35 
 
Guidance for Industry
 
BIOEQUIVALENCE GUIDANCE 
 
(This version of the guidance replaces the version titled Bioequivalence Guidance 
that was made available on October 9, 2002.  This document has been revised to update 
the contact information and typographical errors in the equation) 
Section III.A. of this guidance has been superceded by CDER's guidance entitled "Bioanalytical Method 
Validation".  Any general questions regarding the application of the Bioanalytical Method Validation guidance to 
new animal drugs should be directed to Marilyn Martinez, Center for Veterinary Medicine, Food and Drug 
Administration, 7500 Standish Pl., Rockville, MD  20855, (240)276-8357, marilyn.martinez@fda.hhs.gov.  Any 
questions regarding analytical methods for tissue residues should be directed to Valerie Reeves, 7500 Standish Pl., 
Rockville, MD 20855, (240)276-8207, valerie.reeves@fda.hhs.gov . 
 
This  document  is  intended  to  provide  guidance  for  the  design  and  analysis  of  in  vivo 
bioequivalence studies. This revision to the version that was made available in April 1996 adds 
an illustrative example of how to calculate confidence bounds when log transformed data are 
used. 
Comments and suggestions regarding this guidance document should be submitted to the Divison 
of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 
1061, Rockville, MD 20852.  All comments should be identified with the Docket Number (94D-
0401). Additional information on the 1996 guidance document can be found in the Federal 
Register (Vol. 61, No. 102, May 24, 1996).  Comments will be accepted at any time. 
For  questions  regarding  this  guidance  document,  contact  Ken  J.  Harshman,  Center  for 
Veterinary  Medicine  (HFV-104),  Food  and  Drug  Administration,  7500  Standish  Place, 
Rockville, MD 20855. 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine (CVM) 
November 8, 2006   
BIOEQUIVALENCE GUIDANCE 
Docket No. 94D-0401 
 
This guidance document represents the agency's current thinking on this matter. It 
does not create or confer any rights for or on any person and does not operate to bind 
the FDA or public. An alternative approach may be used if such approach satisfies the 
requirements of the applicable statute, regulations or both.  
 
PREAMBLE 
In 1996, the Center for Veterinary Medicine (CVM) revised a document entitled April 1990 
Bioequivalence Guideline.  The revised document, Bioequivalence Guidance (Final) 1996, 
was issued in final form following notice and comment.  
Many of the changes in the Bioequivalence Guidance (Final) 1996 were based upon reports 
from panel presentations at the 1993 Veterinary Drug Bioequivalence Workshop in Rockville, 
Maryland, sponsored by the Center for Veterinary Medicine (CVM), the Animal Health Institute 
(AHI), the American Academy of Veterinary Pharmacology and Therapeutics (AAVPT), and the 
Animal Drug Alliance1. Some new topics were introduced into the guidance as a result of issues 
identified in the review of bioequivalence protocols and studies.  
The major new topics in the guidance were as follows: 
1.  Higher than approved dose bioequivalence studies.  
2.  Bioequivalence testing for multiple strength solid oral dosage forms.  
3.  Assay considerations for bioequivalence studies.  
4.  AUC and CMAX as the pivotal parameters for bioequivalence determination.  
5.  Blood level bioequivalence studies to be accompanied by tissue residue depletion studies 
for generic products for food-producing animals. 
 
CVM has revised the 1996 Bioequivalence Guidance to add an illustrative example of how to 
calculate confidence bounds when log transformed data are used.  The guidance has also been 
revised in accordance with FDAs Good Guidance Practices (GGPs, found in the Federal 
Register of February 27, 1997, 62 FR 8961).  With the exception of the addition of information 
on how to calculate confidence bounds when log transformed data are used, minor revisions 
made to comply with the GGPs (e.g., addition of a cover sheet), and revisions to the Preamble, 
  2 the document is the same as the document issued in 1996.  In September 2000, FDA revised the 
guidance to clarify sources of information more clearly. 
A person may follow the guidance or may choose to follow alternate procedures or practices. If a 
person chooses to use alternate procedures or practices, that person may wish to discuss the 
matter further with the agency to prevent an expenditure of money and effort on activities that 
may later be determined to be unacceptable to FDA. Although this guidance document does not 
bind the agency or the public, and it does not create or confer any rights, privileges, or benefits 
for or on any person, it represents FDA's current thinking on bioequivalence testing for animal 
drugs. When a guidance document states a requirement imposed by statute or regulation, the 
requirement is law and its force and effect are not changed in any way by virtue of its inclusion 
in the guidance.  
  3 TABLE OF CONTENTS 
 
I. INTRODUCTION...............................................................................................6 
II. GENERAL CONSIDERATIONS .....................................................................7 
A. Selection of Reference Product for Bioequivalence Testing..........................7 
B. Criteria for Waiver of In Vivo  Bioequivalence Study....................................7 
C. Selection of Blood Level, Pharmacologic End-point, or Clinical End-point 
Study....................................................................................................................8 
D. Species Selection............................................................................................8 
E. Dose Selection ................................................................................................8 
F. Multiple Strengths of Solid Oral Dosage Forms............................................9 
G. Manufacturing of Pilot Batch ("Biobatch")...................................................9 
III. BLOOD LEVEL STUDIES..............................................................................9 
A. Assay Consideration.......................................................................................9 
B. General Experimental Design Considerations.............................................12 
1. Dosing by Labeled Concentration..............................................................12 
2. Single Dose vs Multiple Dose Studies.......................................................12 
3. Subject Characteristics...............................................................................12 
4. Fed vs Fasted State.....................................................................................13 
C. Pharmacokinetic and Statistical Considerations in Study Design...............13 
  4 1. Sampling Time Considerations..................................................................13 
2. Protein Binding ..........................................................................................14 
3. Subject Number..........................................................................................14 
4. Cross-over and Parallel Design Considerations.........................................14 
5. Duration of Washout Time for Cross-over Study......................................15 
6. Pivotal Parameters for Blood Level Bioequivalence.................................16 
D. Statistical Analysis.......................................................................................17 
IV. PHARMACOLOGIC END-POINT STUDIES..............................................22 
A. General Design Aspects................................................................................22 
B. Statistical Analysis........................................................................................22 
V. CLINICAL END-POINT STUDIES...............................................................23 
A. General Design Aspects................................................................................23 
B. Subject Characteristics and Data Collection...............................................23 
C. Statistical Analysis .......................................................................................23 
VI. HUMAN FOOD SAFETY CONSIDERATIONS..........................................24 
APPENDIX:  CONFIDENCE BOUNDS ON THE LOGARITHM SCALE.........................26 
BIBLIOGRAPHY/ENDNOTES....................................................................................28 
  5  
I. INTRODUCTION  
This document is intended to provide guidance for the design and analysis of in vivo  
bioequivalence studies. The guidance is an update of the April 12, 1990 Bioequivalence 
Guideline. Many of the changes in the guideline are based upon reports from panel presentations 
at the 1993 Veterinary Drug Bioequivalence Workshop1.  
Two products are considered to be bioequivalent when they are equally bioavailable; that is, 
equal in the rate and extent to which the active ingredient(s) or therapeutic ingredient(s) is (are) 
absorbed and become(s) available at the site(s) of drug action.  
The Generic Animal Drug and Patent Term Restoration Act (GADPTRA) signed into law on 
November 16, 1988, permits sponsors to submit an Abbreviated New Animal Drug Application 
(ANADA) for a generic version of any off-patent approved animal drug (with certain exceptions 
noted in the law) regardless of whether the drug was approved prior to 1962 and subject to the 
National Academy of Sciences / National Research Council / Drug Effectiveness Study Implementation 
(NAS/NRC/DESI) review.  
Bioequivalence studies are used in a variety of situations, most often when a sponsor proposes 
manufacturing a generic version of an approved off-patent product. A bioequivalence study may 
also be part of a new animal drug application (NADA) or supplemental NADA for approval of 
an alternative dosage form, new route of administration, or a significant manufacturing change 
which may affect drug bioavailability. 
The Center has concluded that the tissue residue depletion of the generic product is not 
adequately addressed through bioequivalence studies. Therefore, sponsors of ANADA's for drug 
products for food-producing animals will generally be asked to include bioequivalence and tissue 
residue studies (21 USC 360 b (n) (1) (E)). A tissue residue study should generally accompany 
clinical end-point and pharmacologic end-point bioequivalence studies, and blood level 
bioequivalence studies that can not quantify the concentration of the drug in blood throughout 
the established withdrawal period (21 USC 360 b (n) (1) (A) (ii)). 
Bioequivalence studies (i.e., blood level, pharmacologic end-point, and clinical end-point 
studies) and tissue residue depletion studies should be conducted in accordance with good 
laboratory practice (GLP) regulations (21 CFR Part 58). 
Whereas the focus of the guidance is bioequivalence testing for ANADA approval, the general 
principles also apply to relative bioavailability studies conducted for NADA's. 
Sponsors should consult with the Center early in the product development process to facilitate 
the design of studies adequate for drug approval. The Center urges sponsors to submit protocols 
for review prior to conducting studies. 
  6  
II. GENERAL CONSIDERATIONS  
A. Selection of Reference Product for Bioequivalence Testing  
As a general rule, the proposed generic product should be tested against the original pioneer 
product. 
If the original pioneer product is no longer marketed, but remains eligible to be copied, then the 
first approved and available generic copy of the pioneer should be used as the reference product 
for bioequivalence testing against the proposed new generic product. 
If several approved NADA's exist for the same drug product, and each approved product is 
labeled differently (i.e., different species and/or claims), then the generic sponsor must clearly 
identify which product label is the intended pioneer. Bioequivalence testing should be conducted 
against the single approved product which bears the labeling that the generic sponsor intends to 
copy. 
The generic sponsor should consult with CVM regarding selection of the appropriate reference 
product before conducting the bioequivalence study. 
B. Criteria for Waiver of In Vivo  Bioequivalence Study  
The requirement for the in vivo bioequivalence study may be waived for certain generic products 
(21 USC 360 b (n) (1) (E)).  Categories of products which may be eligible for waivers include, but 
are not limited to, the following:  
1.  Parenteral solutions intended for injection by the intravenous, subcutaneous, or 
intramuscular routes of administration.  
2.  Oral solutions or other solubilized forms.  
3.  Topically applied solutions intended for local therapeutic effects. Other topically applied 
dosage forms intended for local therapeutic effects for non-food animals only.  
4.  Inhalant volatile anesthetic solutions. 
In general, the generic product being considered for a waiver contains the same active and 
inactive ingredients in the same dosage form and concentration and has the same pH and 
physico-chemical characteristics as an approved pioneer product. 
However, the Center will consider bioequivalence waivers for non-food animal topical products 
with certain differences in the inactive ingredients of the pioneer and generic products. 
If a waiver of the in vivo  bioequivalence and/or the tissue residue study/studies is granted for a 
food animal drug product, then the withdrawal period established for the pioneer product will be 
assigned to the generic product. 
  7 Sponsors may apply for waivers of in vivo  bioequivalence studies prior to submission of the 
ANADA's. 
C. Selection of Blood Level, Pharmacologic End-point, or Clinical End-point Study  
In vivo  bioequivalence may be determined by one of several direct or indirect methods. 
Selection of the method depends upon the purpose of the study, the analytical method available, 
and the nature of the drug product. Bioequivalence testing should be conducted using the most 
appropriate method available for the specific use of the product.  
The preferred hierarchy of bioequivalence studies (in descending order of sensitivity) is the 
blood level study, pharmacologic end-point study, and clinical end-point study. When absorption 
of the drug is sufficient to measure drug concentration directly  in the blood (or other appropriate 
biological fluids or tissues) and systemic absorption is relevant to the drug action, then a blood 
(or other biological fluid or tissue) level bioequivalence study should be conducted. The blood 
level study is generally preferred above all others as the most sensitive measure of 
bioequivalence. The sponsor should provide justification for choosing either a pharmacologic or 
clinical end-point study over a blood-level (or other biological fluids or tissues) study. 
When the measurement of the rate and extent of absorption of the drug in biological fluids can 
not be achieved or is unrelated to drug action, a pharmacologic end-point (i.e., drug induced 
physiologic change which is related to the approved indications for use) study may be conducted. 
Lastly, in order of preference, if drug concentrations in blood (or fluids or tissues) are not 
measurable or are inappropriate, and there are no appropriate pharmacologic effects that can be 
monitored, then a clinical end-point study may be conducted, comparing the test (generic) 
product to the reference (pioneer) product and a placebo (or negative) control.  
D. Species Selection  
A bioequivalence study generally should be conducted for each species for which the pioneer 
product is approved on the label, with the exception of "minor" species (as defined in section 
514.1 (d) (1) of Title 21 of the Code of Federal Regulations)  on the label.  
E. Dose Selection  
Dose selection will depend upon the label claims, consideration of assay sensitivity, and 
relevance to the practical use conditions of the reference product. A blood level bioequivalence 
study should generally be conducted at the highest dose approved for the pioneer product.  
However, the Center will consider a bioequivalence study conducted at a higher than approved 
dose in certain cases. Such a study may be appropriate when a multiple of the highest approved 
dose achieves measurable blood levels, but the highest approved dose does not. In general, the 
study would be limited to 2-3x the highest dose approved for the pioneer product. The pioneer 
product should have an adequate margin of safety at the higher than approved dose level. The 
generic sponsor should also confirm (e.g., through literature) that the drug follows linear 
kinetics. A higher than approved dose bioequivalence study in food animal species would be 
accompanied by a tissue residue withdrawal study conducted at the highest approved dose for the 
  8 pioneer product.  
For products labeled for multiple claims involving different pharmacologic actions at a broad 
dose range (e.g.,  therapeutic and production claims), a single bioequivalence study at the highest 
approved dose will usually be adequate. However, multiple bioequivalence studies at different 
doses may be needed if the drug is known to follow nonlinear kinetics. The sponsor should 
consult with CVM to discuss the bioequivalence study or studies appropriate to a particular drug.  
F. Multiple Strengths of Solid Oral Dosage Forms  
The generic sponsor should discuss with CVM the appropriate in vivo  bioequivalence testing 
and in vitro  dissolution testing to obtain approval for multiple strengths (or concentrations) of 
solid oral dosage forms. 
CVM will consider the ratio of active to inactive ingredients and the in vitro  dissolution profiles 
of the different strengths, the water solubility of the drug, and the range of strengths for which 
approval is sought. 
One in vivo  bioequivalence study with highest strength product may suffice if the multiple 
strength products have the same ratio of active to inactive ingredients and are otherwise identical 
in formulation. 
In vitro  dissolution testing should be conducted, using an FDA approved method, to compare 
each strength of the generic product to the corresponding strength of the reference product. 
G. Manufacturing of Pilot Batch ("Biobatch")  
A pilot batch or "biobatch" should be the source of the finished drug product used in the pivotal 
studies (i.e., bioequivalence studies and tissue residue studies), stability studies and the 
validation studies for the proposed analytical and stability indicating methods (refer to CVM's 
guidance number 42, "Animal Drug Manufacturing Guidelines"). 
III. BLOOD LEVEL STUDIES  
Blood level bioequivalence studies compare a test (generic) product to a reference (pioneer) 
product using parameters derived from the concentrations of the drug moiety and/or its 
metabolites, as a function of time, in whole blood, plasma, serum (or in other appropriate 
biological fluids or tissues). This approach is particularly applicable to dosage forms intended to 
deliver the active drug ingredient(s) to the systemic circulation (e.g., injectable drugs and most 
oral dosage forms). Generally, the study should encompass the absorption, distribution, and 
depletion (elimination) phases of the drug concentration vs time profiles.  
A. Assay Consideration  
A properly validated assay method is pivotal to the acceptability of any pharmacokinetic study. 
Sponsors should discuss any questions or problems concerning the analytical methodology with 
CVM before undertaking the bioequivalence studies. The ANADA submission should contain 
adequate information necessary for the CVM reviewer to determine the validity of the analytical 
  9 method used to quantitate the level of drug in the biological matrix (e.g., blood). 
The following aspects should be addressed in assessing method performance: 
1.  Concentration Range and Linearity  
The quantitative relationship between concentration and response should be adequately 
characterized over the entire range of expected sample concentrations. For linear 
relationships, a standard curve should be defined by at least 5 concentrations. If the 
concentration response function is non-linear, additional points would be necessary to 
define the non-linear portions of the curve. Extrapolation beyond a standard curve is not 
acceptable.  
2.  Limit of Detection (LOD)  
The standard deviation of the background signal and LOD should be determined. The 
LOD is estimated as the response value calculated by adding 3 times the standard 
deviation of the background response to the average background response.  
3.  Limit of Quantitation (LOQ)  
The initial determination of LOQ should involve the addition of 10 times the standard 
deviation of the background response to the average background response. The second 
step in determining LOQ is assessing the precision (reproducibility) and accuracy 
(recovery) of the method at the LOQ. The LOQ will generally be the lowest 
concentration on the standard curve that can be quantified with acceptable accuracy and 
precision (see items 5. and 6. below).  
4.  Specificity  
The absence of matrix interferences should be demonstrated by the analysis of 6 
independent sources of control matrix. The effect of environmental, physiological, or 
procedural variables on the matrix should be assessed. Each independent control matrix 
will be used to produce a standard curve, which will be compared to a standard curve 
produced under chemically defined conditions. The comparison of curves should exhibit 
parallelism and superimposability within the limits of analytical variation established for 
the chemically defined standard curve. 
5.  Accuracy (Recovery)  
This parameter should be evaluated using at least 3 known concentrations of analyte 
freshly spiked in control matrix, one being at a point 2 standard deviations above the 
LOQ, one in the middle of the range of the standard curve ("mid-range") and one at a 
point 2 standard deviations below the upper quantitative limit of the standard curve. The 
accuracy of the method, based upon the mean value of 6 replicate injections, at each 
concentration level, should be within 80-120% of the nominal concentration at each level 
(high, mid-range, and LOQ).  
  10 6.  Precision  
This parameter should be evaluated using at least 3 known concentrations of analyte 
freshly spiked in control matrix, at the same points used for determination of accuracy. 
The coefficient of variation (CV) of 6 replicates should be  10% for concentrations at or 
above 0.1 ppm (0.1  g/mL). A CV of  20% is acceptable for concentrations below 0.1 
ppm.  
7.  Analyte Stability  
Stability of the analyte in the biological matrix under the conditions of the experiment 
(including any period for which samples are stored before analyses) should be 
established. It is recommended that the stability be determined with incurred analyte in 
the matrix of dosed animals in addition to, or instead of, control matrix spiked with pure 
analyte. Also, the influence of 3 freeze-thaw cycles at 2 concentrations should be 
determined.  
Stability samples at 3 concentrations should be stored with the study samples and 
analyzed through the period of time in which study samples are analyzed. These analyses 
will establish whether or not analyte levels have decreased during the time of analysis. 
8.  Analytical System Stability  
To assure that the analytical system remains stable over the time course of the assay, the 
reproducibility of the standard curve should be monitored during the assay. A minimal 
design would be to run analytical standards at the beginning and at the end of the 
analytical run. 
9.  Quality Control (QC) Samples  
The purpose of QC samples is to assure that the complete analytical method, sample 
preparation, extraction, clean-up, and instrumental analysis perform according to 
acceptable criteria. The stability of the drug in the text matrix for the QC samples should 
be known and any tendency for the drug to bind to tissue or serum components over time 
should also be known. 
Drug free control matrix, e.g., tissue, serum, etc. that is freshly spiked known quantities 
of test drug, should be analyzed contemporaneously with test samples, evenly dispersed 
throughout each analytical run. This can be met by the determination of accuracy and 
precision of each analytical run (Items 5 and 6).  
10. Replicate and Repeat Analyses  
Single rather than replicate analyses are recommended, unless the reproducibility and/or 
accuracy of the method are borderline. Criteria for repeat analyses should be determined 
prior to running the study and recorded in the method SOP.  
11. Summary of Samples to Be Run With Each Analysis  
  11 a. Accuracy estimate (Item 5) 
b. Precision estimate (Item 6) 
c. Analytical system stability (Item 8) 
d. Analyte stability samples (Item 7)  
B. General Experimental Design Considerations  
1.  Dosing by Labeled Concentration  
The potency of the pioneer and generic products should be assayed prior to conducting 
the bioequivalence study to ensure that FDA or compendial specifications are met. The 
Center recommends that the potency of the pioneer and generic lots should differ by no 
more than 5% for dosage form products. 
The animals should be dosed according to the labeled concentration or strength of the 
product, rather than the assayed potency of the individual batch (i.e., the dose should not 
be corrected for the assayed potency of the product). The bioequivalence data or derived 
parameters should not be normalized to account for any potency differences between the 
pioneer and generic product lots. 
2.  Single Dose vs Multiple Dose Studies  
A single dose study at the highest approved dose will generally be adequate for the 
demonstration of bioequivalence. A single dose study at a higher than approved dose may 
be appropriate for certain drugs (refer to the section on Dose Selection). 
A multiple dose study may be appropriate when there are concerns regarding poorly 
predictable drug accumulation, (e.g. ,a drug with nonlinear kinetics) or a drug with a 
narrow therapeutic window. A multiple dose study may also be needed when assay 
sensitivity is inadequate to permit drug quantification out to 3 terminal elimination half-
lives beyond the time when maximum blood concentrations (CMAX) are achieved, or in 
cases where prolonged or delayed absorption2 exist. The determination of prolonged or 
delayed absorption (i.e., flip-flop kinetics) may be made from pilot data, from the 
literature, or from information contained with FOI summaries pertaining to the particular 
drug or family of drugs.  
3.  Subject Characteristics  
Ordinarily, studies should be conducted with healthy animals representative of the 
species, class, gender, and physiological maturity for which the drug is approved. The 
bioequivalence study may be conducted with a single gender for which the pioneer 
product is approved, unless there is a known interaction of formulation with gender.  
An attempt should be made to restrict the weight of the test animals to a narrow range in 
order to maintain the same total dose across study subjects.  
The animals should not receive any medication prior to testing for a period of two weeks 
or more, depending upon the biological half-life of the ancillary drug.  
  12 4.  Fed vs Fasted State  
Feeding may either enhance or interfere with drug absorption, depending upon the 
characteristics of the drug and the formulation. Feeding may also increase the inter- and 
intrasubject variability in the rate and extent of drug absorption. The rationale for 
conducting each bioequivalence study under fasting or fed conditions should be provided 
in the protocol. 
Fasting conditions, if used, should be fully described, giving careful consideration to the 
pharmacokinetics of the drug and the humane treatment of the test animals.  
The protocol should describe the diet and feeding regime which will be used in the study.  
If a pioneer product label indicates that the product is limited to administration either in 
the fed or fasted state, then the bioequivalence study should be conducted accordingly. If 
the bioequivalence study parameters pass the agreed upon confidence intervals, then the 
single study is acceptable as the basis for approval of the generic drug.  
However, for certain product classifications or drug entities, such as enteric coated and 
oral sustained release products, demonstration of bioequivalence in both the fasted and 
the fed states may be necessary, if drug bioavailability is highly variable under feeding 
conditions, as determined from the literature or from pilot data. A bioequivalence study 
conducted under fasted conditions may be necessary to pass the confidence intervals. A 
second smaller study may be necessary to examine meal effects. CVM will evaluate the 
smaller study with respect to the means of the pivotal parameters (AUC, CMAX). The 
sponsors should consult with CVM prior to conducting the studies. 
C. Pharmacokinetic and Statistical Considerations in Study Design  
1.  Sampling Time Considerations  
The total number of sampling times necessary to characterize the blood level profiles will 
depend upon the curvature of the profiles and the magnitude of variability associated with 
the bioavailability data (including pharmacokinetic variability, assay error and 
interproduct differences in absorption kinetics).  
The sampling times should adequately define peak concentration(s) and the extent of 
absorption. The sampling times should extend to at least 3 terminal elimination half-lives 
beyond TMAX. The sponsor should consult with CVM prior to conducting the pivotal 
bioequivalence study if the assay is unable to quantify samples to 3 half-lives. 
Maximum sampling time efficiency may be achieved by conducting a pilot investigation. 
The pilot study should identify the general shapes of the test and reference curves, the 
magnitude of the difference in product profiles, and the noise associated with each blood 
sampling time (e.g., variability attributable to assay error and the variability between 
subjects, for parallel study designs, or within subjects, for crossover study designs). This 
information should be applied to the determination of an optimum blood sampling 
  13 schedule. Depending upon these variability estimates, it may be more efficient to cluster 
several blood samples rather than to have samples which are periodically dispersed 
throughout the duration of blood sampling.3
2.  Protein Binding  
In general, product bioequivalence should be based upon total (free plus protein bound) 
concentrations of the parent drug (or metabolite, when applicable). However, if nonlinear 
protein binding is known to occur within the therapeutic dosing range (as determined 
from literature or pilot data), then sponsors may need to submit data on both the free and 
total drug concentrations for the generic and pioneer products.  
Similarly, if the drug is known to enter blood erythrocytes, the protocol should address 
the issue of potential nonlinearity in erythrocyte uptake of the drug administered within 
the labeled therapeutic dosing range.  
The bioequivalence protocol or completed study report should provide any information 
available from the literature regarding erythrocyte uptake and protein binding 
characteristics of the drug or drug class, including the magnitude of protein binding and 
the type of blood protein to which it binds. 
3.  Subject Number  
Pilot studies are recommended as a means of estimating the appropriate sample size for 
the pivotal bioequivalence study. Estimated sample size will vary depending upon 
whether the data are analyzed on a log or linear scale. Useful references for sample size 
estimates include Westlake4, Hauschke5, and Steinijans6. 
4.  Cross-over and Parallel Design Considerations  
A two-period cross-over design is commonly used in blood level studies. The use of 
cross-over designs eliminates a major source of study variability: between subject 
differences in the rates of drug absorption, drug clearance, and the volume of drug 
distribution.  
In a typical two-period cross-over design, subjects are randomly assigned to either 
sequence A or sequence B with the restriction that equal numbers of subjects are initially 
assigned to each sequence. The design is as follows:  
 
  
 
 
 
                 Sequence A      Sequence B  
  _________________________________________        
 
   Period 1      Test            Reference 
 
   Period 2      Reference       Test  
  14  
 
 
A crucial assumption in the two-period cross-over design is that of equal residual effects. 
Unequal residual effects may result, for example, from an inadequate washout period. 
Another assumption of the cross-over (or extended period) design is that there is no 
subject by formulation interaction. In other words, the assumption is that all subjects are 
from a relatively homogeneous population and will exhibit similar relative bioavailability 
of the test and reference products. If there are subpopulations of subjects, such that the 
relationship between product bioavailability is a function of the subpopulation within 
which they are being tested, then a subject by formulation interaction is said to exist.  
A one-period parallel design may be preferable in the following situations: 
a. The drug induces physiological changes in the animal (e.g., liver microsomal enzyme 
induction) which persist after total drug clearance and alter the bioavailability of the 
product administered in the second period. 
b. The drug has a very long terminal elimination half-life, creating a risk of residual drug 
present in the animal at the time of the second period dosing. 
c. The duration of the washout time for the two-period cross-over study is so long as to 
result in significant maturational changes in the study subjects. 
d. The drug follows delayed or prolonged absorption (flip-flop kinetics2), where the slope 
of the [[beta]]-elimination phase is dictated by the rate of drug absorption rather than the 
rate of drug elimination from one or both products. 
Other designs, such as the two-period design with four treatment sequences (Test/Test, 
Reference/Reference, Test/Reference and Reference/Test) or the extended period design 
may be appropriate depending on the circumstances. The use of alternative study designs 
should be discussed with CVM prior to conducting the bioequivalence study. Pilot data or 
literature may be used in support of alternative study designs. 
5.  Duration of Washout Time for Cross-over Study  
For drugs which follow a one or two compartment open body model, the duration of the 
washout time should be approximately 10x the plasma apparent terminal elimination 
half-life, to provide for 99.9% of the administered dose to be eliminated from the body. 
If more highly complex kinetic models are anticipated (e.g., drugs for which long 
withdrawal times have been assigned due to prolonged tissue binding), or for drugs with 
the potential for physiologic carryover effects, the washout time should be adjusted 
accordingly. 
The washout period should be sufficiently long to allow the second period of the cross-
over study to be applicable in the statistical analysis. However, if sequence effects are 
noted, the data from the first period may be evaluated as a parallel design study.  
  15 6.  Pivotal Parameters for Blood Level Bioequivalence  
The sponsor is encouraged to calculate parameters using formulas which involve only the 
raw data (i.e., so-called model independent methods).  
a. Area Under the Curve (AUC) Estimates 
The extent of product bioavailability is estimated by the area under the blood 
concentration vs time curve (AUC). AUC is most frequently estimated using the linear 
trapezoidal rule. Other methods for AUC estimation may be proposed by the sponsor and 
should be accompanied by appropriate literature references during protocol development. 
For a single dose bioequivalence study, AUC should be calculated from time 0 (predose) 
to the last sampling time associated with quantifiable drug concentration AUC(0-LOQ). 
The comparison of the test and reference product value for this noninfinity estimate 
provides the closest approximation of the measure of uncertainty (variance) and the 
relative bioavailability estimate associated with AUC(0-INF), the full extent of product 
bioavailability7. 
The relative AUC values generally change very little once the absorption of both 
products has been completed8, 9.  However, because of the possibility of multifunctional 
absorption kinetics, it can not always be determined when the available drug has been 
completely absorbed. Therefore, CVM recommends extending the duration of sampling 
until such time that AUC(0-LOQ)/AUC(0-INF) > = 0.80. Generally, the sampling times 
should extend to at least 3 multiples of the drug's apparent terminal elimination half-life, 
beyond the time when maximum blood concentrations are achieved. 
AUC(0-INF)should be used to demonstrate that the concentration-time curve can be 
quantitated such that AUC(0-LOQ)/AUC(0-INF) >= 0.80. The method for estimating the 
terminal elimination phase should be described in the protocol and the final study report. 
The AUC(0-LOQ)/AUC(0-INF) is calculated to determine whether AUC(0-LOQ) 
adequately reflects the extent of absorption.  
The sponsor should consult with CVM if AUC(0-LOQ)/AUC(0-INF) is determined to be 
< 0.80. If AUC(0-LOQ)/AUC(0-INF) is <0.80, then a multiple dose study to steady state 
may be needed to allow an accurate assessment of AUC(0-INF) (where AUC(0-INF) = 
AUC(0-t) at steady state and t is the dosing interval). 
In a multiple dose study, the AUC should be calculated over one complete dosing interval 
AUC(0-t). Under steady state conditions, AUC(0-t) equals the full extent of 
bioavailability of the individual dose AUC(0-INF) assuming linear kinetics. For drugs 
which are known to follow nonlinear kinetics, the sponsor should consult with CVM to 
determine the appropriate parameters for the bioequivalence determination.  
b. Rate of Absorption  
The rate of absorption will be estimated by the maximum observed drug concentration 
  16 (CMAX) and the corresponding time to reach this maximum concentration (TMAX).  
When conducting a steady-state investigation, data on the minimum drug concentrations 
(trough values) observed during a single dosing interval (CMIN) should also be collected. 
Generally, three successive CMIN values should be provided to verify that steady-state 
conditions have been achieved. Although CMIN most frequently occurs immediately 
prior to the next successive dose, situations do occur with CMIN observed subsequent to 
dosing. To determine a steady state concentration, the CMIN values should be regressed 
over time and the resultant slope should be tested for its difference from zero.  
c. Determination of Product Bioequivalence 
Unless otherwise indicated by CVM during the protocol development for a given 
application, the pivotal bioequivalence parameters will be CMAX and AUC(0-LOQ) (for 
a single dose study) or AUC(0-t) (for a multiple dose study). To be indicative of product 
bioequivalence, the pivotal metrics should be associated with confidence intervals which 
fall within a set of acceptability limits (see Statistical Analysis section of this Guidance.  
The sponsor and CVM should agree to the acceptable bounds for the confidence limits 
for the particular drug and formulation during protocol development.  
If studies or literature demonstrate that the pioneer drug product exhibits highly variable 
kinetics, then the generic drug sponsor may propose alternatives to the generally 
acceptable bounds for the confidence limits.  
TMAX in single dose studies and CMIN in multiple dose studies will be assessed by 
clinical judgment.  
D. Statistical Analysis  
CVM advocates the use of 90% confidence intervals, as the best available method for 
evaluating bioequivalence study data. Papers by Schuirmann10 and Westlake4 compare various 
methodologies for assessing drug product equivalence and describe the confidence interval 
approach.  
The confidence interval approach should be applied to the individual parameters of interest 
(e.g., AUC and CMAX). The sponsor may use untransformed or log transformed data. 
However, the choice of untransformed or log transformed data should be made by the sponsor 
with concurrence by the Center prior to conducting the study. 
1.  Untransformed Data 
A discussion of how the confidence interval approach should be applied to (normally 
distributed) untransformed data from a two-period crossover design is given below.  
  17 If we let X  be the mean for the test drug in period 1,  X the mean for the test drug 
T1 T2
in period 2, and  X and  X  the respective means for the reference drug, then the 
R1 R2
( )
estimates for the drugs averaged over both periods are X =(1/2) X + X  for the 
T T1 T2
( )
test drug and  X =(1/2) X + X  for the reference drug. Although both sequence 
R R1 R2
groups usually start with the same number of animals, the number of animals in each 
sequence group (n  and n ) that successfully finish the study may not be equal. The 
A B
formulas above utilize the marginal or least squares estimates of   and  , the 
T R
corresponding means in the target population.   These means are not a function of the 
sample size in each sequence. 
An analysis of variance is needed to obtain the estimate of 2, the error variance. The estimator, 
s2, which will be used in the calculation of the 90% confidence interval should be obtained from 
the "error" mean square term found in the following ANOVA table. 
  
 
 
 
 
        Source                          Degrees of Freedom 
       ______________________________________________________________ 
 
        Sequence                        1   
 
        Animal (Sequence)               nA+nB-2  
 
        Period                          1   
 
        Formulation                     1  
 
        Error                           nA+nB-2  
 
        Total                           2nA+2nB-1  
 
 
      
Lower and upper 90% confidence intervals are then found by formulas based on Student's 
t-distribution.  
( ) 1  1 1 
L = X  X t s  + 
T R n +n 2;0.05 2n n   
A B  
A B
  18 ( ) 1  1 1 
U = X  X +t s  + 
T R n +n 2;0.05 2 n n   
A B  
A B
The procedure of declaring two formulations bioequivalent if the 90% confidence 
interval is completely contained in some fixed interval, is statistically equivalent to 
performing two one-sided statistical tests ( = .05) at the end-points of the interval.  
Consider the following example with L = 3, U = 17,  X = 110 and  X  = 100. By the 
T R
traditional hypothesis testing approach, the result would be considered statistically 
significant since the confidence interval does not include 0. Using the confidence interval 
approach, the entire confidence interval lies within 17% of   X . (The lower end of the 
R
confidence interval lies within L/ X  = 3/100 = 3% of  X , while the upper end of the 
R R
confidence interval lies within U/ X  = 17/100 = 17% of  X  .) If it were determined 
R R
by CVM that only differences larger than 20% were biomedically important, then using 
the confidence interval approach the results of this study would be considered adequate to 
demonstrate bioequivalence. 
Now consider an example with L = -4, U = 24,  X  = 110 and  X  = 100. In this case, 
T R
by the traditional hypothesis testing approach the result would not be considered 
statistically significant since the confidence interval includes 0. However, the confidence 
interval extends as far as 24% from  X  . (The lower end of the confidence interval lies 
R
within L/ X  = -4/100 = -4% of  X  , while the upper end of the confidence interval 
R R
extends to U/ X  = 24/100 = 24% of  X  .) If it were determined by CVM that only 
R R
differences larger than 20% were biomedically important, then the results of this study 
would be considered inadequate to demonstrate bioequivalence, since the entire 
confidence interval is not within 20% of  X  .  
R
2.  Logarithmically transformed data 
This section discusses how the 90% confidence interval approach should be applied to 
log-transformed data.  In this situation the individual animal AUC and CMAX values are 
log-transformed and the analysis is done on the transformed data.  For a two-period 
crossover study, as described in D.1, the ANOVA model used to calculate estimates of 
the error variance and the least square means are identical for both transformed and 
untransformed data.  The procedural difference comes after the lower and upper 90% 
confidence intervals are found by formulas based on Students t-distribution. 
The lower and upper confidence bounds of the log-transformed data will then need to be 
  19 back-transformed in order to be expressed on the original scale of the measurement.   One 
thing to keep in mind when moving between the logarithm scale and the original scale is 
that the back-transformed mean of a set of data that has been transformed to the 
logarithm scale is not strictly equivalent to the mean that would be calculated from the 
data on the original scale of measurement.  This back-transformed mean is known instead 
as the geometric mean.   
It may help to see the calculations involved.  If the AUC from each animal has been 
transformed to the logarithm scale, we can express the transformed AUC as LnAUC.  
Then the mean on the logarithm scale is as follows: 
n LnAUC
LnAUC =  it   where the subscript t represents the AUC determinations for 
t n
i=1
the test article, i is the AUC of the ith animal, and n is the total number of animals 
receiving the text article.  When this mean is back-transformed, it becomes the geometric 
( )
e LnAUCt
mean:   .  This geometric mean will be on the original scale of the 
measurement.  It will be close to but not exactly equal to the mean obtained on the 
original scale of the measurement. 
The back-transformation of the confidence bounds is accomplished in the following way: 
 
Lower bound (expressed as a  =  (eL1)100
percentage)   
Upper bound (expressed as a  =  (eU 1) 100
percentage)   
Where  L  is the lower 90% confidence interval as 
given in Section III D 1 and calculated on 
the log-transformed data;  
  U  is the upper 90% confidence interval as 
given in Section III D 1 and calculated on 
the log-transformed data. 
 
As an example, consider the data for AUC from a hypothetical crossover study in the 
following table: 
    Reference Article  Test Article 
Animal  Crossover  AUC  LogAUC  AUC  LogAUC 
  20 Sequence 
1  1  518.0  6.25  317.8  5.76 
2  1  454.9  6.12  465.0  6.14 
3  1  232.8  5.45  548.4  6.31 
4  1  311.1  5.74  334.8  5.81 
5  2  340.4  5.83  224.7  5.41 
6  2  497.7  6.21  249.2  5.52 
7  2  652.0  6.48  625.4  6.44 
8  2  464.1  6.14  848.7  6.74 
Mean 433.8  6.03  451.7  6.02 
Standard deviation 133.3  0.33  214.3  0.47 
Geometric mean   414.7    410.5 
 
 
 
 
 
 
The statistics for AUC will be calculated from the log-transformed data. In this example, 
L, the lower 90% confidence interval calculated on the log scale is -0.395.  U, the upper 
90% confidence interval calculated on the log scale is 0.372.   To back-transform these 
intervals and express them as percentages, we do the following: 
 
Back-transformed lower bound:
( )
  e0.3951 100=(0.6741)100=(0.326)100=32.6% 
Back-transformed upper bound: 
( )
  e0.3721 100=(1.4511)100=(0.451)100= 45.1% 
  21 Therefore the lower end of the confidence bound lies within -32.6% of the geometric 
mean of the reference article, while the upper end of the confidence interval lies within 
45.1% of the geometric mean of the reference article.   If it were determined by CVM 
that the acceptable confidence bound was 80% to125% of the geometric mean of the 
reference article in order to demonstrate bioequivalence, then the back-transformed lower 
bound can be as low as -20% and the back-transformed upper bound can be as high as 
25%.    In this example, we would determine that the study had not demonstrated an 
acceptable level of bioequivalence between the test article and the reference article. 
A more detailed derivation of these expressions for upper and lower confidence bounds is 
found in the Appendix. 
The width of the confidence interval is determined by the within subject variance (between 
subject variance for parallel group studies) and the number of subjects in the study. In general, 
the confidence interval for untransformed data should be 80-120% (the confidence interval 
should lie within  20% of the mean of the reference product). For logarithmically transformed 
data, the confidence interval is generally 80-125% (the confidence interval should lie within       
20% to +25% of the mean of the reference product). The sponsor and CVM should determine 
the acceptable bounds for confidence limits for the particular drug and formulation during 
protocol development.  
 
IV. PHARMACOLOGIC END-POINT STUDIES  
Where the direct measurement of the rate and extent of absorption of the new animal drug in 
biological fluids is inappropriate or impractical, the evaluation of a pharmacologic end-point 
related to the labeled indications for use will be acceptable.  
A. General Design Aspects  
Typically the design of a pharmacologic end-point study should follow the same general 
considerations as the blood level studies. However, specifics such as the number of subjects or 
sampling times will depend on the pharmacologic end-point monitored. The parameters to be 
measured will also depend upon the pharmacologic end-points and may differ from those used in 
blood level studies. As with blood level studies, when pharmacologic end-point studies are used 
to demonstrate bioequivalence, a tissue residue study will also be required in food-producing 
animals. 
B. Statistical Analysis  
For parameters which can be measured over time, a time vs  effect profile is generated, and 
equivalence is determined with the method of statistical analysis essentially the same as for the 
blood level bioequivalence study.  
For pharmacologic effects for which effect vs  time curves can not be generated, then alternative 
procedures for statistical analysis should be discussed with CVM prior to conducting the study.  
  22 V. CLINICAL END-POINT STUDIES  
If measurement of the drug or its metabolites in blood, biological fluids or tissues is 
inappropriate or impractical, and there are no appropriate pharmacologic end-points to monitor 
(e.g., most production drugs and some coccidiostats and anthelmintics), then well-controlled 
clinical end-point studies are acceptable for the demonstration of bioequivalence.  
A. General Design Aspects  
Generally, a parallel group design with three treatment groups should be used. The groups should 
be a placebo (or negative) control, a positive control (reference/pioneer product) and the test 
(generic) product. The purpose of the placebo (or negative) control is to confirm the sensitivity 
or validity of the study.  
Dosage(s) approved for the pioneer product should be used in the study. Dosage(s) should be 
selected following consultation with CVM and should reflect consideration for experimental 
sensitivity and relevance to the common use of the pioneer product. 
B. Subject Characteristics and Data Collection  
Studies should generally be conducted using the target animal species, with consideration for the 
sex, class, body weight, age, health status, and feeding and husbandry conditions, as described on 
the pioneer product labeling. In general, the length of time that the study is conducted should be 
consistent with the duration of use on the pioneer product labeling.  
In general, the response(s) to be measured in a clinical end-point study should be based upon the 
labeling claims of the pioneer product and selected in consultation with CVM. It may not be 
necessary to collect data on some overlapping claims (e.g., for a production drug which is added 
at the same amount per ton of feed for both growth rate and feed efficiency, data from only one 
of the two responses need be collected).  
C. Statistical Analysis  
When considering sample size, it is important to note that the pen, not the individual animal, is 
often the experimental unit.  
As with blood level bioequivalence studies, CVM is advocating the use of 90% confidence 
intervals as the best method for evaluating clinical end-point studies. The bounds for confidence 
limits (e.g.,  20% of the improvement over placebo [or negative] control) for the particular drug 
should be agreed upon with CVM prior to initiation of the study.  
The analysis should be used to compare the test product and the reference product. In addition, a 
traditional hypothesis test should be performed comparing both the test and reference products 
separately to the placebo (or negative) control. The hypothesis test is conducted to ensure that the 
study has adequate sensitivity to detect differences when they actually occur. If no significant 
improvement ( = .05) is seen in the parameter (i.e., the mean of the test and the mean of the 
reference products are each not significantly better than the mean of the placebo [or negative] 
control), generally, the study will be considered inadequate to evaluate bioequivalence. 
  23 Assuming that the test and reference products have been shown to be superior to the placebo (or 
negative) control, the determination of bioequivalence is based upon the confidence interval of 
the difference between the two products.  
Some clinical end-point studies may not include a placebo (or negative) control for ethical and/or 
practical considerations. If the placebo is omitted, then the response(s) to the test and reference 
products should each provide a statistically significant improvement over baseline. 
If the results are ordered categorical data (e.g., excellent, good, fair or poor), a non-parametric 
hypothesis test of no difference between test product and placebo (or negative) control and 
between the reference product and placebo (or negative) control should be performed. As above, 
if these tests result in significant differences between the test product and control and the 
reference product and control, then a non-parametric confidence interval on the difference 
between the test and reference products is calculated.  
Another acceptable approach for categorical data is to calculate the confidence interval on the 
odds ratio between the test and reference products after showing that the test and reference 
products are significantly better than the control11.  
VI. HUMAN FOOD SAFETY CONSIDERATIONS  
The toxicology and tolerance developed for the pioneer animal drug are applied to generic copies 
of the drug. 
The Panel on Human Food Safety at the 1993 Veterinary Drug Bioequivalence Workshop 
addressed tissue residue depletion studies for generic animals drugs1. The Center has concluded 
that in addition to a bioequivalence study, a tissue residue depletion study should be conducted 
for approval of a generic animal drug product in a food-producing species. Two drug products 
may have the same plasma disposition profile at the concentrations used to assess product 
bioequivalence, but may have very different tissue disposition kinetics when followed out to the 
withdrawal time for the pioneer product. Therefore, to show the withdrawal period at which 
residues of the generic product will be consistent with the tolerance for the pioneer product, a 
tissue residue depletion study is necessary. 
The results of a bioequivalence study or tissue residue depletion study in one animal species can 
not generally be extrapolated to another species. Possible species differences in drug partitioning 
or binding in tissues could magnify a small difference in the rate or extent of drug absorbed into 
a large difference in marker residue concentrations in the target tissue. Therefore, for a pioneer 
product labeled for more than one food-producing species, a bioequivalence study and a tissue 
residue depletion study will generally be requested for each major food-producing species on the 
label. 
A traditional withdrawal study, as described in CVM's guidance number 3, "General Principles 
for Evaluating the Safety of Compounds Used in Food-Producing Animals," is considered the 
best design for collecting data useful for the calculation of a preslaughter withdrawal period for 
drugs used in food-producing animals. In the traditional withdrawal study, twenty animals are 
divided into four or five groups of four to five animals each. Groups of animals are slaughtered 
  24 at carefully preselected time points following the last administration of the test product and the 
edible tissues are collected for residue analysis. A statistical tolerance limit approach is used to 
determine when, with 95% confidence, 99% of treated animals would have tissue residues below 
the codified limits.  
For purposes of calculating a withdrawal period for a generic animal drug, only the generic 
product would be tested (i.e., not the pioneer product), and only the marker residue in the target 
tissue would be analyzed.  
Other study designs will be considered on a case-by-case basis. Sponsors are encouraged to 
submit the proposed tissue residue depletion protocol for CVM concurrence before proceeding 
with the withdrawal study. 
The generic animal drug will be assigned the withdrawal time supported by the residue depletion 
data, or the withdrawal time currently assigned to the pioneer product, whichever is the longer.  
The generic animal drug sponsor may request a shorter withdrawal period for the generic product 
by supplementing the ANADA and providing tissue residue data necessary to support the shorter 
withdrawal period request. Such a supplement will be reviewed under the agency's policy for 
Category  II  supplements.  For  a  Category  II  supplement,  a  reevaluation  of  the  safety  (or 
effectiveness) data in the parent application (i.e., the pioneer NADA) may be required (21 CFR 
514.106 (b) (2)).  The Center will ordinarily approve a request for a shorter withdrawal period 
when the residue data are adequate and  when no other human food safety concerns for the drug 
are evident. 
Under 21 CFR 514.1(b)(7), applications are required to include a description of practicable 
methods for determining the quantity, if any, of the new animal drug in or on food, and any 
substance formed in or on food because of its use, and the proposed tolerance or withdrawal 
period or other use restrictions to ensure that the proposed use of the drug will be safe.  For 
certain drug products, a tissue residue depletion study is not needed to ensure that residues of the 
test product will be consistent with the codified drug tolerance at the withdrawal time assigned to 
the reference product. These include but may not be limited to products for which a waiver of in 
vivo  bioequivalence testing is granted, and products for which the assay method used in the 
blood level bioequivalence study is sensitive enough to measure blood levels of the drug for the 
entire withdrawal period assigned to the reference product. Other requests for waiver of the 
tissue residue study will be considered on a case-by-case basis. 
CVM will not request that the assay methodology used to determine the withdrawal period for 
the generic product be more rigorous than the approved methodology used to determine the 
existing withdrawal period for the pioneer product. If an analytical method other than the 
approved method of analysis is used, the generic sponsor should provide data comparing the 
alternate method to the approved method. 
  25  
APPENDIX:  Confidence Bounds on the Logarithm Scale 
We want to develop an expression for the confidence bound of the difference between the 
pharmacokinetic parameter for the test treatment and the reference treatment, expressed as a 
percentage of the reference treatment.   This bound is derived from the 90% confidence interval 
of the difference between the mean of the test treatment and the mean of the reference treatment.  
This appendix addresses the case when the data analysis used to calculate the 90% confidence 
interval has been done with the natural log of the pharmacokinetic parameter as the dependent 
variable. 
For purposes of this illustration we will use Area Under the Curve (AUC) as the pharmacokinetic 
parameter. 
 
Notation and Distributional Assumptions:
Area under the Curve for Reference Treatment  AUC (1) 
 
R
Area under the Curve for Test Treatment  AUC (2) 
 
T
Natural log of AUC  LnAUC (3) 
 
LnAUC  ~ N(,2)      and    LnAUC  ~ N(,2)  (4) 
T R
T R
n LnAUC (5) 
LnAUC =  iT  
T  which estimates  , similarly for   
n
T R
i=1
(6) 
e(LnAUCT ) AUC &
 is the geometric mean of  T  to be denoted by  , 
T

&
similarly for   
R
 
Calculation of the Confidence Interval:
( ) (7) 
The 90% Confidence Interval of    is denoted by (L, U) and is 
T R
calculated from 
  26 ( ) t s 1  1 1 
L = LnAUC  LnAUC   + 
  T R n + n  2;.05 2n n    
A B  
A B
( ) t s 1  1 1 
U = LnAUC  LnAUC +  + 
T R n + n  2;.05 2n n   
A B  A B 
Manipulating This Expression Gives:
 L  <    <  U  (8) 
  
T R
eL <  e <  eU (9) 
  T R    
eL <  & <  eU (10) 
 
  T  

&
R
eL 1 <  & <  eU 1 (11) 
   
T 1 

&
R
eL 1 <  & & <  eU 1 (12) 
  T R    

&
R
Expressed As A Percentage:
(eL1)100 <  & &  <  (eU 1)100 (13) 
   T R 100   
  
&
 
R
 
 
 
  27 Bibliography/Endnotes 
1. Martinez, M.N. and Riviere, J.E. 1994. "Review of the 1993 Veterinary Drug Bioequivalence 
Workshop." J. Vet. Pharm. Therap.,17:85-119.  
2. Byron, P.R. and Notari, R.E. 1976. "Critical Analysis of "Flip-flop" Phenomenon in Two-
Compartment Pharmacokinetic Model." J. Pharm. Sci., 65: 1140-1144.  
3. D'Argenio, D.A 1981. "Optimal Sampling Times for Pharmacokinetic Experiments." J. 
Pharm. Biopharm., 9: 739-756. 
4. Westlake, W.J. 1988. "Bioavailability and Bioequivalence of Pharmaceutical Formulations." 
Biopharmaceutical Statistics for Drug Development, Peace, K. (ed), Marcel Dekker, Inc., pp. 
329-352. 
5. Hauschke, D., Steinijans, V.W., Diletti, E. and Burke, M. 1992. "Sample Size Determination 
for Bioequivalence Assessment Using A Multiplicative Model." J. Pharm. Biopharm., 20: 557-
561. 
6. Steinijans, V.W., Hauck, W.W., Diletti, E., Hauschke, D. and Anderson, S. 1992. "Effect of 
Changing the Bioequivalence Range From (0.80, 1.20) to (0.80, 1.25) on the Power and Sample 
Size." J. Clin. Pharm., Therap. and Toxicol., 30: 571-575. 
7. Martinez, M.N. and Jackson, A.J. 1991. "Suitability of Various Noninfinity Area Under the 
Plasma Concentration-Time Curve (AUC) Estimates for Use in Bioequivalence Determinations: 
Relationship to AUC From Zero to Time Infinity (AUC(0-INF))." Pharm. Res., 8: 512-517. 
8. Ronfeld, R.A. and Benet, L.Z. 1977. "Interpretation of Plasma Concentration-Time Curves 
After Oral Dosing." J. Pharm. Sci., 66: 178-180. 
9. Lovering, E., McGilveray, I., McMillan, I. and Tostowaryk, W. 1975. "Comparative 
Bioavailabilies From Truncated Blood Level Curves." J. Pharm. Sci., 64: 1521-1524. 
10. Schuirmann, D.J. 1987. "A Comparison of the Two One-Sided Tests Procedure and the 
Power Approach for Assessing the Equivalence of Average Bioavailability." J. Pharm. 
Biopharm., 15: 657-680. 
11. Mehta, C.R., Patel, N.R., and Tsiatis, A. A. 1984. Biometrics, 40:819-825. 
  
  28