ATLA::Alternatives to Laboratory Animals

Volume 26, Supplement 1

MEIC Evaluation of Acute Systemic Toxicity

Part IV In Vitro Results from 67 Toxicity Assays Used to Test Reference Chemicals 31-50 and a Comparative Cytotoxicity Analysis

ATLA 26, 93-129, March/April 1998

Cecilia Clemedson,¹ Marianee Andersson,² Yasunobu Aoki,³ Frank A. Barile,⁴ Anna Maria Bassi,⁵ Marbel C. Calleja,⁶ Argelia Castano,⁷ Richard H. Clothier,⁸ Paul Dierickx,⁹ Barbro Ekwall,¹ Margherita Ferro,⁵ Geirid Fiskesjö,¹⁰ Lourdes Garza-Ocañas,¹¹ Maria José Gómez-Lechón,¹² Michael Gülden,¹³ Tony Hall,¹⁴ Koichi Imai,¹⁵ Boris Isomaa,^L6 Anne Kahru,¹⁷ Gustaw Kerszman,^L8 Per Kjellstrand,¹⁹ Udo Kristen,²⁰ Manabu Kunimoto,³ Sirpa Kärenlampi,²¹ Lillemor Lewan,² Henrik Lilius,¹⁶ Anatoly Loukianov,²² Francesca Monaco,²³ Tadao Ohno,²⁴ Guido Persoone,⁵ Lennart Romert,²⁵ Thomas W. Sawyer,²⁶ Helmut Segner,²⁷ Hasso Seibert,¹³ Ravi Shrivastava,²⁸ Michael Sjöström,²⁹ Annalaura Stammati,³⁰ Noriho Tanaka,³¹ Ann Thuvander,³² Oscar Torres-Alanis,¹¹ Matteo Valentino,²³ Shinobu Wakuri,³¹ Erik Walum,²⁵ Xianhai Wang,²⁴ Anders Wieslander,¹⁹ Flavia Zuuco³³ and Björn Ekwall¹

¹CTLU, Pavals, När, 620 13 Stånga, Sweden; ²Department of Animal Physiology, University of Lund, Helgonavägen 3D, 223 62 Lund, Sweden; ³Environmental Health Sciences Division, National Institute for Environmental Studies, (NIES), 16-Z Onogawa, Tsukuba, Ibaraki 305, Japan; ⁴Department of Natural Sciences, York College, City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, New York, NY 11451, USA; ⁵Institute of General Pathology, Via L.B. Alberti, 2, 16132 Genova, Italy; ⁶Laboratory for Biological Research in Aquatic Pollution, University of Ghent, 22 J Plateaustraat, 9000 Ghent, Belgium; ⁷Environmental Toxicology, Centro de Investigacion en Sanidad Animal, Valdeolmos, 28130 Madrid, Spain; ⁸School of Biomedical Sciences, Queen's Medical Centre, Nottingham NG7 2UH, UK; ⁹Instituut voor Hygiene en Epidemiologie, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium; ¹⁰Department of Genetics, University of Lund, Sölvegatan 29, 223 62 Lund, Sweden; ¹¹Departement de Farmacologia y Toxicologia, Facultad de Medicina, Universidad Autonoma de Nuevo Leon, Apartado Postal No. 146, Col. del Valle, Nuevo Leon, Mexico; ¹²Cultivos Celulares, Centro Investigacion, Hospital La Fe, Av. Campanar 21, 46009 Valencia, Spain; ¹³Institut für Toxikologie, Zelltoxikologie, Christian-Albrechts-Universität, Weimarer Strasse 8, Haus 3, 24106 Kiel, Germany; ¹⁴MD Laboratories, BP 30, 68870 Bartenheim, France; ¹⁵Department of Biomaterials, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka 573, Japan; ¹⁶Department of Biology, Åbo Akademi University, Biocity, Artillerigatan 6, 20520 Åbo, Finland; ¹⁷Laboratory for Molecular Genetics, Institute of Chemical Physics & Biophysics, Estonian Academy of Sciences, Akadeemia tee 23, 0026 Tallinn, Estonia; ¹⁸Institute for Life Science and Chemistry, University of Roskilde, P.O. Box 260, 4000 Roskilde, Denmark; ¹⁹Gambro AB, Box 10101, 220 10 Lund, Sweden; ²⁰Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany; ²¹Department of Biochemistry and Biotechnology, University of Kuopio, P.O. Box 1627, 702 11 Kuopio, Finland; ²²Centre for the Ethical Treatment of Animals (CETA), 39-3-23, Volzsky Bulvar, 109462 Moscow, Russia; ²³Universitá di Ancona, Clinica del Lavoro, Ospedale Regionate, 60020 Ancona, Italy; ²⁴RIKEN Cell Bank, Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba City, Ibaraki 305, Japan; ²⁵Pharmacia & Upjohn Co., 112 87 Stockholm, Sweden; ²⁶Biomedical Defence Section, Defence Research Establishment Suffield - DRES, Box 4000, Medicine Hat, Alberta T1A 8K6, Canada; ²⁷Umweltforschungs-zentrum Leipzig-Halle GmbH - UFZ, Sektion für Chemische Ökotoxikologie, PF2, 04301 Leipzig, Germany; ²⁸Vitro-Bio, Biopôle Clermont Limaque, 63360 St. Beauzire, France; ²⁹Research Group for Chemometrics, Department of Organic Chemistry, Umeå University, 901 87 Umeå, Sweden; ³⁰Department of Comparative Toxicology and Ecotoxicology, Instituto Superiore di Sanita, Viale Regina Elena 299, 00161 Roma, Italy; ³¹Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257, Japan; ³²National Food Administration, Box 622, 751 26 Uppsala, Sweden; ³³Istituto Tecnologie Biomediche, Consiglio Nationale delle Recherche, Via G.B. Morgagni 30/E, 00161 Roma, Italy

SUMMARY

Results from tests on the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals 31-50 in 67 different in vitro toxicity assays are presented in this paper as a prerequisite to in vitro/in vivo comparisons for all MEIC in vitro toxicity data in forthcoming papers, i.e. the final MEIC evaluation of the relevance of the tests. With the aim of increasing knowledge about the relative significance of some in vitro methodological factors, the strategies and methods of the preceding parts in the MEIC series (Parts II and III) were again employed to enable comparative cytotoxicity analysis of the new in vitro results presented in this paper. A principal components analysis (PCA) of the results from tests of the 20 chemicals in 67 assays demonstrated a dominating first component describing as much as 74% of the variance in the toxicity data, indicating a similar ranking of the cytotoxicities of the chemicals in most of the tests. The influence on the general variability of the results of a few, key methodological factors was also evaluated by using linear regression comparisons of the results of all pairs of methods available in the study, i.e. methods which were similar in all respects except for the factor being analysed. Results from this "random probe" analysis were: a) the cytotoxicities of 11 of the 20 chemicals increased considerably with exposure time (> 10 times over 4-168 hours); b) in general, human cell line toxicity was well predicted by cytotoxicity in animal cells; c) prediction of human cell line toxicity by most ecotoxicological tests was only fairly good; d) 14 comparisons of similar assays with different cell lines showed similar toxicities (mean R² = 0.83); e) nine comparisons of similar assays employing different primary cultures and cell lines shared similar toxicities (mean R² = 0.71), and f) 16 comparisons of similar assays with different growth/viability endpoints showed similar toxicities (mean R² = 0.71). Results b, d, e and f must contribute to the PCA-documented high general similarity of the in vitro toxicity data. Results a and c, together with factors which were not analysed, such as different protocols and interlaboratory variability of tests, could explain the 26% dissimilarity. To provide background information to the planned final MEIC evaluation of the relevance of the 61 methods in which all 50 chemicals have been tested, an additional PCA was made of the 50 chemical-61 assay in vitro database (from Parts II and III and the present paper). This supplementary PCA demonstrated an 80% similarity of results. Compared with the previous analysis of the tests of the first 30 MEIC reference chemicals (MEIC Part III), the present analysis of the tests of the last 20 MEIC chemicals indicates a somewhat higher variation in the results. Correspondingly, some deviating endpoint measurements and cell line responses were demonstrated by the pair-wise comparisons in the present study. As a result, the analysis revealed a high correlation (R² = 0.73) between the average human cell line toxicity and the results from a new protein denaturation test. These preliminary results suggest that intracellular protein denaturation may be a frequently occurring mechanism in basal cytotoxicity.

Keywords: alternatives, basal cytotoxicity, comparative cell toxicology, cultured cells, cytotoxicity, differential cytotoxicity, in vitro toxicology, MEIC, multivariate analysis, protein denaturation, toxicity testing, validation