Toxicological Principles for the Safety Assessment of Food Ingredients
Chapter IV.C.6. Carcinogenicity Studies with Rodents
Return to Redbook 2000 table of contents
- Good Laboratory Practice
- Test Animals
- Test Substance
- Experimental Design
- Observations and Clinical Tests
- Necropsy and Microscopic Examination
This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic. Scientifically justified changes to the 1993 "draft" Redbook version of this section have been made following consultation with other authoritative guidelines and publications.
Carcinogenicity studies (bioassays) in two rodent species (usually rats and mice) are recommended for food ingredients with the highest levels of concern (e.g.,Concern Level III direct food additives, food contact substances with cumulative exposure at or greater than 1 ppm). The carcinogenicity studies (preferably in rats) may be combined with chronic toxicity studies (see Chapter IV.C.7). The Agency recommends that in utero exposure be included in carcinogenicity studies due to the fact that exposure to food ingredients occurs during all stages of life (more information is included in Chapter IV.C.8). These studies are designed to determine whether a food ingredient possesses carcinogenic activity when administered to rodents in regularly repeated oral doses for the "lifetime" of the test animal.
I. Good Laboratory Practice
Nonclinical laboratory studies discussed in this chapter should be conducted according to U.S. FDA good laboratory practice (GLP) regulations, issued under Part 58 Title 21 Code of Federal Regulations. This document may be obtained from the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402, (toll free 866-512-1800). Studies performed under other international/national guidelines may be considered equivalent to those conducted under U.S. FDA GLP regulations. Specific area(s) of non-compliance with FDA GLP regulations should be discussed and justified.
II. Test Animals
A. Care, Maintenance and Housing:
Recommendations about the care, maintenance, and housing of animals contained in the National Research Council, Guide for the Care and Use of Laboratory Animals should be followed unless they conflict with specific recommendations in this chapter.
B. Selection of Species and Strains:
Guidelines contained within this chapter are for studies with mice and rats; if other rodent species are used, modifications may be necessary. Both male and female test animals, which are healthy and have not been subjected to previous experimental procedures, should be used.
It is important to consider the test animals' general sensitivity and the responsiveness of particular organs and tissues of test animals to toxic chemicals when selecting rodent species, strains, and substrains for toxicity studies. The selection of inbred, out-bred, or hybrid rodent strains for toxicity tests should be based upon the scientific questions to be answered. Additionally, it is important that test animals come from well-characterized and healthy colonies. Because recent information suggests survivability problems exist for some strains of rats, test animals should be selected that are likely to survive for the recommended duration of the study (see discussions under sections II.D: Number and Sex and IV.A: Duration of Testing). The Agency encourages petitioners/notifiers to consult with Agency scientists before toxicity testing has begun if they have questions about the appropriateness of a particular species, strain, or substrain.
Another Center within the Agency (Center for Drug Evaluation and Research), as part of a pilot program, accepts safety data from six month studies employing genetically modified mice (i.e., transgenic mice) as a replacement for one of the rodent carcinogenicity studies5. The Office of Food Additive Safety will consider this type of information only as supplemental data but does not consider such studies to be substitutes for the two (2-year) rodent carcinogenicity bioassays. Data from transgenic rodent carcinogenesis or mutagenesis assays may be useful in evaluating compound-specific questions relating to mechanism of action or tissue distribution. For the determination of carcinogenic risk of certain kinds of food ingredients (i.e., constituents and/or contaminants), the transgenic mouse model is inappropriate in that it does not provide quantitative dose-response data. It also has not, as of this date, been fully validated or accepted by most national and international validation organizations (e.g., Scientific Advisory Committee on Alternative Toxicological Method of the Interagency Coordinating Committee on the Validation of Alternative Methods12) or testing laboratories. At this time, there is no large repository of historical control data to establish baseline parameters. Given the nature of consumption patterns of food ingredients (i.e., chronic, lifetime exposures), it is necessary to require the chronic safety testing of food ingredients that would be representative of lifetime exposure in humans, in addition to requiring that only quantitative data derived from fully validated test systems be used in their safety assessment.
C. Age (start of dosing):
D. Number and Sex:
Experimental and control groups should have a sufficient number of animals at the beginning of the study to ensure that at least 25 rodents per sex per group survive to the end of the study. Having sufficient animals survive to the end of the study allows for objective assessment of test substance-related tumor development. Survival can be improved by reducing non-compound related animal pathology, which may occur as a result of excessive weight gain (e.g., obesity-related pituitary changes), or as sequelae to other stressors (e.g., parasitic infection).
The Agency recommends that petitioners/notifiers carefully consider their choice of rat strains for carcinogenicity bioassays, since some strains have more serious problems with survivorship than other strains. It is recommended that carcinogenicity studies begin with at least 50 animals per sex per group. Petitioners/notifiers are encouraged to begin bioassays with more than 50 animals per sex per group if survivorship is expected to be a problem with the rat strain used in the study. If fewer than 25 animals per sex per group are expected to survive to the end of the study (24 months, see section IV.A: Duration of Testing), petitioners/notifiers should take particular care to ensure and document early detection of dead animals through attentive and frequent cage-side observations, thus minimizing the loss of tissues from autolysis. In addition, they should consult with the Agency as soon as a problem with survivorship in a carcinogenicity study becomes apparent.
If interim necropsies are planned, the total number of rodents of each sex per group should be increased by the number scheduled to be sacrificed before completion of the study. A minimum of 10 rodents per sex per group should be available for each interim necropsy.
E. Infected Animals:
Generally, it is not possible to treat animals for infection during the course of a study without the possibility of interaction between the compound used for treatment and the test substance. This interaction may seriously confound or complicate the interpretation of study results. However, if problems with infection do occur, the sponsor for the study should use their best judgment in proceeding with the study and inform the Agency of their decision. In addition, the Agency requests that they provide a full and detailed description of the justification and possible implications of the infection, and if applicable, the justification and possible implications for treatment of the infection.
F. Animal Identification:
Test animals should be characterized by reference to their species, strain (and substrain), sex, age, and weight. Each animal should be assigned a unique identification number (e.g., ear tag, implanted identification chip, tattoo).
Animals should be housed one per cage. This recommendation reflects three points of consideration:
- The amount of feed consumed by each animal in the study cannot be determined when more than one animal is housed in each cage. This information is necessary in the determination of feed efficiency (relationship of feed consumed to body weight gained).
- Minimizing the possibility of confounding analyses in determining whether decreases in body weight gain are due to decreased palatability or test substance mediated toxicity.
- Organs and tissues from moribund and dead animals which are single-caged would not be lost due to cannibalism.
In general, feed and water should be provided ad libitum, and the diets should meet the nutritional requirements for the species13 for normal growth and longevity. Unless special circumstances apply which justify otherwise, care should be taken to ensure that the diets of the test substance treated groups of animals contain the same levels of calories and nutrients (e.g., fiber, micronutrients) as the diets of the control group. Inadequately controlled dietary variables may result in nutritional imbalances or caloric deprivation that could confound interpretation of the toxicity study results (e.g., lifespan, background rates of tumor incidences) and alter the outcome and reproducibility of the studies. However, the Agency is also aware of some beneficial effects on the survivability of certain animal species that have been on calorie-restricted14,24, or low-protein diets15,16. The Agency may accept such study results if the sponsor provides sufficient historical control data on the diet, and the study is well-conducted.
The following issues are important to consider when establishing diets for animals in carcinogenicity studies:
When the test substance has no caloric value and constitutes a substantial amount of the diet (e.g., more than 5%), both caloric and nutrient densities of the high dose diet would be diluted in comparison to the diets of the other groups. As a consequence, some high dose animals may receive higher test substance doses than expected because animals fed such diluted diets ad libitum may eat more than animals in other dosed groups to compensate for the differences in energy and nutrient content of the high dose diets. Such circumstances make it especially important that feed consumption of these animals be as accurately and closely monitored as possible in order to determine whether changes observed could be due to overt toxicity of the test substance or to a dietary imbalance. To further aid in this assessment, two control groups can be used; one group would be fed the undiluted control diet and a second group would be fed the control diet supplemented with an inert filler (e.g., methylcellulose) at a percentage equal to the highest percentage of the test substance in the diet.
When the vehicle for the test substance is expected to have caloric and/or nutritional values, which are greater than that of the control ration, an adjustment in the caloric and/or nutritional components may be necessary.
When administration of the test substance is expected to have an effect on feed intake because of its unpleasant taste or texture, other feeding regimens or experimental designs may be necessary. Consultation with the Agency is recommended when alternatives are being considered.
When the test substance interferes with the absorption of nutrients, leading to nutritional deficiencies or changes in nutrient ratios, this can confound assessment of the toxicological endpoints under consideration. For example, fat soluble vitamins may preferentially partition with a mineral oil or fat substitute which is largely unabsorbed, such that a potential deficiency in these vitamins may result. This potential may be eliminated by additional nutrient fortification of the feed for those groups receiving the test substance. Appropriate levels of nutrient fortification should be determined experimentally.
Other related issues (e.g., advantages and disadvantages of using natural ingredient versus purified diets) are discussed in the National Research Council publication on nutrient requirements of laboratory animals13.
Animals should be assigned to control and compound treated groups in a stratified random manner. This will help minimize bias and assure comparability of pertinent variables across compound treated and control groups. In general, mean body weights and/or body weight ranges are used as a basis of randomization. If other characteristics are used as the basis for randomization, they should be described and justified.
Animals in all groups should be placed on study on the same day. If this is not possible because of the large number of animals in a study, animals may be placed on study over several days. When the latter recommendation is followed, a preselected portion of the control and experimental animals should be placed on the study each day in order to maintain concurrence.
Excessive mortality due to poor animal management is unacceptable and may be a cause to repeat the study.
Adequate animal husbandry practices should result in considerably less than 10 % loss of animals and tissues or organs in a study because of autolysis. Autolysis in excess of this standard may be a cause to repeat the study.
Necropsy should be performed soon after an animal is sacrificed or found dead, so that loss of tissues due to autolysis is minimized. When necropsy cannot be performed immediately, the animal should be refrigerated at a temperature that is low enough to prevent autolysis (i.e., between 4oC and 8oC), but not so low as to cause cell damage. If histopathological examination is to be conducted, tissue specimens should be taken from the animals and placed in appropriate fixatives when the necropsy is performed.
III. Test Substance
The test substance used in carcinogenicity studies should be the same substance that the petitioner/notifier intends to market or, when appropriate, the test substance may be a constituent chemical or an impurity. A single lot of test substance should be used throughout the study. When this is not possible, lots that are as similar as possible in purity and composition should be used. It is the responsibility of the petitioner/notifier to notify the animal test facility of the purity of the test substance, as well as the identity and concentration of any impurities that might be present.
The identity of the test substance or mixture of substances to be tested should be known. We urge the petitioners/notifiers to consult with the Agency regarding the determination of the test compound, and to provide all relevant Chemical Abstract Service (CAS) Registry numbers.
The composition of the test substance should be known including the name and quantities of all major components, known contaminants and impurities, and the percentage of unidentifiable materials.
C. Conditions of Storage:
The test samples should be stored under conditions that maintain their stability and purity until the studies are complete.
D. Expiration Date:
A. Duration of Testing:
The test animal should be exposed to the test substance 7 days per week for 104 consecutive weeks (two years), or for the life span of the animal. In general, the Agency does not recommend early termination of carcinogenicity studies due to decreased survivorship (see section II.D: Number and Sex). Carcinogenicity bioassays should be conducted for a major portion of the test animal's lifetime. While it is desirable to have an optimum number of animals survive to the end of the study, the Agency believes there is more benefit, as well as added sensitivity, to be gained by conducting carcinogenicity bioassays for as long as possible, or for the full 24 months that is recommended in these guidelines.
B. Route of Administration:
The route of administration of the test substance should approximate that of normal human exposure, and if possible, the oral route should be used. A justification should be provided when using other routes. The same method of administration should be used for all test animals throughout the study. The test substance should be administered in one of the following ways:
- In the diet, if human exposure to the test substance is likely to be through consumption of solid foods or a combination of solid and liquid foods. If the test substance is added to the diet, animals should not be able to selectively consume either basal diet or test substance in the diet on the basis of color, smell, or particle size. If the compound is mixed with ground feed and pelleted, nothing in the pelleting process should affect the test substance (for example, heat-labile substances may be destroyed during pellet production by a steam process). When the test substance is administered in the diet, dietary levels should be expressed as mg of the test substance per kg of feed.
- Dissolved in the drinking water, if the test substance is likely to be ingested in liquid form by humans (for example, in soft drinks or beer), or if administration in the diet of rodents is inappropriate. The amount of test substance administered in drinking water should be expressed as mg of test substance per ml of water.
- By encapsulation or oral intubation (gavage), if the two previous methods are unsatisfactory or if human exposure is expected to be through daily ingestion of a single, large bolus dose instead of continual ingestion of small doses. If the test substance is administered by gavage, it should be given at approximately the same time each day. The maximum volume of solution that can be given by gavage in one dose depends on the test animal's size; for rodents, the volume ordinarily should not exceed 1 ml/100 g body weight. If the gavage vehicle is oil, then the volume should be no more than 0.4 ml/100 g of body weight, and the use of a low-fat diet should be considered. If the test substance should be given in divided doses, all doses should be administered within a 6 hour period. Doses of test substance administered by gavage should be expressed as mg of test substance per ml of gavage vehicle. Finally, the petitioner/notifier should provide information that can allow the Agency to conclude that administration of the test compound by encapsulation or gavage is equivalent in toxicologically important respects to administration in the diet or drinking water. Alternatively, metabolic information on both modes of administration should be provided so that appropriate interpretation of data can be accomplished.
C. Dose groups:
1. Selection of Treatment Doses:
Dose selection for long-term toxicity studies should be based on results from subchronic studies and other related test substance information.
Three to five dose levels of the test substance and a concurrent control group should be used in carcinogenicity bioassays. When designing and conducting carcinogenicity bioassays the following should be considered: 1) the high dose (maximum tolerated dose) should be sufficiently high to induce toxic responses in test animals, and should not cause fatalities high enough to prevent meaningful evaluation of the data from the study; 2) the low dose should not induce toxic responses in test animals; and 3) the intermediate dose(s) should be sufficiently high to elicit minimal toxic effects in test animals (such as alterations in enzyme levels or slight decreases in body weight gains. Administration of the test substance to all dose groups should be done concurrently (see Section II.I: Assignment of Control and Compound Treated Animals).
High Dose: The high dose should be the maximum tolerated dose (MTD).
It is not acceptable to select doses for carcinogenicity bioassays based on information unrelated to the toxicity of the test substance. For example, the highest dose in a carcinogenicity study should not be selected so as to provide a pre-determined margin of safety over the maximum expected human exposure to the test substance, assuming that the results of testing at that dose will be negative.
These guidelines recommend that the highest dose in carcinogenicity bioassays should be the MTD. In evaluating the results of carcinogenicity bioassays of a substance in food, Agency scientists will consider the question of whether the substance was tested at the MTD as one of several factors that may affect interpretation of the results of the bioassay. The bioassay should include a description of the process used to select the MTD for the study.
The MTD is defined by the National Toxicology Program (NTP) as "that dose which, when given for the duration of the chronic study as the highest dose, will not shorten the treated animals' longevity from any toxic effects other than the induction of neoplasms"17 The Office of Science and Technology Policy provides the following advice, "The highest dose should be selected after an adequate prechronic study and after evaluating other relevant information, as necessary, to determine the highest dose consistent with predicted minimal target organ toxicity and normal life span, except as a consequence for the possible induction of cancer."18 In addition, the NTP cautions that the MTD should not cause morphologic evidence of toxicity of a severity that would interfere with the interpretation of the study results.17
In general, the MTD is estimated following a careful analysis of data from appropriate subchronic toxicity tests. As the scientific community's experience with toxicity testing has accumulated, the need to consider a broad range of biological information when selecting the MTD has become increasingly clear. For example, data concerning changes in body and organ weight and clinically significant alterations in hematological, urinary and clinical chemistry measurements, in combination with more definitive toxic, gross or histopathologic endpoints, can be used to estimate the MTD.
Although the high dose in a carcinogenicity study should be selected to achieve the MTD, the Agency recognizes that this goal may not always be met. There are uncertainties in predicting the MTD for long-term bioassays from the results of shorter-term studies. Because working definitions of the MTD require the use of scientific judgment, it is sometimes possible for competent investigators looking at the same set of data to arrive at significantly different estimates of the MTD. Such disagreement may be based on different interpretations of the results of metabolic studies or different conclusions about whether an organ alteration is adaptive or toxicological. In situations such as these, when it is unclear what dose of the test substance is the MTD, the petitioner/notifier should consult with the Agency to determine an appropriate high dose (MTD) for the carcinogenicity bioassay.
The Agency recognizes that use of the MTD in carcinogenicity bioassays has several advantages; these include:
- Compensating for the inherent lack of sensitivity of the bioassay, including the relatively small number of rodents used in the study;
- Providing consistency with other models used in toxicology (e.g., high enough doses should be used in order to elicit evidence of the presumed toxicity or increase probability of detecting rare tumors and identifying weak carcinogens); and
- Permitting comparison of carcinogenic potencies of substances tested at the MTD, even when the data are collected from different studies19.
The Agency acknowledges that its recommendation to conduct carcinogenicity studies at the MTD may result in the use of doses that are so high as to be unrepresentative of the toxicity of the test substance at lower doses in animals or humans. For example, excessively high doses of a test substance can saturate enzyme systems involved in detoxification of the test substance. Given the above, after thorough internal assessment and in an agreement with other authoritative bodies 9,10, the Agency concludes that the MTD is still the best choice for selecting the high dose for carcinogenicity studies. It should be noted that this is also in line with the conclusions of the International Conference on Harmonization which recommends the use of the MTD in choosing the high dose for drug safety testing 7,8.
The low dose level should not interfere with the normal growth, development, and lifespan of test animals, nor should it produce any signs of toxicity.
The intermediate dose should produce minimal signs of toxicity. The exact dose selected as the intermediate dose may depend on the pharmacokinetic properties of the test substance.
Optional Fourth Dose Level:
If significant differences exist in the pharmacokinetic or metabolic profiles of the test substance administered at high and low doses, an optional (fourth) dose level may be included in the study. This dose level should be the highest dose that produces a pharmacokinetic or metabolic profile similar to profiles obtained at lower doses. The number of test animals in the optional group should be selected to provide approximately the same sensitivity for the detection of the carcinogenic effects of the test substance as the higher dose group provides.
A concurrent control group of test animals fed the basal diet is required. A carrier or vehicle for the test substance should be given to control animals at a volume equal to the maximum volume of carrier or vehicle given to any dosed group of animals. Sufficient toxicology information should be available on the carrier or vehicle to ensure that its use will not compromise the results of the study. If there is insufficient information about the toxic properties of the vehicle used to administer the test substance, an additional control group that is not exposed to the carrier or vehicle should be included. In all other respects, animals in the control group should be treated the same as animals in dosed groups. (See also section II.H: Diet.)
D. Computerized systems
Computerized systems that are used in the generation, measurement, or assessment of data should be developed, validated, operated, and maintained in ways that are consistent with the intention of the Good Laboratory Practice principles20. The FDA has endorsed the use of the Standard for Exchange of Nonclinical Data (SEND) format for electronic transmission of animal study data. Contact the Agency for more information on this electronic protocol.
V. Observations and Clinical Tests
A. Observations of Test Animals:
Routine cage-side observations should be made on all animals inside the cage once or twice a day throughout the study for signs of departure from normal activity, morbidity and mortality. The usual interval between multiple periods of observations should be at least 6 hours. Individual records should be maintained for each animal and, as possible, the onset and progression of any effects should be recorded, preferably using a scoring system. If grossly visible tumors develop, the following parameters should be recorded; time of onset, location, dimensions, appearance and progression.
B. Body Weight and Feed Intake Data:
Accurate individual body weight, feed, and water consumption measurements are critical in the objective evaluation of the effect of a compound on test animals, since changes in these variables are often the first signs of toxicity. Complete records for these parameters are essential in assessing the time-related occurrence of toxicity-induced changes. When these data are not carefully recorded the evaluation of the overall cancer-inducing potential for a test substance may be compromised. A discussion of some of the variables that affect feed consumption and weight gain/loss can be found under sections II.H: Diet and IV.B: Route of Administration.
Body weights for all test animals should be recorded weekly for the first 13 weeks, and monthly thereafter for the duration of the study. Feed consumption (or water consumption if the test substance is administered in the drinking water) should be measured at the same interval as body weights; petitioners/notifiers should also attempt to quantify spillage of feed by test animals. When it is suspected that test compound administration may be affected by any of the following conditions; 1) feed palatability issues, 2) marked changes in body weight, or 3) increased numbers of animal deaths, the petitioners/notifiers should measure weights and feed (water) consumption more frequently after the initial 13 week period (e.g., every two weeks). Petitioners/notifiers should also use this accumulated information to calculate intake of the test substance.
C. Clinical Testing:
Ophthalmological examination, hematology profiles, clinical chemistry tests, and urinalyses should be performed as described in the following sections:
1. Opthalmological Examination:
This examination should be performed by a qualified individual on all animals before the study begins and on control and high-dose animals at the end of the study. If the results of examinations at termination indicate that changes in the eyes may be associated with administration of the test substance, ophthalmological examinations should be performed on all animals in the study.
Hematological tests should be performed on at least 10 animals per sex in each group should be made during the first 2 weeks of study, and at 3, 6 and 12 months during the study. The time of the first sampling may be based on test results from short-term studies. If data trends or significant parameter changes (biological or statistical) that are of concern are observed at the 12-month measurement, then an 18-month measurement should be included. Additional hematology testing should be conducted at the end of the study if data trends or significant parameter changes are observed at 18 months to be consistent with other clinical testing.
Ideally, the same rodents should be sampled at each collection time point. Blood samples should be analyzed individually, and not pooled. If, due to the large number of animals, it becomes necessary to draw blood samples on more than one consecutive day at each sampling point, the samples should be obtained at approximately the same time each day.
The following determinations are recommended: hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte counts, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet count, and a measure of clotting potential (e.g., clotting time, prothrombin time, activated partial thromboplastin time).
Test compounds may have an effect on the hematopoietic system and therefore appropriate measures should be employed so that evaluations of reticulocyte counts and bone marrow cytology may be performed if warranted. Reticulocyte counts should be obtained for each animal using automated reticulocyte counting capabilities, or from air-dried blood smears. Bone marrow slides for cytological evaluation should be prepared from each animal. These slides only need to be microscopically examined when effects on the hematopoietic system are noted.
3. Clinical Chemistry:
Clinical chemistry tests should be performed on at least 10 animals per sex per group during the first 2 weeks of study, and at 3, 6 and 12 months during the study. The time of the first sampling may be based on test results from short-term studies. If data trends or significant parameter changes (biological or statistical) that are of concern are observed at the 12-month measurement, then an 18-month measurement should be included. Additional clinical chemistry testing should be conducted at the end of the study if data trends or significant parameter changes are observed at 18 months.
Ideally, the same rodents should be sampled at each collection time point. Blood samples should be drawn at the end of the fasting time and before feeding. Fasting duration should be appropriate for the species and the analytical tests to be performed. Blood samples should be analyzed individually, and not pooled. If animals are sampled on more than one day during a study, blood should he drawn at approximately the same time each sampling day.
Clinical chemistry tests that are appropriate for all test substances include measurements of electrolyte balance, nutrients metabolism, and liver and kidney function. Specific determinations should include:
Hepatocellular evaluation (at least 3 of the following 5)
- Alanine aminotransferase (SGPT, ALT)
- Aspartate aminotransferase (SGOT, AST)
- Sorbitol dehydrogenase
- Glutamate dehydrogenase
- Total bile acids
Hepatobiliary evaluation (at least 3 of the following 5)
- Alkaline phosphatase
- Bilirubin (total)
- Gamma-glutamyl transpeptidase (GG transferase)
- 5' nucleotidase
- Total bile acids
Other markers of cell changes or cellular function
- Cholesterol (total)
- Globulin (calculated)
- Glucose (in fasted animals)
- Protein (total)
- Triglycerides (fasting)
- Urea nitrogen
- The Agency understands that the specific nature of the test substance may warrant the consideration of alternative tests. Appropriate justification for alternative tests should be presented in study reports.
In spite of standard operating procedures and equipment calibration, it is not unusual to observe considerable variation in the results of clinical chemistry analyses from day to day21. Ideally, clinical chemistry analyses for all dose groups should be completed during one day. If this is not possible, analyses should be conducted in such a way as to minimize potential variability.
The determination of volume of urine collected, urine specific gravity, pH, glucose, and protein, as well as microscopic analysis of urine for sediment and presence of blood and/or blood cells, are recommended before dosing, and at 3, 6 and 12 months during the study22. If data trends or significant parameter changes (biological or statistical) that are of concern are observed at the 12-month measurement, then an 18-month measurement should be included. Additional urinalysis testing should be conducted at the end of the study if data trends or significant parameter changes are observed at 18 months. These tests should be performed on at least 10 animals per sex per group.
VI. Necropsy and Microscopic Examination
A. Gross Necropsy
All test animals should be subjected to complete gross necropsy, including examination of external surfaces, orifices, cranial, thoracic and abdominal cavities, carcass, and all organs. The gross necropsy should be performed by, or under the direct supervision of, a qualified pathologist, preferably the pathologist who will later perform the microscopic examination.
B. Organ Weight
Organs that should be weighed include the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thyroid/parathyroid, thymus if present, ovaries and uterus. Before being weighed, organs should be carefully dissected and trimmed to remove fat and other contiguous tissue. Organs should be weighed immediately after dissection to minimize the effects of drying on organ weight.
C. Preparation of Tissues for Microscopic Examination
Generally, the following tissues should be fixed in 10 % buffered formalin (or another generally recognized fixative) and sections prepared and stained with hematoxylin and eosin (or another appropriate stain) in preparation for microscopic examination. Lungs should be inflated with fixative prior to immersion in fixative.
- Bone (femur)
- Bone marrow (sternum)
- Brain (at least 3 different levels)
- Corpus and cervix uteri
- Gall bladder (if present)
- Harderian gland
- Lung (with main-stem bronchi)
- Lymph nodes (1 related to route of administration and 1 from a distant location)
- Mammary glands
- Nasal turbinates
- Ovaries and fallopian tubes
- Salivary gland
- Sciatic nerve
- Seminal vesicle (if present)
- Skeletal muscle
- Spinal cord (3 locations: cervical, mid-thoracic, and lumbar)
- Thymus (if present)
- Urinary bladder
- Zymbal's gland
- All tissues showing abnormality
D. Microscopic Evaluation
All gross lesions should be examined microscopically. All tissues from the animals in the control and high dose groups should be examined. If treatment related effects are noted in certain tissues, then those specific tissues in the next lower dose level tested should be examined. Successive examination of the next lower dose level continues until no effects are noted. In addition, all tissues from animals that died prematurely or were sacrificed during the study should be examined microscopically. If there are questions related to the review and interpretation of pathological lesions and statistical results, additional discussion may be found in Chapters IV.B.3 and IV.B.4 of the Redbook 2000.
E. Histopathology of Lymphoid Organs
Histopathology evaluation of the lymphoid organs should be performed as described in the section on immunotoxicity testing (see Chapter V.C. of the 1993 draft Redbook II). A recent publication provides further discussion on this subject23.
1 Organization for Economic Cooperation and Development (OECD), Environment, Health and Safety Publications, Series on Testing and Assessment No. 35 and Series on Pesticides No. 14, Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies, Paris, 2002.
2 OECD, Guideline for Testing of Chemicals, No. 451, Carcinogenicity Studies, Adopted in 1981.
3 National Toxicology Program (NTP)/Department of Health and Human Services (DHHS), Descriptions of NTP Study Types, 2-Year Study Protocol, 2004.
4 Office of Prevention, Pesticides and Toxic Substances (OPPTS)/United States Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.4200: Carcinogenicity, 1998.
5 Center for Drug Evaluation Research/Food and Drug Administration (FDA)/DHHS, Guidance for Industry, S1B Testing for Carcinogenicity of Pharmaceuticals, 1997.
6 International Conference on Harmonization (ICH), Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline, S1B: Testing for Carcinogenicity of Pharmaceuticals, 1997.
7 ICH, Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline, S1C(R): Addendum: Addition of a Limit Dose and Related Notes, 1997.
8 ICH, Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline, S1C: Dose Selection for Carcinogenicity Studies of Pharmaceuticals, 1994.
9 National Research Council (NRC)/National Academy of Sciences (NAS), Issues in Risk Assessment, National Academy Press, pp.15-19&43-96, 1993.
10 International Life Sciences Institute (ILSI)/Health and Environmental Sciences Institute (HESI)), Committee on Carcinogenicity of Chemicals in Food, Consumer Products and Environment, Statement on ILSI/ HESI Research Programme on Alternative Cancer Models (Letter to the Editor), Toxicologic Pathology, 31(2):254-257, 2003.
11 NRC/NAS, Institute of Laboratory Animal Resources, Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, D.C., 1996.
12 Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) of the Interagency Coordinating Committee on the Validation of Alternative Methods, Summary Minutes for March 10 - 11 Meeting, Bethesda, Maryland, 2004.
13 NRC/NAS, Subcommittee on Laboratory Animal Nutrition, Committee on Animal Nutrition, Board on Agriculture, Nutrient Requirements of Laboratory Animals, Fourth Revised Edition, National Academy Press, Washington, D.C., 1995.
14 Keenan K.P., J.B. Coleman, C.L. McCoy, C.M. Hoe, K.A. Soper and P. Laroque, Chronic Nephropathy in ad libitum Overfed Sprague-Dawley Rats and its Early Attenuation by Increasing Degrees of Dietary (Caloric) Restriction to Control Growth, Toxicologic Pathology, 26(6):788-798, 2000.
15 Rao G.N. and P.W. Crockett, Effect of Diet and Housing on Growth, Body Weight, Survival and Tumor Incidences of B6C3F1 Mice in Chronic Studies, Toxicologic Pathology, 31(2):243-250, 2003.
16 Clinton S.K., A.L. Mulloy, S.P. Li, H.J. Mangian and W.J. Visek, Dietary Fat and Protein Intake Differ in Modulation of Prostate Tumor Growth, Prolactin Secretion and Metabolism, and Prostate Gland Prolactin Binding Capacity in Rats, The Journal of Nutrition, 127:225-237, 1997.
17 NTP/DHHS, Board of Scientific Counselors, Report of the NTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation, 1984.
18 Office of Science and Technology Policy/Executive Office of the President, Chemical Carcinogens; A Review of the Science and its Associated Principles. The Federal Register 15(50):10372-10442, March 14, 1985.
19 McConnell, E.E., The Maximum Tolerated Dose: the Debate. Journal of the American College of Toxicology, 8(6):1115-1120, 1989.
20 FDA/DHHS, Draft Guidance for Industry on Computerized Systems Used in Clinical Trials; Availability, The Federal Register, 69(191):59239-59240, October 4, 2004.
21 Gaylor D.W., R.L. Suber, G.L. Wolff and J.A Crowell, Statistical Variation of Selected Clinical Pathological and Biochemical Measurements in Rodents, Proceedings of the Society for Experimental Biology and Medicine, 185:361-367, 1987.
22 Ragan H.A. and R.E. Weller, Markers of Renal Function and Injury, In: The Clinical Chemistry of Laboratory Animals, Second Edition, Taylor&Francis, Philadelphia, PA, pp. 520-533, 1999.
23 Society of Toxicologic Pathology (STP) Immunotoxicology Working Group (P. HALEY et al.), STP Position Paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System, Toxicologic Pathology, 33:404-407, 2005.
24Keenan K.P., C.M. Hoe, L Mixson, C.L. McCoy, J.B. Coleman, B.A. Mattson, G.A. Ballam, L.A. Gumprecht, and K.A. Soper, Diabesity: A Polygenic Model of Dietary-Induced Obesity from ad libitum Overfeeding of Sprague-Dawley Rats and its Modulation by Moderate and Marked Dietary Restriction, Toxicologic Pathology, 33:650-674, 2005.
Submit comments on this guidance document electronically via docket ID: FDA-2013-S-0610 - Specific Electronic Submissions Intended For FDA's Dockets Management Staff (i.e., Citizen Petitions, Draft Proposed Guidance Documents, Variances, and other administrative record submissions)
If unable to submit comments online, please mail written comments to:
Food and Drug Administration
5630 Fishers Lane, Rm 1061
Rockville, MD 20852
All comments should be identified with the title of the guidance.