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Http://pediatrics.aappublications.org/cgi/reprint/113/4/s1/984.pdf

Utilization of Animal Studies to Determine the Effects and Human Risks of
Environmental Toxicants (Drugs, Chemicals, and Physical Agents)
2004;113;984-995 This information is current as of January 26, 2006
The online version of this article, along with updated information and services, is located on the World Wide Web at: PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthlypublication, it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, ElkGrove Village, Illinois, 60007. Copyright 2004 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275. by on January 26, 2006 Utilization of Animal Studies to Determine the Effects and
Human Risks of Environmental Toxicants (Drugs, Chemicals,
and Physical Agents)
Robert L. Brent, MD, PhD Toxicology studies using animals and in
drugs and are more vulnerable to the toxic effects of
vitro cellular or tissue preparations have been used to
environmental chemicals, there are exceptions that indi-
study the toxic effects and mechanism of action of drugs
cate that infant and developing animals may be less
and chemicals and to determine the effective and safe
vulnerable and more resilient to some drugs and chemi-
dose of drugs in humans and the risk of toxicity from
cals. In other words, the generalization indicating that
chemical exposures. Studies in pregnant animals are
developing animals are always more sensitive to envi-
used to determine the risk of birth defects and other
ronmental toxicants is not valid. For animal toxicology
reproductive effects. There is no question that whole
studies to be useful, animal studies have to use modern
animal teratology studies are helpful in raising concerns
concepts of pharmacokinetics and toxicokinetics, as well
about the reproductive effects of drugs and chemicals,
as method-of-action studies to determine whether animal
but negative animal studies do not guarantee that these
data can be used for determining human risk. One exam-
agents are free from reproductive effects. There are ex-
ple is the inability to determine carcinogenic risks in
amples in which drug testing was negative in animals
humans for some drugs and chemicals that produce tu-
(rat and mouse) but was teratogenic in the human (tha-
mors in rodents, because the oncogenesis is the result of
lidomide), and there are examples in which a drug was
peroxisome proliferation, a reaction that is of diminished
teratogenic in an animal model but not in the human
importance in humans. Scientists can use animal studies
(diflunisal). Testing in animals could be improved if
to study the toxicokinetic and toxicodynamic aspects of
animal dosing using the mg/kg basis were abandoned
environmental toxicants, but they have to be performed
and drugs and chemicals were administered to achieve
with the most modern techniques and interpreted with
pharmacokinetically equivalent serum levels in the ani-
the highest level of scholarship and objectivity. Thresh-
mal and the human. Because most human teratogens
old exposures, maximum permissible exposures, and
have been discovered by alert physicians or epidemiol-
toxic effects can be estimated but have to be interpreted
ogy studies, not animal studies, animal studies play a
with caution when applying them to the human. Well-
minor role in discovering teratogens. In vitro studies play
performed epidemiology studies are still the best method
an even less important role, although they are helpful in
for determining the human risk and the effects of envi-
describing the cellular or tissue effects of the drugs or
ronmental toxicants. Pediatrics 2004;113:984 –995; methods
chemicals. One cannot determine the magnitude of hu-
of evaluation, environmental toxicology, pharmacokinetics,
man risks from these in vitro studies. Performing toxi-
pharmacodynamics, toxicokinetics, toxicodynamics, MOA
cology studies on adult animals is performed by phar-
(method of action), deterministic, threshold phenomenon,
maceutical companies, chemical companies, the Food and
stochastic, biologic plausibility, in vitro systems, in vivo
Drug Administration, many laboratories at the National
Institutes of Health, and scientific investigators in labo-
ratories throughout the world. Although a vast amount of
animal toxicology studies are performed on pregnant
ABBREVIATIONS. MOA, method of action; FDA, Food and Drug animals and numerous toxicology studies are performed
Administration; CNS, central nervous system.
on adult animals, there is a paucity of animal studies
using newborn, infant, and juvenile animals. This defi-
ciency is compounded by the fact that there are very few
toxicology studies performed in children. That is why
tant issue. Can the magnitude and type of en- pregnant women and children are referred to as "thera-
vironmental risks to the embryo, child, and peutic orphans." When animal studies are performed
adolescent be determined from animal studies, and with newborn and developing animals, the results dem-
how different are these risks when compared with onstrate that generalizations are less applicable and less
adults? In many instances, environmental agents will predictable than the toxicology studies in pregnant ani-
exploit the vulnerabilities and sensitivities of devel- mals. Although many studies reveal that the infant and
oping organisms. In other instances, there will be no the developing animal have difficulty in metabolizing
difference between the developing organism and theadult when exposed to toxicants, and in some in-stances, children and adolescents may even with- From the Thomas Jefferson University, Alfred I. duPont Hospital for Chil-dren, Laboratory of Clinical and Environmental Teratology, Wilmington, stand the exposures with less insult. The difficulty that we have at this time is that in many situations, Received for publication Oct 7, 2003; accepted Oct 20, 2003.
we do not have enough data and/or scholarly tech- Reprint requests to (R.L.B.) Rm 308, R/A, Alfred I. duPont Hospital for niques to arrive at a conclusion about the relative Children, Box 269, Wilmington, DE 19899. E-mail: [email protected] (ISSN 0031 4005). Copyright 2004 by the American Acad- sensitivity of the developing organism to some envi- emy of Pediatrics.
ronmental agents. Rather than arrive at conclusions PEDIATRICS Vol. 113 No. 4 April 2004 www.pediatrics.org by on January 26, 2006 about environmental agents or exposures for which newborns, infants, juvenile animals, and adults; and there are insufficient data, we need to initiate inves- 3) oncogenic effects of environmental toxicants.
tigative approaches to obtain the necessary data con-cerning agents and exposures that have not beenclarified, so it is important that we initiate and ex- USE OF ANIMAL STUDIES TO DETERMINE
pand quality research in environmental toxicology.
REPRODUCTIVE RISKS IN HUMANS
Although chemicals and drugs can be evaluated (TERATOGENESIS, GROWTH RETARDATION,
for their toxic potential by using in vivo animal stud- PREGNANCY LOSS, STILLBIRTH, AND
ies and in vitro systems, it should be recognized that these testing procedures are only 1 component in the Pediatricians and other clinicians have little train- process of evaluating the potential toxic risk of drugs ing on how to interpret animal toxicology studies and chemicals. The evaluation of the toxicity of during medical school and residency training. This is drugs and chemicals should include, when possible, probably more true of reproductive toxicology stud- data obtained from a number of investigative ap- ies than in any other area of animal testing. Unfor- proaches1: 1) epidemiologic studies1,2; 2) secular tunately, for physicians, the most frequent source trend or ecological trend analysis; 3) animal stud- and contact with animal testing information is in the ies3–7; 4) pharmacokinetic, toxicokinetic, pharmaco- package insert or the Physician's Desk Reference.10 dynamic, and toxicodynamic studies; 5) mechanism The Physician's Desk Reference uses the FDA's clas- of action (MOA) studies; and 6) basic science studies sification of reproductive risks, partly based on ani- that pertain specifically to the agent, such as MOA mal testing. The categories are A, B, C, D, and X. The studies, which include receptor affinity, cytotoxicity, A category includes drugs that have no risk for re- genotoxicity, organ toxicity, neurotoxicity, etc.1,4,6,8 productive effects. The B, C, and D categories have Human studies are expensive and take years to com- increasing risks, and the X category includes drugs plete. Therefore, scientists have asked whether ap- such as methotrexate, Accutane, and thalidomide propriate animal models are available to evaluate the that should not be used in pregnant women or risks of environmental toxicants to the embryo, in- women of reproductive age who are not on contra- fant, child, and adolescent. This is not an easy task.
ceptives. These categories are misleading more than There are a few toxicologic principles that should they are helpful. Teratologists, obstetricians, and precede the specific discussion. Frequently, drugs or other clinicians who counsel pregnant women have chemicals are grouped into categories (pesticides, been very critical of the FDA classification11 because trihalomethanes, organochlorines, solvents, proges- the classification ignores the basic principles of tera- tins, heavy metals, chemotherapeutic agents). It isimportant to note that this type of classification may tology12 and the importance of modern pharmacoki- be useful for some purposes but not for concluding netics when evaluating animal studies.5 In 1990, a generalizations about the toxic effects of all of the published article indicated that of the 200 most fre- agents in the group. As an example, the Food and quently prescribed drugs, none of them represented Drug Administration (FDA)9 published a report in a significant teratogenic risk,13 yet only a small pro- the Federal Register disclaiming the term "progestins" portion of these drugs were placed in category A by to describe a group of drugs with identical effects the FDA. There are many reasons for these misclas- and toxicity. Second, chemicals may be referred to as sifications, but the most important reason is the mis- "poisons." This is not a useful label because every application of animal testing results. Let me give you known chemical or drug has an exposure that is some examples.
toxic. Paracelsus stated in the 16th century, "What is When a new drug is marketed or a new environ- there that is not poison? All things are poison and mental toxicant is discovered, frequently the only nothing is without poison. Solely, the dose deter- information that is available is the animal data. Three mines that a thing is without poison." examples are used to emphasize the difficulties that Three areas of animal testing are discussed: 1) occur: 1) meclizine produces cleft palate at very high reproductive effects from exposures during embry- exposures in the rat; 2) leflunomide and its MOA; onic and fetal development; 2) toxic effects of drugs and 3) radiation produced mental retardation; a de- and chemicals administered to animals after birth as terministic or stochastic effect (Table 1)? Stochastic and Threshold (Deterministic) Dose–Response Relationships of Diseases Produced by Environmental Agents Some risk exists at The incidence of the all dosages; at low disease increases, but doses, risk is less the severity and nature than spontaneous risk of the disease remainthe same Multiple, variable No increased risk below Both the severity and growth retardation, the threshold dose incidence of the disease death, toxicity, etc increase with dose Modified from Brent.12 by on January 26, 2006 Meclizine Produces Cleft Palate at Very High
rabbits, malformation of the head and bilateral dys- Exposures in the Rat
plasia of the spine of the scapula. The no-effect level Meclizine is an antihistamine with a lengthy his- for embryotoxicity and teratogenicity in rats and rab- tory and like most antihistamines has not been dem- bits was 1 mg/kg body weight, which resulted in onstrated to have reproductive toxicity in multiple serum levels of 3.7 and 4.1 ␮g/mL, respectively.
epidemiologic studies, yet its pregnancy category The active metabolite of leflunomide, which is the classification is B, primarily because "reproductive pyrimidine antagonist, is maintained at a blood level studies in rats have shown cleft palates at 25 to 50 of 40 ␮g/mL in patients being treated. The decision times the human dose." Actually, what the clinician to label leflunomide as having a teratogenic risk was needs to know is what the blood level is in the rat based on the fact that the human serum level was in and mouse when teratogenesis is produced and how the range of the teratogenic blood level in the animal that blood level compares with the level in patients models, so the initial labeling was an appropriate who receive therapeutic doses of the medication.
precaution to prevent birth defects.
Without this information, the animal experiments are After 4 years of treatment of patients with rheu- meaningless. There are hundreds of drugs in catego- matoid arthritis and no indication of an increase in ries B and C with animal studies using the archaic teratogenesis in a very small group of pregnant pa- mg/kg dose. This same failing has occurred in toxi- tients who were treated and continued their preg- cologic studies with environmental toxicants (lead, nancy to term, we can reanalyze the animal data as mercury, polychlorinated biphenyls, pesticides, fun- follows. This is referred to as the MOA approach.
gicides), namely, using mg/kg exposures in rodents The potential mechanisms of teratogenicity for le- or other animals rather than determining serum lev- flunomide are as follows: els in the animal and the human population forwhich there was concern. Fortunately, more recent 1. Suppression of DNA synthesis by interfering with environmental toxicology studies have been using pyrimidine synthesis based on the presumption modern toxicokinetic techniques, but serum levels of that suppression is equal in the rat, rabbit, and these toxicants are not always available in humans.
human at the same serum levels of the activemetabolite of leflunomide. This was the basis of Leflunomide and Its MOA
the X category labeling.
Leflunomide8 is a relatively new drug (1998) that is 2. The susceptibility of the enzyme to the active le- used to treat rheumatoid arthritis. It has a box warn- flunomide metabolite that is involved in pyrimi- ing for reproductive effects (teratogenesis) and has dine incorporation into DNA in the human and been placed in category X. Because there were no animal models.
human data available at the time of marketing, the 3. The ability of the active metabolite of leflunomide label was based on the animal studies: "There are no to interfere with cell proliferation in the human adequate and well-controlled studies evaluating and animal models.
Arava (leflunomide) in pregnant women. However,based on animal studies, leflunomide may cause fetal If all 3 mechanisms of action were operative to the death or teratogenic effects when administered to a same degree at the same serum level in the animals and the patients, then there would be concurrence Leflunomide is a novel isoxazole immunomodula- and the human risks would be determined to be tory agent that inhibits de novo pyrimidine synthesis identical from studying all 3 mechanisms. In vitro and has antiproliferative activity. In vitro, after mi- studies of the active metabolite of leflunomide re- togen stimulation, the active metabolite of lefluno- vealed that the rat was 40 times more sensitive to the mide inhibits T-cell proliferation, DNA synthesis, suppression of dihydroorotate dehydrogenase than and the expression of certain cell surface and nuclear the human and that the rat was 328 times more antigens directly involved in T-cell activation and sensitive to the active metabolite of leflunomide than proliferation. It inhibits mitogen-stimulated prolifer- was the human in suppressing cell proliferation.
ation of human peripheral blood mononuclear cells What this means is that if enzyme suppression or and proliferation in transformed murine and human antiproliferative activity is the MOA of teratogenicity cell lines in a dose-dependent manner. It has been in the rat, then the clinical use of leflunomide in demonstrated that the active metabolite binds to and pregnant women would probably not be teratogenic, is a potent inhibitor of dihydroorotate dehydroge- but no one would act on these findings without nase, an enzyme in the de novo pyrimidine synthesis confirmation from the ongoing epidemiologic sur- pathway important for DNA synthesis. Together, veillance of this drug. This is an example of modern these data suggest that at serum concentrations pharmacokinetic studies having improved risk as- achievable in patients, leflunomide inhibits de novo sessment and made epidemiologic studies under- pyrimidine synthesis in activated lymphocytes and other rapidly dividing cell populations, resulting inreversible cell cycle arrest.
In Utero Effects of Ionizing Radiation on the Risk of
In oral embryotoxicity and teratogenicity studies in rats and in rabbits, leflunomide was embryotoxic Here is an example in which animal behavioral (growth retardation, embryolethality, and terato- studies and concomitant pathology were helpful in genicity) in rats, consisting of malformations of the resolving an important issue with regard to in utero head, rump, vertebral column, ribs, and limbs; and in radiation–induced mental retardation. The main is- CHILD/ADULT PHARMACOKINETIC DIFFERENCES by on January 26, 2006 Effect of In Utero Ionizing Radiation on Developmental and Neurologic Parameters in Dose of X-ray (Gy) Embryonic or Fetal Age Growth retardation at term Growth retardation postpartum Developmental parameters (4) sue with regard to the risk of mental retardation after one has to be careful in extrapolating animal data to an in utero exposure to ionizing radiation pertains to humans, the lack of neurobehavioral effects from in whether the risk from exposure is stochastic (no utero irradiation supports the other findings that threshold) or deterministic (threshold effect; Table 1).
indicate that mental retardation is a threshold (de- The possibility that 0.01 Sv (1 rad) might double the terministic) effect (Tables 1 and 2).
risk of mental retardation was suggested in 1984.14 Once a drug, chemical, or other agent is suspected From the perspective of biological plausibility of producing congenital malformations or other re- and the results of animal studies, it seems that productive effects, appropriate use of in vitro and in the data favor the viewpoint that mental retardation vivo testing can be helpful in evaluating the specific is a deterministic effect with a threshold above 0.2 allegation and in determining the mechanism of ac- Sv.12,15–22 Histologic examination of the irradiated tion of the agent. Whole animal testing, although brain exposed to 0.01 Sv reveals no pathologic con- serving important and useful purposes, can still be sequences that could account for severe mental re- improved so that they can be better used to estimate tardation.23 That would mean that the pattern of human reproductive risks. These improvements are effects produced by ionizing radiation that accounts listed in Table 3.
for mental retardation when the fetus is exposed to In vitro tests can be used to study the mechanisms 0.5 to 2 Sv does not occur at very low exposures.
of teratogenesis and embryogenesis and for prelim- Furthermore, additional studies by Schull and inary screening procedures, but in vitro studies will Otake24 revealed that these authors were able to never be able to be predictive of human teratogenic quantify the risk of reduced intellect after in utero risks at particular exposures without the benefit of ionizing radiation exposures. They estimated that data obtained from whole animal studies (Table 3) there was a reduction in intellect of approximately 30 and epidemiologic studies.5,7,25–27 Despite the ad- IQ points per Sv in their studies. Even if there were vances in in vitro and in vivo testing for teratogenic- a linear relationship between the dose and IQ reduc- ity, human epidemiologic surveillance by various tion, one could predict that 0.01 Sv could not account methods is and will be our most powerful tool for for a doubling of the incidence of mental retardation, discovering human reproductive toxins and terato- because a linear extrapolation of Otake and Schull's gens. It may be difficult for experimental teratolo- data would represent only a maximum reduction of gists to accept that alert physicians and scientists 0.3 of an IQ point at 0.01 Gy. Behavioral studies in have been the most prominent contributors to the animals were unable to demonstrate neurobehav- discovery of the environmental causes of birth de- ioral effects below 0.02 Gy15–17 (Table 2). Although fects2 (Table 4).
A Whole-Animal Teratology-Reproductive Toxicity Protocol Should Include the Fol- lowing Parameters and Goals 1. Determine the reproductive effects at stages of gestation that may have markedly different endpoints, namely, preimplantation, organogenesis, and early fetal and late fetal stages.
2. The importance of various reproductive endpoints may vary considerably by the gestational stages being evaluated, and exposures at one stage may exaggerate, modify, or eliminate effectsthat occur at another stage 1. Teratogenesis2. Embryolethality3. Growth retardation4. Postnatal physiologic, biochemical, developmental, and behavioral effects 3. Determine the no-effect dose for the parameters mentioned in item 2 at various stages of 4. Determine the ratio of the no-effect dose to the human therapeutic dose, usual exposure dose, or maximal permissible exposure for the parameters mentioned in item 2.
5. Determine the quantitative relationship between the human and animal model pharmacokinetics and toxicokinetics concerning the dose and the blood levels and the metabolism in the animalmodel and human.
6. Determine the MOA of the environmental toxicant.
7. Determine the ratio of the LD/50 for the mother and the embryo.
by on January 26, 2006 How Some Human Teratogens Have Been Discovered Major Means of Discovery Hydantoins 1963Trimethadione 1970 Valproic acid 1982 Isotretinoin 1983 Penicillamine 1971 Chorionic villous sampling PCB indicates polychlorinated biphenyl.
EFFECTS OF ENVIRONMENTAL TOXICANTS THAT
apparent that comparisons of toxicity or therapeutic ARE ADMINISTERED TO ANIMALS AFTER BIRTH
effects bore a closer relationship to the 0.7 power of AS NEWBORNS, INFANTS, JUVENILE ANIMALS,
body weight, and this figure was closely related to AND ADULTS FOR DETERMINING HUMAN RISKS
surface area.30,31 Many physiologic functions are pro- It is obvious that animal experiments cannot be portional to surface area because the extracellular planned to consider all of the variables that occur in volume in humans is constant on a surface area basis.
the human. In fact, there are situations in animal Therefore, the serum concentrations obtained from studies that differentiate the animal species from the administration or exposure to drugs and chemicals human. For example, coprophagy and other behav- on a surface area basis would result in serum con- iors in rodents and other species can markedly alter centrations that would be similar. This is more the dynamics of toxicity studies. Differences in ab- closely related to the 0.73 power of the weight at all sorption, metabolism, and excretion of drugs and ages in humans.32,33 If, however, drugs or chemicals chemicals represent the greatest barrier to applying are also distributed in the total body water, then risks obtained from animal studies directly to the neither the mg/kg nor the surface area model will be accurate, because total body water is not a constant It is hoped that regulatory agencies and toxicolo- using the surface area constant or the mg/kg rela- gists who deal with issues of developmental toxicity tionship. It is obvious that no one method of dose will develop animal models that will predict toxico- calculation for the young will be satisfactory for eval- logic effects in children and adolescents from expo- uating appropriate therapeutic doses or for deter- sure to drugs and chemicals. Although this is an mining toxic risks. If that is the case for human optimistic view, Done28 pointed out that although exposures, then animal toxicology studies that are the number of drug hazards that have proved to be based on the mg/kg or surface area will not be unique in the infant have proved to be small: "With- universally appropriate for determining human risks out exception, recognition of the proved hazard has or proper doses. In fact, the fields of pharmacokinet- come about only after widespread use, and then ics and toxicokinetics have demonstrated that animal usually when tragic consequences focused attention toxicology experiments have to be performed know- ing the serum level of the drug or chemical in thehuman and using those levels in animal toxicology Animal Toxicology Studies
Historically, the administration of drugs and We are interested in the usefulness of information chemicals to humans and animals in experimental obtained from animal toxicology studies, using studies has used the mg/kg exposure method. Even drugs and chemicals for determining the risks to in the 1800s, there was recognition that there was not children and adolescents from these exposures. The a proportional relationship between body weight largest literature in this field pertains to animal tox- and dose between the infant and the adult human.29 icology studies using newborn and infant animal It was apparent that appropriate infant doses would models. Most of these studies are acute toxicology in some cases be toxic in the adult and appropriate studies and use the mg/kg method of dosing the adult doses would be inadequate for the infant if the adult and infant animals. Much of the information is mg/kg approach were used. Animal investigators simply the determination of the lethal exposure or have long been aware of this dilemma. It became the effect on growth. The most important finding in CHILD/ADULT PHARMACOKINETIC DIFFERENCES by on January 26, 2006 these studies is that newborn and infant animals are important aspects in designing these animal studies not always more sensitive or more deleteriously af- are the application of modern toxicokinetics and fected by drugs and chemicals when compared with pharmacokinetics. Exposure levels should include exposures that occur in the environment, and a major Urethane, an anesthetic that is no longer used for effort should be made to determine the no-effect that purpose, was unable to anesthetize newborn animals at exposures that anesthetized adults, The developmental events that can be affected by whereas ether altered reflexes at lower concentration environmental exposures to drugs, chemicals, and in newborn animals than in adults.34 Newborn mice physical agents include the following developmental and other animal species have demonstrated a toler- events that occur during childhood and adolescent ance to hypoxic conditions that is not present in adult animals.35–39 Newborn mice continued tobreathe for a longer period when exposed to ether Interference With Growth, Epiphyseal Development, and than adult mice.40 Newborn mice had a prolonged survival when compared with adults that were ex- Alterations in growth from exposure to environ- posed to asphyxia as a result of exposure to CO, mental toxicants can result in accelerated growth or HCN, CO2, H2, and CH3. Longer exposures to strych- growth retardation. Accelerated growth and matura- nine, curare, cyanide injection, strangulation, hyp- tion can result in larger stature or smaller stature.
oxia, or nitrobenzol were necessary to produce respi- Smaller stature can result from the combination of ratory arrest in newborn mice as compared with growth acceleration and earlier epiphyseal closure.
Drugs and chemicals that are cytotoxic or interfere In summarizing this information, Done28 was cau- with normal hormonal and endocrine relationships tious, pointing out the multiplicity and variability of have the potential for altering growth and develop- experimental details in these studies. He concluded, ment, but the exposure has to be above the threshold "Some tentative generalizations and observations for producing results. Useful information about the may be worth making. First, it is apparent that im- effect of environmental toxicants can be obtained by maturity does not necessarily entail greater sensitiv- exposing animals during various developmental ity. A notable example is thiourea, which is 50 to 400 stages before puberty.
times as toxic in the adult as in infant rats."41,42Conversely, the animal experiments with chloram-phenicol clearly demonstrated that this drug was Reproductive and Hormonal Effects more toxic in the infant rat than in the adult. Animal Do exposures during childhood and adolescence toxicity studies corroborated the toxicity reported in from environmental agents having hormonal activ- ity, cytotoxicity, or other effects alter the timing of In Done's28 review of developmental toxicology, puberty, alter the maturation of sexual organs in- he indicated that the newborn or infant animal was cluding breast development, or affect fertility or the more sensitive to many drugs (eg, chloramphenicol, normalcy of spermatogenesis and oogenesis?53 Ga- morphine, some other opiates, picrotoxin, tetracy- mete production in both the male and female begins cline, novobiocin, some organophosphate anticho- at puberty: spermatogenesis in the male and ovula- linesterases, atropine, histamine, sodium salicylate) tion in the female. Immature and pubertal rats seem and less sensitive to others (eg, ethanol, strychnine, to be more sensitive than adults to testicular toxicity metrazol, codeine, acetocycloheximide, thiourea, induced by phthalate esters,54–56 but the primate thyroid hormone). Many other drugs had sensitivi- does not have the same susceptibility as the rat. The ties that were similar in the neonate and adult ani- pesticide 1,2-dibromo-3chloropropane affects the im- mal, but, of course, most of these data were based on mature rat testes more severely than the adult, al- the mg/kg dosage and the endpoints were simplis- though the testes of the adult are also affected, as tic, ie, death or cessation of respiration.
1,2-dibromo-3chloropropane was banned becauseoccupational exposure in adult males resulted in in- Development Toxicity Studies in Juvenile Animal
fertility.57,58 Lemasters et al59 pointed out that imma- Models: Relevance for Estimating Developmental Risks
ture animals are not always more sensitive than in Humans
adults. Fetal Leydig cells are less sensitive than adult Can concerns about developmental problems from Leydig cells to ethane dimethane sulfonate, a known exposure to developmental toxicants in children and Leydig cell toxicant.60 Before the onset of puberty, adolescent be evaluated with appropriately designed rats are insensitive to testicular toxicity after expo- animal studies? Selevan et al46 indicated "that little sure to 1,3-dinitrobenzene, a Sertoli cell toxicant, and concrete information exists on critical windows for young adults are less sensitive than mature male exposure during the postnatal period."47,48 However, adults.61 Although spermatogenesis has many simi- a systematic examination has not been done of avail- larities among mammalian species, oogenesis varies able data on critical windows of vulnerability during considerably. Even the number of primordial ova postnatal development. Most available data are fo- varies in different mammalian species.53 Exposure of cused on prenatal exposures. Postnatal exposures female rats to 4-vinylcyclohexene diepoxide results have been examined for only a few agents (eg, lead, in destruction of oocytes in small follicles, and adult pesticides, radiation),49–52 and it can be stated that rats are less sensitive to the ovatoxicity of this com- the pesticide analysis is far from definitive. The most pound.62 Would the onset of menopause be affected by on January 26, 2006 by certain chemical and drug exposures during developmental events have occurred before the pe- childhood and adolescence? riod of CNS development during childhood and ad- Environmental toxicants can affect thyroid devel- olescence. Rice and Barone79 raised the question as to opment and therefore have a direct impact on neu- whether schizophrenia, dyslexia, epilepsy, and au- rologic normalcy, because normal thyroid function is tism may be caused by environmental influences.
crucial for normal central nervous system (CNS) de- Weiss and Landrigan80 speculated that attention-def- velopment.63 The most common environmental icit/hyperactivity disorder and Parkinson's disease cause of mental retardation in the world is endemic may be attributable to exposures that occurred dur- cretinism as a result of iodine deficiency and is not an ing development. We know that epilepsy can be environmental toxicity in the usual sense.64,65 Con- caused by trauma, infection, and genetic abnormali- versely, children's thyroids have been demonstrated ties and that autism can be produced by an insult to to be more sensitive to the oncogenic effect of exter- the nervous system very early in embryonic devel- nal ionizing radiation exposure as well as radioactive opment.81,82 Rice and Barone79 also raise the question iodine localization in the thyroid.66–69 as to whether early exposures to toxicants can cause With regard to environmental toxicity, questions acceleration of age-related decline in CNS function.
have been raised about the effect of organochlorine Some of these questions are amenable to animal compounds (polychlorinated biphenyls, dioxins) on studies in both rodents and primates, but these stud- thyroid function.70–75 It is difficult to determine the ies are neither easy to perform nor inexpensive, es- magnitude of the risk of these compounds on thyroid pecially in the primate. Two important problems function with the data that are available. The world- exist with regard to evaluating the risk of neurotox- wide problem of endemic cretinism from iodine de- icity of environmental toxicants at various stages of ficiency is without question a real problem. Few development using animal models: 1) we do not studies have evaluated the risk of environmental have precise information that equates various stages toxicants on thyroid function and other endocrine of prepartum and postpartum brain development in organs when exposed during childhood and adoles- the human and animal models,83 and 2) we cannot be certain of our ability to identify and recognize the In the article by Pryor et al53 dealing with repro- most important neurologic diseases in animal mod- ductive effects, the authors stated, "Although it is the els (eg, attention-deficit/hyperactivity disorder, dys- dose that makes the poison, there is no doubt that lexia, autism, schizophrenia, Parkinson's disease).
timing of the exposure may be as important as dose In the publication by Adams et al,84 a number of in determining the potential toxicity of a compound important concepts are discussed. The authors indi- to the reproductive system." This is not a rare state- cated, "Inherent in the brain's protracted period of ment in the "environmental literature," but it is not development is also the phenomenon of neuroplas- correct. Timing of exposure is important, but it is not ticity, and the nervous system's consequent potential important if the actual exposure is below the thresh- for compensation after insult." This is probably the old. If the threshold dose for an effect at any stage of most difficult area to investigate in both human and development is not exceeded, then timing is irrele- animal models. In fact, it is such a difficult area that the authors indicated that it was beyond the scope oftheir review, but it is an area that could be investi- Do Exposures to Environmental Agents During Childhood gated using animal models. Adams et al84 specifi- and Adolescence Affect the Normalcy of the Adult Immune cally discussed the topic of the "vulnerability during the adolescent period of development." They indi- Although it is true that many chemicals can affect cated that the brain of the adolescent undergoes the immune system at high exposures, the question "striking" transformations, which is observed in of whether environmental exposures play any role in many mammalian species. These regions include ar- altering the immune system has not been answered.
eas of remodeling of the prefrontal cortex and other It has not even been determined whether this is a forebrain regions that receive projections of the me- high priority area to be studied using appropriate solimbic dopaminergic terminal projections. In addi- animal models. In the review on this subject by Hol- tion, there is a decline in the volume of the prefrontal laday and Smialowicz,76 the authors stated, "The cortex in humans85 and the rat.86 According to Ad- possibility that developmental exposure to immuno- ams et al,84 there is also substantial synapse elimina- toxicants may play a role in inducing or exacerbating tion of presumed glutaminergic excitatory input to hypersensitivity or autoimmune responses needs to the motor cortex,87 whereas dopaminergic input to be investigated in laboratory animals." the prefrontal cortex increases during adolescence toreach levels higher than that seen earlier or later in Vulnerability of the Nervous System to Environmental Agents life.88 Estimates of basal synthesis and turnover of During Childhood and Adolescence dopamine decline in prefrontal cortex during adoles- Critical developmental processes during the devel- cence in rats, which contrasts with the increase in opment of the CNS include 1) the development of the these measures reported in the nucleus accumbens germ layers, 2) neurulation, 3) the closure of the and striatal dopamine terminal region of adolescent neural tube, 4) neuronal proliferation, 5) neuronal rats.89,90 Maturational events have also been reported migration, 6) differentiation, 7) synaptogenesis, 8) in a variety of other areas, including the hippocam- myelination, and 9) apoptosis. These processes can pus in humans91 and rodents92 and in the hypothal- be studied in animal models.77,78 The first 5 or 6 amus.93 Adams et al84 suggested that the adolescent CHILD/ADULT PHARMACOKINETIC DIFFERENCES by on January 26, 2006 brain may be especially vulnerable during this pe- laboratory animals but not all laboratory animals, riod of remodeling and referred to the publications but the converse is not true, namely, that all agents of Salimov et al,94 who reported the toxic influence of that have been demonstrated to be carcinogenic in alcohol exposure during this stage of development in animals are carcinogenic in humans.97–99 When the the rat. All of these studies are of interest and inform MOA of a carcinogenic agent is understood, the rel- the reader about the developmental processes that evance of the animal studies can be placed into may be occurring in the brain of adolescents, but few proper perspective. The following 2 examples are of these studies reveal whether environmental toxi- animal studies that indicated a carcinogenic poten- cants have any effects on these developmental pro- tial, but when the MOA was understood, these agents were determined not to have human carcino-genic potential.
USE OF ANIMAL STUDIES TO DETERMINE THE
Animal studies have revealed marked differences ONCOGENIC RISKS OF ENVIRONMENTAL
among species with regard to the oncogenic suscep- tibility to environmental chemicals and drugs as There is a truism in medicine that indicates that exemplified by the phthalates.100–108 For example, children are at greater risk for the induction of can- chemicals such as the phthalates induce peroxisome cer than adults from exposure to agents that are proliferation in the rodent resulting in hepato- mutagenic or have demonstrated oncogenic poten- carcinogenicity, but there is less responsiveness in tial.67–69,95 That is certainly proved for high doses of primates or human liver cells.109–119 There is much ionizing radiation and for exposures to radioactive discussion and controversy in the literature regard- 131I.51,66–69 Studies of the oncogenic effects of radia- ing the mechanism of this carcinogenic effect, namely, tion in Hiroshima and Nagasaki demonstrated that the role of increased cell division as the cause of mu- children have a higher risk of cancer after whole- tation and eventual carcinogenicity.120–125 Whether body irradiation. However, this increased risk is the carcinogenicity is the result of mutation or some magnified by the higher proportion of acute lympho- other mechanism related to peroxisome proliferation cytic leukemia in children and the increased risk of is of interest, but the important aspect of this topic is this disease in radiated children. The calculated over- the marked difference in oncogenic susceptibility in all risk of cancer in irradiated children has 95% con- various species.101,107,114,126 Animal carcinogenicity fidence limits of 1.0 to 1.8.68,69 studies using the phthalates and other chemicals that There are very few cancer studies in animals that stimulate the peroxisome proliferation response may expose the animals during a narrow window of time not be appropriate models to determine human can- that would be equivalent to childhood or adoles- cence. Most animal cancer studies using environ- The second agent that received much attention is mental chemicals and drugs involve life-long expo- saccharin, which produced bladder cancer when sures. The children who were exposed to high doses high doses of saccharin were administered to ro- of ionizing radiation in Hiroshima and Nagasaki did dents. At high doses, precipitates of saccharin de- have an increased incidence of leukemia to a greater velop in the rodent bladder, producing inflammation extent than did the exposed adults.67–69 There are and proliferation that ultimately result in bladder studies involving children and adolescents who have tumors.127,128 Other experiments indicated that hu- been treated for cancer with chemotherapeutic drugs man exposures of saccharin would never result in the and radiation, and these survivors are at an in- situation that occurred in the rodent.
creased risk of second cancers. However, when they The phthalate and saccharin experiences indicate become parents, they do not have offspring with an that when the MOA for carcinogenesis is determin- increased incidence of cancer.96 Animal studies that istic (a threshold effect), the risk may not be present would involve only short exposures to proven hu- at lower exposures and that species differences in man carcinogens during the equivalent of childhood metabolism and response may make it difficult to or adolescence could be performed. The most appro- apply animal risks to human risks. Conversely, when priate first approach would be to select agents that the oncogenic effect is related to a mutagenic agent, have been demonstrated to be positive in a life-long the theoretical risk for a no-threshold or stochastic animal study or agents that are definitely mutagenic effect exists (Table 1).
as a first approach to determine the oncogenic sen- For determining whether the oncogenic risk for sitivity to environmental toxicants during various drugs and chemicals is greater during postpartum animal development, protocols would have to be There are extensive reports concerning the onco- developed during these stages of animal develop- genic effect of drugs and chemicals in life-long ani- ment. Before embarking on the initiation of such mal studies. Many of these cancer studies have eval- testing, it would be important to determine whether uated environmental chemicals (eg, organochlorine these studies would be of benefit for human assess- chemicals, ethylene oxide, pesticides, organic sol- ment of oncogenic risks. Pilot studies could be per- vents, phthalates, acrylonitriles, trihalomethanes).
formed using known mutagenic or carcinogenic Most of these cancer studies have used rodents and agents. The increased costs and possible benefits of have also exposed the animals at relatively high ex- the new information would have to be evaluated to determine whether we should initiate these develop- Most agents that have been demonstrated to be mental oncogenic studies. This is a difficult issue to carcinogenic in humans will produce cancer in some settle. It might be better to perform research on MOA by on January 26, 2006 Protocol for Environmental Toxicant Studies Using Animals During Developmental Stages (Neonatal, Infant, and Juvenile Animals) 1. The toxicant exposure should occur by the same route in the animal as it occurs in the human.
2. Exposure should include a wide range and include the level to which humans are exposed.
3. Serum or tissue concentrations of the toxicant or its active metabolite should be determined, whichever is more appropriate.
4. Metabolism, half-life, turnover, mechanism of detoxification, and excretion should be 5. Biomarkers for evaluating the effects of toxicants in developing organisms should include growth, maturation, time of puberty, neurobehavioral effects, fertility, specific organ and tissuetoxicity, and pathology at windows during various stages of development.
6. The no-effect or threshold exposure should be determined for all toxic or detrimental findings.
7. The concentration of the toxicant should be determined in the sera or tissues of humans to determine whether the human is being exposed to concentrations that deleteriously affect theanimal model.
8. Mechanism of action studies should be initiated to determine the active metabolites that result in deleterious effects and determine whether the animal and human respond similarly or muchdifferently to the toxicant and its metabolites.
of carcinogenic agents and use that information in combination with the usual animal carcinogenicity 1. Brent RL. Evaluating the alleged teratogenicity of environmental agents. In: Brent RL, Beckman DA, eds. Clinics in Perinatology. Vol 13.
Philadelphia, PA: WB Saunders; 1986: 609 – 613 2. Brent RL. Protecting the public from teratogenic and mutagenic haz- There are a number of important observations that ards. J Clin Pharmacol. 1972;12:61–70 3. Brent RL. Drug testing in animals for teratogenic effects: thalidomide one could derive from reviewing the literature on in the pregnant rat. J Pediatr. 1964;64:762–770 using in vivo animal studies for studying the effects 4. Brent RL. The prediction of human diseases from laboratory and of environmental toxicants in developing fetuses and animal tests for teratogenicity, carcinogenicity and mutagenicity. In: postpartum developing animals. A few federal agen- Lasagna L ed. Controversies in Therapeutics. Philadelphia, PA: WB cies are requesting protocols to improve animal test- Saunders; 1980:134 –150 ing to study the sensitivity of neonatal, infant, and 5. Brent RL. Predicting teratogenic and reproductive risks in humans from exposure to various environmental agents using in vitro tech- juvenile animals to determine the effects of environ- niques and in vivo animal studies. Congenit Anom Kyoto. 1988; mental toxicants. Attempting to expose animals during narrow windows of development is more 6. Christian MS, Brent RL. Teratogen update: evaluation of the reproduc- difficult and more expensive, but because of the dif- tive and developmental risks of caffeine. Teratology. 2001;64:51–78 ferences in animals and humans, infant and juvenile 7. Brent RL. Book review: Scientific Frontiers in Developmental Toxicol- ogy and Risk Assessment by the National Research Council, National studies have to be designed to correct for these dif- Academy of Sciences, National Academy Press, Washington, DC, 327 ferences. It is true that the multigenerational studies pp., 2000. Teratology. 2002;65:88 –96 performed in animals are expensive, but they pro- 8. Brent RL. Teratogen update: reproductive risks of leflunomide vide information on growth, reproductive capacity, (Avara); a pyrimidine synthesis inhibitor: counseling women taking cancer, and lethality, and these studies would have leflunomide before or during pregnancy and men taking leflunomidewho are contemplating fathering a child. Teratology. 2001;63:106 –112 to be performed before embarking on selected tar- 9. FDA, Progestational Drug Products for Human Use; Requirements for geted studies at various stages of development.
Labeling Directed to the Patient. Proposed Rules, Department of There is no question that animal studies can provide Health and Human Services, Public Health Service, Food and Drug valuable information pertaining to human and ani- Administration, 21 CFR Part 310 [Docket No. 99N-0188], 64 FR 17985, mal vulnerability to environmental toxicants at dif- Tuesday, April 13, 1999; Federal Register, Vol. 64, No. 70 ferent stages of development. If risk estimates and 10. Physicians Desk Reference. 57th ed. Montvale, NJ: Medical Economics maximum permissible exposures are to be deter- 11. Brent RL. Drugs and pregnancy: are the insert warnings too dire? mined, then they have to be based on quality studies Contemp OB-GYN. 1982;20:42– 49 in animals and humans using modern pharmacoki- 12. Brent RL. Utilization of developmental basic science principles in the netics and toxicokinetic methods, as well as MOA evaluation of reproductive risks from pre- and post-conception envi- studies. Protocols for such studies are contained in ronmental radiation exposures. Teratology. 1999;59:182–204 13. Friedman JM, Little BB, Brent RL, Cordero JF, Hanson JW, Shepard Tables 3 and 5. One useful aspect of animal studies is TH. Potential human teratogenicity of frequently prescribed drugs.
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