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So far Anne Jeffery has created 261 blog entries.

Optimisation of the Bovine Whole In Vitro Embryo System as a Sentinel for Toxicity Screening: A Cadmium Challenge

Ellen P.A. Jorssen, Lucia Vergauwen, Karen Goossens, An Hagenaars, Mario Van Poucke, Evi Petro, Luc Peelman, Dries Knapen, Jo L.M.R. Leroy and Peter E.J. Bols

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more ‘cadmium-typical’ transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and ‘cadmium-typical’ manner to our reference toxicant.
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The EpiOcular Eye Irritation Test (EIT) for Hazard Identification and Labelling of Eye Irritating Chemicals: Protocol Optimisation for Solid Materials and the Results after Extended Shipment

Yulia Kaluzhny, Helena Kandárová, Yuki Handa, Jane DeLuca, Thoa Truong, Amy Hunter, Paul Kearney, Laurence d’Argembeau-Thornton and Mitchell Klausner

The 7th Amendment to the EU Cosmetics Directive and the EU REACH Regulation have reinforced the need for in vitro ocular test methods. Validated in vitro ocular toxicity tests that can predict the human response to chemicals, cosmetics and other consumer products are required for the safety assessment of materials that intentionally, or inadvertently, come into contact with the eye. The EpiOcular Eye Irritation Test (EIT), which uses the normal human cell-based EpiOcular™ tissue model, was developed to address this need. The EpiOcular-EIT is able to discriminate, with high sensitivity and accuracy, between ocular irritant/corrosive materials and those that require no labelling. Although the original EpiOcular-EIT protocol was successfully pre-validated in an international, multicentre study sponsored by COLIPA (the predecessor to Cosmetics Europe), data from two larger studies (the EURL ECVAM–COLIPA validation study and an independent in-house validation at BASF SE) resulted in a sensitivity for the protocol for solids that was below the acceptance criteria set by the Validation Management Group (VMG) for eye irritation, and indicated the need for improvement of the assay’s sensitivity for solids. By increasing the exposure time for solid materials from 90 minutes to 6 hours, the optimised EpiOcular-EIT protocol achieved 100% sensitivity, 68.4% specificity and 84.6% accuracy, thereby meeting all the acceptance criteria set by the VMG. In addition, to satisfy the needs of Japan and the Pacific region, the EpiOcular-EIT method was evaluated for its performance after extended shipment and storage of the tissues (4–5 days), and it was confirmed that the assay performs with similar levels of sensitivity, specificity and reproducibility in these circumstances.
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How Long Must They Suffer? Success and Failure of our Efforts to End the Animal Tragedy in Laboratories

Roman Kolar

Scientific findings have revealed how much we have dramatically underestimated the intellectual, social and emotional capabilities of non-human animals, including their levels of self-consciousness and ability to suffer from psychological stress. In the 21st century, the field of animal ethics has evolved as a serious scientific discipline, and nowadays largely advocates that the way we treat animals, both legally and in practice, is morally wrong. Politics and legislation have reacted to these facts, to some extent, but neither current legislation nor current practice reflect the scientific and moral state-of-the-art. Too often, the will to change things is watered down in the decision-making process, e.g. in the drafting of legislation. In the field of animal experimentation there have been many genuine efforts by various players, to advance and apply the principles behind the Three Rs. However, the fundamental problem, i.e. the overall number of animals sacrificed for scientific purposes, has increased. Clearly, if we are serious about our will to regard animal experimentation as an ethical and societal problem, we have to put much more emphasis on addressing the question of how to avoid the use of animals in science. To achieve this goal, certain issues need to be considered: a) the present system of ethical evaluation of animal experiments, including testing for regulatory purposes, needs to be reformed and applied effectively to meet the legal and moral requirements; b) animal testing must be avoided in future legislation, and existing legislation has to be revised in that regard; c) resources from animal-based research have to re-allocated toward alternatives; and d) the academic curricula must be reformed to foster and integrate ethical and animal welfare issues.
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Applications of the Neutral Red Cytotoxicity Assay to In Vitro Toxicology

Harvey Babich and Ellen Borenfreund

A concerted effort is currently in progress to develop alternatives to the use of live animals for the acute toxicity testing of xenobiotics. To this end, the neutral red in vitro cytotoxicity assay was developed which, although initially based on the use of mammalian cells in culture, has also been adapted for ecotoxicity studies using fish cells in culture. The neutral red assay is based on the binding of neutral red, a weakly cationic supra vital dye, to the lysosomal matrix of viable cells after their incubation with toxic agents. Spectrophotometric quantitation of the extracted dye with a scanning microtitre well reader at 540nm was found to be linear with the number of surviving, viable cells. The assay has been used to determine the relative acute cytotoxicities of a broad spectrum of chemical test agents, to establish structure-toxicity relationships for series of related chemicals, to study metabolism-mediated cytotoxicity, to evaluate interactions between combinations oftest agents, to evaluate differential and selective toxicities of cancer chemotherapeutics and other pharmaceuticals, and to study temperature-toxicity interactions.
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IIVS News & Views

ATLA staff writers

New Management Roles at IIVS
Slides Available from the PISC/Chemical Watch Webinar Series on the Use of Alternative Methods for REACH
2015 Practical Methods for In Vitro Toxicology Training Course
IIVS’ Industry Council for the Advancement of Regulatory Acceptance of Alternatives (ICARAA): Training at NIFDC Beijing, China
IIVS-Hosted Workshop: Assessment of In Vitro COPD Models for Tobacco Regulatory Science
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2017-01-09T06:03:19+00:00 Tags: , |

Human Embryonal Carcinoma Cells in Serum-free Conditions as an In Vitro Model System of Neural Differentiation

Jovana Jasnic-Savovic, Andrijana Klajn, Milena Milivojevic, Marija Mojsin and Gordana Nikcevic

Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, six days of RA treatment resulted in a marked increase in SOX3 protein levels in serum-free media compared to serum-containing media, indicating that serum might have an inhibitory effect on the expression of this neural differentiation marker. This finding is important for both basic and translational studies that hope to exploit cell culture conditions that are free of animal-derived products.
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A Step Forward in the Quality Control Testing of Inactivated Rabies Vaccines — Extensive Evaluation of European Vaccines by Using Alternative Methods to the In Vivo Potency Tests

Alexandre Servat, Sébastien Kempff, Valère Brogat, Estelle Litaize, Jean-Luc Schereffer and Florence Cliquet

The mouse challenge test still remains the reference method for the potency determination of human and animal inactivated rabies vaccines, and it is still widely used throughout the world. This test suffers from many disadvantages — it is expensive and time consuming, uses a large number of mice, causes significant animal distress, and suffers from high variability. Recently, the European Pharmacopoeia has recognised the use of a serological potency assay (SPA) as an alternative method to the challenge test. This new test is based on the determination of rabies neutralising antibody titres in vaccinated mice, by using the modified Rapid Fluorescent Focus Inhibition Test (mRFFIT). With the objective of adopting this new method for the batch release of inactivated rabies vaccines, we evaluated its performance on a large collection of rabies vaccines currently assessed in our laboratory. The Fluorescent Antibody Virus Neutralisation test (FAVNt) was used in parallel with the mRFFIT, and the results were compared to the mouse challenge test. Our results demonstrate that the SPA is capable of estimating the potency of vaccines formulated with a potency margin well above the minimum of 1IU/dose. For low potency vaccines, this new method demonstrated some limitations, due to the recurrent invalidation of the assay. We have also demonstrated the superior sensitivity of the FAVNt when compared to the mRFFIT, and the importance of minimising the risk of detecting non-responders in vaccinated mice.
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Better Science with Human Cell-based Organ and Tissue Models

Tuula Heinonen

At present, animal-based models are the major test systems for assessing the tolerability and safety of chemical substances for regulatory purposes, and also for pivotal efficacy testing in pharmaceutical development. In spite of the high genetic similarity between many laboratory animals and humans, animal models are very poor predictors of human health effects and pathophysiological processes. Thus, models and testing strategies that are more relevant to human biology, are needed for these purposes. The best predictability is achieved with human organotypic models that mimic the microenvironment of human tissues. During their development, such models have to be characterised at the structural, genetic and functional levels, and compared to the respective human tissues. Their predictivity should be confirmed by using known reference chemicals with corresponding human data. The use of these methods in safety assessment and biomedical research, combined with the knowledge gained of the underlying biological processes on gene and protein expression, as well as on cellular signalling, will ultimately lead to better human science and animal welfare.
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