United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1-85/005 Mar. 1985
Project Summary
Experiments in Microwave
Exposure in the Rat:
Body Temperature, Serum
Chemistry, and the Use of
Chemical Restraint
Ezra Berman and Hershell Carter
A series of experiments was conduct-
ed in rats to observe their responses to
2450-MHz (CW) microwaves. Colonic
temperatures were measured after ex-
posure to 0, 20, or 30 mW/cm2 for up
to 4 h. Exposures of 30 mW/cm2 caused
a regular and significant increase in
colonic temperature. A plateau of
38.9°C was reached in 90 min and
maintained for the rest of the 4-h
exposure. Rats exposed to 20 mW/cm2
for 4 h exhibited patterns of colonic
. temperature similar to sham-exposed
rats. Acclimation for 90 min had no
effect on these patterns. Measurements
(or calculations) of 26 serum chemistry
values produced similar results in rats
exposed to 30 mW/cm2 for 120 min
and rats exposed to 0 or 20 mW/cm2
for 120 min. Only serum corticosterone
was significantly increased in the dose-
related response of naive rats to this
acute exposure.
Various injectable anesthetics were
evaluated as restraints in microwave
exposure experiments. Colonic temper-
ature was a measure of the response to
exposure. I mmobilization was accompa-
nied by decreased colonic temperature.
After exposure to 30 mW/cm2 for 90
min, mean colonic temperatures in
chemically restrained rats ranged from
39-40.4°C, and increased up to 5.5°C
from anesthetically depressed pre-ex-
posure levels. Limitations on the use of
chemical restraint for this kind of
experiment and on the use of multi-
value serum chemistry screening tests
were determined during this research.
This Project Summary was developed
by EPA's Health Effects Research Labo-
ratory, Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Extrapolating from the results of micro-
wave-exposure studies to human health
effects depends mainly upon the develop-
ment of a base of knowledge from labo-
ratory animal experimentation. Because
such experiments are conducted under a
great variety of conditions, attempts at
replication in other laboratories often fail.
Variation in microwave-exposure condi-
tions may account for some of these
failures, but experimental techniques
may also vary among laboratories in ways
less obvious to the researcher. Apparent-
ly, even minor variations in the tech-
niques of handling and restraint can
cause untoward reactions and results in
laboratory animals.
The physiologic consequences of ex-
posure to microwave irradiation have
been measured by many methods. Per-
haps the most common standard of
assessment of an effective level of ex-
posure is the measurement of body or
colonic temperature. Temperature is usu-
-------
ally taken just before and just after an
exposure. Measurements taken during
an exposure have had limited interpret-
ability because of errors introduced by
metallic temperature sensors. However,
reliance on post-exposure body temper-
ature values is not justified without
measurements of temperature variations
encountered during the exposure itself.
The rat was chosen as the experimental
subject for these studies because it is one
of the most commonly exployed whole
animal models in the microwave bioef-
fects field as well as in.this laboratory. For
instance, in a review of the results from
17 reports of microwave teratogenesis in
animals, about half of the studies were
conducted in rats. Another important
rationale for the use of rats in these
studies is that the rat may randomly
orient itself in a 2450-MHz frequency
microwave field without the whole-body
specific absorption rate (SAR) being af-
fected, whereas, in mice, the SAR varies
according to orientation
The full report presents data from rats
exposed to microwave radiation under
"typical" experimental conditions. These
results are intended for use as reference
or baseline data.
Discussion
Body Temperature
Experiments 1 and 2 measured body
temperatures in the rat after exposures to
varying power densities and durations up
to 4 h. Experiment 1 compared power
densities of 0, 20, and 30 mW/cm2, the
latter being a common high level of power
density used in rats in this laboratory. The
curve of mean body temperatures after
increasing exposure durations showed a
rapid (15 min) response to this high power
density. Within 60 mm, the mean tem-
perature had risen to essentially 39°C,
and remained there during the longer
exposures for up to 4 h. Extended expo-
sure to a power density of 30 mW/cm2
produced a response within the thermal
physiologic capacity of the rat, as evi-
denced by a platea u of body temperature.
How long the plateau could be maintained
in the face of continued absorption of this
energy (SAR = 6.6 mW/g) is not known
and is beyond the scope of the full report.
On the other hand, a power density of
20 mW/cm2 applied under identical
conditions had virtually no effect on body
temperature. The curve of body temper-
ature at various exposure durations at
this power density (SAR = 4.4 mW/g)
approximates that of sham exposures, ex-
cept for the initial period of up to 1 h, in
which lower body temperatures were
registered for shams. Apparently, 20
mW/cm2 did not constitute a significant
absorption of energy by these rats, and
was conveniently handled by these
animals.
The rats in all these experiments were
naive to the procedures used There are
limitations on the interpretation of re-
sponses in rodents in naive situations. To
help avoid these obstacles, some re-
searchers use a period of acclimation to
the experimental techniques on the basis
that a stress response to a new exper-
imental situation can generate a consid-
erable amount of physiologic "noise." In
turn, it is possible to misinterpret the
"noise" as being the rats' response to
microwave exposure and, thus, a more
subtle response may be entirely masked.
To some degree, acclimation was provid-
ed in Experiment 2 Naive rats were
acclimated to the exposure conditions
(handled, caged in the constramer, and
exposed in the environmental chambers
at 0 mW/cm2) for 90 mm Body temper-
atures were not measured, but were
expected to follow a decreasing curve like
that of rats sham-irradiated in Experiment
1. What seems like a dramatic decrease
in the sham-irradiated rats' temperatures
results from the contrast with their
initially high mean temperatures (approxi-
mately 38.2°C; pre-exposure value).
Those high temperatures were caused by
the excitement from being awakened in
the maintenance cage, and handled and
transported to the exposure facility. With-
in 60 min, the mean temperature of
sham-irradiated rats fell below37°C, and
the animals were quiet and usually
dozing.
Rats that were acclimated for 90 mm,
then microwave-irradiated for 90 min at
30 mW/cm2 (Experiment 2) had essential-
ly the same body temperatures as those
irradiated with 30 mW/cm2 with no
acclimation. Acclimation of 90 min did
not appear to influence the final body
temperature reached by rats exposed to
30 mW/cm2. Even when acclimation
reduced the temperature to <37°C and at
least 1.0°C below the initial pre-exposed
value, the final temperature still reached
levels seen in non-acclimated rats. There-
fore, the temperature at the beginning of
the exposure did not appear to be a
significant influence on the final temper-
ature after exposure to microwave radia-
tion.
Anesthetics
The measurements made in Experi-
ments 1 and 2 were taken m animals that
had normal thermoregulation. The rats
used in Experiment 3 were administered
six anesthetics, all of which altered phys-
iologic thermal control Used commonly
in veterinary and laboratory animals
procedures, these chemical restraints
were explored for their suitability for
microwave experiments (immobilization
for 1-2 h, without effect on physiologic
thermal mechanisms). The compounds
used were anileridme, a synthetic nar-
cotic, chloral hydrate and pentobarbital,
both CNS anesthetics; promazine, atran-
quilizer; and ketamine and xylazme, both
dissociative drugs.
We administered the chemicals at
doses suggested in the literature on
laboratory animals or veterinary medi-
cine. These doses provided starting points
from which the lowest dose producing
practical immobilization was determined.
For example, the recommended anesthet-
ic dose of pentobarbital in the rat is 25-40
mg/Kg i.p. or 50 mg/Kg i.p. We found
that 35 mg/kg i.p. was an effective
anesthetic dose. The effective tranquil-
izmg dose for promazine in the rat is given
as 1 -20 mg/Kg i.m. The deepest immobil-
ization was obtained from 40 mg/Kg i.m
Recommended doses of anileridine in
animals were not available, as the drug
was not recommended for veterinary use.
(This drug is no longer manufactured
commercially (The doses of drugs used to
obtain the greatest immobilization and
least response to touch were1 anileridine,
10 mg/Kg s c., chloral hydrate, 300
mg/Kg i.p; ketamine, 160 mg/Kg i.m.;
pentobarbital, 50 mg/Kg i.p.; promazine,
40 mg/Kg i m ; and xylazme, 0.75 mg/Kg
i.p.
Anileridine caused little loss of wake-
fulness and produced little immobiliza-
tion The response to touch was still
present during the measurement of tem-
perature, but quiet handling was possible.
Colonic temperatures during the test
period remained as high as in active
animals, and we suspect that some mild
hyperthermia had resulted from the dose.
Chloral hydrate produced profound
sleep and immobilization at 300 mg/Kg
and depressed the response to touch for
more than 90 mm after injection Colonic
temperature means were steadily de-
pressed by this drug, falling below 36°C
near the end of the effective period, a
decrease of 1.5°C. All anesthetized rats
were observed while in open-topped
plastic cages shaped like shoe boxes with
pine-shaving bedding, without this insul-
ation, temperatures might have decreased
even further than observed.
-------
Ketamine depressed effectively the
response to touch, but caused almost no
immobilization. The body temperature
depression was, like chloral hydrate,
decreased approximately 1.5°C. The de-
pression of temperature with no effective
immobilization suggests that ketamine
alone is not a useful drug for restraint
during microwave exposure.
Pentobarbital is very commonly used as
an animal anesthetic and has been
thoroughly researched. Rats responded
with deep and constant immobilization
and with a lack of response to touch for at
least 90 min after a single i.p. injection of
35 mg/Kg. During this period, body
temperatures were down to 35.5°C, a
loss of 2-2.5°C. All three measures
(response, sleep, temperature) were af-
fected for almost 3 h. The use of male rats
and the bedding may have offset each
other in this experiment; male rats are
reported to sleep longer than females,
and pine-shaving bedding is reported to
reduce the drug-induced sleep time
Promazine (chlorpromazine; Sparine)
was relatively ineffective in reducing
touch-response. There was a long delay
(> 60 min) before any depth of sleep
occurred, and the sleep was erratic. The
drug was effective (within its small effec-
tive dose range) for the entire 3-h period.
Mean body temperatures by that time
were almost 2°C below initial tempera-
ture Promazine's delayed and erratic
effect suggests that this drug is not useful
as a restraint for microwave exposures.
Xylazine was ineffective. No sleep
(immobilization) occurred, and response
to touch was normal. Even so, tempera-
tures at 90 min after injection were 2°C
below initial temperatures
When rats were given one of the four
chosen chemicals, and then irradiated
with 2450-MHz microwaves at a power
density of 30 mW/cm2 for 90 min, rectal
temperatures showed a sharp increase.
Mean temperatures at the end of expo-
sure were 39 to < 40°C. These values
were as much as 5.5°C above those
encountered with the drug but without
microwave exposure. Because body tem-
peratures also exceeded those of rats in
similar microwave-exposure situations
(Experiment 1), we suspect that all the
tested drugs interfered with normal
thermal regulation. Therefore, we do not
recommend the use of these drugs as
restraining agents in microwave exposure
when body temperatures are used to
determine physiologic response, unless
the compromised thermal regulatory
functions are taken into account.
Serum Chemistries
Chemical analyses of serum constitu-
ents also are common experimental
techniques for the assessment of physio-
logic response to microwave exposure in
laboratory animals. The use of serum
chemistries in this respect may well
increase because of the economy and
convenience associated with modern
automated analytic techniques. For ex-
ample, the SMAC-24 tests used in Experi-
ment 4 reported 24 replicated values and
ratios of 21 constituents on one serum
sample of less than 1-ml volume. Only
one additional milliliter was required to
do the serum Ta and T4 and corticosterone
tests using radio-immune assay tech-
niques. The cost of this battery of tests is
insignificant when compared to the cost
incurred by manual analyses used just a
few years ago.
Because these tests are convenient
and relatively economical, one can lose
sight of their limited applicability. Data
values are often subjected to no more
evaluation than statistical tests of con-
fidence (t-test; analysis of variance). A
warning of the hidden problems associ-
ated with indiscriminate use of serum
chemistry values has suggested consid-
eration of age, sex, strain, environment,
and physiologic status (diurnal cycle,
postural stance, age) of the animal when
interpreting values derived from blood. It
has also been stressed that in human
medicine, in which these batteries of
tests find their most effective use and for
which they are designed, the test results
should be confined to decisions of diag-
nosis, treatment, and prognosis, and that
the critical aspect is interpretation. Also,
any interpretation should be strongly
limited when there are no baseline values
derived from the same population that
can also be extrapolated to other popula-
tions.
More important, perhaps, is the sug-
gestion that a Bayesian approach should
be used in interpreting these diagnostic
tests. Paraphrased for toxicologic re-
search, e.g., microwave-exposure studies,
the requirements might be. (a) clear-cut
definition of the disease (effect) being
sought; (b) delimitation of the stage of the
effect being evaluated by the test; and (c)
knowledge of true-positive and true-
negative results of the test. More simply
stated, one should have a firm prior
hypothesis.
Statistically, the determination of 21,
24, or more values at once in one subject
is expected to very much reduce the
independence of these values. Appropri-
ate adjustments should be made when
applying ordinary statistical tests. In our
research, the method we used extended
acceptable differences to p <.01. Another
related aspect of statistical methodology
is that serum chemistry values may not
assume normal distributions, and statis-
tical tests that require normality (t-test)
may not be applicable here. Bilirubin
values, for instance, were expressed only
as 0,0.5,1.0, or 1.5; chi-square statistics
are more appropriate than t-test statistics
in the cases of creatinine and bilirubin
values.
Only three serum constituents (alkaline
phosphatase, corticosterone, and creati-
nine), exceeded the limitations of the
analytic criteria (p < .01). However,
further evaluative tests should be put to
these values. It is reasonable to expect
the values to follow some consistent
direction or trend. The application of
microwaves should cause an increasing
or decreasing trend in the results.
Biphasic (up-then-down, or down-then-
up) trends are not to be reasonably
expected from increasing exposure. How-
ever, alkaline phosphatase increased
from 250 mg/dl serum in the 0 mW/cm2
group to 315 in the 20 mW/cm2 group,
and then decreased in the 30 mW/cm2
group. Such an up-then-down pattern is
unexpected and more than difficult to
rationalize as resulting only from expo-
sure to microwaves. Serum alkaline
phosphatase increases in response to
extrahepatic blockage or to bone disease.
As these values did not continue to
increase with the increased power den-
sity, the rationale of microwave exposure
as the cause of the observed changes in
serum alkaline phosphatase does not
bear scrutiny.
Serum creatinine values, on the other
hand, did show a trend in one direction.
They showed a sharp increase in animals
exposed at 20 mW/cm2 and 30 mW/cm2
as compared to sham animals(p = .0001).
Creatinine is elevated when renal blood
flow is reduced; however, an increase,
not a decrease, of renal blood flow would
be expected along with raised tempera-
tures due to microwave exposure (30
mW/cm2). Therefore, no physiologic
rationale is available to explain the in-
crease in serum creatinine. We suspect
that we observed a sampling error,
especially in the rather low values of 0.3-
0.4 mg/dl in the sham group. The values
are well within normal limits for rats
when the values of all animals are
averaged (0.47 mg/dl); the average is
very close to the mean of normal rats
(0.45 mg/dl).
-------
A corticosterone increase due to
microwave irradiation is the only one of
the statistically different serum constitu-
ents which may lend itself to a reasonable
physiologic rationale. Adrenal corticoid
secretion is stimulated as a result of
handling of animals in naive situations.
As the exposure to 30 mW/cm2 was
sufficient to increase colonic tempera-
tures to almost 39°C, it may also have
caused an increase in adrenal corticoster-
oid hormone secretion. The additional
response to 30 mW/cm2 exposure, as a
stress reaction, may account for the more
than doubling of corticosterone concen-
tration in the high dose group as com-
pared to shams (260 vs. 116 Aig/ml,
respectively).
Conclusions
When naive young adult male rats are
exposed to 2450-MHz microwaves at a
power density of 30 mW/cm2 with a
duration of up to 4 h, their colonic
temperatures rise to 39°C. Twenty
mW/cm2 causes no obvious temperature
increase in rats. However, acclimation to
the same conditions, without microwaves,
does not alter the pattern of increased
temperature. Except for a significant rise
in corticosterone concentration, serum
chemistry values in animals exposed to
30 mW/cm2 are not acutely altered.
Anesthetics are not recommended for
restraint in animals during microwave
exposure unless it is appreciated how
they limit normal thermal physiologic
mechanisms.
The EPA authors Ezra Rerman and Hershell Carter are with the Health effects
Research Laboratory. Research Triangle Park, NC 27711.
The complete report, entitled "Experiments in Microwave Exposure in the Rat:
Body Temperature, Serum Chemistry, and the Use of Chemical Restraint,"
(Order No. PB 85-156 834/AS; Cost: $8.50, subject to change) will be available
only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at'
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
US GOVERNMENT PRINTING OFFICE 1985-559-016/27003
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
OC00329 PS
U S ENVIR PROTECTION HGINCY
REGION 5 LIBRARY
230 S DEARBORN STREfT
CHICAGO IL
------- |