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

-------
     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.

-------
     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.

-------
     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

-------
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.

-------
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

-------
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.

-------
     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-

-------
                              8





     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

-------
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.

-------
                              10
     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.

-------
                              11
     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.

-------