Bioelectromagnetics 25:114^117 (2004)

Teratological Studies of Prenatal Exposureof Mice to a 20 kHz Sawtooth Magnetic Field

Sung-Ho Kim,1 Ji-Eun Song,2 Se-ra Kim,1 Heon Oh,1 Youn-Myoung Gimm,3

Done-Sik Yoo,4 Jeong-Ki Pack,5 and Yun-Sil Lee2*1College of VeterinaryMedicine, ChonnamNational University, Kwangju, SouthKorea2Laboratory of Radiation Effect, Korea Cancer Center Hospital, Seoul, SouthKorea

3EMFSafety, DankookUniversity, Seoul, SouthKorea4EMEResearchTeam, Radio & BroadcastingTechnologyLab, ETRI, Daejon, SouthKorea

5Department of Radio Sciences & Engineering, College of Engineering, Daejon, SouthKorea

In order to evaluate the importance of gestational age in possible effects due to exposure to a 20 kHzsawtooth magnetic field, pregnant ICR mice at gestational 2.5–15.5 days post-coitus, which is themost sensitive stage for the induction of major congenital malformations, were exposed in a carrouselirradiator. Themicewere exposed to a 20 kHz intermediate frequency (IF) sawtoothmagnetic field hada 6.5 mT peak intensity for 8 h/day. The animals were sacrificed on the 18th day of gestation; and thefetuses were examined for mortality, growth retardation, changes in head size, and other morphol-ogical abnormalities. From the above conditions, it is concluded that the exposure to a 20 kHz sawtoothmagnetic field with 6.5 mT peak intensity does not inflict any adverse effect on fetuses of pregnantmice. Bioelectromagnetics 25:114–117, 2004. � 2004 Wiley-Liss, Inc.

Key words: 20 kHz sawtooth; intermediate frequency; gestation; mice; congenitalmalformation

INTRODUCTION

The possibility of previously unrecognized repro-ductive adverse effects of exposure to intermediatefrequency (IF) magnetic is one of the important publicconcerns. Epidemiological studies as well as labora-tory experiments have shown inconsistent results[Wertheimer and Leeper, 1986a,b; Goldhaber et al.,1988; McDonald et al., 1988; Juutilainen et al., 1993].Significantly increased placental resorptions or inci-dence of external malformations have been observed inmice exposed to pulsedmagnetic fields [Tribukait et al.,1987; Frolen et al., 1993]; however, Zecca et al. [1985]found no adverse effects on rat fetuses. Zusman et al.[1990] exposed pre implantation mouse embryos orearly somite rat embryos to 50 Hz pulsed IF magneticfield in vitro and found growth retardation in mice andincreased cranial abnormalities in rats. However, apulsed IF magnetic field exposure study might be irre-levant to power frequency concernes because of dif-ferent induced currents due to different wave forms. Inthis study, we exposed rats to a 20 kHz IF sawtoothmagnetic fields, a frequency used in TV sets and per-sonal computers, and examined the fields’ effect as afunction of the gestational age of pregnant mice.

MATERIALS AND METHODS

Magnetic field generation and monitoring equip-ment was designed and constructed in collaborationwith EMF & Environment Research Team, Radio &Broadcasting Technology Lab, ETRI, Daejeon, Korea.A 2 axis magnetic field exposure equipment wasdesigned to produce the magnetic field strength of6.25 mT peak intensity, which is the regulated exposurelimit of themagnetic field for the public at 20 kHz [Yoo,2000]. The exposure setupwasmade ofwood in order tominimize the field perturbations, and the size was1.5� 1.5� 1.5 m. As shown in Figure 1, the exposuresetup consists of four horizontal and four vertical woodframes on which coils were wound. Inside the frames,

�2004Wiley-Liss, Inc.

——————*Correspondence to: Yun-Sil Lee, Laboratory of Radiation Effect,Korea Cancer Center Hospital, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, South Korea. E-mail: yslee@kcch.re.kr

Received for review 7 November 2002; Final revision received4 June 2003

DOI 10.1002/bem.10164Published online in Wiley InterScience (www.interscience.wiley.com).

two wood panels (1� 1 m) were placed and used asan exposure shelf where cages were positioned. Thefrequency was set to 20 kHz, which is the frequency ofTV sets and personal computer monitors. A sawtoothcurrent waveform was applied. A high voltage squarewave generator was designed and constructed in orderto supply and adjust input power (Vp) and the currentwaveform. For the magnetic field generation, coils of1.2 mm diameter enamel coated copper wirewere used.The maximum approved current of the coil was 3 A.

Magnetic field strength and uniformity at two (topand bottom) panels in the exposure equipment (seeFig. 1) were measured using a 3 axis magnetic fieldprobe (HI-3637, Holaday Industries, Cedar Park, TX).Each panel surface was divided into 25 regions (fivehorizontal and five vertical divisions) in order toestimate the field uniformity in the exposure chamber.The Vp was adjusted so that the magnetic field strengthreached 6.25 mT peak at the center of the top panel.Measurement results of the magnetic field strength atthe various positions on top and bottom panels in theexposure chamber are represented in Tables 1 and 2.The field strength was 6.17� 0.16 mT at the top paneland 6.14� 0.20 mT at the bottom panel. The fielduniformity was 4� 7% with respect to the center of the

top panel. The Vp was 250 V and the output magneticfield waveform was the sawtooth wave of 20 kHz. Thedistance the animals were from the coils was 30 cmfrom the side panel and 35 cm from the top panel. Sixcages were placed on each panel in the exposurechamber, and therefore, a total of 12 cages were ex-posed in the study. The animals were exposed to themagnetic field for 8 h a day.

Studies were conducted under guidelines for theuse and care of laboratory animals, whichwas approvedby the Institutional Animal Care and Use Committeeof the Korea Institute of Radiological and MedicalSciences. ICRmice (SLC, Shizuoka, Japan)weremain-tained on standard mouse food and water, ad libitum,under controlled temperature and light conditions.Virgin females and males, 10–12 weeks of age, wererandomlymated overnight. Femaleswith a vaginal plugwere separated in the morning and marked as 0 dayspregnant. Pregnant ICR mice at gestational days 2.5–15.5 post-coitus, which is the most sensitive stage forthe induction of major congenital malformations, wereexposed in the carrousel irradiator to 20 kHz inter-mediate frequency magnetic field at 6.5 mT for 8 h/day.All the mice were killed on the day 18 by cervicaldislocation.

Uterine horns were opened, and the total numberof implantations, including resorption, embryonicdeath, and fetal death, were examined. Resorptionsincluded (1) implantation failure inwhich the implanta-tion site was marked by a rudimentary fleshy mass, butnot a full placentum, and (2) cases where only a placen-tum was present, with no attached embryonic rudi-ments. Embryonic death was a partly formed embryoattached to the placental disc. Fetal deaths were fullyformed dead fetuses which were distinguished by adarker color, macerated fetuses which were pale incolor, ormacerated fetuseswhichwere pale in color andsoft to touch. Preimplantation loss with no identifiablemark on the uterine wall, if any, was not included inthis study.

Live fetuses were removed from the uterus,cleaned, and observed for any externally detectabledevelopmental anomalies. Fetuses were weighed indi-vidually, and the mean fetal weight of the individual

Fig. 1. Schematic diagram of the 2 axis magnetic field exposureequipment.

TABLE 1. Measurement Results of Magnetic Field Strength (mT) at Vp¼ 250 V for theTop Panel

X far-left X mid-left X center X mid-right X far-right

Y far-front 6.00 6.25 6.50 6.50 6.25Y mid-front 6.00 6.00 6.25 6.00 6.00Y center 6.00 6.00 6.25 6.25 6.25Y mid-back 5.90 6.00 6.25 6.25 6.25Y far-back 6.00 6.25 6.25 6.25 6.25

Teratological Studies of 20 kHz 115

litter group was calculated. Fetuses weighing less thantwo standard deviations of themean control group bodyweight were considered as growth retarded. Bodylengthwasmeasured from the tip of the snout to the baseof the tail. The longitudinal distance from the tip of thesnout to the base of the skull was recorded as headlength, and the distance between the two ears wasrecorded as head width. Measurements were madewitha vernier caliper. All fetuses were checked for externalmalformations under a dissection microscope.

Fetuses were fixed in Couin’s solution and thenstored in 70% ethanol. The presence of visceralmalformations was examined by the Wilson’s cross-sectional technique [Wilson, 1965]. Alizarin red-S andalcian blue staining were used to examine skeletalmalformations [Kimmel and Taummell, 1982].

Statistical comparisons were made using Stu-dent’s t-test, and a null hypothesis was rejectedwhenever a P value of 0.05 or less was found.

RESULTS

As illustrated in Table 3, therewere no statisticallysignificant differences in the numbers of implantation,

embryonic death, fetal death, resorption, prenatalmortality, and live fetuses between the control and theIF exposed group. The number of growth retardedfetuses (GRF) was slightly increased in the IF exposedmice; however, there was no significant differencebetween the groups. Also, body weight, body length,head length, and head width of fetuses were not differ-ent between the groups. There was no group differencein the incidence of external abnormalities (Table 4).One hematoma was observed in the IF exposed fetusand one digit was observed in the control fetus.However, analysis of the total frequencies of externalabnormalities by the group revealed no statisticalsignificance. In the case of internal abnormalities, onecleft palate and one dilatation of renal pelvis were ob-served in the IF exposed group, but it was not significant(Table 5). The incidence of skeletal abnormalitiesshowed no statistically significant group differences(Table 6); there was one hypoplasia of sternebrae in theIF exposed fetus with no statistical significance. Thenumber of total malformed offspring was four in the IFexposed group and one in the sham control group.

DISCUSSION

In the present study, groups of mated female micewere simultaneously exposed to sham and 20 kHzsawtooth magnetic fields with 6.25 mT peak intensityduring the 2.5–15.5 days of gestation, the period ofmouse fetal organogenesis. None of the endpoints

TABLE 2. Measurement Results of Magnetic Field Strength (mT) at Vp¼ 250 V for theBottom Panel

X far-left X mid-left X center X mid-right X far-right

Y far-front 6.25 6.50 6.25 6.00 5.80Y mid-front 6.25 6.25 6.25 6.00 5.80Y center 6.25 6.25 6.00 6.00 5.80Y mid-back 6.25 6.25 6.25 6.00 5.80Y far-back 6.25 6.25 6.50 6.25 6.00

TABLE 3. Observations on the 18th Day Mouse FetusesExposed to EMF From Day 2.5–15.5 of Gestation

Observations Sham EMF

No. of mother 18 16No. of implants 240 219No. of embryonic death 2 0No. of fetal death 0 3No. of resorption 0 0Prenatal mortality no. (%) 2 (0.83) 3 (1.37)Live fetuses 238 216GRF no. (%)a 15 (6.30) 23 (10.65)Body weight (g) 1.45� 0.12b 1.34� 0.08Body length (cm) 2.94� 0.10 2.85� 0.08Head length (cm) 1.03� 0.03 0.99� 0.02Head width (cm) 0.65� 0.02 0.67� 0.02

aGRF, growth retarded fetuses, calculated as the number of GRF/total number of live fetuses. Fetusesweighing less than two standarddeviations of mean body weight of the control group wereconsidered as growth retarded.bMean� SD.

TABLE 4. External Malformations in 18th DayMouse FetusesExposed to EMF From Day 2.5–15.5 of Gestation

Sham EMF

Fetus examined 238 216Omphalocele 0 0Kinky tail 0 0Brachyury 0 0Club foot 0 0Digits (%) 1 (0.42) 0Dwarf 0 0Anal atresia 0 0Hematoma (%) 0 1 (0.46)

116 Kim et al.

examined revealed any statistically significant differ-ence between the exposed and the sham control group.Therefore, the results described here do not provideany support to the hypothesis that the IF magnetic fieldexposure under the conditions presently employedaffects mouse fetuses.

There are a few reports to show the effects of the IFmagnetic field exposure on fetal body weights.Kowalczuk et al. [1994] reported a significant increasein the body weight of CD-1 mice fetuses exposed to50 Hz, 20 mT magnetic field. Contrary to the above,Kato et al. [1998] found a small but statistically signi-ficant reduction in the body weights of male and femaleSD rat fetuses among the 60Hz, 200mTexposure group,but not in the 2 mT and 1 mT exposure groups. Theyconcluded that the reduction in the mean body weightsin the 200 mT group was a sporadic finding, notconsidered to be biologically significant, becauseexposure to the IF magnetic field had no field strengthrelated effect on fetal body weights.

Although all of the earlier data by others wereobtained by employing extremely high dose level ofmagnetic field, this study used the usual environmentalexposure of 6.25 mT magnetic field. Thus our resultsexcluded minimum toxic effects of the magnetic fieldunder the environmental exposure situation. From theabove conditions, it is concluded that the exposure of20 kHz sawtooth magnetic field with 6.25 mT peakintensity does not inflict any adverse effect on pregnantmice fetuses.

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TABLE 5. Internal Malformations in 18th Day Mouse FetusesExposed to EMF From Day 2.5–15.5 of Gestation

Sham EMF

Fetuses examined 119 108Dilatation of cerebral ventricle 0 0Stenosis of nasal cavity 0 0Cleft palate (%) 0 1 (0.93)Levorotation of heart 0 0Abnormal lobation of lung 0 0Dilatation of renal pelvis (%) 0 1 (0.93)

TABLE 6. Skeletal Malformations in 18th Day Mouse FetusesExposed to EMF From Day 2.5–15.5 of Gestation

Sham EMF

Fetuses examined 119 108Fusion of cervical vertebrae 0 0Deformity of occipital bone 0 0Splitting of cervical vertebrae 0 0Separating of cervical vertebrae 0 0Abnormal ossification of coccygeal vertebrae 0 0Fusion of lumbar vertebrae 0 0Fusion of thoracic vertebrae 0 0Absence of ribs 0 0Fusion of ribs 0 0Wavy ribs 0 0Hypoplasia of ribs 0 0Dysplasia of sternebrae 0 0Missing of sternebrae 0 0Hypoplasia of sternebrae (%) 0 1 (0.93)Curvature of tibia 0 0Absence of metatarsal bone 0 0Absence of metacarpal bone 0 0

Teratological Studies of 20 kHz 117