Volume 43, Issue 3 - June 2015

Application of non-viscous liquids for  the EpiOcular-EIT


The European Citizens’ Stop Vivisection Initiative

Michael Balls

The European Commission’s considerate response to the controversial European Citizens’ Stop Vivisection Initiative promises to accelerate progress toward eliminating the need for animal experimentation in research and testing.
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News and Views

ATLA Staff Writer

In Vitro Model of Varicella zoster Virus Reactivation
‘Sleep’ Recreated In Vitro
C. elegans model of ALS
Name Changes
Air France–KLM to Continue to Transport Primates for Research
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CAAT News & Views

ATLA Staff Writer

Workshop on Dynamic modeling of metabolic responses in neurotoxicology (Dynametox)
Thomas Hartung Presented at the Mid-Atlantic Society of Toxicology Webinar
Good Cell Culture Practice (GCCP) for Induced Pluripotent Stem Cells Workshop
François Busquet Presents at Three Rs Workshop in Romania, Interviewed on French Radio
Mardas Daneshian Radio Interview on GM Animals
Joint Information Day on Biology Inspired Microsystems — Status and Future
Discover Magazine Covers CAAT’s Brain-on-a-Chip Research
Webinar The Human Toxome Project — a Test Case for Pathway Identification by Multi-omics Integration Available for On-demand Streaming
New Paper in Archives of Toxicology
Recent Publications
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The Protozoan, Paramecium primaurelia, as a Nonsentient Model to Test Laser Light Irradiation:

The Effects of an 808nm Infrared Laser Diode on Cellular Respiration

Andrea Amaroli, Silvia Ravera, Steven Parker, Isabella Panfoli, Alberico Benedicenti and Stefano Benedicenti

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm2) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly (p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more expensive mammalian cellular cultures.

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The Ex Vivo Eye Irritation Test as an Alternative Test Method for Serious Eye Damage/Eye Irritation

Felix Spöler, Oya Kray, Stefan Kray, Claudia Panfil, and Norbert F. Schrage

Ocular irritation testing is a common requirement for the classification, labelling and packaging of chemicals (substances and mixtures). The in vivo Draize rabbit eye test (OECD Test Guideline 405) is considered to be the regulatory reference method for the classification of chemicals according to their potential to induce eye injury. In the Draize test, chemicals are applied to rabbit eyes in vivo, and changes are monitored over time. If no damage is observed, the chemical is not categorised. Otherwise, the classification depends on the severity and reversibility of the damage. Alternative test methods have to be designed to match the classifications from the in vivo reference method. However, observation of damage reversibility is usually not possible in vitro. Within the present study, a new organotypic method based on rabbit corneas obtained from food production is demonstrated to close this gap. The Ex Vivo Eye Irritation Test (EVEIT) retains the full biochemical activity of the corneal epithelium, epithelial stem cells and endothelium. This permits the in-depth analysis of ocular chemical trauma beyond that achievable by using established in vitro methods. In particular, the EVEIT is the first test to permit the direct monitoring of recovery of all corneal layers after damage. To develop a prediction model for the EVEIT that is comparable to the GHS system, 37 reference chemicals were analysed. The experimental data were used to derive a three-level potency ranking of eye irritation and corrosion that best fits the GHS categorisation. In vivo data available in the literature were used for comparison. When compared with GHS classification predictions, the overall accuracy of the three-level potency ranking was 78%. The classification of chemicals as irritating versus non-irritating resulted in 96% sensitivity, 91% specificity and 95% accuracy.

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The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations:

Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes

Susanne N. Kolle, Maria Cecilia Rey Moreno, Winfried Mayer, Andrew van Cott, Bennard van Ravenzwaay and Robert Landsiede

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the
identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury) and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.

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Non-animal Replacements for Acute Toxicity Testing

Carol Barker-Treasure, Kevin Coll, Nathalie Belot, Chris Longmore, Karl Bygrave, Suzanne Avey and Richard Clothier

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.
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Dorothy Hegarty Award

ATLA Staff Writer

The Dorothy Hegarty Award for the best article published in the 2014 ATLA, has been won by Zhuo Li, Ka Yee Lok and Changhe Yu (Bio S&T, Montreal, Canada), and Tong Guo (Goodman Institute of Investment Management, John Molson School of Business, Concordia University, Montreal, Canada). Their paper, Lung fibrosis: Drug screening and disease biomarker identification with a lung slice culture model and subtracted cDNA library, appeared in ATLA 42, pp. 235–243.
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<h3>Alim Louis Benabid, Mahlon Delong & Marwan Hariz

Jarrod Bailey</h3>

Letters re: Bailey, J. (2014). Monkey-based research on human disease: The implications of genetic differences. ATLA 42, 287–317.
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FRAME is the Fund for the Replacement of Animals in Medical Experiments. It publishes ATLA. It also promotes the replacement of animals in laboratories through better science. Visit FRAME website
PiLAS is Perspectives in Laboratory Animal Science. It improves the quality of discussion about animal experimentation by giving scientists in all fields a place to exchange views. Read PiLAS