Toxicological screening

REGULATORY

TOXICOLOGY

AND

PHARMACOLOGY

4,275-286

Toxicological G. ZBINDEN,

(1984)

Screening

J. ELSNER, AND U. A. BOEISTERLI

Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, Schwerzenbach, Switzerland

Received December 19, 1983

Well-defined dose- and time-related toxic effects of chemicals can often be detected using simple tests with small numbers of animals. The strategies for the establishment of toxicological screening tests are discussed. The most important steps are the definition of the targets, the selection of the methods, and the setting of the test criteria. Screening tests must then be validated with standard reference chemicals, and the test criteria must be adjusted so that the standards can be detected regularly. Maximal flexibility is allowed in the design of the tests. For evaluation, the results obtained in treated animals can be compared with those of controls, using conventional concepts of biostatistics. It is also possible to base the evaluation on preset test criteria. Toxicological screening tests do not replace conventional safety studies, but they help in selecting the most promising candidates in a series of related chemicals and in establishing priorities for further testing. As an example, a screening for the hemolytic effects of chemicals is presented.

1. INTRODUCTION In recent years mounting public concern about laboratory animal welfare has induced many toxicologists to think anew about the justification of their activities and to explore the possibilities for reducing the use of experimental animals. It is evident that considerations of human safety must remain the primary concern of toxicology. However, the question must be raised whether methods currently used for safety evaluation of chemicals represent the optimum, or whether equal or even better results could be obtained with fewer animals (Zbinden and FIury-Roversi, 1981). This paper was motivated by the experience that many relevant toxic effects of chemical substances can be readily recognized and measured in experimental models. If high precision of the dose-effect relationship is not essential, the experiments can be conducted with a surprisingly small number of subjects. It is often possible to design test procedures which reliably recognize adverse effects of standard reference substances, and to use these techniques to identify comparable properties in new compounds. Results from such pilot experiments can help in devising the most appropriate testing strategies and may also lead to the development 275 0273-2300/84 $3.00 Copyright Q 1984 by Academic Press. Inc. All rights of reproduction in any form rexwed.

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of a toxicological screening procedure. A significant reduction in the number of experimental animals can ensue, if the screening tests permit one to eliminate compounds identified as hazardous from further extensive investigation. 2. DEFINITION

AND

GOALS

OF THE

TOXICOLOGICAL

SCREENING

Pharmacologists use the term “screening” to describe the practice of subjecting new chemicals to a battery of tests, designed to detect therapeutically useful properties. What passes through this filter is discarded. Compounds which show an effect, however weak, are looked at as potentially interesting, and serve as “leads” for the synthesis of more potent or more specifically active derivatives. In a similar manner, toxicological screening tests must detect chemicals which possess undesirable properties. The procedures should also provide quantitative information so that the test compound may be compared with standard reference agents. If several related substances are investigated it should be possible to establish a ranking order for each of the toxicological qualities under study. In the case of environmental pollutants, by-products of industrial processes, and naturally occurring chemicals, toxicological screening serves as a means to detect potential hazards and helps in setting priorities for more extensive testing of the most dangerous representatives. Its results are also useful in planning the extensive toxicological studies conducted for the establishment of safety and for regulatory purposes. It is clear that screening procedures can only detect dose and time-related toxic reactions. They are not useful for the discovery of the so-called low-incidence responses where idiosyncratic and allergic reactions and genetically determined metabolic peculiarities of the subjects arc of primary importance (Plaa, 1978). Moreover, for the extrapolation of the results to man, the limitations and precautions imposed by species differences must be considered in the same manner as for conventional preclinical safety data. 3. SCREENING

STRATEGIES

In contrast to the standard studies which follow predetermined protocols, the toxicological screening uses a flexible approach. The researcher is free to look for as little or as much as he wishes, he alone decides what the purpose of the experiment shall be, and he determines the research strategy. The development of a screening test is a stepwise procedure which requires the following actions: Selection and definition Decision on strategy Selection of method Validation of method

of targets

3.1 Selection and Definition of Targets The need for a toxicological screening often emerges from the observation of an adverse effect of a chemical in man. In an effort to make new compounds which

TOXICOLOGICAL

SCREENING

277

are better tolerated, a biological test is developed and used as a screening device. An example would be an animal model to assessthe ulcerogenic effect of nonsteroidal anti-inflammatory drugs. However, the target must not necessarily be related to an established untoward effect in man. It can also be a biological property deemed to be potentially harmful based on experimental results, e.g., high affinity of chemicals to melanin pigment (Meier-Ruge, 1972).

3.2. Decision on Strategy There are usually many ways with which a toxicological response could be investigated. The possibilities range from whole animal models to in vitro biochemical assays. The screening strategy is mainly influenced by prior knowledge of the biological properties of the compounds submitted for study. If we deal with substances of which little, if anything, is known, we must primarily attempt to detect hazard, i.e., presence or absence of undesirable effects in one or more target organs. On the other hand, if we know more or less what to expect from a series of chemically related compounds a model designed to detect a defined toxic reaction can be developed. Experience shows that the selection of an appropriate testing approach is a key step in setting up a successful toxicological screening. In some cases it is useful to develop test batteries which cover a broad spectrum of toxic reactions. A scientifically more challenging and often more economical approach is the design of a hierarchical screening strategem. Ideally, this would start with a highly sensitive, broad-based general screening test which would detect all compounds having a toxic action on the organ under study but would not be very specific with regard to mechanisms of action. Chemicals which are positive in this test would either be dropped or would move on to a sequence of secondary studies in which information on potency, site and mechanism of action would be gathered. A proposal for such a strategy in behavioral toxicology has recently been published (Elsner, 1983).

3.3. Selection of Method There are many factors which influence the method selection in toxicological screening. An obvious one is the available infrastructure, together with limitations on manpower, equipment, and funds. A very important influence is the basic training of the scientist and his subsequent professional experience. However, the scientist must learn to experiment with a variety of experimental approaches and must critically evaluate many options in order to assure that the most sensitive and the most discriminating methods are favored.

3.4. Validation of Method The first step in the validation procedure is the collection of data on normal responses, reproducibility, interindividual and day-today variations in the same subject, and physiological variations as they may be related to age, sex, time of the day, estrus cycle, feeding pattern, extraneous stresses, etc.

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Critical evaluation obtained:

ELSNER,

AND

BOELSTERLI

for the success is the selection of reference compounds and their in the test procedure. Answers to the following questions must be

How reliably can the toxic effect of the reference substances be detected? What is the minimally effective dose or concentration? Is there a dose-effect relationship? What are the optimal experimental conditions to demonstrate the effects of the reference substance? What is the minimal number of animals necessary to demonstrate the effects of the reference substances? 4. TESTING

OF NEW

COMPOUNDS

In many instances, toxicological screening procedures differ little from the conventional toxicity studies: Groups of animals are treated with graded doses. Test results are recorded and compared with those of a control group using standard statistical methods. In other instances different approaches may be used. This is particularly the case when savings of time, money, and lives of animals are of paramount concern, and when high precision results are not required. The most important features of these alternative approaches include: comparison of the test results with historical control values or “normal ranges” rather than with data from an actual control group; predetermination of the biologically relevant changes, i.e., setting of test criteria, by the toxicologist; acceptance of descriptive terms to identify toxicologically relevant changes whenever conventional statistics cannot be used; and flexibility in manipulating experimental conditions, e.g., with regard to dosing schedules, administration routes, etc. (this may include a sequential design experiment, whereby new groups of animals using the same or different dosing schedules are added as more experience is gained; in other cases the dose may be increased periodically or treatment may be discontinued permanently in order to evaluate the reparative processes). 5. EVALUATION

OF TOXICOLOGICAL

SCREENING

TESTS

As described in Section 3.2., one of the important goals of toxicological screening is the discovery of hazard, i.e., the presence or absence of a particular toxic effect. If the experiment was designed with a sufficient number of animals and appropriate controls, the evaluation is based on the conventional concepts of biostatistics. On the other hand, if small numbers of animals are used and if control groups are not regularly included, different approaches are necessary. The most important step is the definition of a test criterion (xc) for each screening procedure. This may be done purely arbitrarily, taking into account past experience. Another possibility is based on the distribution of the measured values in the historical controls. The threshold or test criterion can be set at any desired probability of error I (a) for one sample. Assuming normal distribution, the test criterion (xc) can then be calculated according to the formula

TOXICOLOGICAL

Measure

value

in

279

SCREENING

standard

deviation

units

RG. 1. Determination of criterion (threshold) based on a predetermined control distribution. example the error I probability was selected to be .05.

In this

where m and s are the mean and the standard deviation of the control population respectively, and z is the standard normal distribution abscissa at the selected CY. Using the same formula, it is possible to calculate the probability associated with the test criterion which was set arbitrarily. In Fig. 1 the setting of a test criterion is shown graphically. The error I probability CYwas selected at 0.05. The threshold is thus located at a distance of 1.64 standard deviations from the mean of the control population. In Table 1 false positive error probabilities are calculated for groups of 2, 4, and 6 animals with ar values of 0.05, 0.01, and 0.001. For example, if the test criterion is set at cr = 0.01, and if we are using 4 animals of which 3 reach the test criterion, the false positive error probability is 1:250,000.

TABLE 1 ERROR I F%OBABILITIE~ (%), IN F~NCIION OF THE NUMBER N OF INDEPENDENT SAMPLES AND THE NUMBER K OF TIMES THE CRITERION Is REACHED, DETERMINED ACCORDING TO THE ONE SAMPLE ERROR I PROBABILI’IY a

1%

5% KN:

0.1%

2

4

6

2

4

6

2

4

6

90.3 9.5 .250

81.5 17.1 1.35 0.0475 0.0006

73.5 23.2 3.05 0.214 0.0085 0.0002
98.0 1.98 0.0100

96. I 3.88 0.0588 0.0004
94.1 5.71 0.144 0.0019
99.8 0.200 0.0001

99.6 0.400 0.0006
99.4 0.597 0.0015 <0.0001
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It is sometimes useful to evaluate the results of screening tests using descriptive terms such as “level of suspicion I to III” (LOS I to III), and “level of virtual certainty” (LVC). The LOS I to III indicate an increasing confidence of the toxicologist that the compound has the adverse effect measured by the screening test. When a LVC is reached, there is little doubt left about the toxic properties of the substance. In Table 2 LOS and LVC are assigned arbitrarily to test results using groups of 2, 4, and 6 animals. This classification was made in accordance with the error Z probabilities of Table 1. From Table 1 it is clear that the error Z probabilities depend very much on the location of the test criterion. The further away the test criterion is set from the mean of the controls, the lower is the probability that a false positive result will be obtained. It should be stressed again that the procedures are set-up in such a way that the standard reference compounds used for validation always reach the test criterion. It is thus quite possible that the test would fail to detect a compound whose toxic properties are considerably weaker than, or qualitatively different from those of the standards. Up to this point we have only considered whether or not a test compound reaches the preset screening criterion. However, other observations may also be relevant. Important among them is the magnitude of the response. If the response of an animal treated with a new compound not only reaches the test criterion, but exceeds it greatly, the toxicologist often feels much more confident that the substance possesses the toxic properties measured in the screening procedure. Therefore, it may be useful to define a “highly pathological response” (HPR) which, when present in one or more animals, increases the LOS or enhances it to a LVC. Similarly, a clear-cut dose-effect relationship or good evidence of a cumulative effect can often add valuable information to arrive at a final evaluation of a toxicological screening test. TABLE 2 ARBITRARY

EVALUATION

OF SCREENING

No. of animals reaching criterion/ No. of animals tested

o/2 112 212

014 114 214

TEST RESULTS

Evaluation“

LOS I LOS II LOS I

314

LOS II

414

LOS III

016 116 216 316 416 516 616 ’ LOS, level of suspicion; LVC, level of virtual certainty.

-

LOS LOS LOS LOS LVC

I II II III

TOXICOLOGICAL

281

SCREENING

From this discussion it is evident that the toxicological screening is, as pointed out in the definition of the goals, primarily concerned with the detection of compounds having a certain level of toxicity. It is, of coutse, much more difficult to prove that a test compound lacks a toxic effect. However, if desired, a screening procedure designed to find compounds not having the toxic characteristics of a standard agent can be developed. The test criterion would then be set based on the distribution in a relatively large group of animals treated with the standard agent, and the same calculations as described above, but this time for false negative results, can be made. Once a compound has been identified as presenting a certain hazard, the screening test should also give information on potency in comparison with the reference substances and often also with respect to other agents included in the screening procedure. Whenever the data available are sufficient to determine the precise location of the test compound’s dose-effect curve along the dose axis, conventional biostatistical methods can be applied. With small numbers of animals and limited information on dose-effect relationships, comparisons with reference compounds and with other test substances must often remain tentative, e.g., in the form of an approximate ranking order. However, the confidence in such comparative assertions mounts as the potency differences between test substances and reference agents increase. 6. TOXICOLOGICAL

SCREENING VERSUS SAFETY EVALUATION

STANDARD

In discussing the merits of toxicological screening it is essential to contrast it with the concepts on which routine toxicity studies are based. Screening tests are certainly most appropriate if well-defined toxic effects must be detected. They are less useful for a general evaluation of the toxicological characteristics of a chemical compound. For this reason they cannot and should not replace standard toxicological test procedures. One of the important aspects of the screening approach is the acquisition of toxicological information with small numbers of animals. The best results can be expected if the normal range of the parameter measured is distinctly separated from the value selected as screening criterion. For such clear-cut, dose- and time-related toxic reactions experiments with small numbers of animals are capable of identifying active chemicals. The reproducibility of such test methods is often excellent, although the precision of the results does, of course, not match that of studies conducted with large numbers of subjects. For this reason the toxicologist must decide, before he embarks on a screening test, whether or not he requires high precision data. He must only proceed if the answer to this question is negative. Experiments with large numbers of animals are necessary if one wishes to detect rare events. However, the confidence one usually attaches to the group sizes included in conventional standard toxicity experiments is somewhat exaggerated. If the “true” incidence of a toxic reaction is 1 in 10, one must use 29 animals to have a 95% probability of finding just one subject exhibiting the toxic reaction; if the “true” incidence were 1 in 100, then 299 animals per group would be needed (Zbinden, 1973). The probability of finding such an event in a group of 20 animals (15.6) is only slightly better than that calculated for a group of 6 (1: 17).

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A particularly critical problem in toxicology is the establishment of safety. It is clear that unambiguous proof of safety, i.e., lack of a toxic effect, is never obtainable. Therefore, large numbers of animals are often needed to reach at least a satisfactory confidence that a chemical substance is devoid of a particular toxic effect. Clearly, screening tests with small numbers of animals are not very useful to provide this type of evidence and peace of mind. However, they are not totally meaningless. As it was pointed out earlier, screening tests are validated in such a way that they reproducibly detect the toxic action of reference substances. Thus, if a new compound gives a negative result under the same experimental conditions, the statement can be made that it does not have the same adverse properties as the substance used to validate the test. And this information can, at times, be quite important. Each toxic effect has characteristic dose-effect and time-effect relationships. It follows that the probability to detect an abnormal reaction can vary greatly depending on the timing of the test procedure, the dosing schedule, the magnitude of the doses, the effectiveness of the repair processes, and the adaptability of the subjects. Because routine toxicity tests are rigidly standardized, it is not possible to study each adverse reaction when the chances for its detection are most favorable, and to adjust the experimental procedures to obtain optimal conditions for the induction of each toxic effect. But this is exactly what toxicological screening tests can do. It is evident, therefore, that they are well suited to fill in for deficiencies of the standard toxicological test procedures. 7. EXAMPLE:

TOXIC

7.1. Screening Approach and Experimental

HEMOLYSIS

Methods

An increased rate of destruction of red blood cells (RBC) is caused by chemicals that injure the cell membrane, cause oxidative damage to hemoglobin, or suppress the antioxidant defense system of the erythrocytes. Even if a compound has only weak hemolytic properties it represents a hazard, particularly in subjects with inherited intraerythrocytic metabolic defects (Beutler, 1969). The hemolytic properties of chemicals can be investigated by observing RBC lysis in vitro or by determination of osmotic fragility of the RBC after incubation with the test compounds. However, many chemicals stabilize the RBC membrane at low concentrations and increase its fragility at high concentrations, and the shape of the concentration-effect curve is unpredictable (Seeman, 1972). Moreover, the in vitro systems do not appropriately reflect the pharmacokinetic and metabolic peculiarities of the test compounds. For these reasons it was decided to use an in vivo model system. The experimental approach is summarized in Fig. 2. Groups of six male rats were treated for 3 days with a low dose selected arbitrarily. One hour after the third administration, blood was taken for the determination of methemoglobin (Met-Hb). Twenty-three hours later another sample of blood was obtained for the determination of Met-Hb, reticulocytes (RC), Heinz bodies (H Bad), siderocytes (SC), RBC count, hematocrit (Hkt), hemoglobin (Hb), mean corpuscular fragility (MCF), and mean corpuscular volume of the RBC ghosts (MCVg). Details of the experimental

TOXICOLOGICAL TEST ( DAY

ww1

283

SCREENING

DRUG

:

: 2

/...”

; 3

:

;

4 I

5

0 :-e-e

; 6

7

----

8

FIG. 2. Screening procedurefor toxic hemolysis.

procedures, including strain of rats, age, housing conditions, blood sampling technique, and laboratory methods, have been published previously (Boelsterli et al., 1983a,b). As test criteria the arithmetic means of the controls f 1.64 X standard deviation was selected. If no evidence of hemolysis was observed, the experiment was repeated in the following week with a higher (usually double) dose. The study was terminated when hemolytic changes occurred, or when signs of general toxicity (e.g., poor weight gain, diarrhea, soiled fur, etc.) were recorded. In order to monitor the reversibility of the changes, the rats were subjected to the tests once more after 1 week without treatment. The normal values of the laboratory parameters and the test criteria are given in Table 3. For RC, H Bod, Met-Hb, and SC a test threshold representing a HPR was selected arbitrarily. TABLE 3 NORMAL

VALUES OF SCREENING

Parameter Reticulocytes (RC) (per 103) Heinz Bodies (H Bod) (per 10’1 Methemoglobin (Met-Hb) (%) Red blood cells (RBC) (X 10’2/1) Hematokrit (Hkt) Hemoglobin (Hb) (g/100 ml) Mean corpuscular fragility (MCF) (9%NaCl) Volume of RBC ghosts (MCW (rtm”) Siderocytes (SC) (per 10’)

Mean 41

PARAMETERS

SD 7

N” 36

-

AND TEST CRITERIA

Test criteria >52

0.64

0.25

38 36

z-0 >1.15

6.33 0.38 14.1

0.45 0.02 0.8

38 38 38

<5.5 <0.35 <12.8

0.012

22

5

38 38

0

0.464 125 0

-

Highly pathological response (HPR) >lOO >lO Z-5

z-O.484 <117 20

110

’ N, number of independent values from control rats comprising data recorded at the start and at the termination of each experiment.

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ZBINDEN,

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7.2. Validation of the Screening Test Three compounds known to cause oxidative RBC damage, sodium nitrite, menadione diphosphate, and isoniazid (Zbinden et al., 1957; Kiese, 1974; Boelsterli et al., 1983b) and ethanol, which supresses the RBC antioxidant system (Cape1 et al., 1980), were selected as reference substances. The results of the screening tests are summarized in Table 4. It is evident that the hemolytic properties of the four standard agents were detected unambiguously by the screening procedure. The most reliable test was the RC count which was positive with all compounds. It was interesting that the RC decreased when the highest isoniazid dose was reached, and that it was normal with the highest dose of ethanol. However, after 1 week of recovery RC increased again TABLE 4 RESULTS OF SCREENING

TESTS

Met-Hb Test drug and dose Sodium nitrite bwlbJh9 10 20 50 I week recovery

RC

HBod

O/6

60 min

24 hr

MCF

MCVg

ndh

O/6 O/5 O/4 O/5

O/5 2~5 l/6 O/5

O/6 O/5 O/5 O/5

O/6” 315 515 O/5

O/5 O/5 O/5

516

OJ6

nd

OJ5

OJ6

OJ6

10

6/6

20 50

515 5J5 315

016 2J5

nd nd nd nd

316 115 3J5 l/5

O/6 O/5 OJ5 O/5

O/6 O/5 OJ5 115

416 6J6 516

3J3 414 5J5 (5)

RBC, Hb, Hkt, !3C

016 O/5 O/5 O/5

Menadione diphosphate bW&W 5

1 week recovery Isoniazid (mgJkgJday) 100 200 400

1 week recovery

5J5

216

nd

RBC l/6 Hb l/6 Hkt l/6 O/6

Hb l/6 OJ5 O/5

216

O/6 O/6

616

016

nd nd nd nd

616

nd

nd

‘36

O/6

116

Hb l/6 Hkt l/6

6J6

nd nd nd

nd nd nd

OJ6 O/5 O/5

O/6 O/6 o/5

3J6 O/5 O/5

O/6 O/5 OJ5

516 5J6

016

316

016

O/6 O/6 (4/W

O/6 O/6 l/6

SC l/5 SC 6J6 (6) SC 616 (6) O/6

Ethanol (ml/kg/day) 1 2 4

1 week recovery

O/5 415

’ Number of animals reaching test criterion/number animals with HPR. ’ nd, not determined. cDecrease.

of animals tested. In parentheses is the number of

TOXICOLOGICAL

SCREENING

285

with both compounds, indicating that the replenishment of the destroyed erythrocytes with reticulocytes was depressed by the highdose treatment. Met-I% elevation was found with the three compounds causing oxidative RBC damage, but only with sodium nitrite was it a more sensitive indicator than the RC count. With this compound, Met-Hb disappeared within 24 hr, whereas with menadione diphosphate and isoziazid Met-Hb concentrations were still increased 24 hr after the last dose. H Bod were present only in rats treated with the two higher doses of menadione diphosphate. The measurements of osmotic fragility gave disappointing results. MCF was only increased in a few animals treated with sodium nitrite. It decreased in 4/6 animals 1 week alter the end of treatment with isoniazid. This was probably due to the presence of a population of young RBC (Marks and Johnson, 1958). The same effect was observed in phenacetin-treated rats (Boelsterli et al., 1983a). SC were conspicuous only in rats treated with isoniazid, but in these animals the increase was marked, and often represented a HPR. No evidence of anemia was observed, and the MCVg remained unchanged. 7.3. Conclusions The example of toxic hemolysis illustrates well the steps involved in the development of toxicological screening. The circulating erythrocytes were selected as the target of toxicity. After considering various testing approaches a short-term in vivo model with three daily doses, followed by a 4day rest period and additional 3day treatment periods with increased doses, was selected. To assess the hemolytic properties of the test compounds various laboratory parameters were used, i.e., measurements of direct toxic effects on the RBC (MetHb, H Bod, MCF, MCVg), the consequences of increased cell destruction (RBC count, Hb, Hkt), and the regenerative response of the bone marrow (RC, MCVg). For validation of the screening test, a potent inducer of Met-Hb (sodium nitrite), a drug known to induce hemolysis in susceptible individuals (menadione diphosphate), a drug with hydrazine structure which rarely causes hemolysis in humans when used at therapeutic doses (isoniazid) (Gierling, 1982), and a compound affecting mainly the antioxidant defense system of the RBC (ethanol) were selected. The experiments showed that determination of RC and Met-I% gave the most reliable information on the hemolytic properties of the reference compounds. H Bod were occasionally increased, but provided no additional evidence for the toxic effects investigated. Determination of MCF, MCVg, RBC count, Hb, and Hkt proved to be too insensitive to be of any use for the toxicological screening. The stepwise increase of the doses was useful, not only because it permitted the observation of a dose-effect relationship, but also because it provided evidence of bone marrow depression at high doses of isoniazid and ethanol. Finally, with the addition of the simple determination of SC counts, information on toxic effects on hemoglobin formation could be obtained (Yunis and Salem, 1980). Based on these findings a toxicological screening test using groups of not more than six rats is proposed. Treatments are limited to periods of 3 days with increases of the dose at weekly intervals until evidence of hemolysis is obtained or until other signs of intolerance are noted. Primary screening parameters are RC count,

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Met-Hb, H Bod, and the count of SC. Further validation experiments of the screening test with known or potentially hemolytic substances of different chemical structures are underway. ACKNOWLEDGMENTS This work was supported by a research grant from the Ministerium Rir Jugend, Familie und Gesundheit, Federal Republic of Germany. We thank Dr. R. Buxxi for his help in the development of the statistical concepts for the evaluation of screening tests.

REFERENCES BEUTLER, E. (1969). Drug-induced hemolytic anemia. Pharmacol. Rev. 21,73-103. BOELSTERLI, U. A., MAYER, E., AND ZBINDEN, G. (1983b). Screening for hemolytic e&c& paper read at the Symposium “Situation actuelle et orientation future en toxicologic des medicaments” Paris, May 9-11. BOELSTERLI, U. A., SHIE, K. P., BUNDLE, E., AND ZBINDEN, G. (1983a). Toxicological screening models: Drug-induced oxidative hemolysis. Toxicol. Lett. 15, 153-l 58. ELSNER, J. (1983). Systematic information acquisition in behavioral toxicology. In Application of Behavioral Pharmacology in Toxicology (G. Zbinden, V. Cuomo, G. Racegni, and B. Weiss, eds.), pp. 45-56. Raven Press, New York. GIERLING, D. J. (1982). Adverse effects of antituberculosis drugs. Drugs 23, 56-74. K~ESE, M. (1974) Methemoglobinemia: A comprehensive treatise. Causes, consequences, and correction of increased contents ofjkrrihemoglobin in blood. CRC Press, Cleveland, Ohio. MARKS, P. A., AND JOHNSON, A. B. (1958). Relationship between the age of human erythrocytes and their osmotic resistance: A basis for separating young and old erythrccytes. J. Clin. Invest. 37, 15421548. MEIER-RUOE, W. (1972). Drug-induced retinopathy. CRC Crit. Rev. Toxicol. 1, 325-360. PLAA, G. L. (1978). The problems of low-incidence response. In Proceedings of the First International Congress on Toxicology: Toxicology as a Predictive Science (G. L. Plaa and W. A. M. Duncan, eds.) pp. 207-219. Academic Press, New York. SEEMAN, P. C. (1972). The membrane action of anesthetics and tranquilizers. Pharmacol. Rev. 24. 583655. YUNIS, A. A., AND SALEM, Z. (1980). Drug-induced mitochondrial damage and sideroblastic change. Clin. Haematol. 9, 607-619. ZBINDEN, G. (1973). Progress in Toxicology: Special Topics, Vol. 1. Springer-Verlag, Heidelberg@erlin/ New York. ZBINDEN, G. AND FLURY-ROVERSI, M. (1981). Significance of the LD50 test for the toxicological evaluation of chemical substances. Arch. Toxicol. 47, 77-99. ZBINDEN, G., SCHKRER,K., AND STUDER, A. (1957). Experimentelle Untersuchungen iiber ErythrocytenSchiidigung durch Menadionderivate im Vergleich zu Vitamin K,. Schweiz. Med. Wochenschr. 87, 1238-1241. ZBINDEN, G., AND STUDER, A. (1959). Experimental pathology of iproniaxid and related compounds. Ann N. Y. Acad. Sci. 80, 873-884.