4.4 Non-cancer health effects in experimental animals

4.4.1 Immunological effects

In general, the experimental models used to study interactions with EMF have been guided by methods and end-points developed to assess the effects of drugs, chemicals, and ionizing radiation on the immune system. Specific assays have been developed to measure suppression and/or stimulation (immunopotentiation) of a specific immune organ (thymus, spleen, lymph nodes), cell (helper and suppresser T cells, antibody-producing cells, natural killer cells, and macrophages), or function (antibody synthesis, cytokine production, cytotoxic activity, host resistance to infectious and/or malignant disease) (Luster et al., 1990; Luster et al., 1992a; Luster et al., 1994; Luster et al., 1993; Luster et al., 1992b). Changes in the results of these assay have been correlated with host resistance. Alterations in antibody-producing cells, NK cell activity, and delayed hypersensitivity are significantly related to altered disease resistance (Luster et al., 1993). Multiple end-points must be examined in order to clearly establish an interaction between an outside influence and the multiple-faceted immune system.

The exposure, biological end-points, and responses observed in a number of studies are summarized below and in Table 4.27.

4.4.1.1 Magnetic and electric fields

In a second study, eight baboons were field exposed (30 kV/m, 100 µT) and eight were sham exposed, and the responses in mitogen stimulation assays and the distribution of cell types as identified by surface markers and interleukin-2 receptor activity were compared. The decreases seen in the pilot study were not seen in this follow-up study.

4.4.1.2 Magnetic fields

Two separate studies (Tremblay et al., 1996) were conducted in which Fischer 344/N pregnant rats were exposed to 60 Hz, sinusoidal magnetic fields of 2, 20, 200, or 2000 µT for six weeks with sham-exposed controls, beginning on gestational day 20. At weaning, pups were separated from their mothers and held under the same field intensity for the duration of exposure of six weeks. In both studies, a cage control was included. Duplicate treatment groups of eight animals per group per study were included. The biological end-points were innate immunity (NK cell and peritoneal macrophage activities) and T and B cell counts in spleen based on specific surface markers (CD5⁺, CD4⁺, CD8⁺, and Ig⁺). The distribution of CD5⁺ cells (all T cells) was reduced in rats exposed to 200 (p < 0.25) or 2000 µT (p < 0.05), and the numbers of CD4⁺ cells (T helper cells) and CD8⁺ cells (T cytotoxic/suppressor cells) were decreased significantly in rats exposed to 2000 µT (p < 0.005). A significant reduction in all B cells was observed in rats exposed to 20 or 200 µT (p < 0.05). An enhanced response of NK cells was observed in rats exposed to 2000 µT when compared with cage controls (p < 0.05). Regression analysis of the data indicated a significant positive relationship between NK activity and magnetic field intensity (p < 0.05). The authors concluded that 60 Hz magnetic fields have significant effects on the immune responses of exposed animals. [The significant effects were found in comparisons with cage controls; no effects were seen when comparisons were made with sham-exposed animals.]

An extensive series of studies in mice and rats (House et al., 1996) was conducted to measure body and tissue weights, cellularity and lymphocyte subtypes in spleen, and functional activity, delayed hypersensitivity, and host resistance of the immune system in vivo. Mice and rats received actual or sham exposure to 2, 200, or 1000 µT continuously or 1000 µT intermittently and to a 60 Hz magnetic field. No significant difference in the distribution of lymphocyte subsets in the spleens of exposed mice was observed when compared with controls.

NK cell activity was measured by ⁵¹Cr release in female mice exposed to 1000 µT magnetic fields continuously or 1000 µT intermittently. A positive control was included to validate the responsiveness and repeatability of the assay system. Significantly (p < 0.05), consistently enhanced responses were seen in spleen-cell preparations from mice four weeks after exposure to 1000 µT magnetic fields continuously or intermittently, but the response was suppressed after 13 weeks of exposure to 200 or 1000 µT continuously or 1000 µT intermittently (p < 0.05). The studies were repeated three times for six weeks of exposure; the results were inconsistent.

The authors also conducted a series of exposures in Fischer 344 rats exposed for six weeks to sham, 2, 200, or 1000 µT continuous magnetic fields or an intermittent 1000 µT intensity. No consistent differences were observed in comparison with sham-exposed controls. Studies of cellular immunity (delayed hypersensitivity to oxazolone) in male and female mice exposed or sham-exposed for 4 or 13 weeks did not show a clear pattern or trend. No significant differences in mortality or survival time were observed in exposed mice administered Listeria monocytogenes when compared with appropriate sham-exposed controls.

House et al. (House et al., 1996) concluded that continuous exposure of male and female mice to linearly polarized, pure sinusoidal 60 Hz magnetic fields at field strengths up to 1000 µT for up to 90 days did not significantly affect a broad range of immune effector functions. Moreover, this exposure regimen had no observable effect on the ability of mice to resist infection to a bacterial infection. [The results provide little support for the hypothesis that magnetic fields can alter immune responses.]

4.4.1.3 Magnetic fields and 7,12-dimethylbenz[a]anthracene

4.4.1.4 Summary

There is no evidence in experimental animals for effects of ELF EMF on the immune system.

[This conclusion was supported by 13 members of the Working Group; there were 6 votes for 'weak' evidence, 1 abstention, and 9 absent.]

Table 4.27 Summary of immunological studies of exposure to EMF in experimental animals

4.4.2 Hematological effects

Hematological studies include measurement of the distribution of erythrocytic indices (red blood cells, hemoglobin concentration, packed red cell volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hematocrit, and number of reticulocytes), platelets, total counts of nucleated leukocytes and red blood cells, and total and differential leukocyte counts (lymphocytes, neutrophils, basophils, monocytes, and eosinophils). Assessments of bone-marrow cellularity and the impression of bone-marrow smears complete the picture of the distribution of these cell types in peripheral blood.

4.4.2.1 Magnetic and electric fields

In a follow-up study (Picazo et al., 1995b), OF1 mice were exposed for two generations to a 50 Hz 15 µT sinusoidal magnetic field. The first generation (mothers: 12 control and 12 exposed) was exposed for 17 weeks, and the second generation (daughters: 30 control and 30 exposed) was born and exposed for 14 weeks. Comparisons of hemological parameters in exposed and control mice showed no statistically significant change. [This study involved only females.]

Three groups of 18 mice were used to assess peripheral blood characteristics after exposure to a 20 mT magnetic field for 24 h/d for seven days (Lorimore et al., 1990). The leukocytes were counted 0, 2, 4, 7, 10, and 18 days after exposure in three mice at each time. The authors stated that there were no significant differences between the field-exposed and sham-exposed groups. [The groups were extremely small.]

Margonato et al. (Margonato et al., 1993) examined numerous hematological and serum chemical parameters in groups of 20 adult male Sprague-Dawley albino rats sham-exposed or field-exposed to 25 or 100 kV/m 50 Hz electric fields for 8 h/d for 280, 440, or 1240 h. No statistically significant effects were observed.

Margonato et al. (Margonato et al., 1995) assigned 512 adult male Sprague-Dawley albino rats to sham or field exposure to a 50 Hz, 5 µT magnetic field for 22 h/d for a total of 32 weeks. Hematological and serum chemical variables were measured at weeks 0, 12, 24, and 32. No statistically significant differences were detected.

Groups of 10 male and 10 female Fischer 344/N rats were sham-exposed or exposed to 60 Hz magnetic fields at 20, 200, or 1000 µT continuously or 1000 µT intermittently for 18.5 h/d for seven days per week (Boorman et al., 1997). After eight weeks, the animals were anesthetized with CO₂, and blood was collected. Hematological and clinical chemical parameters did not differ between sham-exposed and field-exposed animals.

Zecca et al. (Zecca et al., 1998) used three groups of 64 adult male Sprague-Dawley rats; one group was sham exposed; a second group was exposed to a 50 Hz, 5 µT, 1 kV/m field; and a third group was exposed to a 100 µT, 5 kV/m field. Blood samples were collected before exposure and after 12 weeks. No statistically significant difference was observed in numerous hematological and serum variables.

[The seven studies reviewed provide no evidence that exposure to power-frequency EMF affects the hematological or clinical chemical picture of rodents. These studies were generally short and some included a limited number of animals.]

4.4.2.2 Summary

[This conclusion was supported by 17 members of the Working Group; there was 1 abstention and 11 absent.]

4.4.3 Effects on the nervous system

4.4.3.1 Field detection

There is no doubt that animals can perceive electric fields, as previously reviewed by the National Research Council (NRC et al., 1997). Briefly, Graves et al. (Graves et al., 1978), Cooper et al. (Cooper et al., 1981), and Graves (Graves, 1981) used conditioned response suppression techniques to assess perception. The data suggest that pigeons can detect 50 kV/m and chickens, 32 kV/m fields. Stern et al. (Stern et al., 1983) and Stern and Laties (Stern & Laties, 1985) reported electric field detection thresholds of 3-10 kV/m in psychophysical experiments with rats, but the authors tended to emphasize the best performance of the animals. Sagan et al. (Sagan et al., 1987) observed mean thresholds of 8 and 13 kV/m, depending on the psychophysical method used.

Stell et al. (Stell et al., 1993) reported that the mean electric field detection threshold in rats was 8 kV/m (Table 4.28). In a series of experiments with six baboons, Rogers et al. (Rogers et al., 1995c) demonstrated that electric fields of 22-64 kV/m can serve as a discriminative stimulus and a secondary reinforcer, implying detection. Orr et al. (Orr et al., 1995) used psychophysical methods to measure electric field detection by six baboons: the mean threshold was 12 kV/m; however, one animal with an estimated threshold of 5 kV/m was able to detect electric fields of less than 4 kV/m, the lowest intensity achievable.

Weigel et al. (Weigel et al., 1987) recorded single-unit activity in the sensory nerves of a cat's forearm; exposure to 600 kV/m (60 Hz) increased the firing rate, and shaving and application of mineral oil reduced it. Weigel and Lundstrom (Weigel & Lundstrom, 1987) showed that relative humidity affected vibration of the whiskers of anesthetized rats in a 50 kV/m electric field, suggesting that charges trapped on the hair led to vibration at 60 Hz. In dry air, there was less vibration. Stell et al. (Stell et al., 1993) suggested, however, that hair vibration does not play a critical role in the detection of electric fields (0-25 kV/m, 50 Hz) by rats, and Graves (Graves, 1981) argued that electric field (50 kV/m, 60 Hz) detection by pigeons did not involve vibration of feathers. Hair vibration is thus probably sufficient but not necessary for detection of electric fields.

[Detection of electric fields is a well-established phenomenon, and the detection thresholds for mammals appear to be similar. Differences within species appear to be about as large as differences between species.]

(b) Magnetic fields

Smith et al. (Smith et al., 1994) reported that a very strong magnetic field could serve as a cue for five Long-Evans female rats in a conditioned suppression paradigm (Table 4.29). Five frequency- and flux-dependent pairs of stimuli, ranging between 1900 µT at 7 Hz and 200 µT at 65 Hz, were used. The effect did not vary with different magnetic field intensity or frequency combinations. Stern (Stern & Justesen, 1995) suggested that the design of this study was inadequate to support the conclusion reached; no sham-field tests were completed, and temporal contingencies might have supported responses. [There is no experimental evidence that mammals can perceive magnetic fields at an environmentally relevant flux density.]

4.4.3.2 Avoidance and aversion

(a) Electric fields

It was reported in some early studies that exposure of rodents to electric fields was 'stressful' (see section 4.4.3.4 for a discussion of the endocrine effects of exposure to electric fields). Stimuli detectable at low intensities become aversive when present at high levels. The microshocks that animals can receive when exposed to high-voltage electric fields could elicit adverse behavioral or physiological responses. Thus, perception and/or aversion might be an 'indirect' causative mechanism in experiments using supra-threshold electric fields. These possibilities were not carefully considered in the early studies of exposure to electric fields, in which very high electric field strengths were used in an effort to address the problem of scaling between rodents and humans (see section 3.4 for a discussion of electric field scaling factors; size, shape, and orientation are all important).

Hjeresen et al. (Hjeresen et al., 1980; Hjeresen et al., 1982) reported that exposure to electric fields (60 to 105 kVm, 60 Hz) induced avoidance of the exposure area (p < 0.05) during the inactive portion of the day-night cycle: eight sham-exposed and 32 exposed rats showed avoidance during the day, and 15 sham-exposed and seven exposed pigs (30 kVm, 60 Hz) showed avoidance at night. Rosenberg et al. (Rosenberg et al., 1981) demonstrated that deer mice (21-34 per group) responded to strong electric fields (100 kV/m) with a transitory increase in activity. Rosenberg et al. (Rosenberg et al., 1983) indicated that the increase in activity appeared at about 50 kV/m, and Blackwell and Reed (Blackwell & Reed, 1985) reported that 20 sham-exposed and 20 mice exposed to 400 V/m (50 Hz) or less did not show changes in activity. Easley et al. (Easley et al., 1991) demonstrated that social stress was induced in baboons (eight per group) exposed to 60 kV/m (60 Hz) electric fields. These results, in several species, implied that exposure to electric fields might be aversive; however, Creim (Creim et al., 1984) reported that exposure to as much as 133 kV/m electric fields did not induce taste aversion in male rats (three per group), and Stern and Laties (Stern & Laties, 1989) established that five male rats would not turn off a 100 kV/m (60 Hz) electric field but would turn off a light. [These results suggest that exposure to electric fields up to 133 kV/m is not highly aversive.]

Coelho et al. (Coelho et al., 1991) reported that exposure to electric fields at 30 kV/m (60 Hz) increased the frequency of occurrence (p < 0.05) of three of ten categories of social behavior of baboons (eight per group) during a six-week exposure, in comparison with the equivalent rates observed in six-week pre- and post-exposure periods (Table 4.29). The effects on passive affinity, tension, and stereotypy were of the same magnitude with exposure to 30 or 60 kV/m (Easley et al., 1991).

Easley et al. (Easley et al., 1992) reported a replication of the behavioral effect at 30 kV/m. The means for the entire exposure period differed (p < 0.05) from those for the pre- and post-exposure periods, which were equivalent; analyses of weekly means indicated that the effect occurred almost exclusively during the first week of exposure (p < 0.05). The effect also occurred during the first week in the experiments reported by Easley et al. (Easley et al., 1991) and Coelho et al. (Coelho et al., 1991).

Rogers et al. (Rogers et al., 1995c) found that six male baboons would not turn off an electric field above their detection thresholds, suggesting that exposure to electric fields is not aversive. [This outcome is generally similar to those of several studies in rats.]

Rogers et al. (Rogers et al., 1995b) examined the effects of exposing two groups of six male baboons to 30 kV/m (60 Hz) electric fields. The baboons had been trained, under signal control, to respond on either FR30 (fixed ratio 30) or DRL 20 (differential reinforcement of low rate, 20 s) schedules. They were assigned randomly to field-exposed and sham-exposed groups and entered into a six-week pre-exposure, exposure, and post-exposure schedule. On the first day of exposure, the field-exposed baboons showed 'work stoppage' (p < 0.05) or did not respond. Most began responding on the second day of exposure; once responding began, performance was normal. In a cross-over experiment, the effect was examined a second time. When the former controls received their first 30-kV/m exposure, they showed work stoppage. The original experiment was repeated at 60 kV/m with a new set of baboons, and the same work stoppage effect was observed (p < 0.05). Because exposure to a novel stimulus usually interrupts operant response, the authors did not regard this as an adverse effect.

[Introduction of a perceptible electric field can change the behavior of mice, rats, and nonhuman primates; however, the changes are transitory, appear to be secondary to detection of a novel stimulus, and do not suggest acute adverse effects.]

(b) Magnetic fields

Smith and Justesen (Smith & Justesen, 1977) reported that exposure to magnetic fields (1.7 mT, 60 Hz) increased activity in mice. Rudolph et al. (Rudolph et al., 1985) reported that exposure to 40 µT (50 Hz) increased activity in male Wistar rats (24 per group) but only at the beginning of tests conducted in the presence of illumination. Lovely et al. (Lovely et al., 1992) reported that a 60 Hz magnetic field of 3.03 mT did not induce place avoidance in adult male rats (eight sham- and 24 field-exposed). [Too few experiments are available to reach a conclusion on the effect of magnetic fields on avoidance and aversion reactions.]

(c) Electric and magnetic fields

Groups of eight field-exposed (6 kV/m, 50 µT, 60 Hz) and eight sham-exposed baboons were tested by the approach described previously (Coelho et al., 1991). When the same animals were subsequently exposed for six weeks to 30 kV/m, 100 µT, 30 kV/m electric fields did not elicit the initial increases in the frequency of passive affinity, tension, and stereotype. The authors speculated that the presence of the magnetic field might have blocked the response normally elicited by strong exposure to electric fields.

Orr et al. (Orr et al., 1995) examined the effects of exposure to EMF in two groups of five male baboons trained to perform a match-to-sample operant task. In the first experiment, initial exposure to 6 kV/m and 50 µT did not induce work stoppage. When the same animals were exposed to a perceptible 30 kV/m field accompanied by 100 µT, no work stoppage occurred. A short cross-over experiment was completed in which the unexposed controls were exposed to 30 kV/m and 100 µT for the first time; once again, exposure to EMF did not induce work stoppage. Inclusion of the 100 µT field prevented the occurrence of work stoppage seen with 30 kV/m alone.

[The available evidence, including that from the series of experiments conducted with nonhuman primates in a well-controlled exposure facility suggests that exposure to EMF is not aversive] (Rogers et al., 1995a; Rogers et al., 1995b). [This series of experiments also suggests that addition of a magnetic field can modulate the acute behavioral response of animals to exposure to perceptible electric fields.]

4.4.3.3 Learning and performance

(a) Electric fields

Several investigators have examined the ability of power-frequency, sinusoidal electric fields to affect learning and/or performance. Gavalas et al. (Gavalas et al., 1970) and Gavalas-Medici and Day-Magdelano (Gavalas-Medici & Day-Magdaleno, 1976) reported that exposure to very weak electric fields (7-100 V/m) at 4-10 Hz (in the range of electroencephalogram (EEG) frequencies) disrupted the 'timing' behaviors of macaques (3-5 animals) performing a DRL-like operant schedule. Frey and Wesler (Frey & Wesler, 1984) reported that exposure to a relatively weak electric field (3.5 kV/m) induced a reduction response in suppression (Table 4.30). [This brief report is lacking many important details.]

Eight measures of the operant behavior of baboons on a FR30/DRL20 schedule were studied at 30 kV/m and at 60 kV/m (Rogers et al., 1995b) and analyzed by analysis of variance. Other than the initial work stoppage, no effect on operant performance was seen. In a series of seven experiments with a set of six baboons, Rogers et al. (Rogers et al., 1995c) observed that exposure to electric fields had no effect on response rate, number of errors, or extinction of a simple, appetitively motivated operant task.

[There is relatively little information on the possibility that exposure to power-frequency electric fields can affect performance of learned behavior. The available evidence does not suggest major, persistent effects.]

(b) Magnetic fields

As reviewed by the National Research Council (NRC et al., 1997), Davis et al. (Davis et al., 1984) reported that exposure to a 1.65 mT (60 Hz) magnetic field did not affect passive avoidance learning by male mice. Thomas et al. (Thomas et al., 1986) reported that a 30-min treatment with a combined horizontal 60 Hz magnetic field (26 µT) and static magnetic field adversely affected performance of five male rats on a DRL schedule, increasing DRL response. Fixed ratio performance was unaffected. The DC field was used to 'cancel' the Earth's geomagnetic field. The key behavioral observation was that fixed ratio response was unaffected but DRL performance was adversely affected (p < 0.05), suggesting an effect on cognitive function. The key physical concept is that the Earth's geomagnetic field can interact with a power-frequency magnetic field to produce an effect. The authors reported that neither DC exposure nor sinusoidal exposure to magnetic fields alone produced the effect. Liboff et al. (Liboff et al., 1989) reported further studies in the same animals, which showed a threshold at 27 µT. A step-function 'threshold', rather than a monotonically increasing function over a range of values, was observed. [These reports by themselves are not convincing; they were based on data from only five rats and a few sessions conducted at the end of a series of experiments.]

Details of the following studies on learning and behavioral are presented in Table 4.30.

Trzeciak et al. (Trzeciak et al., 1993) reported that exposure to magnetic fields (50 Hz, 18 mT) had no effect on locomotor activity or on open-field behavior of groups of 10-12 adult male and female Wistar rats; however, 'irritability', defined as response to blowing air on the back, touching the whiskers and back with a glass rod, and holding the animal in the hand was reduced. [The behavioral testing and scoring methods were not adequately explained.]

Sienkiewicz et al.(Sienkiewicz et al., 1994) exposed CD1 mice to 20 mT (50 Hz) fields nearly continuously throughout pregnancy; dams and pups were removed from the magnetic field less than 18 h after birth. Many behavioral indices were examined at multiple times in 168 sham-exposed pups from 21 litters and 184 pups from 23 litters as the pups developed. Three significant effects (p < 0.05) were detected: exposed animals acquired the righting reflex earlier; exposed males (but not females) were lighter, only at 30 days of age; and exposed animals performed less well on the rotorod, only at days 30 and 45 of age. The authors concluded that no major effects occurred.

Kavaliers et al. (Kavaliers et al., 1996) reported that exposure of groups of five male and five female deer mice to 100 µT, 60 Hz for 5 min during acquisition and retention of a learned task (Morris water maze task) improved performance and acquisition. Similar results were found with meadow voles (Kavaliers et al., 1993). Lai et al. (Lai & Singh, 1997b) presented data suggesting that exposure to a magnetic field (1 mT, 60 Hz) for 1 h immediately before testing adversely affected some aspects of the performance of male Sprague-Dawley rats in a Morris water-maze (five cage controls, 10 sham-exposed, 9 field-exposed). The motor component of this task was not affected by exposure; however, spatial reference memory was diminished. The authors also noted that the deficit was related to a decrease in motivation rather than to a deficit in learning ability.

Sienkiewicz et al. (Sienkiewicz et al., 1996b) field-exposed (5 mT, 50 Hz) or sham-exposed CD-1 pregnant mice until just before parturition. The offspring were allowed to develop without further exposure to magnetic fields. Beginning at 83 days of age, 10 males per group began training on an eight-arm radial maze. No differences in performance were observed. Sienkiewicz et al. (Sienkiewicz et al., 1996a) studied the effects of exposure to a 50 Hz magnetic field at intensities of 5-5000 µT on acquisition of an eight-arm radial arm maze task by adult male CD-1 mice (10 per group). Exposure occurred only while the animals were in the maze. The authors concluded that concurrent exposure to magnetic fields had no effect on spatial learning.

Groups of 4-8 male Sprague-Dawley rats exposed for 45 min to a 0.75 mT 60 Hz magnetic field immediately before training on a 12-arm radial maze made more errors than did the sham-exposed controls (p < 0.05) (Lai, 1996). The running speeds of the two groups did not differ. Pretreatment with the cholinergic agonist physostigmine blocked the effect but had no effect on animals not exposed to magnetic fields.

Sienkiewicz et al. (Sienkiewicz et al., 1998) present data from four experiments showing that exposure to 0.75 mT (60 Hz) for 45 min before training in an eight-arm radial maze slowed acquisition of the task by male C57Bl/6J mice (six animals per group). The results of all four experiments were similar. Inter-group differences were most apparent in sessions 2-4 and were gone by session 6 or 7. These results confirm the observation of Lai (Lai, 1996) that exposure to magnetic fields just prior to training sessions slows acquisition but not final performance of the task.

[One interpretative difficulty with these studies is that changes in performance can be due to a variety of causes, including level of arousal. Thus, in the absence of additional experiments, the conclusion of an effect on spatial reference memory is only a testable hypothesis for additional experiments.]

Stern et al. (Stern et al., 1996) conducted two experiments in an effort to replicate the findings of Thomas et al. (Thomas et al., 1986) and Liboff et al. (Liboff et al., 1989), who suggested that cyclotron resonance could affect the operant behavior of rats. None of the several exposure conditions tested had an effect on the operant behavior of the 13 rats examined. [No obvious reasons are apparent for the differing results of the two groups.]

[Ten experiments indicate that exposure to power-frequency magnetic fields can affect acquisition or performance of learned behavior. The effects are either adverse, beneficial, or absent, depending on the task and the timing of exposure to magnetic fields.]

(c) Electric and magnetic fields

Few investigators have examined the effects of combined EMF. Wolpaw et al. (Wolpaw et al., 1989) exposed six macaques to electric fields of 3-30 kV/m in combination with magnetic fields of 10-90 µT for 18 h/d for three weeks. The performance of a simple food-motivated operant task was not affected as compared with sham-exposed controls. Salzinger et al. (Salzinger et al., 1990) exposed rats to 60 Hz 30 kV/m and 100 µT. In a complex operant experiment with multiple extinctions and testing at various times during the diurnal cycle, there was some evidence (p < 0.05) for a slower response at the mid-point of the light portion of the light-dark cycle.

de Lorge and Grissett (de Lorge & Grisset, 1977) studied the effects of EMF on the operant behavior of 10 male and female monkeys (Table 4.30) but detected no effects on match-to-sample, fixed interval, or reaction time response. [This brief publication summarizes a complicated series of experiments performed with various exposure paradigms and dependent behavioral variables; the information provided is insufficient for detailed review of this work.]

Orr et al. (Orr et al., 1995) studied the effects of exposure to EMF on a match-to-sample operant task with two groups of six male baboons. As in the studies of social behavior, described previously (Coelho et al., 1995) imperceptible 6 kV/m 50 µT fields had no effect on any aspect of operant performance. When the same animals were exposed to 30 kV/m and 100 µT, no effects on operant performance were observed.

[All of the experiments in which nonhuman primates were used to examine the effects of combined exposure to electric and magnetic fields did not detect effects on operant performance. The single experiment in rats showed a subtle effect. Overall, no major adverse effects on operant performance have been reported as a result of exposure to power-frequency EMF.]

4.4.3.4 Neurophysiology

Some of the early studies of the effects of exposure to electric fields on growth and development (Burack et al., 1984; Marino et al., 1977; Marino et al., 1976; Marino et al., 1980) suggested that it was 'stressful'. Thus, investigators have sought endocrine changes that are the sine qua non of stress.

As reviewed by the National Research Council (NRC et al., 1997), Marino et al. (Marino et al., 1977) measured blood corticosterone concentrations in rats exposed to a 15 kV/m, 60 Hz electric field for 10 months; they reported reductions in six of the ten experiments. Hackman and Graves (Hackman & Graves, 1981) reported that adult mice (5-15 per group) exposed to electric fields of up to 50 kV/m, 60 Hz showed reductions in plasma corticosterone concentrations, but only at the onset (15 min) of exposure. Free et al. (Free et al., 1981) no increase in corticosterone concentration was found at 30 or 120 days. Quinlan et al. (Quinlan et al., 1985) exposed four Long-Evans hooded rats to 100 kV/m (60 Hz) for 1 or 3 h. Plasma corticosterone concentrations were not elevated in the four field-exposed rats relative to four sham-exposed controls. Portet and Cabanes (Portet & Cabanes, 1988) reported that rabbits (seven per group), but not rats (25 per group) exposed to 50 kV/m (50 Hz) showed reduced adrenal gland cortisol content. Changes were not observed in blood corticosterone concentrations.

Rommereim et al. (Rommereim et al., 1990) observed chromodacryorrhea in female rats (68 per group) exposed continuously to 130 kV/m (60 Hz) and interpreted this as a stress response. Leung et al. (Leung et al., 1990) quantified the chromodacryorrhea effect, reporting that it was increased in incidence and severity at 65 and 130 kV/m; the group at 10 kV/m did not differ from the sham-exposed control group.

Margonato et al. (Margonato et al., 1993) exposed groups of 20 rats to 50 Hz electric fields of either 25 or 100 kV/m for 8 h/d for 280, 440, or 1240 h. The plasma concentrations of lutenizing hormone, follicle-stimulating hormone, and testosterone differed widely among animals within groups, and no differences between field-exposed and sham-exposed groups could be detected. No effects on testis were reported. (see Table 4.31)

In three experiments, Kato et al. (Kato et al., 1994a) measured plasma testosterone concentrations in adult male Wistar king rats (10-12 per group) after 42 days of exposure to a 50 Hz circularly polarized magnetic field of 0.02, 0.1, 1, 5, or 50 µT. There was no effect. Picazzo et al. (Picazo et al., 1995a) report that groups of 30 male mice continuously exposed to a 15 µT 50 Hz magnetic field had a 27% increase (p < 0.05) in serum testosterone concentration, accompanied by a 7% increase (p < 0.05) in testis weight with a 12% increase in the area of the interstitium.

de Bruyn and de Jager (de Bruyn & de Jager, 1994) suggested that continuous exposure of mice to electric fields (50 Hz, 10 kV/m) acts as a stressor (Table 4.31). Their conclusion is based on two positive observations (out of a larger set) in the offspring of breeding pairs exposed to 10 kV/m, 22 h/d during gestation until either 35 days, 6 months, or 18 months of age. Mice of each sex and three ages were examined in daytime or night-time for several different variables. In one study, the serum corticosterone concentration was determined both day or night. In the exposed adult males (5-13 per group), the mean daytime corticosterone concentration was higher than in the controls (p < 0.02). Lipid staining of the adrenal cortex in the zona glomerulosa but not in the zona fasiculata or zona reticularis was elevated (p < 0.05) only in adult males.

Picazo et al. (Picazo et al., 1996) report that the second generation of OF-1 mice (30 exposed and 30 controls) continuously exposed to a 15 µT 50 Hz magnetic field showed significant changes (p < 0.05) indicative of adrenocortical effects: the normal diurnal rhythms in plasma cortisol rhythms and adrenal cortex thickness were lost. The authors indicated that 15-20% of the exposed animals showed changes associated with adrenocortical hyperplasia; however, extensive histopathological examination did not reveal statistically significant differences. [The controls were not sham exposed, and the pathology assessments were not conducted in a blind fashion.]

Thompson et al. (Thompson et al., 1995) exposed 10 sheep beneath a 500 kV (60 Hz) transmission line; the 10 controls were housed away from the line. The average field strengths were 6 kV/m and 4 µT. Blood samples were obtained at intervals of 0.5-3 h over a 48-h periods on eight occasions over eight months. After the pre-exposure sampling, blood was sampled two weeks after initiation of exposure. No change in plasma cortisol concentration was detected. Burchard et al. (Burchard et al., 1996) studied numerous aspects of the physiology of 16 lactating, pregnant Holstein cows during periods with and without exposure to EMF. Cortisol was sampled twice a week and showed no changes, while progesterone was measured weekly and was increased by 11% (p < 0.05).

[Some of these studies present evidence that exposure to electric fields can be stressful, but the available evidence does not clearly establish that such an effect occurs.]

(b) Effects on opioid action

Ossenkopp and Kavaliers (Ossenkopp & Kavaliers, 1987b) examined the effects of 60 Hz magnetic fields of 2, 100, and 150 µT intensity; 30-min exposure of groups of 10-16 CF-1 mice produced a field intensity-dependent reduction (p < 0.05) in the analgesic effect of morphine. Ossenkopp and Kavaliers (Ossenkopp & Kavaliers, 1987a) also described a field intensity-dependent reduction (p < 0.05) in morphine analgesia in two experiments conducted at 50-150 µT (Table 4.31). Morphine was given at the mid-point of a 60-min exposure and testing was done at the end of exposure to magnetic fields. In the hot-plate analgesia test, the latency to forepaw lick was 56% of the control time in mice at 50 µT, 45% at 100 µT, and 23% at 150 µT. [The latency cut-off of 150 s was excessive for studies of morphine analgesia and raises concern about the method, especially with regard to determining a relationship with the endogenous opioid system. It limits interpretation of the effects on analgesia.]

Lai and Carino (Lai & Carino, 1998) exposed 6-10 male Sprague-Dawley rats to a 2 mT, 60 Hz magnetic field for 1 h and repeated their earlier observation of decreased (p < 0.05) cholinergic activity in the frontal cortex and hippocampus. They also showed that pretreatment of rats with the µ-opiate receptor agonist 3-funaltrexamine or the d-opiate receptor agonist naltrindole through an intracerebroventricular cannula 24 h before exposure to magnetic fields blocks the induced decrease in sodium-dependent, high-affinity choline uptake in the frontal cortex and hippocampus. Zecca et al. (Zecca et al., 1998) exposed groups of 64 male Sprague-Dawley rats to 5 µT and 1 kV/m or 100 µT and 5 kV/m for eight months and measured µ-opiod receptors in the frontal and parietal cortex and hippocampus in subgroups of 10-21 rats; no change in µ-opiod receptor concentration was found in the hypothalamus, striatum, or cerebellum.

(c) Neurotransmitters

Vasquez et al. (Vasquez et al., 1988) measured monoamines in the hypothalamus and striatum of groups of six male Sprague-Dawley rats after exposure to 39 kV/m (60 Hz) for 20 h/d for 30 days; measurements were made every 4 h throughout the light-dark cycle. Changes in phase, rather than amplitude, were reported for norepinephrine, dopamine, and the serotonin metabolite 5-hydroxindole acetic acid in the hypothalamus and for 3,4-dihydroxyphenyl acetic acid in the striatum. Seegal et al. (Seegal et al., 1989) measured monoamine concentrations in the cerebrospinal fluid of macaques after exposure to 60 Hz 10 µT, 3 kV/m or 30 µT, 10 kV/m or 90 µT, 30 kV/m for 20 days. The concentrations of the dopamine metabolite homovanillic acid and 5-hydroxindole acetic acid were reduced in field-exposed animals.

Zecca et al. (Zecca et al., 1991) measured norepinephrine, serotonin, 5-hydroxindole acetic acid, dopamine, and it metabolites homovanillic acid and 3-4-dihydroxyphenyl acetic acid in striatum of animals exposed to electric fields of 25 and 100 kV/m (50 Hz) for various times. The amino acid neurotransmitters taurine, glutamine, aspartic acid, glutamic acid, glycine, alanine, and -aminobutyric acid were also measured. Numerous t tests were performed, and some small (10% or less) differences (p < 0.05) were observed (Table 4.31).

Lai et al. (Lai et al., 1993) examined the effects of 45 min exposures to a 60 Hz magnetic field of 500, 750, and 1000 µT intensity on sodium-dependent, high-affinity choline uptake in the brains of six male rats. Brains were collected immediately after exposure. Decreased (p < 0.05) sodium-dependent high-affinity choline uptake was demonstrated; dose-dependent reductions were observed in frontal cortex and hippocampus. The authors also showed that the opioid antagonist naltrexone, administered just before exposure to magnetic fields, blocked these effect s, but naloxone did not. [Concern was raised by the differential effects of these two narcotic antagonists, since both inhibit central opioid receptors.]

Sakamoto et al. (Sakamoto et al., 1993) exposed Sprague-Dawley dams and sires to 60 Hz, 50 µT circularly polarized magnetic fields before mating and dams during gestation. Six to 12 embryos per group were harvested at days 12, 14, 16, 18, and 20 of gestation and choline transferase activity in brain was measured. In an additional experiment, groups of six animals were exposed for three months before mating. The brains of offspring were analyzed 10 days after birth. In both studies, normal age-related changes were found but no effects of exposure.

Margonato et al. (Margonato et al., 1995) measured the brain neurotransmitter concentrations in 256 male Sprague-Dawley rats (groups of 15) after 5000 h of exposure to a 50 Hz, 5 µT field for 20 h/d. Striatum, hypothalamus, hippocampus, and cerebellum were analyzed for norepinephrine, dopamine, 3,4-dihydroxyphenyl acetic acid, homovanillic acid, serotonin, and 5-hydroxindole acetic acid. No group differences were detected. Zecca et al. (Zecca et al., 1998) determined norepinephrine, dopamine, 3,4-dihydroxyphenyl acetic acid, homovanillic acid, serotonin, and 5-hydroxindole acetic acid in the frontal cortex, parietal cortex, striatum, hypothalamus, and cerebellum; D2 receptor concentrations were measured in striatum and frontal cortex. No effects of an eight-month exposure to 5 µT and 1 kV/m or to 100 µT and 5 kV/m were found. [In both experiments, neurotransmitter concentrations were assessed at the end of long-term exposure, and any acute temporal trends would not have been determined.]

[Although a few studies have shown effects of magnetic fields on neurotransmitters, the results are mixed and the effects noted are relatively small. The design of many of these studies included terminal sampling at the end of long-term exposure, and limited end-points were evaluated to assess the functioning of the neurotransmitter system. The inherent difficulty in associating changes in regional neurotransmitter concentrations with functional or behavioral alterations makes interpretation of the biological significance of these findings difficult.]

4.4.3.5 Electrophysiology

Jaffe et al. (Jaffe et al., 1983) indicated that exposure of groups of 2-9 rats exposed in vivo to electric fields (60 Hz, 65 kV/m) increased the synaptic excitability, measured ex vitro, of the excised superior cervical ganglion. Jaffe et al. (Jaffe et al., 1981) reported that exposure to electric fields (65 kV/m, multiple frequencies) increased the fatiguability at the rat neuromuscular junction (13-22 animals per group). Jaffe et al. (Jaffe et al., 1980) measured visual evoked potentials repeatedly in developing rats exposed to 60 kV/m; exposure to electric fields had no effect. Blackwell (Blackwell, 1986) measured the firing rates of 51 single units in rat cortex. Electric fields of 100 V/m had no effect on the overall firing rate; at 15 or 30 Hz, some synchrony with the applied waveform was observed, but the effect was not observed at 50 Hz.

Gavalas et al. (Gavalas et al., 1970) and Gavalas-Medici and Day-Magdelano (Gavalas-Medici & Day-Magdaleno, 1976) suggested that exposure of macaques to electric fields within the range of normal EEG frequencies, typically 30 Hz or less, affected neuronal activity. ELF-modulated VHF fields appear to be a particularly effective stimulus for such effects (Bawin et al., 1973).

(b) Magnetic fields

As reported by the National Research Council (NRC et al., 1997) Ossenkopp and Cain (Ossenkopp & Cain, 1988) found that groups of 17 adult male rats field-exposed or sham-exposed to a 100 µT magnetic field at 60 Hz showed attenuation of seizure discharges in a kindling model. Ossenkopp and Cain (Ossenkopp & Cain, 1991) presented data from six experiments with group sizes of 11-20 rats in which a 60 Hz magnetic field of 50-185 µT was applied for 1 h before induction of seizures with pentylenetetrazol (Table 4.32). In only two of six studies was seizure duration decreased (p < 0.05), and fewer seizures (p < 0.05) were observed in only one experiment. Mortality due to seizures was diminished by exposure but not in relation to field intensity. [Overall, exposure to 60 Hz magnetic fields does not aggravate seizures; however, this paper does not convincingly demonstrate that exposure to electric fields diminishes their number.]

Lyskov et al. (Lyskov et al., 1993a) exposed 12 female Sprague-Dawley rats to magnetic fields of 126 µT or 1.26 mT for 24 h (multiple frequencies) and then recorded an EEG. An average response (based on measurements taken immediately and 15 and 30 min after exposure) of four statistically significant effects was observed after sham exposure. The observation of 4-fold (1.26 mT) and 8.5-fold (126 µT) increases in the number of statistically significant (p <0.05) changes in EEF variables was taken by the authors as evidence of an effect of exposure to magnetic fields on the EEG. Numerous other comparisons led the authors to the generalizations that power in the delta (1-4 Hz) and theta (4-8 Hz) bands was decreased and power in the beta (12-20 Hz) and high frequency (30-60 Hz) bands was increased. [They analyzed 31 EEG parameters for four electrode combinations and three times, so that many statistical comparisons were made, and the probability that one or more of the results would be significant by chance alone (Type I errors) was high. The study is statistically invalid and has other methodological short comings. Use of pre-implanted metallic screws as EEG electrodes might be an issue.]

(c) Electric and magnetic fields

Dowman et al. (Dowman et al., 1989) measured auditory, visual, and somatosensory evoked potentials twice a week during three-week exposures in six adult macaques exposed to EMF combinations of 3 kV/m and 10 µT, 10 kV/m and 20 µT, and 30 kV/m and 30 µT; there were four sham-exposed controls. Evoked potentials were measured during the daily 6-h periods without exposure to EMF. There were some signs that the amplitudes of the later components of somatosensory evoked potentials were reduced in the two groups exposed to higher field intensities.

[A few papers indicate that exposure to power-frequency electric and/or magnetic fields can change the characteristics of EEGs or evoked potential activity in animals. The effects on the EEG are more prominent at frequencies less than 30 Hz, where the imposed field is in the same frequency range as endogenous neural activity; however, none of the results suggests that such effects are hazardous. The available data suggest that exposure to magnetic fields inhibits, rather than stimulates, epileptic activity.]

4.4.3.6 Summary

[This conclusion was supported by 18 members of the Working Group; there were 2 abstentions and 9 absent.]

There is weak evidence for the neurobehavioral, neuropharmacological, neurophysiological, and neurochemical effects of electromagnetic fields.

[This conclusion was supported by 9 members of the Working Group; there were 8 votes for 'moderate' evidence, 3 abstentions, and 9 absent.]

Table 4.28 Perception in experimental animals exposed to EMF

Table 4.29 Summary of experiments assessing aversion in experimental animals fields

Table 4.30 Learning and performance in experimental animals exposed to EMF

Table 4.31 Neurophysiological effects of EMF in experimental animals

Table 4.32 Electrophysiological effects of EMF in experimental animals

4.4.4 Reproductive and developmental effects

Assessments of the effects of magnetic fields on reproduction and development have included a wide spectrum of biological end-points, including gametogenesis, fertilization, implantation, embryogenesis, and pre- and postnatal development. A series of reviews (Brent et al., 1993; Chernoff et al., 1992; Huuskonen et al., 1998; Juutilainen, 1991; Juutilainen & Lang, 1997) have been published on the reproductive and developmental toxicity of EMF. This section addresses studies of the effects of magnetic fields on reproduction and development in avian and mammalian systems; these are summarized in Table 4.33. Studies of avian systems involving pulsed magnetic fields are not considered in this section.

4.4.4.1 Birds

Pafkova et al. (Pafkova et al., 1994) reported the effects of 50 Hz magnetic fields at field intensities of 6 µT or 10 mT on avian embryonic development at 2, 6, 16, 20, and 40 h. No effects were observed on mortality or structural malformations evaluated on the ninth day.

In a related study, Pafkova and Jerabek (Pafkova & Jerabek, 1994) combined magnetic fields with ionizing radiation (X-ray). Chick embryos were pre-exposed to a 50 Hz, 10 mT magnetic field from the second to fortieth hour of incubation and exposed to X-rays (4 or 5 Gy) on the third or fourth day of embryonic age. In a second series, chicks were exposed to magnetic fields before or after X-ray treatment on day 3 or 4. The authors found a statistically significant reduction in teratogenic development in embryos exposed to magnetic fields before ionizing radiation (p < 0.003) when the results of all studies were combined. Exposing the embryos to magnetic fields after ionizing radiation resulted in a potentiation of adverse developmental effects (p < 0.02).

Pafkova et al. (Pafkova et al., 1996) conducted a series of studies in which chick embryos were pre-exposed to magnetic fields (50 Hz, 10 mT) for eight 2-h sessions for the first 48 h of incubation. After two or three days of incubation, the eggs were injected through a window in the shell with 10 µl of insulin (0.00-0.05 µg) or tetracycline (10-30 µg). Controls were injected with 10 µl of water. Exposure of eggs to magnetic fields before treatment with insulin reduced the embryotoxic effects (p < 0.01). Exposure to magnetic fields and tetracycline conclusion inconsistent with paper.

The effects of an intermittent horizontal sinusoidal 50 Hz magnetic field was reported by Veicsteinas et al. (Veicsteinas et al., 1996). In these studies, eggs were exposed to an intermittent (2 h on/ 22 h off) magnetic field of 200 µT. The eggs were exposed or sham-exposed for 48 h to magnetic fields and incubation was continued in an artificial brooder. The embryos were examined for developmental anomalies and maturity at the end of the 48 h. Other end-points included extracellular membrane components on day 7 and histological malformations in the brain, liver, and heart on days 2, 7, 12 and 18 of incubation. Egg fertility and weight was examined on days 2, 7, 12, and 18. Additionally, eggs from each treatment group were allowed to hatch and the chicks were followed, being weighed at intervals, for 90 days, when the organs were examined histologically. No effects were seen on any of the end-points of embryo development, body weight, or organs.

Farrell et al. (Farrell et al., 1997) reported the effects of 60 Hz, 4 µT sinusoidal magnetic fields on 545 developing chick embryos. A significant increase (p < 0.01) in morphological abnormalities was observed in eggs exposed to the sinusoidal magnetic field.

[Development abnormalities were observed in three of five studies in which chick embryos were exposed to sinusoidal 50 or 60 Hz magnetic fields, but the relevance of these results to mammalian growth and development is unclear.]

4.4.4.2 Mice

In studies with CD-1 mice, Kowalczuk et al. (Kowalczuk et al., 1994) exposed or sham-exposed pregnant mice on days 0-17 of gestation to a 50 Hz sinusoidal magnetic field at 20 mT. Pre- and postimplantation survival was recorded, and fetuses were examined for the presence of gross external, internal, and skeletal abnormalities. There were 90 exposed and 86 sham-exposed mice, and 2167 fetuses were examined. The study was conducted over a two-year period. No association was found between exposure to magnetic fields and these end-points, but an association was found between exposure and longer, heavier fetuses at term.

Spermatogenesis was evaluated in groups of five male hybrid (C57Bl/Cne x C3H/Cne) F1 mice (de Vita et al., 1995), 8-10 weeks of age, which were exposed to 50 Hz magnetic fields (1.7 mT) for 2 or 4 h and analyzed 7, 14, 21, 28, 35, and 42 days after exposure. Flow cytometric DNA histograms were prepared on testicular cell suspensions to determine the relative frequencies of the various spermatogenetic cell subpopulations. In groups exposed for 2 h, no effects were observed at any time. In the groups exposed for 4 h, a statistically significant effect (p < 0.001) was observed only on day 28.

[Exposure to EMF at power frequencies has not been found to have Teratogenic effects in mice.]

4.4.4.3 Rats

Mevissen et al. (Mevissen et al., 1994) exposed female Wistar rats on days 1-20 of gestation to a 50 Hz sinusoidal magnetic field (30 mT). Three sets of exposures were conducted in which 12 rats per group were exposed or sham-exposed. On gestational day 20, the dams were sacrificed for reproductive and teratological assessment. No major malformations were seen in any groups but a number of fetuses had minor skeletal malformations.

In a study by Rommereim et al. (Rommereim et al., 1996), pregnant Sprague-Dawley rats were weighed and randomly assigned to field- or sham-exposed groups. The rats were exposed to a 1 or 0.61 mT 60 Hz horizontal magnetic field, on days 1-20 of gestation, 20 h/d, 7 d per week. The studies were conducted as two replicate, with 96 rats per treatment group. On day 20 of gestation, the dams were sacrificed for assessment of reproductive and teratogenic end-points. The litters were evaluated for the numbers of implantations, fetal deaths, and resorptions and for gross external, visceral, and skeletal malformations and fetal weights. A total of 7903 fetuses from 519 litters were examined. In the first replicate study, the number of fetuses per litter was decreased in the group exposed to 1 mT (p < 0.05). This decrease was not observed in the replicate study. No major malformations were correlated with exposure to magnetic fields.

Ryan et al. (Ryan et al., 1996) exposed groups of 46-55 timed-pregnant Sprague-Dawley rats to linearly polarized, 60 Hz magnetic fields on gestational days 6-19. The field strengths were 2 µT, 200 µT, and 1 mT with concurrent sham-exposed controls. A positive control group of 15 rats treated with ethylenethiourea was included. The animals were exposed or sham-exposed for 18.5 h/d, 7 d per week. On gestation day 20, the dams were sacrificed for reproductive and tertalogical assessments. No evidence of maternal toxicity was observed as a result of exposure. A battery of fetal examinations did not demonstrate any significant difference in the incidence of fetal malformations or anomalies between field-exposed and sham-exposed rats. The positive control caused malformations and body-weight reductions in 100% of treated animals. [No malformations were seen in rats exposed to 50 or 60 Hz magnetic fields.]

In a study of similar design, Ryan et al. (Ryan et al., 1998) failed to show any effect of exposure to magnetic fields in multigeneration studies of reproductive toxicity in groups of 40 male and female Sprague-Dawley rats.

4.4.4.4 Hamsters

4.4.4.5 Summary

There is no evidence for the reproductive or developmental effects of exposure to sinusoidal magnetic fields.

[This conclusion was supported by 17 Working Group members; there were 3 votes for 'weak' evidence, 8 abstentions, and 1 absent.]

Table 4.33 Studies on EMF exposures and reproductive and developmental effects of EMF in experimental animals and birds

4.4.5 Effects on melatonin

The studies discussed in this section are summarized in Table 4.34. Laboratory experiments on suppression of nocturnal melatonin in human volunteers are described in section 4.6.4.1.

4.4.5.1 Electric fields

Groups of 20 adult male Sprague-Dawley rats, eight weeks old, with long day cycles (14 h light, 10 h dark) were sham-exposed or exposed to 39 kV/m linearly polarized 60 Hz electric fields continuously for four weeks (Wilson et al., 1986), as described above. After each week of exposure, 10 field-exposed and 10 sham-exposed rats were sacrificed immediately after field termination (dark phase), and pineal melatonin and N-acetyltransferase activity were measured. Additional sacrifices and measurements were made three and 14 days after exposure. Significant decreases in nocturnal pineal melatonin and N-acetyltransferase activity were found after three or four weeks of exposure [no p value given]. The levels returned to normal within 3 d after exposure.

Groups of six to eight pregnant rats with long day cycles (14 h light, 10 h dark) were exposed throughout gestation, and their pups were exposed from birth to day 23 to 0, 10, 65, or 130 kV/m linearly polarized 60 Hz electric fields for 19 h/d (Reiter et al., 1988) by a parallel-plate system, with the rats in electrical contact with the reference ground electrode. Sham-exposed rats were housed identically except for the electric field. At the end of exposure, male pups from six to eight litters per time were sacrificed at 22:00, 02:00 (2 h after lights off), 04:00, 06:00, and 10:00 (lights on), and their pineal melatonin levels were measured. Significant decreases in pineal melatonin were found at 02:00 (p < 0.001), when the concentration of melatonin would be expected to be highest.

Groups of 7-16 eight-week-old male Sprague-Dawley rats with long day cycles (14 h light, 10 h dark) were sham-exposed or exposed to 65 kV/m linearly polarized 60 Hz electric fields for 20 h/d for 30 d (Grota et al., 1994). The exposure apparatus consisted of three parallel plates: a top electrified plate and grounded intermediate and bottom plates. The animals between the top two plates were exposed to a 60 Hz electric field, and the animals between the bottom two grounded plates were sham exposed. Harmonics were not detected, and the spillover to the sham condition between the lower plates was 1% or less. The effective field strength was estimated to be 82% in clean cages, which was reduced to 43% in cages in which rats had lived for more than four days. Clean cages were provided twice a week, and husbandry was done during the daily field-off period. Immediately after exposure, at 04:00 (light) or 16:00 (dark), groups of field-exposed and sham-exposed rats were sacrificed, and the plasma and pineal melatonin concentrations and pineal N-acetyltransferase and hydroxyindole-O-methyltransferase activities were measured. Significant decreases were found in nocturnal plasma melatonin (p < 0.05) but not in pineal melatonin or in N-acetyltransferase or hydroxyindole-O-methyltransferase activity.

[In all of these studies except that of Grota et al. exposure to electric fields had a statistically significant effect on at least one observation of pineal melatonin concentration. They speculated that the difference in the results of their study and others may be due to the sensitivity of the measurements to time. In particular, they considered that the dark phase was not the most sensitive for measurements of pineal function. In some experiments, the pineal and blood melatonin concentrations decreased in rats exposed to linearly polarized 60 Hz electric fields.]

4.4.5.2 Exposure to magnetic fields for < 1 h

Adult Djungarian hamsters with long day cycles (16 h light, 8 h dark) were exposed once to a 10 or 100 µT linearly polarized 60 Hz magnetic field for 15 min beginning 4 h before or 4 h after lights off (Truong & Yellon, 1997). Other hamsters were exposed to a 100 µT linearly polarized 60 Hz intermittent (1 min on, 1 min off) magnetic field for 15 or 60 min between 1 and 2 h before lights off. The exposure system consisted of two Merritt coils modified to produce highly uniform fields across two perpendicular axes. Six hamsters per time per group were sacrificed 1, 1.75, 2.5, 3, and 4 h after lights off, and their sera were harvested and pineal glands obtained for radioimmunoassay of melatonin. In sham-exposed controls, i.e. hamsters placed in an adjacent coil system but without current (< 0.6 µT), the pineal and plasma melatonin concentrations increased from a low baseline (1 h after lights off) to concentrations that were typical of the night-time peak by 3 h after darkness. Exposure to the continuous magnetic field for 15 min either 4 h before or 4 h after lights off did not disrupt the nocturnal rise in pineal or plasma melatonin concentration. Similarly, the onset of the melatonin rhythm was not suppressed in comparison with that in sham controls by intermittent exposure to magnetic fields. Thus, several paradigms of exposure to magnetic fields failed to alter the rising phase of the melatonin rhythm in pineal gland content or in the circulation. The authors conclude that the biological clock mechanism that mediates photoperiodic time measurement in this seasonally breeding rodent is resistant to a variety of continuous or intermittent exposures to magnetic fields.

Adult Siberian hamsters in constant darkness were exposed to a 100 µT linearly polarized 60 Hz magnetic field for 15 min (Yellon & Truong, 1998). Sham-exposed hamsters were simultaneously placed in an adjacent coil system but without current (< 0.6 T). Groups of five to seven animals per group were sacrified 0, 0.75, 1.5, 2.25, and 3 h after lights off, and their sera were harvested and pineal glands obtained for radioimmunoassay of melatonin. The increase in the pineal and the circulatory content of melatonin during subjective night was not affected by exposure to magnetic fields; regression of the testes occurred in both sham-exposed controls and in hamsters in constant darkness exposed daily to magnetic fields. The authors concluded that magnetic fields are unlikely to serve as a zeitgeber for the circadian changes in the melatonin rhythm; rather, the photoperiod is a predominant cue for the rising phase of melatonin production in the pineal gland and concentration in circulation.

In juvenile hamsters exposed to a 100 µT magnetic field for 15 min daily for nine days, statistically significant reductions in nocturnal melatonin concentrations were observed at some times, but pubertal development was normal. A replicate study showed no reduction in nocturnal melatonin (Truong et al., 1996).

Adult Djungarian hamsters were exposed to a 100 µT 60 Hz magnetic field for 15 min 2 h before dark daily for three or six weeks. The night-time rise in melatonin concentration was delayed, and the duration was reduced in the animals exposed for six weeks but not in those exposed for three weeks (Yellon, 1996).

Groups of male Siberian hamsters, four to six months of age, were exposed to a 100 µT 60 Hz linear magnetic field. Exposure depressed the pineal melatonin concentration at 4 h but not 2 h after the onset of darkness in male but not female hamsters. The hamsters with a short photoperiod were more sensitive to the magnetic field-induced response than those with a long photoperiod. The depression in melatonin was not seen after 42 days of exposure to magnetic fields (Wilson et al., 1998).

[The apparent contradiction between the effects seen in the Yellon's 1994 study and the lack of effects in the 1997 and 1998 studies from his laboratory may be due to diurnal cycling (long days in 1994 and 1997, constant darkness in 1998), time of exposure (2 h before darkness in 1994, 4 h before or after darkness in 1997), or continuity of exposure (continuous in 1994 and 1998, continuous or intermittent in 1997). The effect of a magnetic field may be influenced by the length of the day of the exposed animals. Truong et al. found that the 1994 exposure schedule resulted in changes in melatonin but not to the expected downstream effects in reproductive development. Additionally, Yellon was unable to reproduce the effects that he obtained in 1994. A single continuous exposure to a 100 µT linearly polarized 60 Hz magnetic field does not reproducibly shorten or blunt night-time plasma melatonin concentrations in Djungarian hamsters.]

4.4.5.3 Exposure to magnetic fields for 1-24 h

Groups of eight male Sprague-Dawley rats, 12 weeks old, with standard day cycles (12 h light, 12 h dim red light) were exposed through Helmholz coils to 1 mT intermittent (1 min on, 1 min off cycles) linearly polarized 60 Hz magnetic fields for 1 h, starting 2 h before darkness (John et al., 1998). Sham-exposed rats were simultaneously placed in an adjacent coil system but without current (< 0.17 µT). Urine was collected at 2-h intervals. The circadian profile of urinary 6-sulfatoxymelatonin was examined before, during, and after exposure; no significant effect on excretion was observed.

Groups of 10 male and 10 female adult Wistar rats with short day cycles (9 h light, 15 h dark) were exposed to either 5 or 500 µT linearly polarized 50 Hz magnetic fields through a pair of double-wound coils embedded in epoxy resin for 24 h (Bakos et al., 1995). Sham-exposed rats were simultaneously placed outside the coil system (5 µT stray fields). The fields were homogeneous to within 1%. The animals were all housed in metabolic cages. The urine of the animals was collected twice per day for five consecutive days, and the concentration of 6-sulfatoxymelatonin was measured by ¹²⁵I-radioimmunoassay. The urinary excretion of 6-sulfatoxymelatonin did not change significantly from baseline during or after exposure.

Groups of five adult male Wistar rats with short day cycles (9 h light, 15 h dark) were exposed to either 1 or 100 µT linearly polarized 50 Hz magnetic fields for 24 h (Bakos et al., 1997). Sham-exposed rats were simultaneously placed outside the coil system (1 µT stray fields). The animals were all housed in metabolic cages. The urinary excretion of 6-sulfatoxymelatonin was not statistically significantly decreased during or after exposure to either flux density. Excretion of rats at 100 µT was significantly increased the day after exposure (p < 0.02) in comparison with the value during exposure, but was not significantly different from the baseline value before exposure.

[These studies neither directly support nor contradict each other because of confounding effects of strain (Wistar vs. Sprague-Dawley), field characteristics (50 Hz continuously vs. 60 Hz intermittently), and effects measured (plasma melatonin, pineal N-acetyltransferase activity, or urinary 6-sulfatoxymelatonin excretion). The difference between the two studies by Bakos, one indicating a potential for increased 6-sulfatoxymelatonin excretion, is confusing. The results of Selmaoui remain suggestive, although the study suffers from the lack of a well-characterized exposure system. The biological significance of small changes in melatonin concentrations is not clear.]

4.4.5.4 Exposure to magnetic fields for > 24 h

In a study with similar exposure, groups of 22-30 adult (11-18 weeks old) male, pigmented Long-Evans rats with standard day cycles were exposed to 0.02 or 1 µT circularly polarized 50 Hz magnetic fields continuously for six weeks (Kato et al., 1994a). Significant decreases in plasma melatonin concentration (> 20%, p < 0.01) were found at the mid-points of the dark and light phases at both 0.02 and 1 µT. Significant decreases in pineal melatonin concentration (p < 0.01) were found during the light phase at both intensities and during the dark phase at 1 µT.

In a further study, groups of eight adult (13-21 weeks old) male Wistar-King rats with standard day cycles were exposed to either 0.02 or 1 µT circularly polarized 50 Hz magnetic fields continuously for six weeks (Kato et al., 1994b). Animals were sacrificed at the end of exposure, and the plasma melatonin concentrations were determined at that time and one and four weeks after cessation of exposure. The nocturnal melatonin concentration was reduced by 25% (p < 0.05), but the concentration one week after the end of exposure was normal, and no further change was observed four weeks later.

In yet another study, groups of 23-30 adult (11-20 weeks old) male Wistar-King rats were exposed to 0.02 or 1 µT linearly polarized 50 Hz magnetic fields continuously for six weeks (Kato et al., 1994c). No significant change in plasma melatonin or pineal melatonin concentration was found.

Groups of 36 six-week-old female Sprague-Dawley rats with standard day cycles were either sham-exposed or exposed to a gradient of 0.3-1 µT linearly polarized 50 Hz magnetic field continuously through six cylindrical coils for 91 days beginning eight to nine weeks after intragastric administration of 20 mg (5 mg/week) DMBA (Löscher et al., 1994). Sham-exposed rats were housed in the same room and experienced stray fields of 0.02-0.04 µT. The animals were sacrificed at the end of exposure, and the concentrations of plasma and pineal gland melatonin were determined by radioimmunoassay. The nocturnal melatonin levels were reduced by 20% (p < 0.05) at all intensities.

Groups of 99 six-week old female Sprague-Dawley rats with standard day cycles were either sham exposed or exposed to a 50 µT linearly polarized 50 Hz magnetic field continuously for 91 days after intragastric administration of 20 mg (5 mg/week) DMBA (Mevissen et al., 1996b). The sham-exposed rats were housed in the same room and experienced stray fields of 0.05 µT. The animals were sacrificed at the end of exposure, and the concentrations of plasma and pineal gland melatonin were determined by radioimmunoassay. Determination of nocturnal plasma melatonin after 9 and 12 weeks of exposure to 50 µT showed no significant difference between field-exposed and sham-exposed rats. In another study with the same experimental design but with exposures to 10 µT, a significant decrease was observed in serum but not in pineal melatonin concentration (Mevissen et al., 1996a).

Groups of 12 male five-week-old Wistar rats with standard day cycles were exposed to either 1, 10, or 100 µT linearly polarized 50 Hz magnetic fields from rectangular coils for 18 h/d for 30 days (Selmaoui & Touitou, 1995). Sham-exposed animals were kept in a similar environment. The animals were sacrificed under dim-red light at the end of exposure during their dark phase. The nocturnal plasma melatonin concentration and pineal N-acetyltransferase activity were depressed at both 10 (27%, p < 0.05) and 100 µT (42%, p < 0.05). No effect was seen on hydroxyindole-O-methyltransferase activity.

Groups of 45 male Djungarian hamsters with long days (16 h light, 8 h dark) were exposed to a 300 µT linearly polarized 50 Hz magnetic field from Helmholz coils continuously for 56 days (Niehaus et al., 1997). Sham-exposed rats were simultaneously placed outside the coil system and experienced < 3 µT stray fields. No effects on nighttime plasma or pineal melatonin concentrations were found as determined by radioimmunoassay.

Groups of eight adult male Sprague-Dawley rats with standard day cycles were exposed to a 1 mT linearly polarized 60 Hz magnetic field for 20 h/d for 10 d or six weeks or to 1 mT intermittently (1 min on, 1 min off) of a linearly polarized 60 Hz magnetic field for 20 h/d beginning 1 h before darkness for two days (John et al., 1998). Sham-exposed rats were simultaneously placed in an adjacent coil system but without current (< 0.17 µT). Urine was collected at 2-h intervals. The circadian profile of urinary 6-sulfatoxymelatonin was examined before, during, and after exposure. No significant effect on excretion was observed during exposure.

Groups of 10 seven-week-old female Sprague-Dawley rats with standard day cycles were either sham-exposed or exposed to 100 or 500 µT linearly polarized 50 Hz magnetic fields or to a 100 µT linearly polarized 60 Hz magnetic field for 18.5 h/d for 4, 8, or 12 weeks, with intragastric administration of 8, 10, or 20 mg DMBA (NTP, 1998a). Control rats were housed in a separate room. The animals were sacrificed at the end of exposure, and the concentrations of plasma and pineal gland melatonin were determined by liquid chromatography and tandem mass spectrometry. No significant differences were found between field-exposed and sham-exposed rats. [The data varied considerably.]

[As in the studies of short-term exposure, confounding variables such as species (hamster vs. rat), strain (Wistar vs. Sprague-Dawley), sex, co-exposure (DMBA), field characteristics (polarization, intermittence), and measured outcome (plasma melatonin, pineal melatonin, pineal N-acetyltransferase activity, urinary 6-sulfatoxymelatonin) complicate interpretation of the database. Long-term exposure to circularly polarized 50 Hz magnetic fields may decrease dark-phase melatonin concentrations in rats, and long-term exposure to 10 or 100 µT linearly polarized 50 Hz magnetic fields decreases plasma and pineal melatonin concentrations in male Wistar rats.]

4.4.5.5 Exposure to electric and magnetic fields

Groups of 10 female Suffolk lambs, eight weeks old and housed outdoors, were exposed to mean fields of either 4 µT and 6 kV/m or 3.77 µT and 6.3 kV/m from the electrical environment of a 500 kV transmission line and < 3 µT and < 0.01 kV/m or 0.02 µT and < 0.01 kV/m from a nearby pen (Lee et al., 1995; Lee et al., 1993). The sheep remained in these pens for eight months, and the fields were monitored continuously. Plasma samples were collected at 0.5-3 h intervals over eight 48-h periods from the same animals throughout the study. The melatonin concentrations in the plasma were determined by radioimmunoassay. The characteristic pattern of melatonin secretion during night-time (amplitude, phase, and duration) did not differ between control and treated groups.

Groups of three male baboons, aged seven to eight years, with long day cycles (15.5 h light, 8.5 h dark) were exposed to either 50 µT, 6 kV/m or 100 µT, 30 kV/m linearly polarized 60 Hz EMF without transients for three blocks of 4 h (08:30-12:30, 13:00-17:00, 17:30-21:30) each day for six weeks (Rogers et al., 1995d). Homogeneous vertical electric fields were generated between a bus-suspended at 2.5 m and a large, nonferrous metal gate. Homogeneous magnetic fields were generated by a series of conductors beneath the grate. The field strengths were logged at 15-min intervals. Plasma melatonin concentrations were determined at 2-h intervals from the same animals throughout the day on days before, during, and after the six weeks of exposure period. No changes in plasma melatonin concentration were observed.

In a similar study, groups of two adult male baboons with long day cycles were exposed from the same sources to a complex schedule of 50 µ T, 6 kV/m or 100 µT, 30 kV/m linearly polarized 60 Hz electric and magnetic fields for 0-24 h/d for 30 d (Rogers et al., 1995e). Transients were purposely not avoided, and 300 Hz transients were observed. The night-time plasma melatonin concentrations during exposure were decreased by more than 80% from pre-exposure levels.

[The studies of mixed exposure, unlike the others summarized here, were all done on non-rodent species. Changes in plasma melatonin concentrations were found only by Rogers et al.; however, only two animals were used.]

4.4.5.6 Summary

It was reported that long-term exposure to magnetic fields (100-1000 µT) can reduce the nocturnal pineal or blood concentrations of melatonin in rodents, but other laboratories did not find similar results.

Field orientation and linear or circular polarization may be important. Reductions in melatonin levels were not found in sheep or baboons. Similarly, studies of the effects of acute exposure to magnetic fields in hamsters showed reductions in melatonin; nonsignificant results were obtained more frequently than significant ones, but all those that were significant showed suppression. The intensities used were relatively high, even when human-rodent scaling issues are considered. The biological significance of melatonin reduction is not clear.

There is weak evidence that exposure to electric and magnetic fields alters the levels of melatonin in rodents.

[This conclusion was supported by 14 members of the Working Group; there were 9 votes for 'moderate' support; 4 abstentions, and 2 absent.]

There is no evidence that exposure to electric and magnetic fields alters the levels of melatonin in sheep or baboons.

[This conclusion was supported by 14 members of the Working Group; there were 13 abstentions and 2 absent.]

Table 4.34 Studies of the effect of exposure to EMF on melatonin in experimental animals

4.4.6 Bone and tissue repair and adaptation

4.4.6.1 Clinical bone healing with pulsed electromagnetic fields

The transition to reliance on magnetic induction required identification of a dynamic flux pattern to induce the appropriate electrical currents. Unlike the studies of hazard, which are based largely on sinusoidal fields, the development of clinical devices has focused almost exclusively on use of pulsed fields. This approach was primarily pragmatic, as high rates of change could easily be implemented for relatively low flux densities, permitting the induction of currents similar in magnitude to those observed during development or the mechanical deformation of bone structures (i.e. 0.1-1 V/m). The clinical PEMF signals typically consist of peak flux densities in the range of 0.1-5 mT with rise times on the order of hundreds of microseconds. This results in a typical dB/dt in the range of 1-50 T/s and corresponding peak induced electric fields of 0.1-1 V/m. The energy distribution in such complex fields is centered near the pulse repetition rate and the harmonics of this rate. The pulse repetition rates range from 15 Hz up to several kilohertz; however, many of the clinical signals involve a pulse burst pattern, in which a short burst of pulses is repeated at a low-duty cycle. Burst repetition rates are always in the ELF range, which can be as low as 1 Hz and rarely extend beyond several tens of hertz. Such pulse-burst signals have extremely complex energy distribution spectra which cover the frequency range starting at the burst repetition rate and extending to higher-order harmonics associated with the pulse width (McLeod & Rubin, 1990).

EMF-induced healing differs in two main ways from the most studies of the hazards of EMF. While the major emphasis in the development of these devices has been on induced electric fields, the preferred technique is to center the induction coils over the injury. The effect of this strategy is to minimize the magnitude of the induced electric field at the focus of the injury while maximizing the induced field in the periphery. The maximum reported induced electric fields in the central region of healing are therefore far below the reported peak induced electric fields, perhaps by an order of magnitude or more. This exposures is, however, spatially far more consistent than those experienced in any study of whole-body exposure, in that the induction coils are generally strapped to the body, ensuring a fixed orientation of the flux to the body part and, correspondingly, a fixed electric field distribution. This spatial coherence may be a critical aspect of field-induced healing.

Pulsed electromagnetic field therapy for tibial fractures

PEMF have been used to promote bone healing under conditions of non-union, delayed union, osteotomy, and bone fusion. By far the majority of the published studies are retrospective analyses of patient populations. Representative of these reports is the multicenter review of cases of tibial non-union compiled shortly after the Food and Drug Administration approved PEMF devices as a safe and effective for the treatment of non-unions and failed fusions (Bassett et al., 1981). In this report, the cases of 125 adult patients who had undergone PEMF therapy for delayed union (no evidence of union four to nine months after fracture) or non-union (no union nine months after fracture) were reviewed; true pseudoarthroses were specifically excluded from the study. The treatment consisted of cast immobilization and treatment of the fracture site for at least 10 h/d by exposure to a 15 Hz pulse-burst signal. A peak flux of 1.5 mT, decaying in 25 µs, was used, with pulses repeated at a 4-kHz rate over the burst duration of 5 ms. This resulted in an induced electric field pulse in the bone tissue estimated to be on the order of 0.1-0.2 V/m. The reported overall success rate in this study was 87%, requiring an average duration of treatment of 5.2 months (range, 2-22 months). Failure to promote healing was attributed by the authors to absence of rigid immobilization, fracture gaps greater than 1 cm, use of the stimulus for less than 10 h/d, or off-center or skewed placement of the induction coils.

In 1984, the first of a series of double-blind, randomized, placebo-controlled trials of pulsed magnetic field therapy was published (Barker et al., 1984), in which 16 patients with tibial fractures that had been un-united for at least 52 weeks were enrolled, immobilized in casts, and randomly assigned to treatment with either an active or placebo (no coil current) device. Patients were excluded if the fracture gap was greater than 0.5 cm, internal or external fixation was present at the fracture site, sepsis was present, or the patient was undergoing steroid treatment. Patients were instructed to use the device for 12-16 h/d with a minimum of 1 h/d. Satisfactory compliance was considered to be an average of 10 h/d, with less than 6 h/d use on no more than 7 d in each six-week period. The patients were monitored every six weeks. At 24 weeks, the fractures of five of the seven patients with placebo devices had healed as compared with five of nine patients with active devices. While the group with active devices was significantly older than the placebo group (38 years vs. 29 years), the authors concluded that conservative management of true tibial non-unions is as effective as magnetic field therapy.

At the same time, a randomized double-blind trial on delayed unions was undertaken (Sharrard, 1990), in which adult patients with tibial fractures that had not united at 16 weeks but had occurred less than 32 weeks previously and who had not undergone surgical treatment were enrolled. After the fractured limbs had been immobilized in long-leg plaster casts, those with apposition over less than 50% of the fracture surface or with a fracture gap greater than 0.5 cm were excluded. Over a period of six years, 51 patients were identified who met the study criteria. Patients were instructed to undergo therapy each day for a total of 12 h/d; treatment continued for 12 weeks, with the same exposure system as described by Barker et al. (Barker et al., 1984). The study protocol was completed by 45 patients. Despite randomization, the age distribution was found to be significantly different in the two study populations, the mean age of patients in the active group being 35 years (median, 28), while that of the control group was 45 (median, 45). Radiographic assessment showed that five of the actively treated fractures had healed within 10 weeks, with progress in five and no progress in 10. Conversely, in the placebo group, one fracture had healed, progress was seen in one patient, and no progress was observed in 23 patients. The results were interpreted as demonstrating a significant effect of magnetic field therapy (p < 0.002). [The extended duration required to recruit this cohort and the significant age difference between the two groups raise serious concern about the actual efficacy of magnetic field therapy for this condition.]

Pulsed electromagnetic field therapy for osteotomies

Osteotomies are a distinctly different type of fracture from others. Normally, fractures progress through the process of secondary bone formation or endochondral ossification; that is, in a normally displaced fracture, healing is a sequential process starting when the hematoma is replaced by a fibrous fracture callus, which is replaced by cartilage, which subsequently calcifies. In a minimally displaced fracture (e.g. a hairline fracture), however, hematoma and callus do not form, and healing occurs by primary bone formation or the direct bridging of the fracture site by new bone. This primary healing process also occurs after osteotomies, the surgical procedure of transecting a bone in order to realign an articulating surface.

Promotion of osteotomy healing by magnetic field therapy has been investigated in two randomized, placebo-controlled clinical trials. In the first of these (Borsalino et al., 1988), 32 patients treated by intertrochanteric osteotomy for degenerative arthritis of the hip were randomly assigned into treatment and placebo groups. Treatment consisted of exposure to a 75 Hz repetitive pulse signal consisting of a 1.8 mT flux with a rise time of 1.3 ms. The peak induced electric field intensities can be estimated to have been about 50 mV/m. Induction coils were not energized in the placebo devices. Exposure was for 8 h/d for 90 d. Healing was evaluated radiographically at days 40 and 90 in the 31 patients who completed the treatment protocol. Trabecular bridging was found to be significantly increased in the active treatment group at both 40 days (p < 0.02) and 90 days (p < 0.001).

Subsequently, a study of tibial osteotomy was undertaken (Mammi et al., 1993), in which 40 consecutive patients treated with vagus tibial osteotomy for degenerative arthrosis of the knee were randomly assigned to placebo or treatment by order of admission. The distributions of sex and age were similar in the two groups. Exposure was given from a device similar to that used by Borsalino et al. (Borsalino et al., 1988), consisting of a 75 Hz pulsed signal of 1.8 mT with a 1.3 ms pulse width, with instructions to use it for 8 h/d; the output current was shorted in the placebo devices. Healing was assessed radiographically by four independent orthopedic surgeons on a four-point scale. A total of 37 patients completed the protocol. At 60 days, the average score for the actively treated population was 3 vs. 2.1 for the placebo-treated group, reflecting a significant enhancement in healing rate (p < 0.006 by Fisher exact test).

Pulsed electromagnetic field therapy for lumbar spinal fusion

While lumbar spinal fusion is a common surgical procedure, the intervertebral space represents a wide vascular region, making it difficult to ensure revascularization of the bone graft. As lumbar fusions are therefore slow to achieve union, the potential for PEMF therapy to accelerate fusion has also been investigated. Mooney (Mooney, 1990) studied the efficacy of this therapy to stimulate fusion in a randomized double-blind trial involving 195 patients with autogenous, cadaverous, or combination grafts and patients with and without internal fixation hardware. Exposure was given from a 15 Hz pulse-burst signal, 1.5 mT in magnitude, with a 25 µs rise time and 4 kHz pulse repetition rate for bursts of 5 ms. Patients in both the active and placebo groups were fitted with a brace that they were instructed to wear for 8 h/d. Radiographic analysis by the orthopaedic surgeon was used to determine time to fusion, with confirmation by an independent radiologist. The overall fusion rate was 92% in the 65 patients in the active group and 65% in the 98 patients on placebo. The spines of the 34 patients who used the treatment device sporadically (< 4 h/d) fused with the same success rate as those on placebo. The author interpreted the results to represent a significant increase in success rate with PEMF therapy (p < 0.005).

The most recent of the double-blind clinical trials addressing the clinical efficacy of PEMF on bone healing involved administration of pulse-field therapy during limb lengthening procedures (Eyres et al., 1996). In this orthopedic procedure, an osteotomy is performed and external fixation hardware is inserted to obtain progressive distraction of the proximal and distal bone ends. Bone then regenerates in the distraction gap. After distraction is complete, the gap undergoes consolidation, similar to that seen in fracture healing. In this study, patients were randomly assigned to active treatment or placebo, with instructions to use the induction devices for 4 h/d. The characteristics of the field exposure were similar to those described above: 15 Hz pulse-burst field, 1.5 mT pulses with a 25 ms rise time, repeated 4 kHz pulse rate in a 5 ms burst. The effectiveness of the treatment was quantified by dual energy X-ray analysis, which provides bone density measurements at the mid-point of the distraction gap and proximal and distal to the osteotomy site. Densities were normalized to those of the contralateral, non-operated limb. The patients were followed for 12 months. No significant difference was observed in limb-lengthening rate or in distraction gap bone density due to PEMF; however, large differences were observed in the proximal and distal segments of the bone. In the proximal segment, a significant (p < 0.0001) increase in bone mineral density was observed as compared with placebo controls, an effect which began after three months of stimulation and resulted in 20% greater bone mineral density than in controls at 12 months. On the distal side, the control bone mineral density dropped by 46% (such bone loss due to disuse is expected in this procedure), whereas the field-exposed population had a loss of less than 15% (p < 0.0001).

4.4.6.2 Experimental studies

Initial attempts to develop non-invasive electric field therapy involved capacitive coupling, although the effectiveness of this technique in the ELF regime was limited and electromagnetic coupling rapidly supplanted this simpler technique.

Exogenously induced field effects on bone tissue

After the demonstration of the piezoelectric properties of bone (Fukada & Yasuda, 1957), numerous investigators pursued measurements of electrical potentials in bone during deformation in order to determine whether the distribution of currents or charge might account for the adaptive responses of bone tissue. In 1968, McElhaney and Stalnaker hypothesized the converse, that application of electric fields results in a reverse piezoelectric effect, leading to deformation of the tissue sufficient to inhibit bone loss due to disuse. In their study, the right legs of 48 male Sprague-Dawley rats weighing about 100 g were immobilized in casts after 3/4 x 1/2 x 1/16-inch [19 x 12.7 x 1.6-mm] insulated and waterproofed copper electrodes had been fastened to the medial and lateral surfaces. The animals were assigned to four groups: sham exposure (no voltage applied to plates), DC control (100 V DC applied to plates), 3 Hz stimulus at 200 V, or 30 Hz stimulus at 200 V. [The induced electric field due to the time-varying electric field can be estimated to be 0.1-1 V/m. The DC field would induce no field in the tissue.] All animals were treated for 1 h every 12 h for 28 d. The bone cross-sectional area, hardness, and compressive strength were measured, and chemical and histomorphometric analyses were performed on the left and right femurs of the 24 animals that survived the protocol. On the basis of this broad array of measures, the authors concluded that the most robust exposure, as compared with sham exposure, was the 30 Hz stimulus, the 3 Hz stimulus appearing less effective in maintaining the bone normally lost due to disuse; the DC exposure was the least effective. Eight apparent bone tumors or prolific new bone formation were seen in the 18 electrically treated animals. Five of these tumors occurred in the group exposed to 30 Hz, two in those at 3 Hz, and one in those exposed to DC. No tumors were found in the sham-exposed group.

In a study to specifically address the incidence of bone tumors reported by McElhaney et al. (McElhaney et al., 1968) undertook a similar study of disuse osteoporosis in 50 male Sprague-Dawley rats weighing 150 g with limbs immobilized in casts. Four study groups were used: a control group with no implanted electrodes, a sham-exposed group with implanted electrodes (19 x 13 x 1.6 mm copper with epoxy insulation) but no excitation, a group exposed to a 30 Hz stimulus at 200 V peak-peak applied to the plates for 1 h every 12 h, and a group exposed to the same stimulus but for 8 h per weekday and 1 h/d on weekends. At 28 days, the animals were sacrificed, and the cortical area, mass, density, and percent ash of the femurs were determined. A much smaller effect on disuse was seen than in the previous study. [The greater size (and therefore age) of the animals may account for much of this difference.] A strong effect of exposure to 30 Hz was again observed, with a duration dependence, as the group exposed for 8 h/d had a greater increase in cross-sectional area than those exposed for 2 h. No evidence of tumors or hypertrophic growth was found in any of the animals examined. [Of concern in this trial is the asymmetric loss of animals in the various groups, prohibiting comparison of the groups exposed for 2 and 8 h with regard to three of the four properties assayed.]

Cruess et al. (Cruess et al., 1983) revisited the question of osteoporosis, using PEMF to treat disuse osteoporosis in a rat model. Two groups of 250 g male rats underwent surgical removal of the gastrocnemius and soleus muscles bilaterally and were then placed in plaster casts. One of the groups was treated continuously by whole-body exposure to a repetitive 65-72 Hz pulse of 325 µs in width, sufficient to induce an electric field of 150 mV/m at the tibia; the second group remained untreated. A third group of free-roaming normal rats was included in the study. After 14 days of exposure, the tibias were removed and morphological and biochemical assays performed. While the surgical procedure resulted in significant loss of body mass over the two-week period as compared with the free roaming controls, no significant difference in wet weight or in percent ash weight was observed between treated and untreated animals. Biochemical assays showed significantly (p < 0.05) higher collagen synthesis rates, lower collagenase activity, and higher mineral uptake in the treated group than in the operated group with no field exposure.

Enzler et al. (Enzler et al., 1984) completed a study in a canine ulna non-union model system to test the efficacy of PEMF to accelerate fracture healing. Twelve female beagle dogs aged four to six years underwent bilateral surgical procedures for osteotomy of the ulna. The right limbs of six animals and the left limbs of the remaining six were exposed to PEMF for 24 h/d, the contralateral limb being used as control. The pulse waveform was similar to that used in clinical studies: a 2-ms burst comprised of 10 pulses, with the burst repeated at a 10 Hz rate. [The magnitude of the peak flux density was not reported, although it can be expected to have been 1-2 mT, consistent with the other experimental devices produced by ElectroBiology, Inc. During the 1970s and 80s, this company was in the forefront of development of PEMF therapy and provided exposure systems to a large number of research groups. Two systems were commonly used: a 70-75 Hz repetitive pulse signal and a 1.5-15 Hz pulse-burst signal waveform, both with a peak flux range of 1-2 mT. In these studies, induced electric field intensity is estimated as the product of the peak time-rated change of the flux and a characteristic dimension of the exposed tissue. In this specific study, the approximate 30 µs rise time would be consistent with a dB/dt on the order of 50 T/sec, corresponding to peak induced electric fields in the range of 1 V/m.] After sacrifice 22-30 days after the start of exposure, the fractures were tested for torsional stiffness and examined histologically. None of the osteotomies healed during the experimental period, no histological changes were evident, and no statistical differences in the torsional stiffness of the fracture calluses were observed between the treated and untreated limbs.

Bone tissue healing can be classified as comprising three distinct processes: fracture healing, appositional growth, and epiphyseal plate growth (lengthening) by endochondral ossification. While most early studies of the effects of EMF on bone healing emphasized the first two of these processes, Smith and Nagel (Smith & Nagel, 1983) investigated the effect of PEMF on bone elongation in the hind limbs of rabbits. In order to study the time of growth-plate closure, female rabbits aged 12 weeks at the beginning of the protocol were exposed for 18 weeks. To study bone elongation, the animals were exposed for eight weeks starting at the age of six weeks. Exposure was for 24 h/d to a 72 Hz repetitive pulse signal generated by hardware supplied by Electrobiology, Inc. [The peak flux densities were not reported but are presumed to be in the range of 1-2 mT.] Induction coils were attached to the left and right sides of the animals by a coil support, which permitted some movement. In addition to limb length and growth-plate closing time, blood flow was evaluated by technetium scanning, and glycosaminoglycan distribution was analyzed in extracted articular cartilage. No effect of exposure on the pattern of epiphyseal plate closure was observed, either with a 12-h on/off cycle of exposure or with continuous exposure (n = 6). Similarly, no effect on morphological measures of the tibia or on blood flow patterns was evident; however, the growth rate of the femurs of the animals experiencing continuous exposure was slightly inhibited (n = 18; p < 0.06), and the glycosaminoglycan content was significantly elevated (22%, p < 0.003). The authors interpreted their results as indicating that PEMF inhibits cartilage maturation.

It is not uncommon in orthopedic surgery for segmental, autogenous cortical bone grafting operations to fail due to graft-host non-union. Given the extensive anecdotal reports of healing of traumatic non-unions following PEMF therapy, Miller et al. (Miller et al., 1984) studied the potential use of this therapy to promote healing in autogenous grafts. Twenty adult mongrel dogs underwent bilateral surgery to remove 4 cm long fibular segments, which were then inverted and replaced in the graft bed with no internal fixation. One week post-operatively, one leg of the animal was exposed to a pulsed magnetic field from an orthosis incorporating a Helmholtz coil pair producing a 5 ms burst of 22 30 µs wide pulses repeated at a 15 Hz rate. The estimated peak induced electric field was reported to be 1.5 V/m. All of the animals were exposed for 20 h/d, with 10 animals treated for two months and 10 for six months. All animals were sacrificed six months post-operatively and histological and biomechanical analyses performed. No statistically significant differences were seen in biochemical or biomechanical measures or in terms of time to union.

Aaron et al. (Aaron et al., 1989) also studied the effect exposure to EMF on endochondral ossification, but in a model system based on subcutaneous implantation of decalcified bone matrix. In an early report, the effect of field exposure on collagen synthesis and matrix calcification was studied. Decalcified bone matrix (25 mg) obtained from the tibia and femur of mature male CD rats was implanted along the thoracic musculature in immature male CD rats. The animals were randomly assigned to one of three groups: free-roaming controls, sham-exposed, and treated groups. Sham- and field-exposed groups were confined in restraining boxes for 8 h/d during the animal's normal sleep cycle. The treated group was given a whole-body exposure to a pulse-burst magnetic stimulus consisting of a 4.5-ms burst containing 20 20 µs pulses of 2.0 mT. The burst was repeated at a rate of 15 Hz. This waveform therefore produced a peak dB/dt of 100 T/s, corresponding to an estimated peak induced electric field on the order of 1 V/m. The animals were sacrificed at 2-d intervals over two weeks, and the ossicles were evaluated biochemically, histologically, and histomorphometrically. Exposure had no significant effect on the total volume of calcified tissue in the ossicles at any time. A transient increases in sulfate incorporation and in the volume of cartilage were observed in association with field exposure, peaking at day 8, and the calcium content of the developing ossicles was consistently higher than that in restrained control animals (p < 0.05). [A confounding aspect of this study is that the restrained controls had a significantly inhibited ossicle development pattern as compared with the free-roaming controls, such that the effect of field exposure was essentially to return the development process to normal.]

In a study of the ability of magnetic fields to promote appositional bone growth, McLeod and Rubin (McLeod & Rubin, 1992) coupled observations made with pulse fields to those based on exposure to sinusoidal ELF fields. Bone resorption and formation were studied in an avian model of disuse osteopenia. Bone loss due to disuse was initiated by proximal and distal osteotomies on the left ulna of adult male turkeys, and Delrin caps were installed over the bone ends to prevent re-union. Exposure to magnetic fields was accomplished from a Helmholtz-like coil pair strapped to the wing of the bird. Exposure was for 1 h/d, five days per week. Six experimental groups were used, representing disuse, sham exposure (unenergized coils), exposure to a 75 Hz pulsed field (0.2 mT peak with rise time of 380 µs), and exposure to three sinusoidal fields at 75 and 150 Hz (representing the fundamental and second harmonics of the pulsed waveform) and 15 Hz. The flux densities of the sinusoidal fields were established to provide the same dB/dt as in the fundamental component of the 75 Hz pulsed stimulus. The electric fields induced in the preparation were estimated to be on the order of 10-20 mV/m with the pulsed field and less than 1 mV/m with the sinusoidal fields. Morphological assessment after eight weeks showed a clear frequency dependence of the bone remodeling response, 150 Hz being less effective than 75 Hz, which was significantly (p < 0.05) less effective than 15 Hz. The 15 Hz stimulus was found to be more effective than the pulsed field exposure in initiating new bone formation, despite the similar peak flux density used (0.24 mT) and the much lower peak induced electric field. [As the peak dB/dt was kept constant for the sinusoidal fields, the current flow into the coils would have had to increase by 10-fold between the 150 Hz and 15 Hz stimuli. In the absence of an active sham-exposed control, this dose dependence may, to some degree, simply reflect a difference in heating of the induction coils.]

Takano-Yamamoto et al. (Takano-Yamamoto et al., 1992) investigated the ability of magnetic fields to promote bone formation in a bone defect model. A 2 mm non-healing bone defect was surgically prepared in the premaxilla of 180 g male Wistar rats, and they were either treated with 7 mg demineralized bone matrix or not, with or without field exposure, yielding a total of five groups with the sham-operated group, with 4-10 animals per group. Animals were exposed in a 25 x 20 x 60 cm plastic container which was placed in a solenoid 30 cm in diameter and 60 cm in length. Exposure was for 12 h/d to a pulse-burst waveform composed of a 10 ms burst of 100 µs pulses, with a burst repetition rate of 15 Hz. The peak flux densities were 0.15-0.18 mT. After sacrifice of the animals at days 0, 21, and 35, the defect sites were examined for histological alterations, alkaline phosphatase activity, and Ca⁴⁵ incorporation. Defects treated with demineralized bone matrix graft and PEMF had significantly greater alkaline phosphatase activity and calcium incorporation (p < 0.05) than those with any of the four alternative treatment protocols. In addition, histological examination showed almost complete osseous bridging of the defect by day 35.

In a another test of the efficacy of PEMF to promote bone growth, Buch et al. (Buch et al., 1993) studied in-growth into a titanium bone harvest chamber. The titanium chamber (6 x 10 mm) was surgically implanted into the proximal tibia metaphyses of six male and female rabbits and became anchored within four weeks. Bone tissue was then harvested at three-week intervals with the implant in situ, providing a repeated measure of in-growth. Bone tissue was harvested once before field exposure, six times during the exposure period, and then twice after field exposure was terminated. The animals were restrained and underwent whole-body exposure 2 h/d from a Helmholtz-like coil pair. The waveform consisted of a 72 Hz repetitive pulse pattern with a peak flux density of 3 mT. [Because of the geometry and the presence of a metal chamber, the induced electric field cannot be easily calculated.] The mass of bone harvested from the bone chamber declined significantly after the first three-week harvest (p < 0.003 by repeated measures analysis). [While this result may well be due to the inability of the tissue to sustain a high level of bone in-growth, the decline coincides with the period of field exposure, resulting in an apparent inhibitory effect.] After termination of the field exposure at the time of the sixth harvest, bone in-growth into the chamber declined further (p < 0.004). The authors interpreted these results to indicate that field exposure sustains a level of in-growth that would have continued to decline in the absence of stimulation. [Without concurrent sham-exposed controls, this conclusion is difficult to support.]

Pienkowski et al. (Pienkowski et al., 1994) attempted to optimize PEMF therapy in a rabbit fibular osteotomy model. This study is unique in that it was fully blinded, with placebo devices for the controls. Fibular osteotomies were performed on the right limbs of 399 immature (2.8-3.2 kg) male rabbits, which underwent 20 experimental conditions. Field exposure was accomplished from a saddle-shaped induction coil placed around the right leg and centered over the osteotomy site. A pulse-burst waveform pattern was used in a 5-ms burst with a burst repetition rate of 15 Hz. The peak flux density and pulse width were varied in an attempt to identify optimal exposure conditions. The peak flux densities were 0.028-1.1 mT and the pulse widths were 0.5-10 µs. All of the animals were sacrificed on day 16 after the operation, and the efficacy of treatment was evaluated by a three-point bending test of the mechanical stiffness of the fibula. While certain combinations of pulse width and amplitude appeared to result in a significantly stiffer (p < 0.05) callus, replication experiments did not confirm this observation. No clear pattern of dose- or duration-response was evident in this partially factorial experiment, suggesting that no consistent improvement in fracture healing resulted from field exposure.

In an extension of their studies on the acceleration of endochondral ossification by exposure to magnetic fields, Aaron and Ciombor (Aaron & Ciombor, 1996) investigated the effect of phased exposure. Immature (80 g) male rats received demineralized bone matrix pellets in the thoracic musculature and were exposed to a magnetic field stimulus for only a fraction of the 20-d experimental protocol. There were four exposure groups: on days 1-3, corresponding to the mesenchymal phase of ossicle development; on days 4-8, corresponding to the chondrogenic phase; on days 9-19, corresponding to the calcification phase; and for the full 20 days. Exposure was accomplished as described above, with a pulse-burst waveform of 5 ms bursts repeated at a 15 Hz rate, and a peak flux of 2 mT. Animals underwent whole-body exposure while confined in exposure containers. All of them were sacrificed on day 20, and glycosaminoglycan synthesis and Ca⁴⁵ uptake were measured. While all of the exposed animals showed significantly increased calcium uptake and glycosaminoglycan synthesis rates as compared with unexposed controls, stimulation for just the first three days (mesenchymal phase) was found to be as effective in promoting ossicle development as exposure for the full 20 days.

McLeod and Rubin (McLeod & Rubin, 1998) also continued their studies on the response of the disuse avian ulna model to exposure to magnetic fields in an attempt to identify the frequency and dose-response characteristics of bone tissue. They continued to use proximal and distal osteotomies of the left ulnas of adult male turkeys to create a bone disuse situation; however, in this study the bone ends were capped with stainless-steel (non-magnetic) caps to prevent re-union, and external fixation hardware was used to prevent any inadvertent mechanical loading of the bone. In addition, a solenoid induction coil was used to impose the magnetic field along the long axis of the bone. The induced electrical currents therefore had an essentially uniform two-dimensional distribution, permitting more accurate calculation of the induced current and field intensities. Groups of three to four treated ulnas were exposed for 1 h/d under one of 10 exposure conditions, which spanned the frequency range of 5-150 Hz (all at a constant dB/dt of 0.025 T/s) and a flux density ranging up to 2.5 mT at a frequency of 15 Hz. The bone cross-sectional area at the ulna mid-diaphysis was assayed after sacrifice of the birds eight weeks after operation. A distinct frequency response was observed, with sensitivity peaking at 15 Hz. The investigators noted that the very low sensitivity observed at 5 Hz removes concern about a possible heating effect, although no sham-exposed control was used. A dose-dependent response was observed for flux densities up to 250 µT, with a significant effect (p < 0.05) as compared with no field treatment for flux densities as low as 2.5 µT at 15 Hz. The authors considered that the lack of an increased response at 2.5 mT over that observed at 250 µT confirms the non-thermal origin of the effect. The distributions of the induced electric fields, determined by two-dimensional impedance network techniques, indicate that the peak-induced 15 Hz electric field in the bone tissue at 250 µT would be approximately 0.3 mV/m.

Effects of exogenous induced fields on nerve and skin healing

In principle, the same electrokinetic processes that give rise to electrical currents in bone tissue will also produce electrical currents in soft connective tissues. In fact, the normal (i.e. non-destructive) mechanical deformations of soft tissues during typical physiological activities far exceed that seen in bone tissue; however, with the exception of cartilage, the charge density in these tissues is far lower than that of bone. In addition, soft connective tissues lack the highly organized collagen structure that can permit a large piezoelectric coefficient. Thus, the predominant electrical currents in skin, tendon, and ligament probably arise through the extracellular currents generated during muscle contraction. The approval and extensive use of pulsed magnetic fields in the clinical setting for bone healing naturally led to investigations on the efficacy of pulsed fields to affect soft connective tissue healing, particularly over the last 10 years.

Following the report of several studies demonstrating effects of ELF fields on fibroblasts in vitro (Liboff et al., 1984; McLeod et al., 1987c; Ottani et al., 1988) undertook a study to determine whether pulsed magnetic fields could affect skin wound healing in vivo. In this study, a 3 x 3-cm square of skin (with the cutaneous muscle) was surgically removed from the dorsal thoracolumbar region of four-month-old male Wistar rats (weighing 350-370 g). The animals were then exposed or sham-exposed, and eight animals from each group were sacrificed at days 6, 12, 21, and 42 for histological and ultrastructural analysis of the wounds. Field exposure was implemented in a 40 cm diameter solenoid driven by a 50 Hz triangular wave to a peak flux of 8 mT, providing a dB/dt of 0.8 T/s, sufficient to induce electric field intensities on the order of 10-20 mV/m. The animals were exposed for 30 min every 12 h. Rats undergoing sham exposure spent the same time in the exposure apparatus with no excitation. The rate of healing, as assayed by area of wound vs. time, was found to be significantly higher (p < 0.001 by linear regression) in exposed animals, reflecting an approximate halving of the healing time. The investigators noted the appearance of a network of blood vessels in the wound as early as six days post-operatively. [The investigators also referred to unreported data collected at a variety of other exposure frequencies (e.g. 60 and 400 Hz) with which similar results were obtained and suggested that the waveform is probably not critical to ensure a physiological effect.]

Lin et al. (Lin et al., 1993) reported similar studies using a ligament defect model in rabbits. In their investigation, 80 male rabbits underwent square resections (4 x 4 mm) of both the left and the right patellar ligaments. They were then assigned to one of four groups representing exposure to a 0.2, 1, or 5 mT peak or control. A pulse magnetic field was generated by a device of undescribed design, but which induced a 10 Hz electric field pulse of 25 µs in width, consistent with a maximum dB/dt of 200 T/s, sufficient to induce electric field intensities in the range of 10-100 V/m. The animals were exposed for 6 h/d, and five animals from each group were sacrificed each week for four weeks. Blood flow, collagen synthesis, collagen typing, and histological assays were performed. Significant (p < 0.05) increases in the collagen content of the wounds were observed in all treated groups as compared with controls, and significant (p < 0.05) increases in blood flow and cross-sectional area were observed within two weeks in the group exposed to 5 mT, an effect sustained throughout the four-week experimental protocol. No differences in collagen type distribution were observed between the exposed and control groups. The authors noted that these results are consistent with those of their earlier studies.

Patino et al. (Patino et al., 1996) reported the results of a study in which flux densities of 20 mT were used. A circular lesion was made on the backs of 22 male Wistar rats weighing 250-360 g, and the animals were then used as controls, treated with nitrofurazone, or treated with PEMF (35 min twice a day to a 20 mT, 50 Hz flux from a 23-cm diameter solenoid 50 cm in length). Planimetry of the wounds was obtained every 7 d over one month. Both the area and the perimeter of the wounds of the field-treated animals were significantly reduced as compared with sham-exposed controls (p < 0.01). Field treatment resulted in significantly smaller wounds on day 21 as compared with the nitrofurazone-treated group (p < 0.01).

Effects of induced fields on nerve tissue

As for bone and soft connective tissue healing, early investigations on the potential use of electric fields to promote nerve healing began with studies of DC fields in the 1970s. Investigations on the use of pulsed magnetic fields to promote nerve healing date from the 1980s, although these studies have been pursued by a relatively small group of investigators. O'Brien et al. (O'Brien et al., 1984) made one of the earliest efforts to determine the efficacy of magnetic field therapy to enhance regeneration in the peripheral nervous system. The peroneal nerves of 12 cats were exposed, and compound action potentials were recorded to provide a baseline measure. The nerve was then transected and repaired by microsurgery. Five days post-operatively, the animals were assigned to one of four groups, representing untreated controls, sham-treated controls, magnetic field-treated with a pulse-burst waveform (15 Hz burst repetition rate), or treated with a 72 Hz repetitive pulse waveform. All treated animals were exposed for 10 h/d, 6 d per week, for 12 weeks. The peak flux density is not reported, but the exposure hardware was provided by Electrobiology, Inc. [The peak flux was probably in the range of 1-2 mT.] Electrophysiological and histological assays were performed at 12 weeks. Both the area of the compound action potential and the retrograde transmission of horseradish peroxidase to the anterior horn of the spinal cord were significantly greater (p < 0.01 by ANOVA) in animals receiving the pulse-burst field than in the other three groups at 12 weeks. [This is a remarkable outcome, given that only three animals were available in each group.] The authors noted that no functional assays were incorporated to ensure successful regeneration.

A more thoroughly described study on nerve regeneration was later reported by Sisken et al. (Sisken et al., 1989), in which a crush lesion was used rather than nerve transection. Male Sprague-Dawley rats weighing 300 g underwent a surgical procedure to produce a crush lesion in the right sciatic nerve. At days 3, 4, and 6, a pinch test (pinching the exposed nerve to produce muscle twitches) was performed under anesthesia to assay the extent of regeneration. Field exposure was accomplished with a 30 cm diameter Helmholtz coil pair, the rats being either restrained or free-roaming in plastic cages. A 2 Hz square wave drive was applied to the coils, creating a peak flux of 0.3 mT with a rise time of approximately 1 ms, thereby producing a peak induced electric field proportional to 0.3 T/s or on the order of 10 mV/m. The animals were exposed for 4 h/d; sham-exposed animals were placed in the exposure chamber without current excitation. No significant effect of restraint as compared with free roaming was observed in the control or the field-exposed group; however, field exposure was found to result in a significant (20%; p < 0.001) increase in regeneration rate. In collateral studies, no effect of magnetic field orientation (horizontal vs. vertical) or exposure duration (1 vs. 4 vs. 10 h/d) was observed, all variations resulting in significantly enhanced nerve regeneration rates. Similarly, exposure of the animals to fields for 4 d before creating the crush lesion also resulted in an increased regeneration rate post-operatively.

The frequency dependence of enhanced nerve regeneration capability was subsequently reported by Rusovan et al. (Rusovan et al., 1992). A crush lesion of the sciatic nerve was induced in female Sprague-Dawley rats weighing 200 g. The animals were randomly assigned to one of eight groups corresponding to controls or exposure to one of seven 0.1 mT sinusoidal fields at a frequency of 50, 100, 250, 500, 1000, 1500, or 2000 Hz. The induced fields were estimated by the authors by assuming an average radius of 5 cm per rat, suggesting an induced electric field intensity of 15 mV/m at 1 kHz. The regeneration distance was evaluated by the pinch test at days 3, 4 and 6. While exposure to 50 or 100 Hz had no effect on regeneration, exposure to 250, 500, or 1000 Hz resulted in significantly enhanced regeneration rates (p < 0.001). The maximal effect was a 24% increase at 1 kHz, while the responses at 1.5 and 2 kHz were similar to that at 100 Hz. The authors noted that the increase in response as a function of frequency is consistent with an effect of the induced electric field, as the magnetic flux was held constant; however, they also note that similar decreases in sensitivity at higher field frequencies have been observed in numerous in-vitro systems.

This group of investigators continued their studies on the effects of magnetic fields on nerve regeneration, pursuing their observations of pretreatment effects (Kanje et al., 1993). Female Sprague-Dawley rats weighing 200 g underwent a variety of pretreatment exposures, ranging from 15 min/d for 2 d to 4 h/d for 4 d. The sciatic nerves of the animals were then submitted to a crush injury, and the regeneration distance was assayed on days 1, 2, 3, 4, 7, and 14. Field exposure was accomplished with the apparatus described by Sisken et al. (Sisken et al., 1989), consisting of a 30 cm diameter Helmholtz coil pair creating a vertical flux. Two peak flux densities, 60 and 300 µT, were used with a 2 Hz pulsed signal and a rise time of 0.8 ms, corresponding to peak induced electric fields on the order of 10 mV/m. The animals were allowed to roam freely throughout exposure. While the 60 µT pretreatment had no effect on subsequent regeneration rates, all 300 µT pretreatments, including treatments as short as 15 min/d for 2 d were found to significantly enhance the regeneration rates. Animals pretreated for 4 h/d for 2 d had an increased regeneration rate for at least 10 d.

4.4.6.3 Summary

If retrospective reports of the efficacy of PEMF in promoting the healing of fractures are ignored, there is little clinical evidence of a real effect of exposure to fields in accelerating or improving the probability of successful fracture healing. Two prospective, blinded trials of the treatment of non- and delayed unions were undertaken, but both have serious design flaws and furthermore show no or only a weak effect. The trial on limb lengthening appears to be both well designed and definitive in showing a lack of effect on bone fracture callus formation or on the process of secondary bone healing. In clinical use of PEMF devices, however, it has been the rule to center the induction coil over the fracture site. This technique may well have ensured a low success rate by minimizing the induced electric field intensity at the distraction or fracture gap.

Conversely, there appears to be substantial, accumulating evidence that complex clinical exposures to PEMF have a significant effect on primary bone healing processes. The studies of both osteotomy and spinal fusion show a robust effect. While quantification and analysis were weak in these two studies, they are prospective, randomized, double-blind trials, a rarity in the field of orthopedics. Perhaps the most convincing trial is that of the response of bone tissue during limb lengthening. While no effect on secondary bone healing was observed, there was significant inhibition of bone resorption and evidence of new bone formation. Unlike secondary bone healing, primary bone healing, new bone formation, and inhibition of bone resorption depend mainly on the state of vascularization of the tissue. The results of the two studies therefore indicate that the predominant effect of exposure to PEMF is on the vascular supply or on cells capable of stimulating revascularization, rather than a direct effect on the bone cell population. Of course, the issue of coil placement cannot be ignored in the study of limb lengthening, in which the proximal and distal segments of the bone would, in fact, experience the maximum induced electric field intensities. The responses observed in these two regions may therefore simply reflect much higher field intensities.

In capacitive coupling, with application of a 3 Hz field, the distribution of the induced electrical currents is readily determined by the placement of the external plates, and the maximal field intensity can be expected to occur in the central region between the coupling plates. In electromagnetic coupling, however, the issue is more complicated, as the induced electric field will actually reach a maximum at the periphery of the magnetically exposed area. In clinical trials in which local exposure is desired, the investigators have typically centered the exposure coils over the site of interest, essentially ensuring that the induced electric fields in the region will not be maximal.

Studies in experimental animals

Although early trials suggested that non-union tissue responds to EMF, and despite extensive clinical use of magnetic field therapy for treatment of this condition, studies in animals in vivo indicate only limited efficacy. Magnetic field therapy appears incapable of enhancing the healing of osteotomies, in-growth of bone into a defect, bone elongation, or graft healing and, in at least one case (in-growth), may inhibit the normal process. The results obtained in a model of endochondral ossification after exposure of whole animals suggest, however, that magnetic field therapy can be effective. This raises two possibilities. One is that neither magnetic fields nor induced electric fields have any direct effect on the endochondral ossification process but have an effect at the systemic level manifested by accelerated growth processes. Alternatively, the ossification model differs critically from bone healing in situ in that the induced electric fields are distributed in both the healing tissue and the surrounding tissue. Exposure to local fluxes can result in minimal induced electric field intensities at the healing site. As the distribution of induced fields is quite complex, however, the effect of this variable may be beyond interpretation.

Conversely, magnetic fields appear to have a strong, reproducible effect on the process of appositional (surface) bone growth and on inhibition of bone resorption. Moreover, the fact that exposure to pure electric fields and time-varying magnetic fields can inhibit bone loss suggests that this effect is mediated by the induced electric field. In the studies reviewed here, the frequency range within which maximal bone tissue responses are observed is consistent, induced fields in the range of 15-75 Hz appearing to be optimal in all of the studies. The effect of field intensity is much less clear; however, in a study that specifically addressed this factor (McLeod & Rubin, 1998), a threshold intensity of < 0.1 mV/m was established for exposure at 15 Hz.

Consistent effects on the healing of soft connective tissue have been reported by numerous groups. Perhaps the most notable feature is that remarkably high flux densities (5-20 mT) are required to ensure an enhanced healing rate. Because no active sham-exposure device was used in any of these studies, there is clearly potential concern about warming of the animals. In addition, the fact that whole-animal exposure was used in these studies suggests the possibility of neuroendocrine stimulation.

Consistent responses were also seen in the studies of nerve regeneration, although they usually represented only about a 20% enhancement in growth rate. These studies confirm the results of the studies of soft and hard connective tissue healing, particularly with regard to the sensitivity of the nerve healing process to frequency. In addition, attention was drawn to the angiogenic properties of stimulation of nerve regeneration by magnetic fields, as has been observed repeatedly in studies of both skin and bone healing. This observation may be critical with regard to the role of magnetic fields in the promotion of tumor growth, which is known to depend on angiogenesis.

As in the clinical studies, the nature of the exposure of the target tissue is not precisely defined. Whole-body exposure has commonly been used in studies of small animals, which can result, for sites of interest near the periphery of the body, in maximal induced electric field intensity. This type of exposure may also affect many other sites in the body, producing systemic responses that could influence tissue healing. For example, calcitonin, a growth hormone which has strong effects on bone growth in young animals, is produced by the C cells of the thyroid. The location of the thyroid at the periphery of the body ensures maximal exposure to electric fields under the conditions of either vertical or horizontal exposure to magnetic fields. Stimulation of the thyroid could therefore result in enhanced bone growth or healing, independent of any local effect on the bone or surrounding tissue. This Working Group did not address the possible adverse side-effects of exposure to electromagnetic fields on bone and tissue repair and adaptation.

There is strong evidence that exposure to electric and magnetic fields affects bone repair and adaptation.

[This conclusion was supported by 14 Working Group members; there were 5 votes for 'moderate' evidence, 8 abstentions, and 2 absent.]

The Working Group could not reach a conclusion about whether exposure to electric and magnetic fields affect nervous and non-bone connective tissue repair and adaptation in vertebrates.

[This conclusion was supported by 12 Working Group members; there were 10 votes for 'moderate' evidence, 6 votes for 'weak' evidence, and 1 absent.]