GUIDANCE DOCUMENT

Redbook 2000: IV.C.8. In-Utero Exposure Phase for Addition to Carcinogenicity Studies or Chronic Toxicity Studies with Rodents July 2007

Final

Issued by:: Guidance Issuing Office

Human Foods Program

Toxicological Principles for the Safety Assessment of Food Ingredients

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This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.

Good Laboratory Practice
Test Animals
Test Substance
Experimental Design
Observations and Clinical Tests
Necropsy and Microscopic Examination
References

Scientifically justified changes to 1993 "draft" Redbook version of this section have been made after consulting with other authoritative guidelines[3] [4] [5] [10] [11] [12] [19] [20] [22] [23]and publications (see the relevant sections below).

The FDA recommends including an in-utero exposure phase in a carcinogenicity or a chronic toxicity study conducted with rodents for the safety assessment of potential 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 animal toxicity studies recommended in this chapter are designed to determine whether a test food ingredient has early developmental effects that may increase the incidence of cancers and/or chronic disease outcomes (e.g., altered glucose tolerance, diabetes mellitus, cardiovascular disorders) when administered in regularly repeated oral doses for the duration of the study in the test animals.

An in-utero exposure phase should be added to one of the two recommended rodent carcinogenicity studies (or bioassays; see Chapter IV.C.6.). In general, the in-utero phase should be added to a bioassay study with rats since the rat is the recommended species for reproduction studies (see Chapter IV.C.9.a.) and the FDA has a larger database on carcinogenicity bioassays with in-utero exposure in rats than in mice. When chronic toxicity studies are the only long-term studies in support of the safety of a food ingredient, the FDA recommends on a case-by-case basis that an in-utero exposure phase be added to at least one of the studies.

The purpose of this chapter is to provide specific guidance for the design and conduct of an in-utero exposure phase addition to bioassay or chronic toxicity studies of food ingredients. However, these general procedures may also be applied to a combined chronic toxicity/carcinogenicity study or shorter-term toxicity studies with modifications (e.g., duration, dose, etc). The FDA encourages petitioners and/or notifiers to consult with the appropriate FDA scientists before toxicity testing has begun if they have questions about the appropriateness of adding an in-utero exposure phase to any of these studies. 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.

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 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.

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 [24] 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. Strains selected should not have low fecundity and should be sensitive to teratogens and embryotoxins. 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 FDA encourages petitioners/notifiers to consult with the appropriate FDA scientists before toxicity testing has begun if they have questions about the appropriateness of a particular species, strain, or substrain.

Another Center within the FDA (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 studies.[20] 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 test substances (i.e., constituents and/or contaminants in food ingredients), 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 Methods [16]) 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 important to provide the chronic safety testing of food ingredients that would be representative of lifetime exposure in humans, in addition to also providing that only quantitative data derived from fully validated test systems be used in their safety assessment.

C. Age (start of dosing):

Following a suitable acclimation period of at least 5 days, parental animals should receive the test substance. Females should receive the test substance for a minimum of four weeks prior to mating and males should receive the test substance for at least ten weeks prior to exposure to cover the full spermatogenic cycle. Dosing of all test and control pups (F1) should begin at weaning (see also section IV.A: Duration of testing).

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 effects including 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 FDA recommends that petitioners/notifiers carefully consider their choice of rat strains for bioassays or chronic toxicity studies, since some strains have more serious problems with survivorship than other strains. It is recommended that these studies begin with at least 70 animals per sex per group. Petitioners and/or notifiers should begin bioassays with more than 70 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 (e.g., 1-year or longer in chronic study and 2-years in bioassay, 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 FDA as soon as a problem with survivorship in a carcinogenicity or chronic study becomes apparent.

One male and one female per litter are preferred; no more than two males and two females per litter should be included in any group. For example, if the petitioner decides that each group should contain 70 animals per sex, at least 70 litters/group should be produced in the in-utero phase. Thus, for this example the number of parental animals per sex for the in-utero phase should be sufficient to ensure at least 70 litters 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. Mating Procedures:

For each mating, a female should be placed with a single randomly selected male from the same dose group until pregnancy occurs or two to three weeks have elapsed. Animals should be separated as soon as possible after evidence of copulation has been observed. If mating has not occurred after two to three weeks, the animals should be separated without further opportunity for mating. Mating pairs should be clearly identified in the data. Sibling matings should be avoided. Each morning, all females should be examined for the presence of sperm in the vaginal lavage or the presence of a vaginal plug; if sperm and/or a vaginal plug are found, this is considered day zero of gestation. Near parturition, pregnant females should be caged separately in delivery or maternity cages that contain nesting materials. Pregnant females in test and control groups should be allowed to litter naturally.

F. Standardizing the Number of Pups per Litter:

Standardization of the number of pups per litter through culling is optional. Litters may be standardized to 10 or 8 based on historical litter size for the strain. It is recommended that standardization be performed on postnatal day 4 by reducing all litters of more than 10 to 10 (or more than 8 to 8) in a random manner. If possible, the retained litter-mates should consist of equal numbers of males and females; excess males or females should be randomly selected out. Random selection is important to guard against the human tendency to keep the fit animals in the study.

G. Selection of Pups (F1):

One animal per sex per litter, or up to two animals for single sex litters, should be randomly selected.

H. 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 FDA 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.

I. 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).

J. Caging:

Animals should be single-caged during the study, except during mating and lactation. 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.

K. Diet:

In general, feed and water should be provided ad libitum, and the diets should meet the nutritional requirements to support pregnancy in the test species as well as of the species for normal growth and longevity.[25] 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 FDA is also aware of some beneficial effects on the survivability of certain animal species that have been on calorie-restricted, [7] [8] or low-protein diets. [1] [15] The FDA 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 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.[25]

L. Assignment of Control and Compound Treated 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.

M. Mortality:

Excessive mortality due to poor animal management is unacceptable and may be a cause to repeat the study.

N. Autolysis:

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.

O. Necropsy:

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 or chronic toxicity studies with an in-utero exposure phase 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.

A. Identity:

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 FDA regarding the method(s) of determination of the test compound, and should provide all relevant Chemical Abstract Service (CAS) Registry numbers.

B. Composition/Purity:

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.

IV. Experimental Design

A. Duration of Testing:

The parental animals should receive the test substance starting at a minimum four weeks (or ten weeks exposure is preferable for males to cover full spermatogenic cycle) prior to mating. Exposure should be continued throughout pre-mating, mating, gestation, and lactation until the F1 animals have been weaned. Dosing of all test and control F1 animals should begin at weaning, and continue for 7 days per week for the duration of the study (e.g., 1-year or longer in chronic study and 2-years in bioassay).

In general, the FDA does not recommend early termination of carcinogenicity studies due to decreased survivorship (see discussions under 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 FDA believes there is more benefit, as well as added sensitivity, to be gained by conducting carcinogenicity bioassays for as long as possible, or for no longer than the full 24 months that is recommended in this guidance.

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 FDA 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:

1. Controls:

A concurrent control group of test animals fed the basal diet is necessary for all studies. 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 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.K: Diet.)

2. Selection of Treatment Doses for Carcinogenicity Studies with an in-utero exposure phase:

It is recommended that a minimum of three dose levels of the test substance be used in carcinogenicity bioassays with an in-utero exposure phase. As a result of maternal or fetal toxicity, it may be necessary to use lower doses during the in-utero phase of the studies in order to produce sufficient offspring for the post-weaning phase. Data justifying this protocol modification should be provided; it is recommended that pilot studies be performed to select doses. Results from metabolism and pharmacokinetic studies should also provide guidance in selecting an appropriate dosage regimen.

When designing and conducting carcinogenicity bioassays with an in-utero exposure phase 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 discussions under section II.L: 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 with an in-utero exposure phase based on information unrelated to the toxicity of the test substance. For example, the highest dose 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.

This guidance recommends that the highest dose in carcinogenicity bioassays with an in-utero exposure phase should be the MTD. FDA 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 bioassays. The bioassays 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".[21] 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.".[13] 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.[21]

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 neurological, 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 with an in-utero exposure phase should be selected to achieve the MTD, the FDA 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 FDA to determine an appropriate high dose (MTD) for the carcinogenicity bioassay with an in-utero exposure phase.

The FDA recognizes that use of the MTD in carcinogenicity bioassays with an in-utero exposure phase 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 studies. [9]

The FDA acknowledges that its recommendation to conduct carcinogenicity studies with an in-utero exposure phase 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,[6] [26] the FDA concludes that the MTD is still the best choice for selecting the high dose for carcinogenicity studies even with an in-utero exposure phase. It should be noted that this is also in line with the principles discussed by the International Conference on Harmonization which recommends the use of the MTD in choosing the high dose for drug safety testing.[4] [5]

Low Dose:

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.

Intermediate Dose:

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.

3. Selection of Treatment Doses for Chronic Toxicity Studies with an in-utero exposure phase:

It is recommended that a minimum of three dose levels of the test substance be used in chronic toxicity studies with an in-utero exposure phase. Dose selection should be based on results from subchronic studies and other related test substance information (i.e., metabolism and pharmacokinetic studies). As a result of maternal or fetal toxicity, it may be necessary to use lower doses during the in-utero phase of the chronic-feeding studies in order to produce sufficient offspring for the post-weaning phase. Data justifying this protocol modification should be provided.

The FDA acknowledges that it is complicated and difficult to conduct a combined chronic toxicity/carcinogenicity rodent study with an in-utero exposure phase due to difficulty in setting and administering appropriate dose levels for both types of studies concurrently. However, when pre-chronic studies provide reasonable estimates of toxicity to predict the information on treatment doses to be used in a single bioassay, a chronic toxicity study may be combined with a carcinogenicity study with an in-utero exposure. It is recommended that the petitioner/notifier consult with the FDA before conducting a combined study.

The following is a general consideration in selecting the treatment dose levels for chronic toxicity studies with an in-utero exposure phase: 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).

High Dose:

The high dose in a chronic toxicity study should produce toxicity so that a toxicological profile of the test substance can be obtained. We do not recommend that petitioner/notifiers use information unrelated to the toxicity of the test substance as a basis for dose selection. For example, the highest dose 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. 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.

Low Dose:

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).

Intermediate Dose:

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.[18] 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 FDA for more information on this electronic protocol.

V. Observations and Clinical Tests

A. Observations of Parental Animals:

Routine cage-side observations of all parental animals should be made for general signs of departure from normal activity, morbidity and mortality once or twice a day until F1 animals are weaned. 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.

If reproductive parameters (e.g., fertility index, gestation length, gestation index, live-born index, etc) are collected, they should be included in the study report.

B. Observations of F1 Animals:

These animals should be observed carefully for signs of departure from normal activity, morbidity and mortality at least twice daily throughout the study period. The usual interval between multiple periods of observations should be at least 6 hours. Observations of general appearance and the presence of dead pups should be recorded. The total number of pups per litter and the number of pups per sex should be recorded. 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.

In a chronic toxicity (or combined chronic/carcinogenicity) study with an in-utero exposure phase, an expanded set of clinical evaluations should be carried out on animals inside and outside of the cage 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 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.

C. 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. 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.K: Diet and IV.B: Route of Administration.

Parental animals should be weighed immediately before the first dose of the test substance is administered, and weekly throughout gestation and lactation. If the substance is given by gavage, animals should be weighed every 1-3 days. Feed consumption should be measured weekly. Water consumption should be measured weekly if the test substance is administered in the water.

Body weights for all F1 animals should be recorded weekly for the first 13 weeks after weaning, 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 experimental 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 as mg/kg body weight/day.

D. Clinical Testing:

Ophthalmological examination, hematology profiles, clinical chemistry tests, and urinalyses should be performed in all F1 animals as described in the following sections:

1. Ophthalmological Examination:

This examination should be performed by a qualified individual on all F1 animals during the first 2 weeks of study 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 F1 animals in the study.

2. Hematology:

Hematological tests should be performed on at least ten F1 animals per sex per group during the first 2 weeks of study, 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, an 18-month measurement should be included.

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 ten F1 animals per sex per group during the first 2 weeks of study, 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, an 18-month measurement should be included.

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 be 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

Albumin
Calcium
Chloride
Cholesterol (total)
Cholinesterase
Creatinine
Globulin (calculated)
Glucose
Phosphorous
Potassium
Protein (total)
Sodium
Triglycerides
Urea nitrogen
The FDA 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. [2] 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.

4. Urinalyses:

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[14] during the first 2 weeks of study, 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, an 18-month measurement should be included. These tests should be performed on at least ten F1 animals per sex per group.

VI. Necropsy and Microscopic Examination

A. Gross Necropsy

Termination of Parental and F1 Animals not Selected for the Post-Weaning Phase

These animals should be killed after selection of the F1 animals to be continued on studying. If toxic signs or reproductive toxicity are observed, these animals should be subject to a complete gross necropsy.

Termination of F1 Animals Selected for the Post-Weaning Phase

All of these F1 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.

Adrenals
Aorta
Bone (femur)
Bone marrow (sternum)
Brain (at least 3 different levels)
Cecum
Colon
Corpus and cervix uteri
Duodenum
Epididymides
Esophagus
Eyes
Gall bladder (if present)
Harderian gland
Heart
Ileum
Jejunum
Kidneys
Liver
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
Pancreas
Pituitary
Prostate
Rectum
Salivary gland
Sciatic nerve
Seminal vesicle (if present)
Skeletal muscle
Skin
Spinal cord (3 locations: cervical, mid-thoracic, and lumbar)
Spleen
Stomach
Testes
Thymus (if present)
Thyroid/parathyroid
Trachea
Urinary bladder
Vagina
Zymbal's gland
All tissues showing abnormality

D. Microscopic Evaluation

All gross lesions should be examined microscopically. All tissues from the F1 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 both parental and F1 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.D. of the 1993 draft Redbook II). A recent publication provides further discussion on this subject.[17]

VII. References

1 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.

2 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.

3 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.

4 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.

5 ICH, Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline, S1C: Dose Selection for Carcinogenicity Studies of Pharmaceuticals, 1994.

6 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.

7 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.

8 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.

9 McConnell, E.E., The Maximum Tolerated Dose: the Debate. Journal of the American College of Toxicology, 8(6):1115-1120, 1989.

10 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.

11 OECD, Guideline for Testing of Chemicals, No. 451, Carcinogenicity Studies, Adopted in 1981.

12 OECD, Guideline for Testing of Chemicals, No. 452, Chronic Toxicity Studies, Adopted in 1981.

13 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.

14 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.

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 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.

17 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.

18 U.S. Department of Health and Human Services (DHHS), Food and Drug Administration (FDA), Center for Drug Evaluation Research (CDER), Draft Guidance for Industry on Computerized Systems Used in Clinical Trials; Availability, The Federal Register, 69(191):59239-59240, October 4, 2004.

19 U.S. DHHS, National Toxicology Program (NTP), Descriptions of NTP Study Types, 2-Year Study Protocol, 2004.

20 U.S. DHHS, FDA, CDER, Guidance for Industry, S1B Testing for Carcinogenicity of Pharmaceuticals, 1997.

21 U.S. DHHS, NTP, Board of Scientific Counselors, Report of the NTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation, 1984.

22 U.S. Environmental Protection FDA (EPA), Office of Prevention, Pesticides and Toxic Substances (OPPTS), Health Effects Test Guidelines, OPPTS 870.4200: Carcinogenicity, 1998.

23 U.S. EPA, OPPTS, Health Effects Test Guidelines, OPPTS 870.4100: Chronic Toxicity, 1998.

24 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.

25 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.

26 U.S. NAS, NRC, Issues in Risk Assessment, National Academy Press, pp.15-19 & 43-96, 1993.

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