Guideline No. 49
Guidance Document For Target Animal Safety And Drug Effectiveness Studies For Anti-Microbial Bovine Mastitis Products (Lactating and Non-Lactating Cow Products)
Revised April 4, 1996 (Revises the February 1, 1993 Guideline)
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
PUBLIC HEALTH SERVICE
FOOD AND DRUG ADMINISTRATION
CENTER FOR VETERINARY MEDICINE
Docket No. 93D-0025
II. GENERAL PRINCIPLES FOR DRUG EFFECTIVENESS STUDIES
IV.DATA PRESENTATION AND STATISTICAL ANALYSIS
The Food and Drug Administration's Center for Veterinary Medicine (CVM) has revised its GUIDANCE DOCUMENT FOR ANTI-INFECTIVE BOVINE MASTITIS PRODUCT DEVELOPMENT. This guidance document serves to interpret statutory and regulatory requirements and outlines general procedures for conducting evaluations for an antimicrobial being considered for approval. This guidance document does not preclude alternative procedures which document animal safety and effectiveness of an anti-microbial.
This Guidance Document represents the agency's position on a procedure or practice at the time of its issuance. This Guidance Document is not a legal requirement. A person may follow the Guidance Document or may choose to follow alternative procedures or practices. If a person chooses to use alternate procedures or practices that person may wish to discuss the matter further with the FDA/CVM to prevent an expenditure of money and effort on activities that may later be determined to be unacceptable. This Guidance Document does not bind the agency, and it does not create or confer any rights, privileges, immunities, or benefits for or on any person. When a Guidance Document states a requirement imposed by statute or regulation, however, the requirement is law and its force and effect are not changed in any way by virtue of its inclusion in the guidance document.
Introduction:
This guidance document has been assembled to inform the pharmaceutical industry of the types of data that will demonstrate that an anti-microbial bovine mastitis product is safe and effective for the cow. This guidance document is by no means complete; it deals with the product-specific issues listed above. Other useful general information may be found in CVM guidance documents for Human Food Safety, Drug Stability, etc., which are available from the Communications and Education Branch (HFV-12), FDA/Center for Veterinary Medicine, 7500 Standish Place, Rockville, MD 20855.
Regulations:
Before a new animal drug is approved, the drug's sponsor must demonstrate, among other relevant factors, that the proposed drug is safe and effective for use as recommended in the proposed labeling. The authority upon which these requirements are based can be found in Section 512 of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 360b). Applicable Federal regulations are Title 21 of the Code of Federal Regulations, Part 514 which covers New Animal Drug Applications.
The sponsor must demonstrate that the product which it proposes to market is safe to the target animal. The safety study listed below will demonstrate target animal safety. In addition, any adverse drug reactions occurring during efficacy trials should be reported.
A. IRRITATION STUDY
Animal Selection:
a. Lactating cow products
Select 6 normal multiparous cows and 6 normal primiparous cows free of mastitis. Half (3) of each group should have daily milk production ABOVE 60 lb (early lactation) and half (3) BELOW 35 lb (late lactation). A description of each test animal should be submitted stating the animal's age, lactation stage and number, and daily milk production.
b. Non-lactating cow products
Select 6 normal multiparous (second lactation or more completed) cows and 6 primiparous cows completing their first lactation. A description of each test animal should be submitted stating the animal's age, number of lactations and, in case of multiparous cows, milk production for the previous lactation.
Data collection: Information to be documented for the pre-treatment, treatment, withdrawal, and post-treatment periods should include animal identification, farm site, trial number, date of observation, test day and hour, individual milk production, and body temperature. Parameters to be measured twice daily for all four quarters include palpation results indicating absence of swelling, redness and soreness. Somatic cell counts should be determined by quantitative count methods as described under each treatment period. Electronic cell counting may also be used. It is advised that quantitative somatic cell counts (QSCC) be determined consistently in aliquot samples taken from the total milk production of each quarter. Milk samples used in the determination of milk quality may also be taken from this sample. Animal data should include a copy of the laboratory analyses (QSCCs) with the technician's signature and the dates of the analyses.
B. TREATMENT PERIODS
The PRE-TREATMENT period includes at least four (4) milkings (Milking intervals are assumed to be 12 hours) before the drug product is administered. Base line observations are made during this time to establish that test animals are healthy. During this period two milk samples should be taken at a 24-hour interval for bacterial culture and QSCC determinations from all four quarters. This procedure will help distinguish udder irritation due to the treatment in presence or in absence of a pathogenic organism.
The TREATMENT period begins at the fifth (5th) milking, or 60 hours from the beginning of the pre-treatment period. The labeled dose schedule should be followed, and all four quarters should be treated with test article. During this period two milk samples should be taken, at a 24-hour interval, from all four quarters for bacterial culture and QSCC determinations.
The WITHDRAWAL period begins with the last recommended treatment and continues for the labeled milk withdrawal time when the milk is discarded. During this period two milk samples should be taken, at a 24-hour interval, from all four quarters for bacterial culture and QSCC determinations.
The POST-TREATMENT period consists of the twelve (12) milkings1 after the proposed labeled milk withdrawal time. During this period two milk samples should be taken, at a 24-hour interval, from all four quarters for bacterial culture and QSCC determinations.
The sponsor must demonstrate final product efficacy, consistent with labeled use and indications against infectious bovine mastitis. For clinical treatment claims, the presence of an invading udder pathogen, a cytologic reaction, and either abnormal milk or clinical signs (such as swelling, redness and soreness) or both are the conditions under which the proposed drug should be tested. For sub-clinical treatment claims, the presence of an invading udder pathogen and a cytologic reaction are the conditions under which the proposed drug should be tested.
A. In the clinical study, the distribution of mastitis pathogens from the study will be utilized to determine the label efficacy statement. A sufficient number of major udder pathogens will be required to demonstrate effectiveness.
B. The degree of cytologic reaction (QSCC) should be reported. This information will be used as a check of the numerical trend between the means of QSCC for "cured" and "not cured" cows within each treated group to determine if other studies are needed for inflammation and safety.
C. Data should be collected using animals representative of the target population/herd. Therefore, all lactating or non-lactating cows in the target herd, depending on the label claim(s), should be surveyed. All eligible (infected) cows in the herd should be available for inclusion in the trial.
D. General herd/cow descriptive data should be submitted including total adult herd size, number of cows currently lactating, number and percentage of lactating cows sampled in the herd, number and percentage of lactating cows affected by infectious mastitis, approximate age of each cow, breed of each cow, current daily milk production, lactation number and stage of lactation.
E. A pre-trial culture/sample survey is recommended for sub-clinical infectious mastitis studies. It should include milk samples from all lactating quarters of all cows in a herd. All infected quarters of cows in the herd should be available for inclusion in the study.
F. The establishment of the efficacy of a combination product for the treatment of clinical mastitis should follow the current requirements for drug combinations. CVM strongly recommends that sponsors intending to develop combination products discuss data requirements as well as the merit of a particular drug combination before initiating studies.
G. Any concurrent antibiotic or non-antibiotic therapy administered to study animals should be described together under a separate section, and reported in the NADA submission. However, data from these animals should not be included in the sponsor's analysis of the data.
H. Test animals should have no history of vaccination with products intended to cause or induce an immune-mediated anti-mastitis response. Such products include but are not limited to vaccines, bacterins, immunomodulators, serum antibodies, or antitoxins.
I. Herds participating in a clinical study must have a sufficient number of clinical mastitis cases to fill an adequate number of blocks. The number of herds and the number of blocks in each herd should be specified prior to initiating the study to avoid potential problems with sequential testing.
J. The study should be blinded and the details of the blinding method used should be provided.
A. DOSE DETERMINATION will be based on a control and at least three non-zero drug levels consisting of a less effective lower dose and a higher dose that is no more effective than the dose selected for field trials in cows with clinical mastitis. These trials should be designed to define the target dose. Where dose determination trials do not clearly demonstrate a target dose, it may be appropriate for the sponsor to repeat the trial with different doses or to take more than one dose into field confirmation studies.
Dose determination should be conducted in at least two geographic locations by at least two different investigators, where the investigator is defined as that individual receiving the drug shipment and supervising the trial. These trials should involve a minimum of six herds. The selection of geographic locations should be discussed with CVM prior to initiation of trials. Quarters of individual cows should be treated with a single drug dose level.
In some cases, an appropriate experimentally induced mastitis infection (a model infection) could be used for dose determination. This option should be discussed with CVM prior to initiation of dose determination trials.
B. DOSE CONFIRMATION will be based on an appropriate dose selected from the dose determination studies. These studies must contain a control (preferably negative) group.
Dose confirmation studies should be conducted in at least two different geographic (climatologic) locations. Each location should utilize a different investigator, where an investigator is defined as that individual receiving the drug shipment and supervising the trial. These trials should involve a minimum of six herds. The sponsor has the responsibility to provide an adequate number of cows to demonstrate the claimed treatment response. Quarters of an individual cow should be treated with a single drug for which a claim is sought.
If a sponsor is considering a positively-controlled study, the sponsor should provide a basis for the need to have a positively-controlled study and discuss it with CVM prior to initiating the study.
C. The PIVOTAL PARAMETERS for the effectiveness of the drug product are the results of clinical response and bacteriological cultures. Investigators should refer to Microbiological Procedures for the Diagnosis of Bovine Mastitis, National Mastitis Council (NMC) 1990.
ENROLLMENT IN STUDY
a. The experimental unit will be the lactating dairy cow with clinical mastitis. Only cows with a single quarter with clinical mastitis will be enrolled. CVM will use this single quarter data base to infer efficacy to all cows with mastitis in 1 or more quarters.
b. Cows with a diagnosis of clinical mastitis (visually abnormal milk with clots, flakes, watery appearance, and/or udder clinical signs with swelling, redness, and soreness) will be needed for enrollment in the study. Diagnosis of clinical mastitis should be made at the time of sampling by the investigator, but at all other times may be assessed by farm personnel.
PRE-TREATMENT SAMPLING (Investigators should screen herds in advance of the pre-treatment samplings to encourage equal distribution of pathogens among treatment groups.)
a. Prior to treatment, single milk samples for microbiologic and quantitative somatic cell count (QSCC) will be obtained. The minimum inhibitory concentration (MIC) and MIC breakpoint for the isolated mastitis pathogens will be determined by using the National Committee for Clinical Laboratory Standards (NCCLS) to evaluate the susceptibility of the organisms to the new antimicrobial product.
b. Milk from each enrolled quarter should be examined at the time of sampling for signs of CLINICAL mastitis (abnormal milk with clots, flakes or watery appearance). Positive findings should be recorded on barn data record forms and pooled with data from daily examinations at each milking.
TREATMENT
Only the single affected quarter will be treated. Any cow developing mastitis in additional quarters during her enrollment will be dropped from the study and not considered a failure. Cows requiring and/or receiving treatment in an additional mastitic quarter will be excluded from consideration of efficacy, but daily observations should continue. Control group options include: pretrial, approved positive control drug treatment; milking every 3 or 4 hours; or delayed treatment.
POST-TREATMENT SAMPLING
A minimum of two single microbiology test samples will be obtained at least 5 days apart during the assessment period (14 to 28 days post-treatment). Two single QSCC samples will be obtained at the same time.
ASSESSMENT OF CURE
a. Cure will be assessed between 14 and 28 days post-treatment based on negative control study design.
b. Clinically, a cured quarter must have normal milk and no clinical signs of mastitis in that quarter (See C.1.b., above).
c. Microbiologically, the mastitis pathogen isolated in the pre-treatment sample must be absent from two post-treatment test samples. If no pre-treatment pathogen is isolated, the post-treatment sample must culture negative.
d. QSCC will not be used in the determination of cure for the individual cow.
e. QSCC results will only be used as a check of the numerical trend between the means of QSCC for "cured" and "not-cured" cows within each treatment group to determine if other studies are needed for inflammation and safety.
f. Only clinical cases of mastitis in which a mastitis pathogen (reference list to be determined) is isolated in the pre-treatment sample will be used to calculate cure rate. It will be necessary to submit to FDA/CVM the pre- and post-treatment bacteriological culture data with the MIC profile from those cows that were initially enrolled in the study but subsequently cultured negative on the pre-treatment sample.
g. The distribution of mastitis pathogens from the clinical study will be utilized to determine the label efficacy statement. An example for an effective antibiotic for staphylococcal and streptococcal mastitis pathogens will be: "Effective for the treatment of clinical mastitis caused by Staphylococcus species such as Staphylococcus aureus, and Streptococcus species such as Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis."
ENROLLMENT IN STUDY
a. All new anti-microbial products for mastitis in the lactating dairy cow must show efficacy for clinical mastitis. No new product will be approved solely on the basis of sub-clinical data as in the old guidelines.
b. To obtain a sub-clinical indication, additional sub-clinical data will be required. With acceptable clinical mastitis efficacy results, the subsequent sub-clinical mastitis study will require that the new therapy demonstrate efficacy but at a lower probability level (e.g., p<0.10). Fewer cows may be necessary for the sub-clinical study since elimination of the pre-treatment pathogen is required in the clinical study. Sub-clinical trial(s) will select cows with a positive quarter, thus fewer cows may be needed.
PRE-TREATMENT SAMPLING (Investigators should screen herds in advance of the pre-treatment samplings to encourage equal distribution of pathogens among treatment groups.)
The sub-clinical study will be a randomized study. Prior to treatment, two single microbiology and QSCC samples will be obtained at a 24-hour interval (Note: Investigators should screen herds in advance of the pre-treatment sampling to encourage equal distribution of pathogens among treatment groups).
TREATMENT
In the sub-clinical study, only one quarter from any cow will be treated. For cows infected in multiple quarters, the quarter to be treated will be randomly selected. The other quarters will not be treated. If additional quarters of clinical mastitis require additional treatment, the cow will be ineligible for inclusion in the study.
POST-TREATMENT SAMPLING
At 14 to 28 days post-treatment, two single microbiology and QSCC samples will be obtained at least 5 days apart.
ASSESSMENT OF CURE
a. Cure will be assessed between 14 and 28 days post-treatment based on negative control study design. QSCC results will be used similarly in sub-clinical studies as for clinical studies to detect changes and perhaps indicate possible safety problems.
b. Microbiologically, the mastitis pathogen isolated in the pre-treatment samples must be absent from two post-treatment test samples.
c. Products with acceptable efficacy data from both clinical and sub-clinical studies will receive an indication similar to the following: "Effective for the treatment of clinical and sub-clinical mastitis caused by Staphylococcus species such as Staphylococcus aureus, and Streptococcus species such as Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis."
Separate studies will be necessary to obtain a treatment and prevention claim for products intended for use in dry cow therapy. Treatment would mean the elimination of infections which exist at the time of dry cow therapy. Prevention would mean the "protection" from establishment of new infection during the dry cow period.
For the prevention claim, it will be necessary to establish, through a negatively-controlled group, the rate of new infection (estimates are approximately 2 to 3 percent).
ENROLLMENT IN STUDY
Cows in late lactation period with decreased milk production and ready for drying off should be enrolled in the study.
PRE-TREATMENT SAMPLING
TREATMENT For both treatment and prevention claims, all quarters should be treated after the second milk sample is collected.
POST-TREATMENT SAMPLING
ASSESSMENT OF CURE
For each organism for which a claim is sought, the cure rate data should be sorted, summarized, and submitted by herd for each investigator. Due to the large variation between herds in treatment response and organism prevalence, the statistical analysis should be conducted using statistical methods for discrete data that account for the differences due to herds and investigators. The specific statistical analysis used should be proposed and agreed to during protocol development. If herd and investigator differences are not accounted for in the statistical analysis, the true treatment effect can be confounded with the treatment response and sample size differences in the various herds tested. Although the herds and investigators are a random effect and should be analyzed as such, many of the available statistical software packages do not facilitate such an analysis. Therefore, until appropriate methods become generally available and accepted, it is permissible to consider algorithms that treat herds and investigators as fixed effects for the statistical analysis of these data.
Table 1. Treatment of CLINICAL Infectious Mastitis
Pre-treatment Post-treatment
Culture Results1 Culture Results2 Claim Status
SA3 - - - Cure for SA
STA4 - - - Cure for STA
SA SA - - Cure for SA
SA SA STA STA Cure for SA5
SA - STA STA Cure for SA5
SA SA STA - Cure for SA5
SA - STA - Cure for SA5
STA STA - - Cure for STA
STA STA SA SA Cure for STA5
STA STA SA - Cure for STA5
STA - SA SA Cure for STA5
STA - SA - Cure for STA5
SA SA SA SA Fail for SA
SA SA SA - Fail for SA
SA - SA - Fail for SA
STA STA STA STA Fail for STA
STA STA STA - Fail for STA
STA - STA - Fail for STA
1For contagious mammary gland pathogens (Staphylococcus aureus
and Streptococcus agalactiae), only a single positive pre-treatment
result qualifies the animal for trial entry. For all other pathogens, both
of the consecutive pre-treatment samples must be positive for the pathogen
to qualify for trial entry.
2For all pathogens, a single post-treatment positive result (the
same pathogen cultured in the pre-treatment sample) is a treatment failure.
3Staphylococcus aureus.
4Streptococcus agalactiae.
5Cases in which one pathogen is isolated from pre-treatment samples,
and a different pathogen from post-treatment samples should be recorded.
The incidence of this occurrence should be presented in the final trial report.
Note: Pre-treatment and post-treatment signs of abnormal milk, and
clinical signs of udder inflammation should be recorded on raw data sheets.
Table 2. Treatment of SUB-CLINICAL Mastitis
Pre-treatment Post-treatment
Culture Results1 Culture Results2 Claim Status
SA3 - - - Cure for SA
STA4 - - - Cure for STA
SA SA - - Cure for SA
SA SA STA STA Cure for SA5
SA - STA STA Cure for SA5
SA SA STA - Cure for SA5
SA - STA - Cure for SA5
STA STA - - Cure for STA
STA STA SA SA Cure for STA5
STA STA SA - Cure for STA5
STA - SA SA Cure for STA5
STA - SA - Cure for STA5
SA SA SA SA Fail for SA
SA SA SA - Fail for SA
SA - SA - Fail for SA
STA STA STA STA Fail for STA
STA STA STA - Fail for STA
STA - STA - Fail for STA
1For contagious mammary gland pathogens (Staphylococcus aureus
and Streptococcus agalactiae), only a single positive pre-treatment
result qualifies the animal for trial entry. For all other pathogens,
both of the consecutive pre-treatment samples must be positive for the
pathogen to qualify for trial entry.
2For all pathogens, a single post-treatment positive result
(the same pathogen cultured in the pre-treatment sample) is a treatment failure.
3Staphylococcus aureus.
4Streptococcus agalactiae.
5Cases in which one pathogen is isolated from pre-treatment samples,
and a different pathogen from post-treatment samples should be recorded.
The incidence of this occurrence should be presented in the final trial report.
Table 3. Treatment of SUB-CLINICAL Infectious Mastitis
Pre-treatment Post-treatment
Culture Results1 Culture Results2 Claim Status
SA3 - - - Cure for SA
STA4 - - - Cure for STA
SA SA - - Cure for SA
SA SA STA STA Cure for SA5
SA - STA STA Cure for SA5
SA SA STA - Cure for SA5
SA - STA - Cure for SA5
STA STA - - Cure for STA
STA STA SA SA Cure for STA5
STA STA SA - Cure for STA5
STA - SA SA Cure for STA5
STA - SA - Cure for STA5
SA SA SA SA Fail for SA
SA SA SA - Fail for SA
SA - SA - Fail for SA
STA STA STA STA Fail for STA
STA STA STA - Fail for STA
STA - STA - Fail for STA
1For contagious mammary gland pathogens (Staphylococcus aureus
and Streptococcus agalactiae), only a single positive pre-treatment result
qualifies the animal for trial entry. For all other pathogens, both of the
consecutive pre-treatment samples must be positive for the pathogen to
qualify for trial entry.
2For all pathogens, a single post-treatment positive result (the same
pathogen cultured in the pre-treatment sample) is a treatment failure.
3Staphylococcus aureus.
4Streptococcus agalactiae.
5Cases in which one pathogen is isolated from pre-treatment samples,
and a different pathogen from post-treatment samples should be recorded.
The incidence of this occurrence should be presented in the final trial report.
Table 4. Prevention of SUB-CLINICAL Infectious Mastitis
Pre-treatment Post-treatment
Culture Results1 Culture Results2 Status
- - - - No new infection
- - SA3 - New infection
- - - SA New infection
- - SA SA New infection
- - STA4 - New infection
- - - STA New infection
- - STA STA New infection
1 For demonstration of prevention claim, all pre-treatment
samples should be cultured negative.
2For all pathogens, a single post-treatment positive result
(the same pathogen cultured in the pre-treatment sample) is a treatment failure.
3Staphylococcus aureus.
4Streptococcus agalactiae.
Note: 1. The prevention of SUB-CLINICAL infectious mastitis is measured
by at least a 50% reduction in the rate of new infection.
2. A QSCC should be determined from one of the pre-treatment milk culture samples.
Sponsors are referred to FDA's GENERAL PRINCIPLES FOR EVALUATING THE SAFETY OF COMPOUNDS USED IN FOOD-PRODUCING ANIMALS, 1986, for generating residue data and establishing a milk discard time.
a) grossly abnormal milk and presence of flakes, clots and/or discoloration.
b) evidence of inflammation with apparent clinical tissue changes, swelling, heat and/or pain in the affected quarters.
c) evidence of leukocytosis in milk.
d) isolation of pathogenic microorganism in pure culture from fresh plating of milk sample is generally possible.
e) drop in milk production.
f) fever (especially in cases of peracute mastitis)
1. positive control - Approved drug used as a treatment in the control quarters. The data documenting currently approved mastitis drug products have not been evaluated in accordance with the clinical mastitis parameters discussed in this guideline.
2. milk out - Control quarters are milked out every 3 to 4 hours for 36 hours.
1. no treatment
2. placebo (vehicle only)
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