explanation is that changes in the gluten-rich cereals themselves were the principal causes. Celiac-triggering gluten proteins are indeed expressed to higher levels in modern cereals while non-triggering proteins are expressed less. Sophisticated hybridization techniques have been used to produce new strains of modern wheat, the most high-yielding of which have since made their way into human foods in the absence of animal or human safety testing. The dramatic changes in the clinical presentation of celiac disease and NCGS have taken place when new cereal hybrids were introduced into human foods. This is a critical medical and environmental issue which needs to be investigated by appropriate studies.