Toxicological Principles for the Safety Assessment of Food Ingredients
Chapter IV.C.5.a. Chronic Toxicity 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
The FDA has made scientifically justified changes to 1993 "draft" Redbook Chapter IV.C.7., Combined Chronic Toxicity/Carcinogenicity Studies with Rodents, and developed Chapter IV.C.5.a., Chronic Toxicity Studies with Rodents, after consulting other authoritative guidelines   and publications (see also relevant sections below). This section of Redbook 2000 supersedes the 1993 "draft" Redbook Chapter IV.C.7.
The FDA acknowledges that it is complicated and difficult to conduct a combined study due to difficulty in setting and administering appropriate dose levels for both types of studies concurrently. In addition, the general objectives of these two types of studies are different. However, when pre-chronic studies provide reasonable estimates of toxicity to predict the information (e.g., treatment doses) to be used in a single bioassay, a chronic toxicity study may be combined with a carcinogenicity study and reveal information about an ingredient’s potential to be a carcinogen as well as the maximum dose that produces no adverse effects. On a case-by-case, an in-utero exposure phase may also be added to a chronic toxicity study (or combined chronic toxicity/carcinogenicity study) to determine early developmental effects that may increase the incidence of chronic disease outcomes (and/or cancers). Sponsors/submitters of petitions/notifications are encouraged to become familiar with the Carcinogenicity Studies with Rodents (Chapter IV.C.6.) and In-utero Exposure Phase for Addition to Carcinogenicity Studies or Chronic Toxicity Studies with Rodents (Chapter IV.C.8.) during the development of a combined study design. The petitioner/notifier should also consult with the FDA before conducting a combined study.
Chronic toxicity studies with rodents should be conducted for a minimum of 12 months (one-year). Results of these tests can be used, 1) to characterize the toxicity of a food ingredient following prolonged and repeated exposure, and 2) to determine toxicological dose-response relationships needed to establish the maximum dose that produces no adverse effects (i.e., NOEL or NOAEL). The following guidance is written primarily for rats or mice, if other non-rodents are used, modifications to the guidance may be necessary. Sponsors/submitters of petitions/notifications are encouraged to also become familiar with the Guidelines for Reporting Results of Toxicity Studies (Chapter IV.B.2.), Pathology Considerations in Toxicity Studies (Chapter IV.B.3.), Statistical Considerations in Toxicity Studies (Chapter IV.B.4.), during the development of study design.
Nonclinical laboratory studies discussed in this chapter should be conducted according to U.S. FDA good laboratory practice (GLP) regulations, issued under Part 58 of Title 21 of the 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 or DC area 202-512-1800). 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.
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:
Guidance contained within this chapter is 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. The petitioners/notifiers should consult with the Agency scientists before toxicity testing has begun if they have questions about the appropriateness of a particular species, strain, or substrain.
C. Age (start of dosing):
Dosing of rodents should begin after weaning, and following a suitable acclimation period of at least 5 days, and before they are approximately 6-8 weeks old.
D. Number and Sex:
Both experimental and control groups should have at least 20 rodents per sex per group. 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 therapeutic agent 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 FDA requests that they provide a full and detailed description of the justification for study continuation 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 with sufficient accuracy 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 of the species 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-restricted,  or low-protein diets.  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 chronic toxicity 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 FDA 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 animals.
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 4°C and 8°C), 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.
The test substance used in chronic toxicity 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 (e.g., either a single component or a mixture of components) should be known. The petitioners/notifiers are encouraged to consult with the Agency regarding the method(s) of determination of the test substance, and should 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:
The expiration date of the test material should be known and easily available. Test materials should not be used past their expiration date.
A. Duration of Testing:
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 test substance 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. It is best to adjust the volume every 1-3 days based on the animal’s body weight response. 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 substance 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:
Dose selection for chronic toxicity studies should be based on results from subchronic studies and other related information. Appropriate dose selection should enable one to predict the maximum dose that produces no adverse effects, also known as the NOEL or NOAEL. A rationale should be provided for the dose selection. Administration of the test substance to all dose groups should be done concurrently (see section II.I: Assignment of Control and Compound Treated Animals).
A concurrent control group of test animals fed the basal diet is necessary. 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 group of treated animals. Sufficient toxicological 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 carrier or 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.)
2. Selection of Treatment Doses:
We recommend that a minimum of three dose levels of the test substance should be used in chronic toxicity studies. The following is a general consideration in selecting the treatment dose levels: 1) the high 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 biologically significant toxic responses in test animals; and 3) the intermediate dose 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).
We do not recommend that petitioner/notifiers use information unrelated to the toxicity of the test substance as a basis for dose selection of chronic toxicity studies. For example, the highest dose in a chronic toxicity 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.
The high dose in a chronic toxicity study should produce toxicity so that a toxicological profile of the test substance can be obtained. When no toxicity is observed in other studies, however, the high dose could be subject to some preset limits such as the highest percent of the test substance in the diet that could be fed without compromising nutritional balance with other nutrients (e.g., about 5%, see also ‘section II.H: Diet’ for other important dietary issues).
In general, the high dose tested 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 high dose has become increasingly clear. For example, data from a subchronic (90-day) study concerning changes in body and organ weight and clinically significant alterations in neurological, hematological, urinary and clinical chemistry measurements, in combination with more definitive exposure-related toxic, gross or histopathologic endpoints, can be used to estimate the high dose in a chronic toxicity study.
Although the high dose in a chronic toxicity study should be selected to achieve toxic responses in test animals, and should not cause fatalities high enough to prevent meaningful evaluation of the data from the study, the Agency recognizes that this goal may not always be met. In situations such as these, when it is unclear what dose of the test substance is the high dose, the petitioner/notifier should consult with the Agency to determine an appropriate high dose for the chronic toxicity study.
The low dose level should not interfere with the normal growth, development, and lifespan of test animals, nor should it produce any other biologically significant signs of toxicity (e.g., NOEL or NOAEL).
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.
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 principles. The FDA has endorsed the use of the Standard for Exchange of Nonclinical Data (SEND) format for electronic transmission of animal study data. You are encouraged to contact the Agency for more information on this electronic protocol.
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 or palpable tumors develop, the following parameters should be recorded; time of onset, location, dimensions, appearance and progression.
An expanded set of clinical evaluations performed on animals inside and outside of the cage, should be carried out to enable detection not only of general signs of departure from normal activity, morbidity and mortality but also of neurologic disorders, behavioral changes, autonomic dysfunctions, and other signs of nervous system toxicity. Specific information about the systematic clinical tests/observations is contained in Chapter IV.C.10. This expanded set of clinical examinations, conducted on animals inside and outside the cage, should be age appropriate and performed on all animals at least once prior to initiation of treatment, and periodically during treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions or other evidence of autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Additionally, changes in gait, posture and response to handling, as well as the presence of clonic or tonic seizures, stereotypic (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilating, walking backwards) should be recorded. During the course of a study, toxic and pharmacologic signs may suggest the need for additional clinical tests or expanded post-mortem examinations.
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 test substance on experimental 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 including tumor formation. 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 substance 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 as mg/kg body weight/day.
C. Clinical Testing:
Ophthalmological examination, hematology profiles, clinical chemistry tests, and urinalyses should be performed as described in the following sections:
1. Ophthalmological 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 all animals during the first 2 weeks of study, 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) are observed that are of concern at the 12-month measurement and the study lasts longer than one-year, additional hematological testing should be conducted at the end of the study.
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 all animals 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) are observed that are of concern at the 12-month measurement and the study lasts longer than one-year, additional clinical chemistry testing should be conducted at the end of the study.
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)
- Protein (total)
- 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 day. 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 study. If data trends or significant parameter changes (biological or statistical) are observed that are of concern at the 12-month measurement and the study lasts longer than one-year, additional urinalyses testing should be conducted at the end of the study. These tests should be performed on all animals.
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 at minimum include the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, prostate, 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
Histological evaluation of the lymphoid organs should be performed as described in the section on immunotoxicity testing (see Chapter V.D. of the 1993 draft Redbook II). A recent publication provides further discussion on this subject.
1 Chhabra, R.S., J.E. Huff, B.S. Schwetz, and J. Selkirk, An Overview of Prechronic and Chronic Toxicity/Carcinogenicity Experimental Study Designs and Criteria Used by the National Toxicology Program, Environmental Health Perspectives, 86:313-321, 1990.
2 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.
3 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.
4 Keenan 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.
5 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.
6 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.
10 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.
11. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation Research, Draft Guidance for Industry on Computerized Systems Used in Clinical Trials; Availability, The Federal Register, 69(191):59239-59240, October 4, 2004.
13. U.S. National Academy of Sciences (NAS), National Research Council (NRC), Institute of Laboratory Animal Resources, Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, D.C., 1996.
14. U.S. NAS, NRC, 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.
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