"Experimental Groups

The rats in Groups 1–5 were exposed to EMF as detailed in Table 1. Group 1 was a complete control without any treatment. Group 2 received ENU alone and was kept in cages throughout the experiment. Group 3 was given ENU and set in exposure boxes in the same manner as in Groups 4 and 5 but with no actual exposure of EMF (sham exposure group). Groups 4 and 5, given ENU, then were exposed to EMF at low (specific absorption rate (SAR): 0.67 W/kg) and high (SAR: 2.0 W/kg) levels, respectively.

Exposure Apparatus for EMF

The exposure apparatus was specially designed for this study and is diagrammatically illustrated in Figure 1a. The exposure box was 90x90x60 cm, the inside walls except for the ceiling being covered with electromagnetic absorber (Fig. 1b). Ten rats, fixed individually in plastic tubes, were set on a disk-shaped plate like a carrousel with the nose direction to the center. The disk-like plate of 65 cm in diameter was supported by the center hollow cylinder pole and the height of the plate was adjustable. Electric fan-forced ventilation air was fed into the box through the pole. A 1/4 wavelength monopole antenna was set from the ceiling of the exposure box in order to fit in the center of the disk-shaped plate. The distance between the antenna and each rat nose was set at 30 mm and the distance between the ceiling and rat back was 5 mm (Fig. 1a). It was revealed in a preliminary experiment that the size of the rat holders must be frequently changed according rapid growth of animals for the first 3 months for both sexes, and rat plastic holders with four different sizes (inner diameter: 4.0, 4.7, 5.5, and 6.2 cm, respectively) were actually employed.

EMF Dosimetry and Exposure Protocol

A 1.439 GHz time division multiple access (TDMA) signal for the Personal Digital Cellular (PDC), Japanese standard cellular system was used. Long term intermittent exposure to the head of the rats (90 min a day, 5 days a week) was started at 5 weeks of age and continued for 104 weeks (started on May 23, 000 and terminated on May 21, 2002). Usually the exposure to EMF was daily on Monday through Friday. Setting of 10 exposure boxes in a room made it possible for a total of 100 rats to be in the exposure boxes at one time, resulting in a total of 300 (150 males and 150 females) in Groups 3–5 in a day by rotating schedule of three exposure times (twice in the morning and once in the afternoon). All exposures, including sham exposure, were controlled with the assistance of a computer system; and all exposure data, such as input power level of each antenna and temperature inside each exposure box, were checked every day to confirm that exposure conditions in each box were achieved as scheduled. If the number of rats in an exposure box became less than 10 due to loss through mortality, dummy rat(s) were used to maintain symmetrical arrangement of animals and scheduled specific absorption rate (SAR) of EMF exposure. SAR distributions in rats were evaluated by both numerical dosimetry with Finite Difference Time Domain (FDTD) method and experimental dosimetry with thermographic camera [Watanabe et al., 2000]. During this experiment, rats grew from under 100 g to over 400 g. Since the size and the shape of animals influences the absorption of EMF energy, we evaluated the relationship between the weight of rats and SAR using different sized rat models.We made three original rat models based on X-ray computed tomography data of 126, 263, and 359 g rats in plastic holders and then created other sized models by numerical scaling. Brain averageSARwas set at levels, 0.67 and 2.0 W/kg, with a time peak SAR of 2.0 and 6.0 W/kg, respectively, and the whole-body average SAR was estimated to be less than 0.4 W/kg.

Animal Handling and Termination of the Experiment

Rats were observed clinically twice a day (morning and evening) throughout the experiment for deaths and signs of illness. Body weights and food consumption were measured weekly for the first 14 weeks and thereafter biweekly. Food consumption rates per animal were calculated from actual food consumption for 4 days per cage. Animals found dead or killed due to moribund condition were autopsied. Five rats in each group were used for collecting blood from the eye pit for measurement of two hormones, corticosterone and melatonin, 2 days before the termination of the experiment. Blood collection was performed 3 h after lights off, i.e., at 10 p.m., in a consideration of the diurnal rhythm of melatonin secretion. Another group of five rats in each group was lightly anesthetized with ethyl ether for collection of aorta blood for measurement of adrenocortical stimulating hormone (ACTH) after overnight starvation. Blood collection for ACTH measurement was started at 9 a.m. with a random selection of animals among the groups, and the time between anesthesia and blood collection was less than 3 min. The interval for each animal was about 2 min. All animals surviving after the 104 week exposure to EMF were lightly anesthetized with ethyl ether and then killed by collecting aortal blood for hematology analysis. Then a complete autopsy was done. Blood smear samples were also prepared to investigate leukemia development. Organ weights were measured for the brain, heart, liver, kidney, spleen, pituitary gland, adrenal glands, testes, and ovaries. All organs and macroscopically observable lesions were fixed in 10% buffered formalin and embedded in paraffin, and sections (4 mmthickness) were stained with H&E for histological examination. Rats that died or became moribund during the experiment were also autopsied, and all organs were collected for histological examination. The brains (cerebrum and cerebellum) were cut into around 10 slices at intervals of 2 mm with a Rodent brain matrix (Harvard Apparatus, Holliston, MA) and embedded in paraffin, avoiding setting the same cut surfaces for sectioning. Paraffin sections of the spinal cords were also made at intervals of 1 cm.

Histological diagnosis of lesions, including tumors of the CNS,was performed by Dr. Kawabe,Dr. Ichihara, Dr. Tamano, and Dr. Shirai, who are all toxicological pathologists certified by the Japanese Association of Toxicological Pathology, without knowing to which group each slide belonged to. CNS tumor diagnosis underwent peer reviewbyDr. Michihito Takahashi who agreed with the results and conclusion. Other biological measurements such as weight, food consumption, hormone analysis were carried out according to the protocol of GLP Standards of the Japanese Ministry of Health and Welfare Audience No. 21 (March 26, 1997). Pathological surveillance in the course of the study was monitored by checking the presence of microorganisms in the sentinel rats housed in the same animal room with experimental rats. These animals (one male and one female) were killed at 6 month intervals and samples were sent to the Monitoring Center of Experimental Animal Central Institute (Kanagawa, Japan) for testing for infection with microorganisms.

Data Analysis Methods

Kaplan–Meyer survival analysis was used to compare between Groups 1 and 2, Groups 3–5; females and males were independently analyzed. Differences in body and organ weights and hormone levels between Groups 1 and 2 were compared by t-test when there was homogeneity of variance, analyzed by the F-test, and by the Welch method when there was not. These differences among Groups 3–5 were analyzed by Dunnett’s test when there was homogeneity of variance, as analyzed by Barlett’s, and by the Steel’s test when there was not. The tumor incidences were compared by Fisher’s exact test. The level of significance used in all test procedures was P<0.05. Animal Care and Protocols The animal facilities at Daiyu-kai Institute of Medical Science are fully accredited as compliant with the GLP Standards by the Ministry of Health and Welfare of Japan, the Organization for Pharmaceutical Safety and Research, and the Ministry of Agriculture, Forestry, and Fisheries of Japan."

Fisher 344 rats, female

HAWC