Role of maternal toxicity in assessing developmental toxicity in animals: A discussion

REGULATORY

TOXICOLOGY

AND

PHARMACOLOGY

16,189-201 (1992)

Role of Maternal Toxicity in Assessing Developmental in Animals: A Discussion DANIEL L. BLACK’ AND THOMAS

Toxicity

A. MARKS

Safely Pharmacology, The Upjohn Company, Kalamazoo, Michigan 49001

Received April 13, 1992

The beliefthat any drug or chemical, when administered at a high enough dose, can be expected to produce fetal malformations is not consistent with the facts. However, the stress associated with maternally toxic doses can be expected to result in associated, often transient, fetal abnormalities that may not be the result of deviant organogenesis. Sometimes the toxicity toward the pregnant animal, including her embryos/fetuses since they are hardly in a sanctuary, is severe enough to result in resorption of the embryo or abortion of the fetus. Thus, it is possible that the embryolethality and other indications of developmental toxicity, produced by some drugs and chemicals, may be the result of a mechanism(s) other than selective toxicity toward the embryo. Also, some test materials have been shown to affect maternal homeostasis, thereby disrupting support to the embryo, without causing significant overt toxicity to the embryo or dam; e.g., the endocrine system of the dam is altered. Routine testing has thus far revealed a relatively limited number of true teratogens, although a large number of drugs and chemicals have resulted in fetal effectssuch as developmental variations when administered at doses that approach lethal levels. Such effects on the fetus should be expected when the maternal animals are stressed by the high dosages usually employed. A better understanding of the etiology and biological relevance of the embryo/fetal deviations often seen in developmental toxicology studies might help to avoid the sometimes unjustified withholding of potentially useful drugs and chemicals from the marketplace. 0 1992 Academic

Press, Inc.

INTRODUCTION

Although much of the controversy surrounding the employment of high doses in drug/chemical safety studies has focused on carcinogenicity studies (Carr and Kolbye, 199 1; McConnel, 1989), such arguments also pertain to developmental toxicity studies. As in carcinogenicity studies, regulatory guidelines for developmental toxicology studies (U.S. EPA, 1989; U.S. FDA, 1982) emphasize the importance of employing the maximum tolerated dose (MTD). Thus, most developmental toxicity studies are performed at doses that are very close to toxic levels. The difficulty in extrapolating the results of studies employing high doses to the lower pharmacological doses employed in the ’ To whom correspondence should be addressed at Safety Pharmacology, The Upjohn Company, 7224300-4, Kalamazoo, MI 4900 1. 189 0213-2300192 $5.00 Copyi& 0 1992 by Academic Press, Inc. All rights of reproduciion in any form reserved.

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clinics, or to chemical exposure levels found in the environment/workplace, is generally recognized. No generally accepted formula for relating the no observed adverse effect level (NOAEL) found in animals to potential risk in humans has been identified. Since we are dealing with such an inexact science, every effort should be made to avoid imprecise labels and to use qualifiers and descriptive statements when interpreting developmental toxicology studies in animals. In order to maximize the chances of finding signs of developmental toxicity, while establishing as high a NOAEL as possible, in our laboratory we routinely employ the MTD and multiples of the MTD, usually 0.5 and 0.25 (Marks, 1985). At least 90% of the more than 150 pharmaceuticals and chemicals that we have tested over the past 15 years were not teratogenic in standard Segment II teratology studies in mice and/ or rats and/or rabbits. In fact, only four pharmaceuticals have shown evidence of teratogenicity in rats at The Upjohn Company in the past 12 years; none of the substances tested was found to be teratogenic in rabbits. However, what we have classified as indications of “fetotoxicity” (Black and Marks, 1986) have frequently been observed in these studies at the MTD. Thus, we have no reason to conclude that we are finding a high number of teratogens. If other companies doing developmental toxicity studies are experiencing similar results, there clearly is little concern that large numbers of animal teratogens are being identified by the pharmaceutical industry. However, the term developmental toxicity (Wilson, 1977) has come to be more frequently employed in teratology studies than the word teratogenicity. Although we do not wish to discourage this practice, we feel it is important that some effort be made to prevent the phrase “developmental toxicant” from becoming “buzzwords,” as has been the case with the word “teratogen” (Johnson and Christian, 1984). The possible role of maternal toxicity in teratogenic findings (Khera, 1984, 1985) has met with much resistance (Kimmel et al., 1987). However, since what we call fetotoxicity (Black and Marks, 1986) can be classified as growth retardation, and thus one of the four manifestations of abnormal development, and since it has been our experience that such indications of developmental toxicity can be expected at doses that approach maternally lethal levels, it is important that a substance not be termed a developmental toxicant before thoroughly addressing the effects of the test substance on the maternal animal during her pregnancy. One must remember that virtually everything is toxic at some level and that exposure levels are important as all substances can be expected to have thresholds for developmental toxicity. The term stress has been commonly used to describe various phenomena. Thus, stresses include a variety of physical, psychological, chemical, or infectious influences that may be modified by intrinsic and extrinsic factors. However, in this discussion this term has been used to describe an adaptive response by an animal to threats to its homeostasis (Dohms and Metz, 199 1). Here we are primarily concerned with indirect effects on in utero development that result from the maternal animal’s attempts to return to normal homeostasis after being exposed to a substance at a toxic level. As Khera (1984) has pointed out, “any serious alteration in maternal homeostasis or physiology during pregnancy is liable to influence embryonal and/or fetal development. It is well accepted that almost any test chemical given at high doses could, by altering maternal homeostasis, adversely affect fetal development.” Other papers also have discussed the link between maternal toxicity and stress to dose-related increases in embryotoxicity and fetal abnormalities (Chernoff et al., 1989; Khera, 1985, 1991; Johnson and Christian, 1984). However, maternal effects produced by test sub-

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stances are often overlooked in reporting fetal effects. Khera (1984) pointed out that 33 of the 85 studies that he evaluated did not present data on maternal toxicity that would be sufficient for meaningful appraisal. Although one could assume that there were no indications of maternal toxicity in any of these 33 studies, it is likely that at least some of the authors cited in the above manuscripts believed that the maternal effects were not important, particularly when there were fetal effects on which to focus. It is hoped that such a viewpoint is no longer widely held, as evidenced by the recent consensus workshop and the subsequent publications in the special issue (Volume 7, Number 3, 1987) of Teratogenesis,Carcinogenesisand Mutagenesis on the evaluation of maternal toxicity. As emphasized in manuscripts coming out of this workshop, as well as in another recent publication (Hood, 1989), a substance’s NOAEL for developmental toxicity is an important element in assessing its potential risk to the human conceptus; likely human exposure is another important element in assessing risk. However, for synthetic chemicals that cannot be classified as drugs or pesticides, reproductive and developmental toxicology studies may not be necessary in most cases (Johnson, 1987; Marks, 199 1). Thus, if people of reproductive potential can be expected to be exposed to such a chemical only at a level significantly below the NOAEL in an acute toxicity test on the substance, then the extremely low risk to the human may make further testing unnecessary. We suggest that the risk is de minirnis if exposure levels to such chemicals are less than one-tenth of the NOAEL. INFLUENCE OF MATERNAL TOXICITY ON DEVELOPMENTAL TOXICITY It is likely that, in at least some developmental toxicity studies, the maternal effects resulting from exposure to substances at high doses may be the only direct effect of the test substance, with the embryonic/fetal effects being secondary to disruptions in maternal homeostasis. Since the embryos/fetuses are generally at least as susceptable to toxic insult as the maternal host, one would expect that they also would be experiencing comparable and even worse effects. Animals, intoxicated as the result of exposure to a test substance, can be expected to be less efficient in normal cellular pr3cesses while their tissues are taking steps to eliminate a toxic substance and “rep$iring” any pathological and/or physiological damage in an attempt to return to normalcy. During the evolutionary process it is likely that survival of the maternal animalwould take precedence over survival of the developing embryo/fetus; otherwise both would be lost. Pregnant animals that lose their developing embryo/fetus can be expected to become pregnant again if they survive their exposure to a toxic substance. Thus, it isnot surprising that deprivation of the food supply can, in the absence of exposure to toxic substances, induce fetal wastage and/or fetal abnormalities (Kalter and Warkany, 1959; Shrader and Zeman, 1973). In spite of the evidence relating drug/chemical-induced maternal effects to adverse embryonic/fetal effects, there is little evidence that maternal toxicity necessarily results in teratogenicity. However, drugs and chemicals may be judged to be teratogenic on the basis of the total array of fetal effects without sufficient regard to the mechanisms involved in the effects seen, or the confounding toxicity toward the maternal animal. A teratogenic effect should be interpreted as a defect in normal organogenesis that results in one or more permanent structural alterations, and/or defective functioning

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of an organ or tissue, and sometimes embryonic death as a consequence of such abnormalities. The commonly observed fetal abnormalities seen in many developmental toxicology studies appear not to be the result of direct effects on organogenesis, but rather the consequence of the toxicity resulting from exposure to a substance at high doses. Such anomalies include delayed normal bone and organ development, as well as reduced fetal body weight. Unfortunately, the lack of a consistent categorization scheme sometimes leads to a consolidation of all fetal abnormalities as the result of their having been given equal importance with respect to their teratologic significance (Black and Marks, 1986). The substitution of the phrase “developmental toxicant” in such studies may appear to some to be an appropriate compromise. However, as long as such studies are carried out at doses that are between 10 and 100% of a lethal level for at least some of the exposed maternal animals, any resultant developmental effects should be closely weighed against maternal effects. It has been our experience that drugs and chemicals that are tested at maternally toxic levels often affect the developing embryo/fetus in some way. Such effects are generally observed when the maternal animal has been significantly stressed by highdose intoxication, especially if the slope of the dose-response curve is not steep. However, it is not unusual for such increases in the incidence of effects on the embryo/ fetus to be interpreted as evidence of “teratogenicity” or at least developmental toxicity. Fetal effects that are commonly associated with maternal toxicity include the following: reduced fetal body weight, delayed ossification, and delayed organ development, e.g., renal papillae (Woo and Hoar, 1972), bent ribs, associated with delayed ossification and the pull of developing musculature (Khera, 198 l), embryolethality unassociated with malformations, and “genetic” variations such as accessory ribs (Kavlock et al., 1985; Chemoff et al., 1987). Some investigators have further divided such abnormalities according to the stage of development in which they were seen. For example, embryolethality might be considered to be an embryotoxic effect, while developmental delays such as reduced fetal body weight and incomplete ossification might be considered fetotoxic effects (Black and Marks, 1986). Although the testing of a drug or chemical at a level that produces overt maternal toxicity may lead to treatment-related increases in one or more fetal variations, this is not always the case and the same indicators of developmental toxicity may not be consistently observed (Chernoff et al., 1990). However, a drug/chemical should not be classified as a potential human developmental hazard if adverse effects on the conceptuses are found only at doses that are maternally toxic, unless women are regularly exposed to toxic levels of the substance, e.g., alcohol. In fact, exposure to substances that are relatively less acutely toxic can be expected to result in signs of developmental toxicity, when given to pregnant animals at doses between 10 and “lOO”% of a lethal level, in contrast to substances that are highly potent; exposure to the latter substances often results in embryolethality and maternal lethality over a narrow dose range, with few surviving fetuses to examine. Thus, studies on substances that do not have a sharp dose-effect curve often result in indications of developmental toxicity since the offspring are more likely to be alive when the uterus is opened. For such substances, we should expect to see developmental delays in offspring of significantly intoxicated pregnant animals because the embryo and fetus generally are more vulnerable to toxicity than the maternal animal due to the immaturity or absence of defense systems, including metabolic enzymes. Thus the pregnant animal may be able to handle high levels of less acutely toxic substances, whereas her embryos/fetuses cannot.

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Developmental toxicologists often have to evaluate the hazard potential of drugs and chemicals that, in animal tests, result in delays in normal fetal development at dose levels that also produce signs of maternal toxicity. In such situations, we should not ignore the probability that when the dam’s normal primary pathways for detoxification and excretion of a chemical are saturated, secondary pathways come into play, and the type and concentration of metabolites may differ from those produced by the primary pathways (Subcommittee on Food Technology, 1990). Thus, even when there is metabolic equivalency across species, the result of such exposures may not truely form a basis for predicting effects at the lower concentrations typical of human use levels. Test results, especially those found at the maximum tolerated dose and higher, may be misleading. Therefore, one must keep in mind that adverse effects, observed only at high doses, are not valid for high-to-low dose extrapolation. In the absence of clear dose-response effects and/or comparable effects in a second species, we shouldn’t be overly concerned about indications of a delay in fetal development/ increases in fetal variations unless we also see increases in the incidences of permanent structural and/or functional alterations, and/or behavioral abnormalities, and/or reproductive deficiencies, especially if found in the absence of overt maternal effects. CLASSIFICATION

OF FETAL

ANOMALIES

For some time many developmental toxicologists have segregated fetal abnormalities into categories according to the perceived adverse affects on the survivability of the offspring (Kimmel and Wilson, 1973). However, others continue to tabulate all fetal effects together. We do not believe the latter method of tabulating fetal abnormalities is appropriate since exposure to any chemical or drug at a high enough dose can be expected to result in fetal effects of one kind or another. Although we do not believe that all drugs or chemicals are potential teratogens (Karnofsky, 1965), it is likely that rigorous testing of any substance will result in increases in at least one of the four manifestations of abnormal development (Wilson, 1977) and thus could be classified as a developmental toxicant. Therefore, we feel that it is important that developmental toxicology study reports include detailed information on maternal effects while categorizing fetal effects according to level of concern, i.e., variations, malformations, functional effects, deaths. Although we do not encourage the use of the term malformation to describe any and all developmental anomalies, including transient developmental delays in normal organogenesis, we certainly recognize that the occurrence of malformations, as opposed to developmental delays, may be the result of embryonic/fetal-specific “teratogenic” effects, not attributable to maternal toxicity. However, few substances truly are teratogenic such that exposure results in permanent fetal malformations, with or without maternal effects. Those of us who routinely perform range-finding studies in pregnant animals usually include doses that are highly toxic, even lethal to some of the animals. In such studies, severe maternal toxicity usually does not result in an increased incidence of malformations, although dose-related increases in the incidences of fetal variations are almost always seen (Black and Marks, 1986). BENT (WAVY)

RIBS: VARIATION

OR MALFORMATION?

Some anomalies fall into a gray area in that they could be categorized as a malformation or a variation. For example, some developmental toxicologists consider bent

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or wavy ribs to be a malformation resulting from defective embryogenesis, since the architecture of the bone has been altered and the defect appears to be permanent. However, other investigators consider that wavy ribs are the manifestation of nonspecific damage to the fetus after exposure to maternally toxic doses because of the large variation in chemical structure and biological activity of compounds that produce them (Joosten et al., 1980; Khera, 198 1). Such effects may be related to a developmental delay in rib ossification associated with maternal toxicity (Khera, 1970). Support for this latter classification is the finding that bent ribs, apparent at birth, often straighten out by weaning (Nishimura et al., 1982). However, Aliverti et al. (1980) reported that glycerol formal administration resulted in wavy ribs in rat fetuses in the absence of any overt maternal toxicity. Myometrial constriction also has been reported as an inducer of wavy ribs in rat fetuses (Nakatsuka, 1988). We would be inclined to classify multiple, moderately to severely, bent ribs as a malformation while classifying anything less in this category to be a variation. We would be very reluctant to call a substance a teratogen if bent ribs was the only “malformation” found, especially if found only in the group receiving the maximum tolerated dose. GENETIC

VARIATIONS

Another area of contention involves anomalies that often are termed “genetic variations.” These variations are not transient, as are developmental variations, but occur at a high incidence in untreated control animals and appear to be related to the genetic characteristics of the species or strain used. Severe maternal toxicity can be expected to influence the expression of these effects, as increases in the incidences of variations are frequently observed at toxic dose levels. However, Kimmel and Wilson (1973) suggest a possible link between increases in some of these variations to possible teratogenicity, i.e., the occurrence of malformations at a higher, as yet untested, dose level. Other investigators suggest that such abnormalities are a response to nonspecific maternal toxicity and/or stress and thus are similar in etiology to developmental delays (Beyer and Chernoff, 1986; Chernoff et al., 1987; Kavloch et al., 1985; Kenel et al., 1984). TOXICITY

AND CELLULAR

HOMEOSTASIS

Although malformations probably are the result of a disruption(s) in normal organogenesis, most likely at the cellular or molecular level, indirect effects also have been implicated as possible causes of malformation, including temporary interruption of the blood supply to the pregnant uterus (Brent, 1990) and stress (Scialli, 1988). Certainly the stress associated with exposure to toxic or near-toxic levels of any substance can be expected to affect the homeostasis of pregnant, as well as nonpregnant, animals. Alterations at the cellular, let alone the molecular, level usually are not reported in developmental toxicology studies, although the relationship between cellular ion deregulation and acute and chronic toxicity has been studied (Orrenius et al., 1988; Trump et al., 1989); such effects would be expected to also influence embryonic development. Recent research on the role of intracellular pH in the production of malformations has led to the hypothesis (Scott et al., 1987) that disruptions in intracellular

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homeostasis can all&t embryogenesis. Khera (199 1) also has studied the possible development of fetal anomalies as the result of chemically induced alterations in maternal homeostasis traceable to maternal acid-base-electrolyte imbalance and histological changes in the matemal/extraembryonic tissues. On the basis of his studies, he hypothesized that maternal factors such as metabolic acidosis, hyperosmolality, hemorrhages in the ectoplacental cone, and necrosis of decidua basalis may directly or indirectly reduce fetal nutrition and matemoembryonic gaseous exchange, ultimately resulting in altered fetal development. Drug-related pharmacological effects on the dam have been shown to result in fetal malformations. For example, vasodilators may decrease uteroplacental blood flow and result in digit malformations (Danielsson et al., 1990). Prostaglandin E, (alprostadil) administration (s.c.) also has resulted in congenital anomalies at levels that also had been shown to cause uterine contractions and likely to retard uterine blood flow (Marks et al., 1987). Although substances known to be mutagenic have been found to be teratogenic, many substances that have shown evidence of mutagenicity and/or carcinogenicity have not been shown to cause malformations. In addition, mutagens that are teratogenic may act via cytotoxicity to cells in the target organ (Giavini, 1988). Such teratogenic effects probably are not the result of permanent genetic changes that persist and affect embryonic development for many cycles (Beckman and Brent, 1984). If most fetal malformations were the result of cytotoxicity and/or interference with programmed cell death, one would expect more generalized tissue destruction. However, malformed fetuses typically display effects involving particular organs or anatomical areas while other locations appear developmentally unaffected. This phenomenon is possibly the result of cells acquiring stage-specific properties during embryogenesis (Chandebois and Brunet, 1988). Malformations among organs need not be obvious by visualization. Some malformations may be so specific as to have affected only certain cell types without distorting the external architecture of the organ involved. For example, malformations could result in altered metabolic pathways, or abnormal endocrine function, and may be detectable only by functional changes and/or adverse reproductive performance in the offspring. Such malformations, although unusual, hopefully will be detectable in the behavioral and reproductive tests that are included in some of the developmental toxicology protocols presently employed. However, delays in the achievement of developmental landmarks including pinna detachment, eye opening, etc., generally correlate with reduced pup body weight (Christian, 1989; Lochry et al., 1986). Thus, since reduced fetal/neonatal weight often results from severe maternal toxicity, and is generally transient, we believe that these effects represent delayed normal development. Such effects should be considered as evidence of selective developmental toxicity only in the absence of maternal toxicity. STRESS AND HEAT

SHOCK

PROTEINS

A prime example of how exposure to toxic and near-toxic levels of a substance can lead to major disruptions in homeostasis, and in some instances teratogenicity, involves the release of stress proteins. For example, hyperthermia in pregnant animals has been shown to lead to terata (Edwards, 1986). Heat shock proteins also have been shown to be released during the stress associated with other insults (Lindquist and Craig, 1988). Thus, the stress involved with exposure to cadmium (Kapron-Bras and Hales,

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199 l), retenoic acid (Anson et al., 199 I), and other substances known to be teratogenic (Li et al., 1982) appears to result in the release of stress or heat shock proteins. Although other factors may be involved in thermotolerance (Boon-Niermeimer et al., 1987) it appears certain that the stress associated with exposure to toxic, or near-toxic, levels of at least some of the substances/conditions identified as being teratogenic does lead to biomarkers, indicating that the stressed animal is attempting to deal with the situation on the cellular, if not the molecular, level. Although mildly elevated temperatures are cytotoxic to cells and the cell-killing effect of many anticancer drugs is vastly enhanced at elevated temperatures, cells also can be made transiently heat resistant by exposure to heat itself or to a variety of other environmental stresses (Meyer et al., 1991). Hyperthermia has been shown to affect a variety of cellular structures and functions, causing changes in mitochondria and nuclear structure, denaturation of soluble and membrane proteins, the disassembly of actin microfilament bundles, and alterations in major biochemical pathways (Carper et al., 1989). The heat shock proteins that have been associated with hyperthermia are just one group of stress proteins, the other group being glucose-regulated proteins, which exhibit increased synthesis in cells either deprived of glucose or oxygen or following treatment with agents that perturb calcium homeostasis (Welch et al., 1989). These relatively acidic polypeptides generally are synthesized at moderate or even high levels in cells incubated under normal growth conditions, but in cells experiencing stress, these proteins stand out as the syntheses of most of the proteins normally being produced in the unstressed cell are curtailed. Since exposure of cells to mild sublethal heat shock, leading to recovery at normal temperature, results in the cells acquiring resistance to a second, otherwise lethal, shock, the resultant thermotolerance suggests that such cells have attained the ability to function normally under conditions that are toxic to the cell (Welch et al., 1989). Similarly, heat has been shown to induce tolerance to the cytotoxic effects of drugs such as Adriamycin (Li et al., 1982) and Bleomycin (Hahn et al., 1989). Thus, it is apparent that even individual cells are capable of protecting themselves against stress. It is also clear that such stress leads to major alterations in normal cellular homeostasis. As pointed out earlier in this article, one would expect the maternal animal to be better able to deal with stress than the developing embryo. Thus, one would expect that most agents with steep dose-response curves would tend to kill the developing embryos at doses that are highly toxic to the maternal animal, whereas substances that are less acutely toxic would be more tolerable to the embryo, as well as to the maternal animal, but still capable of causing malformations that may or may not result in embryolethality. However, one would further expect that environmental or pharmacological exposure to such substances, at levels likely to be encountered by humans, would not generally be teratogenic. TERATOGENICITY

AND EMBRYOLETHALITY

Teratogenicity is only one of the indicators of developmental toxicity; thus, the occurrence of in utero deaths, especially in the absence of maternal toxicity, is of equal concern. However, a significant treatment-related increase in embryonic deaths is not necessarily a teratogenic effect, related to drug- or chemical-induced defects in organogenesis, if no concurrent malformations are present. Although severe malformations

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can be expected to result in embryonic death, malformed fetuses often survive until term. In fact, exposure of pregnant animals to some teratogens may result in severe fetal malformations without producing any embryonic death. For example, cyclophosphamide, when administered to pregnant mice, has been shown to produce severe malformations, including cleft palate, absent/malformed digits, micromelia, exencephaly, and fused/malformed bones, in more than 70% of the fetuses without producing a corresponding increase in embryonic deaths and/or decreased litter size (Francis et al., 1990). In our laboratory we have found that drugs or chemicals, which produce significant embryolethality at levels that also produce substantial maternal toxicity, frequently do not also produce malformations in surviving embryos. Thus, when one observes embryolethality in the absence of identifiable malformations in any surviving fetuses and when concurrent evidence of pronounced maternal toxicity is found, one may consider that the embryolethal effects were consistent with maternal effects; i.e., the effects probably were not selective to the embryo. Although the biological significance may be of equal concern, because of their disparity we feel that malformations and embryolethal effects should not be equated when assessing the developmental toxicity of a substance, unless the effects on the conceptuses occur at doses that do not result in signs of maternal toxicity. EMBRYOTOXICITY:

SELECTIVE

VERSUS

SECONDARY

One often assumes that embryotoxicity is the result of a direct effect on the embryo by the drug or chemical being tested. Embryo-specific toxicity can be expected to result in cytotoxic, or other, effects including teratogenicity. However, as mentioned above, embryotoxicity can be the result of secondary effects related to maternal effects, either toxic or consistent with the pharmacologic actions of the agent on the dam, rather than direct toxicity towards the embryo. Thus, if a drug or chemical is administered at a level that is high enough to threaten the dam’s survival, embryonic resorption, or fetal death and/or abortion could result. However, exposure to a substance may result in embryotoxicity in the absence of overt maternal toxicity. In the face of such findings, the decision to discontinue further development of the compound because of its perceived developmental toxicity potential may result. However, such effects may not be related to selective developmental toxicity, but may have been a manifestation of the maternal effects, such as altered maternal endocrine homeostasis resulting in a disruption in hormonal support of the embryo. For example, administration of the immunomodulators bropirimine (Marks et al., 1990) or tilorone has resulted in embryolethal effects, apparently by disrupting the synthesis and/or release of progesterone by the corpora lutea, as such effects can be prevented by exogenous administration of progesterone (Black et al., 199 1; Terry et al., 1992). Interestingly, at least with bropirimine, these effects occurred at dosages that produced only transient, non-life-threatening, maternal toxicity. Thus, exposure to drugs/chemicals may lead to embryonic death without any direct effect on organogenesis or even pronounced maternal toxicity. ASSESSING

POTENTIAL

HUMAN

RISK

The primary reason for conducting developmental toxicology studies in animals is to obtain information that can be used to assess the potential risk of a particular

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substance to the developing human embryo/fetus/neonate. It is important when assessing risk that each of the four manifestations of developmental toxicity (death, malformation, growth retardation, and functional deficit) be monitored and that each developmental anomaly should be carefully categorized, i.e., malformation vs developmental or genetic variation. Magnitude of difference is estimated between the lowest observed adverse effect level (LOAEL) and the expected exposure level or therapeutic dose In addition, one should determine the A/D ratio: The lowest dose causing toxicity in the pregnant adult divided by the highest dose causing developmental toxicity (Johnson, 1984). The lack of a dose-response effect, especially when the developmentally toxic dose is between 10 and 100% of the LDso for the test substance, is an important factor in assessing risk. Whether or not the substance is a developmental toxicant in two or more species is also important. Finally, information on the mechanism for developmental toxicity is highly valuable in risk assessment. It is hoped that, in the process of assessing risk, the marketing of potentially useful substances will not be unnecessarily delayed or curtailed. Decisions to suspend commercial development or to require further testing can be expected if one ignores the probability that any substance can be expected to show evidence of developmental toxicity, at some dosage, if maternal toxicity is not considered and if all embryonic/ fetal/neonatal effects are given equal biological consideration. Since regulatory agencies generally recommend that substances subjected to developmental toxicology studies be tested, such that at least the high-dose level demonstrates maternal toxicity, it becomes very difficult for a new agent to undergo such testing without showing some developmental effects. However, regulatory agencies are aware of this likelihood (Chernoff et al., 1989); thus, commercial development should not be suspended on the sole basis that a substance gave indications of developmental toxicity at doses close to those that are maternally toxic. The occurrence of malformations in animal studies does not necessarily indicate a threat to human in utero development, especially if such effects occur in the presence of significant concurrent maternal effects and the margins of exposure (Hart, et al., 1988) are acceptable. Thus, although pregnant women may encounter some substances such as alcohol at intoxicating levels, most substances found to be teratogenic in animals, including aspirin, caffeine, and vitamin A, are likely to be a threat to humans only if taken at doses far above normal exposure levels (Marks, 199 1). In our opinion, a substance has a high potential of being a human teratogen primarily on the basis of the following: (1) there is a dose-related statistically significant increase in the incidence of fetal anomalies, as compared to the vehicle control group, in an experimental animal model; (2) at least one of the fetal anomalies seen in animals is the result of deviant embryogenesis and is likely to be permanent; (3) acute maternal toxicity, at dosages found to produce fetal malformations, is not substantial, or humans can be expected to be regularly exposed to toxic levels; and (4) human exposure levels can be expected to be greater than one-tenth of the NOAEL for developmental toxicity in the most sensitive species. SUMMARY

AND CONCLUSIONS

Some developmental toxicologists believe that any drug or chemical, when administered at a high enough dose, will produce fetal malformations, as well as other adverse

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effects on fetal development. However, although the presence of fetal anomalies suggests a selective effect on in utero development, we feel that such selectivity does not occur after test exposure to most substances. We believe that in experimental animal studies, many of the fetal abnormalities that result from exposure to drugs and chemicals during pregnancy are the result of alterations in homeostasis in the embryo/fetus and/ or pregnant animal associated with dosages that show evidence of maternal toxicity. Such fetal abnormalities are often transient and are seldom the result of deviant organogenesis. Sometimes the resultant toxicity is so severe as to lead to in utero deaths, resorptions, and even abortions of the embryos/fetuses. Additionally, the pharmacological actions of some drugs may interfere with normal maternal support to the developing embryos. Categorizing individual fetal abnormalities according to what is known about their etiology and biological relevance, and taking into consideration the confounding influence of any maternal toxicity lead us to conclude that, at usual human exposure levels, very few of the substances reported to be developmental toxicants in animals are likely to be a threat to the human conceptus. This conclusion is supported by the finding that only 30 of the 1200 substances that produced “congenital anomalies” in experimental animals are known to cause defects in the human (Shepard, 1989). In many cases, the adverse fetal effects observed in developmental toxicity studies are developmental variations that should have been expected when maternal animals are stressed by the high dosages employed. By segregating fetal abnormalities according to the criteria discussed in this paper, we might avoid unnecessarily discontinuing the development of potentially useful drugs and chemicals, while more accurately characterizing any potential developmental hazards. Additionally, by studying the etiology of particular embryonic/fetal effects, such as embryonic death, a better understanding of the mechanism of action of the agent being tested could result. REFERENCES ALIVERTI, V., BONANOMI, L., GIAVINI, E., LEONE, V. G., AND MARIANI, L. (1980). Effects of glycerol formol on embryonic development in the rat. Toxicol. Appl. Pharmacul. 56,93-100. ANSON, J. F., LABORDE, J. B., PIPKIN, J. L., HINSON, W. G., HANSEN, D. K., SHEEHAN, D. M., AND YOUNG, J. F. (199 1). Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice. Teratology 44, 19-28. BECKMAN, D. A., AND BRENT, R. L. (1984). Mechanisms of teratogenesis. Annu. Rev. Pharmacol. Toxicol. 24,485-500. BEYER,P. E., AND CHERNOFF,N. (1986). The induction of supernumerary ribs in rodents: Role of maternal stress. Teratogen. Carcinogen. Mutagen. 6, 4 19-429. BLACK, D. L., AND MARKS, T. A. (1986). Inconsistent use of terminology in animal developmental toxicology studies: A discussion. Teratology 33, 333-338. BLACK. D. L., MARKS, T. A., BRANSTETTER, D. G., AND KIRTON, K. T. (1991). Reversal of bropirimine developmental toxicity with progesterone. Toxicol. Appl. Pharmacol. 108, 12 1-l 28. BOON-NIERMEIJER, E. K., SOUREN, J. E., AND VANWIJK, R. (1987). Thermotolerance induced by 2.4dinitrophenol. Int. J. Hyperthermia 3, 133-141. BRENT, R. L. (1990). Relationship between uterine vascular clamping, vascular disruption syndrome and cocaine teratogenicity. Teratology 41, 757-760. CARPER, S. W., HARARI, P. M., FULLER, D. J. M., AND GERNER, E. W. (1989). Biochemical and cellular responses to hyperthermia in cancer therapy. In Stress-Znduced Proteins (M. L. Pardue, J. R. Feramisco. and S. Lindquist, eds.), pp. 247-256. A. R. Liss, New York. CARR, C. J., AND KOLBYE, A. C., JR. (1991). A Critique of the use of the maximum tolerated dose in bioassays to assesscancer risks from chemicals. Regul. Toxicol. Pharmacol. 14, 78-87.

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