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Behavioural teratology of exogenous substances: regulation aspects
G. J. Boer, M.G. P. Feenstra, M. Mirmiran, D. F. Swaab and F. Van Haaren Progress in Brain Research, Vol. 73 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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CHAPTER 5
Behavioural teratology of exogenous substances: regulation aspects Herman B. W.M. Koljter Department of Biological Toxicology, TNO-CIVO Toxicology and Num'tion Institute, Zeist. Zhe Netherlandr
Introduction It is a basic principle of teratology that a system is especially vulnerable during the period of differentiation. This implies in particular to the central nervous system (CNS), which consists of a number of different parts and cell types, each with its own developmentalprogram. The susceptibility of the CNS to teratogens became evident as exposure to a number of exogenous substances during development resulted in structural central nervous defects. However, it took until the early seventies before it was understood that the same sorts of factors that had long been known to disturb the development of structures are also able, not infrequently at much lower dose levels, to disturb functional development and thus behaviour (Butcher, 1985). One of the reasons that behavioural teratology gained acceptancein the 1970s was that the investigators of this period related their research to the principles and concepts of toxicology. Thus, several principles of behavioural teratology are framed in a toxicological perspective (Vorhees and Butcher, 1982). Firstly, behavioural teratogenicity is expressed as delayed behavioural maturation and impaired or abnormal behaviour. This 6rst principle is analogous to the principle in developmental toxicology that developmental disorders are expressed as retarded development, embryo- and feto-
toxicity and anomalies and malformations. Although the behavioural manifestations mentioned in this lirst principle are thus clearly appropriate, other indices may be added in the future. The second principle is that the period of susceptibility to behavioural teratogenesis is isomorphic with the period of CNS development. This principle refers to the biological basis of behaviour and indicates that all behaviour arises from structural and functional interactions residing within the CNS. It also indicates that the severity as well as the type of behavioural effects correlate with the period of CNS development during which exposure took place. This relationship has clearly been demonstrated by Rodier and co-workers with exposures to 5-azacytidine (Rodier 1976, 1977; Rodier et al., 1979). The third principle is that the type and magnitude of the response are a function of (a) the type of agent administered, (b) the dose of the agent, (c) the time and duration of exposure, (d) the environment of the target organism, and (e) the genetic background of the target organism. This principle is not unique to behavioural teratology but originates from pharmacology and toxicology (Vorhees and Butcher, 1982): the unique characteristics of the chemical compound determines its biological activity and consequently its toxicity. The principle that the type and magnitude of the response are a function of the dose suggests that,
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as in toxicology and teratology, a no-adverseeffect level might be established. In behavioural teratology, as in developmental toxicology, time and duration of exposure are extremely important, since the susceptibilityof the developing organism to exogenous substances varies considerably with the stage of development of the CNS. The environment and the genetic background of the fetus or neonate evidently play an important role in the type and magnitude of the behavioural effect. Although behavioural teratology gained wide acceptance since most of the research was based on these principles, there were still a number of problems to be solved. These were, among others: (a) how is ‘behavioural teratology’ defined, (b) should behavioural teratogenicity studies be requested upon safety evaluation of (new) substances and, if so, for which types of substance, (c) what type of behavioural functions should be assessed, (d) what is the relevance of the results of animal tests to the human situation?
Definition of bebavioural teratology The World Health Organisation (WHO) proposed the following definition: ‘Behaviour teratology refers to the study of effects on behaviour onceptus at any which result from damage to time during its development’ (W ;1986). The most important aspect of this definition is that it says ‘at any time during its development’, indicating that the inquction of behavioural teratogenic effects is not at all restricted to the period of gestation but could also occur after birth (Health Council of the Netherlands, 1985). Another interesting aspect of this definition is that it does not make any restrictions regarding maternal toxicity. Unfortunately, ‘damage to the conceptus’is not specified and implies that, according to the definition, the study of behavioural alternations resulting from structural damage of organs other than the CNS may also be considered as behavioural teratology! In practice, however, behavioural indices will merely be used as fine
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tools for the detection of CNS damage that would otherwise not have been observed in (classical) teratology studies. An alternative approach is to use the term ‘functional neuroteratology’, indicating that one should not regard behaviour as the sole endpoint of non-morphological anomalies but one should include all other physiological aspects subject to central regulation.
Regulations involved The next question was: ‘Should behavioural teratogenicity studies be requested upon safety evaluation of new substances’. It should be emphasized that upon safety evaluation of a compound, reproduction toxicity data and data on behavioural teratogenicity are only one aspect among many others. Therefore it was considered, especially by industry, a drastic measure when in 1974/1975 the British and Japanese authorities incorporated behavioural assessments in their guidelines for reproduction studies of pharmaceuticals. Moreover, at that time behavioural teratology was still very much in its infancy and nobody knew exactly how to tackle the problem of routine screening. The U.K. was the first to introduce a requirement. The guideline specifies that ‘late effects on the progeny in terms of auditory, visual and behavioural function should be assessed‘. The guidance note was deliberately designed not to be restrictive and, although auditory, visual and behavioural development are specifically mentioned in order to give some idea of the types of test which could be done, a wide range of postnatal tests have in practice been accepted in drug submissions (Barlow, 1985). At present, these rather arbitrary British guidelines are still effective. The early Japanese guideline for the testing of drugs stating that ‘as to behavioural studies, appropriate tests for locomotion, learning, sensory functions or emotionality shall be done’ were updated in 1984. The specifications as to which behavioural functions should be assessed have been replaced by a more general and open-ended guidenote, reading: ‘for obser-
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vations of growth and development, morphological, functional and behavioural examinations should be made' (Ministry of Health and Welfare Japan, 1984). Although this new guideline leaves sufficient room for the investigator to design the most appropriate test battery for a particular drug, the industry is now more than ever uncertain whether the chosen test approach is acceptable to the registration authorities. Although scientists in industry are generally in favour of the new, more liberal guidelines, their colleagues in the financial department and management may not be. For them, it is easier to live with very strict and welldefined requirements, so diminishing the chance of refusal or delay upon notification of their new drug. Moreover, strict requirements usually do not lead to unexpected and unpleasant surprises with respect to the budget of the safety assessment studies. Apart from the U.K. and Japan, today only Segment I: m y M y . -Exposure before conception and during pregnancy - Emluotion of :gonadal function, oestrus cycle, mating. conception and early. aestation -
1
I
-Exposure during embryonic development -Evaluation of: embryonic and foctal development
-Exposure from post-organogenesis through lactation
UK
France USA
Preweaning period
i
I
T
Mating
'
I
Period of organogenesis
'
1
,
+ I
Period of
' I
lactation 1 Birth Weaning
Fig. 1 Phases of testing drugs for their developmental toxicological potential and exposure periods as recommended in guidelines for developmental toxicity testing of drugs.
France has included behavioural aspects in their guidelines for the testing of drugs. Since their guidelines are essentially similar to the British ones, at present there are really only two sets. Apart from the differences between the two sets in detailing specific behavioural functions, the Japanese and British/French guidelines also differ in another important aspect: they require behavioural evaluations in different phases of the reproductive assessment process. Three phases of testing drugs for their developmental toxicological potential are currently distinguishable (Fig. 1). In Segment I studies, test compounds are administered to animals prior to and during the mating period. Japanese guidelines recommend administration to females throughout early gestation, whereas European and U.S.A. guidelines suggest doing so throughout a substantial part of the period of organogenesis (Segment Ia, Fig. l), even throughout weaning of the offspring. Apart from the evaluation of gonadal function, oestrus cycle, mating and implantation, the British/French guideline requires behavioural testing in this study, whereas the Japanese guideline does not. In the Segment I1 study, the test compound is administered to pregnant females during the period of organogenesis. This test has traditionally been applied for the assessment of morphological developmental anomalies. The Japanese guideline, however, also expects the assessment of functional developmental disorders in this test. In Segment I11 studies the occurrence of malformations which become apparent in the postnatal period is studied. In these studies, animals are dosed from late pregnancy throughout parturition and lactation until weaning. During lactation and after weaning the offspring are examined for structural and functional developmental disorders. Both the Japanese and the British/French guidelines include behavioural testing in this type of study. Overlap of exposure periods is required by the
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British/French guideline and the U.S.A. guideline. It is difficult to determine which phase of testing is optimal for the detection of behavioural deficits, but one working principle that has emerged from behavioural teratology does provide some insight, namely that the period of greatest vulnerability to malformations of the central nervous system is also the period of greatest vulnerability to behavioural abnormalities (Vorhees et al., 1978). This principle leads to the conclusion that applying behavioural testing in Segment I studies as required by the British/French guideline is only feasible when dosing is continued during organogenesis, since during this period the CNS is most susceptible to adverse effects. As can be seen in the diagram, this is indeed the case in the U.K. guideline. Since the Segment I11 study covers more of the final development of the CNS,when not only are neurotransmitter and other factors of functionalorganization presumably predominant (Vorhees, 1982) but also the cerebellum is still in an important stage of structural development (Rodier, 1980), it seems appropriate to include behavioural testing in this type of study as well. Summarizing, at present the regulatory involvement in behavioural teratology is such that Japanese and British/French guidelines include some general requirements for behavioural testing which differ with respect to specification of behavioural indices that should be measured as well as to the type of study in which behaviour testing on offspring should be conducted. It may be added here that behavioural teratological testing is only required for pharmaceuticals. In order to decide whether behavioural teratogenicity studies are necessary upon safety evaluation, and as a consequence should be incorporated in guidelines, the sensitivity and reliability of the methods used should be defined. Although the differences between the existing guidelines show that an international consensus on the methods used has not yet been reached, a number of compounds have positively been identified as behavioural teratogens.
TABLE 1 Compounds reported and confirmed as experimental behavioural teratogens ClaSS
Agent
Alcoholic beverages Anesthetics Drugs
Ethanol Halothane, nitrous oxide Anticonvulsants, antimitotics, antineurotics, antipsychotics Antioxidants, MSG Lead, methylmercury Methadone, morphine Amphetamines, caffeine Vitamin A excess
Food additives Metals Narcotics Stimulants Vitamins
(Source: Vorhees and Butcher, 1982)
In Table 1 some examples of compounds reported as being experimental behavioural teratogens are listed. For each of the agents mentioned in this table behavioural teratogenicity has been observed by several investigators in Merent laboratories (Vorhees and Butcher, 1982). Although internationally most attention is paid to drugs, it is interesting to learn from this table that several compounds other than drugs have also been shown to be behavioural teratogens. It becomes even more interesting when compounds reported as causing behavioural deficits in humans are tabulated (Table 2). For each compound, behavioural teratogenic findings have been confirmed by a number of investigators. It is surprisingto see TABLE 2 Compounds reported as causing behavioural deficits in humans Alcohol Hydantoin Lead Methylmercury Tobacco (Source: Tanimura, 1980; WHO, 1986)
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that almost all compounds are substances other than drugs! Therefore, the requirement for including tests of behavioural teratogenicity in guidelines for the safety assessment of drugs only does not accurately reflect the available data and should therefore be reconsidered. On the other hand, it would probably be unrealistic to recommend that behavioural teratology screening should be included as part of the screening procedure for all chemicals. To date, there is a consensus amongst the majority of scientists active in the field of (behavioural) teratology that behavioural teratology testing is a valid approach for assessing the presence of nervous system anomalies in the developing organism (Adams and Buelke-Sam, 1981; Sobotka and Vorhees, 1985, Kimmel et al., 1985). Consequently, some behavioural screening should indeed be required for drugs where it is considered essential to learn as much as possible about effects on development. With regard to other types of chemical, such as food additives, agrochemicals, industrial chemicals and toiletries and cosmetics, there was consensus among experts during the 1985 workshop on the collaborative behavioural teratology study of the National Center for Toxicological Research (NCTR) that ‘behavioural teratology testing in some fashion should be carried out but that the selection of chemicals for testing should be based on certain criteria which generally indicate a potential nervous system involvement’ (Sobotka and Vorhees, 1985). During the same workshop it was agreed that all compounds that are suspected of interference with the CNS should at least be subjected to some sort of behavioural teratology testing. It should be emphasized that this approach implies that for most of the novel chemicals it cannot be decided beforehand whether behavioural teratology screening is appropriate. In The Netherlands, the Health Council has recommended that a decision on reproduction toxicity testing of novel chemicals should depend on the results of a pilot study (Health Council, 1985). Such a pilot study should be designed so that, in addition to information with respect to
gametogenesis,mating, fertility and general reproductive performance, some indication of possible (functional) developmental toxicity can be obtained (Koeter, 1983). Apart from the decision as to further testing for general reproduction toxicity, the results of such a pilot screening could also serve as a basis for decision-making with respect to behavioural teratology testing. In addition to this, priorities for such testing could also be set on the basis of other criteria. For the testing of current, insaciently tested chemicals, the Health Council of The Netherlands has proposed the following criteria, which could also be used for settingfurther priorities with respect to behavioural testing (Health Council, 1985): (a) bioactivity of the agent, (b) number of men, women and children likely to be exposed either voluntarily or involuntarily, (c) available biological data such as suspected human teratogenicity, teratogenic effects in domestic animals or wildlife and toxicity in the adult at low doses, and (d) available information on ecotoxicity such as biodegradation and bioaccumulation. Summarizing, some behavioural teratogenicity studies should indeed be included in safety evaluation for a greater number of substances. However, guidelines should not be rigid in this respect and the decision as to whether behavioural teratology screening is necessary should depend on a variety of factors and criteria, most of which are related to toxicity data derived from other studies. But also the type of application and the environmentalconditions should be taken into account. The best approach for industry would be to discuss proposals for toxicity study programmes with the relevant registration authorities, prior to the start of these programmes. Of course, the authorities responsible should be willing to advise in this matter.
Behavioural functions to be assessed When it is thus recommended that for certain substances behavioural teratology screening should be considered for safety evaluation, the
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next problem is how to make a choice with respect to type of behavioural functions that should be assessed. At present, no single approach or behavioural test battery has been identified as the most reliable, sensitive and economical means of detecting behavioural dysfunction following developmental insult. Moreover, the current state of knowledge is such that although various approaches and test batteries have been described (Adams, 1986; Adams and Buelke-Sam, 1981; Kimmel et al., 1985; Rodier, 1978; Spijper Cranmer and Goad, 1983). Mechanisms underlying behavioural deficits are not fully understood (WHO, 1984, 1986). Therefore, at present the majority of scientists in the field are of the opinion that fixed protocols should not yet be included in routine experimental animal studies. There is, however, much agreement with respect to the strategy of testing. A well-accepted strategy is that behavioral teratology tests should be performed at two levels of sensitivity and complexity. Initial testing of chemicals should ensure that behavioural effects are not missed. It should be broad and comprehensivewith regard to the types of function assessed. This is necessary because many types of function, including fertility, may be adversely affected by an agent and these effects cannot be reliably predicted (WHO, 1984). Secondary testing should be performed if behavioural alterations are observed in initial testing. More sophisticated and selective behavioural techniques, perhaps also in other species, should be used to help delineate the type(s) and extent of effects produced and the possible mechanisms involved. Also, at this stage, more selective exposure regimens and testing schedules should be employed. Secondary behavioural evaluations should be flexibly designed with agent specificity in mind (Adams and Buelke-Sam, 198 1). With respect to initial testing, two different concepts are distinguishable. Firstly, the so-called ‘apicaltest strategy’ (Grant, 1976; Butcher, 1976). In essence, this strategy is based on the assumption that successful completion of a multi-
behavioural test by an animal implies that all contributory functions are normal. On the other hand, it is assumed that if one or more of the component functions is deficient, then the overall performance of the animal will be impaired. This approach, however, is not widely recommended, since there is insufficient knowledge of the extent to which experimental animals are able to compensate for specific deficienciesin such a way that the overall performance remains satisfactory. It is also unclear to what degree a particular function need be affected in order to produce a specific behavioural effect (Vorhees and Butcher, 1982). The second concept, and at present still the most adequate strategy for initial testing, is to employ a test system that combines a wide exposure period with a series of developmental tests which sample a broad range of CNS functions (Adams and Buelke-Sam, 1981). For each category of behavioural functions separate tests should be conducted. In general, measures of both early neurological, physical and behavioural development and behaviour at adulthood should be included. With respect to the categories of function to be assessed in the initial phase of testing the consensus opinion of experts in the field is that the following should receive sufficient attention (WHO, 1984; Kimmel et al., 1985): (a) Physicalgrowth and maturation. Although these parameters are not really behavioural indices, they are essential for evaluation of behavioural test results and as such are used as co-variables in statistical analyses. (b) Reflexive behaviour. As a measure of neurological development reflexes are very important and this category should include both reflexes that develop early and those that develop late during the postnatal period. (c) Sensory-motorevaluation. Tests of this category are based on the localization or orientation responsiveness to stimuli. Although preferably sensory functions should be assessed separately from motor functions, in practice this appears to be very dficult as the response to stimuli is usually expressed by some sort of motor activity.
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(d) Emotionality or motivational state. These states of the animal, which are difficult to define, are usually measured as spontaneous activity, reactivity, habituation and exploratory behaviour. In addition, sleep testing is occasionally applied. (e) Learning evaluations. Learning tests include habituation as an example of non-associative learning. This category further includes relatively simple associative learning (Pavlovian conditioning) as well as complex forms of learning. For each of these categories a number of tests are available and well-documented (Health Council of The Netherlands, 1985; WHO, 1984). The selection should be made by the investigator and will depend on practical aspects, knowledge and experience with certain tests and the nature of the agent to be tested. However, upon selection it is strongly recommended to consider the so-called ‘roofing-tile principle’. This implies that tests should have a certain overlap, through which it is possible to confirm a positive finding with respect to a certain behaviour in a different test, conducted under different conditions. Consequently, a positive finding should only be considered of toxicological significance when the finding is confinned in another test. Depending on the results of the initial testing phase and on the (social) importance of the substance tested, it could be decided whether or not secondary testing is appropriate. As mentioned earlier, secondary testing should be performed in a ‘tailor-made fashion’, based on earlier findings and application of the agent.
Relevance of behavioural effects in animals to man The number of examples in which there is a correspondence between developmental behavioural defects observed in humans and effects seen in animal studies is limited to a few agents such as methylmercury, lead and some psychoactive drugs (Lorente, 1981; Needleman et al., 1984; Vorhees and Butcher, 1982). As developmental neurobehavioural toxicology is a recently developed field of research, only limited evidence has so
far accumulated to convincingly demonstrate parallel effects in animals and humans. In addition, there is considerable disagreement concerning the importance or meaning of behavioural teratology data. In particular, the importance of transient changes in behaviour and accelerated or delayed maturation of reflexes is sometimes considered questionable. A better understanding can only be achieved when more information from animal and human data has been accumulated. Further, more research with respect to possible correlations between behavioural changes and structural or biochemical CNS alterations is urgently needed. In this respect, the research of Rodier and colleagues (Rodier, 1977; Koeter and Rodier, 1985; Rodier et al., 1986) on cell proliferation in the developing brain after exposure to behavioural teratogens as well as that of others (see other chapters in this volume) significantly contribute to a better understanding of the importance of developmental behavioural effects. It should be clear from the above that there is still a lot to learn before deviant behaviours are really understood and before behavioural effects in (young) animals can be extrapolated to the human situation with sufficient confidence.
Summary It has been shown that the same factors that have long been known to disturb the structural development are also able to disturb functional development and thus behaviour. At present, behavioural teratogenicity studies are only required by registration authorities for drugs. Although it is generally accepted that at least some data on behavioural teratogenicity should be available for all new drugs, the majority of compounds known as being experimental, behavioural teratogens are chemicals other than drugs. Therefore, the rationale for including behavioural teratogenicity testing in guidelines for the safety assessment of drugs only is unclear. As it would probably be unrealistic to recommend that behavioural teratogenicity screening
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should be included as part of the reproduction toxicity study programme for all chemicals, a procedure for the selection of priority chemicals needs to be developed. Criteria for such a selection procedure are proposed: (a) indications of a potential nervous system involvement, (b) positive results from a pilot reproduction study, (c) bioactivity of the chemical, (d) expected human exposures and (e) available information on ecotoxicity. When behavioural testing is considered relevant, a well-accepted strategy is to perform tests at two levels of sensitivity and complexity. The most adequate approach for initial testing is to employ a test system that combines a wide exposure period with a series of tests, sampling a broad range of CNS functions. Furthermore, tests should overlap. This should facilitate confirmation of a positive finding by a Merent test, conducted under different conditions. Depending on the results of the initial testing phase it can be decided whether secondary testing, to be performed in a ‘tailor-madefashion’, is appropriate. References Adams, J. (1986)Methods in behavioral teratology. In E.P. Riley and C.V. Vorhees (Eds.), Handbook of Behavioral Teratology, Plenum Press, New York. Adams, J. and Buelke-Sam, J. (1981) Behavioural assessment of the postnatal animal: testing and methods development. In C.A. Kimmel and J. Buelke-Sam (Eds.), Developmental Toxicology,Raven Press, New York, pp. 233-258. Barlow, S.M.(1985)United Kingdom: Regulatory attitudes towards behavioural teratology testing. Neurobehav. Toxicol. Teratol., 7:643-646. Butcher, R.E.(1976)Behavioural testing as a method for assessing risk. Environm. Health Perspct., 18: 75-78. Butcher, R. E.(1985)A historical perspective on behavioural teratology. Neurobehav. Toxicol. Teratol., 7: 537-540. Grant, L. D.(1976)Research strategies for behavioural teratology studies. Environm. Health Perspect., 18: 85-94. Health Council of The Netherlands (1985)The evaluation of the teratogenicity of chemical substances. Report No. 1985/6E. Kimmel, C. A., Buelke-Sam, J. and Adams, J. (1985)Collaborative BehaviouralTeratology Study: implications, current
applications and future directions. Neurobehav. Toxicol. Teratol., 7:669-673. Koeter, H.B. W.M. (1983)Relevance of parameters related to fertility and reproduction in toxicity testing. Am. J. Znd. Med., 4:81-86. KoZIter, H.B.W.M. and Rodier, P.M. (1985) Behavioural effects in rats and morphologic and behavioural effects in mice after exposure to inhalant anesthetics during early development. Neurobehav. Toxicol. Teratol., 7: 676. Lorente, C. A., Tassinari, M. S. and Keith, D. A. (1981)The effects of phenytoin on rat development: an animal model system for fetal hydantoin syndrome. Teratology, 24: 169-180. Ministry of Health and Welfare, Japan (1984)Information on the guidelines of toxicity studies required for application for approval to manufacture (import) drugs. Notification No. 118 of the Pharmaceutical Mairs Bureau, Ministry of Health and Welfare. Needleman, H.L., Rabinowitz, M., Leviton, A., Luin, S. and Schoenbaum, S. (1984)The relationship between prenatal exposure to lead and congenital anomalies. J. Am. Med. ASSOC.,251:2956-2959. Rodier, P.M. (1976) Critical periods for behavioural anomalies in mice. Environm. Health Perspect., 18:79-83. Rodier, P.M. (1977) Correlations between prenatally induced alternations in CNS cell populations and postnatal function. Teratology, 16:235-246. Rodier, P.M. (1978)Behavioral teratology. In Wilson and Frazier (Eds.), Handbook of Teratology, Vol. 4 , Plenum Press, New York. Rodier, P.M. (1980) Chronology of neuron development: animal studies and their clinical implications. Dev. Med. Chiki Neurol., 22: 525-545. Rodier, P. M., Reynolds, S. S. and Roberts, W. N. (1979)Behavioural consequencesof interference with CNS development in the early fetal period. Teratology, 19: 327-336. Rodier, P.M., Aschner, M., Lewis, L.S. and Koeter, H.B.W.M. (1986)Cell proliferation in developing brain after brief exposure to nitrous oxide or halothane. Anesthesiology, 64: 680-687. Sobotka, T. J. and Vorhees, C. V. (1985)Application of behavioural teratology testing procedures. Neurobehav. Toxiwl. Teratol., 7: 665. Spijper Cranmer, J. and Goad, P.T. (1983)Validation of selected behavioral tests for evaluating low-level toxicity. In G. Zbinden (Ed.), Application of Behavioural Pharmacology in Toxicology, Raven Press, New York. Suter. K.E. and SchBn, H. (1985)Possibilities and limitations of current methods in behavioural teratology screening:an industrial view-point. In Proceedings of the symposium: ‘Aktuelle Probleme der Biomedizin’, Salzburg, September 18-20,1985. Tanimura, T. (1984)Prospects of the reproductive toxicology with special referenceto the developmentalhazards due to
67 the treatment at the stages from prefertilization to implantation. Congenital Anomalies, 24: 319-328. Vorhees, C. V., Brunner, R. L., MacDaniel, C. R. and Butcher, R.E. (1978) The relationship of gestational age to vitamin A induced postnatal dysfunction. Teratology, 17: 27 1-276. Vorhees, C.V. and Butcher, R.E. (1982) Behavioural teratogenicity. In K. Snell (Ed.), Developmental Toxicology, Croom Helm, London, pp. 247-298. W. H.O. (1984) Principles for evaluating health risks to progeny associated with exposure to chemicals during pregnancy. Environm. Health Criteria, 30. W. H. 0. (1986) Draft Guidelines for the assessment of drugs and other chemicals for behavioural teratogenicity, WHO Regional Office for Europe, Copenhagen.
Discussion A. J. Friedhoff We know that very many non-psychotropes as well as all psychotropic drugs enter the brain, and it is likely that many of both types of drug affect brain development in ways we do not yet know. As we find out more about behavioral teratogenicity of drugs that enter the brain it is going to be necessary to learn now to differentiate among positive, negative and neutral effects, or we will probably have to ban very many drugs, because they have some effect on development. H.B.W.M. K&ter: Behavioral effects should never be extrapolated from the period of development to the adult situation. Since behavioral teratogenicity studies are only part of the developmental toxicity study program for the compound, and, moreover, even reproductive toxicity is only one aspect of the toxicology profile, there is always more information needed and often available. Only on the basis of this full toxicity profile should a compound be evaluated.
T.D. Yih: A number of drug classes were mentioned that
should be submitted to behavioral teratological testing, among which are CNS-active drugs. Do you mean drugs developed for treatment of CNS disorders or drugs which affect CNS physiology (eg. neurotransmitter levels)? The majority of newly developed drugs have to some extent such side effects. Should they all be tested? H.B.W.M. K&ter: In my opinion all drugs intended to be active at the CNS level should indeed be tested. It should be emphasized, however, that results of behavioral tests should only be evaluated within the context of the full toxicological profile. P.M. Rodier: Where guidelines are flexible, will regulators emphasize the use of positive controls and soforth, to ensure that tests have some validity? H.B.W.M. K d t e r : To date, not so many compounds have been evaluated by regulatory authorities. To my knowledge, there are no requirements as to positive controls or other means to ensure the validity of the tests applied. Authorities should be encouraged to implement such controls. C. V. Vorhees: I agree that more information is needed on the relevance of animal to human behavioral change. However, I would not like to have your comments leave the impression that this problem is any more difficult than with malformations, or cancer, where also one cannot precisely extrapolate specific effects from animals to human. H.B.W.M. Koeter: Although your argument is valid, I feel that upon the introduction of any new discipline in toxicology the same mistake (made some 25 years ago when the majority of toxicity test methods were introduced) of being merely descriptive should not be made again. As in pharmacology, one should strive after understanding mechanisms rather than describe gross findings only.
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CHAPTER 5
Behavioural teratology of exogenous substances: regulation aspects Herman B. W.M. Koljter Department of Biological Toxicology, TNO-CIVO Toxicology and Num'tion Institute, Zeist. Zhe Netherlandr
Introduction It is a basic principle of teratology that a system is especially vulnerable during the period of differentiation. This implies in particular to the central nervous system (CNS), which consists of a number of different parts and cell types, each with its own developmentalprogram. The susceptibility of the CNS to teratogens became evident as exposure to a number of exogenous substances during development resulted in structural central nervous defects. However, it took until the early seventies before it was understood that the same sorts of factors that had long been known to disturb the development of structures are also able, not infrequently at much lower dose levels, to disturb functional development and thus behaviour (Butcher, 1985). One of the reasons that behavioural teratology gained acceptancein the 1970s was that the investigators of this period related their research to the principles and concepts of toxicology. Thus, several principles of behavioural teratology are framed in a toxicological perspective (Vorhees and Butcher, 1982). Firstly, behavioural teratogenicity is expressed as delayed behavioural maturation and impaired or abnormal behaviour. This 6rst principle is analogous to the principle in developmental toxicology that developmental disorders are expressed as retarded development, embryo- and feto-
toxicity and anomalies and malformations. Although the behavioural manifestations mentioned in this lirst principle are thus clearly appropriate, other indices may be added in the future. The second principle is that the period of susceptibility to behavioural teratogenesis is isomorphic with the period of CNS development. This principle refers to the biological basis of behaviour and indicates that all behaviour arises from structural and functional interactions residing within the CNS. It also indicates that the severity as well as the type of behavioural effects correlate with the period of CNS development during which exposure took place. This relationship has clearly been demonstrated by Rodier and co-workers with exposures to 5-azacytidine (Rodier 1976, 1977; Rodier et al., 1979). The third principle is that the type and magnitude of the response are a function of (a) the type of agent administered, (b) the dose of the agent, (c) the time and duration of exposure, (d) the environment of the target organism, and (e) the genetic background of the target organism. This principle is not unique to behavioural teratology but originates from pharmacology and toxicology (Vorhees and Butcher, 1982): the unique characteristics of the chemical compound determines its biological activity and consequently its toxicity. The principle that the type and magnitude of the response are a function of the dose suggests that,
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as in toxicology and teratology, a no-adverseeffect level might be established. In behavioural teratology, as in developmental toxicology, time and duration of exposure are extremely important, since the susceptibilityof the developing organism to exogenous substances varies considerably with the stage of development of the CNS. The environment and the genetic background of the fetus or neonate evidently play an important role in the type and magnitude of the behavioural effect. Although behavioural teratology gained wide acceptance since most of the research was based on these principles, there were still a number of problems to be solved. These were, among others: (a) how is ‘behavioural teratology’ defined, (b) should behavioural teratogenicity studies be requested upon safety evaluation of (new) substances and, if so, for which types of substance, (c) what type of behavioural functions should be assessed, (d) what is the relevance of the results of animal tests to the human situation?
Definition of bebavioural teratology The World Health Organisation (WHO) proposed the following definition: ‘Behaviour teratology refers to the study of effects on behaviour onceptus at any which result from damage to time during its development’ (W ;1986). The most important aspect of this definition is that it says ‘at any time during its development’, indicating that the inquction of behavioural teratogenic effects is not at all restricted to the period of gestation but could also occur after birth (Health Council of the Netherlands, 1985). Another interesting aspect of this definition is that it does not make any restrictions regarding maternal toxicity. Unfortunately, ‘damage to the conceptus’is not specified and implies that, according to the definition, the study of behavioural alternations resulting from structural damage of organs other than the CNS may also be considered as behavioural teratology! In practice, however, behavioural indices will merely be used as fine
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tools for the detection of CNS damage that would otherwise not have been observed in (classical) teratology studies. An alternative approach is to use the term ‘functional neuroteratology’, indicating that one should not regard behaviour as the sole endpoint of non-morphological anomalies but one should include all other physiological aspects subject to central regulation.
Regulations involved The next question was: ‘Should behavioural teratogenicity studies be requested upon safety evaluation of new substances’. It should be emphasized that upon safety evaluation of a compound, reproduction toxicity data and data on behavioural teratogenicity are only one aspect among many others. Therefore it was considered, especially by industry, a drastic measure when in 1974/1975 the British and Japanese authorities incorporated behavioural assessments in their guidelines for reproduction studies of pharmaceuticals. Moreover, at that time behavioural teratology was still very much in its infancy and nobody knew exactly how to tackle the problem of routine screening. The U.K. was the first to introduce a requirement. The guideline specifies that ‘late effects on the progeny in terms of auditory, visual and behavioural function should be assessed‘. The guidance note was deliberately designed not to be restrictive and, although auditory, visual and behavioural development are specifically mentioned in order to give some idea of the types of test which could be done, a wide range of postnatal tests have in practice been accepted in drug submissions (Barlow, 1985). At present, these rather arbitrary British guidelines are still effective. The early Japanese guideline for the testing of drugs stating that ‘as to behavioural studies, appropriate tests for locomotion, learning, sensory functions or emotionality shall be done’ were updated in 1984. The specifications as to which behavioural functions should be assessed have been replaced by a more general and open-ended guidenote, reading: ‘for obser-
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vations of growth and development, morphological, functional and behavioural examinations should be made' (Ministry of Health and Welfare Japan, 1984). Although this new guideline leaves sufficient room for the investigator to design the most appropriate test battery for a particular drug, the industry is now more than ever uncertain whether the chosen test approach is acceptable to the registration authorities. Although scientists in industry are generally in favour of the new, more liberal guidelines, their colleagues in the financial department and management may not be. For them, it is easier to live with very strict and welldefined requirements, so diminishing the chance of refusal or delay upon notification of their new drug. Moreover, strict requirements usually do not lead to unexpected and unpleasant surprises with respect to the budget of the safety assessment studies. Apart from the U.K. and Japan, today only Segment I: m y M y . -Exposure before conception and during pregnancy - Emluotion of :gonadal function, oestrus cycle, mating. conception and early. aestation -
1
I
-Exposure during embryonic development -Evaluation of: embryonic and foctal development
-Exposure from post-organogenesis through lactation
UK
France USA
Preweaning period
i
I
T
Mating
'
I
Period of organogenesis
'
1
,
+ I
Period of
' I
lactation 1 Birth Weaning
Fig. 1 Phases of testing drugs for their developmental toxicological potential and exposure periods as recommended in guidelines for developmental toxicity testing of drugs.
France has included behavioural aspects in their guidelines for the testing of drugs. Since their guidelines are essentially similar to the British ones, at present there are really only two sets. Apart from the differences between the two sets in detailing specific behavioural functions, the Japanese and British/French guidelines also differ in another important aspect: they require behavioural evaluations in different phases of the reproductive assessment process. Three phases of testing drugs for their developmental toxicological potential are currently distinguishable (Fig. 1). In Segment I studies, test compounds are administered to animals prior to and during the mating period. Japanese guidelines recommend administration to females throughout early gestation, whereas European and U.S.A. guidelines suggest doing so throughout a substantial part of the period of organogenesis (Segment Ia, Fig. l), even throughout weaning of the offspring. Apart from the evaluation of gonadal function, oestrus cycle, mating and implantation, the British/French guideline requires behavioural testing in this study, whereas the Japanese guideline does not. In the Segment I1 study, the test compound is administered to pregnant females during the period of organogenesis. This test has traditionally been applied for the assessment of morphological developmental anomalies. The Japanese guideline, however, also expects the assessment of functional developmental disorders in this test. In Segment I11 studies the occurrence of malformations which become apparent in the postnatal period is studied. In these studies, animals are dosed from late pregnancy throughout parturition and lactation until weaning. During lactation and after weaning the offspring are examined for structural and functional developmental disorders. Both the Japanese and the British/French guidelines include behavioural testing in this type of study. Overlap of exposure periods is required by the
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British/French guideline and the U.S.A. guideline. It is difficult to determine which phase of testing is optimal for the detection of behavioural deficits, but one working principle that has emerged from behavioural teratology does provide some insight, namely that the period of greatest vulnerability to malformations of the central nervous system is also the period of greatest vulnerability to behavioural abnormalities (Vorhees et al., 1978). This principle leads to the conclusion that applying behavioural testing in Segment I studies as required by the British/French guideline is only feasible when dosing is continued during organogenesis, since during this period the CNS is most susceptible to adverse effects. As can be seen in the diagram, this is indeed the case in the U.K. guideline. Since the Segment I11 study covers more of the final development of the CNS,when not only are neurotransmitter and other factors of functionalorganization presumably predominant (Vorhees, 1982) but also the cerebellum is still in an important stage of structural development (Rodier, 1980), it seems appropriate to include behavioural testing in this type of study as well. Summarizing, at present the regulatory involvement in behavioural teratology is such that Japanese and British/French guidelines include some general requirements for behavioural testing which differ with respect to specification of behavioural indices that should be measured as well as to the type of study in which behaviour testing on offspring should be conducted. It may be added here that behavioural teratological testing is only required for pharmaceuticals. In order to decide whether behavioural teratogenicity studies are necessary upon safety evaluation, and as a consequence should be incorporated in guidelines, the sensitivity and reliability of the methods used should be defined. Although the differences between the existing guidelines show that an international consensus on the methods used has not yet been reached, a number of compounds have positively been identified as behavioural teratogens.
TABLE 1 Compounds reported and confirmed as experimental behavioural teratogens ClaSS
Agent
Alcoholic beverages Anesthetics Drugs
Ethanol Halothane, nitrous oxide Anticonvulsants, antimitotics, antineurotics, antipsychotics Antioxidants, MSG Lead, methylmercury Methadone, morphine Amphetamines, caffeine Vitamin A excess
Food additives Metals Narcotics Stimulants Vitamins
(Source: Vorhees and Butcher, 1982)
In Table 1 some examples of compounds reported as being experimental behavioural teratogens are listed. For each of the agents mentioned in this table behavioural teratogenicity has been observed by several investigators in Merent laboratories (Vorhees and Butcher, 1982). Although internationally most attention is paid to drugs, it is interesting to learn from this table that several compounds other than drugs have also been shown to be behavioural teratogens. It becomes even more interesting when compounds reported as causing behavioural deficits in humans are tabulated (Table 2). For each compound, behavioural teratogenic findings have been confirmed by a number of investigators. It is surprisingto see TABLE 2 Compounds reported as causing behavioural deficits in humans Alcohol Hydantoin Lead Methylmercury Tobacco (Source: Tanimura, 1980; WHO, 1986)
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that almost all compounds are substances other than drugs! Therefore, the requirement for including tests of behavioural teratogenicity in guidelines for the safety assessment of drugs only does not accurately reflect the available data and should therefore be reconsidered. On the other hand, it would probably be unrealistic to recommend that behavioural teratology screening should be included as part of the screening procedure for all chemicals. To date, there is a consensus amongst the majority of scientists active in the field of (behavioural) teratology that behavioural teratology testing is a valid approach for assessing the presence of nervous system anomalies in the developing organism (Adams and Buelke-Sam, 1981; Sobotka and Vorhees, 1985, Kimmel et al., 1985). Consequently, some behavioural screening should indeed be required for drugs where it is considered essential to learn as much as possible about effects on development. With regard to other types of chemical, such as food additives, agrochemicals, industrial chemicals and toiletries and cosmetics, there was consensus among experts during the 1985 workshop on the collaborative behavioural teratology study of the National Center for Toxicological Research (NCTR) that ‘behavioural teratology testing in some fashion should be carried out but that the selection of chemicals for testing should be based on certain criteria which generally indicate a potential nervous system involvement’ (Sobotka and Vorhees, 1985). During the same workshop it was agreed that all compounds that are suspected of interference with the CNS should at least be subjected to some sort of behavioural teratology testing. It should be emphasized that this approach implies that for most of the novel chemicals it cannot be decided beforehand whether behavioural teratology screening is appropriate. In The Netherlands, the Health Council has recommended that a decision on reproduction toxicity testing of novel chemicals should depend on the results of a pilot study (Health Council, 1985). Such a pilot study should be designed so that, in addition to information with respect to
gametogenesis,mating, fertility and general reproductive performance, some indication of possible (functional) developmental toxicity can be obtained (Koeter, 1983). Apart from the decision as to further testing for general reproduction toxicity, the results of such a pilot screening could also serve as a basis for decision-making with respect to behavioural teratology testing. In addition to this, priorities for such testing could also be set on the basis of other criteria. For the testing of current, insaciently tested chemicals, the Health Council of The Netherlands has proposed the following criteria, which could also be used for settingfurther priorities with respect to behavioural testing (Health Council, 1985): (a) bioactivity of the agent, (b) number of men, women and children likely to be exposed either voluntarily or involuntarily, (c) available biological data such as suspected human teratogenicity, teratogenic effects in domestic animals or wildlife and toxicity in the adult at low doses, and (d) available information on ecotoxicity such as biodegradation and bioaccumulation. Summarizing, some behavioural teratogenicity studies should indeed be included in safety evaluation for a greater number of substances. However, guidelines should not be rigid in this respect and the decision as to whether behavioural teratology screening is necessary should depend on a variety of factors and criteria, most of which are related to toxicity data derived from other studies. But also the type of application and the environmentalconditions should be taken into account. The best approach for industry would be to discuss proposals for toxicity study programmes with the relevant registration authorities, prior to the start of these programmes. Of course, the authorities responsible should be willing to advise in this matter.
Behavioural functions to be assessed When it is thus recommended that for certain substances behavioural teratology screening should be considered for safety evaluation, the
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next problem is how to make a choice with respect to type of behavioural functions that should be assessed. At present, no single approach or behavioural test battery has been identified as the most reliable, sensitive and economical means of detecting behavioural dysfunction following developmental insult. Moreover, the current state of knowledge is such that although various approaches and test batteries have been described (Adams, 1986; Adams and Buelke-Sam, 1981; Kimmel et al., 1985; Rodier, 1978; Spijper Cranmer and Goad, 1983). Mechanisms underlying behavioural deficits are not fully understood (WHO, 1984, 1986). Therefore, at present the majority of scientists in the field are of the opinion that fixed protocols should not yet be included in routine experimental animal studies. There is, however, much agreement with respect to the strategy of testing. A well-accepted strategy is that behavioral teratology tests should be performed at two levels of sensitivity and complexity. Initial testing of chemicals should ensure that behavioural effects are not missed. It should be broad and comprehensivewith regard to the types of function assessed. This is necessary because many types of function, including fertility, may be adversely affected by an agent and these effects cannot be reliably predicted (WHO, 1984). Secondary testing should be performed if behavioural alterations are observed in initial testing. More sophisticated and selective behavioural techniques, perhaps also in other species, should be used to help delineate the type(s) and extent of effects produced and the possible mechanisms involved. Also, at this stage, more selective exposure regimens and testing schedules should be employed. Secondary behavioural evaluations should be flexibly designed with agent specificity in mind (Adams and Buelke-Sam, 198 1). With respect to initial testing, two different concepts are distinguishable. Firstly, the so-called ‘apicaltest strategy’ (Grant, 1976; Butcher, 1976). In essence, this strategy is based on the assumption that successful completion of a multi-
behavioural test by an animal implies that all contributory functions are normal. On the other hand, it is assumed that if one or more of the component functions is deficient, then the overall performance of the animal will be impaired. This approach, however, is not widely recommended, since there is insufficient knowledge of the extent to which experimental animals are able to compensate for specific deficienciesin such a way that the overall performance remains satisfactory. It is also unclear to what degree a particular function need be affected in order to produce a specific behavioural effect (Vorhees and Butcher, 1982). The second concept, and at present still the most adequate strategy for initial testing, is to employ a test system that combines a wide exposure period with a series of developmental tests which sample a broad range of CNS functions (Adams and Buelke-Sam, 1981). For each category of behavioural functions separate tests should be conducted. In general, measures of both early neurological, physical and behavioural development and behaviour at adulthood should be included. With respect to the categories of function to be assessed in the initial phase of testing the consensus opinion of experts in the field is that the following should receive sufficient attention (WHO, 1984; Kimmel et al., 1985): (a) Physicalgrowth and maturation. Although these parameters are not really behavioural indices, they are essential for evaluation of behavioural test results and as such are used as co-variables in statistical analyses. (b) Reflexive behaviour. As a measure of neurological development reflexes are very important and this category should include both reflexes that develop early and those that develop late during the postnatal period. (c) Sensory-motorevaluation. Tests of this category are based on the localization or orientation responsiveness to stimuli. Although preferably sensory functions should be assessed separately from motor functions, in practice this appears to be very dficult as the response to stimuli is usually expressed by some sort of motor activity.
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(d) Emotionality or motivational state. These states of the animal, which are difficult to define, are usually measured as spontaneous activity, reactivity, habituation and exploratory behaviour. In addition, sleep testing is occasionally applied. (e) Learning evaluations. Learning tests include habituation as an example of non-associative learning. This category further includes relatively simple associative learning (Pavlovian conditioning) as well as complex forms of learning. For each of these categories a number of tests are available and well-documented (Health Council of The Netherlands, 1985; WHO, 1984). The selection should be made by the investigator and will depend on practical aspects, knowledge and experience with certain tests and the nature of the agent to be tested. However, upon selection it is strongly recommended to consider the so-called ‘roofing-tile principle’. This implies that tests should have a certain overlap, through which it is possible to confirm a positive finding with respect to a certain behaviour in a different test, conducted under different conditions. Consequently, a positive finding should only be considered of toxicological significance when the finding is confinned in another test. Depending on the results of the initial testing phase and on the (social) importance of the substance tested, it could be decided whether or not secondary testing is appropriate. As mentioned earlier, secondary testing should be performed in a ‘tailor-made fashion’, based on earlier findings and application of the agent.
Relevance of behavioural effects in animals to man The number of examples in which there is a correspondence between developmental behavioural defects observed in humans and effects seen in animal studies is limited to a few agents such as methylmercury, lead and some psychoactive drugs (Lorente, 1981; Needleman et al., 1984; Vorhees and Butcher, 1982). As developmental neurobehavioural toxicology is a recently developed field of research, only limited evidence has so
far accumulated to convincingly demonstrate parallel effects in animals and humans. In addition, there is considerable disagreement concerning the importance or meaning of behavioural teratology data. In particular, the importance of transient changes in behaviour and accelerated or delayed maturation of reflexes is sometimes considered questionable. A better understanding can only be achieved when more information from animal and human data has been accumulated. Further, more research with respect to possible correlations between behavioural changes and structural or biochemical CNS alterations is urgently needed. In this respect, the research of Rodier and colleagues (Rodier, 1977; Koeter and Rodier, 1985; Rodier et al., 1986) on cell proliferation in the developing brain after exposure to behavioural teratogens as well as that of others (see other chapters in this volume) significantly contribute to a better understanding of the importance of developmental behavioural effects. It should be clear from the above that there is still a lot to learn before deviant behaviours are really understood and before behavioural effects in (young) animals can be extrapolated to the human situation with sufficient confidence.
Summary It has been shown that the same factors that have long been known to disturb the structural development are also able to disturb functional development and thus behaviour. At present, behavioural teratogenicity studies are only required by registration authorities for drugs. Although it is generally accepted that at least some data on behavioural teratogenicity should be available for all new drugs, the majority of compounds known as being experimental, behavioural teratogens are chemicals other than drugs. Therefore, the rationale for including behavioural teratogenicity testing in guidelines for the safety assessment of drugs only is unclear. As it would probably be unrealistic to recommend that behavioural teratogenicity screening
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should be included as part of the reproduction toxicity study programme for all chemicals, a procedure for the selection of priority chemicals needs to be developed. Criteria for such a selection procedure are proposed: (a) indications of a potential nervous system involvement, (b) positive results from a pilot reproduction study, (c) bioactivity of the chemical, (d) expected human exposures and (e) available information on ecotoxicity. When behavioural testing is considered relevant, a well-accepted strategy is to perform tests at two levels of sensitivity and complexity. The most adequate approach for initial testing is to employ a test system that combines a wide exposure period with a series of tests, sampling a broad range of CNS functions. Furthermore, tests should overlap. This should facilitate confirmation of a positive finding by a Merent test, conducted under different conditions. Depending on the results of the initial testing phase it can be decided whether secondary testing, to be performed in a ‘tailor-madefashion’, is appropriate. References Adams, J. (1986)Methods in behavioral teratology. In E.P. Riley and C.V. Vorhees (Eds.), Handbook of Behavioral Teratology, Plenum Press, New York. Adams, J. and Buelke-Sam, J. (1981) Behavioural assessment of the postnatal animal: testing and methods development. In C.A. Kimmel and J. Buelke-Sam (Eds.), Developmental Toxicology,Raven Press, New York, pp. 233-258. Barlow, S.M.(1985)United Kingdom: Regulatory attitudes towards behavioural teratology testing. Neurobehav. Toxicol. Teratol., 7:643-646. Butcher, R.E.(1976)Behavioural testing as a method for assessing risk. Environm. Health Perspct., 18: 75-78. Butcher, R. E.(1985)A historical perspective on behavioural teratology. Neurobehav. Toxicol. Teratol., 7: 537-540. Grant, L. D.(1976)Research strategies for behavioural teratology studies. Environm. Health Perspect., 18: 85-94. Health Council of The Netherlands (1985)The evaluation of the teratogenicity of chemical substances. Report No. 1985/6E. Kimmel, C. A., Buelke-Sam, J. and Adams, J. (1985)Collaborative BehaviouralTeratology Study: implications, current
applications and future directions. Neurobehav. Toxicol. Teratol., 7:669-673. Koeter, H.B. W.M. (1983)Relevance of parameters related to fertility and reproduction in toxicity testing. Am. J. Znd. Med., 4:81-86. KoZIter, H.B.W.M. and Rodier, P.M. (1985) Behavioural effects in rats and morphologic and behavioural effects in mice after exposure to inhalant anesthetics during early development. Neurobehav. Toxicol. Teratol., 7: 676. Lorente, C. A., Tassinari, M. S. and Keith, D. A. (1981)The effects of phenytoin on rat development: an animal model system for fetal hydantoin syndrome. Teratology, 24: 169-180. Ministry of Health and Welfare, Japan (1984)Information on the guidelines of toxicity studies required for application for approval to manufacture (import) drugs. Notification No. 118 of the Pharmaceutical Mairs Bureau, Ministry of Health and Welfare. Needleman, H.L., Rabinowitz, M., Leviton, A., Luin, S. and Schoenbaum, S. (1984)The relationship between prenatal exposure to lead and congenital anomalies. J. Am. Med. ASSOC.,251:2956-2959. Rodier, P.M. (1976) Critical periods for behavioural anomalies in mice. Environm. Health Perspect., 18:79-83. Rodier, P.M. (1977) Correlations between prenatally induced alternations in CNS cell populations and postnatal function. Teratology, 16:235-246. Rodier, P.M. (1978)Behavioral teratology. In Wilson and Frazier (Eds.), Handbook of Teratology, Vol. 4 , Plenum Press, New York. Rodier, P.M. (1980) Chronology of neuron development: animal studies and their clinical implications. Dev. Med. Chiki Neurol., 22: 525-545. Rodier, P. M., Reynolds, S. S. and Roberts, W. N. (1979)Behavioural consequencesof interference with CNS development in the early fetal period. Teratology, 19: 327-336. Rodier, P.M., Aschner, M., Lewis, L.S. and Koeter, H.B.W.M. (1986)Cell proliferation in developing brain after brief exposure to nitrous oxide or halothane. Anesthesiology, 64: 680-687. Sobotka, T. J. and Vorhees, C. V. (1985)Application of behavioural teratology testing procedures. Neurobehav. Toxiwl. Teratol., 7: 665. Spijper Cranmer, J. and Goad, P.T. (1983)Validation of selected behavioral tests for evaluating low-level toxicity. In G. Zbinden (Ed.), Application of Behavioural Pharmacology in Toxicology, Raven Press, New York. Suter. K.E. and SchBn, H. (1985)Possibilities and limitations of current methods in behavioural teratology screening:an industrial view-point. In Proceedings of the symposium: ‘Aktuelle Probleme der Biomedizin’, Salzburg, September 18-20,1985. Tanimura, T. (1984)Prospects of the reproductive toxicology with special referenceto the developmentalhazards due to
67 the treatment at the stages from prefertilization to implantation. Congenital Anomalies, 24: 319-328. Vorhees, C. V., Brunner, R. L., MacDaniel, C. R. and Butcher, R.E. (1978) The relationship of gestational age to vitamin A induced postnatal dysfunction. Teratology, 17: 27 1-276. Vorhees, C.V. and Butcher, R.E. (1982) Behavioural teratogenicity. In K. Snell (Ed.), Developmental Toxicology, Croom Helm, London, pp. 247-298. W. H.O. (1984) Principles for evaluating health risks to progeny associated with exposure to chemicals during pregnancy. Environm. Health Criteria, 30. W. H. 0. (1986) Draft Guidelines for the assessment of drugs and other chemicals for behavioural teratogenicity, WHO Regional Office for Europe, Copenhagen.
Discussion A. J. Friedhoff We know that very many non-psychotropes as well as all psychotropic drugs enter the brain, and it is likely that many of both types of drug affect brain development in ways we do not yet know. As we find out more about behavioral teratogenicity of drugs that enter the brain it is going to be necessary to learn now to differentiate among positive, negative and neutral effects, or we will probably have to ban very many drugs, because they have some effect on development. H.B.W.M. K&ter: Behavioral effects should never be extrapolated from the period of development to the adult situation. Since behavioral teratogenicity studies are only part of the developmental toxicity study program for the compound, and, moreover, even reproductive toxicity is only one aspect of the toxicology profile, there is always more information needed and often available. Only on the basis of this full toxicity profile should a compound be evaluated.
T.D. Yih: A number of drug classes were mentioned that
should be submitted to behavioral teratological testing, among which are CNS-active drugs. Do you mean drugs developed for treatment of CNS disorders or drugs which affect CNS physiology (eg. neurotransmitter levels)? The majority of newly developed drugs have to some extent such side effects. Should they all be tested? H.B.W.M. K&ter: In my opinion all drugs intended to be active at the CNS level should indeed be tested. It should be emphasized, however, that results of behavioral tests should only be evaluated within the context of the full toxicological profile. P.M. Rodier: Where guidelines are flexible, will regulators emphasize the use of positive controls and soforth, to ensure that tests have some validity? H.B.W.M. K d t e r : To date, not so many compounds have been evaluated by regulatory authorities. To my knowledge, there are no requirements as to positive controls or other means to ensure the validity of the tests applied. Authorities should be encouraged to implement such controls. C. V. Vorhees: I agree that more information is needed on the relevance of animal to human behavioral change. However, I would not like to have your comments leave the impression that this problem is any more difficult than with malformations, or cancer, where also one cannot precisely extrapolate specific effects from animals to human. H.B.W.M. Koeter: Although your argument is valid, I feel that upon the introduction of any new discipline in toxicology the same mistake (made some 25 years ago when the majority of toxicity test methods were introduced) of being merely descriptive should not be made again. As in pharmacology, one should strive after understanding mechanisms rather than describe gross findings only.