ATLA 27.4, July 1999

//ATLA 27.4, July 1999

Animal Experiments: Letting the People Speak

Michael Balls

“Make no mistake, the debate over animal experimentation has become deadly serious. In Britain, we’ve seen bombings, hunger strikes and death threats. The scientists involved, labelled ‘torturers’, live in fear of becoming the next target. Peaceful campaigners for animal rights find themselves branded as ‘terrorist sympathisers’. Yet amidst the violence and rhetoric, one thing is missing: no one really knows what the public think because they haven’t been asked where they would draw the line on animal research.
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The Potential Use of Non-invasive Methods in the Safety Assessment of Cosmetic Products

Vera Rogiers, Michael Balls, David Basketter, Enzo Berardesca, Christopher Edwards, Peter Elsner, Joachim Ennen, Jean Luc Lévêque, Marie Lóden, Philippe Masson, José Parra, Marc Paye, Gérald Piérard, Luis Rodrigues, Hans Schaefer, David Salter and Valérie Zuang

This is the report of the thirty-sixth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which reduce, refine or replace the use of laboratory animals.
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The Principles of Good Laboratory Practice: Application to In Vitro Toxicology Studies

Robin Cooper-Hannan, John W. Harbell, Sandra Coecke, Michael Balls, Gerard Bowe, Miroslav Cervinka, Richard Clothier, Frauke Hermann, Lynn K. Klahm, Jan de Lange, Manfred Liebsch and Philippe Vanparys

This is the report of the thirty-seventh of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals.
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The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status

Sandra Coecke, Vera Rogiers, Martin Bayliss, José Castell, Johannes Doehmer, Gérard Fabre, Jeffrey Fry, Armin Kern and Carl Westmoreland

In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of interest. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sandwich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process.
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In Vitro Alternatives and Phototoxicity Testing. II. Effects of Reactive Oxygen Species in In Vitro Phototoxicity Assays

Yuuko Okamoto, Akemi Ryu and Kenji Ohkoshi

The effects of reactive oxygen species (including singlet oxygen) in two in vitro phototoxicity assays — the 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) assay and the photohaemolysis assay — were assessed by using scavengers. Fifteen test substances, which had previously been shown to be phototoxic in vitro, were assessed. Eleven of these produced singlet oxygen. The major factor in the photodynamic reaction of bithionol was thought to be a Type I reaction, because bithionol did not produce singlet oxygen and did not react to histidine. Acridine was regarded as a Type II substance, because of the evident effect of histidine as a scavenger. 8-Methoxypsoralen and 5-methoxypsoralen produced singlet oxygen, but their actions were not affected by the scavengers. In this study, we confirmed that reactive oxygen species have great effects in in vitro phototoxicity, and that the 3T3 NRU PT assay can be used to detect effects which are thought to be the direct reaction of an excited photosensitiser to biological substrates (Type III reaction), for example, 8-methoxypsoralen. Therefore, we suggest that photohaemolysis and phototoxicity could be used to evaluate the photodynamic mechanisms of photosensitising chemicals.

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In Vitro Alternatives and Phototoxicity Testing. I. Evaluation of In Vitro Phototoxicity Assays

Yuuko Okamoto, Akemi Ryu and Kenji Ohkoshi

Three in vitro phototoxicity assays — the photohaemolysis assay, the haemoglobin (Hb) photo-oxidation assay, and the 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) assay — were evaluated for use as screening methods in predicting the phototoxicity of test substances. Twenty-seven test substances, including cosmetic ingredients and drugs, were assessed in this study. The phototoxicity predicted in each assay was compared with that in guinea-pigs. In total, nine phototoxic substances used in this study could be detected by some of the in vitro phototoxicity assays. Eleven test substances regarded as non-phototoxic in vivo were predicted as non-phototoxic in the in vitro assays. Of the eight false positives revealed in some of the in vitro assays, five are believed to be photo-allergens. This suggests that in vitro phototoxicity assays might not be able to discriminate clearly between in vivo phototoxicity and in vivo photo-allergy, because the mechanisms in both processes are based on photodynamic reactions. The predictability of the three in vitro assays was comparatively good, but positive predictive values were low. The equivalence values of the photohaemolysis assay, the Hb photo-oxidation assay and the 3T3 NRU PT assay were 81%, 70% and 70%, respectively. From these results, we suggest that the three in vitro phototoxicity assays could be used as screens in predicting phototoxicity.
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An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group

Toshiyuki Tsuchiya, Makoto Umeda, Hiroshi Nishiyama, Isao Yoshimura, Shozo Ajimi, Masumi Asakura, Hiroshi Baba, Yasuaki Dewa, Youji Ebe, Yuichi Fushiwaki, Shuichi Hamada, Tetsuo Hamamura, Makoto Hayashi, Yumiko Iwase, Yoshitsugu Kajiwara, Yasushi Kasahara, Masayoshi Kawabata, Emiko Kitada, Kinya Kubo, Kaori Mashiko, Daisaku Miura, Fukutaro Mizuhashi, Fumio Mizuno, Madoka Nakajima, Yoshiyuki Nakamura, Naoko Nobe, Hidetoshi Oishi, Erina Ota, Ayako Sakai, Miho Sato, Sawako Shimada, Toshie Sugiyama, Chitose Takahashi, Yuko Takeda, Noriho Tanaka, Chikako Toyoizumi, Takeki Tsutsui, Shinobu Wakuri, Satoshi Yajima and Nobuhiro Yajima

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin"transferrin" ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1µg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2µg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.
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Letter from CAAT

Lisa A. Libowitz

Despite protests from the animal rights movement, the US Government’s plan to gather information on 2800 top-selling chemicals moved forward throughout the spring and early summer, as did the campaign to promote the use of the Three Rs in obtaining the data.
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Conference Reports

Elizabeth Jenkins

This joint meeting of the International Council for Laboratory Animal Science (ICLAS) and the Federation of European Laboratory Animal Science Associations (FELASA) was held in Palma, Majorca, on 26–28 May 1999.
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