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 inter-laboratory 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 pairwise 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.

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The cytotoxicity of 48 compounds included in the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) list was determined in cultures of rat hepatocytes, McCoy, and MDBK cells. The average minimum concentration of each compound inducing cytotoxicity was measured in each cell type. The cytotoxicity values were then compared with published oral LD50 values for rats and mice. The logarithmic transformation of in vivo toxic doses and the corresponding in vitro cytotoxic concentrations showed a statistically significant correlation between the in vitro and in vivo values. The results show that an accurate in vivo LD50 dose could be predicted from in vitro data for at least 75% of the selected compounds. It is hoped that this finding will not only stimulate others to pursue in vitro technique but will eventually lead to elimination of the in vivo LD50 test.

The multicentre evaluation of in vitro cytotoxicity (MEIC) study is a programme designed to evaluate the relevance of in vitro toxicity tests for predicting human toxicity, and is organised by the Scandinavian Society for Cell Toxicology. The project started in 1989 and is scheduled to be finished by June 1996. MEIC is a voluntary effort by international laboratories to test the same 50 reference chemicals in their own in vitro toxicity systems. At present, 31 laboratories have submitted results for the first 30 reference chemicals from a total of 68 in vitro cytotoxicity tests. In the definitive evaluation of the MEIC programme, these in vitro results will be compared with human lethal blood concentrations and other relevant acute systemic toxicity data, and the results will be published as a series of articles. This paper, which is the first article in this series, describes and analyses the methodologies used in the 68 tests. The origins and purities of the test chemicals, the biological systems and the toxicity endpoints are also discussed. Since MEIC is not centrally directed, the selection of tests was entirely dependent on the preferences of the individual laboratories. Thus, the collection of tests is not representative of the full range of existing in vitro toxicity tests. In our study, basal cytotoxicity tests and ecotoxicological tests are prevalent, while tests for toxicity to primary cultures of differentiated cells, measured by organotypic toxicity endpoints, are clearly under-represented.

The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme was set up to evaluate the relevance for human acute toxicity of in vitro cytotoxicity tests. At the end of the project in 1996, 29 laboratories had tested all 50 reference chemicals in 61 cytotoxicity assays. Five previous articles have presented the in vitro data and the human database to be used in the evaluation. This article presents three important parts of the final evaluation: a) a comparison of rat and mouse oral LD50 with human acute lethal doses for all 50 chemicals; b) a display of the correlations between IC50 (concentration causing 50% inhibition) values from all 61 assays and three independent sets of human acute lethal blood concentrations, i.e. clinical lethal concentrations, forensic lethal concentrations, and peak concentrations; and c) a series of comparisons between average IC50 values from ten human cell line 24-hour assays and human lethal blood concentrations. In the latter comparisons, results from correlations were linked with known human toxicity data for the chemicals, to provide an understanding of correlative results. This correlative/mechanistic approach had the double purpose of assessing the relevance of the in vitro cytotoxicities, and of testing a series of hypotheses connected with the basal cytotoxicity concept. The results of the studies were as follows. Rat LD50 predictions of human lethal dosage were only relatively good (R2 = 0.61), while mouse LD50s gave a somewhat better prediction (R2 = 0.65). Comparisons performed between IC50 values from the 61 assays and the human lethal peak concentrations demonstrated that human ceil line tests gave the best average results (R2 = 0.64), while mammalian and fish cell tests correlated less well (R2 = 0.52–0.58), followed by non-fish ecotoxicological tests (R2 = 0.36). Most of the 61 assays underpredicted human toxicity for digoxin, malathion, carbon tetrachloride and atropine sulphate. In the correlative/mechanistic study, the 50 chemicals were first separated into three groups: A = fast-acting chemicals with a restricted passage across the blood–brain barrier; B = slow-acting chemicals with a restricted passage across the blood–brain barrier; and C = chemicals which cross the blood–brain barrier freely, while inducing a non-specific excitation/depression of the central nervous system (CNS). The IC50 values for chemicals in group C were divided by a factor of ten to compensate for a hypothetical extra vulnerability of the CNS to cytotoxicity. Finally, the average human cell line IC50 values (24-hour IC50 for groups A and C, and after 48-hour for group B) were compared with relevant human lethal blood concentrations (peak concentrations for groups A and C, and 48-hour concentrations for group B). As a result, in vitro toxicity and in vivo toxicity correlated very well for all groups (R2 = 0.98, 0.82 and 0.85, respectively). No clear overprediction of human toxicity was made by the human cell tests. The human cell line tests underpredicted human toxicity for only four of the 50 chemicals. These outlier chemicals were digoxin, malathion, nicotine and atropine sulphate, all of which have a lethal action in man through interaction with specific target sites not usually found in cell lines. Potassium cyanide has a cellular human lethal action which cannot be measured by standard anaerobic cell lines. The good prediction of the human lethal whole-blood concentration of this chemical was not conclusive, i.e. was probably a “false good correlation”. Another two chemicals in group C resulted in “false good correlations”, i.e. paracetamol and paraquat. The comparisons thus indicated that human cell line cytotoxicities are relevant for the human acute lethal action for 43 of the 50 chemicals. The results strongly support the basal cytotoxicity concept, and further point to the non-specific CNS depression being the obligatory reaction of humans to cytotoxic concentrations of chemicals, provided that the chemicals are able to pass the blood–brain barrier.