An in vitro toxicity testing strategy for the classification and labelling of chemicals according to their potential acute lethal potency

Toxic. in Vitro Vol. 8, No. 4, pp. 847-850, 1994

Pergamon

0887-2333(94)E0080-D

Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00

A N IN VITRO TOXICITY TESTING S T R A T E G Y FOR THE CLASSIFICATION AND LABELLING OF CHEMICALS ACCORDING TO THEIR POTENTIAL ACUTE LETHAL POTENCY H. SEIBERT, M. GOLDEN and J.-U. Voss Universit/it Kiel, Institut ffir Toxikologie, Zelltoxikologie, Weimarer Strasse 8, Haus 3, D-24106, Germany Abstract--An in vitro test battery is described which consists of five systems using different types of cells: (1) bovine sperm cells, (2) Balb/c 3T3 cells, (3) primary cultures of rat hepatocytes, (4) primary cultures of rat muscle cells, and (5) co-cultures of microcarrier-attached rate hepatocytes and Balb/c 3T3 cells. This combination of in vitro systems covers various aspects of cellular toxicity and permits determination of the intrinsic activity of chemicals with respect to general cytotoxicity, selective cytotoxicity and interference with selected cell-specific functions (Seibert et al., 1992). During the current phase of evaluation the different test systems are used in parallel, resulting in in vitro toxicity profiles which are the basis (a) for interpretation with respect to toxic potential, and (b) for the selection of appropriate assays for inclusion in a hierarchical approach to testing. Based on the experience with this test battery, a preliminary stepwise approach is proposed for the classification of chemicals according to their acute lethal potency. The principle steps and candidate tests are: (1) determination of cytotoxic activity (cytolethal and cytostatic)~sperm cells, 3T3 cell line; (2) determination of hepatocyte-specific cytotoxicity and of the role of bioactivation for cytotoxic activity---co-cultures of hepatocytes and 3T3 cells; and (3) determination of the potential of chemicals to interfere with electrically excitable membranes--muscle cell cultures.

Introduction One of the purposes of acute toxicity testing in laboratory animals is to obtain quantitative data for the classification and labelling of new chemicals. The question of whether the number of animals used for this type of toxicity testing could be significantly reduced by the use of in vitro systems is still open. Since acute systemic toxicity can be caused by various mechanisms, testing for basal cytotoxic potency in vitro cannot be sufficient to predict acute lethal doses in vivo. In principle, cellular in vitro systems permit determination of the intrinsic activity of chemicals with respect to general cytotoxicity, selective cytotoxicity, and interference with cell-specific functions (Seibert et al., 1992).

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(3)

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Components of the in vitro test battery A battery of in vitro tests has been established which at present consists of five different systems which are outlined below:

(5)

(1) Bovine spermatozoa. These are used in a shortterm test (exposure time; 1 hr). The endpoints Abbreviations: CK = creatine kinase; EOD = 7-ethoxycoumarin O-deethylation; LDH = lactate dehydrogenase; PCP = penta-chlorophenol.

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used are swimming activity and energy status monitored by determination of ATP contents (Seibert et al., 1989). Balb/c 3T3 cells. The potency of chemicals to inhibit cell proliferation is determined after 24 hr of exposure. The parameters measured are neutral red accumulation and protein content. Primary cultures o f rat hepatocytes. The exposure times used are 1 and 24 hr. After 1 hr ATP contents and the biotransformation of 7-ethoxycoumarin (EOD) are determined. After 24 hr EOD activity is determined again, and leakage of lactate dehydrogenase (LDH) and protein content are also measured as indicators of cytolethal action and cell detachment, respectively (Aschmann et al., 1989). Primary cultures o f rat muscle cell. The exposure times used are I and 24 hr. After I hr, contractility and leakage of creatine kinase (CK) are determined. After 24 hr, contractility, loss of CK and glucose consumption are measured (Giilden et al., 1994). Co-cultures o f microcarrier-attached rat hepatocytes and Balb/c 3T3 cells. Hepatocytes attached to microcarriers are transferred in inserts to culture dishes with growing 3T3 cells. After exposure of the co-cultures for 24 hr the hepatocytes are withdrawn and EOD activity and LDH content are determined. Growth of

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H. SEIBERTet al. Mot1 ATP1 Mot2 ATP2

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Fig. 1. In vitro toxicity profile of pentachlorophenol. Mot, ATP = sperm motility and ATP-content with glucose added (I) or at inhibited glycolysis (2); NRi = cell growth (neutral red uptake); Proti --- cell growth (protein content); EOD = 7-ethoxycoumarin O-deethylation; ATP = ATP content; LDH = leakage of lactate dehydrogenase; Prot = hepatocyte protein; Contr = spontaneous contractility; CK =creatine kinase loss; Glu-i, Glu-d = increase or decrease of glucose consumption. Columns with arrows indicate the highest concentration tested that did not cause half maximum response.

the sperm assay, the decrease in A T P levels in hepatocytes, and the increase in glucose consumption of muscle cells, it is obvious that this compound interferes with mitochondrial A T P supply. PCP is an uncoupler of mitochondrial phosphorylation. A completely different profile results for a compound like nicotine. With the exception of contractility of muscle cells, no response is seen with any of the endpoints even at very high concentrations. Nicotine

3T3 cells is determined by measuring the protein content (Voss and Seibert, 1991 and 1992). In eitro

toxicity profiles

Figures 1 and 2 present examples for the results that are obtained with this battery of in v i t r o systems. The toxicity profile of pentachlorophenol (PCP) indicates that in this case the cytotoxic activity is dominating. F r o m the results of

Mot1

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Fig. 2. In vitro toxicity profile of nicotine. (For abbreviations see legend to Fig. 1.)

Classification by acute lethal potency

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Physicochemical data 1 r

I First order tests

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classification

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Second order tests

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classification

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if the resultsindicatethe lowesttoxicity class

l Limited in vivo confirmation: Fixed-dose procedure of the BTS ( 500 mg/kg; 2000 mg/kg ) Fig. 3. Tier scheme for classification of chemicals according to their potential acute lethal potency. is an example of a compound with a low cytotoxic activity, but a high activity for interference with electrically excitable membranes. Results from the co-culture system are not included in the profiles shown because up until now only model compounds have been studied. However, the results obtained indicate that this system is well suited to detect both toxicity towards hepatocytes and the role of biotransformation in cytotoxic activity using proliferating target cells (Voss and Seibert, 1991 and 1992).

(Pow x V Lb) + Vw.b Dealt ----"Ca (~ow-x ~ Vw.t The lipid and water compartments for the model body were assumed to be 0.1 and 0.6 iitre/kg, respectively; the corresponding compartments in vitro were estimated to be 0.0003 and 1 litre/litre, respectively (M. Giilden et aL, unpublished data, 1993). After conversion, calculated EDs0 values can be compared with LDs0 values and the same toxicity classification scheme can be used for in vitro and in vivo data.

In vivo /in vitro comparisons

Proposal for an integrated approach for toxicity testing

The usual way to judge the relevance of in vitro data is to compare ECs0 values with LDs0 values. A major problem with this type of comparison is that nominal in vitro effective concentrations are compared with in vivo lethal doses and that basic differences in toxicokinetic parameters between the in vitro and the in vivo situation are not taken into account. To enable a more reasonable comparison we have developed the following equation, which represents a first attempt to convert nominal in vitro concentrations (C.) into equivalent doses (Dcalc) for a model body, taking into account the lipophilicity of compounds (octanol-water partition coefficient, Pow) and the relative volumes of lipid and water compartments in vivo (VL.b; Vw,b) and in vitro (VL,t; Vw,t).

Meanwhile numerous model compounds, as well as reference chemicals of the MEIC project (Bondesson et el., 1989) have been studied (Giilden et el., 1994; Seibert et al., 1992). On the basis of the results of quantitative in vivo/in vitro comparisons as outlined above the following principal steps of in vitro toxicity testing can be defined. First order tests. These are used to determine basal cytotoxic activity (i.e. potency for cytolethal action and cell growth inhibition). Candidate systems are spermatozoa and a proliferating cell line (e.g. Balb 3T3 cells). Second order test. This is used to detect hepatocyte specific cytotoxicity and the role of biotransformation for cytotoxic activity. The candidate test system

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H. SEIBERT el al.

is represented by co-cultures of microcarrier-attached rat hepatocytes a n d 3T3 cells. Third order tests. These will provide i n f o r m a t i o n concerning further selective cytotoxic activities a n d interferences with specific, non-vital cell functions. A t present the battery only contains muscle cell cultures as representative of this g r o u p of tests. Contractility as an e n d p o i n t allows the detection o f chemical interference with electrically excitable m e m b r a n e s . F u r t h e r test systems could be added to this step, provided that they have been d e m o n s t r a t e d to add relevant a n d necessary data. Figure 3 presents a proposal for a practical decision-tree a p p r o a c h to toxicity testing for the classification of chemicals. Briefly, testing o f a chemical could be performed in the following way. First order tests are conducted as described. Their results are converted to effective doses as outlined above. If the result is positive, then the c o m p o u n d must be classified as very toxic, and no further testing needs to be done. If not, the second order test has to be performed. If its result is positive, testing is stopped at this stage, If not, third order tests have to be performed. According to their results the chemical is classified as very toxic, toxic, or harmful. If the results indicate the lowest toxicity class (i.e. unclassified), a limited in vivo confirmation must be done. A n appropriate m e t h o d could be the fixed dose procedure o f the BTS (van den Heuvel et al., 1990). These animal studies are mainly to ascertain that no severe underestimation o f toxic potency has occurred, which could be the case if the chemical tested acts by a m e c h a n i s m that is not covered by the in vitro test battery or if toxicokinetics in vivo exert an unexpected influence. However, a m o n g the first 30 M E I C chemi-

cals to be tested, there has not been one case of such a severe underestimation (Gfilden et al., 1994).

REFERENCES

Aschmann C., Stork T. and Wassermann O. (1989) Shortterm effects ofchlorophenols on the function and viability of primary cultured rat hepatocytes. Archives of Toxicology 63, 121-126. Bondesson I., Ekwall B., Hellberg S., Romort L., Stenberg K. and Walum E. (1989) MEIC--a new international multicenter project to evaluate the relevance of human toxicity of in vitro cytotoxicity tests. Cell Biology and Toxicology 5, 331-347. Giilden M., Seibert H. and Voss J.-U. (1994) In vitro toxicity screening using cultured rat skeletal muscle cells. II. Agents affecting excitable membranes. Toxicology in Vitro 8, 197-206. Gfilden M., Seibert H. and Voss J.-U. (1994) The use of cultured skeletal muscle cells in testing for acute systemic toxicity. Toxicology in Vitro 8, Seibert H. Gfilden M., Kolossa M. and Schepers G. (1992) Evaluation of the relevance of selected in vitro toxicity test systems for acute systemic toxicity. A TLA 20, 240-245. Seibert H., Kolossa M. and Wassermann O. (1989) Bovine spermatozoa as an in vitro model for studies on the cytotoxicity of chemicals: effects of chlorophenols. Cell Biology and Toxicology 5, 315-330. van den Heuvel M. J., Clark D. G., Fielder R. J., Koundakjian P. P., Oliver G. J. A., Pelling D., Tomlinson N. J. and Walker A. P. (1990) The international validation of a fixed-dose procedure as an alternative to the classical LDs0 test. Food and Chemical Toxicology 28, 469482. Voss J.-U. and Seibert H. (1991) Microcarrier-attached rat hepatocytes as a xenobiotic metabolizing system in cocultures. Cell Biology and Toxicology 7, 387-399. Voss J.-U. and Seibert H. (1992) Toxicity of glycols and allyl alcohol evaluated by means of co-cultures of microcarrier-attached rat hepatocytes and BALB/c 3T3 cells. A TLA 20, 266-270.