ATLA 43.1, March 2015

//ATLA 43.1, March 2015

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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Toxicity Assessment of Tobacco Products in Vitro

Joseph R. Manuppello and Kristie M. Sullivan

Driven by new regulatory demands to demonstrate risk reduction, the toxicity assessment of tobacco products increasingly employs innovative in vitro methods, including biphasic cell and tissue cultures exposed to whole cigarette smoke at the air–liquid interface, cell transformation assays, and genomic analyses. At the same time, novel tobacco products are increasingly compared to traditional cigarettes. This overview of in vitro toxicology studies of tobacco products reported in the last five years provides evidence to support the prioritisation of in vitro over in vivo methods by industry and their recommendation by regulatory authorities.
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Participation of Brazil in the World Congresses on Alternatives and Animal Use in the Life Sciences: An Increase in Commitment to the Three Rs

Octavio Presgrave, Cristiane Caldeira, Wlamir Moura, Mayara Cruz, Gisele Méier, Elisabete dos Santos and Maria H.V. Boas

Many Brazilian researchers have long been interested in the development and use of alternative methods. Most of their research groups work in isolation, due to the lack of funding for collaborative studies. Despite these problems, since the Third World Congress on Alternatives and Animal Use in the Life Sciences, Brazilian researchers have strongly participated, not only by presenting posters and oral presentations, but also by being involved in the World Congress Committees. The Brazilian Center for the Validation of Alternative Methods (BraCVAM) must play an important role in the development and validation of alternative methods, through the active participation of the National Network of Alternative Methods (ReNaMA). In Brazil, Law 11,794/2008 regulates the use of animals in experimentation and education, and Law 9,605/1998 clearly states that use of the original animal test is not permitted, if an alternative method is available. Therefore, given the current legal framework, it is very important that all the Ministries involved with animal use, and the organisations responsible for funding researchers, strive to increase the financial support of those groups that are involved in the development and use of alternative methods in Brazil.
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