Guideline No. 33
Target Animal Safety Guidelines for New Animal Drugs
Revised June 1989
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
CENTER FOR VETERINARY MEDICINE
Prepared by:
Office of New Animal Drug Evaluation
Center for Veterinary Medicine Food and Drug Administration
JUNE 1989
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
TABLE OF CONTENTS CHAPTER I: INTRODUCTION A. Good Laboratory Practice (GLP) Regulations B. Statistical Considerations C. Animal Food Additive Safety D. Definitions E. Utility of Laboratory Animal Toxicity Studies and Effectiveness Studies F. Final Report CHAPTER II: GENERAL PRINCIPLES CHAPTER III: DRUG TOLERANCE TEST CHAPTER IV: OBJECTIVES OF TOXICITY STUDIES CHAPTER V: TOXICITY STUDIES FOR DOGS AND CATS CHAPTER VI: TOXICITY STUDIES FOR HORSES CHAPTER VII: TOXICITY STUDIES FOR RUMINANTS CHAPTER VIII: TOXICITY STUDIES FOR SWINE CHAPTER IX: TOXICITY STUDIES FOR POULTRY
Prior to the approval of a new animal drug for marketing, the sponsor must show that the drug is safe for use as recommended in the proposed labeling [21 CFR 514.1(b)(8) and section 512(d) of the Federal Food, Drug, and Cosmetic Act (the Act)]. These guidelines were prepared to aid sponsors in developing protocols for safety experiments to meet the Food and Drug Administration's (FDA) requirements for animal drugs.
These guidelines cover many different parameters. The specific information needed for a product depends upon such factors as proposed use regimen, type of product, chemistry, species, breed and/or class of animals, claims, dosage, and/or previous use history. All data mentioned in these guidelines would not necessarily need to be collected for every product. Under special circumstances information not specified in these guidelines may be required for some products. For these special circumstances the requirements should be determined by communication between the sponsor and the Center for Veterinary Medicine (CVM
These guidelines are intended to provide general directions for acquiring essential information pertinent to the evaluation of safety for the target animal species and to promote uniform review of submissions by the Food and Drug Administration. They should remain flexible to allow scientific discretion in the design and conduction of studies which will yield the maximum information on a product.
To use these guidelines to the best advantage, the sponsor should review the applicable sections and develop a protocol for collecting animal safety data. Appropriate protocols can be developed by discussion and agreement between personnel representing the sponsor and CVM. It is recommended that the protocol be submitted to the appropriate review group in the Office of the Associate Director for New Animal Drug Evaluation (HFV-100), Center for Veterinary Medicine 7500 Standish Place, Rockville, MD 20855, for comments and discussion before studies begin.
Existing data on the physiologic, pathologic and toxicologic properties collected from studies conducted with animals, including humans, should be used to focus upon the requirements for toxicity studies of the drug product in the target animal.
It is the position of the Center for Veterinary Medicine that animal testing should derive the maximum amount of useful scientific information using the minimum number of animals necessary. Consideration should be given to the use of accepted alternative methods to whole animal testing. Attempts should be made to eliminate or minimize the degree and duration of suffering in the animals that are used. Pain-relieving medication, including anesthetics, should be considered and employed when such drugs will not interfere with the nature and purpose of the testing. Euthanasia of moribund animals should be considered and employed when the procedure will not interfere with the nature and purpose of the testing. The euthanasia procedure employed should comply with the recommendations of the American Veterinary Medical Association Panel on euthanasia (Journal of the American Veterinary Medical Association, 1986, Vol. 188, No. 3, pp. 252-268, or succeeding revised editions
A. Good Laboratory Practice (GLP) Regulations
Nonclinical laboratory studies in target animals shall be conducted in accordance with GLP regulations, i.e., 21 CFR Part 58.
Nonclinical studies which are relevant to animal safety determinations are subject to the GLP regulations. Since animal husbandry requirements often differ between laboratory animals and domestic animals, FDA does not require that domestic animals, including poultry, be maintained under the same conditions as laboratory animals. The regulations include terms such as "when applicable" and "as required" for those situations where differences in acceptable husbandry practices exist. Guidance may be found in "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching" (available from American Society of Animal Science, 309 West Clark Street, Champaign, IL 61820 Issues regarding animal use and maintenance should be resolved with the FDA during the protocol development stage.
Each nonclinical laboratory study contained in the new animal drug application must be accompanied by a statement declaring whether or not the study was conducted in compliance with the GLP regulations. If the study was not conducted in compliance with such regulations, the statement must describe, in detail, differences between the practices used and those required in the regulations. Although clinical studies contribute data relative to the overall safety assessment of a drug product, GLP regulations do not apply to clinical studies.
B. Statistical Considerations
Studies should be adequately designed, well-controlled and conducted by qualified investigators in order to provide a meaningful inference to the pertinent population under study. When applicable. studies should be blinded to the investigator, toxicologist, pathologist, etc. Treated and control animals should have similar characteristics and should be handled identically except for drug exposure. An experimental unit is the smallest unit in which the treatment application is randomized or varied. The sampling unit is the unit in which the treatment effect is measured. Hence, depending upon the nature of the experiment, the experimental unit might be the individual animal or an entire pen of animals.
Experimental units should be randomly assigned to the treatment groups. The appropriate number of experimental units per treatment and the number of times the experiment should be repeated will depend upon such things as the variability of the observations, the size of the differences among the various treatment groups that the experimenter would like to be able to detect, as well as the significance level and power of the statistical tests employed. In any event, it is the firm's responsibility to ensure that the sample sizes employed are adequate for detecting a difference among the treatment groups in the study with a reasonably high statistical power, if in fact such a difference were actually to exist in the population under study. The sponsor should obtain FDA's concurrence regarding an acceptable power for the study.
When special tests, e.g., gross or microscopic pathology, are to be performed on a subset from each dosage group, animals should be randomly selected prior to beginning an experiment to avoid selection bias. There should be agreement between FDA and the sponsor regarding the number of animals to include in the subset (three is suggested as the minimum number for a subset
The sponsor should discuss these matters with their statistician during the planning phase so that an adequate design can be achieved. Type and design of study vary with the type of drug product being tested.
In some instances statistical analyses can be important in identifying toxicological differences, especially with large data sets. Any preliminary statistical analyses should be agreed upon before they are conducted. When statistical analyses are appropriate, the selection of statistical methods should include a consideration of whether variables will be repeated measurements or single end point measurements. Where appropriate, baseline data should be used to adjust the analyses of data collected during the treatment period for pretreatment differences. There should be agreement between the sponsor and CVM on presentation of the data, especially requirements for tables and graphs. The sponsor must recognize that the purpose of preliminary statistical analyses is to identify parameters which may indicate toxicity. Results of these analyses will require further interpretation, and additional statistical analyses may be required. The sponsor is encouraged to discuss the possibility and means of submitting data and reports electronically (as well as in hard copy
Before initiating any controlled animal safety studies for Investigational New Animal Drug (INAD) or New Animal Drug Application (NADA) submissions, it is suggested that the protocol and the proposed methods of statistical analysis be submitted to FDA for comment. When submitting studies for review, data summaries and complete details of statistical methods utilized, including references, should be provided. This information will facilitate the review.
C. Animal Food Additive Safety
Therefore, these guidelines do not apply per se to animal food additive studies.
D. Definitions
Maximum Tolerated Dose (MTD) - The maximum dose of a test compound that can be administered without overt adverse effect.
Therapeutic Index (Margin of Safety) - The ratio between the effective dose (clinical/optimum dose) and the toxic dose.
Toxic Dose - The lowest dose of a test compound at which the test animal exhibits toxic signs.
Acclimation Period - Time allowed for the animals to become acclimated to the new environment in the test facility.
Baseline Period - Time period in which baseline data are collected.
Baseline Data - Information collected after the acclimation period and before the administration of the drug. This information is used to compare and deduce the effects caused by administration of the test compound.
All prophylactic or other treatments administered for purposes outside of the study protocol should be given at a sufficient time prior to the baseline observation period to allow for possible carry-over effects to subside. Any exceptions for providing treatment during the study should be explained in the protocol.
Treatment in the event of complicating diseases should be avoided, and diseased animals should be removed from the study, if possible. It may be necessary to terminate the study and repeat it with other animals.
Test Period - The time period during which data are collected. This includes baseline, treatment and the post-treatment periods.
Adverse Effect - An undesirable effect reflected in the animal by alterations in structure, function, or behavior. The observations must focus upon drug toxicity and/or sensitivity reactions. If possible, a distinction should be made between reversible and irreversible effects.
Test Animals - Healthy animals (of the species, breed, sex, age, and size for which the drug is intended) that have not been used in previous experiments must be used except in rare situations where use of diseased animals is required. The most sensitive class/breed should be tested when the drug will be used in them.
Diseased animals will not be required routinely for safety studies. The field efficacy studies will be reviewed for safety of the animal drug in diseased animals. Drugs with a narrow margin of safety, which are intended for use in diseased and severely debilitated animals, may require safety studies using animals afflicted with the appropriate disease.
E. Utility of Laboratory Animal Toxicity Studies and Effectiveness Studies
The data requirements to demonstrate the safety of an animal drug in target animals depends upon the amount of historical information that is available on the subject drug, especially the toxicity studies conducted in laboratory animals. Other information that can be used to determine target animal toxicity study requirements includes the following: pharmacokinetic data, pharmacologic action, metabolism studies, treatment regimen, tolerance test in target animal species, and other relevant information. Further, the aforementioned information on a new animal drug is a factor considered in the need for, as well as the rigor of, other target animal safety studies such as the reproduction studies, teratology studies, and multigeneration studies.
Data collected from non-target animals can be used to focus studies in target animals by identifying possible target tissues and potential functional changes. However, the non-target animal studies cannot be used to replace studies in target animals.
Target animal toxicity studies do not require the collection of data on the entire list of tissues and tests contained in these guidelines simply because the tissues and tests are identified in the guidelines. Sound scientific judgment should be used to determine what tissues should be examined and what tests should be conducted.
The classical LD50 test is not required by FDA for determining safety, and its use is not part of agency testing policy. The FDA's full policy regarding the LD50 test is described in the Federal Register of October 11, 1988 (53 FR 39650
F. Final Report
Record keeping is essential and it begins with a protocol which delineates the objectives of the study and outlines the experimental design and methods.
The final report should include:
a. Identification of the animal
b. Nature and severity of disease
c. Date of first observation and duration of disease
d. Nature of treatment and dates
e. Outcome of treatments
Animals used for testing should represent the species in which the drug will be used. The most sensitive breed/class of animal should be selected for testing; however, it will not be necessary to test all breeds/classes of a species. They usually should be free of any disease and not exposed to environmental conditions which could interfere with the purpose or conduct of the study.
Additional experimental parameters should be included in the animal safety studies when they might reveal suspected adverse properties of the test product, e.g., a known species sensitivity to the test product or related drug product, suspected enzyme inhibition, etc.
Animals should be properly acclimated to the study environment.
Studies should be adequately designed, well controlled, and conducted by qualified investigators to provide meaningful results with respect to the total class or animal population.
The product to be evaluated should be identical to the product intended to be marketed, i.e., same chemical, same particle size, and formulation. Discussion between the sponsor and CVM prior to use of an alternative drug product is recommended.
The route(s) of administration should be the same as proposed by labeling; however, the nature of some studies may require modifications in the route of administration, e.g., drench in lieu of medicated feeds.
In order to minimize autolytic decomposition a necropsy should be performed promptly after death on all animals that die during the study. The necropsy should be performed by a qualified person.
A complete physical examination should be performed, and baseline data should be collected by a qualified person. Data should be obtained prior to the start of the trial and at reasonable, predetermined intervals thereafter in accordance with the study protocol.
Clinical observations should be recorded twice daily, seven days a week, during the entire study period, or as provided in a study protocol that is acceptable to CVM.
Appropriate clinical pathologic procedures should be conducted on all test groups. This is required on all animals in each group or, when appropriate, on a representative number (usually one half or a previously agreed upon number) of animals preselected at random from each group at predetermined intervals, as provided in the protocol.
When the studies include histologic examination, tissues should be collected and preserved from all animals or a representative number (usually one half or a previously agreed upon number) from each group as provided in the protocol (if the number of the test animals is large Tissues from test animals in all groups should be collected and saved. Tissue from the highest treatment level and from control groups should be examined histologically. If microscopic lesions are observed, corresponding tissues from the next lower treatment group should be examined until a no-observable-effect-level is determined.
Appropriate diagnostic tests, vaccinations, prophylactic and therapeutic treatments should be completed prior to the baseline period of the test period. Exceptions should be included in the study protocol.
Toxicity studies should document the toxicity that may occur under representative conditions of use for a given drug product.
A. Introduction
The dose response curve of a product in animals is a dynamic continuum ranging from no effect to effective, to toxic effects, and to lethal effects. Our interest is focused upon the effective and toxic doses and the margin of safety represented by the difference between the two.
The drug tolerance test characterizes, under controlled conditions, the target animal response to a toxic dose(s) of a drug. This should be accomplished by inducing toxicity and recording the clinical sign(s) manifested by the test animals following administration of specific doses. The investigator should observe clinical signs, including changes in behavior and appearance, and gross lesions in animals which die during the test. This will provide a clinical picture for the toxic effects of a drug product. Clinical pathologic and histologic data should be collected. These data should reveal the physiological functions most readily affected, and this should aid in focusing the data requirements of the target animal toxicity studies. In many instances, data required to identify the tolerance to a product can be obtained from acute and subchronic toxicity studies.
Drug tolerance testing is required for drug products containing a new chemical entity, and for use of an approved new animal drug in additional animal species. In general, drug tolerance studies will not be required for drugs administered for localized activity (e.g., otic, ophthalmic, intramammary, intraarticular, etc. Conversely, this exemption does not apply to drugs for generalized dermal application and topically administered drugs which act systemically rather than locally.
Drug tolerance testing generally does not apply to:
Supplemental use of an approved new animal drug in the same animal species/class and at equivalent or lower doses.
Previously approved single ingredient drug products (generic applications
Combination drug products containing previously approved drug products provided that safety and efficacy data do not indicate a chemical or physiological reaction between the drug ingredients.
New salts of a moiety with a known toxic syndrome and/or widespread clinical use. Comparability studies should usually suffice for such drug products.
B. Test Design
a. Dosage form
The market formulation of the drug should be used. If this is not possible, the formulation used should be acceptable for adequate testing of the product.
b. Route of administration
The drug being tested should be administered by the recommended route(s) under the proposed conditions of use, unless a different route would be more useful in measuring the particular parameters of drug action. If volume or palatability becomes a limiting factor for administration, special techniques (gavage, intravenous, multiple sites, incremental doses, etc.) may be used. Depending upon the vehicle and the volume to be used, and special techniques to be employed, a vehicle control group may be required.
c. Dose
The characteristics of the drug and its proposed conditions of use are used to determine the dosage regimen.
If toxicity to the drug occurs at one of the levels in the toxicity study (described below), it will meet the requirement for a drug tolerance test. An alternative is to include a 10X treatment regimen in the toxicity study to meet the requirements of a tolerance test.
Studies should be conducted in healthy animals representative of the class/classes/breed(s) of animals (species, size, and age) for which the drug is intended. Generally, no more than four animals are required for the tolerance test. If the situation warrants the testing of fewer than four animals, a minimum of two animals is recommended.
Baseline values for parameters of drug action to be measured during the study should be determined prior to beginning the test. All prophylactic and therapeutic medication should be administered prior to this period so that they do not interfere with the study.
Test animals should be closely monitored to characterize the response to a toxic dose(s) of a drug. The types of observations and examinations depend upon the drug and class of animals being tested. Examples of some of the findings indicating drug toxicity are listed in Addendum No. 1 of each chapter for target animal species except poultry, which findings are listed in Chapter IX.
Clinical pathological tests should be conducted on all animals.
If the animals exhibit toxic signs to a drug, gross and histopathologic examinations should also be conducted. These data will aid in focusing the data requirements of the target animal toxicity study. The parameters to be measured depend upon the drug and class of animals being tested.
Examples of histopathologic examinations and clinical pathologic tests are found in Addenda No. 2 and 3 of each chapter for each target animal species except poultry, which are given in Chapter IX.
The objectives of toxicity studies in target animal species are to document:
A. Toxicity Study
In designing toxicity experiments, consideration should be given to historical data on use of the drug. A literature search should be conducted and combined with results of any preliminary experiments to determine the possible areas of drug toxicity. Drug entities should be characterized as to their chemical and pharmacological properties, and experiments should be designed and conducted that will determine the effect of these properties on the target animal. Testing should evaluate all known or possible problems.
a. Test drug
Dog and cat Safety studies should be conducted with multiple dose levels of the drug administered in the intended market formulation.
b. Test animals
Studies should be performed in healthy dogs and cats representative of the class(es) of dogs and cats (size, weight range, sex or age) for which the drug is intended. If a drug has a narrow margin of safety and is intended for use in severely debilitated dogs and cats, it may be necessary to conduct safety studies in dogs and cats afflicted with the disease for which the drug is indicated.
c. Duration of drug administration
For drugs recommended for long-term administration (15 days or longer), safety studies should be conducted with the drug being administered for the recommended maximum use duration, or longer (minimum of six weeks Drugs proposed for shorter periods of administration should be studied at least at 3X the recommended maximum duration of use.
d. Dose levels
The objective is to determine the margin of safety for the drug for the proposed duration of use.
Dose levels of drugs tested should include at least three levels plus controls (0-level) and they should provide a suitable dose spread. The dosing regimen should include the following experimental groups: Unmedicated--use level--intemediate drug level (higher than use level)--estimated toxic level of the drug; or multiples of the recommended use level--0, 1X, 3X, and 5X.
e. Route of administration
Route of drug administration should be the proposed route(s) of administration which will appear on the label.
f. Acclimation and baseline periods
Two weeks or longer may be required to acclimate test dogs and cats. All prophylactic and therapeutic medications should be administered prior to the baseline study period so that they will not interfere with the study. Baseline values should be established before the study begins. Evaluation of drug response parameters should continue until the study is terminated.
Documentation necessary to evaluate drug toxicity depends upon the type of drug action, potential for toxicity, proposed use of the drug, and class to which the dogs and cats belong.
Evaluation for signs of toxicity should include:
a. Feed and water consumption, clinical observations, and physical examinations (See Addendum No. 1
b. Clinical pathologic tests (Addendum No. 3) conducted either on all animals or on randomly preselected animals and on all dogs and cats showing signs of toxicity.
c. Gross pathologic examinations (Addendum No. 2) on dogs and cats preselected at random and on all animals that die. Histopathological examination of all grossly affected organs, and known or suspected target organs, based on laboratory animal toxicologic studies and other pertinent data.
B. Special Studies
a. Objectives
Reproductive studies should be conducted on both sexes to evaluate possible drug effects in the target species on fertility; ovulation and spermatogenesis; egg and sperm integrity; transportion, implantation and development of the zygote; parturition and the neonate; lactation, weaning, and care of the young; on delayed postnatal deviations and future breeding potential with special interest directed to the teratogenic, fetotoxic, and mutagenic potential of the drug.
The studies are divided into three segments, each of which pertains to a specific phase of the reproductive process.
In this segment, emphasis is placed on determining the effects of a given drug on gonadal function, estrous cycles, mating behavior, conception rates, and the early and middle stages of gestation.
Within this segment, the objective is to determine if a drug has embryotoxic or teratogenic effects. For this purpose, drug administration is restricted to the period of organogenesis.
The purpose of this segment is to study the effects of a drug administered during the last third of pregnancy and through the period of lactation to weaning. Studies should delineate the effects of the drug on late fetal development, labor- and delivery, abortion, lactation, neonatal viability, and growth of the newborn. This segment should also include studies designed to evaluate future breeding potential of young animals exposed to a drug in utero or through suckling.
(a) Study of fertility and general reproductive performances (see Addendum No. 5
For an adequate study of fertility, both males and females should be studied and evaluated. Results should be reported using appropriate reproduction indices.
Males should be treated during the 90-day period prior to mating and at appropriate times during the breeding period. Breeding soundness (semen) evaluations should be conducted just before breeding and at appropriate intervals.
For the study of drug action on female fertility treatment should be conducted through all phases of the estrous cycle (i.e., folliculogenesis, ovulation, pre-implantation, early and mid-gestation
(b) Teratogenic and embryotoxic studies
Drug administration should be repeated regularly (daily) and started early enough and continued long enough to cover the entire period of organogenesis.
(c) Prenatal and postnatal study
The period of drug administration to the dam should cover the final one-third of gestation and continue throughout lactation to weaning. Particular emphasis should be placed on observation of labor and delivery. Abortion, dystocia, agalactia, prolonged labor, and delayed labor are all possibilities to be observed and noted. The duration of gestation should be recorded.
If multiple generation studies are necessary, the offspring should be allowed to reach sexual and physical maturity. This would provide information on delayed effects of the drug on reproductive capacity.
The drug should be administered at a level at least 3X the recommended therapeutic dose; a control group should be included.
Criteria for malformation: The incidence and types of malformations should be recorded. The evaluation of malformations should be based primarily on the results derived from concurrent control groups; however, the incidence of malformations observed in the breed and species being studied should also be considered.
NOTE: All animals used for reproduction studies should be in good health and at optimum reproduction age. For dogs and cats, "this is usually between 1 and 5 years of age."
Multiple generation studies may be required for some drugs, e.g., (a) the drug is known to produce toxic effects following long-term use or (b) the product is recommended for lifetime prophylaxis.
Tissue irritation studies for injectable drugs should include data on the product vehicle and at least 2X the use level concentration of the active ingredient. The same volume of both preparations should be administered to all experimental groups. Consideration should be given to tissue inflammation, swelling, necrosis, etc. This information may aid in establishing the maximum amount of drug that can be injected per site.
a. Topicals
A vehicle control group should be included in the experiments. The drug product should be applied to an extensive area (greater than 10%) of the skin surface unless the drug is known to be very irritating or caustic and is intended for use on small surfaces. Higher concentrations, at least 2X the proposed concentration of the active ingredient, may be needed for some drug products. Depending on the chemical characteristics of the drug and its proposed use, skin irritation studies may be needed. The factors in Addendum No. 4 may be useful in evaluating skin reactions.
b. Intravaginal and/or intrauterine
Volume, concentration of active ingredient, and vehicles should be considered.
This includes absorption, distribution, bioaccumulation, and form and rate of excretion. These studies will be required for some drug products.
The need for additional toxicity studies on a drug combination will be based upon biochemical, chemical, and pharmacologic properties of each single drug. It will be necessary to show that the drugs are compatible in combination when used in dogs and cats.
Depending on the known or suspected properties of the test substances, it may be necessary to conduct additional toxicological tests.
Addendum No. 1
Physical Examinations
An examination should be conducted for the purpose of detecting
any abnormalities that may be drug related. Examples of some methods
and clinical observations which are useful for assessment of toxicity
and health are as follows:
Ocular Integument
Corneal opacities Alopecia
Nystagmus Haircoat condition
Pupillary changes Status of hydration
Blepharospasm Pruritus
Blindness Erythema
Examine eye and adnexa
Iritis Injection site
Chemosis
Photophobia Soreness
Congestion Swelling
Blanching Hardness
Discharge Persistence
Conjunctivitis Inflammation
Necrosis
Equilibrium
Body temperature
Unsteadiness on front legs
Unsteadiness on rear legs Gastrointestinal
Unsteadiness while standing
Unsteadiness while walking Consistency of stools
Incoordination of front legs Propulsive diarrhea
Incoordination of rear legs Hyperactive gut
Anterior ataxia or paresis Colic
Posterior ataxia or paresis Colic and rolling
Abnormal reflexes Abdominal muscles tense
Vomiting
Muscular disturbances Auscultation
Tremors of triceps Cardiovascular
Tremors of front legs
Tremors of rear legs Heart rate
Generalized tremors Heart rhythm
Lip drooping and/or salivation Color of mucous membranes
Paralysis Auscultation (heart
Atony sounds)
Atrophy Blood pressure
ECG
Appetite/General Health
Body weight
Feed consumption/time (amount)
Water consumption/time (amount)
Behavior (mental attitude) Respiratory
Anxious Respirations/minute
Apprehensive Dyspnea
Circling Respiratory sounds
Comatose Color of mucous membranes
Depressed Nasal discharge
Dorso-ventral recumbency Apnea
Lateral recumbency Tidal volume
Lateral recumbency - paddling
Sedated
Restless
Nose or lip wrinkled
Shaking head
Pressing head
Tongue hanging out (panting)
Convulsions
Aggression
Addendum No. 2 Pathology
Gross examinations for pathologic changes should be complete. All
organ systems should be examined and abnormalities should be
recorded. Organ weights should be recorded where relevant.
Histopathology
Histological requirements will depend upon what is known about
the drug being tested. It may not be necessary to examine all tissues
microscopically, but they should always be fixed (e.g., in buffered
formalin) and maintained. Generally, the following tissues should
be considered and specimens of appropriate tissues should be
examined:
Pituitary gland Mammary glands
Thyroid glands Liver
Parathyroid glands Gallbladder
Adrenal glands Kidneys
Pancreas Urinary bladder
Ovaries Heart
Uterus Large and small arteries
Testes Bone (with marrow)
Prostate Marrow smear
Epididymis Spleen
Thymus Stomach
Lymph nodes (cervical, mediastinal Duodenum
mesenteric)
Jejunum
Brain (cerebrum, cerebellum, mid
brain, brain stem) Ileum
Spinal cord (cervical, thoracal Colon
lumbar)
Cecum
Eyes
Other tissues as
Lung indicated by gross
lesions or by drug
activity
Muscle tissue at site of
intramuscular injection
Addendum No. 3
Clinical Pathology
Clinical pathologic tests will depend on the ruminant drug product
being tested. Generaly, the following parameters should be consid-
ered and appropriate clinical pathologic tests should be conducted:
Hematology
Complete blood count
Packed cell volume (PCV)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin concentration (MCHC)
Hemoglobin
Partial prothrombin time
Platelet count
Blood and Serum Chemistries
Amylase
AST (asparate aminotransferase)
ALT (alanine aminotransferase)
SAP (serum alkaline phosphatase)
Total and direct bilirubin
Glucose
Creatinine
Sodium
Chloride
Potassium
Phosphorus
Total protein
Albumin
Globulin
Calcium
BUN (blood urea nitrogen)
LDH (lactic dehydrogenase) with isoenzymes
Other useful tests include CPK (creatinine phosphokinase),
bicarbonate or blood pH, and GGT
(gamma glutamyl transpeptidase
Urinalysis
Visual observations--color, consistency, quantity
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Microscopic examination of formed elements
Fecal Examination
Blood or other signs of hemorrhage
Quantity, color and consistency
Excessive cellular debris
Biliary activity
Parasites
Other abnormalities
Addendum No. 4
EVALUATION OF SKIN REACTION
Erythema and Eschar Formation VALUE
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness to slight
eschar formation (injuries in depth) 4
Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well
defined by definite raising) 2
Moderate edema (raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and
extending beyond the area of exposure) 4
Severe eschar and/or corrosion Note
occurrence
(From, Acute Dermal Toxicity Test, Interagency Regulatory
Liason Group, Testing Standards and Guidelines Work Group, 1981.)
Addendum No. 5
Reproduction Indices
1. Fertility Index number of animals conceived
(conception rate) = --------------------------- X 100
number of total matings
2. Whelping Index = number of total live young
--------------------------- X 100
number of pregnant animals
3. Weaning Index = number of pups weaned
--------------------- X 100
number of pups whelped
A. Toxicity Study
In designing toxicity studies, consideration should be given to historical data on use of the drug. A literature search should be conducted and combined with results of any preliminary experiments to determine possible areas of drug toxicity. Drug entities should be characterized as to their chemical and pharmacological properties, and experiments should be designed and conducted that will determine the effect of these properties on the target animal. Testing should evaluate known or suspected problems.
a. Test drug
Animal safety studies should be conducted with multiple dose levels of the drug administered in the intended market formulation.
b. Test animals
Studies should be performed in healthy horses representative of the class(es)/breeds of horses (size, weight range, sex or age) for which the drug is intended. If a drug has a narrow margin of safety and is intended for use in severely debilitated horses, it may be necessary to conduct safety studies using horses afflicted with the disease for which the drug is indicated.
c. Duration of drug administration
For drugs recommended for long-term administration (15 days or longer), safety studies should be conducted with the drug being administered for the recommended maximum duration of use, or longer (minimum of six weeks Drugs proposed for shorter periods of use should be studied at least 3X the recommended maximum duration of use.
d. Dose levels
The objective is to determine the margin of safety for the drug for the proposed duration of use. Dose levels of drugs tested should include at least three levels plus controls (0 - level), and there should be a suitable spread in doses tested. The dosing regimen should include the following experimental groups: Unmedicated--use level--intermediate drug level (higher than use level)--estimated toxic level of the drug; or multiples of the recommended use level--0, 1X, 3X, and 5X.
e. Route of administration
Route(s) of drug administration should be the proposed route(s) of administration which will appear on the label.
f. Acclimation and baseline period
Two weeks or longer may be required to acclimate test horses. All prophylactic and therapeutic medications should be administered prior to the baseline study period so that they will not interfere with the study. Baseline values should be established before the study begins. Evaluation of drug response should continue until the study is terminated.
Documentation necessary to evaluate drug toxicity depends upon the type of drug action, potential for toxicity, proposed use of the drug, and class/breed(s) to which the horses belong.
Evaluation for signs of toxicity should include:
a. Feed and water consumption, clinical observations, and physical examination. (See Addendum No. 1
b. Clinical pathologic tests (Addendum No. 3) conducted either on all animals or on randomly preselected animals and on all horses showing signs of toxicity.
c. Gross pathologic examinations (Addendum No. 2) on horses preselected at random and on all animals that die. Histopathological examination of all affected organs and known or suspected target organs, based on laboratory animal toxicologic studies and other pertinent data.
B. Special Studies
a. Objectives Reproductive studies should be conducted on both sexes to evaluate possible drug effects on fertility in the target species; ovulation and spermatogenesis; the egg and sperm integrity; transportion, implantation and development of zygote; parturition and the neonate; lactation, weaning, and care of the young; delayed postnatal deviations and future breeding potential, with special interest directed to the teratogenic, fetoxic, and mutagenic potential of the drug.
The studies are divided into three segments, each of which pertains to a specific phase of the reproductive process.
In this segment, emphasis is placed on determining the effects of a given drug on gonadal function, estrous cycles, mating behavior, conception rates, and the early and middle stages of gestation.
Within this segment, the objective is to determine if the drug has embryotoxic or teratogenic effects. For this purpose, drug administration is restricted to the period of organogenesis.
The purpose of this segment is to determine the effects of a drug administered during the last third of pregnancy and through the period of lactation to weaning. Studies should delineate the effects of the drug on late fetal development, labor and delivery, abortion, lactation, neonatal viability, and growth of the newborn. This segment should also include studies designed to evaluate future breeding potential of young animals exposed to a drug in utero or through suckling.
(a) Study of fertility and general reproductive performances
For an adequate study of fertility, both males and females should be studied andevaluated. Results should be reported using the appropriate reproduction indices.
Males should be treated during the 90-day period prior to mating and at appropriate times during the breeding period. Breeding soundness (semen) evaluations should be conducted just before breeding and at appropriate intervals.
For the study of drug action on female fertility, treatment should be conducted through all phases of the estrous cycle (i.e., folliculogenesis, ovulation, pre-implantation, early and mid-gestation
(b) Teratogenic and embryotoxic studies
Drug administration should be repeated regularly (daily) and started early enough and continued long enough to cover the entire period of organogenesis.
(c) Prenatal and postnatal study
The period of drug administration to the dam should cover the final one-third of gestation and continue throughout lactation to weaning. Particular emphasis should be placed on observation of labor and delivery. Abortion, dystocia, agalactia, prolonged labor, and delayed labor are all possibilities to be observed and noted. The duration of gestation should be recorded.
For some drugs, it may be necessary to allow offspring to reach sexual and physical maturity. This would provide information on delayed effects of the drug on alterations of behavioral and reproductive functions and capacity.
The drug should be administered at a level at least 3X the recommended therapeutic dose; a control group should be included.
Criteria for malformation: The incidence and types of malformations should be recorded. An evaluation of malformations should be based primarily on the results derived from concurrent control groups; however, the incidence of malformations observed in the breed and species being studied should also be considered.
Tissue irritation studies for injectable drugs should include data on the product vehicle and at least 2X the use level concentration of the active ingredient. The same volume should be administered to all experimental groups. Consideration should be given to tissue inflammation, swelling, necrosis, etc. This information may aid in establishing the maximum amount of drug that can be injected per site.
a. Topicals
A vehicle control group should be included in the experiments. The drug product should be applied to an extensive area (greater than 10%) of the skin surface unless it is intended for use on small surfaces and is expected to be very irritating or caustic. Higher concentrations, at least 2X the proposed concentration of the active ingredient, may be needed for some drug products.
Depending on the characteristics of the drug and its proposed use, skin irritation studies may be needed. The factors in Addendum No. 4 may be useful in evaluating skin reactions.
b. Intra-articular
The system detects vertical forces. Horses are walked or jogged over the plate and characteristic curves for each limb are recorded on chart paper. The maximum amplitude for each tracing is measured and converted to kg by the use of a standard curve. Several replicate tracings are made of each limb at each recording session and the results are expressed as an average.
Observations and results are determined by clinical response and pre-injection and post-injection synovial fluid evaluation.
(a) Synovial fluid glucose levels
(b) Synovial fluid alkaline phosphatase
(c) Synovial fluid viscosity and hyaluronic acid content
(d) Cytology
Volume, concentration of active ingredient, and vehicles should be considered.
This includes absorption, distribution, bioaccumulation, and form and route of excretion. These studies will only be required for some drug products.
The need for additional toxicity studies on a drug combination will be based upon the biochemical, chemical, and pharmacologic properties of each single drug. It will be necessary to show that the drugs are compatible in combination when used in horses.
Depending on the known or suspected properties of the test substances, it may be necessary to conduct additional toxicological tests.
Addendum No. 1
Physical Examinations
An examination should be conducted for the purpose of detecting any
abnormalities that may be drug related. Examples of some methods and
clinical observations which are useful for assessment of toxicity
and health are as follows:
Ocular Appetite/General Health
Corneal opacities Body weight
Nystagmus Feed consumption/time
Pupillary changes Water consumption/time
Blepharospasm
Blindness Integument
Examine eye and adnexa
Iritis Alopecia
Chemosis Haircoat condition
Photophobia Status of hydration
Congestion Pruritus
Blanching
Discharge Injection site
Conjunctivitis Soreness
Equilibrium Swelling
Hardness
Unsteadiness on front legs Persistence
Unsteadiness on rear legs Inflammation
Unsteadiness while standing Necrosis
Unsteadiness while walking
Incoordination of front legs Body temperature
Incoordination of rear legs
Anterior ataxia or paresis Gastrointestinal
Posterior ataxia or paresis
Abnormal reflexes Consistency of stools
Propulsive diarrhea
Muscular disturbances Colic
Colic and rolling
Tremors of triceps Abdominal muscles tense
Tremors of front legs Perspiration (sweating)
Tremors of rear legs Auscultation
Generalized tremors
Lip drooping and/or salivation
Paralysis Cardiovascular
Atony Heart rate
Atrophy Heart rhythm
Color of mucous membranes
Auscultation (heart
sounds)
ECG
Blood pressure
Behavior (mental attitude) Respiratory
Anxious Respirations/minute
Apprehensive Dyspnea
Circling Respiratory sounds
Comatose Color of mucous membranes
Depressed Nasal discharge
Dorso-ventral recumbency Apnea
Lateral recumbency Tidal volume
Lateral recumbency - paddling
Sedated
Restless
Nose or lip wrinkled
Shaking head
Pressing head
Tongue hanging out (panting)
Convulsions
Reproductive
Mare
Rectal examination
Estrus - acceptance of the advances of the teaser stallion
Diestrus - resistance of the advances of the teaser stallion
Stallion
Time from presentation of mare to erection
Number of mounts per ejaculation
Addendum No. 2 Pathology
Gross examinations for pathologic changes should be complete. All
organ systems should be examined and abnormalities should be
recorded. Organ weights should be recorded where relevant.
Histopathology
Histological requirements will depend upon what is known about
the drug being tested. It may not be necessary to examine all
tissues microscopically, but they should always be fixed (e.g.,
in buffered formalin) and maintained. Generally, the following
tissues should be considered and specimens of appropriate
tissues should be examined:
Pituitary gland Mammary glands
Thyroid glands Liver
Parathyroid glands Gallbladder
Adrenal glands Kidneys
Pancreas Urinary bladder
Ovaries Heart
Uterus Large and small arteries
Testes Bone (with marrow)
Prostate Marrow smear
Epididymis Spleen
Thymus Stomach
Lymph nodes (cervical, mediastinal Duodenum
mesenteric)
Jejunum
Brain (cerebrum, cerebellum, mid
brain, brain stem) Ileum
Spinal cord (cervical, thoracal Colon
lumbar)
Cecum
Eyes
Other tissues as
Lung indicated by gross
lesions or by drug
activity
Addendum No. 3
Clinical Pathology
Clinical pathologic tests will depend on the ruminant drug product
being tested. Generaly, the following parameters should be consid-
ered and appropriate clinical pathologic tests should be conducted:
Hematology
Complete blood count (CBC)
Packed cell volume (PCV)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin concentration (MCHC)
Hemoglobin
Prothrombin time
Partial prothrombin time (PPT)
Platelet count
Blood and Serum Chemistries
Amylase
SDH (sorbital dehydrogenase)
Creatinine
Glucose
Calcium
Sodium
Chloride
Potassium
Phosphorus
ALT (alanine aminotransferase)
AST (asparate aminotransferase)
Total protein
Albumin
Globulin
Total and direct bilirubin
BUN (blood urea nitrogen)
Other useful tests include :
CPK (creatinine phosphokinase),
GLDH (glutamate dehydrogenase), and
GGT (gamma glutamyl transpeptidase
Urinalysis
Visual observations--color, consistency, quantity
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Microscopic examination of formed elements
Fecal Examination
Blood or other signs of hemorrhage
Quantity, color and consistency
Excessive cellular debris
Biliary activity
Parasites
Other abnormalities
Addendum No. 4
EVALUATION OF SKIN REACTION
Erythema and Eschar Formation VALUE
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness to slight
eschar formation (injuries in depth) 4
Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well
defined by definite raising) 2
Moderate edema (raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and
extending beyond the area of exposure) 4
Severe eschar and/or corrosion Note
occurrence
(From, Acute Dermal Toxicity Test, Interagency Regulatory
Liason Group, Testing Standards and Guidelines Work Group, 1981.)
A. Toxicity Study
In designing toxicity experiments, consideration should be given to historical data on use of the drug. A literature search should be conducted and combined with results of any preliminary experiments to determine the possible areas of drug toxicity. Drug entities should be characterized as to their chemical and pharmacological properties, and experiments should be designed that will determine the effect of these properties as they relate to animal safety. Testing should evaluate all known or suspected problems.
a. Test drug
Ruminant safety studies should be conducted with multiple dose levels of the drug given in the intended market formulation.
b. Test animals
Studies should be conducted in healthy ruminants representative of the species and class(es) of ruminants (size, weight range, sex, or age) for which the drug is intended. If a drug has a narrow margin of safety and is intended for use in debilitated ruminants, or the drug may more severely affect animals with a physiological imbalance (e.g., hormone imbalance), it may be necessary to conduct safety studies in ruminants afflicted with the disease for which the test drug is indicated and/or in ruminants affected with a physiological imbalance.
c. Duration of drug administration
For drugs recommended for long-term administration (15 days or longer), safety studies should be performed with the drug being administered for the recommended maximum use duration, or longer (minimum of six weeks Drugs proposed for shorter periods of administration should be studied at least at 3X the recommended maximum duration of use.
d. Dose levels
The objective is to determine the margin of safety for the drug for the proposed duration of use. Dose levels of drugs tested should include at least three levels, plus controls (0 - level The dosing regimen should include the following experimental groups: Unmedicated-use level--intermediate drug level (higher than use level)-- estimated toxic level of the drug; or multiples of the recommended use level--0, 1x, 3X, and 5X.
e. Route of administration
Route of drug administration should be the proposed route(s) of administration which is to appear on the label.
f. Acclimation and baseline periods
Two weeks or longer may be required to acclimate test ruminants, especially if a special diet or feeding regimen is intended. Prophylactic and therapeutic medications should be administered prior to the baseline study period so that they will not interfere with the study. Baseline values should be established before the study begins. Evaluation of drug response should continue until the study is terminated.
Documentation necessary to evaluate drug toxicity depends upon the type of drug action, potential for toxicity, proposed use of the drug, and the species and class of ruminants that will receive the drug.
Evaluation for signs of toxicity should include:
a. Periodic weighing, feed and water consumption, clinical observations, and physical examinations of animals performed in accordance with the protocol (Addendum No. 1
b. Clinical pathologic tests (Addendum No. 3) conducted either on all animals or on randomly preselected animals and on all ruminants showing signs of toxicity.
c. Gross pathologic examinations (Addendum No. 2) on ruminants preselected at random and on all ruminants that die. Examine histopathologically all grossly affected organs and known or suspected target organs, based on laboratory animal toxicologic studies and other pertinent data.
B. Special Studies
a. Objectives
Reproductive studies should be conducted on both sexes to evaluate possible drug effects on fertility in the target species; ovulation and spermatogenesis; egg and sperm integrity; transportation, implantation, and development of the zygote; parturition and the neonate; lactation, weaning, and care of the young; delayed postnatal deviations from normal and future breeding potential, with special interest directed to the teratogenic, embryotoxic, and mutagenic potential of the drug.
Studies are divided into three segments, and each pertains to a specific phase of the reproductive process.
Emphasis is placed on determining the effects of the drug on gonadal function, estrous cycles, mating behavior, conception rates, and the early and middle stages of gestation.
The objective is to determine if the drug has embryotoxic or teratogenic effects. For this purpose, drug administration is restricted to the period of organogenesis.
The purpose of this segment is to determine the effects of a drug administered during the last third of pregnancy, through the period of lactation to weaning. Studies should delineate the effects of the drug on late fetal development, labor and delivery, abortion, lactation, neonatal viability, and growth of the newborn. Studies should be designed to evaluate future breeding potential of young animals. These studies should include animals exposed to a drug in utero, through suckling or by other appropriate routes and methods of drug administration.
(a) Study of fertility and general reproductive
performances
The fertility of both males and females should be studied and evaluated. Results should be reported using the appropriate reproduction indices.
Males should be given the drug during the 90-day period prior to mating and at appropriate times during the breeding period. Breeding soundness (semen) evaluations should be conducted at appropriate intervals. Additional information may be found in, "Manual for Breeding Soundness Examination of Bulls," Journal of the Society for Theriogenology, Volume XII, February, 1983.
For the study of drug action on female fertility, treatment should be conducted through all phases of the estrous cycle (i.e., folliculogenesis, ovulation, pre-implantation, early and mid-gestation
(b) Teratogenic and embryotoxic studies
Drug treatment should be repeated regularly (daily) and started early enough and continued long enough to cover the period of organogenesis in the embryos.
(c) Prenatal and postnatal study
The period of drug administration to the dam should cover the final one-third of gestation and continue through lactation to weaning. Particular emphasis should be placed on observation of labor and delivery. Abortion, dystocia, agalactia, prolonged labor, and delayed labor are all possibilities to be observed and noted. The duration of gestation should be recorded.
For certain drugs, it may be necessary that offspring be allowed to reach sexual and physical maturity. This would provide information on reproductive capacity and delayed effects of the drug on reproduction.
The drug should be administered at a level at least 3X the recommended maximum therapeutic dose; a control group should be included.
Criteria for malformation: The incidence and types of malformations should be recorded. An evaluation should be based primarily on the results derived from concurrent control groups; the incidence of malformations observed in the breed and species being studied should also be considered.
Tissue irritation studies for injectable drugs should establish the time it requires for the tissues surrounding an injection site to return to an acceptable condition. This may aid in the possible development of edible tissue trim-out statements on the label.
The need for additional studies on a drug combination will be based upon the biochemical, chemical, and pharmacologic properties of each single drug. It will be necessary to show that the drugs are compatible in combination when used in ruminants.
Safety studies for these drugs are detailed in CVM Guidelines for the Evaluation of Antimicrobial Drugs for Intra-mammary Infusion. (Single copies of these guidelines are available upon request from, The Dockets Management Branch (HFA-305), Food and Drug Administration, Room 4-62, 5600 Fishers Lane, Rockville, Maryland 20857
Addendum No. 1
Physical Examinations
An examination should be conducted for the purpose of detecting any
abnormalities that may be drug related. Examples of some methods and
clinical observations which are useful for assessment of toxicity
and health are as follows:
Ocular Appetite/General Health
Corneal opacities Body weight
Nystagmus Feed consumption/time
Pupillary changes Water consumption/time
Blepharospasm
Blindness Integument
Examine eye and adnexa
Iritis Alopecia
Chemosis Haircoat condition
Photophobia Status of hydration
Congestion Pruritus
Blanching
Discharge Injection site
Conjunctivitis Soreness
Equilibrium Swelling
Hardness
Unsteadiness on front legs Persistence
Unsteadiness on rear legs Inflammation
Unsteadiness while standing Necrosis
Unsteadiness while walking
Incoordination of front legs Body temperature
Incoordination of rear legs
Anterior ataxia or paresis Gastrointestinal
Posterior ataxia or paresis
Abnormal reflexes Consistency of stools
Colic
Muscular disturbances Colic and rolling
Abdominal muscles tense
Perspiration (sweating)
Tremors of triceps Ruminal activity
Tremors of front legs Auscultation
Tremors of rear legs Propulsive diarrhea
Generalized tremors
Lip drooping and/or salivation Cardiovascular
Paralysis
Atony Heart rhythm
Atrophy Color of musous membranes
Auscultation (heart sounds)
ECG
Heart rate
Behavior (mental attitude) Respiratory
Anxious Respirations/minute
Apprehensive Dyspnea
Circling Respiratory sounds
Comatose Color of mucous membranes
Depressed Nasal discharge
Dorso-ventral recumbency Apnea
Lateral recumbency Tidal volume
Lateral recumbency - paddling
Sedated
Restless
Nose or lip wrinkled
Shaking head
Pressing head
Tongue hanging out (panting)
Convulsions
Addendum No. 2
Pathology
Gross examinations for pathologic lesions should be complete. All
organ systems should be examined and abnormalities should be recorded.
Organ weights should be recorded where relevant.
Histopathology
Histological requirements will depend upon what is known about
the drug being tested. It may not be necessary to examine all
tissues microscopically, but they should always be fixed (e.g.,
in buffered formalin) and maintained. Generally, the following
tissues should be considered and specimens of appropriate tissues
should be examined:
Pituitary gland Mammary glands
Thyroid glands Liver
Parathyroid glands Gallbladder
Adrenal glands Kidneys
Pancreas Urinary bladder
Ovaries Heart
Uterus Large and small arteries
Testes Bone (with marrow)
Prostate Marrow smear
Epididymis Spleen
Thymus Stomach (rumen, reticulum,
omasum and abomasum)
Lymph nodes (cervical, mediastinal Duodenum
mesenteric)
Jejunum
Brain (cerebrum, cerebellum, mid
brain, brain stem) Ileum
Spinal cord (cervical, thoracal Colon
lumbar)
Cecum
Eyes
Other tissues as
Lung indicated by gross
lesions or by drug
activity
Muscle tissue at site of
intramuscular injection
Addendum No. 3
Clinical Pathology
Clinical pathologic tests will depend on the ruminant drug product
being tested. Generally, the following parameters should be consid-
ered and appropriate clinical pathologic tests should be conducted:
Hematology
White blood cell (WBC) count
Red blood cell (RBC) count
Differential white blood cell (WBC)
Packed cell volume (PCV)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin concentration (MCHC)
Hemoglobin
Partial prothrombin time (PPT)
Platelet count
Blood and Serum Chemistries
Amylase
SDH (sorbital dehydrogenase)
Creatinine
Glucose
Calcium
Sodium
Chloride
Potassium
AST (aspartate aminotransferase)
Total protein
Albumin
Globulin
Total and direct bilirubin
Other useful tests include SDH (sorbital dehydrogenase),
GLDH (glutamate dehydrogenase), and
GGT (gamma glutamyl transferase
Urinalysis
Visual observations--color, consistency, quantity
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Microscopic examination of formed elements
Fecal Examination
Blood or other signs of hemorrhage
Excessive cellular debris
Biliary activity
Quantity, color, consistency
Parasites
Other abnormalities
A. Toxicity Study
In designing toxicity experiments, consideration should be given to historical data on use of the drug. A literature search should be conducted and combined with results of any preliminary experiments to determine the possible areas of drug toxicity. Drug entities should be characterized as to their chemical and pharmacological properties, and experiments should be designed that will determine the effect of these properties on the target animal. Testing should evaluate known or suspected problems.
a. Test drug
Swine safety studies should be performed with multiple dose levels of the drug given in the intended market formulation.
b. Test animals
Studies should be conducted in healthy swine representative of the class(es) of swine (size, weight range, sex or age) for which the drug is intended. If a drug has a narrow margin of safety and is intended for use in debilitated swine, it may be necessary to conduct safety studies in swine afflicted with the disease for which the test drug is indicated.
c. Duration of drug administration
For drugs recommended for long-term administration (15 days or longer), safety studies should be conducted with the drug being administered for the recommended maximum use duration, or longer (minimum of six weeks Drugs proposed for shorter periods of administration should be studied at least at 3X the recommended maximum duration of use.
d. Dose levels
The objective is to determine the margin of safety of a drug for the proposed duration of use. Dose levels of drugs tested should include at least 3 levels plus controls (0 - level The dosing regimen should include the following experiment groups: Unmedicated--use level--intermediate drug level (higher than use level)--estimated toxic level of the drug; or multiples of the recommended use level--0, 1X, 3X, and 5X.
e. Route of administration
Route of drug administration should be the proposed route(s) of administration which is to appear on the label.
f. Acclimation and baseline periods
Two weeks or longer may be required to acclimate test swine prior to baseline study. All prophylactic and therapeutic medications should be administered prior to the baseline study period so that they will not interfere with the study. Baseline values should be established before the study begins. Evaluation of drug response should continue until the study is terminated.
Documentation necessary to evaluate drug toxicity depends upon the type of drug action, potential for toxicity, proposed use of the drug, and class to which the swine belong.
Evaluation for signs of toxicity should include:
a. Periodic weighing, feed consumption, water consumption, clinical observations, and physical examinations performed in accordance with the protocol (Addendum No. 1
b. Clinical pathologic tests (Addendum No. 3) conducted either on all animals or on randomly preselected animals and on all swine showing signs of toxicity.
c. Gross pathologic examinations (Addendum No. 2) on swine preselected at random and on all swine that die. Examine histopathologically all grossly affected organs and known or suspected target organs, based on laboratory animal toxicologic studies and other pertinent data.
B. Special Studies
a. Objectives
Reproductive studies should be conducted in both sexes to evaluate possible drug effects on fertility in the target species; ovulation and spermatogenesis; egg and sperm integrity; transportation, implantation and development of the zygote; on parturition and the neonate; lactation, weaning, and care of the young; delayed postnatal deviations from normal and future breeding potential, with special interest directed to the teratogenic, embryotoxic and mutagenic potential of the drug.
Studies are divided into three segments, and each pertains to a specific phase of the reproductive process.
Emphasis is placed on determining the effects of the drug on gonadal function, estrous cycles, mating behavior, conception rates, and on the early and middle stages of gestation (Addendum No. 4
The objective is to determine whether a drug has embryotoxic or teratogenic effects. For this purpose drug administration is restricted to the period of organogenesis.
The purpose is to study the effects of a drug administered during the last third of pregnancy, through the period of lactation to weaning. Studies should include determining the effects of the drug on late fetal development, labor and delivery, abortion, lactation, neonatal viability, and growth of the newborn. Studies should be designed to evaluate future breeding potential of young animals. These studies should include animals exposed to a drug in utero, through suckling or by other appropriate routes and methods of drug administration.
(a) Study of fertility and general reproductive performances.
Fertility in both males and females should- be studied and evaluated. Results should be reported using appropriate reproduction indices.
The drug should be administered to males during the 90-day period prior to mating and at appropriate times during the breeding period. Breeding soundness (semen) evaluations should be conducted at appropriate intervals. Refer to "Reproductive Examination of the Boar," Journal of the Society for Theriogenology, Volume XIII, 1984.
For the study of drug action on female fertility, treatment should be conducted throughout the estrous cycle (i.e. folliculogenesis, ovulation, pre-implantation, early and mid-gestation
(b) Teratogenic and embryotoxic studies
Drug treatment should be repeated regularly (daily) and started early enough and continued long enough to cover the period of organ formation in embryos.
(c) Prenatal and postnatal study
The period of drug administration to the dam should cover the final one-third of gestation and continue throughout lactation to weaning. Particular emphasis should be placed on observation of labor and delivery. Abortion, dystocia, agalactia, prolonged labor, and delayed labor are possibilities to be observed and noted. The duration of gestation should be recorded. The investigator should record the number of live pigs born, dead pigs at birth, and pigs that survive for predetermined periods of time. It may also be necessary to record pig weights at birth and at predetermined time periods. (see addendum #4
For certain drugs, it may be necessary to allow offspring to reach sexual and physical maturity. This would provide information on reproductive capacity and delayed effects of the drug on reproduction.
The drug should be administered at a level at least 3X the recommended therapeutic dose; a control group should be included.
Criteria for malformation: The incidence and types of malformations should be recorded. The evaluation of malformations should be based primarily on the results derived from the concurrent control groups; however, the incidence of malformations observed in the breed and species being studied should also be considered.
Tissue irritation studies for injectable drugs should establish the time it requires for the tissues surrounding the injection site to return to an acceptable condition. This may aid in the possible development of edible tissue trim-out statements on the label.
The need for additional studies on a drug combination will be based upon the biochemical, chemical, and pharmacologic properties of each single drug. It will be necessary to show that the drugs are compatible in combination when used in swine.
Addendum No. 1
Physical Examinations
An examination should be conducted for the purpose of detecting
any abnormalities that may be drug related. Examples of some methods
and clinical observations which are useful for assessment of toxicity
and health are as follows:
Ocular Integument
Corneal opacities Alopecia
Nystagmus Haircoat condition
Pupillary changes Status of hydration
Blepharospasm Pruritus
Blindness Erythema
Examine eye and adnexa
Iritis Injection site
Chemosis
Photophobia Soreness
Congestion Swelling
Blanching Hardness
Discharge Persistence
Conjunctivitis Inflammation
Necrosis
Equilibrium
Body temperature
Unsteadiness on front legs
Unsteadiness on rear legs Gastrointestinal
Unsteadiness while standing
Unsteadiness while walking Consistency of stools
Incoordination of front legs Propulsive diarrhea
Incoordination of rear legs Colic
Anterior ataxia or paresis Colic and rolling
Posterior ataxia or paresis Abdominal muscles tense
Abnormal reflexes Vomiting
Auscultation
Muscular disturbances
Tremors of triceps Cardiovascular
Tremors of front legs
Tremors of rear legs Heart rate
Generalized tremors Heart rhythm
Lip drooping and/or salivation Color of mucous membranes
Paralysis Auscultation (heart
Atony sounds)
Atrophy ECG
Appetite/General Health
Body weight
Feed consumption/time (amount)
Behavior (mental attitude) Respiratory
Anxious Respirations/minute
Apprehensive Dyspnea
Circling Respiratory sounds
Comatose Color of mucous membranes
Depressed Nasal discharge
Dorso-ventral recumbency Apnea
Lateral recumbency Tidal volume
Lateral recumbency - paddling
Sedated
Restless
Nose or lip wrinkled
Shaking head
Pressing head
Tongue hanging out (panting)
Convulsions
Addendum No. 2
Pathology
Gross examinations for pathologic lesions should be complete. All
organ systems should be examined and abnormalities should be
recorded. Organ weights should be recorded where relevant.
Histopathology
Histological requirements will depend upon what is known about
the drug being tested. It may not be necessary to examine all
tissues microscopically, but they should always be fixed (e.g.,
in buffered formalin) and maintained. Generally, the following
tissues should be considered and specimens of appropriate tissues
should be examined:
Pituitary gland Mammary glands
Thyroid glands Liver
Parathyroid glands Gallbladder
Adrenal glands Kidneys
Pancreas Urinary bladder
Ovaries Heart
Uterus Large and small arteries
Testes Bone (with marrow)
Prostate Marrow smear
Epididymis Spleen
Thymus Stomach
Lymph nodes (cervical, mediastina Duodenum
mesenteric)
Jejunum
Brain (cerebrum, cerebellum, mid
brain, brain stem) Ileum
Spinal cord (cervical, thoracal Colon
lumbar)
Cecum
Eyes
Other tissues as
Lung indicated by gross
lesions or by drug
Muscle tissue at site of activity
intramuscular injection
Addendum No. 3
Clinical Pathology
Clinical pathology will depend on the drug product being tested.
Generally the following parameters should be considered, and
appropriate clinical pathologic tests should be conducted:
Hematology
White blood cell (WBC) count
Red blood cell (RBC) count
Differential white blood cell (WBC)
Packed cell volume (PCV)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin concentration (MCHC)
Hemoglobin
Partial prothrombin time (PPT)
Platelet count
Blood and Serum Chemistries
Amylase
Creatinine
Glucose
Calcium
Sodium
Chloride
Potassium
Phosphorus
AST (aspartate aminotransferase)
Total protein
Albumin
Globulin
Total and direct bilirubin
BUN (blood urea nitrogen)
Other useful tests include SDH (sorbital dehydrogenase),
GLDH (glutamate dehydrogenase),
GGT (gamma glutamyl transferase), and
SAP serum alkaline phosphatase
Urinalysis
Visual observations--color, consistency, quantity
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Microscopic examination of formed elements
Urine volume vs. water intake
Fecal Examination
Blood or other signs of hemorrhage
Excessive cellular debris
Quantity, color, consistency
Biliary activity
Parasites
Other abnormalities
Addendum No. 4
Fertility Observations
a. Estrus behavior.
b. Services per conception.
c. Conception rate.
d. Pregnancy rate.
e. Number aborted.
Farrowing Observations
a. Date of onset of parturition.
b. Duration of parturition.
c. Signs of dystocia.
d. Expulsion of placenta.
e. Number of pigs born (alive and dead
f. Condition of piglets at birth.
g. Pig weight at birth.
h. Teratogenicity and embryotoxicity.
i. Lactation.
Litter Performance Observations
a. Time and cause of death of any piglet.
b. Number of pigs weaned.
c. Weaning weight.
d. Date of weaning.
e. Postparturient condition of dam.
A. Toxicity Study
a. Study design
Adequate numbers of chicks or poults in small pens with replicates are necessary. Equal numbers of both sexes are recommended for the following classes of poultry: broiler chickens, roaster chickens, breeding chickens (including broiler breeders), replacement chickens, breeding turkeys and/or growing turkeys (meat turkeys), etc., depending upon product claim(s
Note: Coccidiosis should be no problem provided reasonable precautions are used and fresh clean litter is used and kept dry.
The following principles apply for drug evaluation under simulated use conditions (Phase I) for target animal safety:
Note: Adequate and well controlled floor pen toxicity studies should be conducted tinder simulated use conditions, i.e., if the drug is a water soluble product, it should be given in the drinking water; if the drug is used in pelleted feed, it should be evaluated in pelleted or crumbled feed; if it is to be used in birds that are raised on the floor, it should be tested in floor pens where management practices simulate commercial use conditions.
NOTES: In many cases the target animal safety data are satisfactory for the corresponding classes of growing poultry, i.e., broiler chickens for roaster chickens and broiler breeder replacement chickens, growing turkeys for turkey breeders and Leghorn-type replacement chickens in the case of Leghorn-type laying and breeding chickens. If, however, this is not the case, (3), (4), (5) or (6) above must be started with one day old chicks or poults.
The following experimental groups and dose levels are required:
Notes: The highest level should be overtly toxic. Drug sponsors and FDA have found this approach to be very acceptable for poultry drugs. By using an overtly toxic drug level in toxicity studies, the basic objectives of the drug tolerance test are satisfied. If 10X the recommended level is nontoxic, it is not necessary for the sponsor to demonstrate a toxic level.
Test animals should be closely monitored to characterize the target animal's response to a toxic dose(s) of a drug. Specific observations will depend upon the known properties of the drug and the class of poultry bring tested.
e. Parameters to measure - for each class of chickens or turkeys
Examinations postmortem should be conducted on all birds that die during the experiment. Mortality resulting from or associated with drug toxicity, disease, or other causes should be confirmed by acceptable diagnostic techniques, and dates of death should be maintained. All remaining birds should be killed and examined for gross, drug related lesions. Any bird showing suspected drug related, gross lesions should be examined histologically. Records of all these examinations should be submitted in the NADA.
g. Histopathology
At the termination of experiments, a significant number of test birds preselected at random should be killed for histologic examination. The following tissues should be collected and stored in buffered formalin solution: liver, kidney, heart, adrenal glands, bursa of Fabricius, brain and spinal cord, pancreas, bone and marrow, thyroid glands, thymus, eye, lung, trachea, parathyroid glands, pituitary body, ovaries and oviducts, testes, oesophagus, crop, spleen, proventriculus, ventriculus, intestines (upper, middle, and ceca), and skin. The following tissues should be routinely examined: liver, kidney, heart, bursa of Fabricius, brain, spleen, thymus, bone marrow, ovaries, and testes. Examination of the other tissues might be required depending upon toxicity problems encountered.
If tissues from birds dosed at higher than recommended concentration(s) show no drug related histo-pathology, as compared to non-medicated controls, tissues from loner medicated dosage birds need not be examined histologically. However, tissues from all concentrations should be saved in the event drug toxicity problems arise later. Complete records and dates of all activities should be maintained and submitted in the NADA.
h. Clinical Pathology
Standard hematology measurements, i.e., red blood cell count (RBC), hematocrit or packed cell volume (PCV), differential leukocyte counts, hemoglobin (Hb), and blood clotting time are necessary. NOTE: Leukocyte counts in poultry are subject to error because of nucleated erythrocytes and nucleated thrombocytes in birds.
i. Additional parameters
Eggs from three (3) or more consecutive days of production at the beginning of each 28-day period will be used for egg quality evaluation, e.g., egg weights, shell thickness or shell strength, and interior egg quality, e.g., yolk color, blood and meat spots, and albumin thickness (expressed as Haugh units) and organoleptic tests to determine off flavors, etc. Table eggs are infertile; therefore, fertility, hatchability, or teratology studies are not required.
Note on field trials: Field trials conducted with the drug administered at use level(s) are required for all drugs proposed for continuous use. Nonmedicated controls are not required. Studies are required to be conducted under actual commercial conditions utilizing the proposed product to determine adverse drug effects, if any, under use conditions. For premixes to make finished feed, the appropriate classes of poultry should be fed pellets or crumbled feed. Each batch of feed should be assayed, and in the case of pelleted or crumbled feed, the feed should be assayed before and after pelleting. Birds that die should be necropsied, and an etiologic diagnosis should be attempted.
If the drug causes no adverse effect in a battery or pen experiment after 3X intended duration of use in each class of chicken or turkey for which it is recommended, this is sufficient evidence of safety to the target animal provided the toxic effects are identified in overdose studies. Please refer to the previously itemized "Experimental procedures for drugs for continuous use (Chapter IX. A. 1.)" for procedures and parameters for evaluation.
Drug irritation studies including routine histology and microscopic examination of the injection site are necessary. One injection at use level, estimated toxic level, intermediate level, and control battery study is satisfactory. Parameters to be measured in the study are the same as for broiler chickens or growing turkeys.
B. Special Studies
Data are necessary on hatchability of fertile eggs. Teratology and overdose studies are required. Data on use at the hatchery (field trials) are necessary.
The need for additional studies on a drug combination will be based upon the chemical, biochemical, and pharmacologic properties of each single drug, and uponsufficient documentation to determine that the drugs are compatible in combination when used in poultry.
The requirements for game birds and ducks are described in the FEDERAL REGISTER document entitled, "New Animal Drug Applications; Safety and Effectiveness Data Supporting the Approval of Minor Use New Animal Drugs," and the related guidelines. Refer to 21 CFR 514.1(d), which was published in the FEDERAL REGISTER on January 14, 1983 (48 FR 1922
C. Miscellaneous
Sponsors who propose anticoccidial drugs for intermittent use are advised to address efficacy issues before submitting protocols for target animal safety studies or initiating target animal safety studies.
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