ATLA::Alternatives to Laboratory Animals

Volume 26, Supplement 1

MEIC Evaluation of Acute Systemic Toxicity

Part III In Vitro Results from 16 Additional Methods Used to Test the First 30 Reference Chemicals and a Comparative Cytotoxicity Analysis

ATLA 26, 93-129, March/April 1998

Cecilia Clemedson,¹ Frank A. Barile,² Barbro Ekwall,¹ Maria José Gomez Lechón,³ Tony Hall,⁴ Koichi Imai,⁵ Anne Kahru,⁶ Pam Logemann,⁷ Francesca Monaco,⁸ Tadao Ohno,⁹ Helmut Segner,¹⁰ Michael Sjöström, Matteo Valentino,⁸ Erik Walum,¹² Xianhai Wang⁹ and Björn Ekwall¹

¹CTLU, Pavals, När, 620 13 Stånga, Sweden; ²Department of Natural Sciences, York College, City University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, NY 11451, USA; ³Cultivos Celulares, Centro Investigacion, Hospital La Fe, Av. Campanar 21, 46009 Valencia, Spain; ⁴MD Laboratories, B.P. 30, 68870 Bartenheim, France; ⁵Department of Biomaterials, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka 573, Japan; ⁶Laboratory for Molecular Genetics, Institute of Chemical Physics & Biophysics, Estonia Academy of Sciences, Akadeemia tee 23, EE-0026 Tallinn, Estonia; ⁷Advanced Tissue Sciences, 10933 North Torrey Pines Road, La Jolla, CA 92037, USA; ⁸Universita' di Ancona, Clinica del Lavoro, Ospedale Regionale, 60020 Ancona, Italy; ⁹RIKEN Cell Bank, Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba City, Ibaraki 305, Japan; ¹⁰UFZ - Umweltforschungszentrum, Leipzig-Halle GmbH, Sektion Chemische Ökotoxikologie, PF 2, 04301 Leipzig, Germany; ¹¹Research Group for Chemometrics, Department of Organic Chemistry, Umeå University, 901 87 Umea, Sweden; ¹²Pharmacia & Upjohn, 112 87 Stockholm, Sweden

SUMMARY

Results from tests on the first 30 MEIC reference chemicals in 16 different systems are presented as a prerequisite to the subsequent in vitro/in vivo comparisons of acute toxicity data, i.e. the final MEIC evaluation of all test results of the study. The study is a supplement to the previously published results from 68 methods (including methods 45B and 46B [old numbers]) used to test the same set of chemicals. The strategies and methods of the preceding paper were employed to enable a comparative cytotoxicity analysis of the results from these 68 methods and from the 16 new methods to be made. Principal components analysis (PCA) of 82 assays demonstrated a dominating first component which described as much as 83% of the variance in the toxicity data. This remarkable similarity of all toxicity data was the main finding of the present study, and confirmed the results of the previous study with a less-extensive database. Also, the influence on the general variability of results of several key methodological factors was evaluated by analysis of selected sets of data, including linear regression of the results of pairs of methods, which were similar in all respects except for the factor under analysis. This analysis of the same 82 assays as before also confirmed previous results from the 68 assay database: a) the toxicities of a third of the chemicals increased considerably with exposure time; b) in general, cytotoxicity for human cells was well predicted by cytotoxicity tests with animal cells; c) this prediction was poor for two chemicals, i.e. digoxin and malathion; d) prediction of human cytotoxicity by ecotoxicological tests was only fairly good; e) 25 comparisons of similar assays employing different cell lines showed strikingly similar toxicities (mean R² = 0.86); f) 22 comparisons of similar pairs of assays employing different primary cultures and cell lines also revealed similar toxicities (mean R² = 0.79); and g) 15 comparisons of similar assays with different growth/viability endpoint measurements demonstrated strikingly similar toxicities (mean R² = 0.89). Results b, e, f, and g must be the main causes of the general similarity of results, while results a, c, and d, together with other factors, could explain the 20% dissimilarity. These findings support the basal cytotoxicity concept and may assist in guiding and refining in vitro toxicity testing in the future.

Keywords: alternatives, basal cytotoxicity, biostatistics, comparative cell toxicology, cultured cells, cytotoxicity endpoints, differential cytotoxicity, evaluation of in vitro cytotoxicity, MEIC, multivariate analysis, organ-specific toxicity, toxicity testing, validation