CVM GFI #53 Evaluation of the Utility of Food Additives in Diet Fed to Aquatic Animals May 1994

GUIDANCE DOCUMENT

• Docket Number: FDA-2021-D-0618
• Issued by: Center for Veterinary Medicine

Revised May 1994

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
CENTER FOR VETERINARY MEDICINE

Contents

1. Introduction
2. General Considerations
   1. Protocol Development
   2. Record Keeping
   3. Analytical Assays
   4. Nutritional Content and Preparation of Experimental Diets
   5. Physical Characteristics of Diets
   6. Experimental Procedures
      1. Design
      2. Animals
      3. Feeding
      4. Loading
      5. Laboratory Trials
      6. Field Trials
   7. Investigator Report
3. Definitions
4. References

I. Introduction

A food additive cannot be shipped legally in interstate commerce until a food additive regulation is promulgated by the U.S. Food and Drug Administration (FDA). The basis for a food additive regulation is a food additive petition (FAP) that contains data demonstrating, among other things, the safety and utility of the use of proposed additive. This guideline delineates the types of evidence that may be used by sponsors to demonstrate the utility of food additives used in aquaculture (see food additive definition in Section III).

This guideline is not an exhaustive source of information on this topic. Manufacturers and scientists are encouraged to consult the Federal Food, Drug, and Cosmetic Act, particularly sections 201 (Definitions), 301 (Prohibited Acts), and 409 (Food Additives), and the regulations in Code of Federal Regulations 21 CFR 570.17 (investigational food additive) and 21 CFR 571 (food additive petitions). Sponsors of food additives are also encouraged to consult the Guideline on the Conduct of Clinical Investigations: Responsibilities of Clinical Investigators and Monitors for Investigational New Animal Drug Trials, for additional technical guidance.

Guidelines state practices or procedures that may be useful but are not legal requirements. The guideline represents the agency's position at the time of issuance. A person may follow the guideline or may choose to follow alternate practices or procedures. If a person chooses to use alternate practices or procedures, that person may wish to discuss the matter further with the agency to prevent an expenditure of money and effort on activities that may later be determined to be unacceptable to FDA. The guideline does not bind the agency, and it does not create or confer any rights, privileges, or benefits for or on any person. When a guideline states a requirement imposed by statute or regulation, however, the requirement is law and its force and effect are not in any way changed by virtue of its inclusion in the guideline.

FDA will modify this guideline as needed. We are open to constructive suggestions which will make the guideline a more meaningful tool for developing data for FDA preclearance of aquaculture food additive products.

II. General Considerations

A. Protocol Development

The logical starting point in any experiment is a clear understanding of the questions that need answering. The Center believes the time and effort put into planning an experiment should be a rigorous component of the food additive process. There is no regulatory requirement that sponsors of food additives submit their experimental protocols to CVM, and CVM does not "approve" protocols. However, the CVM strongly encourages submission of protocols prior to initiation of the studies. The sponsor should allot sufficient time for CVM comment and discussion of all issues in the design, well in advance of initiation of the studies.

The Center recognizes the diversity of food additive types. Consequently, protocols may reflect idiosyncrasies in experimental design unique to the product. Regardless of uniqueness, utility should be demonstrated through a combination of laboratory and field trials conducted with the target species. Sponsors are strongly urged to utilize this guideline when developing protocols. For specific guidance regarding antimicrobial food additives, sponsors should consult the "Guidelines for Utility Studies for anti-Salmonella Chemical Food Additives in Animal Feeds."

B. Record Keeping

The integrity of the data collected and reported is a critical component in determining the utility of a food additive. The data should be collected and managed such that they are valid. Valid data should have the following characteristics: 1) They should be signed and dated by the person making the observation entry; 2) They should be original (i.e., they should be the first recording of the observations); 3) They should be legible; 4) They should be contemporaneous; and, 5) They should be accurate. All data should be recorded in a permanent manner. Each original data sheet should be signed by the person making the observation and recording the data. If data are collected electronically, proper system controls should be employed to ensure that data have all characteristics above mentioned in order to be considered valid. Additional information on investigator record-keeping and record retention can be found in the Guideline on the Conduct of Clinical Investigations: Responsibilities of Clinical Investigators and Monitors for Investigational New Animal Drug Trials.

C. Analytical Assays

There must be assurance that the specific amount of the tested additive has been incorporated into the feed. A quantitative approach is necessary to provide this assurance, and it is achieved by chemical analysis. The results of chemical analysis are usually available as a set of numbers representing replicate determinations made under (as near as possible) identical conditions. These numbers might correspond to percentage purity, etc. The interpretation of a raw data set answers two questions: (1) What is the best estimate of the true value of the quantity being measured? and (2) How reliable is the number as an estimator of the true value?

Every analytical procedure possesses three main attribute categories: applicability, reliability, and efficiency. The purposes of the analytical procedure are to determine the composition of matter and the identity of its components and their concentration. The reliability and confidence of these determinations should be stated. The depth and breadth of the description of these attributes depends upon the intended use of the method, i.e., research, regulatory, or screening. In most cases the research method becomes the regulatory one. The following parameters should be evaluated by the petitioner:

1) Applicability - The method of measurement should be specific for the analyte (s) in question. The method should be able to separate or resolve the analyte (s) from other substances and interfering compounds, i.e., it must be selective. Every method has limits of measurement and they should be described in terms of limit of detection and limit of determination (quantitation). The range of use and measurement of the method should be described. In addition, the following questions should be asked: (a) What commodities or matrices will the method be applied to; (b) What is the concentration range of the analyte to be measured; (c) Is the method linear over this concentration range; and (d) Is the method susceptible to interference?

2) Reliability - This parameter addresses the accuracy of the measurement system. Accuracy is described according to its systematic (bias) and random (imprecision) errors. Random error is described as repeatability or analyst-to-analyst, and day-to-day variation, and reproducibility, or lab-to-lab variation. The method should be shown to be rugged or optimized, and under control.

3) Efficiency - The method should be easy and cost efficient to run. The level of training and safety should be addressed.

Once the error associated with the system of measurement is quantified, the system must be validated in a matrix that is representative of field use. After that, the petitioner should demonstrate that the active material can be adequately mixed in a fish ration to be pelleted or extruded. This is best done by mixing a production batch of feed, taking many samples of the feed throughout the process and quantifying the analyte by multiple analysis. This approach is valid also for the research feed batch prepared in the laboratory. In addition, the petitioner should demonstrate the additive stability in the market container, and in the feed. A stability protocol should be developed and CVM concurrence obtained. Additional information on validation of analytical methods can be found in the CVM guideline entitled "NRSP-7: Recommendations for Evaluating Analytical Methods."

D. Nutritional Content and Preparation of Experimental Diets

The Center believes that the experimental data should be collected on animals consuming nutritionally adequate diets, except when the additive is a nutrient, so that observed responses are attributable to the food additive rather than a possible nutritional interaction or to confounding effects due to inadequate or excessive amounts of certain nutrients. Nutrient recommendations for aquatic animals, published by the National Academy of Sciences, National Research Council, can serve as a reference for dietary formulation. However, sponsors should be aware that nutritional information for aquatic animals is being developed at a relatively rapid rate (e.g. Cowey et al. 1985, Lovell 1988, Halver 1989, Takeda and Watanabe 1990). New research findings, particularly those having a logical connection with the food additive, should be included in the experimental design. As an alternative, diets may be formulated to meet predominant commercial practices for the species and class of animal being fed. Diets should be formulated and manufactured with those ingredients currently in use with the target species.

Experimental diets should be prepared from a basal diet which is uniform with regards to nutrient densities and composition. If the basal diet is derived from several mixer batches, the diet corresponding to each treatment group should be composed of aliquots from each mixer batch. For example, if 3000 pounds of each experimental diet is needed and the mixer capacity is only 3000 pounds, the schedule in Figure 1 should be followed. In this example, only two levels of the additive (zero and 1X proposed use level) are used.

When individual batches are fed separately, each batch should be equally divided among treatment groups. All treatments should be switched from batch 1 to batch 2 feed, from batch 2 to batch 3 feed, etc. at the same time. Also, the experimental diets should be switched at the same time across treatment groups (e.g., from starter to grower feeds). Additionally, diets used in related laboratory and field studies should contain similar nutrient densities. The procedures used to prepare the basal diet and collect feed samples should be described in the protocol.

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Figure 1. Example of procedure for development of an uniform basal diet for a food additive study.

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For the three batches, three thousand pounds of feed is mixed, and divided into three (3) aliquots of 1000 pounds. The aliquots will be assigned randomly to the two experimental groups and one reserve (spare) group. The aliquot assigned to the reserve group will be used only if necessary.

	Diet 1	Diet 2	Reserve
Batch 1	000 pounds	1000 pounds	1000 pounds
Batch 2	1000 pounds	1000 pounds	1000 pounds
Batch 3	1000 pounds	1000 pounds	1000 pounds
Totals	3000 pounds	3000 pounds	3000 pounds

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Diet 1- Obtained by combining the three 1000-pound aliquots. No food additive is mixed with this diet. The diet is pelleted (see note below), assayed, and bagged.

Diet 2 - Obtained by combining the three 1000-pound aliquots. The combination is mixed with the food additive ( 1X proposed use level), then pelleted (see note below), assayed, and bagged.

Reserve - Obtained by combining the three 1000-pound aliquots and then remixed (extra diet).

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NOTE: Substitute extruded for pelleted where appropriate.

Proper mixing of feedstuffs and additives will ensure the uniform dispersal of nutrients and food additive(s) in finished feeds. The sponsor and/or investigator should be cognizant of the performance capabilities of the feed mixer used to prepare the experimental diets. One method that can be used to measure mixer performance is a determination of the mixing time required to ensure that assays are either the lesser of a ten percent (or less) coefficient of variation, or two times the analytical variation of the selected assay (Behnke, 1991; Wicker and Poole, 1991). Each investigator should develop standard operating procedures to prevent contamination of experimental feeds.

The form (pellet, extruded pellet, crumble, etc.) of the experimental diets should reflect the predominant conditions of use. It is important to conduct these studies using the predominant feed form because the milling process may affect the utility of the food additive(s). Pelleted and extruded feeds are subjected to varying degrees of temperature and pressure, which may affect the stability of the food additive(s) and microbiological profile of the feed. Manufacturing conditions, particularly for extruded diets, should be closely monitored and reported.

To demonstrate the nutritional adequacy of the experimental diet(s), the sponsor should include in the protocol a complete and detailed list, including amounts therein, of all feeds and vitamin-mineral premixes used in compounding the basal diet. Feeds used in compounding should be those commonly used in each geographical location of production. At a minimum, the calculated nutrient levels of the uniform basal diet should be reported for crude protein, crude fat, lysine, arginine, methionine, methionine + cystine, tryptophan, vitamin C, and phosphorus (total and estimated available). If the food additive is known to interact, either positively or negatively, with other nutrients, then the levels of those nutrients should be reported as well. The sponsor should substantiate nutritional adequacy by demonstrating that the calculated nutrient levels meet the minimum requirement as established by the National Research Council or more recent authoritative source.

To ensure that the diets are within the formulated values specified in the protocols, chemical analyses should be performed on the uniform basal diet and all experimental diets after pelleting or extrusion. A composite sample from each diet, which is representative of the diet, should be assayed for the following; proximate components (crude protein, crude fiber, ash, crude fat and moisture), lysine, methionine, cystine, arginine, tryptophan, phosphorus, vitamin C, and degree of lipid oxidation. The exact number of analytical replicates should be specified in the protocol. The sponsor should indicate whether analyses are reported on an as-fed or dry matter basis and provide sufficient information to determine key nutrient levels on either basis.

The Center recommends using a 90% confidence interval to determine the adequacy of the diet. The Center considers the confidence interval approach to be a means by which the sponsor and/or investigator can be assured that the diets are nutritionally adequate and acceptable to the Center for the nutrients assayed. Nutrient concentrations that fall outside the confidence interval are not necessarily unsatisfactory to the Center. The Center will consider the magnitude of the deviation from the confidence interval and nutritional circumstances when the adequacy of the diet is determined.

The confidence interval approach can be applied in the following manner. The sponsor should select a laboratory that will conduct the chemical analyses of the manufactured feed. Once a laboratory has been selected, the sponsor should obtain the coefficient of variation associated with each laboratory assay procedure. The coefficient of variation and a reference to the methodologies for each assay should be stated in the protocol.

The 90% confidence interval (CI) for a diet formulated to contain a theoretical (calculated) nutrient concentration is calculated as follows:

• CI = X ± t(alpha/2, infinity) *(CV * X),

where,

• t(alpha/2, infinity) = t(.10/2, infinity) = 1.645
• CV - coefficient of variation associated with the chemical assay, and
• X = theoretical (calculated) nutrient concentration.

For example, a CI for a diet formulated to contain 0.50% dietary methionine, given a hypothetical coefficient of variation of 12%, would be as follows:

• CI = 0.50% methionine ± 1.645 x (0.12 x 0.50), or
• CI = 0.50% methionine ± 0.10%.

Therefore, nutrient concentration for methionine may range from 0.40 to 0.60% and will be acceptable to the CVM. Because it is equal to t (0.10/2,infinity), the coefficient of variation used should be based on a large number of samples (greater than 120).

For products labeled for a specific dietary nutrient concentration, the chemically determined nutrient concentration should fall within the confidence interval. When a single assay is conducted, the assay should fall within the confidence interval, as given above. Because multiple assays of a single feed sample provide additional information on the nutrient concentration, the assay values should be averaged and the CI should be adjusted. This is necessary because the CI concept is predicated on a single assay value. In this situation, the sponsor can apply the adjusted CI method to the data. This procedure will verify that the population from which the assay values are collected is similar with respect to the mean and variance as the population used to establish the assay coefficient of variation. The coefficient of variation used in the confidence interval should be adjusted as follows:

• Adjusted CV = CV/n,

where,

• CV = the coefficient of variation, and
• n = the square root of the number of assays conducted on a single feed sample.

E. Physical Characteristics of the Diet

Water stability evaluation of finished feeds, whether of the sinking or the floating form, should be conducted after manufacturing and should include physical and nutritional characterization. Chemical composition of the water used should be thoroughly characterized and reported. Changes in nutrient levels (particularly dry matter, crude protein, crude fat, crude fiber, ash, phosphorus, and vitamin C), and food additive should be recorded from samples removed from the water at 1, 5, 10, 20, and 40 minutes. If a feed contains crystalline amino acids, then their concentrations should also be determined. These studies should be replicated in water at optimal temperature for the target species and at 5°C above and below that temperature. If the target species is euryhaline, then these studies should be conducted in freshwater, brackishwater and saltwater (i.e., 0, 6-10 and 30-40 ppt salinity, respectively). If the target species is stenohaline on either extreme (i.e., either strictly freshwater or saltwater), then evaluations might appropriately be conducted only in the salinity likely to be encountered in commercial settings.

Other food additives, whose intended effect may be on the pellet rather than the target species (e.g., mold inhibitors, antioxidants, etc.), should be evaluated in such a manner that resulting data are scientifically valid and convincing in their claim of utility. It is best for sponsors to submit protocols to the Center for comment as discussed in the Introduction to this document. Whether the intended effect is on the pellet rather than the target species, feeding trials should also be conducted. The combined physical diet characterization and feeding data will help insure the Center of the utility of the additive as well as the effect of feeding it to the target species.

F. Experimental Procedure

All studies designed to evaluate the utility of food additives in diets fed to aquatic animals should follow all applicable local, state and federal regulations, including, but not limited to, environmental regulations regarding discharge of effluents from experimental sites. All personnel involved with the investigations should have adequate scientific training and experience with the species used in the experiments. The personnel responsible for the day-to-day management of the animals and for making and recording observations ideally should be blinded to the experimental treatments. The blinding procedures should be described in the protocol.

1. Design
   A number of different experimental designs have been used in nutritional studies with aquatic animals. Thus, no single design is recommended. Again, sponsors are encouraged to submit experimental protocols to CVM prior to initiation of any study and wait for CVM comments before starting the experiments. Salient points to consider include the following: 1) studies should be conducted in a scientifically valid manner: 2) If a block experiment, blocks should be homogenous with respect to environmental conditions, except in field trials in which environmental conditions will vary; 3) If a Latin Square experiment, it should be completely replicated: and 4) At least three replicates of each experimental group should be used in all evaluations. All procedures relating to experimental design, assignment of animals and treatments, and statistical analyses should be described in the protocol. A completely randomized design should be considered for the evaluation of food stability and leaching of nutrients.

   The protocols should include diagrams of the buildings and identify any blocking and treatment allocation schemes used. Those diagram(s) should be specific for each location and include the following: orientation of the building, type of building, ventilation and lighting systems, dimensions of experimental units, location of feeder, and location of aeration supply.

   The number of studies that will be conducted should be specified in the protocol prior to initiation of any experimentation. For field trials, a minimum of three studies representing different geographical locales should be conducted.

2. Animals
   Studies should be conducted using the target animal for which the food additive is intended. The animals used should be from contemporary, commercial genotypes. Healthy juveniles or adults representing the target age group, sex and species should be used in the experiments. The source of the animals should be documented. The age and health of the broodstock from which the animals originated should also be described. Detailed records should be kept on acquisition of animals, including past health records from the site of origin. Transportation to the experimental site should be designed to minimize stress. Procedures used during transportation should be those found in commercial situations. Typical conditions are withholding feed prior to transport, careful attention to water temperatures, maintenance of adequate dissolved oxygen levels during transport, use of only approved chemicals, and loading rates that do not result in excessive stress. Any compounds used during this period should be documented, including dosages, frequency of application, and any observed responses. After arrival at the experimental site, all animals should be quarantined for a minimum of three weeks in holding units separate from those to be used in the experiment. All observations must be reported in the FAP.

   Stresses outside the scope of the experiment should be minimized. Animals should be handled gently at all times. During the experiment, animals should be observed as frequently as required by good management techniques. The frequency of observation should be specified in the protocol. The Center recognizes that most aquatic animals are not domesticated, and losses of animals may occur. However, those losses, when possible, should be thoroughly evaluated to determine causes and a complete report of the gross necropsy findings and histopathological examination, by a qualified professional, should be included with the FAP. Bacteriology, virology and toxicology reports should be included in those situations that warrant the additional evaluations.

   Vaccination programs, if warranted, should be designed to protect the animals against prevalent infectious disease (s), yet not debilitate the animals or otherwise compromise the experiment. Information concerning any vaccination program should be provided (i.e., method of administration, date of vaccination, age at vaccination, source of vaccine, lot number, type of vaccine, handling of vaccine, expiration date and any other pertinent information).

   Gender of animals should be determined, minimally, on completion of the field trials by post-mortem examination. A subsample of animals is appropriate in large-scale studies, but the procedures for selecting samples should be explained. While determining gender is impractical in some laboratory studies because the animals are too young to have developed testes or ovaries, sex determinations should be attempted in all phases of the evaluations.

3. Feeding
   Feed allotments should be restricted in laboratory studies and ad libitum in field trials. Restricted feed rates are those calculated and expressed as a percent of body weight offered on a daily basis. The rate should be calculated on the basis of dry weight of feed and wet weight of animal. Ad libitum feeding means offering food until all of the animals appear satiated. The Center recognizes that this can vary among sites and individuals judging satiation. Thus, it is important to provide data indicating feed consumption when using this feeding approach. This comparison will allow more precise information regarding effective levels and extrapolation of those data to commercial situations. The Center recognizes the impracticality of restricted feeding studies with juveniles of some species (e.g., fish, crustaceans, mollusks, etc., which, in commercial settings, are generally offered feed in excess and at frequent intervals) and recommends feeding those species in a manner similar to practical situations.

   If automatic feeders are used in field trials, they should be maintained in such a way as to minimize feed wastage and calibrated as closely as possible. If demand feeders are used in field trials, estimates of voluntary consumption should be collected on a regular basis to allow for identification of changes in consumption among treatment groups or as the studies progress.

4. Loading
   An experimental unit is the physical container holding a group of aquatic animals. Common experimental units are aquaria, tanks, cages, net pens, raceways and ponds. Consistent methods should be used in placing the fish in the experimental units to ensure the accuracy of the number of fish per unit. The protocol should include a description of those methods. Extra animals should not be placed in the experimental units. Fish which die during the experiment should not be replaced.

   The loading rate is established by determining the amount (in weight) of fish that can be reared in a experimental unit which receives a known water flow (e.g. gallon/minute). Factors that may influence the loading rate include water quality, water temperature, water volume, rate of flow, rate of change, and kind and size of fish held. The loading rate should be at a level that is considered to elicit no adverse effects on the target species. If appropriate loading rates are not known for the target species, then those considered appropriate for a closely related species should be used. It is imperative that the loading rate be maintained during the study. This may be done, for example, by adjusting the water flow in proportion to the changes in total weight of the fish in an experimental unit.

5. Laboratory Trials
   In the laboratory studies, the animal husbandry techniques used should be designed to minimize stress of the animals and promote maximum weight gain. While the Center recognizes that each species is distinct, some general areas need to be considered in the experimental design and those should be adequately addressed in the protocol. Water quality values should be maintained at levels that will not elicit any adverse effect on the animals. While those values could range widely due to varying susceptibility of the target species, some common considerations are provided. Dissolved oxygen levels vary with temperature, salinity and altitude. Regardless of conditions, dissolved oxygen levels should be maintained at all times at 90% of saturation and monitored at least daily in the experimental units. Nitrogenous compounds (particularly ammonia-N and nitrite-N) should be maintained below levels that elicit adverse response from the target species. If detailed toxicological studies have not been conducted with the target species, then un-ionized ammonia-N and nitrite-N levels should be maintained below 0.02 mg/L (Thurston et al., 1979) and 0.4 mg/L, respectively. Levels of nitrogenous products, including nitrate-N, should be monitored in such a way as to assure minimal chance of transient increases that might not be recorded. For example, experimental systems that have established records of successful studies with the target species would be preferred. Submission of water quality records, historical values if available and those from the actual study, should be submitted with the FAP. Other water quality variables should be closely monitored. Those include hardness (total-, calcium-, and magnesium hardness), alkalinity (total-, carbonate- and bicarbonate alkalinity), pH and carbon dioxide. As with the nitrogenous compounds, values should be determined on a regular basis and the schedule adopted should be described. Temperatures should be maintained at near optimal for the target species and not vary more than ± 1°C from the target temperature. Studies should be conducted for a sufficient period of time to define intended effects. Appropriate time frames for laboratory studies will vary depending on species and food additive, but should be consistent with similar studies published in the peer-reviewed literature.

6. Field Trials
   The size of experimental unit used in field trials should be the same as those used commercially. Further, sites of field trials should be in several areas throughout the range of production of the target species. For example, sites near the center of current production, and at the extremes should be included. With aquatic animals, a range of sites in which water quality (temperature, hardness, etc.) values may vary would provide more detailed information about the food additive in a variety of commercial situations. This approach should be seriously considered in the design. Animals should be grown until they reach marketable size, based on current practices at the experimental site, or until environmental conditions (e.g., temperatures at which the target species stops growing) warrant cessation of the experiment. Stocking densities should be those found in commercial practices in the area and should be reported. In addition to the water quality values mentioned above (laboratory trials), other water quality variables which are considered important in field trials should also be monitored. Those include algal and zooplankton population and species composition, and turbidity. If warranted, pesticide and heavy metal levels should be determined in tissues of aquatic animals from field trials. The water quality collection schedule should be adequately described in the protocol. The CVM recognizes the daily changes in water quality in some production systems for aquatic animals. However, water quality variables should be maintained in a range considered to have no adverse effect on the target species. Protocols for monitoring and maintaining water quality should be carefully documented.

   Additional studies should be conducted if the target species is commercially raised in a variety of production systems, or if the sponsor intends to label the food additive as appropriate for the target species raised in different production systems. For example, if tilapia are the target species and the sponsor desires to include the additive in diets fed in earthen ponds, flow-through tanks, and recirculating systems, then additional studies should be conducted in the distinct production systems.

G. Investigator Report

An investigator should prepare and submit to the sponsor upon completion of each of the investigator's study(ies) a signed and dated, detailed, independent report that evaluates all observations made during the study. The first report submitted to the sponsor is the investigator's "final" report. Any and all subsequent changes to the report should be considered as amendments. The final report should include, but not be limited to, the following:

1. The name, physical location, and mailing address of the investigator and the specific facitity(ies) where each study was performed.
2. The dates on which the study was initiated and completed.
3. The objectives and procedures stated in the study protocol, which should include any changes from the original protocol. A description of all management practices used, which should include a record of all observations. Sufficient detail should be provided to allow reconstruction or replication of the study(ies).
4. The number, species, stock or strain, source of supply, gender if known, size and age, or any change in the health condition of the animals used should be specified. The identification procedures, if used, and disposition records for each animal should be provided.
5. A description of the dosage regimen, and duration of the treatment should be given. Records of the disposition of the unused food additive or animal feeds containing the additive should be provided.
6. Records of all mixing or further dilution of the food additive, and results of the assays in the feed.
7. All adverse reactions observed during the study.
8. A description of all circumstances that may have affected the quality or integrity of the data. This should include specifying the time frame and the extent of the occurrences.
9. The name of the study monitor, associates, colleagues, and employees involved, and the nature and extent of their participation.
10. The location of all original copies of source data, specimens, samples, and study records should be reported.
11. A statement attesting to the accuracy and completeness of the data, which should include a statement acknowledging that the data were collected in compliance with the Federal Food, Drug, and Cosmetic Act.
12. A copy of all source data.

III. Definitions

Adults are defined as a life history stage characterized as time from sexual maturity to expiration.

Animal Feed is defined in the Federal Food, Drug, and Cosmetic Act, Section 201 (321) (x) to mean "an article which is intended for use for food for animals other than man and which is intended for use as a substantial source of nutrients in the diet of the animal, and is not limited to a mixture intended to be the sole ration of the animal."

Aquatic animals are defined as those vertebrates or invertebrates that spend the majority of their life in water regardless of their mode of respiration. Petitions submitted to the Center for approval of food additives in animals that may spend a significant portion of their lives out of water, but could be considered aquatic animals by conventional classification schemes (e.g., alligators, land crabs, newts, etc.), should generally adhere to these guidelines with any appropriate modifications in experimental design or procedures unique to the animal in question.

Coldwater is defined as a temperature range of 0-17°C

Color Additive is defined in the Federal Food, Drug, and Cosmetic Act (FFDCA), Section 201 (321) (t) (1-3) to "mean a material which:

(1)(A) is a dye, pigment or other substance made by a process of synthesis or similar artifice, or extracted, isolated, or otherwise derived, with or without vegetable, animal, mineral, or other source, and

(B) when added or applied to a food, drug, or cosmetic, or to the human body or any part thereof, is capable (alone or through reaction with other substance) of imparting color thereto: except that such term does not include material which the Secretary, by regulation, determines is used (or intended to be used) solely for the purpose or purposes other than coloring.

(2) the term "color" includes black, white and intermediate grays.

(3) Nothing in subparagraph (1) of this paragraph shall be construed to apply to any pesticide chemical, soft or plant nutrient, or other agricultural chemical solely because of its effect in aiding, retarding, or otherwise affecting, directly or indirectly, the growth or other natural physiological processes of produce of the soil and thereby affecting its color, whether before or after harvest."

Coolwater is defined as a temperature range of 18-25°C.

Food additive is defined in the Federal Food, Drug, and Cosmetic Act (FFDCA), Section 201 (s) as "any substance the intended use of which results or may reasonably be expected to result, directly or indirectly, in its becoming a component or otherwise affecting characteristics of any food (including any substance intended for use in producing, manufacturing, packing, processing, preparing, treating, packaging, transporting, or holding food; and including any source of radiation intended for any such use), if such substance is not generally recognized, among experts qualified by scientific training and experience to evaluate its safety, as having been adequately shown through scientific procedures (or, in the case of a substance used prior to January 1, 1958, through either scientific procedures or experience based on common use in food) to be safe under the conditions of its intended use; except that such term does not include:

(1) a pesticide chemical in or on raw agricultural commodities; or

(2) a pesticide chemical to the extent that it is intended for use or is used in the production, storage, or transportation of any raw agricultural commodity; or

(3) a color additive; or

(4) any substance use in accordance with a sanction or approval granted prior to the enactment of this paragraph pursuant to this Act, the Poultry Products Inspection Act (21 U.S.C. 451 and the following) or the Meat Inspection Act of March 4, 1907 (34 Stat. 1260), as amended and extended (21 U.S.C. 71 and the following); or

(5) a new animal drug."

Euryhaline has been defined as those aquatic animals that are capable of normal physiological and biochemical processes (e.g., survival, growth, osmoregulation, reproduction, etc.) in a wide variety of salinities.

Juvenile is defined as a life history stage characterized as the time aquatic animals first consume exogenous food material to the onset of sexual maturity.

Larval is defined with some species to describe early life history stages (prior to complete development of juvenile characteristics).

Stenohaline is defined as those aquatic animals that are capable of normal physiological and biochemical processes (e.g., survival, growth, osmoregulation, reproduction, etc.) in a very narrow range of salinities.

Utility is defined as the effectiveness of the product in meeting claims for labeling.

Warmwater is defined as a temperature range of 26 °C and higher.

IV. References

Behnke, K.C. 1991. Feed uniformity and mixer evaluation. Delmarva Poultry Nutrition Short Course. University of Delaware.

Cowey, C.B., A.M. Mackie, and J.G. Bell. 1985. Nutrition and feeding in fish. Academic Press, New York, New York.

Halver, J.E, editor. 1989. Fish Nutrition, second edition. Academic Press, New York, New York.

Lovell, T. 1989. Nutrition and feeding of fish. Van Nostrand Reinhold, New York, New York.

National Academy of Sciences. 1981. Nutrient requirements of coldwater fish. National Academy of Sciences, Washington, D.C.

National Academy of Sciences. 1983. Nutritional requirements of fish and shellfish, revised edition. National Academy of Sciences, Washington, D.C.

Takeda, M. and T. Watanabe. 1990. The current status of fish nutrition in aquaculture. Japan Translation Center, Ltd., Tokyo, Japan.

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