Reproductive and teratologic effects of electromagnetic fields

Reproductive Toxicology, Vol. 7, pp. 535-580, 1993

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REPRODUCTIVE AND TERATOLOGIC EFFECTS OF ELECTROMAGNETIC FIELDS ROBERT L. BRENT,* WILLIAM E. GORDON,t WILLIAM R. BENNETT,~ and DAVID A. BECKMAN* *Alfred I. duPont Institute, Department of Medical Cell Biology, Wilmington, Delaware, tSpace Physics Department, Rice University, Houston, Texas, and ~:Applied Physics Department, Yale Station, New Haven, Connecticut Abstract m The reproductive risks of electromagnetic fields (EMF) were evaluated based on an extensive review of the scientific literature pertaining to human epidemiologic studies, secular trend data, in vivo animal studies and in vitro studies, and biologic plausibility. The epidemiologic studies involving the reproductive effects of EMF exposures to human populations have included populations exposed to: (1) video display terminals (VDTs), and (2) power fines and household appliances. The clinical use of diagnostic MRI (magnetic resonance imaging) has been increasing, but there are few reports or studies of pregnant women or individuals of reproductive age who have been exposed to MRI, and whose reproductive performance has been evaluated. The population that has been studied most frequently are women exposed to VDTs, but their EMF exposures are extremely low and frequently are at the level of the ambient EMF in a house or office. The results of epidemiologic studies involving VDTs are generally negataive for the reproductive effects that have been studied. Based on the number of studies, the exposure levels, and the fairly consistent results, it can be argued that VDT epidemiologic studies should no longer be given priority. There have been fewer studies concerned with the reproductive risks of power lines, electric substations, and home appliances. In some publications, positive findings for reproductive risks were reported, but the more consistent findings indicate that EMF, even at these higher exposures, do not generate a measurable increase in reproductive failures in the human population. When compared to other fields of human epidemiology, it is obvious that these studies have many difficulties. Exposures are rarely determined. Studies frequently involve small sample sizes and the investigators rarely have a combined expertise in EMF physics, engineering, and reproductive biology. Because of the allegation that there may be particular windows of frequency, wave shape, and intensity that may be deleterious, it is impossible to disregard low frequency EMF exposures as having no deleterious reproductive effects. Yet the epidemiologic data that are available would point in that direction. Secular trend data analysis of birth defect incidence data indicate that increasing generation of electric power during this century is not associated with a concomitant rise in the incidence of birth defects. There are over 70 EMF research projects dealing with animal and in vitro studies that are concerned with some aspect of reproduction and growth. Unfortunately, a large proportion of the embryology studies utilized the chick embryo and evaluated the presence or absence of teratogenesis after 48 to 52 hours of development. The chick embryo studies were of little assistance to the epidemiologist or clinician in determining whether EMF exposure represents a hazard to the human embryo, and the results were, in any case, inconsistent. Embryo culture or cell culture studies are also of fittle assistance in determining the human risk of EMF. In vitro or in vivo studies in nonhuman species can be used to study mechanisms and the effects that have been suggested by human investigations. Only well designed wholeanimal teratology studies are appropriate when the epidemiologists and clinical teratologists are uncertain about the environmental risks. Other aspects of reproductive failure such as abortion, infertility, stillbirth, and prematurity cannot be addressed by in vitro or culture experiments. In fact, it is very difficult to design and interpret nonprimate in vivo studies. Biologic plausability for teratogenesis can be supported if an agent such as EMF can be demonstrated to be cytotoxic or mutagenic. The studies dealing with mutagenesis, ceil death, and cell proliferation using in vitro systems do not indicate that EMF have the potential for deleteriously affecting proliferating and differentiating embryonic cells at the exposures to which populations are usually exposed. Of course, there is no environmental agent that has no effect, deleterious or not, at very high exposures. While there does not appear to be measurable risk of reproductive failure and birth defects from EMF exposures in humans, a modest program of investigation is warranted epidemiologically and in the Address correspondence to Robert L. Brent, Department of Medical Cell Biology, Alfred I. duPont Institute, P.O. Box 269, Wilmington, DE 19899. 535

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laboratory to answer some of the issues that have been raised that need clarification. It does not appear that the risk of EMF exposure is significant and, therefore, although preparing conducted research in the area of E M F risks is appropriate, it does not necessitate a massive expenditure of resources. Key Words: electromagnetic fields; EMF; epidemiology; teratology; in vitro.

INTRODUCTION We live our lives immersed in electric and magnetic fields, both internally and externally, from natural and manmade sources. Natural sources include those associated with the normal physiologic functions of the body and the magnetic field around the earth (Table 1). Man-made electric fields and magnetic fields are produced, for example in the generation, distribution, and consumption of electric power (Table 1 and 2). We tend to take the services and conveniences provided by electric power for granted. Neither the earth's natural fields nor the manmade electric and magnetic fields are visible, although their presence is detectable with appropriate devices. This section describes the fields, indicates the strengths of typical fields in various situations, and outlines the coupling of fields outside the human body to sites inside the body. There is a large variation in the frequencies produced by different sources and appliances we utilize: power line frequencies are 50 or 60 Hz (oscillations per second); video display terminals (VDTs) and television receivers produce time-varying (alternating) electric fields and magnetic fields that include 60 kHz and about 15 kHz, and multiples thereof; and radio frequencies are around one million Hz for AM radio and about I00 times higher for FM radio. At power line and VDT frequencies, the electric field and the magnetic field can be considered separately. The electric field is related to the voltage on

Table 1. Typical electric and magnetic fields encountered in daily life produced at power line frequencies Situation Home wiring At electrical appliances Under distribution lines serving homes Inside railroad cars Under high-voltage transmission lines Earth's static field (for comparison)

Electric field (volts/meter)

Magnetic field (milligauss)

1-10 30-300 10-60

1-5 5-3000 1-10

-1000-7000

10-200 25-100

120

500

the line relative to ground. Voltages are either 110 or 220 volts (V) in homes, about 12,000 V on distribution lines, and much higher on major transmission lines (Table 1 and 2). The magnetic field is related to the flow of current, i.e., electrons, in the line, which is measured in amperes (A). A circuit in a home is usually fused for 15 or 20 A, which limits the current in the circuit. A small household appliance such as a toaster may draw 10 A or less. The currents in distribution lines and transmission lines are higher, since they serve progressively more customers. Tables 1, 2, and 3 present the range of typical values of electric and magnetic fields encountered in daily life. The range clearly illustrates the ordersof-magnitude differences in the electric and magnetic fields associated with various sources of exposure. Substantially higher fields may be encountered in industrial and laboratory situations. The strengths of these electric and magnetic fields decrease rapidly with distance from the source (Tables 2 and 3). A person can be shielded relatively easily from external electric fields by a conducting box (screen room), a house, or an office, but not easily from magnetic fields. In addition, wiring configurations often can be arranged to minimize both the electric and magnetic fields produced by these common sources. The penetration of electric fields and of magnetic fields into and through the human body is very different. The ambient electric field unperturbed by the presence of the human body can be measured or calculated with some precision. However, the body is a complicated conductive dielectric with different tissues having different conductivities and permittivities. Such a conducting medium distorts the original electric field so as to reduce it by enormous (typically, a hundred million at power line frequencies) factors inside the body. At the same time, the field external to the body can be increased by factors of 10 to 50 by the body itself acting as a conductor. The result is that only estimates of the internal field are available, and these estimates vary from about one millionth to one-hundred millionth of the original, unperturbed external electric field. Thus external electric fields are markedly attenuated by the moderate conductivity of the body's surface.

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R e p r o d u c t i v e effects of E M F • R. L. BRENT ET AL.

Table 2. Typical electric and magnetic field strengths at ground level near high-tension lines Distance from line (feet) 500 kV line 230 k v line 115 kV line

Electric field (kV/m) Magnetic field (mG) Electric field (kV/m) Magnetic field (mG) Electric field (kV/m) Magnetic field (mG)

0

50

65

7.0 70 2.0 35 1.0 20

--1.5 15 0.5 5

3 25 -----

100

200

1 25

0.3 3 0.05 1 0.01 0.3

.3 5 0.07 1

Note: The magnetic fields va ry with the current, he nc e the load on the line.

smaller than the unavoidable thermal electric fields across the cell. Even the highest magnetic fields in Table 1 (3000 milligauss [mG] from a spiral hot plate) would not induce current densities comparable to those naturally occurring in the body. Moreover, order-ofmagnitude reductions of these and other magnetic fields produced by electric appliances and distribution lines can often be achieved by simple changes in wiring configurations that reduce the distance between wires carrying oppositely flowing currents. For example, the magnetic fields from electrified railroads have been substantially reduced by the adoption of out-of-phase feeder lines to provide current to the system in opposing directions at frequent intervals along the route; the use of twisted pair wiring in armored cable ("BX cable") can reduce background fields from house wiring well below the 1 mG level; and the magnetic fields from spiral hot plates can be reduced by an order of magnitude by the adoption of bifilar windings. Although there is no firm evidence to indicate that magnetic fields associated with common household appliances are biologically harmful, the use of minimum-field wiring

Because of this enormous attenuation, the electric fields listed in Table 1 are reduced to levels that are negligibly small compared to the normal background electric fields in the body generated by thermal fluctuations. For a representative cell diameter of 20 microns (micrometers) having a resistivity typical of body tissue (about 2 ohmmeters), these unavoidable thermal fields amount to about 0.02 volt/meter (V/m) at body temperature in a bandwidth of 100 Hz. Hence, electric fields of the order of a million V/m would be required outside the body to produce fields across a cell that are of the same order of magnitude as the unavoidable thermal fields. In contrast to the external electric field situation, magnetic fields are unperturbed by the human body and penetrate without attenuation. This transparency of the body to magnetic fields comes about because the body contains almost no magnetic material. Internal electric fields are generated within the body by these time-varying magnetic fields through a well-known mechanism called the Faraday effect. For magnetic fields in the range shown in Table 1, these induced electric fields are also very much

Table 3. RMS magnetic fields in milliguass (mG) D i s t a nc e in c e n t i m e t e r s (cm) Appliance Spiral hot plate M i c r o w a v e oven, rear side Coffee m a k e r F u r n a c e fan Attic e x h a u s t fan 80 watt fluorescent t r a n s f o r m e r 100 a m p circuit b r e a k e r 200 w a t t stereo Washing machine bottom H e a t i n g pad Refrigerator, rear side Four-slice t o a s t e r 100 w a t t light bulb

2

4

10

20

40

10

1570 1050 230 76 45 40 40 39 30 20 13 8 4

1160 820 90 36 21 34 31 27 22 11 11 4 2.5

750 350 26 11 6 26 21 13 11 3 8 1 1.5

31 140 7 5 2 18 12 5 5 -5 ---

4 28 1 1 -6 2 1 2 -1 ---

-5 ------------

Note: The b a c k g r o u n d from the E a r t h ' s static magnetic field is about 500 mG.

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configurations would be prudent and would also minimize interference with other electronic devices. The great majority of exposures of humans to electric and magnetic fields are quite low compared to exposures from natural phenomenon; such as the earth's magnetic field and the electrical fields induced by thermal energy at body temperature. As with all environmental risks, one of the most important criterion utilized in determining the risk is the dose or exposure. In many epidemiologic studies and animal studies presented in this paper, the actual exposures being studied for their biologic effects were below the electric and magnetic fields resulting from unavoidable phenomena.

The coupling of ambient EMF into the human body electric field The ambient electric field, unperturbed by the human body, can be measured and/or calculated with some precision, but it is difficult to measure or calculate the electric field inside the body. The body is a conductor, and the problem is complicated by the fact that different tissues have different conductivities. In general, a conducting medium perturbs the field so theoreticians look for models of the body and experimentalists look for measuring devices. The result is that only estimates of the internal fields are available. The estimates at sites in the electrolytes within the human body range from 10 -5 to 10 -8 of the external field. A range of 3 orders of magnitude in the estimated electric field at a site within the body relative to the field outside the body confounds efforts to define effects and dose-response relationships.

Magnetic field In contrast to the electric field, the magnetic field is essentially unperturbed by the human body; it penetrates the body with little recognition that the body is present. This occurs because the body has almost no magnetic materials that will interact with the field. It will be helpful to consider two different 60-hertz (Hz) magnetic field levels: 1. A magnetic field o f B = 650 gauss (G) acting over the largest diameter (say, 40 cm) loop of tissue in the body will induce current densities (~ 1 ampere/meter 2 [A/m2]) large enough to stimulate cells. Such field levels are usually only found in worst-case industrial or laboratory situations. 2. A field of B = 6.5 G acting over the same area will induce current densities (~0.01 A/m 2) comparable to those produced by natural and unavoidable thermal electric fields at the cell level.

Volume7, Number 6, 1993 This field is at least ten times higher than the largest magnetic fields found under common urban distribution lines and is comparable to the largest fields encountered in very close proximity (< 10 cm) to household appliances such as spiral wound electric hot plates. The current density induced in the body is proportional to the diameter of the body (or the body component), the frequency of the field (for the frequencies being considered), and the conductivity of the tissue. Unlike the coupling of electric fields into the body where the field inside is orders of magnitude below the field outside the body, magnetic fields readily enter the body; therefore, the magnetic fields inside and outside are virtually of the same strength. However, it is difficult to determine the induced currents in the body because of the inhomogeneity of the body as reflected in the conductivities of the various tissues. When the magnetic field is pulsed there is a spectrum of frequencies in the output that includes the fundamental pulse repetition frequency and many of its harmonics. Often, the spectral information is missing or incomplete in the reports of experiments; this makes the results impossible, or at least very difficult to interpret.

BACKGROUND INFORMATION

The epidemiology of reproductive failures and reproductive risks One of the most difficult and complicated areas of epidemiologic research is in the area of reproductive risks. The reason for the complexity of this area of research are the following: 1. Reproductive failures of various types are common, affect a substantial portion of the population, and have occurrence patterns that vary in the general population (Table 4) (1). 2. Reproductive problems include a wide variety of pathologic conditions that may or may not be mechanistically related to each other. 3. The known etiologies for reproductive problems include both genetic and environmental problems. Furthermore, the environmental factors include a host of external chemical, infectious, and physical agents as well as intrinsic disease processes that may be initiated before or during pregnancy in women and before fertilization in men (1). How do investigators interested in determining whether a particular environmental agent such as electromagnetic fields (EMF), presents reproductive

Reproductive effects of EMF • R. L. BRENT ET AL.

Table 4. Reproductive risks in human populations Reproductiverisk Immunologically diagnosed spontaneous abortions/106 conceptions Clinically recognized spontaneous abortion/106 pregnancies Genetic diseases in 106 births Multifactorial or polygenic (genetic-environmental interactions) Dominantly inherited disease Autosomal and sex-linked genetic disease Cytogenetic (chromosomal abnormalities) New mutations Major congenital malformations/106 births (many are genetic in etiology) Prematurity/106 births Fetal growth retardation/106 births Stillbirths/106 pregnancies (>20 wks) Infertility

Frequency 350,000 150,000 110,000 90,000 10,000 1200 5000 3000 30,000 40,000 30,000 20,900 15% of couples

risks to the human population? The study of human populations not only necessitates a knowledge of epidemiology, but also a broad understanding of the field of reproductive failures. Since all types of reproductive problems have multiple etiologies, the epidemiologist has to recognize that differences in the incidence of reproductive problems between exposed and nonexposed groups may be due to differences in the study groups that are unrelated to the EMF exposure. Reproductive failures can be categorized as follows. Infertility may be due to hereditary disease, congenital malformations of the reproductive tract, infection and venereal disease, mechanical problems, psychologic illness, medication, chronic illness, or ignorance. Spontaneous abortion can result from inherited or acquired chromosomal abnormalities, inherited diseases, medically or environmentally produced blighted (malformed) embryos, maternal illness, or lupus anticoagulant factor (2-5). Stillbirth can result from maternal and fetal infections, congenital malformations, mechanical problems with the placenta or umbilical cord, placental disease, maternal illness, chromosomal abnormalities, multiple births, environmental drug, chemical or medication exposure, or poor prenatal care.

Prematurity can result from maternal nutritional problems, maternal illness, genital tract infections, poor prenatal care, congenital malformations, placental abnormalities, placental disease, eclampsia,

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maternal hypertensive-renal-vascular disease, or trauma. Fetal growth retardation can be associated with maternal nutritional problems, maternal illness, embryonic and fetal infections, poor prenatal care, genetic diseases, chromosomal abnormalities, congenital malformations, medical treatments, drug and chemical exposure, including smoking and alcohol, or placental diseases. Hereditary diseases are predominantly inherited, but may be due to spontaneous mutations or to prenatally acquired chromosomal abnormalities. There is no question that many environmental drugs and chemicals have mutagenic potential, yet there is little evidence to suggest that environmental exposure to mutagenic agents contributes very much to the incidence of genetic disease. The offspring of the atomic bomb survivors had no measurable increase in induced mutations following exposure to an agent that has proven mutagenic potential (6). By contrast, there are substantial data to indicate that environmental drugs, chemicals, and ionizing radiation can induce cancer in human populations (7,8). The greater potential for mutagenic agents to induce clinical cancer rather than hereditary disease is very likely due to the loss of mutations and mutated cells during the process of meiosis, fertilization, implantation, and organogenesis, rather than to the fact that environmentally induced mutations do not occur. Congenital malformations. The etiology of congenital malformations can be divided into three broad categories: unknown, genetic, and environmental factors (Table 5). The etiology of the majority of human malformations, approximately 65 to 75%, is unknown (1,9-11). However, a significant proportion of congenital malformations of unknown etiology is likely to be polygenic, that is, due to two or more genetic loci (12,13) or at least has an important genetic component. Malformations with an increased recurrence risk, such as cleft lip and palate, anencephaly, spina bifida, certain congenital heart diseases, pyloric stenosis, hypospadias, inguinal hernia, talipes equinovarus, and congenital dislocation of the hip can fit the category of multifactorial disease, as well as the category of polygenic inherited disease (12,14). The multifactorial/threshold hypothesis (14) involves the modulation of a continuum of genetic characteristics by intrinsic and extrinsic (environmental) factors. Although the modulating factors are not known, they probably include placental blood flow, placental transport, site of implantation, maternal disease states, infections, drugs, chemicals, and spontaneous errors of development.

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Table 5. E t i o l o g y on h u m a n congenital malformations o b s e r v e d during the first y e a r of life a Percent of total

Suspected cause Unknown Polygenic Multifactorial (gene-environment interactions) Spontaneous errors of development Synergistic interactions of teratogens Genetic Autosomal and sex-linked genetic disease New mutations Cytogenetic (chromosomal abnormalities) Environmental Maternal conditions: alcoholism, diabetes, endocrinopathies, phenylketonuria, smoking and nicotine, starvation, nutritional Infectious agents: rubella, toxoplasmosis, syphilis, herpes, cytomegalic inclusion disease, varicella, Venezuelan equine encephalitis, parvovirus B19 Mechanical problems (deformations): amniotic band contrictions, umbilical cord constraint, disparity in uterine size and uterine contents Chemicals, prescription drugs, high dose ionizing radiation, hyperthermia

65-75

10-25

10 4 3

1-2

<1

"Adapted from Brent (1, 10) and Brent and Holmes (33).

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Spontaneous errors of development may account for some of the malformations that occur without apparent abnormalities of the genome or environmental influence. It has been postulated that there is some probability for error during embryonic development, based on the fact that embryonic development is a complicated process, similar to the concept of spontaneous mutations (1,10,15). It has been estimated that up to 50% of all abortuses in the human are lost within the first three weeks of development (16; Tables 4 and 6). The World Health Organization (5) estimated that 15% of all clinically recognizable pregnancies end in spontaneous abortion, and 50 to 60% of the spontaneously aborted fetuses have chromosomal abnormalities (20,21). This means that, as a conservative estimate, 1173 clinically recognized pregnancies will result in approximately 173 miscarriages, and 30 to 60 of the infants will have congenital anomalies in the remaining 1000 live births. The true incidence of pregnancy loss is much higher, but undocumented pregnancies are not included in this risk estimate. The 3 to 6% prevalence of malformed offspring at birth represents the background risk for human maldevel-

Table 6. E s t i m a t e d o u t c o m e of 100 pregnancies versus time from c o n c e p t i o n

Time from conception Preimplantation 0-6 days Postimplantation 7-13 days 14-20 days 3-5 weeks

6-9 weeks

10-13 weeks 14-17 weeks 18-21 weeks 22-25 weeks 26-29 weeks 30-33 weeks 34-37 weeks 38 + weeks

Percent Percent death survival during to term a interval a 25

54.55

55 73 79.5

24.66 8.18 7.56

90

6.52

92 96.26 97.56 98.39 98.69 98.98 99.26 99.32

4.42 1.33 0.85 0.3 l 0.30 0.30 0.34 0.68

Last time for induction of selected malformations b

23rd day cyclopia, sirenomelia 26th day anencephaly 28th day meningomyelocele 34th day transposition of great vessels 36th day cleft lip, limb reduction defects 6th week diaphragmatic hernia, rectal atresia, ventricular septal defect, syndactyly 9th week cleft palate 10th week omphalocele 12th week hypospadias

aData from Kline and Stein (2). An estimated 50 to 70 percent of all human conceptions are lost in the first 30 weeks of gestation (19) and 78 percent are lost before term (23). bModified from Schardein (68).

Reproductive effects of EMF • R. L. BRENTET AL. opment. The lower figure represents severe or debilitating malformations, and the higher figure includes both major and minor malformations. Although we know little about the mechanisms that result in the in utero death of defective embryos (22), it is perhaps more important to understand the circumstances that permit abnormal embryos to survive to term.

Overall teratogenic risk. In order to appreciate the difficulty in predicting the effect that an environmental exposure such as EMF will have on the developing embryo, we will briefly discuss factors that influence this prediction. The baseline risk of human reproduction is based on epidemiologic studies that have determined the occurrence of fetal death and maldevelopment. Substantial numbers of conceptions are lost before term; 50% within the first three weeks (19,23). Of the liveborn infants, 3 to 6% will be recognized as congenitally malformed (Tables 1-3; 1,9). Environmental risk parameters or modifiers. If attention is directed toward environmental influences or agents that interfere with embryonic development, there are several scientific or embryologic principles that have an important impact on assessing the effect of various environmental agents on the developing embryo. These include the impact of: 1) the embryonic stage, 2) the dose or magnitude of the exposure, 3) the threshold concept, and 4) species differences. Other factors, such as pharmacokinetics and metabolism and placental transport are of no importance when evaluating the reproductive risks of an external physical agent such as EMF. Each of these principles will be discussed in some detail below. Additionally, more than 30 drug-related disorders are related to genotype (24), and, while not proven in the human, genetic variations alter drug teratogenicity in experimental animals (25,26). Whether there is a variation in species susceptibility to EMF is a matter for conjecture, but it is evident that the actual exposure to EMF will vary with the size of the organism being exposed within the same EMF. Finally, maternal disease states may produce deleterious effects on the fetus that are difficult to separate from a possible deleterious reproductive effect. This is an especially relevant consideration for long-standing conditions such as diabetes (27-29). When counseling patients, especially in our litigious climate, three confounding influences are at work. The first is the necessity to evaluate reported associations critically because of the anxiety created

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by unfounded reports and misinformation (30). The second is the fact that pregnancy is not without risk and congenital malformations and other reproductive failures occur in the absence of exposures to alleged reproductive toxicants such as EMF. Third, it is a truism that once misinformation has been communicated to the public concerning the cause of reproductive failure, the public is very reluctant to accept the correct information.

Principles of environmentally induced malformations. A basic tenet of environmentally produced malformations is the fact that teratogens or teratogenic milieu have certain characteristics in common and follow certain basic principles. These principles determine the quantitative and qualitative aspects of environmentally produced malformations. Embryonic stage The induction of malformations by environmental agents usually results in a spectrum of malformations and other effects, which vary with the stage and amount of exposure (9,31,32). The developmental period at which an exposure occurs will determine which structures are most susceptible to the deleterious effects of the agent and to what extent the embryo can repair the damage. Furthermore, the period of sensitivity may be narrow or broad, depending on the environmental agent and the malformation in question. Limb defects produced by thalidomide have a very short period of susceptibility, while microcephaly produced by ionizing radiation has a long period of susceptibility (33). Exposure of the embryo to reproductive toxicants during the very early stages of embryonic development, from fertilization through the early postimplantation period, is most likely to result in embryonic mortality (34-37). Surviving embryos have malformation rates similar to the controls, not because malformations cannot be produced at this stage, but because significant induced cell loss or chromosome abnormalities at these stages are most likely to result in malformations that are lethal. Because of the totipotentiality of early embryonic cells, surviving embryos have a much greater potential for normal development than when irradiated later in development. Wilson et al. (38), utilizing ionizing X-irradiation as the experimental teratogen, demonstrated that the "all or none phenomenon" or the marked resistance to teratogenesis, disappears over a period of a few hours in the rat during early organogenesis. The term "all or none phenomenon" has been misinterpreted by some investigators to indicate that malformations cannot be produced at this stage. On the contrary, it is likely that certain drugs,

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Table 7. Comparison of stochastic phenomena and threshold phenomena in the etiology of diseases produced by environmental agents and the risk of occurrence a Relationship

Pathology

Site

Diseases

Risk

Definition The incidence of disease increases with exposure but the severity and the nature of the disease in the patient remain the same Both the severity and incidence increase with higher exposures

Stochastic phenomena

Damage to a single cell may result in disease

DNA

Cancer, mutation

Exists at all exposures, although at low exposure, the risk is below the spontaneous risk

Threshold phenomena

Multiple sites of subcellular and cellular injury

Great variation in etiology, affecting many cells and organ processes

Malformations, death, growth retardation, etc.

Completely disappears below a certain threshold dose

a F r o m B r e n t (52).

chemicals, or other insults during this stage of development can result in an increase in malformed embryos (39,40), but the nature of embryonic development at this stage will still reflect the basic characteristic of the "all or none phenomenon," which is a propensity for the embryos to die, rather than result in surviving malformed embryos. The period of organogenesis (from day 18 through about day 40 of human development), is the period of greatest sensitivity to teratogenic insults and the period when most gross anatomic malformations can be induced (Table 3). Most major malformations are produced before the 36th day of gestation in the human. The exceptions to this rule include malformations of the genitourinary system, the palate, the brain, and deformations due to problems of constraint, disruption, or destruction. Severe growth retardation in the whole embryo or fetus may also result in permanent deleterious effects in many organs or tissues. The fetal period is characterized by histogenesis involving cell growth, differentiation, and migration. Exposure to environmental toxicants during the fetal period may decrease the cell population by producing cell death, or by inhibiting cell division or cell differentiation. Dose or magnitude o f the exposure The dose-response relationship is extremely important when comparing effects among different species. Several considerations affect the interpretation of dose-response relationships: an especially anxiety-provoking concept is that the interaction of two or more reproductive toxicants may potentiate their developmental effects. Although this is an extremely difficult hypothesis to test in the human, it is an important consideration because multiple exposures to environmental agents occur commonly

in human populations. Furthermore, Fraser (41) warns that the actual existence of a threshold phenomenon when nonteratogenic doses of two teratogens are combined could easily be misinterpreted as potentiation or synergism. Threshold dose The threshold dose is the dosage below which the incidence of death, malformation, growth retardation, or functional deficit is not statistically greater than that of the controls. The threshold level of exposure is usually from less than 1 to 3 orders of magnitude below the teratogenic or embryopathic dose for drugs and chemicals that kill or malform half the embryos. An exogenous teratogenic agent, therefore, has a no-effect dose, as contrasted to mutagens or carcinogens, which have a stochastic dose-response curve. Threshold phenomena are compared to stochastic phenomena in Table 7. The severity and incidence of malformations produced by every exogenous teratogenic agent that has been appropriately tested have exhibited threshold phenomena during organogenesis (9). While environmental agents that result in congenital malformations, stillbirth, and fetal growth retardation would be consistent with threshold phenomena, other reproductive failures such as abortion, genetic disease, and infertility might have stochastic relationships with some reproductive toxicants. Since the same principles cannot be applied to all reproductive effects, reproductive epidemiologic research is a very complex field of investigation (Table 7). Placental function, metabolism, and transport. The role of the placenta is extremely important when dealing with the evaluation of drug and chemical reproductive risks to the embryo. Differences in placental function and structure may affect our ability

Reproductive effects of EMF • R. L. B~r~T ET AL.

to apply teratogenic data developed in one species directly to other species, including the human (16,42-45). On the other hand when one is attempting to evaluate the effects of physical agents such as EMF, the impact of species differences in metabolism, function and transport of the placenta becomes much less important (34,46-49). Thus, animal experiments may be more predictive of human reproductive effects for exogenous agents, such as ionizing radiation, ultrasound, microwaves, and EMF because their primary effect is on the developing embryo (50). The most important concept with regard to placental transport of teratogens must be reemphasized. An agent is teratogenic because it affects the embryo directly or indirectly by its ability to produce an effect in the embryo or extraembryonic membranes at exposures that are attained in the human being, not because it crosses the placenta per se.

Species differences The genetic constitution of an organism is an important factor in the susceptibility of a species to a drug or chemical. Even among humans, there is some variability in the response to drugs and chemicals (24).

Mechanisms ofteratogenesis. Based on his review of the literature, Wilson (9) provided a format of theoretical teratogenic mechanisms: 1) mutation; 2) chromosomal aberrations; 3) mitotic interference; 4) altered nucleic acid synthesis and function; 5) lack of precursors, substrates, or coenzymes for biosynthesis; 6) altered energy sources; 7) enzyme inhibition; 8) osmolar imbalance, alterations in fluid pressures, viscosities, and osmotic pressures; and 9) altered membrane characteristics. Even though an agent can produce one or more of these pathologic processes, exposure to such an agent does not guarantee that maldevelopment will occur. Furthermore, it is possible that a drug, chemical, or physical agent can have more than one effect on the pregnant women and the developing conceptus and, therefore, the nature of the agent or its biochemical or physiologic effects will not in themselves predict the existence or the magnitude of the human reproductive effect. The discovery of human teratogens has come primarily from human epidemiologic studies. Animal studies and in vitro studies can be very helpful in determining the mechanism of teratogenesis and the pharmacokinetics related to teratogenesis (57). A list of mechanisms of teratogenic agents includes: 1) cell death beyond the recuperative capacity of

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the embryo/fetus; 2) mitotic delay: increase in the length of the cell cycle; 3) retarded differentiation: slowing or cessation in the process of differentiation or cell migration; 4) physical constraint and vascular insufficiency; 5) interference with histogenesis by processes such as cell depletion, necrosis, calcification, or scarfing; and 6) inhibited cell migration and cell communication (3). However, even if one understands the pathologic effects of an agent, one cannot predict the teratogenic risk of an exposure without taking into consideration the developmental stage, the magnitude of the exposure, and the repairability of the embryo. This would be especially true for physical agents such as EMF. IDENTIFICATION OF H U M A N TERATOGENS A N D R E P R O D U C T I V E TOXICANTS Without decisive evidence of human teratogenicity and reproductive toxicity, it is difficult to provide estimates of the hazard that exposures to specific agents such as EMF present to the human fetus. Single reports suggesting causal associations between suspected agents and human malformations can be misleading (30,53-55). It should be noted that most human teratogens have been identified by alert physicians or scientists. Epidemiologic studies have been most helpful in understanding the frequency, trends, and incidence of congenital malformations, whereas animal studies have been useful in understanding the mechanism of action of known human teratogens as well as lending support to epidemiologic studies by the development of animal models (50,56,57). Although methods of extrapolation are improving, an animal model cannot be extrapolated with certainty to the human condition if one has no information on reproductive toxicity in the human (15,18,56,57). Thus, the study of EMF reproductive risks must involve the development of plausible hypotheses of reproductive risk that are consistent with acceptable reproductive, teratologic, and embryologic principles. Several considerations are helpful in establishing that an environmental exposure causes maldevelopment in the human: 1. Human epidemiologic studies should consistently report that exposure to an agent is associated with an increased incidence of a specific reproductive effect or group of congenital malformations. 2. For common exposures, secular trend data should support the allegation. 3. An animal model mimics the reproductive effort

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Reproductive Toxicology

suspected in humans at exposures that are experienced by pregnant women. 4. The teratogenic effect or reproductive effect should increase with dose. 5. The suggested reproductive effect should be biologically plausible and not contradict established scientific principles (51,56,58,59). This approach is of greatest value when utilized for the evaluation of environmental agents that have been in use for some time or for evaluating new agents that have a similar mechanism of action, function, chemical structure, pharmacology, or physical effect to other agents that have been extensively studied. The evaluation of new drugs, physical agents, or chemicals must depend more on in vivo and in vitro animal testing and the application of the principles of biologic plausibility, since these data are usually not available from human populations. Even if the agent is closely related to a previously used compound, closely related compounds may have markedly different reproductive risks. It is well known that very minor changes in the thalidomide molecule can eliminate its teratogenic effect (60). With regard to EMF exposure there is a concern that certain EMF frequencies may have the ability to resonate or interact in a unique manner with tissues. Thus, there is the possibility that the effects of EMF may be much different at frequencies that are not far apart. This could be interpreted to mean that many experiments could miss combinations of frequency and intensity that present the greatest reproductive risks. The purpose of in vitro and in vivo testing is to determine whether a new environmental agent presents a reproductive risk when a pregnant woman is exposed to this agent: the difficulty with the present in vitro testing systems is that these systems have not been reliable predictors of human teratogenicity (15,57,67). Every drug that has been used for cancer chemotherapy in humans has been teratogenic in animal models; only a small fraction have resulted in human teratogenesis. There are many drugs and chemicals that can produce malformations, embryonic death, or growth retardation at doses far above the usual human exposure. These agents are therefore teratogens, but are not necessarily responsible for human teratogenesis. Similarly, there may be animal experiments that demonstrate some reproductive effect with EMF at frequencies and intensities that are very unlikely to occur in human populations; therefore, such an exposure would not represent a realistic reproductive risk.

Volume 7, Number 6, 1993

Let us define the three categories of drugs or chemicals with regard to their teratogenicity and reproductive toxicity. H u m a n teratogen: An agent or milieu that has been demonstrated to produce permanent alterations in the embryo or fetus following intrauterine exposures that usually occur or are attainable in the human. Potential h u m a n teratogen: An agent or milieu that has not been demonstrated to produce permanent alterations in the embryo or fetus following intrauterine exposures that usually occur or are attainable, but can affect the embryo or fetus if the exposure is substantially raised above the usual human exposure. N o n h u m a n teratogen: An agent or milieu that laas no embryotoxic or fetotoxic potential, because it is nontoxic at any dose or because it is so toxic to the mother that it kills the mother before or at the same dose that it begins to affect the embryo.

In reality, the largest group of drugs and environmental agents are the potential human teratogens because they include all drugs, chemicals, and physical agents that can produce embryotoxic and fetotoxic effects at some exposure. Since these exposures are not utilized or attained in the human, they represent no or minimal risk to the human embryo. The greatest problem facing the regulatory agencies and the teratologist is how to determine the margin of safety that should be required for exposures to potential reproductive toxicants. This can be accomplished if we recognize that the threshold concept of teratogenesis pertains and that even when drugs, chemicals, and environmental agents have toxic effects, there are also safe exposures for these agents. In most instances exposures of 1 to 2 orders of magnitude below the no-effect dose represent safe exposures to the embryo. Furthermore, we could better interpret the animal data if we include in our evaluation the ratio of the no-effect dose to the usual human dose. If one uses the more modern reproductive testing protocols for reproductive testing, one can better approximate safe levels of exposure (15,57,61). Although there have been extensive efforts to improve in vivo animal testing (15,57,62-66) and to design in vitro test systems (57,67), there are still important limitations of our ability to apply these in vitro and in vivo testing models directly to human risk assessment (14,15,17,35,57,67). In spite of the extensive use of animal testing and in vitro tests,

Reproductive effects of EMF • R. L. BRENT ET AL.

Table 8. Human teratogens identified since the thalidomide tragedy by human epidemiologic studies, alert physicians or scientists, and/or animal studies In vivo or in vitro

Teratogenicagents Anticonvulsants (chronic administration) Hydantoins Trimethadione Valproic acid Vitamin analogs Hypervitaminosis A Isotretinoin Etretinate PCB's Coumarin Alcohol Lithium Diethylstilbesterol Penicillamine ACE inhibitors

Year

Human studies

1963 1970 1982

+ + + + + +

1953 1983 1989 1968 1968 1967 1970 1971 1971 1982

+ + + + + + + + +

animal studies

+

+ + + + + + + + +

++ ++

+ + indicates that it was the major factor in the discovery of a human taratogenic effect. + indicates that it contributed to the suspicion that there was a teratogenic effect.

they have been minimally involved in identifying human teratogens since the thalidomide tragedy (Table 8). Most of the human teratogens have been identified since 1960 by means of human epidemiologic studies; i.e., alert physicians reporting clusters, case control studies, or birth registries (35,50,57) (Table 5). In a few instances, animal studies have predicted human teratogenicity (Table 5) and/or supported the suspicion of teratogenicity in the human. In a recent excellent review of the subject of testing for reproductive effects, Schardein (67) pointed out that whole-animal testing has not been modified or improved dramatically from the original two-litter and three-litter tests. He said: The segment II phase of the 1966 FDA (Food and Drug Administration) Guidelines for Reproduction Studies . . . . remains after two decades of use, a valid testing procedure for identifying the potential for teratogenic induction and other developmental toxicity in laboratory animals. Its chief limitation resides in the extent to which such testing procedures are predictive of toxicity in the pregnant human, not in any inherent inadequacy of the testing procedure.

In 1964, it was suggested that every whole animal mammalian testing protocol utilized for predicting teratogenicity in humans should have certain essential features (15). Reproductive toxicity testing should include 1) the determination of the ratio of

545

the maternal LDs0 (the dose that results in a 50% mortality) to the fetal LDs0, 2) the evaluation of embryonic death and growth retardation, as well as teratogenicity, and 3) exposures during fetal stages because of the importance of critical cell loss of important organs (brain, gonads, etc.) during midand late gestation. These suggestions have been only partially included in routine whole-animal testing procedures for reproductive toxicity some 28 years after they were originally suggested (15). Because of advances in science, in general, and teratology, in particular, teratology and reproductive toxicity testing that is more meaningful and more predictive of human effects can be designed. Although the cost of such an evaluation would be greater than for the 1966 FDA segment II reproductive studies, the ability to predict human reproductive risks and even more importantly human reproductive safety, would be improved. A ratio between the no-effect level and the human therapeutic dose that is considered to be safe should be established. If this concept is not emphasized, the reproductive toxicologists will have no incentive to determine the no-effect level, and they will not generate data that will support the value of the ratio of the no-effect level to the therapeutic dose in establishing safe levels of usage or maximum permissible exposures.

In vitro tests

Each test system has some unique features that have been attractive to some investigators. With the proliferation of these systems, it is obvious that the cost of performing a combination of these techniques could be more than a whole-animal reproductive study. More important than these unique features or possible reduced cost, is an important principle, "the nature of these tests indicate that they can NEVER be more predictive of teratogenicity or embryotoxicity than in vivo systems" (67). Schardein (67) eloquently stated, "the real dilemma in their use is eliminating procedures in animals and at the same time making tests more predictive; an incongruity to say the least." In vitro tests offer the experimental embryologists an opportunity to study various facets of their field. They can be used 1) to study normal embryonic development and differentiation, 2) to study some mechanisms of teratogenesis and embryotoxicity, 3) to study pharmacokinetics and the effect of isolated

546

Reproductive Toxicology

or combined metabolic products, and 4) to screen for cytotoxicity and interference with differentiation. It is also obvious that in vitro testing is going to fail to delineate important reproductive toxicity effects, including 1) late central nervous system effects (brain histogenesis, behavior, etc.), 2) unique unpredictable embryotoxic specificity (aspermatogenesis, cardiovascular hemodynamic changes, vascular disruption, yolk sac or chorioplacental effects, 3) differentiating between recuperable versus nonrecuperable growth retardation, 4) transplacental carcinogenesis, 5) lethal effects in the preimplantation period, and 6) effects not previously reported. Before discussing the human epidemiologic studies with regard to the reproductive effects of EMF exposure, some important principles of clinical teratology will be presented. Misconceptions of clinicians and scientists have led to some confusion regarding the potential effects of even proven teratogens. Erroneous dogma include the following concepts: 1. If an agent can produce one type of malformation it can produce any malformation (69). 2. A teratogenic agent presents a risk at any dose, once it has been proven to be teratogenic. Both of these concepts are wrong. The contributions of clinical teratologists and dysmorphologists clearly indicate that proven teratogens do not have the ability to produce every birth defect (49). More importantly, the concept of the syndrome in clinical medicine is probably more appropriate in clinical teratology than in any other area of clinical medicine. Many teratogens can be identified on the basis of the malformations that are produced, although it is true that there is a substantial overlap in malformation syndromes. They may not always be separable and environmentally produced birth defects may be confused with genetically determined malformations. The specificity of some environmental teratogens can sometimes point to the mechanism or site of action. For instance, the predominant central nervous system effects of methyl mercury are understood when one realizes the propensity for organic mercury to be stored in lipid. Some symptoms or signs such as growth retardation or mental retardation appear in many teratogenic syndromes and are, therefore, not very discriminating. On the other hand, rare or specific neurologic effects may point to a specific teratogen, such as deafness, retinitis, or a pattern of cerebral calcifications. Epidemiologic studies could benefit from the input of scientists who are knowledgeable about the nature of birth defect syndromes.

Volume 7, Number 6, 1993

A REVIEW AND ANALYSIS OF THE SCIENTIFIC LITERATURE DEALING WITH THE REPRODUCTIVE RISKS OF EXPOSURE TO EMF

Introduction The principles for the evaluation of human reproductive risks from EMF exposure are summarized as follows (10,51). 1. The results of human epidemiologic studies dealing with EMF exposure should be consistent. 2. The secular trend analysis should demonstrate a relationship between changes in the population exposure to EMF and the incidence of reproductive diseases. 3. In vitro and in vivo animal studies should confirm the reproductive effects that have been suggested to occur in human populations as a result of EMF exposure. 4. Increasing doses should result in an increasing incidence and severity of reproductive effects. 5. The epidemiologic findings should as a rule be biologically plausible, and the suggestion of a reproductive effect should not contradict established biologic principles. The ability to interpret all of these studies properly will depend on some understanding of how organisms and cells interact with electric and magnetic fields. It is important to recognize the vast range in exposures that the population experiences when evaluating the epidemiologic studies, the secular trend data, the animal experiments, and the biologic plausibility of some of the results that have been reported. The following discussion will address the nature of these interactions.

An analysis of human epidemiologic studies dealing with EMF reproductive risks (Table 6) Human epidemiologic studies involve three sources of EMF's: (1) video display terminals (VDTs), (2) power distribution and appliances, and (3) medical diagnostics. The introduction to this paper discussed the complexity and interrelationships of reproductive risks in the human population (Table 1 and 2). Each reproductive problem has a list of known etiologies and usually an unknown category, because many instances of reproductive failure may not be understood. A review of the human epidemiologic literature indicates that these studies have a combination of difficulties that reduce the quality of the studies (4,70-77). A well designed study should have the following qualities 1. The population's exposure should be accurately determined, and the comparison population should be unexposed. This is almost impossible

Reproductive effects of EMF • R. L. BRENTET AL. in EMF research. There is no population that is unexposed. In fact many populations that are evaluated in epidemiologic studies receive exposures from other sources and very few of the reported studies actually measured the exposure of the population they were studying. 2. In a cohort study, the exposed and control populations should be as similar as possible and should include large numbers of subjects because of the low power of this type of study. Some of the EMF cohort studies were very small and, therefore, were unlikely to yield any valid results. The size of the human cohort studies varied from 105 subjects (78) to thousands of subjects (79,80-84). 3. In a case control study, the investigators must guard against memory bias of the subjects or investigator bias, especially when investigating matters pertaining to reproductive problems and birth defects. Because of the lack of consistency in both the cohort studies and case control studies, some investigators suggested memory bias as an explanation for the reported inconsistencies (82,85). 4. The investigators involved in these studies should include individuals with expertise in epidemiology, electric and magnetic field physics and dosimetry, and reproductive biology and teratology. For reasons that are unexplainable, investigators with little experience in the field of teratology and reproductive biology embark on investigative projects that demand expertise in these areas. As an example, epidemiologic investigations of the causes of spontaneous abortions must deal with formidable problems: a. A majority of spontaneous abortions are due to chromosomal abnormalities that are unrelated to environmental exposures that may have occurred during pregnancy. b. The risk of abortion changes with each day of pregnancy (Table 3), so that matching controls is essential in order to eliminate the selection of two populations with different spontaneous abortion rates. c. Attempts to control for the hidden incidence of therapeutic abortions have only limited success (86,89). "The existence of high rates of induced abortion in the population may distort currently employed measures of the rate of spontaneous abortion" (86). The many epidemiologic studies that have been reviewed have dealt with varied sources of electric and magnetic fields. There have been large populations studied as well as very small groups. Studies of drug exposures and associated diseases are mea-

547

surably easier to perform than EMF studies for the above-mentioned reasons. It is important to understand the dosimetry of electric and magnetic fields because they differ from ionizing radiation exposure and drug exposures. The difficulties in interpreting some of the epidemiologic studies can be understood when one realizes how the exposure estimates were determined. Many of the exposure estimates were determined by job classification. Some were determined by questionnaire and an estimate of levels of exposure was determined by asking the participants the number of hours/week (h/week) the employee was exposed. The greatest number of studies dealt with exposure to VDTs rather than to power distribution sources and appliances.

Studies dealing with video display terminals (Table 9) Video display terminals (VDTs) typically expose the operator to maximum magnetic fields of about 50 milligauss (mG) at 10 cm from the screen, 2 mG at 30 cm, and 0.3 mG at 70 cm at frequencies --~ 15.75 kilohertz (kHz) (87,88). The values vary with the manufacturer, and more recent models tend to have smaller fields. The fields are much larger just behind the terminal and at the sides than they are at the front. These fields are comparable to those from home TV sets. At the operator's position, magnetic field strengths are ~ 2 mG and would induce maximum electric fields at 16 kHz of about (1) millivolt/meter (mV/m) in the abdomen of the operator, (2) 50 nanovolts/meter (nV/m) in a 10-micron sperm, (3) 0.4 microvolts/meter (/xV/m) in an 80-micron egg, (4) 10 /zV/m in a 2-millimeter (mm) embryo, and (5) 0.5 mV/m in a 10-cm fetus. For comparison, flying in an airplane through the Earth's magnetic field produces a uniform static electric field of about 10 mV/m throughout the entire human body. There have been a number o f epidemiologic studies dealing with the reproductive risks of EMF (78-80,83,84,87,90-96,202). Table 9 summarizes the human epidemiologic studies dealing with VDT usage. The number of subjects, type of study, source of EMF, reproductive effects examined, and results are presented in some detail. The following reproductive parameters were studied (1) birth defects (nine studies), (2) spontaneous abortions (12 studies), (3) stillbirth (four studies), (4) prematurity (three studies), (5) perinatal deaths (two studies), (6) neonatal deaths (two studies), (7) intrauterine growth retardation (four studies), (8) birth weight (four studies), (9) bleeding during pregnancy (one study), (10)

Reproductive Toxicology

548

Volume 7, Number 6, 1993

T a b l e 9. H u m a n r e p r o d u c t i v e risks o f E M F Author

Year

Locality

Type of study

Exposure source

Bjerkdal & Egenaes (95)

1987

Norway

Secular trend study of pregnancy outcome in 1820 women employed during a period of increasing VDT usage. Data collected from 1967-84.

VDT

Brandt & Nielsen (80)

1990

Denmark

Case control study of commercial and clerical employees; 661 cases & 2252 controls.

VDT

Bryant & Love (92)

1989

Canada

Case control study, using 2 matched controls for each case at 1 of 3 hospitals in Calgary, 1984-85. Matched 333 postpartum & 314 prepartum controls with 334 cases.

VDT

Ericson & K~ll6n (83)

1986

Sweden

Cohort; groups drawn from same socioeconomic strata, but with low, medium, and high occupational exposure. 10,031 in total study. 5136 in '76-'77, 4985 in '80-'81.

VDT

Ericson & Kfill6n (1980-82) (84)

1986

Sweden

Case control; 531 cases & 1032 controls were selected from a birth registry by job classification. Cases had abortions, birth defects, perinatal deaths, & low birth weight.

VDT

Goldhaber et al. (96)

1988

California Kaiser-Permanente Medical Care Program

Case control; 1583 pregnant women attending the Obs and Gyn clinic. Asked to recall exposure in their pregnancy an average of 2.5 yrs after their exposure.

VDT

Kaune et al. (169)

1987

Colorado

Residential magnetic and electric field measurements.

Residential exposure: A magnetic flux density (MFD) and an electric field (E-field) data acquisition system was built for characterizing extremely low-frequency fields in residents.

Knave et al. (97)

1979

Sweden

Exposure-based: 53 workers (men) exposed for over 5 years while working at high voltage substations were compared to 53 unexposed.

Electric substation was described as 50 Hz and 400 kV. Measurements of the electric fields in the areas where the men worked were 10-15 kV/m and in some areas were 20 kV/m.

Reproductive effects of EMF • R. L. BRENT ET AL.

Exposure description

Reproductive effect

549

Results

Remarks

Birth defects Stillbirths Per/natal deaths Neonatal deaths Multiple births Prematurity

No indications that the introduction of VDT's into the working environment has led to adverse outcomes among female employees.

The reproductive outcomes were divided into 3 periods. 1967-72, 1973-78, 1979-84.

VDT exposure at work. Four exposure groups in hours/week (h/week) as determined by questionnaire.

Birth defects (661 birth defects and 2252 controls). Postal questionnaires were used to determine VDT exposure, lifestyle, stress, and solvent exposure.

Negative study. O.R. = 0.96 (0.76-1.2). No assoc, of birth defects with VDT exposure. Mult. log/st, regres, analys, for confounders included mat. age, parity, abortions, job stress, solvent exp., h/week of work, alcohol, smoking, medicines, illness.

The source cases were obtained from 15 to 44 yr. old union employees during 1982-85. Only women who were employed during pregnancy were included. There was a correlation of the amount of VDT use and job stress.

VDT exposure; determined from 3 months before pregnancy to 4 months after the last menstrual period.

Spontaneous abortion was studied and was defined as unintentional cessation of pregnancy before 20 weeks of gestation.

Negative study. Four groups O.R. = 0.74 (0.52-1.06) N = 314 O.R. = .69 (0.45-1.05) O.R. = .87 (0.61-1.25) N = 332 O.R. = .79 (0.52-1.20)

Women had to be admitted and interviewed in the hospital to be included in the study. Exposure based on self hx of h/week of VDT use. Postnatal control group only included liveboru infants. Selection bias discussed. Multivariate logistic regressn, analysis

Exposure estimated by job classification. Distribution of maternal age was the same in all 3 exposure groups.

Birth defects (only significant ones were counted). Stillbirths Neonatal deaths, <7 days of age Birth weight (% low birth weight) Spontaneous abortions

Observed/expected (O/E) for birth defects were not significantly increased, O/E = 0.5 and 1.2 in high exp. group; per/natal death rate, O/E = 1.0 and 0.8; low birth weight O/E = 0.8 and 1.3; and spontaneous malformations were also not increased, O/E = 1.1.

Grouping of all spontaneous abortion. Did not count malformations that were minor or that were variably reported. Population studied at two different times. Secular trend information discussed.

H/week of VDT use was obtained by questionnaire.

Spontaneous abortions Birth defects

Spontaneous abortion: Greater than 10 h/week VDT exposure had) RR = 1.04 (0.9-1.2) Birth defects: Malformations in 44 infants whose mothers were exposed showed no signs of specificity when compared to those in 30 infants who were not exposed in utero.

Authors believe there is no marked risk for spontaneous abortion but they cannot rule out an effect on the rate of birth defects. If present, they believe it is most likely an effect of covarying risk factors, like smoking or stress.

Exposure determined in h/week during the first trimester.

Spontaneous abortion (exposure correlation in adm-clerical workers) Birth defects

N = 1078 >20 h/week, O.R. = 1.8 (1.2-2.8) N = 820:5-20 h/week, O.R. = 1.4 (0.7-2.7) >20 hrs/wk, O.R. = 1.4 (0.7-2.9)

Early pregnancy loss not reported. Grouping of all spontaneous abortions. Exposure to VDT by self hx. Adjusted for age, education, occupation, smoking, alcohol, and other maternal characteristics. Population studied 1981-82.

Houshold wiring evaluated in 43 homes in Colorado.

Evaluated the concept of LCC (low current configuration) and HCC (high current configuration) as possibly responsible for variability in household exposure to magnetic flux densities. Examined for: 1. Central nervous system effects 2. CBC 3. EKG 4. Sedimentation rate 5. Fertility 6. Offspring sex ratio

This research was stimulated by the reports of Wertheimer and Leeper that certain types of electrical transmission and distribution-wiring configurations had an elevated risk for cancer ('79). There were no differences in any of these parameters between the exposed and nonexposed groups.

continued

Reproductive Toxicology

550

T a b l e 9. Locality

Volume 7, Number 6, 1993

(Continued)

Author

Year

Type of study

Exposure source

Kurppa et al. (94)

1985

Finland

Case control; interviewed 1475 women who delivered children with birth defects.

VDT first trimester exposure

McDonald et al. (82)

1986

Canada (Montreal)

Cohort: data obtained VDT from 11 hospitals on 56,012 current and 48,608 past pregnancies. In 17,632 their occupation had substantial use of VDT's.

VDT

McDonald et al. Population studied 1982-84 (81)

1988

Canada (Montreal)

Cohort: VDT usage was recorded in 4712 current and 2164 previous pregnancies in women in full-time employment at conception. Allowances made for 7 confounding variables.

VDT

Mikolajczyk et al. (78)

1987

Poland

Cohort clerks working for Polish airlines. Starting population consisted of a total of 105 employees.

VDT

Nielson & Brandt (79)

1990

Denmark

Case base study: among all women in Denmark who were clerical union members for at least 1 month from 1982-85 (214,108), a subpopulation of 24,352 was identified in the birth register.

VDT

Nordstrom et al. (98)

1983

Sweden

Cohort: retrospective questionnaires given to 542 male workers at power plant with 89% response. The study involved preconception exposures to E & B (magnetic) fields.

Electrical substation workers. 331 exposed to 440 kV, 145 to 220 to 380 V and 66 exposed to a maximum of 130 kV. There were 348 reference unexposed.

Nurmimen & Kruppa (1972-82) (91)

1988

Finland

1475 mothers from a VDT birth defects case control study were divided into office workers (239) and non-office workers (805).

VDT

Schnorr et al. (87)

1991

USA, South

Cohort, 2430 women interviewed; 882 pregnancies met the criteria for inclusion of the study. Compared users to nonusers.

VDT usage in number of h/week.

Singewald et al. (99)

1973

Case reports

Electrical transmission line workers were followed for 9 years

Reproductive effects of EMF • R. L. BRENT ET AL.

Exposure description

Reproductive effect Birth Defects

O.R. = 0.9 (0.6-1.2)

CNS Orofacial cleft Skeletal defect

O.R. = 0.4 (0.2-1.0) O.R. = 0.9 (0.5-1.7) O.R. = 0.8 (0.4-1.9)

CV

O.R. = 1.6 (0.7-3.9)

Birth defects (no difference in incidence between exposed and non-exposed)

Users (3257) 3.31% malf. Users and non-users (8805) 3.52% malf. No difference in rates for prior pregnancies.

Spontaneous abortion (no difference in incidence between exposed and nonexposed)

Exposure for 3 VDT models evaluated in detail. Actual exposure of employees not measured. Ninetyfive percent of the energy was between 10-125 kHz. E = 0.1-10 V/m.

Operators who used VDT's had higher abdominal exposure to 15 kHz EMF. Abdominal exposure to 45-60 Hz was similar in operators and nonoperators.

Results

Spontaneous abortions evaluated in 9442 pregnancies O/E = 1.01 (0.95-1.08) = = = = = = =

0.94 (0.78-1.13) 1.84 (1.07-3.15) 1.19 (1.09-130) 1.06 (0.8-1.4) 0.82 (0.47-1.33) 1.08 (0.98-1.18) 1.03 (0.92-1.15)

551

Remarks Occupational title used for VDT exposure and work description.

Study does not support the suggestion that work with a VDT in pregnancy increases the risk of congenital defects or spontaneous abortions.

Exposure classified by job title. Large study, Authors discuss possibility of recall bias. Interviews conducted at a time of public concern about VDT's. Birth defects with known etiology not excluded.

Birth defects, total Renal defects Spontaneous abortion (individual and grouped) Stillbirth Prematurity Birth weight reduction

O.R. O.R. O.R. O.R. O.R. O.R. O.R.

Spontaneous abortion

Of the t05 clerks evaluated for spontaneous abortion, 40 had therapeutic abortions. Spont. abort, in exposed 20/58 = 34%. Spontaneous abortion in controls; 5/25 = 25%.

A very small population in which to study the incidence of spont, abortion. The authors suggest that the stressful tasks associated with VDT use are contributing factors and have recommended that working with VDT's be contraindicated in pregnant women,

Spontaneous abortion

Source population N = 24,352. There were 2252 abortions identified, and when the VDT cases and control groups were compared: O.R. = 0.94 (0.77-1.14).

Ergonomic factors, life style and job stress were considered in the analysis and did not contribute to the results.

Spontaneous abortion Perinatal mortality Birth defects Birth weight

No significant difference in abortions or perinatal mortality between the three exposed groups and the reference group. There were 12 malformations in the 125 switchyard workers (highest exposure group). There was no malformation pattern.

There was a discordance in the reproductive effects evaluated. Small number of patients in the groups.

Threatened abortion

O.R. = 0.8 (0.5-1.3).

Bleeding

O.R. = 0.9 (0.6-1.6).

Length of gestation Birth weight Placental weight

No difference in pregnancy duration, birth weight, or placental weight.

Used occupational title for VDT exposure. Only mothers who worked throughout the whole pregnancy were included, so 431 women were excluded from the analysis.

Spontaneous abortion

O.R. = 0.93 (0.63-1.38). No dose response relationship. Exposure 1-25 h/week O.R. = 1.04 (0.61-1.79). Exposure >25 h/week. O.R. = 1.00 (0.60-1.64).

Examined 10 men over a period of 9 years for changes in their sperm count and morphology.

No changes observed in sperm morphology and count.

Subclinical loss probably not included. Stress questions excluded. " T h e use of VDTs and exposure to the accompanying EMFs were not associated with an increased risk of spontaneous abortion in this study."

continued

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552

Table 9. Author

Year

Locality

Wertheimer & Leeper (100)

1989

Oregon (Eugene or Springfield) birth record data from 1982-85.

Wertheimer & Leeper (1975-81) (85)

1986

Westerhotm & Ericson (1980-82)

(Continued) Type of study

Exposure source

Cohort study with small numbers,

Electromagnetic field exposure from ceiling electric heat.

Colorado

Cohort: 1296 newborns for whom exposure information had been obtained and 692 earlier births from these same parents.

Electric blankets Heated waterbeds Exposures may be intermittant because they may cycle on and off. In some blankets E may be present when they are not turned on.

1987

Sweden

Cohort; 4117 full term pregnancies of women clerks working for the national social security agency. Questionnaire used to obtain information on population obtained from census data.

VDT

1990

California

Case control: cases were county residents >20 years of age who had a spontaneous abortion before 20 weeks gestation. Controls were obtained from the same source who had normal births.

VDT

(90)

Windham et al. (1986-87) (93)

Volume 7, Number 6, 1993

threatened abortion (one study), and (11) multiple births (one study).

Birth defects. Nine of the epidemiologic studies examined the risk of birth defects. Only one study, a case control study, suggested an increase in the risk of birth defects in the VDT exposed group (84). These investigators also studied the risk of spontaneous abortion and found no association [odds ratio ( O . R . ) = 1.04 (0.9-1.2)]. There were 531 exposed patients and 1032 controls in their study, so the expected number of malformations would be quite small. This prompted them to conclude that their results were most likely due to covarying risk factors. This conclusion was based on the fact that the spectrum of malformations in the exposed and nonexposed population did not differ and therefore it was unlikely that the statistical association was causally related to VDT exposure. The overall re-

suits indicate that pregnant women exposed to VDTs do not have an increased risk of birth defects. Spontaneous abortions. In two of the 12 epidemiologic studies that examined the risk of spontaneous abortion the investigators reported OR that were greater than 1.0 and were statistically significant. The remaining studies did not conclude that there was an increased risk of spontaneous abortions in the population that was exposed to VDTs. It is important to indicate that the negative results were reported in many of the studies that had very large numbers of control and exposed individuals. McDonald et al. (202) examined the hospital records of 56,012 current pregnancies and 46,608 past pregnancies to obtain their study population. They eventually used 9442 patients in their study of spontaneous abortions and obtained an observed to expected ratio (O/E) = 1.01 (0.95-1.08). Nielson and Brandt (79) initiated a case control study from a subpopula-

Reproductive effects of EMF • R. L. BRENTET AL.

553

Reproductive effect

Results

E = 10-50 V/m B = 10mG 60 Hz

Spontaneous abortion before 20 weeks gestation. Analysis was limited to abortions that occurred within 1 year prior to the live birth, which was the proband.

The ratio of abortions to livebirths was identical in a group of 802 women exposed to ceiling heat (7.6% of the mothers had an abortion prior to the year of delivery). This figure was 7.5% of 1077 mothers who were not exposed to ceiling heat.

The investigators then divided the exposed group into 12 monthly groups and noted a correlation of abo~ion with ceiling heat exposure.

B 15 mG

Spontaneous and induced abortion

Increased abortions, P < .05 & .02.

Birth defects Fetal growth

Increased birth defects, P < .05, control 1/335 & exposed 51193. Decreased fetal growth, P < .005 & .05

Information obtained by phone interview of parents who published birth announcements. Of 4271 families with a new child, 1806 (42%) published birth announcements. 1318 were reached by phone and asked whether they used these appliances.

Birth defects Spontaneous abortions Stillbirths Perinatal deaths

Failed to demonstrate convincing deviation in the exposed groups from what one would have expected. As an example the expected frequency of "significant" birth defects was 59 in a population of 3160 exposed. The actual number was 61.

Authors commented that the incidences of spontaneous abortions would be underascertained when utilizing information obtained from hospital records.

Spontaneous abortion

Spontaneous abortion Exposure <20 h/week and Exposure >20 h/week O.R. = 1.2 (0.88-1.05) O.R. = 1.1 (0.52-2.1) O.R. = 1.6 (0.92-2.9)

There were 628 cases and 1308 controls that completed the interviews. Cases obtained by pathology specimens. No exclusion of spontaneous abortion (genetic causes). Exposure information limited. Small numbers in the IUGR and tow birth weight groups.

Exposure description

E 100 V]m, 60 Hz B 4mG E 100 V/m, 60 Hz

Exposure magnitude was determined by the class of work. This classification was performed in cooperation with the national trade union. There were 5 exposure groups.

Low birth weight Intrauterine growth retardation (IUGR)

tion o f 24,352 patients. In this study the OR was 0.94 (0.77-1.14) for the risk o f abortion. In most of the studies the method o f estimating exposure to VDTs was by j o b classification and/or questioning the individuals. It is quite evident that there does not appear to be a risk o f spontaneous abortion from VDT exposure. The remaining reproductive parameters, stillbirth, prematurity, perinatal deaths, neonatal deaths, intrauterine growth retardation, birth weight, bleeding during pregnancy, threatened abortion, and multiple births were evaluated in only a few of the studies. Although none of these risks were evaluated in more than four studies and a few were examined in only one study, it is important to note that none o f these reproductive problems was elevated in the e x p o s e d populations that were studied. Since one o f the key words in epidemiology is consistency or repeatability, it is obvious that the

Remarks

overwhelming majority o f these studies have been negative.

Power distribution and appliances There have been fewer epidemiologic studies dealing with p o w e r lines and electrical appliances than with VDTs (Table 9). These studies not only vary in the type of exposure but also in their reproductive endpoint. Investigators have attempted to examine populations that were exposed to electric blankets (85), electric substations (97,98), electric transmission lines (99), and home heating electromagnetic fields (100). The reproductive effects that were evaluated varied considerably and included the following parameters (1) spontaneous abortion (four studies), (2) birth defects (two studies), (3) threatened abortion (one study), (4) fetal growth (two studies), (5) perinatal mortality (one study), (6) sperm

554

Reproductive Toxicology

morphology (one study), (7) fertility (one study), and (8) sex ratio of offspring (one study). Compared to the epidemiologic data on VDTs, there is meager information concerning the reproductive risks of power distribution and appliance exposure. The paper by Wertheimer and Leeper (85) considered exposure to electric blankets and heated waterbeds. These investigators obtained the data on spontaneous abortion from telephone interviews. They reported an increased risk for spontaneous abortion, birth defects, and decreased fetal growth. While there were serious methodologic concerns about this research, these findings need to be clarified. These same workers also studied the effects of EMF exposure from ceiling radiant heat (85) and reported on the incidence of spontaneous abortions in an exposed and unexposed population. The original data did not demonstrate an increased risk of abortion in the exposed group but when the results were subdivided into seasonal categories, the investigators reported an association of abortion and exposure. Knave et al. (97) examined a number of health parameters in 53 electric substation workers exposed to 50 Hz, 400 kV lines and reported that fertility and sex ratio of the offspring were not affected. Nordstrom et al. (98) reported that there was no increased risk of birth defects, spontaneous abortions, perinatal mortality, and decreased birth weight in the offspring of 542 electrical substation workers when compared to matched controls. Singlewald et al. (99) reported on the sperm morphology of 10 electric transmission line workers over a period of nine years and observed no deleterious effects. These three studies deal with preconception exposures to male populations and are essentially negative. Preconception studies involve the production of genetic changes or infertility (101). The former need a large population base and the latter need high exposures to a toxicant in order to obtain any definitive results. It is extremely difficult to demonstrate an increase in genetic effects in a population because of the high background incidence of these diseases (Tables 1 and 2) and the fact that it has not been possible to demonstrate genetic effects in the offspring of large populations exposed to known mutagens (6). Users of certain appliances (hair dryers, motordriven electric razors, heating coils in coffee makers, or electric ranges) are exposed to higher fields because of the close proximity to the appliances. The reported results, which have inconsistencies and are compounded by known and unknown factors, do not permit definitive conclusions.

Volume 7, Number 6, 1993

Medical resonance imaging (MRI) MRI diagnostic units expose humans to large magnetic fields of about 0.5 to 15 kG. There are no epidemiologic reproductive studies dealing with this type of acute exposure and no anecdotal reports of ill effects except for claustrophobia from confinement in the medical diagnostic unit. Secular trend data A second epidemiologic approach when evaluating environmental hazards is to determine whether there is any correlation of the incidence of the disease or diseases in question with changes in exposure. Secular trend analysis can be performed when: 1. The exposure of the population to the supposedly environmental toxicant has changed substantially over time and this change can be documented. 2. A significant proportion of the population has been exposed to the toxic agent so that if the alleged environmental toxicant had any deleterious effects, it would be plausible to expect an alteration in the incidence of diseases supposedly produced by the toxic agent. 3. There are reliable longitudinal data on the frequency of reproductive diseases that are being studied. EMF exposure meets the criteria for secular trend analysis. There has been an increase in electric power production and consumption, as well as an increase in the number of appliances. Jackson (102) reported the per capita electric power generation and the per capita residential consumption of power during this century. Power generation and consumption has increased by nearly 3 orders of magnitude, thus establishing one criterion for secular trend analysis. It is also quite obvious that a large proportion of the population is exposed, probably approaching 100%. On the other hand, reliable data are not available on all of the reproductive effects that have been studied in the EMF investigations. For example, there have been so many dramatic changes in the prenatal and neonatal care of newborns that data dealing with birth weight, perinatal mortality, neonatal mortality, and length of gestation would not lend themselves to secular trend analysis. The three parameters that would be appropriate for this analysis are birth defects, spontaneous abortions, and stillbirths. Of these three, the data dealing with the incidence of birth defects are the most accurate. The Centers for Disease Control, Congenital Malformation Surveillance Division, has monitored the inci-

Reproductive effectsof EMF • R. L. BRENTETAL.

~101

--

Power 7

110 9

1

~ 10]

g

555

Cleft LIp 7

:-

r 12

= .,,

~O L .11900 1920 1940 1960 1980 2000 YEAR Fig. 1. The increase in electric power generation in megawatthour/year (MWhr/year) per capita has been published by Jackson (1992). During the period from 1900 to 1990, there has been an approximately 100-fold increase in the generation of electric power. Although the incidence of congenital malformations in the United States has only been carefully monitored on a wide scale since 1968, the data have been carefully collected and are reliable (Centers for Disease Control [CDC], Congenital Malformations Surveillance Report 1988). The data in this graph compare the change in electric power generation over time with the reported incidence of limb reduction defects (LRD). It is readily apparent that although the greatest increase in generation of electric power occurred during the 20year period in which the CDC monitored LRD, there was no simultaneous rise in the incidence of LRD. dence of birth defects in the United States since 1968. Other countries also have birth-defect monitoring programs. Canada has had an excellent program in British Columbia since 1951. The incidence of birth defects in the United States is demonstrated in Figures 1 to 4 and is plotted in conjunction with electric power usage. It is obvious that there is no association of electric power usage and the incidence of birth defects. One might ask, how discriminating are these particular secular trend data? In this instance the data are quite helpful because they reflect a relative stability in the incidence of birth defects even before the 1968 initiation date of the Center for Disease Control Surveillance program. Other programs with much earlier starting dates in Canada, Finland, Sweden, and Germany confirm the fact that birth defect incidences have not changed very much. There are some exceptions. For instance, the incidence of neural tube defects have steadily dropped, beginning in the 1930's. There has been no precipitous fall in the incidence in one short period, but rather a steady decline. Two heart malformations,

...

0~ .1 a. 11100 11120 11140 11160 111110 2000 Year Fig. 2. The data in this graph compare the change in electric power generation over time with the reported incidence of cleft lip with or without cleft palate. During the 20-year period in which the CDC monitored cleft palate, there was no rise in the incidence of cleft palate. on the other hand, have had an increase in incidence over the past 30 years; namely, patent ductus arteriosis and ventricular septal defects. The increase in incidence of these malformations is clearly due to the increasing rate of survival of premature infants who have a higher incidence of these heart abnormalities in conjunction with the capabilities of diagnostic ultrasonography. We can therefore conclude that the incidences of most malformations have re-

~, 10 -:]

,

DownsSynd. ~ Power ~

r lO m 8

0

o.

.

1

~

o

a

1900 1920 1940 1960 1980 2000 Year Fig. 3. The data in this graph compare the change in electric power generation over time with the reported incidence of Down's syndrome. During the 20-year period in which the CDC monitored Down's syndrome, there was no rise in the incidence of Down's syndrome.

556

Reproductive Toxicology

~. 10 "~

l -

Anencephaly /la Power

F 10

x o

f, 0 "-

i •

1900

19'20 19'40 1960 Year

19"80 2000

~

Fig. 4. The data in this graph compare the change in electric power generation over time with the reported incidence of anencephaly. During the 20-year period in which the CDC monitored anencephaly, there was no rise in the incidence of anencephaly. In fact, there is actually a reversed association of electric power generation and the incidence of anencephaly.

mained relatively constant. Comparison of the changing incidence of some birth defects with the increase in per capita generation of electric power in the United States allows one to conclude that there is no positive association between them. Is it possible to miss a teratogenic effect from EMF exposure if only the overall malformation rate is examined? The answer is yes. This was the case with maternal rubella. During the rubella epidemic in the early 1960's the malformations associated with rubella did not affect the total incidence of malformations; although when the data were examined, the malformations associated with rubella had increased during that epidemic (l 1). But the circumstances surrounding rubella are quite different. While rubella is a proven teratogenic agent, only a small proportion of pregnant women are susceptible during any one year, even in an epidemic year. The population exposed to EMF is of course much larger, and theoretically, all pregnant women should be susceptible. We know from experience that even proven teratogens do not have the potential for producing all birth defects. We are in the unfortunate position of not knowing which birth defects or which pattern of birth defects to suspect. No epidemiologic study has directed our attention to be suspicious of a certain group or pattern of defects so that more focused studies could be designed. One method to circumvent this problem would be to examine the birth weight of full term babies born at sea level in the

Volume 7, Number 6, 1993

United States. Decreased birth weight is a cardinal sign of reproductive toxicity and may be more readily adapted to secular trend analysis. In summary, the only data presently available that can be satisfactorily used for secular trend analysis are the birth defect data, and the incidence of birth defects does not demonstrate an association with the dramatic increase in electric power generation over the past four decades.

Reproductive effects of EMF in animals (Table 10) The study of reproductive effects in animals has involved the use of the chick embryo, chickens, cows, mice, rats, and Hanford minature swine.

Chick embryo. The most frequently used animal model has been the chick embryo. There have been approximately 15 studies of chick embryos exposed to EMF, which have not yielded consistent results. In fact, the results were in such disagreement that an international study was designed in an effort to resolve the discrepancies (103). Six laboratories located in Europe and the United States utilized the same method of exposing chick embryos just after fertilization (unipolar pulsed magnetic field of 500 microseconds (~sec) with a 2/zsec rise and fall time and a magnetic field (B) of 10 mG, 48-hour exposure). The six laboratories agreed that the EMF had no effect on viability, stage of development, and somite development. They disagreed on the ability of a 10 mG magnetic field to produce malformations. Two of the laboratories reported an increase in malformations in the exposed embryos while the other four laboratories did not. The chick embryo has not been an acceptable model for studying the potential for teratogenic effects of drugs and chemical in humans. It is an excellent model for studying normal and abnormal development and the mechanisms involved in abnormal development. The proponents of the chick embryo model have reasoned that EMF research should be an exception to the rule since EMF characteristics are very much like ionizing radiation in having a direct effect on the embryo (34,46,50,57,104). Since metabolic differences and placental transport differences among species are not likely to affect the results of EMF exposure, the investigators have justified the studies in the chick embryo to be appropriate for determining potential human risks. The controversy over the results in the chick embryo experiments involves two areas. I. Does EMF exposure increase the incidence of congenital malformations? 2. Is there a window of susceptibility that involves

Reproductive effects of EMF • R. L. BgENTET AL. both frequency (Hz) and exposure (G) as suggested by Ubeda et al. (105). The large number of experiments do not answer the question of teratogenic potential because of their design. The initial experiments examined fertilized embryos after approximately 48 hours of exposure. All the other experiments essentially copied this design (Table 10). The embryos were either examined carefully under a dissecting microscope or were prepared for histologic examination. The presence of abnormalities was judged by the individual investigators. There are two serious difficulties with this model. By examining the embryos at such an early stage there is no way to be certain that the variations that were observed are malformations. If they are malformations would the embryos survive? The first step in any series of experiments is to determine the potential for teratogenicity by examining the offspring at the hatching. This tells the investigator whether there is an increase in malformed survivors and/or embryonic death (resorptions in a rodent model). None of the chick embryo investigations address this problem, and therefore, their results, by themselves are difficult to interpret. On the other hand, Krueger et al. (106) exposed chickens to large EMF fields from conception to hatching (E = 1600 V/m, 60 Hz and B = 1.4 G, 60 Hz). The results did not demonstrate an increase in malformed chicks at the time of hatching. The exposures were high, and this relates to the next controversy. Ubeda et al. (105) used the chick embryo model that involved a 48-h exposure after obtaining the fertilized egg. The exposure consisted of a 100 Hz magnetic field with pulses of 500/zsec and four differently shaped pulses. The exposure levels ranged from 4 mG to 1040 mG. The investigators concluded that malformations of many organ systems were produced, and that there was a window of sensitivity for producing teratogenesis between I0 to 139 raG. The serious difficulty with this experiment and many of the others is the small number of chick embryos in each group. Furthermore, there were fourfold differences in the incidence of malformations in the control groups. Using the same data they concluded that the shape of the pulse would also dramatically change the teratogenic effect between 10 to 139 mG. The chick embryo data that are available for analysis are not useful for predicting reproductive effects in humans or for raising concerns about reproductive problems in humans. If, subsequently, the results in the chick embryo were duplicated in appropriate animal models, then the data derived from the chick embryo experiments might be interpretable.

557

We will next examine some of the mammalian data involving E M F exposures. Cows. Algers and Hultgren (107) measured six reproductive parameters in pregnant cows. The cows were exposed for a 4-month period beginning in June. There were 58 cows in the exposed and control groups. The experimental group was exposed to 50 Hz, 400 kV high power lines. The continuous exposure to the cows was E = 4 kV/m and B = 20 mG. There were no changes in any of the reproductive parameters that were studied (fertility, estrous cycle, progesterone levels, intensity of estrous, and viability of the offspring). There was no increase in the incidence of malformations. This study contained too few animals to determine, for example, a twofold increase in malformations. It is of interest that the B exposure was in the middle of the range that was supposed to be most teratogenic in the chick. Mouse. The experiments with pregnant mice are most interesting. There are seven studies summarized in Table 10 as well as others that have been briefly reported (108,109). The experiments varied in size, goals, and quality. For example, the study by Wiley et al. (110), was designed to examine the risk of VDTs, which is indicated by the frequency of the magnetic field they selected. The mice were exposed to 0, 40, 170, and 2000 mG. The study contained more mice than all the other reports combined, 5296 exposed and 1782 controls. The reproductive parameters that were studied included percent of inseminated females that became pregnant, the number of implantations, percent dead embryos, and percent external and visceral malformations. The results are summarized in Table 10 and indicate that there were no reproductive effects in the exposed mice even with a very high magnetic field exposure. In another large pregnant mouse study, Tribukait et al. (111) examined the effects of a wide spectrum of electromagnetic waves on pregnant mice. Two of these exposures included pulsed 100 Hz magnetic fields where B = 10 and 150 mG. These exposures were at the level and frequency reported to produce malformations in the chick embryo. The mice were exposed from conception until the 14th day of development and the following reproductive endpoints were evaluted (1) total implantations, (2) number of living and dead fetuses, (3) fetal weight (after fixation), and (4) percent malformations at term. Most of these parameters were not significantly different from the controls. There may be some benefit in comparing the

558

Reproductive Toxicology

Volume 7, Number 6, 1993

T a b l e 10. R e p r o d u c t i v e risks o f E M F in a n i m a l s a n d in v i t r o s y s t e m s Species & stage of exposure

No. exposed

Author

Year

Controls

Algers & Hultgren (107)

1987

Cows exposed to high tension lines during the breeding season from June to October for a period of 120 days. Swedish white and red breed heifers. Equal number of controls maintained.

58

58

Bardasano et al. (201)

1986

Chick embryos exposed to magnetic field for the first 6 days of development

24

24

Berman et al. (103)

1990

Chick embryos exposed for the first 48 h and immediately evaluated for malformations. Experiments were performed in 6 laboratories around the world using identical equipment and 100 controls and exposed in each laboratory.

600

600

Cameron et al. (170)

1985

Medaka fish eggs. Fertilized eggs (2 and 4 cell embryos) were exposed to E, B, or E and B for 48 hours. Number of control and exposed eggs not provided.

Delgado et al. (171)

1982

Chick embryos exposed during 49 h post conception

43

26

Durfee et al. (172)

1975

Chick embryos exposed f o r t h e duration o f e m bryogenesis.

Hansson (173)

1981

Rabbits exposed from conception to 7.5 weeks post delivery in an outdoor situation in approximation to an electrical substation. Control rabbits reared in outdoor situation but in electrically protected cages.

Hansson et al. (174)

1987

Rabbits, rats and mice exposed to an electric field similar to that reported by Hansson (1981) but in a laboratory situation.

Hendricks et al. (175)

1988

Mice, Balb/c were exposed to B fields produced by magnetic resonance imaging equipment. Exposure occuffed on day 8.75 for 16 h.

231

194

Reproductive effects of EMF • R. L. BRENT ET AL.

Exposure magnitude & type

Reprod effects evaluated

559

Reprod effects results

Remarks

Fertility Estrous cycle normalcy Progesterone levels Intensity of estrus Viability of fetuses

None of the reproductive parameters studied differed from the unexposed group.

Although the incidence of malformations was not increased, a larger group of animals wouM be necessary to identify a small increase. The proportion of live fetuses was 13% greater in the exposed group.

B = 800 mG, 60 Hz

Birth defects Pineal gland of chick embryos was evaluated after 6 days of B exposure. The embryos were evaluated at stage 28.

Birth defects: Gross anatomic examination of the embryos did not reveal any changes. Examination of the pineal gland revealed anomalous Feulgen + cells with multipolar spindles.

Authors suggest that magnetic fields may align microtubules. It was of interest that while other investigators reported "malformations when the embroys are examined at 48 hours, in this experiment they were normal at 6 days."

B: Unipolar pulsed magnetic field of 500 t~sec pulses, with a 2 t~sec rise and fall time. B = 10 m G

Viability (%) Somite (number) Stage

Controls .955 18.3 12.9

While all laboratories agreed on viability, stage, and somite development, there was disagreement on malformations. Four of the laboratories had no increase in malformations, one had a four-fold increase and one a two-fold increase.

B = 1 G , rms, 6 0 H z E = 300 mA/m 2, 60 Hz Combined B and E exposure as well

Birth defects Developmental delay

Birth defects: There was no increase in birth defects in the embryos exposed to the E, B, or combined E + B fields. Developmental delay was observed with the B and B + E fields but not with the E field.

Authors believe B fields of this intensity can retard growth in the vertebrate embryo. (In the mammalian embryo, this early stage is resistant to both the malforming and growth retarding effects of embryotoxic agents).

B: Unipolar pulsed B fields of 500/zsec. There were 6 exposure groups for the 43 embryos: 10, 100, and I000 Hz and 1.2, 12, and 120 mG.

Abnormalities studied grossly and by histoligic section.

Controls: 16% defective Exposed: 78.5% defective

Very few embryos were in each group but investigator concluded that 100 Hz, 12 mG had a "powerful" effect on inducing abnormalities. No group of embryos were allowed to hatch.

B. Exposed developing embryos to 1, 5, 8, 30, 45, 60, & 75 Hz.

Hatchability

There was no difference in hatchability between control and exposed embryos.

If "malformations" observed by other investigators persisted, they should have contributed to an increase in mortality, which did not occur.

E = 14 kV/m, 50 Hz

Histopathology of the developing brain.

Formation of abnormal cytoplasmic structures in Purkinje nerve ceils of the cerebellum.

E = 14 kV/m, 50 Hz

Histopathology of the developing brain.

Changes in glial and Purkinje cells were found in exposed animals, although the appearance of abnormal cytoplasmic structures (lamellar bodies) was infrequent.

No weight differences between exposed and controls.

B = 3500 G, static electric field. The radiofrequency (RF) exposure was 15.05 MHz with a peak power of I W and yielding 2.89 mW/cm 2. The peak RF magnetic field in the coil was 73.8 raG.

Birth defects, stillbirths, birth weight, fetal length at tenn.

There was no alteration in the frequency of birth defects, homeotic skeletal shifts or stillbirths in the exposed embryos. There was a statistical difference (reduction) in fetal length at term.

The fetal length reduction is of interest because weight is usually a more sensitive barometer of fetal effects and there was no difference in weight between the exposed and control term fetuses.

B&E:

Exposed in proximity to 400 kV, 50 Hz high voltage transmission lines. Exposure: E = 4 kV/m B = 20rag

% normal Total Live

.705 .85

Exposed .951 18.2 12.8 .698 .79

560

Reproductive Toxicology T a b l e 10.

Volume 7, Number 6, 1993

(Continued) Species & stage of exposure

No. exposed

Author

Year

Controls

Joshi et al. (176)

1978

Chick embryo exposed to magnetic fields from fertilization to the primitive streak stage. The embryos were cultured in vitro, using New's technique. The embryos were exposed to B field for 1 h.

25

25

Juutilainen (177)

1986

Chick embryos (Makela 16) exposed during first 48 h of development. Each experiment had 10 controls and 10 exposed eggs.

230

224

Juutilainen & Saali (178)

1986

Chick embryos (Makela 16) were exposed from conception to 52 h (about 48 h development time). There were 20 eggs in each control and exposed group.

640

160

Juutilainen et al. (179)

1987

Chick embryos (Makela 16) were exposed from conception to 54 h (about 50 h of development time).

347

77

Juufilainen etal. (180)

1986

Chick embryos (Makela 16) were exposed from conception to 52 h (about 48 h of development time).

Konermann & Monig (181)

1986

Pregnant albino mice were exposed to magnetic fields on days 7, 10, or 13 post-conception.

1,363

319

Kowalczuk & Saunders (142)

1990

Mice; male C3H mice exposed for 2 weeks and then mated to two females each week for a period of 8 weeks.

20

40

Krueger et al. (106)

1975

Chickens; adult laying hens and cocks exposed to various forms of EMF. Exposures were for 3 consecutive 4-week periods.

103

50

Reproductive effects of EMF • R. L. BRENT ET AL.

Exposure magnitude & type

Reprod effects evaluated

561

Reprod effects results

Remarks

B = 5000 oersted

Birth defects Somite development

Birth defects: All 25 exposed embryos exhibited some type of brain abnormality, open neural tube, microcephaly, or both. There were also heart abnormalities reported and diminished somite development.

Authors attributed "malformations" of the neural tube to the B field effect " o n the interkinetic nuclear migration and mitiotic activity." (There were no data indicating that these effects were present at hatching).

B: Sinusoidal 100 Hz magnetic fields. B = 12.5 mG. Groups were divided into exposed and controis, as well as two methods of handling and four different temperatures (36.3-38.5°(2).

Malformation incidence determined at 2 days from fertilization. No eggs were allowed to hatch.

Control: 8% at 36.3°C and 5% at 37°C. Exposed: 23% at 367.3°C and 25% at 37°C. Higher temperatures and storage of eggs for three to four days increased the percentage of abnormal embryos.

" T h e results demonstrate the importance of the handling of the eggs in this kind of experiment."

B: Sinusoidal oscillating magnetic fields of 1, 10, 16.7, 30, 50, lk, 10k, and 100k Hz and at field strengths B = 1.25, 12.5, 125, and 1250 mG.

Malformation incidence determined at 2 days from fertilization. No eggs were allowed to hatch.

Of the 32 exposed groups 15 had a statistically increased incidence of abnormal embryos. Authors concluded a threshold below 16.7 Hz and 12.5 mG. Concluded that biologic effect is result of B field and not the E field.

Authors review data that supports the "existence of intensity windows." Their data suggest 1 and 10 Hz may be less effective. Although there does not appear to be a dose response curve for B there are too few embryos in each group.

B: Field strength of geomagnetic field in lab was 440-550 nag at 70 degrees. Embryos exposed to 50 Hz sinusoidal magnetic fields of 1.25, 3.75, 12.5, and 125 raG. In a 2rid expt. embryos exposed to 5, 7.5, 10.75, and 16.9 mG.

Malformation incidence was determined when the exposure stopped, approximately 2 days after fertilization. No eggs were allowed to hatch.

Control abnormalities, 16% and 17% in the two experiments. % abnormalities in exposed: 1.25 mG-16%; 3.75 raG-14%; 12.5 mG29%; 125 mG-32%

50 Hz magnetic fields causes abnormalities in embryos but none are induced below 11.25-12.5 raG.

B: Sinusoidal, squared, and pulsed waveforms were used with B fields of 1.25 mG to IOO mG at 100 Hz.

Malformations present at the end of the culture period.

Unipolar square waves did not increase the percentage of abnormal embryos at any exposure. Bipolar oscillations increased the rate of malformations above 12.5 mG.

B = 10,000 G, 0 Hz

Birth defects (external), skeletal malformations, embryo lethality, birth weight. Postnatal evaluations of weight, brain weight, diameter of the neocortex, and alignment of the neural cortex.

No developmental effects were observed.

E = 20 kV/m, 50 Hz. Current densities in the mouse testes were estimated to be 1OO/~A/m 2.

Dominant lethal effect in offspring in female mice mated to male mice receiving E exposure prior to fertilization.

There was no effect on the offspring derived from matings of exposed male mice. The pregnancy rate and survival of the offspring was the same in the control and exposed groups.

B&E: E = 1600 V/m (60 Hz) 53 chickens; B = 1.4 G (1.0-2.0) (60 Hz) 50 chickens

Egg production Fertility Egg hatchability Sex ratio Abnormalities in hatched chicks or dead embryos

Birds in E field lowered production; but regained it by 11 weeks, while B field birds dropped to 31% and did not regain it by 12 week. Neither B or E affected fertility. Sex ratio lowered in B field (32.3% males).

In none of the 5 groups (B, E, 260 MHz, 915 MHz, or 2435 MHz) was there an increase in malformations in either the hatched or dead embryos.

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Reproductive Toxicology T a b l e 10.

Volume 7, Number 6, 1993

(Continued) Species & stage of exposure

No. exposed

Author

Year

Controls

Kubiak & Tarkowski (129)

1985

Mice; electrofusion of 2-cell cleaving mouse embryo.

562

0

Levengood (182)

1969

Ambystoma maculatum and Rana sylvata (165 controls and 320 exposed).

128

94

Maffeo et al. (183)

1984

Chick embryo (White leghorn); fertilized eggs were incubated for 48 h. There were two control groups and one 48 h continuous irradiated group. One of the control groups was sham irradiated.

75

150

Maffeo et al. (184)

1988

Chick embryo (white leghorn); fertilized eggs were incubated for 48 h while exposed to a magnetic field. There were 10 separate experiments with 24 eggs in each, 6 exposed, 6 controls, 6 sham exposed, and 6 x-irradiated.

60

180

Marino et al. (185)

1976

Mice; 3 successive generations of mice were raised in a low strength 60 Hz electric field.

331

233

Marino et al. (186)

1980

Mice; 3 successive generations of mice were raised in a low strength 60 Hz electric field. There were 19-24 litters in each generation that was divided into the 2 exposed and 2 control groups.

497

519

Martin (187)

1988

Chick embryos: exposed embryos during the first 48 h of development in order to determine which time period was more sensitive, the first or second 24 h of development.

1,200

300

Martucci et al. (188)

1984

Chick embryos exposed to magnetic fields for 48 h following fertilization.

Reproductive effects of EMF • R. L. BRENT ET AL.

Exposure magnitude & type

563

Reprod effects evaluated

Reprod effects results

E: Direct current electric pulses applied to embryo. Field strength = 100 kV/ m. Two pulses were administered ranging from 20/zs to 1 ms.

Following fusion, the embryos were examined for normal development.

Electrofusion results in fused embryos that are viable and result in normal offspring. This is a very large exposure of a single pulse during an insensitive stage to teratogenesis.

B = 6300 G or 17,700 G, approximate DC current 12 mA. This was a static field.

Birth defects in ambystoma and frog embryos.

Although there were leg deformities in the controls, the incidence was increased in the exposed embryos at the tadpole stage. The incidence of malformations in ambystoma was slightly increased in the exposed.

B: Pulsed square waves of 500 p.s duration and at frequencies of 100 and 1000 Hz. B = 12 and 120 raG.

After exposure, the embryos were scored for the development of eight structural features: primary vesicles, anterior neuropore, optic vesicles, auditory pits, truncal nervous system, somites, and blood vessels.

There was no difference in the development of these structures between exposed, sham-exposed, and control eggs with regard to the development of these structures.

While the exposures were similar to those reported by Delgado, the results contradicted Delgado's results. The embryos were examined very early in development.

B: Pulsed waves of 500 ~s duration and at frequencies of 100 Hz. Exposures of 12 and 129 mG were used. There was a 42 /zsec rise time. B = 10 mG. The positive control embryos received 15.52 Gy (1552 rads).

After exposure the embryos were scored for the development of eight structural features: primary vesicles, anterior neuropore, optic vesicles, auditory pits, truncal nervous system, somites, and blood vessels.

There was no difference in the development of these structures between exposed and control eggs with regard to development of these structures. 100% of the irradiated embryos were abnormal.

Because of discrepancy in the chick embryo experiments among various laboratories with regard to malformation production, the authors "suggest that factors other than field imposition may be operating in these experiments."

E: Vertical field, E = 130 V/cm, 60 Hz. Horizontal field, E = 100 V/cm, 60 Hz.

Mortality during first week postpartum; mortality from 8-35 days postpartum. These parmeters were evaluated for animals exposed to vertical and horizontal fields.

Mice exposed to vertical electric fields exhibited decreased body weight at 35 days postpartum and increased mortality rates for 3 successive generations.

The number of experimental animals are approximated. The results may be due to grounding microcurrents the animals experienced while feeding. These currents were larger in the vertical field.

E = 3.5 kv/m, 60 Hz. There were four experimental groups. Horizontal-exposed Horizontal-control Vertical-exposed Vertical-control

Mortality and weight of animals over 3 generations. There was no examination of fetuses for birth weight or malformation frequency.

The electric fieM was associated with an increased mortality in each generation and an increase in body weight in only the third generation.

Sufficient data regarding mortality was not provided in the paper for independent review and analysis. The number of experimental animals are approximated.

B = 10 mG, 100 Hz with 2 t~s rise and fall times, 500 ~s duration.

Abnormal development following exposure during only the first 24 h of development. The second group of experiments examined the frequency of malformations for exposures during the second 24 h of development.

A significant decrease in the proportion of normal embryos only occurred in the chick embryos exposed during the first 24 h of development. Controls had 93% normal and the exposed had 75% normal.

B: 500 tzs pulsed magnetic field of 12 nag and 100 Hz. The pulse width was 0.5 ms with a 1.5/zs rise time.

Malformations

No difference in the frequency of malformations in the exposed and control groups.

Remarks

continued

564

Reproductive Toxicology T a b l e 10.

Volume 7, Number 6, 1993

(Continued) Species & stage of exposure

No. exposed

Author

Year

Controls

Nahas et al. (127)

1975

Rats; continuous exposure for 28-32 days to magnetic fields.

15

6

Osbakken et al. (189)

1986

Mice (156 mice, total). The mice were raised from 1 to 3 months in a magnetic field. There were 6 experimental groups, each with a control, exposed, and sham exposed. Three groups were adults and 3 groups were offspring.

50

100

Ossenkopp (190)

1972

Rats exposed in utero

Ozil & Modlinski (130)

1986

Rabbit; 2-cell blastomeres fused by electrofusion, utilizing 5 exposures of 1, 1.5, 2.0, 2.5, or 3.0 kV/cm. While many embryos were fused following the application of E field, some were exposed and did not fuse.

38

0

Persinger (191)

1969

Rats exposed during pregnancy to magnetic fields.

Persinger & Foster (192) Persinger & Pear (203)

1971

Rats exposed prenatally.

Prasad et al. (193)

1990

Frog (leopard, Rana pipiens); fertilized eggs were exposed to either the low or high magnetic field for 1 h. Stage: undergoing 2nd meiotic division. A suspension of normal spermatozoa was also exposed for 1 h.

842

822

Reproductive effects of EMF • R. L. BmEr~r ET AL.

565

Exposure magnitude & type

Reprod effects evaluated

Reprod effects results

Remarks

B: 3 exposure groups of pu-

Mortality, growth, and microscopic pathology were evaluated.

These large magnetic fields produced no mortality or histologic effects, but the exposed groups had a dramatic increase in weight when compared to the controls.

In spite of the very small number of animals in the control and experimental groups the results were of interest. Authors state that " n o undesirable effects" were seen.

B = 18,900 G (static field, 0 Hz). Adult groups 1 and 2 exposed for 6.4 h/day for 75 days. Adult group 3 exposed forl2 h/day for 30 days. Three groups of offspring were exposed for 12 h/day and 360, 432, and 624 total h.

Morality, growth microscopic pathology, and blood chemistries were evaluated in exposed adults and offspring. There was no difference in any of the groups with regard to pathology, hematologic findings, and blood chemistries (CBC, etc.).

None of the adults exposed to this very large magnetic field died or became ill. While body weights were lower in exposed group when compared to controls, they were not any different than sham exposed group.

The authors concluded that this large magnetic field had a minimal effect. They attributed their positive findings to the environment created by the method of exposure rather than the magnetic field.

B = magnetic field exposure

Postnatal activity observed postpartum.

Reduced activity levels in both males and females exposed in utero, with the males being more affected.

Replicated Persinger (1969) study, but the activity differences were less marked than in the Persinger study (only a trend).

E = 100-300 kV/m; pulse duration from 35-1000 ;ts. Maximum tolerance of the plasma membrane of a 2 cell embryo is 300 kV/m for 1000 tzs.

Development of nonfused blastomeres exposed to E fields and allowed to develop.

The results suggest that the electric field can be applied successfully in a relatively wide range of magnitude and duration without cansing any visible teratogenic effect on the treated embryos.

B = 3 to 30G, 0.5 Hz

Postnatal activity observed 27 days postpartum.

Reduced activity levels in both males and females exposed in utero, with the males being more affected.

B = 0.5 to 30 G, 0.5 Hz

Behavioral effects evaluated in postnatal animals. Control and exposed animals trained on a operant-avoidance task that required the rat to learn a lever pressing response to avoid a time scheduled shock to the feet.

Evaluation of rats at 80 days of age. Rats exposed prenatally performed the task at a slower rate, but were able to avoid the shock to the same extent as the controis.

In the study the procedure was modified by using a food reward and an auditory stimulus paired with the foot shock. There was no significant difference in the rate of lever pressing, although exposed animals displayed less activity.

B = 1500 G and 45,000 G, 0 Hz for the main field (magnetic resonance imaging). Characteristics of the field were equivalent to the MR imaging exposure conditions that are employed clinically for the 1500 G imager.

Percent of exposed and control eggs cleaving or developing normal tail buds after 1500 and 45,000 G. Percent of embryos fertilized by exposed or normal sperm cleaving or developing normal tail buds.

There was no effect on the development of the embryos whether the fertilized eggs or the sperm were exposed to 1500 or 45,000 G.

The experiments benefitted from large numbers of exposed and control embryos. No evidence of triploidy was observed. Characteristics of the magnetic field was more complicated than a simple static field.

bescent rats were exposed to very high flux magnetic fields of 200 G, 400 G, and 1200 G.

continued

566

Reproductive Toxicology T a b l e 10.

Volume 7, Number 6, 1993

(Continued) Species & stage of exposure

No. exposed

Controls

1987

Rat; at 3 months of age, female rats and their subsequent offspring were exposed to E fields for 19 h/ day for the duration of the experiment. After 4 weeks of exposure, the females were mated to unexposed males.

1,831

1,780

Rommereim et al. (123)

1989

Rat; one month exposure of females to E fields before mating. Exposure for 19 h/d was continued through mating, pregnancy, parturition, and rearing the young.

450

223

Rommereim et al. (124)

1990

Rat; exposure to E fields for 19 h/day was continued through pregnancy, parturition, and rearing the young for 2 generations. There were 3 exposure groups and 1 control group. The litter was the experimental unit.

204

68

Rommereim et al. (124)

1990

Rats in the F1 generation in the previous experiment were mated, continued to be exposed continuously for 19 h/day, and their offspring were evaluated for birth defects. The embryo was the experimental unit in this study.

3,714

1,362

Salzinger et al. (195)

1990

Rats were exposed for 20 h/day to a 60 Hz electromagnetic field for 22 days in utero and the first 8 days postpartum.

21

20

Sandstrom et al. (196)

1987

Chick embryo exposed to B for first 48 h of embryonic development.

136

149

Author

Year

Rommeriem et al. (122)

Reproductive effects of EMF • R. L. BRENT ET AL,

Exposure magnitude & type E = 60 Hz, vertical, 100 kV/m field. The exposure system did not produce detectable levels of corona, audible noise, or ozone and vibration of the cages was less than 1.4 microns (60 Hz, peak to peak).

Reprod effects evaluated Copulatory behavior Intrauterine mortality

Reprod effects results No indication of altered mating behavior. Effect on fertility was not consistent, being present in one group of exposed and absent in another. Fetal death was lower in one exposed group than it was in the controls.

567

Remarks This very large study did find some positive findings but they were not repeated in identical duplicate experiments. These inconsistent results could be due to random variation or because the dose they utilized was just at the threshold dose. The types of malformations were the random pattern of malformations one would see in any control group, and there was no recognized pattern associated with E exposure.

Birth defects

There was an increase in malformations in the exposed group, although not significantly different from the controls.

E = 60 Hz, l l 2 kV/m and 150 kV/m

Litter size, sex ratio, mortality, maternal and fetal weight gain, and growth.

None of the reproductive parameters evaluated differed between the exposed and control litters.

This experiment was initiated to answer one question raised in their 1987 publication. One explanation for the inconsistent results was that their dose was at the threshold dose. This proved not to be correct.

E = 0, 10, 65, and 130 kV/ m and 60 Hz. There were 3 exposed groups and 1 control.

Percent pregnant Gestational weight gain Offspring per litter Neonatal mortality Juvenile mortality Sex ratio Postnatal weight gain

Overall, no evidence of detrimental effects on survival or growth of the offspring was observed in animals exposed to electric fields. Reduced body weight in maternal F0 130 kV group, was not significant.

This very large study was a followup of previous research by these investigators. Even the body weight reduction can be explained by the reduced corpora lutea and number of implantations in the 130 kV/m group at the experiment's start.

E = 0, 10, 65, and 130 kV/ m and 60 Hz. There were 3 exposed groups and 1 control.

Placental weight Number of corpora lutea Number of implantations Number of resorptions Frequency of congenital malformations

In the FI generation, females were unaffected by exposure. "The lack of malformations differences between groups, with the litter as the basis for comparison, indicates that E exposure was not teratogenie."

These studies further substantiated the author's findings that their original equivocal findings were not due to the fact that the exposure they used was just at the threshold for producing an effect.

E: Electromagnetic field: 60 Hz. The electric field component was 30 kV/m rms. B=IG.

Animals exposed in utero were trained to emit an operant response when reinforced with food.

The exposed rats gradually responded at lower rates than did sham exposed controls. Exposed rats did not differ from controls in body mass, appearance, grossly observed activity level, or incidence of disease.

"Although out results with electromagnetic fields do not necessary signify deleterious effect, their robustness over time and changing conditions indicates that they leave a lasting imprint and therefore cannot be ignored."

B: Asymmetrical sawtooth waveform magnetic field. Three exposures of 1, 15, and 160 mG. Time derivatives of 300, 3000, and 33,000 G/sec.

The control (C) and exposed (E) chick embryos were scored after 48 h for embryonic staging, for nonfertilization, and frequency of abnormalities.

Defects + No development 1 mG 15 mG 160 mG E 12.9% 4.9% 17.9% C 12.9% 11.9% 13.5% The mean stage in the 160 mG group was 10.3 in the control and 10.8 in the exposed.

There were 27 to 46 embryos in each of the 6 experimental and control groups and 2 to 6 abnormal embryos in these groups. While there was no statistical increase in abnormalities, the groups were small.

con~nued

568

Reproductive Toxicology T a b l e 10.

Volume 7, Number 6, 1993

(Continued) Species & stage of exposure

No. exposed

Controls

1,346

1,337

Rat: 1) females exposed to (E) 6 days before mating until term, 2) females exposed from conception, and litter and mother exposed for another 8 days, 3) same as 2 except exposure began 17th day and ended 25 days postpartum.

337

128

1987

Hanford miniature swine: F-0 group was exposed for 4 months before breeding and for the first 100 days postpartum for teratology study. This is not a species for which there are extensive birth defect data.

261

114

Sisken et al. (199)

1986

Chick embryo (White Leghorn): fertilized eggs were exposed continuously to a magnetic field for seven days or for 24 h. The embryos were fixed after 7 days of development and blindly evaluated for malformations.

454

344

Soeradi & Tadjudin (128)

1986

Rat: 90 male rats were divided into 10 groups and placed in an electrostatic field. Each group consisted of 7 exposed and 2 controls. There were 6 exposure groups. They were observed 3, 30, 60, and 90 days after exposure for fertility.

152

51

Steen & Oftedal (134)

1967

Drosophila egg hatching. Eggs exposed to static magnetic field for 20 h, and the percent hatching was determined.

400

400

Stern & Laties (117)

1985

Rats: adult female rats, 120 days old, were trained to press a lever if they perceived an electric field.

5

0

Author

Year

Seto et al. (197)

1984

Rats chronically exposed for 21 h/day 60 Hz electric (E) field in which they were conceived, bom, and raised for 4 generations.

Sikov et al. (125)

1984

Sikov et al. (198)

Reproductive effects of EMF • R. L. BRENT ET AL.

569

Exposure magnitude & type

Reprod effects evaluated

Reprod effects results

Remarks

E = 80 kV/m, 60 Hz. Ambient E and B fields were 0.1 V/m and 0.1 mG, respectively. There was a capacity to expose large numbers of animals continuously, and the cages were designed to eliminate the potential for shock in the exposed group.

Fertility, fitter size at birth, litter size at weaning, sex ratio, weight of males and females at weaning, and the frequency of malformations in the control and exposed groups. This study had very large groups of animals in comparison to other studies.

There were no significant differences between the controis and exposed for any of the parameters that were studied. There were not even minimal differences. These results may be due to the magnitude of the study.

While most of the endpoints were carefully studied they did not obtain weights of the newborns, nor did they examine those exposed in utero for malformations in a careful manner. The authors discussed the power of their study.

E: 60 Hz; E = 100 kV/m; exposure for 20 h/day

Fertility, resorptions, viability, sex ratio, birth weight, postpartum growth malformations.

E did not affect any of these reproductive parameters.

Finding occasional significant differences cannot be dismissed, but if enough comparisons are made some will be positive just by chance. Rats can perceive and respond to 60 Hz field strengths below those used in these studies.

Postnatal behavioral tests such as movement, grooming, standing and righting reflex and geotropism.

Transient behavioral changes were recorded in the neonatal period, but were not found when the animals were tested 21 days postpartum.

E = 30 kV/m; uniform, vertical 60 Hz field; exposure for 20 h/day, 7 days/ week.

F 0 and F 1 study were birth defect studies. Birth weight and litter size demonstrated primarily negative findings. Rates of malformations seemed very high in this study, including the controis.

F 0 exposed group had slight increase in malformations but the difference was not significant. The F t groups did have a statistically significant increase in malformations in the exposed group.

The increase in malformations was in the musculoskeletal and digit category. These were not described. There were no CNS or cardiovascular defects described. Increase in malformations not consistant finding in these experiments.

B: Two complicated pulses were used. One signal was similar to the pulse used in the bone healing studies. The B fields of the two pulses were: B = 2.5 G peak, 0.1 G average. The second pulse was B = 16 G peak, 0.5 G average.

Birth defects

No increase incidence of malformations in the embryos exposed for first 7 days or first 24 h, when compared to the controls. Study was able to discern a 30% increase in malformations with a 99% probability.

There was a significant difference in the incidence of malformations in the controls in this experiment and the controls reported by Ubeda and Delgado, but the exposures and experimental design were not identical.

E = 1,2,3,4,5,6,&7 kV. The 7 experimental groups were exposed to increasing increments of E. Animals were exposed for 1 month. Only males exposed.

Fertility, litter size, and birth defect incidence.

All male animals in all exposure groups remained fertile. The litter size was reduced 30% in the exposed. All exposure groups were equally reduced and the reduction was present 3, 30, 60, & 90 days post-exposure.

While litter size was reduced in all dose groups, malformations only appeared in the groups receiving 6 and 7 kV. The malformations were inadequately described and the litter size data were not biologically plausible.

B = 1600 t o 5 0 0 G i n a static magnetic field.

Egg hatching ability was determined in exposed and control eggs. The endpoint was the time required to go from 20% to 80% of the eggs having completed hatching.

There was no difference in the hatching time between the eggs in the magnetic field and the unexposed eggs.

E fields utilized in the study varied between 1 and 30 kV/m. Vertical 60 Hz electric field. Female rats were trained to press a lever in the presence of a 55 kV/m rms electric field.

Recognition of an electric field in adult female rats.

Female rats could detect a vertical electric field. The threshold of detection ranged between 3 and 10 kV/m. These results were indistinguishable from the studies with male rats.

The fact that female rats can perceive E fields adds another dimension to the complexity of evaluating the effects of E fields on reproduction, since changes in behavior could indirectly influence reproductive performance. continued

570

Reproductive Toxicology T a b l e 10.

Volume 7, Number 6, 1993

(Continued) Species & stage of exposure

No. exposed

Author

Year

Stuchly et al. (126)

1988

Rat: Female rats were exposed 2 weeks before conception and throughout pregnancy.

987

340

Tribukait et al. (111)

1986

Mice; pregnant mice were exposed to a pulsed magnetic field from conception to the 14th day of pregnancy. At 18th day of pregnancy the fetuses were examined. There were 4 experimental groups with 2 waveforms and 2 doses.

1,065

517

Tyndall (113)

1990

Mice; C57BL/6J mice were chosen because of their genetic predisposition to eye malformations. Pregnant mice were exposed to both X-ray and a magnetic field on day 7 of pregnancy.

443

116

Ubeda et at. (105)

1983

Chick embryo: Exposed to B during the first 48 h of development. Fixed in Carnoy's and examined under a binocular microscope. At 48 h percent abnormals were determined, although no embryos were observed at term.

259

364

Wiley et al. (110)

1992

Mice; CD-1 mice were exposed to a 20 kHz sawtoothed magnetic field (similar to those associated with a VDT) from day 1 to day 18 of pregnancy. There were at least 140 pregnant dams in each of 4 exposure groups.

5,296

1,782

Zervins (200)

1973

Chick embryo; exposed to 160-G B-field for first 19 days of embryonic development and were then observed for hatchability and the presence of CNS malformations.

62

70

Zusman et al. (112)

1990

Mice; blastocyst development

301

86

Rats; embryotoxicity in 10.5 day cultured rat embryos. In vivo rat teratology study (animal numbers apply to this study).

Controls

Reproductive effects of EMF • R. L. BRENT ET AL.

Exposure magnitude & type

Reprod effects evaluated

Reprod effects results

571

Remarks

B alternating field. Sawtooth waveform (18,000 Hz) similar to a VDT, but at a much higher exposure. Exposure was at 0, 57, 230, and 660 mG for 7 h/day.

Maternal weight gain Fetal and placental weight Litter size Live fetuses and resorptions Major and minor malformations

All reproductive parameters were indistinguishable from control results. There were fewer skeletal variants in higher exposure groups and an increase in minor skeletal anomalies by fetal, but not litter analysis.

This well-designed study utilized appropriately large control and exposed groups. While there were no reproductive effects, there was a reduction of the maternal lymphocyte count, although it was still within the normal range.

B: Sawtooth (ST) and rectangular (R) waveforms were utilized.Ten m G and 150 m G exposures were used with each waveform. Fetuses were not equally distributed among 4 exposed groups.

Total implantations Living and dead fetuses Fetal weight (after fixation) Malformations Skeletal malformations (ribs and vertebrate)

There were no positive resuits from R pulse exposure. The ST pulse group was equally negative. There were 5 umbilical hernias in the 150 mG group, but malformations were not statistically increased.

The authors concluded that ST pulses might have a teratogenic effect. Yet the data indicated that the exposure did not affect growth, mortality or litter size, and the malformation data were not significantly different from the controls.

B = 15,000 G. The magnetic field was generated by a Gyroscan MRI T2 spin echo technique. TE 50 ms, TR 2100 ms. Exposure was on day 7, 30 minutes following 0.3 Gy Xirradiation.

The investigator was examining whether there was an additive or synergistic teratogenie effect of B when combined with X-ray. Teratogenesis of the eye was the specific endpoint evaluated, although data on litter size were obtained.

This very large MRI field did not enhance X-ray teratogenicity of the eye. More important was the fact that at 15,000 G, the exposure did not affect litter size or the incidence of resorptions.

The author concluded that the MRI exposure did not contribute to the incidence of eye malformations, but then he questions whether MRI can lower the threshold for X-ray teratogenesis. His data indicate that this is unlikely.

B exposures were all 100 Hz and had a pulse of 500 /~s. The pulse had 4 different shapes due to rise times of 100/~s (A); 2 t~s (B); 42/zs (C), and 42/~s with ripples (13). There were 5 exposures in each group ranging from 4 mG to 1040 raG.

Malformations that were evaluated included: cephalic nervous system, truncal nervous system, heart, extra embryonic vascularization, somites. (Authors state that earth's B field is 75 times greater than 4 mG)

Teratogenesis was observed with the 100 tzs pulse (A pulse), but with an exposure window of 10-139 mG; not above or below. When 4 and 10 mG were delivered with 2 and 42/.Ls pulses teratogenesis involved all systems.

Authors "confirm sensitivity of chick to E M F at extremely low frequencies and intensity. The pulse shape may be a decisive factor in determining a 'no' or 'severe' effect." There were four-fold shifts in the incidence of malformations in controls.

B = 0, 40, 170, and 2000 mG at 20 kHz. There were 3 exposure groups and a control group.

% pregnant # implants % dead fetuses Ext. malform. Vise. malform.

OG 76.2% 11.2 7.1% 6.4% 13.6%

40 mG 81.2% 11.8 5.3% 11.3% 14.7%

170 mG 81.2% 11.5 8.0% 6.8% 13.6%

2G 77.4% 11.4 6.3% 4.2% 19.4%

This study reflects the impact of large sample sizes on statistical tests. Variations in small samples may be misinterpreted to have biologic meaning. These and all other reproductive parameters that were studied were negative.

B = 160 G, peak to peak, 26 kHz Embryos were exposed to this field for the first 19 days of development.

Hatchability Malformations in hatched eggs

The hypothesis that EMF exposure reduces hatchability was rejected. No gross CNS malformations were observed in any of the hatched embryos.

Of the exposed embryos 64.5% hatched, but only 48.5% of the controls hatched. It would have been helpful to know the status of the embryos at 49 h of development. Just as in the 48 h studies it would have been important to have hatch data.

E: Mouse blastocyst study: EMF pulse of 1, 20, 50, 70, and 100 Hz. E = 0.6 V/m, pulse cycle x 0.1 s. The pulse pause will vary with the frequency. Rat embryo culture and in vivo rat teratology studies had continuous exposure at 20, 50, and 70 Hz.

Mouse blastocyst development

20 and 50 Hz were embryotoxic, inhibiting blastocyst development; 50% did not hatch; 50 (22%) and 70 (30%) Hz affected development of forebrain, optic and otic vesicles, limb bud, and neural tube closure.

In the in vivo teratology study there was a possibility that the birth weight was increased and litter size decreased in the exposed. There was no increase in malformations due to EMF. Authors asked about the nature of the maternal protective effect.

Rat embryo culture In vivo rat teratology study with 20, 50, and 100 Hz examined litter size, fetal growth, birth defects, and postnatal development.

572

Reproductive Toxicology

chick embryo data to the report of Zusman et al. (t12) using embryo cultures exposed to electrical fields. They exposed preimplantation mouse embryos and monitored their development until the blastocyst stage, They also observed the effect of a 0.6 V/m electric field on 10.5 day cultured rat embryos. Both the cultured mouse and rat embryos were affected by the exposure. The mouse zygotes were retarded and the rat embryos exhibited abnormal limb development. But when rats were exposed throughout pregnancy to these same fields at 20, 50, or 70 Hz and the offspring were examined at term, there was no increase in congenital malformations. This is an excellent example of the difficulty of interpreting the significance of embryo culture experiments, if the evaluation is performed early in development and the investigator has no knowledge of the status of the fetuses at the end of pregnancy (mammals) or incubation (chick). Tyndall (113) exposed pregnant mice to magnetic fields that are comparable to magnetic resonance imaging exposures (15,000 G, 0 Hz). Unfortunately these exposures were in combination with a teratogenic dose of x-ray. The author concluded that the magnetic field exposure did not add to the teratogenic effect of X-radiation. Rat. Table I0 summarizes 15 rat studies, in which pregnant or nonpregnant rats were exposed to electric or magnetic fields. Additionally, Utenkotter et al. (114) reported on teratologic effects in rats after exposure to magnetic fields (50 Hz). Investigators have demonstrated that rats and other species can recognize and can be conditioned to an electromagnetic field (115-1 I7). Male and female rats were able to detect 50- to 60-Hz electric fields with a threshold range from 2 kV/m to 10 kV/m. Sagan and Stell (1118) and Sagan et al. (119) demonstrated a threshold for electric fields ranging from 7.9 to 13.3 kV/m. Cooper et al. (120) reported detection thresholds of 12 kV/m, 50 Hz for pigeons and Rogers (121) reported a detection threshold of 12 kV/m for baboons. The fact that female rats can perceive electric fields adds another dimension to the complexity of evaluating the effects of electric fields on reproduction, since changes in behavior could indirectly influence reproductive performance and postnatal rearing practices in animals. The research group at the Battelle Northwest Laboratories that is interested in reproductive toxicants has published five investigations using rats exposed to electric fields (122-125). These five studies were not duplicate experiments but were rather pursuits of unanswered questions raised in their pre-

Volume 7, Number 6, I993

vious studies. They evaluated a battery of reproductive outcomes following the exposure of pregnant animals to electric fields (E). Most of their findings were negative, but they indicated in their report that if a battery of tests were performed, it would not be unexpected to have a small percentage of positive results. In a 1987 study they reported that exposure to an electric field (E = 100 kV/m, 60 Hz) altered mating behavior and fertility, but the results were not consistent. One of their conclusions was that the 100 kV/m exposure may have been just above the threshold dose. They exposed the next groups to I I2 and 150 kV/m and found that none of the reproductive parameters were altered in this experiment, thus refuting their hypothesis. These investigators used a total of 4653 exposed and 3979 control animals in their studies. Their conclusion was that there were some effects that were observed, but they were transient, not repeatable, or were normal variations of biologic behavior. Behavioral effects observed postnatally were transient and the other reproductive effects could not be reproduced consistently. They were not convinced that the electric fields that they studied had a teratogenic effect. Stuchly et al. (126) exposed 987 fetuses to magnetic fields ranging from 57 to 660 mG at I8,000 Hz. They evaluated the following reproductive outcomes: maternal weight gain, fetal and placental weight, litter size, fetal resorptions, and malformations. All reproductive parameters were indistinguishable from the control embryos. Nahas et al. (127) exposed pubescent rats to continuous magnetic fields (B = 200, 400, and 1200 G). There was no increase in mortality at these large exposures, although the exposures were very large and of long duration. The remaining studies were with electric fields and concluded that reproductive effects were minimal or nonexistent (Table I0). One study that relates to conflicting observations reported in humans deals with the effect of an electric field on preconception male rat fertility. Soeradi and Tadjudin (128) exposed male rats for one month to electric fields of 1 to 7 kV. There were seven exposure groups. This was not a pregnancy study, but rather the equivalent of a dominant lethal study or a genetic study. None of the males became infertile but the investigators did report a decrease in litter size that was not dose related and the occurrence of congenital malformations in the offspring of the two groups with the highest exposure. Neither result is plausible. If one observes lethality from preconceptional male exposures, the effects should be dose related. It is not likely that an increase in congenital malformations would be produced following precon-

Reproductiveeffectsof EMF • R. L. BRENTETAL. ception radiation in a small group of offspring since electric fields are not mutagenic.

Exposure of preimplantation embryos One group of observations that has been brought to the attention of investigators interested in the effects of EMF on the embryo is the ability to use electric pulses to fuse embryonic cells. This procedure has been successful in rat, rabbit, and sea urchin embryos (129-13 l). While these investigators report that surviving transplanted embryos did not exhibit an increase in malformations, it should be pointed out that at this early stage of development, mammalian embryos are not susceptible to teratogenic milieu. It is significant that such exposures do not kill the embryos at this stage because of their marked sensitivity to toxic exposures.

Biologic plausibility Because reproductive pathology encompasses so many disparate problems (Table 4), the concept of biologic plausibility must include the consideration of many basic science areas. This is due to the fact that the etiology and mechanisms involved in various reproductive failures will vary with the reproductive problem being considered. As an example, endocrine abnormalities (132) may be an important contributor to prematurity, infertility, and abortion, but may be minimally involved in the production of congenital malformations and stillbirth. Let us examine the biologic plausibility that EMF has a teratogenic effect. Teratogens produce congenital malformations by interfering with developmental events that are quite identifiable. One of the truisms in teratology is that mutagenic agents frequently have teratogenic potential. This is certainly true for ionizing radiation and cancer chemotherapeutic agents. What are the scientific data that pertain to the mutagenic potential of EMF? Because mutagenic agents can affect mitosis, deoxyribonucleic acid (DNA) synthesis, DNA structure at the molecular level, and the chromosomes at the morphologic and submicroscopic level, these agents can affect embryonic and fetal development. It is unlikely that teratogenesis is the result of induced mutations in surviving embryonic cells that have an impact on development. The teratogenic effect of mutagens resides more in their capacity to kill cells at high doses and affect other vital developmental processes. While many mutagens can be demonstrated to produce mutations in vitro or in whole animal models, they may not be teratogenic unless the dose is substantially raised. The mutagenic and DNA-altering potential of

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EMF has been examined by a number of investigators (133-135). Juutilainen and Liimatainen (136) used the Ames test to determine whether external magnetic fields had any mutagenic potential. The investigators utilized field strengths and frequencies that reportedly produced malformations in chick embryos (0.1, l, 10, and 100 A/m Eat 100 Hz). Besides the usual controls, the investigators used a sodium azide positive control. The Ames test was negative for the control groups and all the exposures to magnetic fields. The group exposed to sodium azide exhibited a predictable mutagenic effect. Liboff et al. (137) reported that magnetic fields (15 Hz to 4 kHz, 230 mG to 5.6 G) enhanced DNA synthesis in human fibroblasts grown in culture. The authors report a threshold for this effect and further suggest "the possibility of mutagenic interactions directly arising from short-term changes in the earth's field." This is a surprising conclusion since their experiments did not demonstrate a mutagenic effect. Goodman et al. (138-140) and Goodman and Henderson (141) exposed dipteran salivary gland cells to pulsed electromagnetic fields and noted that different type pulses had different effects. Both types of pulses resulted in an increased specific activity of messenger ribonucleic acid (RNA), but the time necessary for the effect varied with the pulse. The increase in RNA transcription occurred in most of the bands and interbands of the chromosomes. The dominant lethal assay has been used to study one aspect of mutagenesis (142), and the investigators were unable to demonstrate any reproductive effect after exposing male mice to electric fields (50 Hz, 20 kV/m rms) before fertilization. Cohen et al. (143) studied the effect of a 60-Hz EMF with an electric field of 0.3 A/m 2 and a magnetic field of 1 to 2 G. They used human lymphocytes from five men and five women. The lymphocytes were exposed for 69 hours in vitro. The cytologic endpoints that were measured were mitotic rate and chromosome breakage. There were no differences in these parameters between the control and exposed lymphocytes. Another laboratory has examined the effect of EMF on the repair of induced single-stranded breaks (144,145). The experiments reported by this laboratory involved two separate in vitro systems. DNA damage was induced in isolated human lymphocytes with 5 Gray (Gy) ionizing radiation and then exposed to a 60 Hz (electric = 1.0 or 20 V/m or magnetic field = 0.10 mG). None of the exposures interfered with the repair of DNA or the repair of singlestranded chromosome breaks. The investigators used Chinese hamster ovary cells in culture and exposed them for 1 h to 60 Hz magnetic (1 or 20 G)

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and electric fields (1 or 38 V/m) as well as combined electric and magnetic fields. Following the exposures the cells were lysed and examined for singlestranded breaks. There was no evidence of an increase in single-stranded breaks in the exposed cells. Rosenthal and Oboe (146) studied the effect of 50 Hz EMF (1 to 75 G) on cultured human lymphocytes and noted that the exposure did not alter the frequency of sister chromatid exchanges (SCE) or chromosomal aberrations (CA). They also noted that combining EMF with chemical cytotoxic agents increased the frequency of SCE and CA to a level that was greater than the frequency produced by the cytotoxic agents alone. The authors explained that this phenomenon was not due to a mutagenic effect of the EMF, but to alterations of the culture technique. Whitson et al. (147) studied the effect of extremely low-frequency electric fields (60 Hz, 10 kV/ m) on cell growth and DNA repair in human skin fibroblasts grown in vitro. They found that the exposure to this field did not alter cell growth or survival or alter the capacity for DNA excision and repair, when compared to the unexposed control cultures. Kronenberg and Tenforde (148) studied the effect of low-intensity magnetic fields (60 Hz, 23.3 mG) on the growth of EMT6 cells grown in vitro. The cultured cells were exposed for up to 6 days but were examined daily by sampling the flasks. The results "clearly demonstrate the absence of a magnetic field effect on EMT6 cell growth characteristics." Aarholt et al. (149) studied the effect of low frequency magnetic fields (50 and 16.66 Hz, 2 mG to 220 mG) on cultures of E. coli and obtained the following results. They observed that the mean generation time for cultures subjected to alternating magnetic fields is significantly reduced. They observed a threshold dose, below which no effect on generation time was observed. In discussing their results they attempted to resolve the conflicting results in the literature and they concluded that technical errors and experimental variations may be responsible for some of the differences. As an example, a slight change in temperature can change the generation time of a cell culture. They also suggested that their results may not be appropriately applied to multicellular organisms. Ramon et al. (150) also used E. coli as their test organism. They exposed E. coli to 60 to 600 Hz magnetic fields, with a field strength of 220 G and reported a 40% reduction in bacterial count due to lysis of the organisms. Iwasaki et al. (151) studied the effect of magnetic fields (5000 G) on three systems: Chinese hamster ovary cells in culture (cellular growth and multiplicity), cultured

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slime mold (presence of mitotic delay), and fertilized frog eggs (hatchability and the presence of delay in development). The endpoints for each system varied, but none of these systems deviated from the controls. Chandra and Stefani (152) studied the effect of constant and alternating (60 Hz) magnetic fields (1000 G to 10,000 G) on tumor cell growth in vitro and in vivo. There was no regression or retardation in tumor growth when exposed to either constant or alternating magnetic fields. Portnov et al. (153) studied the mutagenic effect of a static electric field on Drosophila females. They reported that fields of 15 kV/m and 33 kV/m induced nondisjunction and sex-linked recessive lethal mutations. There was no dose-response relationship between the magnitude of the effect and the magnitude of the static electric field. Diebolt (154) exposed Drosophila males to electrostatic (3 kV/m) and magnetic fields (10,000 G) for 24 h in order to determine the influence of low-energy fields on the production of sex-linked recessive mutations in immature motile sperm. The frequency of lethals was identical in the exposed and control groups. Tabrah et al. (155) studied the effect of alternating (60 Hz) magnetic fields (60 to 100 G) on Tetrahyrnena pyriformis and neuroblastoma cells. The authors found alternating fields more effective than the permanent magnet in altering growth. The authors observed reduced cell division in Tetrahymena, no effect on the growth of neuroblastoma in culture, but an effect on neuroblastoma cells in vivo. Goodman et al. (138), Greenbaum et al. (156), and Marron et al. (157) investigated the effect of low-frequency electromagnetic fields (45, 60, and 75 Hz; 2 G, 0.7 V/m) on the slime mold. The exposure lasted for more than 700 days. Exposure resulted in mitotic delay, but when the culture was removed from the electric field the mitotic delay disappeared in approximately 40 days. The preponderance of reports indicate that EMF are not mutagenic agents. The literature indicates that a few, but not most, biologic systems can be perturbed by EMF fields if the exposure is sufficiently high. It cannot be determined whether these effects are in anyway deleterious and whether they will occur in vivo or have any effect on the whole organism. In many instances, the authors are concerned that the artificial nature of the culture system has contributed to the results. One has to remember that all environmental agents will have an effect if the exposure is high enough. There is no agent that will not have some effect on living organisms as the exposure increases. The next concept that has to be dealt with is

Reproductive effects of EMF • R. L. BRENT ET AL.

the inference that many of the negative studies are due to the fact that not only is there a threshold for some of these effects but a ceiling as well. Thus the concept has been introduced that EMF effects may have a narrow window of opportunity to produce deleterious biologic effects. This explanation is used to explain why so many studies, both in vivo and in vitro, are negative. The window effect reported by Ubeda using chick embryos has not been confirmed by investigators using the chick embryo as well as other species. But this hypothesis is one of the driving forces behind continued research in this field. A key feature of reproductive toxicity that is missing in the EMF literature is the production of cell death. Cell killing is an important component of many teratogenic agents. Yet even very high exposures fail to result in cell death in rapidly proliferating tissues, thus indicating the relative noncytotoxicity of EMF. One other aspect of teratogenesis that must be addressed is the fact that all proven teratogens have a constellation of effects that enable the teratologist to identify the teratogenic agent with the syndrome. With the extensive studies and the relatively large proportion of the population that is exposed, there is not even the beginning of a conceptualization of the malformations that are part of the EMF syndrome. When one cannot even suggest the components of the syndrome after all these human and animal investigations, it is most likely due to the fact that a syndrome does not exist. It is possible that the diverse and inconsistent findings that have been reported in the animal studies may be due to differences in experimental design and equipment. As an example, few of the investigators measured the food and water intake of the control and exposed animals. Small animals and humans (158-162) have been reported to have an increase in the frequency of birth defects if there is a substantial reduction in food or water intake or the animals are subjected to stress (163-168). The suggested reproductive risks of EMFs are not supported by most of the clinical, animal and basic science studies that pertain to reproduction and teratogenesis. On the other hand, the suggestion that human reproductive risks may have EMF frequencies and exposure combinations that are deleterious has not been adequately investigated. It appears that reproductive problems are not a major consequence of EMF exposure and although the data do not suggest a risk, as yet a reproductive risk from EMF exposure cannot be summarily dismissed. Because of the allegation that there may be

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particular windows of frequency, wave shape, and intensity that may be deleterious, it is impossible to disregard low frequency EMF exposures as having no deleterious reproductive effects. Yet, all the data that are available would point in that direction. Acknowledgments- The authors appreciate the assistance of Yvonne Edney in preparing this manuscript. The preparation of this review was initiated in September 1991 with ORAU (Oak Ridge Associated Universities) and was published ha 1992 for the Committee on Interagency Radiation Research and Policy Coordination (ORAU 92/F8), under the title "Health Effects of Low Frequency Electric and Magnetic fields." Chapter VI was titled "Reproductive and Teratologic Effects of Electromagnetic Fields," by Robert L. Brent, et al. Since the publication of the original report, a number of publications have appeared (204-207). These additional publications do not change the conclusions of the review and in fact strengthen the conclusions in some areas.

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Volume 7, Number 6, 1993 201. Bardasano JL, Meyer AJ, Picazo L. Pineal cells with multipolar spindles in chicken embryos exposed to magnetic fields-first trials. Z Mikrosk Anat Forsch Leipzig. 1986;100: 85-92. 202. McDonald AD, Cherry M, Delorme C, McDonald C. Visual display units and pregnancy: evidence from the Montreal survey. J Occup Med. 1986;28:1226-1231. 203. Persinger MA, Pear J J, Prenatal exposure to an ELF-rotating magnetic field and subsequent increase in conditioned suppression. Devel Psychobiol. 1972;5:269-274. 204. Cheong, HT, Taniguchi T, Hishinuma M, Takahashi Y, Kanagawa H. Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos. Theriogenology. 1991;36:875-885. 205. Klauenberg BJ. Fetal loss associated with two seasonal sources of electromagnetic field exposure. Am. J Epidemiol. 1991 ;134:913-914: 206. Kowalczuk CI, Sienkiewicz ZJ, Robbins L, Butland BK, Haylock RGE, Thomas JM, Saunders RD. Teratological effects of exposure to 20 mT, 50 Hz magnetic fields in CD1 mice. Publication NRPB-R253, National Radiologic Protection Board, Chilton, Canada 1992;!-41. 207. Tikkanen J, Heinonen OP, Kurppa K, Rantala K. Cardiovascular malformations and maternal exposure to video display terminals during pregnancy. Eur. J Epidemiol. 1990;6:61-66.