Cross-species extrapolations and the biologic basis for safety factor determinations in developmental toxicology

REGULATORY

TOXICOLOGY

AND

PHARMACOLOGY

8,22-36 (1988)

Cross-Species Extrapolations and the Biologic Basis for Safety Factor Determinations in Developmental Toxicology E.MARSHALLJOHNSON Department ofAnatomy. Daniel Baugh Institute, Jefferson Medical College, 1020 Locust Street, Philadelphia, Pennsylvania 19107

ReceivedAugust 10. 1987

Designations of agents as teratogenic or nonteratogenic often are inaccurate, as adverse effects are more a reflection ofthe timing and severity oftreatment during pregnancy than agent nature. Careful consideration of both the similarities and the differences between developmental effects in animals and humans and the extent and nature of the data available are essential for protection of the human conceptus. Animal surrogates prove reliable predictors of human developmental effect levels. When the data are evaluated consistent with contemporary concepts of developmental toxicity, for example, where the effect in the embryo is only seen at maternally toxic doses and exposure is below the adult toxic level, relatively modest safety factors are sulficient for safe cross-species extrapolation. Developmental toxicity safety factor magnitude is predicated on data quality and the fact that thresholds of effect exist in mammalian pregnancy. Safety of human concepti is achieved by considering both the developmental hazard index of the chemicals in question and the severity of exposure. o 1988 Academic F-KS, 1~.

I. INTRODUCTION

AND

BACKGROUND

Developmental toxicology is a relatively new field with few precedents for quantitative evaluation of data. It generally is considered that thresholds exist for developmental effects and that there are exposure levels below which no adverse outcomes are expected (Wilson, 1973; Staples, 1974) but quantification of effects above the threshold generally is not attempted (Johnson, 1983a). Definitive quantification of teratogenic potency has been attempted on the basis of an agent’s ability to cause abnormality only at a specific developmental stage versus those that produce effects when applied at any of the multiple stages. This is a difficult and less than adequate undertaking, at least in part because of complex interspecies and intertest pharmacologic considerations (Neubert et al., 1980). Some success at cross-species develop mental toxicity quantification has, however, been obtained by comparing and contrasting levels of treatment needed to produce adverse effects in the conceptus with those affecting the mother (Johnson, 1980, 198 1, 1987; Fabro et al., 1982; Hart et al., 1987). The use of safety factors applied to the no-observed-effect level (NOEL) of a sensi22 0273-2300/88$3.00 CopyrigJa 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tive animal species is a recommended approach (Johnson, 1983a) currently used by the Food and Drug Administration (Frankos, 1985) and as recently as 1986 remained the recommended mode at an EPA-sponsored symposium/workshop (EPA, 1986). The purpose of this paper is to investigate the biologic basis for the use of safety factors in developmental toxicity safety evaluations. The Guidelines for Developmental Toxicology recently published by the U.S. Environmental Protection Agency ( 1986) typifies the pragmatic and potentially more useful melding of many contemporary understandings of abnormal in utero development. Central to this is that developmental toxicity, i.e., any adverse effect on any aspect of development rather than just terata, is now recognized as the best context in which to consider effects of chemicals on the conceptus. Teratogenesis manifested as delivery of live but gross anatomically abnormal young is considered but one of four classes of altered development. The specific cause or causes of most human congenital defects rarely is known. It has been estimated that less than 10% are due to environmental agents such as methyl mercury and radiation and an approximately equal number to gene mutations and chromosomal aberrations (Wilson, 1973). Causation of the remainder is unknown and may be due to multifactorial interactions (Kalter, 1965). One possible cause of congenital defects is the less-frequently discussed stochastic or randomly variable factors intrinsic to development. Ontogeny is one of the more complex of biologic phenomena requiring the timely synchronization of myriad known and unknown events that must interrelate quantitatively, qualitatively and spatially throughout the embryo in order for timely and normal development to occur. Random and uncontrollable variations of developmental patterns could themselves produce or interact with one another to divert an embryo into abnormal developmental sequences. Known human exposure to environmental chemicals and pharmaceuticals accounts for very few of the undesirable pregnancy outcomes, and it is reasonably concluded that human developmental problems will not be eliminated totally by regulatory controls placed on chemicals. Developmental toxicity may be manifested as a structural, functional, or biochemical abnormality that may not become evident until well after delivery. Developmental defects, ranging from those of little clinical significance to those that are potentially life-threatening, are evident in about 3% of the newborn. By the time they reach puberty, about 7% of all children are so affected. Thus, developmental toxicity is a health hazard with a high background incidence rate in humans. According to the March of Dimes Birth Defects Foundation ( 1985) over one million people of all ages are hospitalized each year as a result of developmental problems. Examination of the incidence of a readily ascertained and easily reported birth defect such as an open neural tube illustrates that the occurrence of developmental defects varies with geographic area (CDC, 1982; Fraser et al., 1986), race, and sex (Khoury et al., 1982). Recent data from CDC indicate there is no overall trend, either increasing or decreasing, in the frequency of congenital abnormalities. Some endpoints appear to be increasing, while others are decreasing over the past decade. By the dictionary definition, teratology is the study of monsters or gross anatomically abnormal offspring, but the term was placed into a more contemporary and useful context for safety evaluations when Wilson (1973) considered that there are four manifestations of abnormal development: (1) death of the products of conception, (2) birth of structurally abnormal offspring, (3) decrements of anticipated post-

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natal functional ability, and (4) developmental delays. Death of the human conceptus usually is followed by spontaneous abortion, but the common laboratory animals tend to resorb rather than abort dead concepti. Structural abnormalities range from major malformations such as cleft palate or exencephaly to minor malformations of the ribs, some which may be more appropriately classified as developmental delays. Altered postnatal function is most commonly considered as alteration of normal behavior due to a CNS defect (Butcher et al., 1975), but effects on other functional abilities, e.g., intestinal (Christian, 1983) or respiratory (Newman et al., 1983), may be equally significant. Developmental delays tend to be less precisely considered than frank terata or in utero death. Perhaps this is because, for instance, it can be difficult to determine whether a possible low incidence of retarded skeletal maturation is or is not related to exposure of the experimental animals to the test agent. Developmental delays can be a particularly difficult confounder of data analysis in that experimental findings may be more directly related to maternal stress (Khera, 1985) and/or toxic effects than to primary insult of the conceptus (Johnson, 1987). Indeed, certain skeletal effects are virtually pathognomonic of maternal toxicity (Johnson, 1983b). The adverse outcomes of perturbed in utero development are not listed in hierarchical sequence, perhaps partially because it is not possible to establish that one type of effect necessarily leads to another. There are, however, occasional examples where one type of developmental effect does appear to lead to another. One of these is when, in humans (Nishimura et al., 1966, 1968), or in animals treated with very high levels of a test chemical (Johnson et al., 1963; Johnson, 1984) the most severely abnormal concepti prove nonviable and die or are resorbed prior to term. Interestingly, the opposite also may occur and an increased incidence of abnormal concepti may not be found when exposure is increased above the initial developmental effect level. Turbow ( 1966) and Turbow and Chamberlain ( 1968) reported this phenomenon and considered it to be due to the embryos dying before their developmental pattern could show effects. This is a somewhat uncommon type of observation, but it has been reported by others (Tuchmann-Duplessis, 1970). Bass ( 1975) and Bass et al. ( 1976) described a possible third class of agents that produce either death or abnormal development of concepti but by two different mechanisms. In developmental toxicity studies, terata are but one of several endpoint assays of effect. All adverse effects on development must be considered of concern but they are not necessarily of equal concern in safety evaluations. In particular, developmental effects produced only at maternally overtly toxic exposure levels would not be viewed in the same context as when they occur in the absence of maternal effects. This is predicated, of course, on the assumption that maternally toxic exposure levels to the agent are avoided in humans. Test Method Development The modem era of teratology began with the studies of Hale (1933) and Warkany and Nelson ( 1940), but the breadth of interest increased markedly after the thalidomide tragedy in the early 1960s (McBride, 196 1; Lenz, 196 1). By that time many concepts of teratology as a science were known and later they were placed into clearly stated prose (Wilson, 1973) as general principles. Kamofsky had indicated in 1965 that nearly all substances or agents are capable of adversely affecting the conceptus if

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25

the treatment level is sufficiently high and they are administered by the proper route at a critical stage of development. This interesting concept caused some initial confusion regarding safety evaluations (i.e., why use animal tests or make epidemiologic surveys if everything is teratogenic?). Evaluation of developmental hazards was advanced by the recognition that the primary risk was posed by substances that would adversely affect the embryo at doses significantly below those necessary to produce overt signs of acute toxicity in the mothers (Staples, 1975).

Contemporary Test Methods The most commonly used developmental toxicity safety evaluation is the Segment II type of experiment (Johnson and Gabel, 1983). In this experiment untreated animals are bred, and, as their embryos begin to implant into the uterine lining (the sixth day of pregnancy in rats), treatment of the pregnant dams is begun. Groups of approximately 20 pregnant female rodents are used as a sham-treated control group and as each of at least three treatment groups exposed to different amounts of the test agent. The highest exposure level should cause maternal toxicity and the low dose should be a clear NOEL for both the mothers and the concepti. The intermediate dose is positioned somewhere between those two to provide more information regarding the slope of the dose-response curve. Treatment continues through to the end of major organogenesis (about Day 15 in rats and mice). Gestation is allowed to continue until 1 day prior to anticipated natural delivery when all females are killed and the fetuses retained for evaluation of size, sex, and soft and osseous tissue status. This experiment is a vigorous test of effects on the very sensitive phases of embryonic differentiation and organogenesis. It is not a markedly effective way to produce live term terata (Johnson and Christian, 1984), which are more likely to be observed from a carefully timed (Nelson, 1965), short severe pulse of treatment (Yasuda and Maeda, 1972). This is not invariably the case, however, and a short, severe pulse of a test agent is not always the most disruptive of embryogenesis. A bolus dose of agent (cortisone) has been found to have less of an effect than the same amount administered in fractionated doses over a longer period of time (Isaacson and Chaudhry, 1962). The Segment II test protocol, from the point of view of embryos, is a wholelife exposure and a reliable predictor of effects in development in at least three of the four classes of perturbed in utero development. Thus, the dosing regimen must be considered in evaluating hazard and establishing safety factors. II. STATEMENT

OF PRINCIPLES

Considerations Basic to Data Evaluations In developmental toxicity testing it is essential to use treatment levels extending high enough to produce toxic signs in the mothers. Because of this, some degree of altered development also is likely (Wilson, 1966). Furthermore, because treatment occurs throughout the period of major differentiation and organogenesis, the likelihood is that severely affected concepti are apt to die, rather than live and be present as abnormal pups at term. Thus, both maternal and prenatal effects must be considered in hazard evaluation (EPA, 1986).

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The degree of exposure is a primary consideration in the evaluation of toxicity test data, and particularly of developmental toxicity test data. Exposure of experimental animals to the test substance by the route of most likely human exposure is essential for risk estimation and cross-species extrapolation. since the level needed to produce toxic signs and the very nature of the toxicity is altered by the route of exposure. The route of exposure is not so crucial in the more preliminary stages of testing but, in the final stages preparatory to establishing permissible exposure levels, this is an essential consideration influencing both the NOEL and nature of the developmental outcomes (Kavlock et al., 1982). The duration of exposure during gestation also must be considered because the effects due to either single or repeated exposures may differ from those obtained with a more continuous treatment schedule. Because of the differing susceptibility of the conceptus at various times of development, the time of exposure is often more important than whether there are single or continuous exposures. Both the distribution (Young, 1983) and the metabolism (Chao and Juchau, 1983; York and Manson, 1984) of substances can have marked effects on developmental outcomes. There are marked species and gestational (Neims et al., 1976; Kraues et al., 1980) differences in the way in which some compounds are absorbed and/or distributed. Since the rate of excretion also may vary, the degree and rate of accumulation within the animal (bioaccumulation) may differ from one species to another. Factors such as these merit careful consideration (Kimmel and Young, 1983) because the peak level of a toxicant present for a short period of time may exceed the selfregulatory and repair mechanisms of the conceptus and do more damage than the same amount of the agent applied over a longer period of time. Short transient exposure applied at different stages of embryogenesis tends to produce effects consistent with the types of developmental events occurring at, or just after the time of, insult (Johnson, 1984). Because developmental schedules differ among species, effects may differ from apparently identical treatment schedules. Except in instances where a substance is activated in a unique manner by a species, pregnant animals prove remarkably predictive of human responses and response levels (Clement Assoc., 198 1). Agents demonstrated to adversely affect human development also injure animal development. The anatomical/developmental outcomes of a Segment II evaluation can be similar to those produced in human embryos exposed to the same agent (Khera, 1984). Rarely have human embryos proven to be strikingly more sensitive to a chemical than are animal embryos, and large safety factors applied to the animal NOEL seldom are indicated. There are not yet available extensive detailed comparative evaluations of animal and human effect levels for developmental toxicity. The data on this topic that are available, however (Clement Assoc., 1981), clearly indicate that safety factors larger than 100 have not been necessary when extrapolating from a valid study in animals to human exposure levels. A major consideration in establishing an adequate safety factor must be relevance of the animal model to the human situation. If an agent has been studied in multiple species with concordant effects and toxicokinetics similar to humans, it is reasonable to consider safety factors of less than 100 as reasonable. This view becomes increasingly reasonable as more data become available. It is evident that animals and humans show a great amount of concordance, not just of effect levels but also of the nature of the outcomes produced (OTA, 1985). The better the data base, the smaller need be the safety factor. Determination of safety factor size is a complex undertaking, as additional confounders such as nutritional and disease status, and even season

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(Fraser et al., 1986) of the year, and nature of a carrier substance (Robens, 1969) or route of treatment (Kimmel, 1977; Kavlock et al., 1982) may alter developmental outcomes &alter, 1965; Brent, 1972). Last, but not least, interspecies differences in developmental characteristics and timing also can influence markedly the developmental outcomes of treated animals (Trasler and Fraser, 1977). III.

APPLICATION

OF PRINCIPLES

Since it is more than likely that most chemicals, if administered by the proper route, at a critical developmental stage, and at a sufficiently high dose (e.g., maternal maximum tolerated dose or MTD), can produce adverse effects in the conceptus (Karnofsky, 1965) it is necessary to apply concepts more sophisticated than a binary classification of substances as developmental toxicants or not. Even oxygen (Ferm, 1964), vitamin A (Kochhar and Johnson, 1965) table salt (Nishimura and Miyamoto, 1969) and water can cause developmental toxicity in some circumstances (Turbow et al., 197 l), as can several hundred other commonly used chemicals such as DMSO (Ferm, 1966) and xylene (Marks et al., 1982) already tested in pregnant animals and deemed safe for humans at expected exposure levels. In other words, in a Segment II developmental toxicity safety evaluation many substances have been found capable of injuring the conceptus, yet they are being used safely. Thus, for the purposes of hazard evaluation, a positive animal test does not necessarily mean a hazard exists. To a significant extent this is because the embryo is less vulnerable to a substance’s action than are mothers, and the adult toxic MTD exposure levels necessary in laboratory studies are being avoided by humans (Johnson, 1987).

Developmental Targeting of Chemicals It is of paramount importance in developmental toxicology that the differences between hazard and risk be clearly stated. In the context of this writing, hazard potential is considered as the intrinsic properties of a particular chemical that impart to it the capability to cause a particular effect (e.g., toxicity, explosivity, and flammability). “Risk,” on the other hand, at least in the United States, is defined as the probability of a particular adverse outcome occurring at a particular exposure level. For the sake of completeness, it must be recalled that hazard potential is not purely a matter of chemical molecular content and configuration since form, e.g., droplet or fiber size or even temperature, can markedly influence the capability of some chemical agents to cause some types of effects. In evaluating the risks posed by developmental toxicants, distinctions need to be made between the class of substances that injure embryos at a small fraction of the adult toxic dose and those that do not. These are the primary or direct developmental hazards and, as illustrated in Table 1, their “target” is the conceptus. The other class of hazard to the conceptus (Table 1) would include substances which, though not more toxic to embryos than to mothers, may be used at or very near to, the adult toxic level. These are developmentally coaffective agents (Johnson, 1981) and the conceptus may be injured irrevocably by them while mothers may tolerate or recover from the toxicant. There is a third class of agents of even less concern regarding devel-

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TABLE I DIFFERENTIAL RESPONSESOF ADULTS AND DEVELOPING EMBRYOS TO Two DIFFERENT HYPOTHETICAL TOXICANTS Effect observed Treatment level

Maternal

Fetal

Example No. 1: Adult more vulnerable: A/D ratio < 1 High Intermediate Low Untreated

Severe Moderate NOEL NOEL

Severe” NOEL NOEL NOEL

Example No. 2: Embryo more vulnerable: A/D ratio > 1 High Intermediate LOW Untreated

Moderate NOEL NOEL NOEL

Severe Severe NOEL NOEL

a In some instances effects may be minor developmental delays or, alternatively, no developmental effects may be evident for agents with a very steep dose-response curve if they are also highly toxic or lethal to the mothers before developmental effects become evident.

opmental effects. These are agents so highly toxic to the mothers that they do not attain a disruptive concentration in the conceptus before the mother dies. With a few notable exceptions, agents that are known to adversely affect in utero development do so only at exposure levels high enough to produce overt acute toxicity in the mothers. As described previously, a substance capable of disrupting in utero development only at dose levels also high enough to injure the mother is not considered a hazard to the conceptus unless maternal exposure occurs at or near to the adult toxic dosage. In fact, the mere presence of maternal stress or toxicity (Khera, 1984), altered nutritional status (Hurley et al., 1971; Johnson and Chepenik, 1981), and even hypoxia (Goodlin et al., 1984) alone may be sufficient to interfere with embryonic development. There are, however, agents such as thalidomide that have a marked ability or propensity to interfere with some aspect of developmental biology at doses markedly below those capable of injuring the mother. Substances of this type require careful attention in the workplace, since exposure might not perturb the mother, yet have devastating effects on the conceptus. The National Toxicology Program (NTP) is a major and powerful step forward in adding good science to our understanding of potential toxicants. Its utility is significantly enhanced when it applies contemporary understandings of development and uses the available means to determine the developmental hazard potential of chemical agents (Johnson, 1984, 1987). The structure-activity relationship (SAR) mode sometimes used to identify toxic hazards to adult organisms (Arcos, 1983) is not adequate (Schumacher, 1975; Zimmerman, 1975; Tuchmann-Duplessis, 1980) and, in fact, can be a reverse discriminator for identification of hazards to the conceptus. For instance, we know that the alkylating agents tested thus far interfere with development and, because of this, some would hold that any previously untested alkylating

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agent should be a high-priority candidate for evaluation in a Segment II developmental toxicity safety evaluation. Actually, the reverse is true. A molecule recognized as an alkylating agent is a low- and not a high-priority item for developmental toxicity testing because such agents have a low developmental toxicity hazard index. They are capable of producing all four classes of developmental toxicity, and, because one (when dosage and timing are controlled) of these is production of live abnormal young, they are “teratogens.” They are, however, only coeffective teratogens. They are not primary teratogenic hazards since they injure embryos only at, or very near to, a treatment level high enough to injure the mother. The developmental hazard potential of a chemical is a measure of its propensity to disrupt development without obvious signs in the adult. It can be expressed as the A/D ratio representing the adult and developmental NOELs, respectively, of standard Segment II-type evaluations. This ratio calculated from the data of Segment II experiments is largely independent of species and exposure route and also can be determined in vitro. This relationship of effect levels is a basic principle of developmental toxicology and, when coupled with appropriate considerations of potential exposure levels, facilitates a more clearly defined consideration of developmental hazards and risks (Johnson, 1987).

Safety Factors There is no generally accepted means for extrapolating developmental toxicity effects below the NOEL. Current mathematical models developed for cancer risk assessment are inappropriate for developmental toxicity data (EPA, 1986). Several mathematical models adequately fit parts of the known data and can be used for comparing mechanisms of action and for examining the relationships between maternal weight gain and fetal status (Rands et al., 1982). These appear valid above the NOEL, but it is not possible, by mathematical means, to confidently determine the shape of the dose-response curve at lower treatment levels. Low-dose extrapolation below the lowest observed-effect level (LOEL) that is statistically significantly different from concurrent controls is difficult, and below the NOEL such extrapolations generally are not considered valid in developmental toxicology. Instead, a safety factor approach is used. Human exposure is kept at some fraction of the NOEL as determined in a sensitive, relevant animal model. In addition to the usual uncertainty attendant to extrapolations outside the data points, there is the additional biologic confounder that thresholds exist for developmental toxicity. There are maternal exposure levels below which no measurable adverse effect is produced in the offspring, perhaps due to the fact that some maternal exposures (e.g., formaldehyde) do not gain access to the conceptus. In addition, the embryo itself has significant repair and self-regulating capabilities and small perturbation or even cell loss is overcome (Wilson, 1973). In the animal kingdom, morphogenesis involves considerable migration and displacement of cells as well as their arrangement into primordia groups that eventually will differentiate into specific tissues and organs. The self-regulating ability of developing animal systems is a well-known and classic concept of developmental biology. Many of the studies demonstrating these phenomena predate the era of contemporary teratology, but their nature must not be overlooked. One of the early experiments demonstrating developmental plas-

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ticity was induction of a second amphibian neural plate in an embryo when a second dorsal lip of blastopore was implanted under what would otherwise be surface ectoderm and somotopleure. In response to the inductive signals, a new embryonic axis forms and differentiates (Spemann, 1936). This phenomenon of regulation was an extension of the plasticity demonstrated by transplantation of Hensen’s node in equally accessible avian embryos (Waddington and Schmidt, 1933). More recent experiments have demonstrated that embryonic recovery from damage extends beyond repair and involves actual regulation of induction and response applicable to secondary inductions, as well as those of the primary axis (Wessells, 1977). Experiments of this type have included extirpation and transplantation techniques demonstrating not just metaplastic compliance (Lewis, 1904) but also some of the inductive sequences controlling this phenomenon (Jacobson, 1966). A clear contemporary example is provided by studies of equivalence groups. This phenomenon occurs when groups of cells migrate within, and into, organ fields that have been devoided of their cells and produce normal development. Actually an organ that undergoes abnormal ontogenesis may itself be capable of normal development if it is excised and transplanted (Salaun, 1982). These are but a few of the mechanisms by which organogenesis is monitored and modulated. They illustrate the plasticity of embryonic development far beyond the concepts of morphallaxis and epimorphosis signaled by Morgan (190 l), although in some regards even these may extend beyond the phylogenetic level of the anurans and urodeles. Each pair of identical twins reaffirms the potency of embryonic capacity for regulation. These basic biologic principles provide strong support for the existence of thresholds in developmental toxicity. In view of the intervening maternal, individual, and self-regulative nature of embryonic development, the use of safety factors is the most realistic means for protecting the conceptus from harmful levels of chemical exposure. Their use clearly states that these are exposure levels below which no adverse developmental outcome occurs. A safety factor is a margin of error that allows both for interspecies variability and for degrees of uncertainty in the data. It is applied to animal data to establish exposure levels considered safe for humans. Safety factor size can be difficult to determine and, until recent times, factors of 1000 had been proposed in some quarters and are still considered appropriate by some for some types of developmental effects. Data bases have expanded markedly in recent years, and experience is now sufficient to permit determination that such overly large safety factors for developmental toxicity have been unnecessarily conservative. It is possible to extrapolate to the human from a good animal data base and its NOEL. Exposure levels needed to produce adverse effects in humans are remarkably similar to those of pregnant test animals (Clement Assoc., 198 1). A comparison of A/D ratios among species shows reasonable consistency. This indicates that interspecies variability is often minimal and suggests that large safety factors are not often indicated. Safety factors beyond l/ 100 of the animal NOEL do not appear to ever have been necessary to protect the human conceptus from adverse effects, and safety factors less than 100 would prove protective for most known human developmental toxicants. Determination

of Safety Factor Size

Safety factor size might be set differently for substances causing minor occasional defects, e.g., skin irritation, and those known to be potent carcinogens. It is less than

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logical to recommend that a substance that causes gross structural abnormality have a safety factor different from one causing death of the concepti in a Segment II evaluation. To do so would be an atavism from cancer research where it may be proper and prudent to consider the relevance of safety factors differently for carcinogens and noncarcinogens (U.S. Environmental Protection Agency, 1980). On the other hand, safety factors for certain developmental delays, when associated with maternal toxicity, reasonably may be expected to be lower than those applied to potent developmental toxicants. In developmental toxicology, the terms teratogen and nonteratogen have little, if any, meaning, being more a function of timing and severity of treatment than of substance nature. Furthermore, even changes in the patterns of skeletal maturation would be considered as unacceptable outcomes if produced consistently and if directly attributable to a specific chemical exposure. This is the most pragmatic position, but it serves human safety only if the doses needed to produce this type of effect and those actually existing are considered carefully. Altered skeletal maturation is routinely observed in the high-dose Segment II offspring since, at very high dosage, most substances are capable of inflicting some type of injury on the conceptus. Humans may react similarly if such excessive exposure were to occur. However, it is the object of risk management to control exposure and thereby to prevent developmental toxicity from occurring. The basis for safety factor size has been considered differently by some authors but, in general, the considerations are (Dourson and Stara, 1983) that the NOEL of the most sensitive animal tested is divided by 10 to allow for interspecies differences in sensitivity that might exist between the test animal and man. A second division by 10 is used because within a species (humans in this case) there may be individuals more sensitive than the average person. There are some indications that interspecies variation may be less than 10 (Krasovskii, 1976). The purpose of a safety factor is to compensate for uncertainty. Therefore, an important consideration for this topic is that, as the quality of the data increases, the amount of uncertainty decreases and safety factors can diminish proportionately. There are examples where the safety factor could be reduced because the data from both animals and man indicated marked similarities of absorption, metabolism, excretion, etc. An additional consideration for applying a smaller safety factor may be that workers are less variable than the population at large. That is, permitted human exposure levels are reduced below that level injurious to the average because of population diversity. The very young, old, or infirm may have heightened sensitivity to an agent, so exposure levels tend to be set to protect the more vulnerable individuals. It follows that occasions could exist where local exposure occurs only to people not particularly vulnerable to the substance. There also are instances where effects are produced in experimental animals because they are uniquely vulnerable via an avenue (because of unique anatomy, physiology, or chemistry) that does not exist (Kernis and Johnson, 1969) for a human, so the test compound would be dangerous only to the experimental animals but not to humans (e.g., trypan blue). On the other side of the coin, in the case of thalidomide, it was established that pregnant rats did not prove predictive of effects in humans. Overall, however, test animals have proven to be satisfactory human surrogates for both reproductive and developmental effects (OTA, 1985). The dose-response curve for developmental effects tends to be very steep. When this is established for a test agent, the safety factor magnitude for it reasonably may

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be reduced. However, if the agent proves to be an exception to this generalization and has a flattened dose-response relationship, the safety factor is better kept large. Consideration of the dose-response curve when setting the safety factor is particularly relevant when the agent has been tested by multiple routes of exposure and/or in several species and all produce the same type of outcome. IV. PUTTING

THE

RISKS

INTO

PERSPECTIVE

In tests and evaluations of developmental toxicity it is frequently the dosage employed more than the nature of the chemical that determines the types of toxicologic signs in concepti (Johnson, 1985). All too frequently lists and texts of teratogenic agents focus too much on the outcome but ignore the relevance of the treatment level (e.g., Smith et al., 1983). In considering treatment level relevance, not only is the quantity of test agent needed to produce an effect important, but also maternal status must be considered. It is counterproductive to list a developmental toxicant as a hazard when exposure approaching the maternally lethal level is necessary to achieve the endpoint (Johnson, 1980). Careful reporting of the relevance of developmentally toxic exposure levels to adult acutely toxic exposure levels is essential or the warning provided may precipitate unnecessary responses. The Toxic Substance Control Act (TSCA) requires that chemicals be tested for acute and chronic toxicity as well as for mutagenic, carcinogenic, and teratogenic effects. The terminology “teratogenic” as used in TSCA must be translated consistent with present-day understandings. In the context of regulation of exposure to preclude in utero injury, the term teratogen has lost its meaning due to confusion regarding what really should be termed developmental toxicity and not just teratology, as strictly and narrowly defined. TSCA can lead to an inappropriate regulatory response by identifying all chemicals that test positive (ignoring the A/D ratio and maternal toxicity) as “teratogens.”

False Alarms The context in which information is communicated must be a major consideration regarding public information programs and warning labels. Extreme accuracy and candor, of course, is the only defensible and viable policy to achieve the stated purpose of informing people of potential developmental hazards and the risks involved in specific exposures. However, imprudently disseminated data reported outside of a comprehensible context can do more harm than good. Alerts, warning labels, and even patient package inserts, etc., must be carefully considered because they can do more harm than exposure to low levels of the substance. A test chemical reported simply as teratogenic in experimental animals and, therefore, not recommended for women of child-bearing age can result in tragic scenarios quite unrelated to effects of the substance itself, i.e., precipitate consideration of elective abortion even for trivial exposure (Johnson, 1987). Pregnant women tend to avoid chemical exposure, but unfortunately women who are so exposed do become pregnant, and they are advised to consult their physician and seek advice. If intervention is not recommended, there is a likelihood that litigation will occur. A small percentage of pregnancies unexposed to any known toxicant have undesirable outcomes. If the patient is within this group and an abnormal child is born, the chances are that

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an association with the suspect agent will be made, and it will be claimed that the physician should have known that the test compound was going to produce an adverse effect. Juries can have difficulty understanding the difference between concomitant occurrences and cause and effect as well as the all-important quantification relevance of dosage. In addition, inappropriate regulatory response and “false alarms” that trigger maternal stress and unnecessary abortions is another undesirable aspect of binary labeling of substances as “teratogens” or “nonteratogens.” This leads to improper allocation of limited governmental and private resources that would be more properly targeted toward those substances that present real hazards to the conceptus. Underlying this scenario of sequela of warning label content usage, and all discussions of developmental effects and the application of realistic safety factors from both the perspective of hazard assessment and the risk estimation/management, must be two basic questions. What is the developmental hazard potential or A/D ratio (ratio of the adult NOEL to the developmental NOEL from a standard Segment II experiment) of the substance in question, and what is the severity of the exposure? ACKNOWLEDGMENT This paper was partially supported with funding from the American Industrial Health Council.

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