ATLA 30, 2002

/ATLA 30, 2002

The EU White Paper: Strategy for a Future Chemicals Policy (an Idea that Must Have Seemed Good at the Time!)

Robert Combes

The publication in 2001 of a White Paper on a new European Union (EU) initiative, entitled Strategy for a Future Chemicals Policy,1 has caused great concern among scientists and animal welfare groups about a possibly huge increase in the use of laboratory animals to cope with the further testing of some 30,000 existing chemicals in use in the EU. Also, a burden has been placed on chemical manufacturers and downstream users (for example, companies that use chemicals as ingredients in the manufacture of other products) to provide hazard data and to undertake separate risk assessments, as part of the overall process of risk management.
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2017-01-09T06:28:45+00:00 Tags: |

Alternatives to Animal Testing in the Safety Evaluation of Products

Derek J. Knight and Damien Breheny

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals
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A Comparative Study of the Toxicity of Mercury Dichloride and Methylmercury, Assayed by the Frog Embryo Teratogenesis Assay–Xenopus (FETAX)

Mariangela Prati, Rosalba Gornati, Patrizia Boracchi, Elia Biganzoli, Salvador Fortaner, Romano Pietra, Enrico Sabbioni and Giovanni Bernardini

The Frog Embryo Teratogenesis Assay–Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran amphibian Xenopus laevis. The FETAX can detect xenobiotics that affect embryonic development, when mortality, teratogenicity and growth inhibition are used as endpoints. The FETAX was used to compare the embryotoxic and teratogenic potentials of two chemical species of mercury: inorganic mercury(II) chloride (HgCl2) and organic methylmercury chloride (MeHgCl). MeHgCl, with an estimated median lethal concentration [LC50] of 0.313μM and a median teratogenic concentration [TC50] of 0.236μM, showed a higher toxicity than HgCl2, with estimated LC50 and TC50 values of 0.601μM and 0.513μM, respectively. On the basis of these results, HgCl2 and MeHgCl can be classified as “slightly teratogenic compounds”, as the ratio of LC50/TC50 is less than 1.5. There was a significant deviation from the commonly described monotonic behaviour of the concentration–response curves, suggesting a hormetic effect of both species of mercury. Uptake experiments, followed by neutron activation analysis, showed a higher incorporation of mercury in embryos exposed to MeHgCl compared with those exposed to HgCl2. Interestingly, Hg-exposed embryos showed a higher content of selenium and zinc than did control embryos.
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Use of a Cell Transformation Assay with Established Cell Lines, and a Metabolic Cooperation Assay with V79 Cells for the Detection of Tumour Promoters: A Review

Ayako Sakai, Yumiko Iwase, Yoshiyuki Nakamura, Kiyoshi Sasaki, Noriho Tanaka and Makoto Umeda

Extensive studies on the safety evaluation of chemicals have indicated that a considerable number of non-genotoxic chemicals are carcinogenic. Tumour promoters are likely to be among these nongenotoxic carcinogens, and their detection is considered to be an important approach to the prevention of cancer. In this review, the results are summarised for in vitro transformation assays involving established cell lines, and for an assay for inhibition of gap junctional intercellular communication for the detection of tumour promoters, which involves V79 cells. Although the number of chemicals examined is still too small to permit a full evaluation of the correlation between in vitro cell transformation and in vivo carcinogenicity, it is clear that the sensitivity of the focus formation assay is very high. In the case of the metabolic cooperation assay, the sensitivity appears to be rather poor, but the assay can be considered to be useful because of its simple procedure and its considerable database. These in vitro assays for tumour promoters are recommended as useful tools for the detection of non-genotoxic carcinogens.
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Cystathionine Pathway-dependent Cytotoxicities of Diethyl Maleate and Diamide in Rat and Human Hepatoma-derived Cell Cultures

Paul J. Dierickx, Jacques O. De Beer and Ellen M. Scheers

Glutathione (GSH) plays a role in many toxicologically important metabolic processes. It was previously established that L-buthionine S,R-sulphoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase, reduces the GSH content more efficiently in rat (Fa32) than in human (Hep G2) hepatomaderived cells. We therefore investigated whether the cystathionase inhibitor propargylglycine (PPG) could further decrease the BSO-induced GSH depletion in Hep G2 cells. The influence of the cystathionine precursors N-acetylmethionine, methionine and homocysteine on the cytotoxicity of diethyl maleate (DEM) and diamide [1,1´-azobis(N,N-dimethylformamide)] was also investigated. PPG reduced the GSH content in both cell lines. A further GSH decrease in Hep G2 was obtained when using a BSO + PPG combination containing relatively high concentrations of PPG. BSO diminished the toxicity of PPG. Homocysteine was the most efficacious of the tested cystathionine precursors in increasing the GSH content and reducing the cytotoxicity of DEM and diamide in Fa32 and Hep G2 cells.
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Cytostatic Properties of a Novel Compound Derived from Penicillium pinophilum: An In Vitro Study

Annalaura Stammati, Rosario Nicoletti, Salvatore De Stefano, Franco Zampaglioni & Flavia Zucco

3-O-Methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, inhibits the in vitro growth of plant pathogenic fungi. This specific property suggested that the compound could be used against other fungal pathogenic activities, including dermatological ones. However, for such applications, toxicological side-effects should be taken into account, in order to prevent other types of risk to mammalian cells. Therefore, investigations were made of the basic toxicity of OMF toward a human tumour cell line. The compound was found to have a cytostatic effect, which represents a counter-indication to its use as a therapeutic agent in dermatology, but suggests that it may have potential as an antitumour agent. This study confirmed the validity of in vitro systems for preliminary assays on new compounds, in order to avoid the use of animals in toxicological studies.
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Detection of the Embryotoxic Potential of Cyclophosphamide by Using a Combined System of Metabolic Competent Cells and Embryonic Stem Cells

Susanne Bremer, Cristian Pellizzer, Sandra Coecke, Martin Paparella and Paolo Catalani

In order to develop a method for detecting metabolism-mediated embryotoxicity, differentiating embryonic stem (ES) cells were exposed to the well-known proteratogen, cyclophosphamide (CPA). CPA was tested in a scientifically validated embryonic stem-cell test (EST), and in the newly developed reporter-gene assay for developmental cardiotoxicity. Both assays gave false-negative results. Because no metabolic competence (cytochrome P450 [CYP] activity) was found in the ES cells under the selected culture conditions, a simple biotransformation system was combined with the reporter-gene assay. As the metabolic pathway of CPA is well characterised, the genetically engineered mammalian cell line V79, transfected with CYP2B1 cDNA, was selected as a biotransformation system. CYP2B1 is responsible for transforming CPA into teratogenically active metabolites. The supernatants of genetically engineered V79 cells were analysed in the reporter-gene assay for developmental cardiotoxicity. In preliminary experiments, the combined system was able to detect the embryotoxic potential of the proteratogen, CPA.
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Use of the SOS-chromotest Spot Assay as a Screening System for Detecting Genotoxic Compounds in Crude Plant Extracts

Daniel Arrieta Baez, Gerardo Zepeda Vallejo, Patricia Cano Sánchez, Matilde Breña Valle, Ricardo Reyes Chilpa and Manuel Jiménez Estrada

An SOS-chromotest spot assay was used to detect genotoxic compounds in crude plant extracts. The method allows simultaneous testing of extracts from different species in either a liquid or a solid crystalline form. Extracts from two species of the genus Senna, native to the state of Morelos, Mexico, were assayed. Four genotoxic compounds were isolated, and were identified as quercetin and rutin from S. wislizeni, and 5,7-di-O-methylrutin and 5,7-di-O-methylquercetin from S. skinneri. The SOS-chromotest spot assay proved to be useful for activity-guided fractionation at the beginning of screening for genotoxic compounds in crude plant extracts.
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ECVAM’s Contributions to the Implementation of the Three Rs in the Production and Quality Control of Biologicals

Marlies Halder, Coenraad Hendriksen, Klaus Cussler and Michael Balls

A summary is presented of the activities initiated, and the progress achieved, between April 1993 and December 2001 in implementing the Three Rs in one of the main priority areas of the European Centre for the Validation of Alternative Methods (ECVAM) - the production and quality control of biologicals. These have included organising eight key workshops, and financial contributions to, and sponsorship of, relevant international workshops, symposia and conferences. Noteworthy activities include financial support and/or participation in a number of prevalidation and validation studies. These involved alternative methods for the batch potency testing of: human tetanus vaccines; human and veterinary tetanus antisera and immunoglobulin; rabies vaccines; Leptospira hardjo vaccines; Clostridium perfringens vaccines; and erysipelas vaccines. They also involved a cell culture test for specific toxicity testing of diphtheria toxoid vaccines. In addition, ECVAM funded a study on the use of humane endpoints for vaccine quality control tests involving severe suffering, such as the potency testing of erysipelas, rabies and pertussis vaccines. ECVAM has also contributed financially to the compilation of manuals and expert reports, and to training in test methods. Following the report of an ECVAM Task Force, ECVAM financially supported the prevalidation of some in vitro methods for the potency testing of a recombinant hormone. A proposal is presented for promotion of regulatory acceptance, and suggestions are made for possible future activities.
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