human toxicity

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MEIC Evaluation of Acute Systemic Toxicity

Björn Ekwall, Frank A. Barile, Argelia Castano, Cecilia Clemedson, Richard H. Clothie, Paul Dierickx, Barbro Ekwall, Margherita Ferro, Geirid Fiskesjö, Lourdes Garza-Ocañas, Maria José Gómez-Lechón, Michael Gülden, Tony Hall, Boris Isomaa, Anne Kahru, Gustaw Kerszman, Udo Kristen, Manabu Kunimoto, Sirpa Kärenlampi, Lillemor Lewan, Anatoly Loukianov, Tadao Ohno, Guido Persoone, Lennart Romert, Thomas W. Sawyer, Ravi Shrivastava, Helmut Segner, Annalaura Stammati, Noriho Tanaka, Matteo Valentino, Erik Walum and Flavia Zucco

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 cell 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.
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EDIT: A New International Multicentre Programme to Develop and Evaluate Batteries of In Vitro Tests for Acute and Chronic Systemic Toxicity

Björn Ekwall, Cecilia Clemedson, Barbro Ekwall, Patrik Ring and Lennart Romert

The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme provided a battery of three basal cytotoxicity tests with a good (R2 = 0.77) prediction of human acute lethal blood concentrations. The predictive power of this battery would be considerably improved by the addition of new supplementary in vitro tests. The development of these new tests will be facilitated by a close coupling of test development to evaluation. The Cytotoxicology Laboratory, Uppsala (CTLU), is therefore inviting all interested in vitro toxicologists to take part in the Evaluation-guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). All EDIT activities (subprojects) will be designed on a case-by-case basis, but will follow a common pattern. The CTLU will use the accumulated MEIC/EDIT data, and its experience from the previous MEIC evaluation, to suggest priority areas, i.e. the need for certain in vitro toxicity data/tests as supplements to existing in vitro models/batteries on human systemic toxicity. Detailed research programmes corresponding to these areas will be published on the Internet. The CTLU will also try to raise funds for some projects and will coordinate multilaboratory studies. Interested laboratories developing or already using priority tests are encouraged to join the subprojects and to test specific sets of substances (usually sets of MEIC reference chemicals) in their new assays. The CTLU will provide adequate human reference data and will also evaluate results as single components of complex models, together with the laboratory conducting the test. At present, ten priority areas have been identified: a) repeat dose toxicity in vitro; b) urgent mechanistic information from in vitro studies of protein denaturation, morphology of cell injury, differential toxicity between various rapidly measured endpoints (10–60 minutes) and 24-hour cytotoxicity, toxicity to aerobic cells, and discrimination between rapid and slow cytotoxic mechanisms; c) in vitro tests on vitally important, specific receptor toxicity in humans; d) excitatory cytotoxicity; e) reversibility of cell toxicity; f) in vitro tests on passage across the blood–brain barrier; g) in vitro tests on absorption in the gut; h) protein binding in vitro; i) in vitro tests on distribution volumes (Vd); and j) in vitro tests on biotransformation to more-toxic metabolites (hepatocytes plus target cells). This paper gives a short presentation of the rationale for each subproject and reports on ongoing activities.
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MEIC Evaluation of Acute Systemic Toxicity

Björn Ekwall, Barbro Ekwall and Michael Sjöström

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. A total of 61 assays were used to test all 50 reference chemicals. The results of all the tests and the human database were presented in the first five papers of this series. An evaluation of the relevance for human acute toxicity of all submitted test results with use of hard linear regression modelling was presented in the next two papers, and demonstrated a high relevance of in vitro tests, notably tests involving human cell lines. In the present study, multivariate partial least square (PLS) modelling with latent variables analysis has been used to reach two objectives. The first objective was to study the prediction of human acute toxicity by the 61 assays. The second objective
was to select a practical battery from the 61 assays, with an optimal prediction of lethal blood concentrations from human acute poisonings of the chemicals. A two-component PLS model of all 61 assays predicted three sets of lethal blood concentrations (clinical, forensic and peak concentrations) very well (R2 = 0.77, 0.76 and 0.83, Q2 = 0.74, 0.72 and 0.81, respectively), providing correlative evidence for a high relevance for human acute toxicity of most of the assays. The assays with human cells were highly predictive, whereas assays with very short incubation times and non-fish ecotoxicological assays were least predictive. These findings confirm the previous results from linear regression analysis. To select an optimal battery, 24 successive PLS models of in vitro data were compared with lethal peak concentrations. The battery selection was based on 38 chemicals with reliable and relevant lethal peak concentrations. An initial PLS model of all 61 assays was used to select the 15 most predictive and most distinct assays. Subsequent PLS models were used to measure the decrease in prediction when assays were deleted from the 15-test battery, as well as the increase in prediction when some extrapredictive assays (as identified by the deletion process) were added later to an optimal two-test battery. The most predictive three-test battery (R2 = 0.79 and Q2 = 0.78 for all 50 chemicals) included two circumstantial assays. The most predictive and most cost-effective battery consisted of three human cell line assays, with four endpoints and two exposure times, i.e. protein content (24 hours), ATP content (24 hours), inhibition of elongation of cells (24 hours), and pHchange (7 days). This 1, 5, 9, 16 battery exclusively measures basal cytotoxicity, and is highly predictive (R2 = 0.77 and Q2 = 0.76 for 50 chemicals) of the actual lethal peak blood concentrations from acute poisonings in humans. The battery prediction compares favourably with the prediction of human lethal dose by a PLS model of rat and mouse 50% lethal dose (LD50) values for the 50 chemicals (R2 = 0.65 and Q2 = 0.64). The three assays of the battery and other promising MEIC assays should be formally validated as soon as possible. The battery can be used immediately for several non-
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Development of an In Vitro Test Battery for the Estimation of Acute Human Systemic Toxicity: An Outline of the EDIT Project

Cecilia Clemedson, Marika Nordin-Andersson, Henning F. Bjerregaard, Jørgen Clausen, Anna Forsby, Helena Gustafsson, Ulrika Hansson, Boris Isomaa, Carsten Jørgensen, Ada Kolman, Natalia Kotova, Gunter Krause, Udo Kristen, Kalle Kurppa, Lennart Romert and Ellen Scheers

The aim of the Evaluation-guided Development of New In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R2 = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and
potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that
the development within the EDIT target organ programme should initially be focused on the nervous system.
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The Prediction of Human Acute Systemic Toxicity by the EDIT/MEIC In Vitro Test Battery: The Importance of Protein Binding and of Partitioning into Lipids

Cecilia Clemedson, Paul J. Dierickx and Michael Sjöström

The aim of the two studies presented in this paper was to further improve the predictability of the original Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vitro test battery for acute systemic toxicity. In the first study, whether a protein-free cytotoxicity assay could improve the prediction of human acute systemic toxicity was investigated. The cytotoxicity of 39 MEIC reference chemicals was measured by the neutral red uptake inhibition test after 30 minutes in phosphate-buffered saline (PBS), with hepatomaderived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Mercuric chloride and hexachlorophene were much more cytotoxic in PBS, as was the case, to a lesser extent, for seven other chemicals. Potassium cyanide and eight other chemicals were less cytotoxic in PBS than in complete culture medium, probably because of poor physiological conditions. The correlation between the cytotoxicity measured in PBS and human acute toxicity was rather low, but became of the same order as for other assays, when mercuric chloride and hexachlorophene were withdrawn from the comparison. In the second study, modelling of human lethal blood concentrations by using the results of the three cell line tests of the original MEIC test battery were complemented by logP (octanol–water partition coefficient) values. The introduction of logP into the modelling did not improve the correlations, but some improvement of both R2 and Q2 was obtained by expanding the logP values with logP2 values. The highest R2 (0.84) and Q2 (0.80) values were obtained for a model in which both experimental and calculated (ambiguous) logP values were used. When only experimental logP values were used, the corresponding values were 0.80 and 0.78. These two studies showed that including
protein binding and the partition of chemicals in the MEIC in vitro test battery is important, in order to improve the predictability of the results obtained.
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Prediction of Human Acute Toxicity by the Hep G2/24-hour/Total Protein Assay, with Protein Measurement by the CBQCA Method

Paul J. Dierickx

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r² = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method (r² = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.
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