A POSITION DOCUMENT OF THE U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDE PROGRAMS
SELECTION OF A MAXIMUM TOLERATED DOSE (MTD) IN ONCOGENICITY STUDIES
PREPARED BY
THEODORE M. FARBER, PH.D., D.A.B.T,
CHIEF, TOXICOLOGY BRANCH
OFFICE OF PESTICIDE PROGRAMS
ENVIRONMENTAL PROTECTION AGENCY
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The Toxicology Branch of the Office of Pesticide Programs
frequently encounters oncogenicity and chronic feeding studies
in which the dose levels selected for toxicological evaluation
were not sufficiently high to evoke significant signs of
toxicity in the test animals. The failure to observe toxicity
in an oncogenicity study at the highest dose level may
compromise the hazard identification of the oncogenic activity
of the compound because the test animal may not have been
sufficiently challenged under the conditions of the study.
Despite general agreement among scientists regarding the
requirements for a MTD, there appears to be a lack of consensus
regarding the toxic end points which are adequate for fulfilling
a MTD in an oncogenicity study. In fact, the term MTD has
been described in as many different subjective terms as there
are individuals who use it. This has frequently led to
confusion and differences in the interpretation of results
between regulatory agencies and investigators at the termination
of these studies.
This problem has not gone unnoticed; several efforts,
primarily via the National Toxicology Program and OSTP are
being expended to describe more clearly the MTD concept and
to bring consistency to the definition.
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Within the various guidelines for oncogenicity studies
which have been developed by the Environmental Protection
Agency, there are apparent differences in the description of
the MTD concept. The 1978 proposed guidelines issued by the
Office of Pesticide Programs provided a minimal definition of
the MTD, whereas the 1982 final guidelines do not employ the
term. In response to the need to improve scientific and
regulatory decisions and to provide additional guidance for
the investigators performing oncogenicity studies, this position
statement is presented by the Toxicology Branch of the Hazard
Evaluation Division in the Office of Pesticide Programs.
The Office of Pesticide Programs position statement
should be considered to be an addendum to the Program's 1984
toxicity testing gudielines. This position statement reflects
the scientific thoughts of only the Office of Pesticide
Programs and should not be construed to be guidance from any
other Office within EPA. It should not be assumed that a
study found acceptable by the Office of Pesticide Programs in
terms of the highest dose tested will be automatically acceptable
to other offices in EPA. It is anticipated that a global
Agency definition of MTD will be developed in the future by
the Risk Assessment Forum in EPA.
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The Office of Pesticide Programs believes that in
evaluating the acceptability of chronic/oncogenicity studies,
consideration should be given to the concept that the MTD is
a predicted value derived from observed toxicities in subchronic
studies.
The highest dose to be tested in the oncogenicity study
should be selected below a level which resulted in significant
life-threatening toxicity in the subchronic study. The level
should not be selected too far below a life threatening level
because the highest dose tested in the oncogenicity study
should elicit significant toxicity without substantially
altering the normal life-span of the test species from effects
other than tumor formation. This dose selection process may
be difficult and we advise sponsors to seek some guidance
from the Program before proceedings with the chronic study.
Changes in several toxicological parameters observed in
subchronic studies or chronic studies are important in
determining the highest dose to be tested in the oncogenicity
study or in determining that the highest level of testing in
the chronic study was adequate. These changes involve body
weight gain decrement, hematological effects, histopathologic
changes, alterations in clinical chemistries and urinalysis,
organ weight changes, neurological symptoms and other clinical
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signs. Attempts should be made when possible to correlate
biochemical parameters with clinical signs of toxicity or
histopathological changes in tissues or organs showing a
toxic response to the chemical under test. The Office of
Pesticide Programs would like to present some examples of
some types of toxicity observed in subchronic or chronic
studies which may used to select the highest dose level to be
tested or that the highest dose tested sufficiently expressed
the full range of toxicity of the test substance. These
examples are not intended to be all inclusive by any means.
Body Weight Effects;
The highest dose to be tested can be established on the
basis of a biologically significant decrement in body weight
gain. The Office of Pesticide Programs believes this decrement
in body weight gain should reach 10-15% in subchronic testing.
When a clinical sign such as weight loss is observed,
attempts should be made to determine if this is caused by
anorexia, palatability, dysphagia or secondary effects due to
diarrhea, diuresis or nutritional imbalance. Edema or organ
weight gain, particularly with the liver, may mask a body
weight gain decrement.
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Histopathology;
Consideration of pathological changes in predicting a
MTD and establishing the highest dose to be tested in the
oncogenicity study is imperative especially if these toxicities
may be expected, upon chronic exposure, to result in shortening
the lifespan of the animal, thereby decreasing the time on test.
In the subchronic study dose levels of the agent should
be used which demonstrates a spectrum of histopathlogical
changes in tissues such as fatty metamorphosis, vacuolization,
mineralization, cell proliferation, cell degeneration, cell
necrosis and cell death.
One word of caution should be made at this point. In
several instances during the last two years the Program has
seen the highest dose to be tested in the chronic study
selected on the basis of organ weight increases, cloudy
swelling, fatty metamorphosis and vacuolation, alone or in
combination. In many of these instances the Program believes
that the highest dose selected was inadequate. Fatty
metamorphosis of the liver is often not life-threatening to
the experimental animals and the high dose selected in these
studies was set at the lower end of dosages shown to produce
fatty changes. The Program believes that the highest dose to
be tested for a hepatotoxin should be set at a upper level of
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dosages associated with fatty metamorphosis. If necrosis is
seen along with fatty metamorphosis then a level should be
selected that will probably produce some minimal degree of
necrosis which would not effect the survivability of the
animals on test.
Hematologic effects;
Biologically significant alterations indicating anemia,
changes in prothrombin clotting time, leucopenia, leukemia or
significant alteration in the relative number of critical
formed elements can be used to make a prediction regarding
the highest dose to be tested in the oncogenicity study and
the likelihood of the survivability of the high dose animals
to the termination of the study or to establish that the high
dose employed in the chronic study was adequate.
Clincial chemistries;
Significant depression of at least two of the assayed
cholinesterase enzyme measurements, i.e., plasma, red blood
cell or brain acetylcholinesterase levels can be used to
establish the highest dose to be tested or that the high
level employed in the chronic study was adequate.
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Significant alterations in clinical chemistries such as
aspartate aminotransferase, alanine aminotransferase, alkaline
phosphatase or blood urea nitrogen can be used to support the
selection of the highest dose to be tested when they correlate
with histopathologic changes in seen tissues or organs.
Urinalysis;
Changes in urinalysis can add support to the selection
of the proper high dose level to use in the chronic study or
can support the adequacy of the high dose used in the completed
oncogenicity study.
Organ weight changes;
Biologically significant alterations in organ weights
will support the selection of the high dose to be tested or
the adequacy of the doses used in an oncogenicity if they
correlate with significant histopathology seen in that organ.
The MTD will not be considered to be reached if increases in
relative liver to body weight ratios occur in the absence of
significant histopathology in the liver; these increases are
more likely to result from microsomal enzyme induction,
representing an adaptive or pharmacologic response of the
liver to the test chemical and not likely to effect survivability-
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Basically, the Office of Pesticide Programs will consider
a oncogenicity study to be adequately performed if the highest
dose level studied vigorously tests the agent for oncogenic
potential at levels somewhat below test levels which might
compromise survivability.
After the highest dose to be tested has been selected,
at least two doses lower than the predicted MTD should be
selected not only to help validate responses seen at the
highest dose level as well as to develop dose-response curve
information but also to provide a margin of safety against
overestimating the MTD. Thus, in a three dose study, if
survivability is poor at the highest dose tested, the mid-
dose level, set approximately at one-half the MTD, could
substitute as an adequate level to test the potential
oncogenicity of the test agent.
A MTD level should be determined for each species, sex
and strain of animal to be used in an oncogenicity study. If
the doses selected for each sex are reasonably close, then
the doses should be the same.
In oncogenicity studies of pesticides, a dose of 1 gram/kg
of body weight/day should provide an adequate upper limit for
the testing of most pesticides in laboratory animals. This
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upper limit of 1 gram/kg applies to chemicals which do not
produce toxicological effects in subchronic studies. The dose
of 1 gram/kg/day is equivalent to dietary concentrations of
approximately 20,000 ppm (i.e., 2%) in the rat and 7,000 ppm
(i.e., 0.7%) in the mouse. The dose level of 1 gram/kg/day
is considered an adequate upper limit for testing pesticides
because of exposure is generally limited by their relative
potency and selective toxicity.
There are approximately 250 pesticides that have been
registered for some use on food commodities. In no case does
the exposure of the general U.S. population exceed one
milligram per kilogram of body based on cumulative total
maximum residue concentration values. The cumulative total
maximum residue concentration represents the theoretical
residues levels from all commodities treated with a pesticide
at the time it leaves the farm and assumes the maximum use of
the agent in terms of label recommendations. Reduction in
the level of a pesticide because of decompostion, processing
of the commodity, food preparation and cooking is not considered
in these values. Therefore, these values are often much
higher than the real world human exposure to pesticides. The
vast majority of agents are considerably below a human exposure
level of one milligram per kilogram in terms of their
cumulative total maximum residue concentration.
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The Program believes that the 1000 milligram per kilogram
level established for the rat and mice is a realistic higher
bound for testing of a pesticide for oncogenic potential.
Additionally, in the past three years the Office of Pesticide
Programs' Peer Review Committee has evaluated more than 50 agents
for carcinogenic potential. In only two cases were possible
carcinogenic effects seen only at levels exceeding 1000
milligrams per kilogram. In one situation urinary bladder
tumors were seen at 40,000 ppm probably caused by urinary
caculi. In the other case adenomas/carcinomas were seen in
the male rat kidney at 30,000 ppm and was considered to be an
equivocal finding by the Programs' Scientific Advisory Panel.
If the anticipated toxicity at the properly selected
high dose was not seen because adaptation occurred in the
chronic study, the Office of Pesticide Programs would consider
that chronic study as having met our scientific standards and
it would not have to be repeated because of an absence of
significant toxicity at the termination of the oncogenicity
study. To the contrary, a chronic/oncogenicity study without
a demonstrated effect would be considered unacceptable as an
oncogenicity test if the highest dose was not predicted from
observed toxic effects in a subchronic study.
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It is strongly advised that the sponsor present, in
writing, the scientific rationale for the highest dose to be
tested in an oncogenicity study and whenever possible to meet
with toxicologists in the Office of Pesticide Programs to
reach some concurrence on the doses to be tested in the.study
before the initiation of the study. This interaction between
the Agency and investigators could avoid future problems in
evaluating oncogenicity studies in which inappropriate dose
levels were used for the testing of the carcinogenic potential
of a compound.
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