Björn Ekwall

MEIC—A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests

A new international project to evaluate the relevance for human systemic and local toxicity of in vitro tests of general toxicity of chemicals has been organized by the Scandinavian Society of Cell Toxicology under the title 1Multicenter Evaluation of In Vitro Cytotoxicity (MEIC). The basic assumptions underlying the project, as well as the practical goals and the design of the program are outlined. The list of the first 50 reference chemicals is presented. The chemicals are an otherwise unbiased selection of compounds with known human acutely lethal dosage and blood concentrations, including LD50-values in the rat or mouse. Most agents also have other data on human toxicity and toxicokinetics, including more extensive animal toxicity data. International laboratories already using or developing in vitro tests of various partial aspects of general toxicity are invited to test the substances, the results of which will be evaluated by us. The predictivity of the in vitro results for both partial and gross human toxicity data will be determined with combined use of univariate regression analysis and soft multivariate modeling. The predictivity of the in vitro results will be compared with the predictivity of conventional animal tests for the same chemicals. Finally, batteries of tests with optimal prediction power for various types of human toxicity will be selected. The need for and possible uses of such batteries are discussed.TABLE 1The First 50 Reference Chemicals of the MEIC Project1. Acetaminophen26. Arsenic trioxide2. Aspirin27. Cupric sulfate3. Ferrous sulfate28. Mercuric chloride4. Diazepam29. Thioridazine HCl5. Amitriptyline30. Thallium sulfate6. Digoxin31. Warfarin7. Ethylene glycol32. Lindane8. Methyl alcohol33. Chloroform9. Ethyl alcohol34. Carbon tetrachloride10. Isopropyl alcohol35. Isoniazid11. 1,1,1-Trichloroethane36. Dichloromethane12. Phenol37. Barium nitrate13. Sodium chloride38. Hexachlorophene14. Sodium fluoride39. Pentachlorophenol15. Malathion40. Verapamil HCl16. 2,4-Dichlorophenoxyacetic acid41. Chloroquine pholphate17. Xylene42. Orphenadrine HCl18. Nicotine43. Quinidine sulfate19. Potassium cyanide44. Diphenylhydantoin20. Lithium sulfate45. Chloramphenicol21. Theophylline46. Sodium oxalate22. Dextropropoxyphene HCl47. Amphetamine sulfate23. Propranolol HCl48. Caffeine24. Phenobarbital49. Atropine sulfate25. Paraquat50. Potassium chloride

Comparison of in vivo acute lethal potency and in vitro cytotoxicity of 48 chemicals

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 LDS p 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 LDS p 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.

Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells. I. Comparative in vitro cytotoxicity of 27 drugs.

Combined drug toxicity to HeLa cells was studied in vitro with use of the microtitre MIT-24 test system. Whether drug toxicity to HeLa cells is representative of drug toxicity to other cultivated cells was investigated by a comparison of the MIT-24 toxicity of 27 drugs to HeLa cells with their toxicity to various permanent cell lines and more differentiated primary cell cultures as reported in the literature, together with original recordings of the MIT-24 toxicity of 9 of the drugs to human fetal kidney cells. A similarity of drug toxicity to all cell types was found. Thus the MIT-24 recordings may be representative of a basal drug cytotoxicity, probably corresponding to local drug irritation and to causal systemic drug toxicity.

Toxicity to HeLa cells of 205 drugs as determined by the metabolic inhibition test supplemented by microscopy

The toxicity of 205 drugs to HeLa cells was evaluated by the Metabolic Inhibition Test supplemented by microscopy. Two end points of cyto-inhibition were estimated; total and partial inhibition after 24 h, based on absence or scarcity of spindle-shaped cells, respectively, and total and partial inhibition after 7 days, based on different degrees of basic pH change of the phenol red included in the cell medium. Direct drug-induced pH changes and precipitates in the cultures were also recorded. Many drugs were found to induce a culture zone with a particularly low pH after 7 days of incubation, at concentrations below the cyto-inhibitory concentration. Forty-three drugs regularly caused this hyperacid reaction, while 44 drugs caused the reaction irregularly. Since the reaction was always continuous with the cyto-inhibitory zone, it was provisionally judged to represent excitatory cell injury. Since many of the drugs which regularly induced the reaction are also known to induce proliferation of the endoplasmic reticulum in various cells, the reaction may be related to this and allied effects. Many drugs that are known to accumulate in cells displayed a high inhibitory toxicity, which might have been due to the paucity of cells in the test system. However, the high 7-day inhibitory toxicity shown by antineoplastic and some anti-inflammatory drugs, including triamterene and disulfiram, may constitute a genuine antimetabolic drug action. Seven grand mal antiepileptics were only very slightly toxic as compared with their precipitating tendency, which may be of significance in their therapeutic action.

Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells III. Lethal action to man of 43 drugs related to the HeLa cell toxicity of the lethal drug concentrations

The human lethal plasma concentrations of 46 drugs were divided by their IC50 for HeLa cells in vitro to make up a series of cytotoxic quotients (CQLv). CQLv was then compared with the recorded lethal action to man of 43 of the drugs. While the 7 drugs with the lowest CQLv values produce a non-cytotoxic interference with neuro-transmission, most of the remaining 36 drugs have a known local or systemic cytotoxicity to man. A majority of the 36 drugs induces a non-specific central nervous system (CNS)-depression at lethal dosage, intermingled with function loss from organs outside CNS in proportion to decreasing drug accumulation in CNS cells and increasing CQLv. The remaining drugs which do not penetrate CNS cells and at lethal dosage induce a widespread injury and function loss of tissues outside the CNS, have a CQLv near unity. Non-specific CNS-depression may thus be the primary human reaction to lethal systemic drug cytotoxicity, while widespread drug injury to various tissues outside CNS--conventionally considered to be cytotoxic in origin--may be the obligatory human reaction to drugs that do not penetrate cells well. The present findings indicate a relevance to human toxicity of the HeLa toxicity for most drugs.

Combined toxicity to HeLa cells of 30 drug pairs, studied by a two-dimensional microtitre method

Abstract The complete pattern of the cytotoxic interaction of two drugs may be obtained in a single test, by incubation of the drugs, diluted at a right angle to each other in the same area of a microtitration plate, with HeLa cells, grown at a density of 5.10 4 cells/ml in Parkers medium 199 plus 5% calf serum at 37°C for 7 days, and the recording of cyto-inhibition in all cups of the plate by microscopy after 24 h, supplemented by the study of pH-change of the medium after 7 days. Thirty drug combinations were tested by this method; most developed an additive pattern, while minor groups developed patterns of no interference, potentiative antagonism or supra-additive synergism.

Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells II. Drug toxicity in the MIT-24 system compared with mouse and human lethal dosage of 52 drugs

By a comparison of the 50% inhibitory concentration to HeLa cells in the microtitre Metabolic Inhibition Test supplemented by microscopy of cells after 24 h incubation (the MIT-24 test), of 52 drugs with their mouse i.v. LD50 and available approximate human lethal dosage, 7 were found to have a human LD considerably lower than HeLa IC50, indicating a lethal action to specialized functions of the human body not found in vitro, while 39 were found to have a gross similarity between the human LD and HeLa IC50.

Improved use of the metabolic inhibition test to screen combined drug toxicity to HeLa cells — preliminary study of 61 drug pairs

Abstract A modification of the Metabolic Inhibition Test was used to test the toxicity to HeLa cells of mixtures of two drugs, at equally toxic concentrations, as well as toxicity of the separate drugs in the combination. By a comparison of the toxicity of the combination to the toxicity of both separate drugs, a preliminary determination of the precise type of cytotoxic interaction could be made for 45 out of the 61 combinations, tested as a pilot study of the method. The majority of these 45 combinations interacted synergistically (77%). Used to screen supra-additive interaction, the method revealed 9 such interactions out of 57 combinations.

Preliminary results from the Scandinavian multicentre evaluation of in vitro cytotoxicity (MEIC)

The multicentre evaluation study of in vitro cytotoxicity tests (MEIC) is organized by the Scandinavian Society of Cell Toxicology. All interested laboratories are invited to test a published list of 50 reference chemicals in their various in vitro assays with a bearing on general toxicity. Submitted results will be centrally evaluated for their relevance to human toxicity, including a comparison with the efficiency of conventional animal tests. This brief communication presents the very first preliminary results of the study, that is, prediction of human acute lethal toxicity for the first 10 MEIC chemicals by all the results submitted to date, that is, five in vitro cytotoxicity assays. As a baseline for judging the efficiency of the cytotoxicity tests, rat and mouse LD(50) values were compared with human acute lethal dosage of the chemicals. Rat LD(50) prediction was relatively poor, but mouse LD(50) values correctly predicted the human lethal dose for six out of the 10 substances. A multivariate method of comparison including all cytotoxicity test results, predicted human lethal blood concentrations as well as the mouse LD(50) prediction of dosage. Since the blood concentrations used in the comparison were derived from human lethal dosage with the help of two simple pharmacokinetic factors (absorbed fraction in the intestine and distribution volume of chemicals), the cytotoxicity assays were found also to be able to predict human dosage, as well as did the mouse LD(50) prediction.

Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells. IV. Therapeutic effects and side effects of 50 drugs related to the HeLa toxicity of the therapeutic concentrations.

As a measure of the relevance to human drug effects of drug cytotoxicity in vitro, calculated and actual therapeutic blood concentrations of 50 drugs were divided by their 50% inhibitory concentrations for HeLa cells in vitro to make up three types of cytotoxic quotients (CQTv, CQTd, and CQTr). When the various quotients were compared with known cytotoxic effects of the drugs the parameters correlated well. While most drugs at single dosage reached concentrations with low quotients (CQTv and CQTd) corresponding to few reported cytotoxic effects and side effects, a substantial number of drugs at repeated dosage reached concentrations with high quotients (CQTr) corresponding to known or suspected cytotoxic action in man.