ATLA 30, 2002

/ATLA 30, 2002

Two In Vitro Models for Gas-Phase Exposure to Volatile Compounds

Paola Bonsi, Flavia Zucco and Annalaura Stammati

Two experimental models suitable for the screening of volatile compounds were set up. The first consisted of a glass chamber-slide with eight wells, one carrying the test compound, and the others carrying cells in monolayers. In the second model, the cells were cultured in a glass Petri dish, and the test compound was poured onto a filter lying on a glass cover-slip, supported by a metal ring. Four plant volatiles [carvacrol, S-(+)-carvone, thymol and decanal] and one essential oil (caraway oil) were chosen as test compounds. The toxicity rankings obtained with the two models were compared with that obtained in a previous study performed with the same compounds under conventional culture conditions. Differences in the toxicity ranking were observed between the conventional culture conditions and the gasphase models, confirming the importance of correct exposure conditions for the evaluation of toxicity. Both models have advantages that make them suitable as a preliminary step in the toxicity screening of volatile compounds, or for use in a test battery when combined with conventional approaches.
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Caribbean and Latin American Workshop on Alternative Methods

Krys Bottrill

The first Caribbean and Latin American Workshop on Alternative Methods took place on 4–5 December 2001, in Santiago de Cuba, Cuba. The event was organised by scientists from Toximed (Centre for Toxicology and Biomedicine) as part of their ProAlt initiative to promote the Three Rs in the region. ProAlt is funded by the Foundation for the Promotion of Research on Replacement and Complementary Methods to Reduce Animal Testing (SET), Germany, and additional sponsorship for the workshop was provided by UFAW and Advocates for Animals.
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2017-01-09T06:29:06+00:00 Tags: |

Editorial: Alternatives to Animal Experiments: “Goodbye ECVAM, Hello FRAME”

Michael Balls

By the time this Editorial is published, I will have retired after more than nine years as Head of the European Centre for the Validation of Alternative Methods (ECVAM), and returned from Italy to England, to devote as much as possible of my remaining years to my voluntary role as Chairman of the Trustees of the Fund for the Replacement of Animals in Medical Experiments (FRAME).
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2017-01-09T06:29:06+00:00 Tags: |

News & Views

ATLA Staff Writer

ANZCCART News Goes Electronic
ANZCCART Appoints New Director
New Animal Welfare Legislation in Queensland, Australia
Animal Procedures Committee Research Grants
3T3 NRU In Vitro Phototoxicity Test: Updated Software
European Parliament Votes to Ban Cosmetics Tested on Animals
Human Research Ethics
In Vitro Pyrogenicity Test Commercialised
Angela Eagle leaves Home Office
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2017-01-09T06:29:07+00:00 Tags: |

Organotypic Brain-slice Cultures from Adult Rats: Approaches for a Prolonged Culture Time

Eckbert Wilhelmi, Ulrich H. Schöder, Akilah Benabdallah, Frank Sieg, Jörg Breder and Klaus G. Reymann

Animal experiments are widely used in neurobiological and neuropharmacological research. Today, juvenile brain organotypic slice cultures have partially replaced in vivo experiments, but there is no adequate in vitro counterpart for the adult brain. The present study was aimed at the long-term culture of physiologically intact hippocampal slices from adult rats, by improving the conditions for preparation and culture, and the development of a new culture medium. A cerebrospinal fluid (CSF)-like medium was used, which was modified with a variety of supplements, including energy precursors, free-radical scavengers, and compounds known to inhibit neurotoxicity. The population spike amplitude (PSA) was used as a measure of viability, and amplitudes larger than 1mV indicated viable cultures. The addition of MK-801 during slice preparation improved PSA values during the first two days in vitro (DIV). Ascorbic acid and insulin prolonged the culture time up to DIV 4. FK-506 and vitamin E, alone or in combination, supported slice culture up to DIV 5. An increase in ATP, unless combined with vitamin E, and/or insulin, increased culture time up to DIV 6. Vitamins B1, B2, B12 and D2 had no effect. The modified CSF-like medium developed in this study permits the culture of adult hippocampal tissue for at least 6 days.
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Evaluation of a Rule Base for Identifying Contact Allergens by using a Regulatory Database: Comparison of Data on Chemicals Notified in the European Union with “Structural Alerts” Used in the DEREK Expert System

Stephan Zinke, Ingrid Gerner & Eva Schlede

To assess the suitability of the use of structural alerts to identify the skin-sensitising properties of chemicals, the 40 originally published structural alerts for the prediction of skin-sensitisation properties used by the DEREK system (which now contains 59 structural alerts), have been evaluated against a database developed in the German Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), which contains data submitted under the procedure for notifying new chemicals within the European Union. The evaluation of the 40 structural alerts used in DEREK revealed that eight of the 40 structural alerts for the prediction of skin-sensitising potential could be used without any further refinement. Ten structural alerts may need further specifications or refinements in order avoid false-positive predictions — proposals for refinement are discussed. Not enough substances were found within the BgVV database (containing data for more than 1000 substances) to evaluate ten of the DEREK substructures; hence, for these structural alerts, a judgement on their suitability for prediction of skin-sensitisation properties in expert systems is not possible. For 12 structural alerts, no comparative result could be obtained, because these rules did not “fire” for any of the examined chemicals. As a general result of the evaluation process, the approach of using structural alerts for the prediction of skin-sensitising properties of chemicals proved to be reliable. Proposals are given for a refinement of the structural alerts for prediction of contact allergy used in the DEREK system. In addition, advice and several preconditions are given, that apply when trying to teach a computer system to use structural alerts to predict toxicological properties.
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Is it Possible to Replace Stimulus Animals by Scent-filled Cups in the Social Discrimination Test?

Ruud van den Bos, Klaske J. van der Horst, Annemarie M. Baars and Berry M. Spruijt

A study in which the rat social discrimination test was refined is described. This test measures social memory by using, in general, juvenile rats as stimulus animals. Rats are offered a first juvenile to investigate (learning trial), and after a specified interval, the rats are offered the same rat and a second juvenile rat to investigate again (retrieval trial). When the rats sniff the second juvenile in the retrieval trial more than the first, social memory for the first juvenile is said to be present. This test is mainly based on scents from the juvenile. Attempts were made to refine the test to reduce the number of animals used, to enhance the scope of the test, and to improve its validity. Firstly, the stimulus animals were replaced by the scent of juveniles, in the form of cups filled with sawdust taken from cages of juvenile rats. Similar results to those in the original test were obtained when using these scents. Furthermore, male and female scents were tested, and showed the same results as for the juvenile scents. Secondly, rats were also given two cups (one scent-filled and one filled with plain sawdust) in the learning trial, to determine which allowed a more-precise delineation of motivational, discriminatory and memory components. Overall, it is possible to replace stimulus animals by scent-filled cups in the social discrimination test, to enhance the scope of the test, and to draw more-valid conclusions with respect to social memory.
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In Vitro Toxicity: Mechanisms, Alternatives and Validation — A Report from the 19th Annual Scientific Meeting of the Scandinavian Society for Cell Toxicology

Anna Forsby

The Scandinavian Society for Cell Toxicology (SSCT) has arranged annual scientific meetings since 1983. These workshops were the forum for the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. Along with the MEIC programme, which was completed in 1998, a wide range of topics relating to cytotoxicity have been discussed. The meetings have also given an opportunity for graduate students and young scientists to present their work to an international audience. At the same time, experts in the fields of in vitro toxicity have been invited as speakers. The 19th SSCT scientific meeting, which was held in 2001 at Sørup Manor in Ringsted, Denmark, was no exception. The meeting consisted of four sessions: mechanisms of toxicity; environmental toxicological testing; alternatives to animal experiments; and validation of in vitro tests.
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Cytotoxicity of Amino Alcohols to Rat Hepatoma-derived Fa32 Cells

Ellen M. Scheers, Anna Forsby and Paul J. Dierickx

Amino alcohols are used as emulsifying agents in dry-cleaning soaps, wax removers, cosmetics, paints and insecticides. The cytotoxicities of 12 amino alcohols, which differed in chain length, position of the amino and alcohol groups, and the presence of an additional phenyl group, were determined by the neutral red uptake inhibition assay with normally cultured, glutathione-depleted or antioxidant-enriched Fa32 rat hepatoma-derived cells. Glutathione depletion and antioxidant enrichment were achieved by including 50μM L-buthionine-S,R-sulphoximine (BSO) or 100μM α-tocopherol acetate (vitamin E) in the culture medium for 24 hours before and during the assay. The cytotoxicity of the amino alcohols observed after treatment for 24 hours was expressed as the concentration of compound needed to induce a 50% reduction in neutral red uptake (NI50). The observed NI50 values ranged from 3mM to 30mM. The individual stereoisomers and a racemic mixture of 1-amino-2-propanol exhibited similar cytotoxicities (with normally cultured Fa32 cells, and vitamin E- and BSO-treated cultures). Similar NI50 values for D-(+)-2-amino-1-propanol, 3-amino-1-propanol and the L-, D- or DL- forms of 1-amino-2-propanol, indicated that the position of the amino group had little influence on the cytotoxicities of the amino alcohols. In contrast, the position of the hydroxyl group appeared to play an important role for the toxicity of the compound, as indicated by the significantly different NI50 values for 4-amino-1-butanol and 4-amino-2-butanol. An additional phenyl group greatly increased the cytotoxicity of 2-amino-1,3-propanediol. For most of the compounds, cytotoxicity increased when GSH was depleted, and decreased when the cells were enriched with vitamin E. This indicated that most of the tested chemicals interact with GSH, either directly or indirectly, by processes which generate oxygen free-radicals. Decreased toxicity was found for most of the chemicals administered to vitamin E-enriched cells, indicating that reactive oxygen species could be involved in the toxicity of the amino alcohols.
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Development of an In Vitro Test Battery for the Estimation of Acute Human Systemic Toxicity: An Outline of the EDIT Project

Cecilia Clemedson, Marika Nordin-Andersson, Henning F. Bjerregaard, Jørgen Clausen, Anna Forsby, Helena Gustafsson, Ulrika Hansson, Boris Isomaa, Carsten Jørgensen, Ada Kolman, Natalia Kotova, Gunter Krause, Udo Kristen, Kalle Kurppa, Lennart Romert and Ellen Scheers

The aim of the Evaluation-guided Development of New In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R2 = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and
potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that
the development within the EDIT target organ programme should initially be focused on the nervous system.
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