Bioavailability Studies 
Submitted in NDAs or 
INDs  General 
Considerations 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
April 2022 
Clinical Pharmacology 
 
    
Bioavailability Studies 
Submitted in NDAs or 
INDs  General 
Considerations 
Guidance for Industry 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
April 2022 
Clinical Pharmacology 
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION ...................................................................................................... 1 
II.  BACKGROUND ........................................................................................................ 2 
A.  General BA Considerations ........................................................................................... 3 
B.  Pre-approval Changes ................................................................................................... 4 
C.  Postapproval Changes ................................................................................................... 4 
III.  STUDY DESIGN CONSIDERATIONS..................................................................... 5 
A.  In Vivo Studies ............................................................................................................. 5 
B.  Other Approaches to Determine the BA of a Drug..........................................................10 
IV.  ASSESSING BA AND DEMONSTRATING BE FOR VARIOUS DOSAGE 
FORMS................................................................................................................................ 11 
A.  Solutions and Other Solubilized Dosage Forms ..............................................................12 
B.  IR Drug Products.........................................................................................................12 
C.  MR Drug Products.......................................................................................................13 
V.  ADDITIONAL INFORMATION ON IN VITRO APPROACHES ......................... 17 
A.    General Considerations ................................................................................................17 
B.    In Vitro Studies Conducted in Support of BA ................................................................18 
VI.  SPECIAL TOPICS................................................................................................... 21 
A.  Enantiomers Versus Racemates ....................................................................................21 
B.  Drug Products With Complex Mixtures as the Active Ingredients ...................................21 
C.  Drugs With Long Half-Lives .........................................................................................22 
D.  Orally Administered Drugs Intended for Local Action....................................................22 
E.  Combination and Co-administered Drug Products .........................................................22 
F.  Endogenous Substances ................................................................................................23 
G.  Narrow Therapeutic Index Drugs..................................................................................24 
H.  Characterizing the Effects of Alcoholic Beverages on MR Drug Products.........................24 
APPENDIX A:  GENERAL STUDY DESIGN AND DATA HANDLING ......................... 25 
APPENDIX B:  GUIDELINES FOR CONDUCTING FED OR FASTED STUDIES ........ 28 
APPENDIX C:  GUIDELINES FOR CONDUCTING AN IN VITRO ALCOHOL DOSE-
DUMPING STUDY ............................................................................................................. 30 
i  Contains Nonbinding Recommendations 
 
Bioavailability Studies Submitted in NDAs or INDs  General 
Considerations 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
 
I.  INTRODUCTION  
   
This guidance provides recommendations to sponsors and applicants2 submitting bioavailability 
(BA) information for drug products in investigational new drug applications (INDs), new drug 
applications (NDAs), and NDA supplements.  This guidance contains recommendations on how 
to meet the BA requirements set forth in 21 CFR part 320 as they apply to dosage forms intended 
for oral administration.  These dosage forms include tablets, capsules, solutions, suspensions, 
conventional (e.g., immediate-release (IR) drug products) and modified-release (MR) (e.g., 
extended-release (ER), delayed-release (DR)) drug products.  The guidance is also applicable to 
non-orally administered drug products when it is appropriate to rely on systemic exposure 
measures to determine the BA of a drug (e.g., transdermal delivery systems and certain vaginal, 
rectal, and nasal drug products).  The guidance provides recommendations on conducting BA 
studies during the investigational period for a drug intended to be submitted for approval in an 
NDA and bioequivalence (BE) studies during the postapproval period for certain changes to drug 
products with an approved NDA.3    
 
This guidance does not discuss information for demonstrating BE for drug products in 
abbreviated new drug applications (ANDAs) and ANDA supplements.  In August 2021, the FDA 
issued a separate draft guidance on this topic entitled Bioequivalence Studies with 
Pharmacokinetic Endpoints for Drugs Submitted Under an ANDA.4  Furthermore, this guidance 
does not provide recommendations on studies conducted in support of demonstrating 
 
1 This guidance has been prepared by the Office of Clinical Pharmacology with contributions from the Office of 
Pharmaceutical Quality in the Center for Drug Evaluation and Research at the Food and Drug Administration.   
 
2 Hereafter, the term sponsor will be used to refer to both sponsors and applicants. 
 
3 Bioequivalence (BE) is defined in 21 CFR 314.3(b). 
 
4 When final, this guidance will represent the FDAs current thinking on this topic.  We update guidances 
periodically.  For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
  1  Contains Nonbinding Recommendations 
 
comparability or biosimilarity for biological products licensed under section 351 of the Public 
Health Service Act (see the FDA guidances entitled Clinical Pharmacology Data to Support a 
Demonstration of Biosimilarity to a Reference Product (December 2016) and Considerations in 
Demonstrating Interchangeability With a Reference Product (May 2019) for more information). 
  
This guidance finalizes the FDA guidance entitled Bioavailability Studies Submitted in NDAs or 
INDs  General Considerations (February 2019).  The February 2019 draft of this guidance 
revised and replaced the draft guidance Bioavailability and Bioequivalence Studies Submitted in 
NDAs or INDs  General Considerations (March 2014).  The FDA considered comments 
received on the March 2014 guidance when issuing the February 2019 draft of this guidance.  
The FDA recognizes that this guidance cannot address every issue pertaining to the assessment 
of BA studies for INDs and NDAs.  Therefore, sponsors are encouraged to contact the 
appropriate review division with specific questions not addressed by this guidance. 
 
The contents of this document do not have the force and effect of law and are not meant to bind 
the public in any way, unless specifically incorporated into a contract.  This document is 
intended only to provide clarity to the public regarding existing requirements under the law.  
FDA guidance documents, including this guidance, should be viewed only as recommendations, 
unless specific regulatory or statutory requirements are cited.  The use of the word should in 
Agency guidances means that something is suggested or recommended, but not required. 
 
 
II.  BACKGROUND 
 
Determining the BA of formulations is important during the life cycle of drug products and aids 
in the FDAs evaluation of the safety and effectiveness of a product in an IND, NDA, or NDA 
supplement.  To determine the safety and efficacy of a drug product for the proposed indication, 
the FDA reviews all submitted information, including BA data, exposure-response evaluations, 
and clinical trial results. 
 
BA is defined as the rate and extent to which the active ingredient or active moiety is absorbed 
from a drug product and becomes available at the site of action.5  For drug products that are not 
intended to be absorbed into the bloodstream, BA can be assessed by scientifically valid 
measurements intended to reflect the rate and extent to which the active ingredient or active 
moiety becomes available at the site of drug action (see section III. Study Design 
Considerations).6  BA data provide an estimate of the amount of the drug absorbed as well as 
information related to the pharmacokinetics of the drug, the effects of food on the absorption of 
the drug, and dose proportionality or linearity in the pharmacokinetics of the active moieties. 
 
Sponsors can determine the BA for orally administered drug products by comparing a plasma 
exposure profile to that of a suitable reference product.7  A systemic exposure profile can be 
 
5 21 CFR 314.3(b).  
 
6 21 CFR 314.3(b). 
 
7 21 CFR 320.25. 
  2  Contains Nonbinding Recommendations 
 
generated by measuring the concentration of active ingredients and/or active moieties over time, 
and when appropriate, active metabolites over time in samples collected from the systemic 
circulation (see section III.A.8).  Systemic exposure profiles reflect both the release of the drug 
substance from the drug product as well as presystemic or systemic modifications to the drug 
substance after its release.  Conducting a BA study with an intravenous (IV) reference product 
helps assess the impact of the route of administration on BA and defines the absolute BA of the 
drug released from the drug product.  Conducting a BA study comparing one formulation to 
another enables an assessment of relative BA.   
 
For an evaluation of relative BA, a test product might result in different plasma concentration-
time profiles in comparison to a reference product because of a different rate or extent of 
absorption.  These differences can impact the FDAs assessment of the benefits and risks of the 
new formulation or condition of administration.  For example, if the test product leads to a 
significantly higher systemic exposure than the reference product, the test product could result in 
safety concerns associated with the higher systemic concentrations.  If the test product results in 
a significantly lower systemic exposure than the reference product, the test product could be less 
effective.  When the variability of the test product is greater than that of the reference product, 
both the safety and efficacy of the test product could be affected.  This increased variability 
could indicate that the performance of the test product is not comparable to the reference 
product, and the efficacy and safety of the test product are too variable for the product to be 
clinically useful. 
 
A.  General BA Considerations 
 
BA studies comparing two formulations or two test conditions are usually conducted using a 
crossover design.  For a drug with a long half-life, a parallel design could be more scientifically 
appropriate.8 
 
Determining the BA for new drug products submitted under an IND or an NDA can use the 
principles of BE.  Demonstrating equivalent or similar BA of two products during the 
development of a new drug could be needed to evaluate the safety or effectiveness of a product. 
See section 505 of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C. 355) for 
more information.  In general, a 90 percent confidence interval (CI) with predefined CI 
boundaries should be used in situations such as comparing two dosage forms during drug product 
development (e.g., the to-be-marketed formulation versus the clinical trial formulation) or 
interpreting the effect of food on a drug product.   
 
When similarity in BA is not demonstrated in a comparative BA assessment, the sponsor should 
demonstrate that the differences in the rate and extent of absorption do not meaningfully affect 
the safety and efficacy of the drug product based on the available dose-response or 
concentration-response data.  In the absence of this evidence, the sponsor should consider 
reformulating the test product, changing the method of manufacture for the test product, or 
obtaining additional safety or efficacy data for the test product.   
 
 
8 21 CFR 320.26.   
  3  Contains Nonbinding Recommendations 
 
In some cases, conclusions of similarity in BA based on the peak drug concentration (C ) and 
max
area under the plasma concentration-time curve (AUC) between the test product and the 
reference product could be insufficient to demonstrate that there is no difference in safety or 
efficacy.  An example of this scenario is when differences in the shape of the systemic 
concentration-time profile between the test and reference products imply that the test product 
might not produce the same clinical response as the reference product (e.g., the time to reach the 
peak drug concentration (T ) for analgesic drug products).  In such cases, further data analysis 
max
(e.g., partial AUCs), exposure-response evaluation, or clinical studies could be more 
scientifically appropriate to evaluate the differences in the BA of the two products.  
 
 
B.  Pre-approval Changes 
 
The relative BA of formulations used in drug development should compare:  (1) the early and 
late clinical trial formulations; (2) the formulations used in clinical trials and stability studies, if 
different; (3) the clinical trial formulations and to-be-marketed drug products, if different; (4) the 
equivalence of product strengths; and (5) the comparison of two different products in support of 
an NDA described in section 505(b)(2) of the FD&C Act.  For purposes of this guidance, in each 
comparison, the new formulation, the formulation produced by a new method of manufacture, or 
the new strength is the test product, and the prior formulation, the product made using the prior 
method of manufacture, or the product with the prior strength is the reference product.  See 
sections II.A, General BA Considerations; II.C, Postapproval Changes; and III.B.1, In Vitro 
Studies in this guidance for further discussion on considerations related to when changes in the 
components, the composition, or the method of manufacture suggest that further in vitro or in 
vivo studies should be performed. 
 
C.  Postapproval Changes  
 
In the presence of certain major changes in the components, composition, manufacturing site, or 
method of manufacture of a drug after its approval, the sponsor must demonstrate the in vivo BE 
for the drug product after the change compared to the drug product before the change.9  Certain 
postapproval changes that require BE studies must be submitted in a supplement and approved 
by the FDA before distributing a drug product made with the change.10 
 
Information on the types of recommended in vitro dissolution and in vivo studies for 
demonstrating the BE for IR and MR drug products approved as NDAs for specified 
postapproval changes is provided in the following FDA guidances: 
 
  SUPAC-IR:  Immediate Release Solid Oral Dosage Forms:  Scale-Up and Postapproval 
Changes:  Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, and In 
Vivo Bioequivalence Documentation (November 1995) 
 
 
9 21 CFR 320.21 and 320.22. 
 
10 Section 506A(c)(2) of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C. 356a(c)(2)); 21 CFR 
314.70.  
 
  4  Contains Nonbinding Recommendations 
 
  SUPAC-MR:  Modified Release Solid Oral Dosage Forms:  Scale-Up and Postapproval 
Changes:  Chemistry, Manufacturing, and Controls; In Vitro Dissolution Testing, and In 
Vivo Bioequivalence Documentation (October 1997) 
 
Alternatively, a quality risk assessment and mitigation approach could be used to support 
postapproval changes.  Sponsors should discuss proposals for alternate approaches with the 
appropriate review division. 
 
 
III.  STUDY DESIGN CONSIDERATIONS 
 
Sponsors shall use the most accurate, sensitive, and reproducible method available among those 
listed at 21 CFR 320.24(b) to measure the BA or demonstrate the BE of a product.11  Several in 
vivo and in vitro methods can be used to determine BA and establish BE.  In descending order of 
accuracy, sensitivity, and reproducibility, these methods include, but are not limited to, 
pharmacokinetic (PK) studies, in vitro tests that are predictive of human in vivo BA (in vitro-in 
vivo correlation (IVIVC)), pharmacodynamic (PD) studies, well-controlled clinical trials that 
establish the safety and efficacy of the drug product, and other in vitro studies as deemed 
appropriate by the FDA.12  In addition, where in vivo data are appropriate to determine the BA 
of a drug product, the regulations provide guidelines on the specific types of in vivo BA 
studies.13  This guidance primarily focuses on the use of in vivo studies to determine the BA of a 
drug. 
 
A.  In Vivo Studies 
 
1.  General Considerations 
 
For in vivo studies, the regulations allow the use of PK measures in an accessible biological 
matrix such as the blood, plasma, or serum to indicate the release of the drug substance from the 
drug product into the systemic circulation.14  For the purpose of this guidance, the terms for  
biological matrices (i.e., blood, plasma, and serum) are used interchangeably.  If serial 
measurements of the drug or its metabolites in plasma, serum, or blood cannot be accomplished, 
then measurement of urinary excretion should be considered.15   
 
BA frequently relies on PK measures such as the AUC to reflect the extent of systemic 
absorption and the C and T  to reflect the rate of systemic absorption.  PK-based 
max  max
comparisons to describe relative BA assume that measuring the active moiety at the site of action 
is not possible and that some relationship exists between the concentration of the active moiety 
 
11 21 CFR 320.24(a). 
 
12 21 CFR 320.24(b).  
 
13 21 CFR 320.25 through 320.29. 
 
14 21 CFR 320.24(b)(1)(i). 
 
15 21 CFR 320.24(b)(1)(i). 
  5  Contains Nonbinding Recommendations 
 
in the systemic circulation and the safety and efficacy of the drug.  A typical PK study to 
determine comparative BA is conducted as a crossover study.  The crossover design reduces 
variability in PK measures that are caused by subject-specific factors, thereby increasing the 
ability to discern differences in PK measures that are caused by different formulations. 
 
2.  Pilot Study 
 
If the sponsor chooses, a pilot trial with a small number of subjects can be carried out before 
proceeding with a full-scale BA study.  The results of a pilot study can: 
 
  Assess the variability in PK measures 
 
  Determine the sample size that achieves adequate power to conduct BA analysis in the 
full-scale study 
 
  Optimize the time intervals for sample collection 
 
  Determine the length of the washout period needed between treatments 
 
For conventional IR products, careful timing of the collection of the initial PK samples can 
ensure that the first sample collection occurs before the C , thereby informing the optimal 
max
sample collection schedule for a full-scale study.16  In some circumstances, the results of a pilot 
trial can be used as the sole basis to determine the BA of a drug, if the design and execution of 
the study are suitable, and if enough subjects have completed the study with evaluable PK 
measurements. 
 
3.  Full-Scale Study 
 
General recommendations for a standard BA or BE study based on PK measurements are 
provided in appendix A.  Non-replicate, crossover study designs are recommended for BA 
studies of IR and MR dosage forms.  Sponsors have the option of using replicate designs for BA 
or BE studies, where the reference treatment is repeated, or both the test and the reference 
products are given on multiple occasions.  Replicate crossover designs are used to estimate:  (1) 
the within-subject variance for the reference product or for both the test and reference products; 
and (2) the subject-by-formulation interaction variance component.  These designs account for 
the interoccasion variability that can confound the interpretation of a BE study as compared to a 
non-replicate crossover approach.   
 
In addition to the traditional approach and the use of average BE with replicate designs, the use 
of a reference-scaled BE approach using a replicate design can be considered.  This is an 
approach in which the BE acceptance limits are scaled to the variability of the reference product. 
This reference-scaled BE approach is typically used for drugs with a high intrasubject variability 
 
16 21 CFR 320.25 through 320.27 (for information on guidelines for the conduct and design of an in vivo 
bioavailability or bioequivalence study). 
 
  6  Contains Nonbinding Recommendations 
 
(greater than or equal to 30 percent) or drugs with a narrow therapeutic index.17,18  The 
appropriate review division should be consulted when planning the use of the reference-scaled 
BE approach. 
 
To determine the absolute BA, single-period studies using isotopic labeling approaches are a 
scientifically acceptable alternative. 
 
4.  Study Population 
 
In general, BA studies should be conducted in healthy subjects 18 years of age or older who are 
capable of giving informed consent.  When safety considerations preclude the use of healthy 
subjects, it might be preferable and more appropriate to evaluate the BA of a drug in individuals 
with the disease or condition being studied.19  In this situation, sponsors should attempt to enroll 
individuals whose disease and drug treatment are expected to be stable for the duration of the 
study.  Male and female subjects should be enrolled in BA studies unless there is a specific 
reason to exclude one sex (e.g., the drug product is indicated in only one sex, or there is a greater 
potential for adverse reactions in one sex compared to the other).  Female subjects enrolled in the 
study should not be pregnant or lactating at the beginning of the study and should not become 
pregnant during the study.  
 
5.  Single-Dose and Multiple-Dose (Steady-State) Testing 
 
Consistent with the regulations,20 this guidance generally recommends single-dose, in vivo 
studies to assess the BA of a drug because they are generally more sensitive than steady-state 
studies in assessing the rate and extent of release of the drug substance from the drug product 
into the systemic circulation.  The sponsor is referred to section IV.C for a discussion on the 
conduct of studies to determine the BA of a drug of an MR product. 
 
The regulations also provide guidelines on the design of a multiple-dose, in vivo BA study and 
when such studies are required.21  If a multiple-dose study is performed, the sponsor should dose 
the product to achieve steady-state concentrations of the drug.22  The sponsor should provide 
evidence that steady-state concentrations of the drug were achieved. 
 
17 Davit B, D Conner, 2010, Reference-Scaled Average Bioequivalence Approach, In:  I Kanfer, L Shargel, editors, 
Generic Drug Product Development  International Regulatory Requirements for Bioequivalence, Informa 
Healthcare, 271-272. 
 
18 Jiang, W, F Maklouf, DJ Schuirmann, X Zhang, D Conner, LX Yu, R Lionberger, 2015, A Bioequivalence 
Approach for Generic Narrow Therapeutic Index Drugs: Evaluation of the Reference-Scaled Approach and 
Variability Comparison Criterion, AAPS J, 17(4):891-901. 
 
19 21 CFR 320.25(a).  
 
20 21 CFR 320.26. 
 
21 21 CFR 320.27. 
 
22 21 CFR 320.27(c). 
  7  Contains Nonbinding Recommendations 
 
 
6.  Bioanalytical Methodology 
 
Sponsors must use bioanalytical methods for BA studies that are accurate, precise, specific, 
sensitive, and reproducible.23  A separate FDA guidance entitled Bioanalytical Method 
Validation (May 2018) is available to assist sponsors in validating bioanalytical methods. 
 
7.  Administration Under Fasted or Fed Conditions 
 
The sponsor should determine the BA of the test product under fasted conditions because it is 
generally a more sensitive method to assess differences between formulations.  The effect of 
food on the BA of the test product should also be assessed.24  If BA is determined using an 
approved product as a reference, the reference product should be administered as described in the 
labeling.  If tolerability issues or serious adverse events are anticipated under fasted conditions 
(for either the test or the reference product), the sponsor should conduct the study only under fed 
conditions.  See appendix B for additional guidance. 
 
8.  Moieties to Measure  
 
The active ingredient that is released from the dosage form or its active moiety and, when 
appropriate, its active metabolites,25 should be measured in the biological matrix collected during 
the BA study. 
 
The concentration-time profile of the active ingredient or the active moiety is more sensitive to 
changes in performance of the formulation.  In contrast, the metabolite is more affected by 
metabolite formation, distribution, and elimination.  The following scenarios are instances when 
an active metabolite(s) should be subjected to CI analyses for BA assessment:  
 
  If the metabolite is formed by pre-systemic metabolism (e.g., gut metabolism) and 
contributes to efficacy or safety:  In this case, the active ingredients or the active moiety 
and the active metabolite should be measured. 
 
  When the active ingredient or the active moiety concentrations are too low to allow 
reliable bioanalytical measurements in the appropriate biological matrix:  In this 
case, the metabolite should be measured in lieu of the active ingredient or active moiety.   
 
9.  PK Measures of Systemic Exposure  
 
 
23 21 CFR 320.29 (for information on bioanalytical methods). 
 
24 See the FDA draft guidance entitled Assessing the Effects of Food on Drugs in INDs and NDAs  Clinical 
Pharmacology Considerations (February 2019) for more information.  When final, this guidance will represent the 
FDAs current thinking on this topic. 
 
25 21 CFR 320.24(b)(1)(i). 
  8  Contains Nonbinding Recommendations 
 
When available, sponsors should use clinically relevant systemic exposure measures to 
determine BA.  Exposure measures are defined relative to the peak, partial, and total exposures 
of the drug concentration-time profile in the appropriate biological matrix as described below. 
 
a.  Peak exposure 
 
The sponsor should determine the peak exposure of the drug by measuring the C  obtained 
max
directly from the systemic drug concentration data without interpolation.  The T  can provide 
max
important information about the rate of absorption.  The first point of a drug concentration-time 
curve based on blood or plasma measurements is sometimes the highest concentration, raising 
concerns that the first sampling time was too late to accurately determine the C and T .  A 
max  max
carefully conducted pilot study can help to avoid this problem.  For example, collection of a 
sample at an early timepoint for IR products, between 5 and 15 minutes after dosing, followed by 
additional sample collections (e.g., two to five) in the first hour after dosing, could be sufficient 
to assess early peak concentrations.   
 
b.  Total exposure (extent of absorption) 
 
For single-dose studies, the sponsor should calculate the total exposure using the following: 
 
  The area under the concentration-time curve from time zero to time t (AUC ) from an 
0-t
appropriate biological matrix, where t is the last time point with a measurable 
concentration. 
 
  The area under the concentration time curve from time zero to time infinity (AUC ) 
0-INF
from an appropriate biological matrix, where AUC  = AUC  + Ct/z.  Ct is the last 
0-INF 0-t
measurable drug concentration, and z is the terminal elimination rate constant calculated 
by an appropriate method. 
 
  For drugs with low intrasubject variability (when available) in distribution and clearance 
(i.e., less than 30 percent) and a long half-life, a truncated AUC should be used (see 
section VI.C). 
 
For steady-state studies, the sponsor should calculate the total exposure using the area under the 
concentration-time curve from time zero to time TAU in an appropriate biological matrix over a 
dosing interval at steady state (AUC ), where TAU is the length of the dosing interval. 
0-TAU
 
c.  Partial exposure 
 
In addition to peak and total exposure, for certain classes of drugs (e.g., analgesic drug products), 
an evaluation of the partial exposure could be scientifically appropriate to support the 
determination of the relative BA of the drug product.  The FDA recommends the use of partial 
AUC as a partial exposure measure.  The time to truncate the partial AUC should be related to a 
clinically relevant response measure.  The sponsor should collect sufficient quantifiable samples 
to allow an adequate estimation of the partial AUC.  Sponsors should consult the appropriate 
  9  Contains Nonbinding Recommendations 
 
review division for questions on the suitability of the response measure or the use of partial 
exposure.   
 
10.  Comparison of Drug Exposure Measures in BA Studies   
 
A CI approach is recommended for BA comparisons.  Log-transformation of exposure measures 
before statistical analysis is recommended.  This guidance recommends the use of the BE 
criterion to compare systemic exposure measures for replicate and non-replicate BA studies of 
both IR and MR products.  For additional information on data analysis, refer to appendix A and 
to the FDA guidance entitled Statistical Approaches to Establishing Bioequivalence (February 
2001). 
 
B.  Other Approaches to Determine the BA of a Drug  
 
In certain circumstances, other approaches are recommended to determine the BA of a drug.  
Below are some general considerations regarding these other approaches.     
 
1.  In Vitro Studies 
 
Under certain circumstances, BA can be assessed using in vitro approaches (e.g., dissolution, 
drug-release testing) during the pre-approval and postapproval phases.26  The following FDA 
guidances provide recommendations on developing dissolution methodology, setting 
specifications, and the regulatory applications of dissolution testing:27 
 
  Dissolution Testing of Immediate-Release Solid Oral Dosage Forms (August 1997) 
 
  Extended-Release Oral Dosage Forms: Development, Evaluation, and Application of In 
Vitro/In Vivo Correlations (September 1997) 
 
  Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release 
Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System 
(December 2017) 
 
  M9 Biopharmaceutics Classification System-Based Biowaivers (May 2021) 
 
  Dissolution Testing and Acceptance Criteria for Immediate-Release Solid Oral Dosage 
Form Drug Products Containing High Solubility Drug Substances (August 2018) 
 
2.  In Vitro Tests Predictive of Human In Vivo BA 
 
 
26 21 CFR 320.24(b)(5) and (6). 
 
27 See also the FDA draft guidance entitled The Use of Physiologically Based Pharmacokinetic Analyses - 
Biopharmaceutics Applications for Oral Drug Product Development, Manufacturing Changes, and Controls 
(September 2020).  When final, this guidance will represent the Agencys current thinking on this topic. 
  10  Contains Nonbinding Recommendations 
 
IVIVC is an approach to describe the relationship between an in vitro attribute of a dosage form 
(e.g., the rate or extent of drug release) and a relevant in vivo measure (e.g., the plasma drug 
concentration or the amount of drug absorbed).  Modeling of this relationship facilitates the 
rational development and evaluation of ER dosage forms, and less commonly, of other dosage 
forms.  Once an IVIVC is validated, the in vitro test serves as a surrogate for BA testing as well 
as a tool to screen formulations and set the dissolution and drug-release acceptance criteria. 
 
Specifically, in vitro dissolution and drug-release characterization are recommended for all ER 
product formulations (including prototype formulations), particularly when used to define the in 
vivo absorption characteristics for different product formulations.  Such efforts can enable the 
establishment of an IVIVC.  When an IVIVC or in vitro-in vivo relationship (IVIVR) is 
established, the in vitro test can serve not only as a quality control specification for the 
manufacturing process but also as an indicator of how the product will perform in vivo.28 
 
Additional information on the development and validation of an IVIVC can be found in the FDA 
guidance entitled Extended Release Oral Dosage Forms: Development, Evaluation, and 
Application of In Vitro/In Vivo Correlations (September 1997).  Sponsors should contact the 
appropriate review division regarding approaches for establishing IVIVC. 
 
3.  PD Studies 
 
PK endpoints are preferred because they are generally the most accurate, sensitive, and 
reproducible endpoint.  However, in instances where a PK endpoint is not possible, a well-
justified PD endpoint should be used to determine BA or to demonstrate BE.   
 
4.  Comparative Clinical Studies 
 
In limited cases, the measurement of the active ingredients or active moieties in an accessible 
biological matrix (i.e., the PK approach) or a PD approach is not possible for orally administered 
drug products; in such cases, clinical endpoints can be used.29  These clinical trials would 
generally involve larger sample sizes compared to PK and PD studies due to variability in the 
measurement of the endpoints.30  Because these circumstances do not occur very often, use of 
this approach is expected to be rare (section VI.D).   
 
 
IV.  ASSESSING BA AND DEMONSTRATING BE FOR VARIOUS DOSAGE 
FORMS 
 
 
28 21 CFR 320.24(b)(1)(ii). 
 
29 21 CFR 320.24(b)(4).  
 
30 21 CFR 320.24(b)(4).  
  11  Contains Nonbinding Recommendations 
 
This section summarizes the recommendations for assessing BA and demonstrating BE based on 
the specific dosage forms.  It also describes when BA studies or BE studies should occur (pre- or 
postapproval). 
 
A.  Solutions and Other Solubilized Dosage Forms 
 
For oral solutions, elixirs, syrups, tinctures, or other solubilized dosage forms, in vivo BA is 
generally self-evident, and a requirement of in vivo BA data for a product can be waived based 
on other data in the application.31  Even when a comparative study is not needed, 
characterization of the pharmacokinetics of the drug is required.32  In addition, in vivo BA 
studies that compare different solution formulations are waived based on the assumptions that:  
(1) the release of drug substance from the drug product is self-evident; and (2) that the solutions 
do not contain any excipients that significantly affect drug absorption.  However, there are 
certain excipients that can alter the BA (e.g., sorbitol can reduce the BA of drugs, and vitamin E 
can enhance the BA) in amounts sometimes used in oral, liquid dosage forms.  In these cases, 
determining the in vivo BA of the drug could be required.33  For solutions that contain co-
solvents or are buffered to maintain the drug in solution, precipitation can occur when the 
solution is exposed to gastric contents.  Formulation changes to such products can result in drug 
precipitation; in such cases, an in vivo study could be required.34 
 
B.  IR Drug Products 
 
Included in this discussion are capsules, tablets (including conventional, buccal, chewable, orally 
disintegrating, and sublingual dosage forms), and suspensions.   
 
1.  Pre-approval: BA Studies 
 
For BA studies, the FDA recommends a single-dose, fasted study.35  Under certain 
circumstances, multiple-dose BA studies (see section III.A.5) could be scientifically 
recommended to assess the systemic exposure of the drug.36  Unconventional dosage forms (e.g., 
buccal, chewable, orally disintegrating, and sublingual dosage forms) should be administered per 
the proposed labeling.  In addition, the sponsor should determine the BA of the unconventional 
dosage form when swallowed intact to assess the impact of accidental swallowing of the intact 
product.  Sampling should adequately capture the T  and C  in addition to the total exposure.  
max max
 
31 21 CFR 320.22(b)(3). 
 
32 21 CFR 314.50(d)(3).   
 
33 21 CFR 320.22(b)(3)(iii). 
 
34 21 CFR 320.22(b)(3)(iii). 
 
35 See generally 21 CFR 320.26. 
 
36 For information about food-effect studies see the FDA draft guidance entitled Assessing the Effects of Food on 
Drugs in INDs and NDAs  Clinical Pharmacology Considerations (February 2019).  When final, this guidance will 
represent the FDAs current thinking on this topic.  Also, see generally 21 CFR 320.27. 
  12  Contains Nonbinding Recommendations 
 
The sponsor should evaluate in vitro dissolution for all orally administered solid, oral dosage 
forms, and suspensions.   
 
2.  Postapproval Changes 
 
An FDA guidance entitled SUPAC-IR:  Immediate Release Solid Oral Dosage Forms Scale-Up 
and Postapproval Changes:  Chemistry, Manufacturing, and Controls; In Vitro Dissolution 
Testing, and In Vivo Bioequivalence Documentation (November 1995) provides 
recommendations on in vitro dissolution and in vivo BE studies for postapproval changes.  For 
postapproval changes, the sponsor should compare the results of in vitro or in vivo studies 
between the products before and after the change. 
 
C.  MR Drug Products 
 
MR products include ER (e.g., controlled-release, sustained-release)37 and DR products.   
 
ER products are dosage forms that are designed to extend or prolong the release of the active 
ingredient or the active moiety from the drug product.  ER products can reduce dosing frequency 
and reduce fluctuations in plasma concentrations when compared to an IR product.  ER products 
can be capsules, tablets, granules, pellets, beads, or suspensions. 
 
DR products are dosage forms that release the active ingredient or active moiety at a time later 
than immediately after administration (i.e., there is a lag between the time of administration and 
the first quantifiable plasma concentration).  Typically, coatings (e.g., enteric coatings) are used 
to delay the release of the drug substance until the dosage form has passed through the acidic 
medium of the stomach.   
 
Even though DR drug products are defined as MR products, many DR products behave like IR 
products after accounting for the delay; hence, the FDA considers the requirements and 
recommendations for the BA study of a DR product to be identical to those of an IR product.  In 
cases where the DR product leads to a complex in vivo profile, the relevant review division 
should be contacted for additional information.  The remainder of this section focuses on 
considerations for ER drug products. 
 
1.  Pre-approval:  BA Studies   
 
Regulations address the purpose and requirements of a BA study for an ER product and stipulate 
that the reference material(s) for such a BA study shall be chosen to permit an appropriate 
scientific evaluation of the ER claims made for the drug product. 38,39  Appropriate reference 
products must be one of the following or any combination thereof:   
 
37  For the purposes of this guidance, the terms extended, controlled, and sustained are used interchangeably. 
 
38 21 CFR 320.25(f)(1). 
 
39 21 CFR 320.25(f)(2). 
 
  13  Contains Nonbinding Recommendations 
 
 
  A solution or suspension of the active drug ingredient or therapeutic moiety 
 
  A currently marketed noncontrolled-release drug product containing the same active drug 
ingredient or therapeutic moiety and administered according to approved labeling of the 
noncontrolled-release drug product 
 
  A currently marketed ER drug product containing the same active drug ingredient or 
therapeutic moiety and administered according to the dosage recommendations in the 
labeling of the currently marketed ER product 
 
  A reference material other than one described above that is appropriate for valid scientific 
reasons40 
 
In addition, under the regulations, the purpose of an in vivo BA study involving a drug product 
for which an ER claim is made is to determine if all of the following conditions are met:41  
 
(i) The drug product meets the ER claims made for it.  
 
(ii) The BA profile established for the drug product rules out the occurrence of any dose 
dumping.42  
 
(iii) The drug product's steady-state performance is equivalent to a currently marketed 
non-extended release or extended-release drug product that contains the same active drug 
ingredient or therapeutic moiety and that is subject to an approved full NDA.  
 
(iv) The drug product's formulation provides consistent PK performance between 
individual dosage units. 
 
Therefore, based on the criteria outlined above, the individual PK profiles will be considered.  
These considerations can apply more broadly to products with complex release characteristics.  
 
The FDA recommends that the following BA studies and food-effect studies be conducted for an 
ER drug product submitted as an NDA for the scenarios described below.  In certain cases, 
nonequivalent doses of the ER and IR products can be evaluated.43  The appropriate review 
division should be contacted for additional information. 
 
a.  A new ER formulation compared to an IR product that is already approved 
 
 
40 21 CFR 320.25(f)(2).  
  
41 21 CFR 320.25(f). 
 
42 21 CFR 320.25(f)(1)(ii). 
 
43 21 CFR 320.25(f)(2)(i) and (iv). 
  14  Contains Nonbinding Recommendations 
 
  For drugs with linear pharmacokinetics over the therapeutic dose range, a fasted study 
should compare the ER product administered as a single dose at the highest strength to 
the IR reference product administered over the same interval used for the ER product to 
achieve the same total dose as the ER product.  If for safety reasons the highest strength 
cannot be used, a lower strength should be used.  
  
Consider in the following example, that a 150-milligram (mg) ER product administered 
once daily (QD) is being developed given an approved 50-mg IR reference product 
administered three times a day (TID) or a 75-mg product administered two times a day 
(BID).  For relative BA purposes, the 150-mg ER product administered as a single dose 
could be compared to either the 50-mg IR reference product administered TID or the 75-
mg IR reference product administered BID.    
 
  For drugs with nonlinear pharmacokinetics over the therapeutic dose range, at a 
minimum, a single dose of the highest and lowest strengths of the ER product should be 
compared to their corresponding IR reference products administered over the same time 
period as the ER dosing interval.  If the relative BA of intermediate ER strengths cannot 
be inferred based on the above studies, a single-dose fasted study for the intermediate 
strength or strengths of the ER product should be compared to the corresponding IR 
reference products administered over the ER dosing interval. 
 
  If multiple ER strengths are being developed, and the ER strengths are not proportionally 
similar in composition, a single-dose fasted dosage strength equivalence assessment 
study or a dosage strength proportionality study for the ER product should be conducted. 
Examples of each are: 
 
  If three strengths, 10, 25, and 50 mg, are being developed for a new ER dosage 
o
form, the dosage strength equivalence study should be conducted using 510 mg 
strength, 225 mg, and 150 mg to achieve constancy of dose. 
 
  If three strengths, 10, 25, and 50 mg, are being developed for a new ER dosage 
o
form, the dosage strength proportionality study should be conducted using 110 
mg, 125 mg, and 150 mg. 
 
  When the ER strengths are proportionally similar in composition, and in vitro release 
testing demonstrates different release-rate profiles, a single-dose, fasted, dosage-strength 
equivalence assessment study or a dosage-strength proportionality study for the ER 
product should be conducted. 
 
  A single-dose, high-fat, food-effect study should be conducted on the highest strength of 
the new ER product (ER ).44 
new
 
 
44 See the FDA draft guidance entitled Assessing the Effect of Food on Drugs in INDs and NDAs  Clinical 
Pharmacology Considerations (February 2019) for more information.  When final, this guidance will reflect the 
FDAs current thinking on this topic. 
  15  Contains Nonbinding Recommendations 
 
  A steady-state study should be conducted on the highest strength of the ER product 
compared to an approved IR reference product and dosed to achieve the equivalent total 
dose of the ER product. 
 
b.  New ER product (ER ) comparison to an approved ER product (ER ) 
new old
with a different dosing interval (i.e., where ER  and ER  have unequal dosing 
new old
intervals)  
 
  The recommendations for the development of a new ER product given an approved ER 
product with a different dosing interval are the same as outlined in the previous section 
C.1.a. (i.e., development of a new ER formulation given an already approved IR product) 
except for the choice of the reference product.  In this case, the reference product could 
be either the approved ER  or the IR product. 
old
 
c.  New ER product (ER ) comparison to an approved ER product (ER ) 
new old
with the same dosing interval. 
 
  The sponsor should conduct a single-dose, fasted, BE study on the highest strength of the 
ER  product compared to the ER  product.  If the ER  and ER products are 
new old new old 
different strengths, the sponsor should compare the ER  versus ER  products using the 
new old
highest strengths of these products and the same molar dose. 
 
  A single-dose, high-fat, food-effect study should be conducted using the highest ER  
new
strength. 
 
  When the ER  strengths are not proportionally similar in composition, a single-dose, 
new
fasted dosage-strength equivalence assessment study or a dosage-strength proportionality 
study for the ER  product should be conducted. 
new
 
  If the PK profiles of the two ER products are different (e.g., the shape of the time versus 
concentration profile is different), demonstrating BE between the new and old ER 
products might not be sufficient to ensure that there is no difference in safety or efficacy.  
Additional clinical studies could be scientifically recommended to ensure that the two 
products have the same clinical effect and safety profile. 
 
2.    Postapproval Changes 
 
An FDA guidance entitled SUPAC-MR:  Modified Release Solid Oral Dosage Forms:  Scale-Up 
and Postapproval Changes:  Chemistry, Manufacturing, and Controls; In Vitro Dissolution 
Testing, and In Vivo Bioequivalence Documentation (October 1997) provides recommendations 
on the types of in vitro dissolution and in vivo BE studies for MR drug products, including ER 
drug products, to support specific postapproval changes.  For postapproval changes, the FDA 
recommends that the sponsor conduct in vitro or in vivo comparisons between the product made 
before the change and the product made after the change. 
 
 
  16  Contains Nonbinding Recommendations 
 
V.  ADDITIONAL INFORMATION ON IN VITRO APPROACHES 
 
A.    General Considerations 
 
The regulations indicate that if in vivo BA or BE data are required for a product, a sponsor can 
seek a waiver of these requirements under certain circumstances.45,46  For example, sometimes in 
vivo BA or BE is self-evident based on certain characteristics of the drug product,47 and no 
additional in vivo data are required.  In other circumstances, a requirement for in vivo BA or BE 
data can be waived, and in vitro data can be accepted instead.48  For example, the requirement 
for in vivo data will be waived for different strengths of an IR drug product when:  (1) the drug 
product is in the same dosage form, but in a different strength; (2) this different strength 
formulation is proportionally similar in its active and inactive ingredients to another drug 
product for which the same manufacturer has obtained approval; and (3) the new strength  
formulation meets an appropriate in vitro test as outlined in the regulations.49,50  In addition, to 
obtain a waiver for higher strengths, the sponsor should demonstrate that the pharmacokinetics 
over the therapeutic dose range are linear. 
 
Characteristics that illustrate that formulations are proportionally similar include: 
 
  All active and inactive ingredients are in identical proportions between different strengths 
(e.g., a tablet of 50-mg strength has exactly half of the active ingredients of a tablet of 
100-mg strength and twice the active ingredients of a tablet of 25-mg strength). 
 
  For drug substances with high potency where the amount of the active drug substance in 
the dosage form is relatively low (i.e., the amount of the active substance is less than 5 
percent of the tablet core weight or the weight of the capsule content), then:  (1) the total 
weight of the dosage form remains nearly the same for all strengths (i.e., within plus or 
 
45 21 CFR 320.22(a). 
 
46 In addition to waiver of an in vivo BA or BE requirement under 21 CFR 320.22, there are certain circumstances in 
which BA or BE can be evaluated using in vitro approaches under 21 CFR 320.24(b)(6).  The scientific principles 
described in this guidance regarding waiver of an in vivo requirement also apply to consideration of in vitro data 
under that regulation.  In such circumstances, an in vivo data requirement is not waived, but rather, FDA has 
determined that in vitro data is the most accurate, sensitive, and reproducible approach for establishing BA or BE, as 
required under 21 CFR 320.24(a).  Nonetheless, for ease of the reader, this guidance refers to either the decision to 
waive an in vivo BA or BE requirement under 21 CFR 320.22 or the decision to accept in vitro BA or BE data in 
accordance with 21 CFR 320.24(a) as a waiver. 
 
47 21 CFR 320.22(b). 
 
48 21 CFR 320.22(d). 
 
49 21 CFR 320.22(d)(2). 
 
50 See also 21 CFR 322.22(d)(3) and (4) for additional reasons for a waiver.  Also, the FDA, for good cause, could 
waive or, for NDAs, defer a requirement for the submission of evidence of in vivo BA or BE if the waiver or 
deferral is compatible with the protection of the public health.  See 320.22(e). 
 
  17  Contains Nonbinding Recommendations 
 
minus 10 percent of the total weight of the strength used in the BA study); (2) the same 
inactive ingredients are used for all strengths; and (3) the change in any strength is 
obtained by altering the amount of the active ingredients and one or more of the inactive 
ingredients.   
 
  Bilayer tablets are considered a single formulation even though they consist of two 
separate layers with different compositions.  In assessing the proportional similarity of 
different strengths of bilayer tablets, all components of both layers should be 
proportionally similar.  The fact that only one layer is proportionally similar and the other 
is not indicates that the products (i.e., the whole tablet) are not proportionally similar.   
 
  Active and inactive ingredients are not in identical proportions between different 
strengths as stated above, but the ratios of the inactive ingredients to the total weight of 
the dosage form are within the limits defined by the FDAs SUPAC-IR and SUPAC-MR 
guidances for industry up to and including Level II changes.51,52 
 
Sponsors should contact the appropriate review division and provide adequate justification if 
they seek to demonstrate that a product is proportionally similar to another drug product using 
another approach. 
 
B.    In Vitro Studies Conducted in Support of BA  
  
The FDA could determine that an in vitro approach is the most accurate, sensitive, and 
reproducible method to determine BA.53  Additional recommendations on the conduct of such 
studies is provided below.   
 
1.  IR Formulations (Capsules, Tablets, and Suspensions) 
 
In vitro data can be used to compare formulations of drug products under certain circumstances.  
If a sponsor seeks to determine the BA of IR formulations for capsules, tablets, and suspensions 
using in vitro data, the FDA recommends that sponsors generate dissolution profiles for all 
strengths using an appropriate dissolution method (see III.B.2 for more information on IVIVC).  
If the results indicate that the dissolution characteristics of the product are not dependent on the 
pH or product strength, then dissolution profiles in one medium are usually sufficient to waive 
the need to assess the in vivo BA.  If these criteria are not met, the sponsor should collect 
dissolution data in at least three media (e.g., pH 1.2, 4.5, and 6.8).  Similarity tests should be 
used to compare dissolution profiles from the different strengths of the product (see the FDA 
guidance entitled Dissolution Testing of Immediate Release Solid Oral Dosage Forms (August 
 
51 SUPAC IR:  Immediate-Release Solid Oral Dosage Forms:  Scale-Up and Postapproval Changes: Chemistry, 
Manufacturing, and Controls, In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation (November 
1995) 
 
52 SUPAC-MR: Modified Release Solid Oral Dosage Forms: Scale-Up and Postapproval Changes: Chemistry, 
Manufacturing, and Controls; In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation (October 
1997) 
 
53 21 CFR 320.24(b)(5) and (6). 
  18  Contains Nonbinding Recommendations 
 
1997)).  A similarity factor (f ) value greater than or equal to 50 indicates a sufficiently similar 
2
dissolution profile to determine the drugs in vivo BA.  For f  values less than 50, the FDA 
2
recommends discussing with the appropriate review division whether an in vivo study is 
recommended.  The f  approach is not suitable for drug products that dissolve very rapidly (i.e., 
2
greater than or equal to 85 percent of the drug product is dissolved in 15 minutes or less).  
Alternative statistical approaches should be used in situations where the f  test is not suitable. 
2
 
a.  Over-encapsulation of clinical trial formulations 
 
Blinding of drug products used in clinical trials can be done by over-encapsulation of the dosage 
form.  The sponsor should assess the impact of this over-encapsulation on the release of the drug 
substance from the drug product.  Dissolution can be used to assess the impact of over-
encapsulation, provided that:  (1) no excipients beyond those that are already in the dosage form 
are added to the capsule; and (2) the dissolution profiles between the over-encapsulated and non-
over-encapsulated products are comparable in three media at pH 1.2, pH 4.5 and pH 6.8.  
However, if other excipients are added, then an in vivo study should be conducted unless the 
sponsor can provide a justification as to why the excipients added do not alter the BA of the 
over-encapsulated product.  These recommendations apply equally to both the drug product 
under investigation as well as any product used as a comparator or reference product in the same 
clinical study.  Enzymes could be added to the dissolution medium to better understand the effect 
of over-encapsulation on drug release. 
 
b.  Scale-up and postapproval changes 
 
Following approval, drug products can undergo formulation or manufacturing changes for a 
variety of reasons.  Formulation changes can occur in components and composition, and 
manufacturing changes can occur in scale-up, manufacturing site, manufacturing process, or 
equipment.  Depending on the possible impact of the manufacturing change on the release of the 
active ingredient from the drug product and the BA of the active ingredient, certain 
manufacturing changes for IR products can be approved based solely on the similarity of the 
dissolution profiles between the formulation after the change and the formulation before the 
change.54,55  Information on recommendations for using in vitro dissolution and in vivo BE 
studies for IR drug products in such circumstances is provided in the FDAs guidance entitled 
SUPAC IR:  Immediate-Release Solid Oral Dosage Forms:  Scale-Up and Postapproval 
Changes: Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, and In Vivo 
Bioequivalence Documentation (October 1997).  The same principles described in this guidance 
can be applied to pre-approval changes such as when the to-be-marketed formulation differs 
from the clinical trial formulation.   
 
 
54 21 CFR 320.21(c)(1), 314.70(b)(2), 320.22(d) (for information on submitting a supplement for FDA pre-
approval). 
 
55 21 CFR 320.22(d) (for information on waivers of evidence of in vivo bioavailability or bioequivalence). 
 
  19  Contains Nonbinding Recommendations 
 
2.  MR Formulations 
 
The use of in vitro data could be acceptable for MR drug products with specific postapproval 
changes.56  Specific information on the use of in vitro data for postapproval changes to MR drug 
products is delineated in the FDAs guidance entitled SUPAC-MR: Modified Release Solid Oral 
Dosage Forms: Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls; 
In Vitro Dissolution Testing, and In Vivo Bioequivalence Documentation (October 1997).  The 
same principles described in the guidance might also apply to pre-approval changes.  Additional 
considerations for the use of in vitro data in support of determining a drugs BA are described 
below. 
 
a.  Beaded capsules   
 
Per 21 CFR 320.24(b)(6), in vivo BA studies for higher strengths of beaded capsules (e.g., a 
strength that is developed after initial BA studies of lower strengths) might not be necessary 
based on:  (1) the clinical safety or efficacy data of the proposed dose and the need for the higher 
strength; (2) the linearity of the pharmacokinetics over the therapeutic dose range; and (3) 
whether the same dissolution procedures were used for all strengths and yielded similar 
dissolution results.  The f  similarity test can be used to demonstrate similar profiles among the 
2
different strengths of the product.  The sponsor can determine the in vivo BA of one or more 
lower strengths by comparing the dissolution profiles and conducting an in vivo BA study only 
on the highest strength (unless safety reasons preclude the administration of the highest strength 
to subjects).  The dissolution profiles for each strength should be generated using the 
recommended dissolution method.  If the dissolution method has not been finalized, dissolution 
profiles should be generated in at least three media (e.g., pH 1.2, 4.5, and 6.8).   
 
b.  Other MR dosage forms   
 
For other MR dosage forms, the sponsor should conduct an in vivo BA study using the highest 
strength.  The sponsor can determine the BA for lower strengths by comparing the dissolution 
profiles using f  evaluation when the drug product is in the same dosage form but in a different 
2
strength, and:  (1) the drug exhibits linear pharmacokinetics; (2) the various strengths are 
proportionally similar in their active and inactive ingredients; and (3) the mechanism of release 
of the drug is the same.57  If the formulations of all the strengths are not compositionally 
proportional, in vitro data can be submitted for the middle strengths if the following data are 
acceptable:  (1) BA or BE data, as appropriate, for both the highest and the lowest strengths; and 
(2) comparisons of in vitro multimedia dissolution profiles using f  evaluation.  Alternatively, 
2
waivers can be granted for lower strengths that are not proportional to the highest strength if a 
dissolution safe space has been established for the drug product via either IVIVCs or IVIVRs 
combined with virtual BE.58   
 
 
56 21 CFR 320.24(b)(6). 
 
57 21 CFR 320.24(b)(6). 
 
58 21 CFR 320.24(b)(6). 
  20  Contains Nonbinding Recommendations 
 
The dissolution profiles for each strength should be generated using the recommended 
dissolution method.  If the dissolution method has not been finalized, dissolution profiles should 
be generated in at least three media (e.g., pH 1.2, pH 4.5, and pH 6.8).  The dissolution profiles 
should be generated on the test and reference products of all strengths using the same dissolution 
test conditions. 
 
 
VI.  SPECIAL TOPICS 
 
A.  Enantiomers Versus Racemates  
 
During the development of a racemic drug product, the racemate should be measured in BA 
studies using an achiral assay.  It could also be important to measure the individual enantiomers 
of the racemate to characterize the pharmacokinetics of the enantiomers.  For the development of 
a specific enantiomer, chiral inversion should be assessed.   
 
Measuring individual enantiomers in BA is recommended only when all the following conditions 
are met:   
 
  The enantiomers exhibit different PD characteristics 
 
  The enantiomers exhibit different PK characteristics 
 
  Primary efficacy and safety activities reside with the minor enantiomer 
 
  At least one of the enantiomers exhibits nonlinear absorption (as expressed by a change 
in the enantiomer concentration ratio with change in the input rate of the drug) 
 
In such cases, the sponsor should apply BE criteria to the enantiomers separately.   
 
B.  Drug Products With Complex Mixtures as the Active Ingredients 
 
Certain drug products can contain complex drug substances (i.e., active moieties or active 
ingredients that are mixtures of multiple synthetic or natural source components).  The chemical 
structure or biological activity of some or all of the components of these complex drug 
substances might not be fully characterized.  Quantification of all active or potentially active 
components in BA studies might not be possible.  In such cases, sponsors should use a select 
number of components in BA studies.  The criteria for selecting the components should typically 
include the amount of the moiety in the dosage form, the plasma or blood levels of the moiety, 
and the biological activity of the moiety.  When PK approaches are not feasible to assess the rate 
and extent of absorption of a drug substance from a drug product, the sponsor can consider PD, 
clinical, or in vitro approaches.59  In such cases, sponsors should consult the appropriate review 
division on the approach and moieties for conducting BA studies. 
 
59 21 CFR 320.24(b). 
 
  21  Contains Nonbinding Recommendations 
 
 
C.  Drugs With Long Half-Lives 
 
In a BA or a PK study involving an IR, oral product with a long half-life (i.e., greater than or 
equal to 24 hours), characterization of the products half-life should include blood sampling over 
an adequate period of time.  To determine the BA of a drug product containing a drug with a 
long half-life, a single-dose crossover study should be conducted if an adequate washout period 
is used.  If the crossover study is problematic, a study with a parallel design should be used.  For 
either a crossover or parallel study, the sample collection time should ensure that the drug 
product completely moves through the gastrointestinal tract so that the absorption of the drug 
substance (C ) and a suitably truncated AUC (i.e., for drugs that do not exhibit flip-flop 
max
kinetics and drugs that do not have high intra-subject variability) can be used to characterize the 
peak and total drug exposures, respectively.  In these cases, the sponsor should consult the 
appropriate review division on the duration of sampling and the choice of the PK measures for 
determining BA. 
 
D.  Orally Administered Drugs Intended for Local Action 
 
Determining BA when the drug substance produces its effects by local action in the 
gastrointestinal tract can be achieved either by using pharmacokinetics, an acceptable PD 
endpoint, clinical efficacy and safety studies, or suitably designed and validated in vitro studies, 
as appropriate.60  In these cases, sponsors should consult the appropriate review division 
regarding the approach for assessing BA.   
 
E.  Combination and Co-administered Drug Products 
 
Two or more active ingredients can be formulated as a single drug product, which is referred to 
for the purposes of this guidance document as a fixed combination product.  Generally, the 
purpose of an in vivo BA study involving a fixed combination product is to compare the rate and 
extent of absorption of each active drug ingredient or therapeutic moiety in the combination drug 
product to the rate and extent of absorption of each active drug ingredient or therapeutic moiety 
administered concurrently as separate, single-ingredient preparations.61 
 
A two-arm, single-dose, crossover, fasted study of the fixed combination versus the single-
ingredient drug products administered concurrently or an approved combination product 
containing the same active ingredients is recommended.  This study should use the highest 
strength of the fixed combination with matching doses of the individual drug products.  Certain 
alternative study designs could also be considered depending on the specific situation.  For 
instance, when there are no drug interactions between the components of a fixed combination 
consisting of two components, a three-arm study design comparing the combination drug product 
versus the single-ingredient drug products administered separately could be appropriate. 
 
 
60 21 CFR 320.24. 
 
61 21 CFR 320.25(g). 
  22  Contains Nonbinding Recommendations 
 
In addition, evaluation of the effect of a high-fat meal on the new drug product can be useful to 
support labeling of the fixed combination.  A single-dose, high-fat, food-effect study design 
should be used.   
 
Sponsors should consult with the appropriate review division to discuss their specific situation. 
 
BA studies for the fixed combination product should include the measurement of systemic 
concentrations of each active ingredient.  The CI approach for BA assessment should be applied 
to each measured entity of the fixed combination and its reference product.   
 
In specific cases, drug products are given in combination (but are not co-formulated) with the 
objective of increasing the exposure of one of the drugs (i.e., the subject drug).  The second, 
booster drug is not intended to have a direct therapeutic effect and is given only to increase the 
systemic exposure of the subject drug.  When both the subject drug and booster drug are new 
molecular entities, the BA of each should be determined individually and when administered in 
combination.   If there is a change in the subject drug product formulation that results in the need 
for a BA study, the subject drug should be administered with the booster drug for both post-
change and pre-change products.  The corresponding PK measures, including CIs, should be 
determined and reported for the subject drug.  It is not recommended to measure the 
concentrations of the booster drug.  BA studies for the booster drug should be conducted only 
with the booster drug; the subject drug should not be dosed with the booster drug.  When the 
combination (which is not co-formulated) includes a new molecular entity and an approved 
booster drug product, only the BA of the new molecular entity should be assessed.  It is assumed 
that the BA of the approved booster product has been previously evaluated.    
 
F.  Endogenous Substances 
 
Drug products can contain compounds that are also endogenous to humans (e.g., testosterone).  
When the endogenous substances are identical to the drug that is being administered, it can be 
difficult to determine the amount of drug released from the dosage form and absorbed.  In most 
cases, it is important to measure and approximate the baseline endogenous concentrations of the 
compound in the matrix of choice and subtract these levels from the total concentrations 
measured from each subject after the drug product is administered.  Using this approach, the 
sponsor can estimate the true availability of the drug from the drug product and accurately 
determine BA and demonstrate BE.  Endogenous substances can have homeostatic processes that 
affect their production and therefore impact their systemic concentrations.  To reduce the impact 
of this variability in the concentrations of endogenous substances and to potentially avoid the 
need for baseline correction, an alternative approach could be to enroll individuals in BA studies 
with low or no production of the endogenous substances instead of healthy subjects. 
 
Baseline concentrations of the endogenous substance produced by the body should be measured 
in the period before administration of the study drug.  Depending on the proposed indication, it 
could be advisable to subtract the time-averaged baseline or time-matched baseline from the 
post-dose concentration for each subject.  When the concentrations of endogenous substances are 
influenced by diet, restricting the dietary intake of the substance before and during the study 
could also be appropriate.  To achieve a stable baseline measurement, subjects should be housed 
  23  Contains Nonbinding Recommendations 
 
at the study site for a sufficient period of time before the study and served standardized meals 
with similar content to that of the meals served on the day that PK sampling will take place. 
 
Baseline concentrations should be determined for each dosing period, and baseline corrections 
should be period-specific.  PK and statistical analyses should be performed on both uncorrected 
and corrected data.  Because of the complexities associated with endogenous compounds, 
sponsors should contact the appropriate review division for additional information and to discuss 
topics such as diurnal variations and sample timing. 
 
G.  Narrow Therapeutic Index Drugs 
 
In specific circumstances where knowledge of exposure measures of drugs (AUC or C ) are 
max
critical for the safe and effective use of the drug product, or where therapeutic drug monitoring is 
an essential tool for drug product dosing, the acceptable criteria for demonstrating BE might 
need to be narrowed.62  Because of the complexities associated with narrow therapeutic index 
drugs, sponsors should contact the appropriate review division for additional information. 
 
H.  Characterizing the Effects of Alcoholic Beverages on MR Drug Products 
 
The consumption of alcoholic beverages can affect the release of a drug substance from an MR 
formulation, leading to a more rapid release of the drug and altered systemic exposure.  The FDA 
recommends that sponsors who are developing certain MR, solid, oral dosage forms conduct in 
vitro studies to determine the potential for dose dumping from alcohol in vivo.  In vitro 
assessments of the drug release from the drug product using media with various alcohol 
concentrations should be conducted.  Based on these in vitro study results, an in vivo BA study 
co-administering the drug product with alcohol could be needed.63  See appendix C for details 
regarding designs for in vitro studies that evaluate the effect of alcohol on MR drug products. 
 
 
 
62 Yu, LX, W Jiang, X Zhang, R Lionberger, F Makhlouf, DJ Schuirmann, L Muldowney, M-L Chen, B Davit, D 
Conner, and J Woodcock, 2014, Novel Bioequivalence Approach for Narrow Therapeutic Index Drugs, Clinical 
Pharmacol Ther, 97(3):286-291. 
 
63 21 CFR 320.25(f)(1)(ii). 
  24  Contains Nonbinding Recommendations 
 
APPENDIX A:  GENERAL STUDY DESIGN AND DATA HANDLING 
 
The following general approaches are recommended, recognizing that the elements can be 
adjusted for certain drug substances and drug products. 
 
A.  Study Conduct 
 
  Generally, the BA or BE study should be conducted under fasted conditions (i.e., after an 
overnight fast of at least 10 hours).64 
 
  The test and reference products should be administered with about 8 ounces (240 
milliliters) of water to the study subjects. 
 
  Generally, the highest marketed strength should be administered as a single unit.  If the 
highest strength is not deemed safe for healthy subjects, then the study can be performed 
in individuals with the disease or condition being studied, or a lower strength might be 
appropriate in healthy subjects.  If bioanalytical sensitivity is a limitation, multiple units 
of the highest strength should be administered, if the total single dose remains within the 
labeled dose range, and the total dose is safe for administration to the study subjects. 
 
  An adequate washout period (e.g., greater than or equal to five half-lives of the moieties 
to be measured or until the drug concentration is less than or equal to 5 percent of the 
C  in all subjects) should separate each treatment. 
max
 
  The lot numbers of both the test and reference listed products and the expiration date for 
the reference product should be stated.  The sponsor should include a statement of the 
composition of the test product and, if possible, a side-by-side comparison of the 
compositions of test and reference listed products.  Under 21 CFR 320.63, the study drug 
test article of the test and reference products must be retained for 5 years following the 
date on which the application is approved, or if such application is not approved, at least 
five years following the date of completion of the bioavailability study in which the test 
article was used.65  Samples of the test and reference listed product should be retained in 
accordance with the FDA guidances entitled Handling and Retention of Bioavailability 
BA and Bioequivalence BE Testing Samples (May 2004) and Compliance Policy for the 
Quantity of Bioavailability and Bioequivalence Samples Retained Under 21 CFR 320.38 
(c) (August 2020). 
 
  Before and during each study phase, we recommend that subjects:  (1) are allowed water 
as desired except for 1 hour before to 1 hour after drug administration; (2) are provided 
standard meals no less than 4 hours after drug administration; and (3) abstain from 
 
64 See the FDA draft guidance entitled Assessing the Effects of Food on Drugs in INDs and NDAs  Clinical 
Pharmacology Considerations (February 2019) for more information.  When final, this guidance will represent the 
FDAs current thinking on this topic. 
 
65 See 21 CFR 320.38(e). 
  25  Contains Nonbinding Recommendations 
 
alcohol for 24 hours before each study period and until after the last sample from each 
period is collected. 
 
B.  Sample Collection and Sampling Times 
 
Under normal circumstances, sponsors should collect blood, rather than urine or tissue.  In most 
cases, the drug or metabolites should be measured in serum or plasma.  However, in certain 
cases, such as when an assay of sufficient sensitivity cannot be developed for plasma, whole 
blood might be more appropriate for analysis.  We recommend that sponsors draw blood samples 
at appropriate times to describe the absorption, distribution, and elimination phases of the drug.  
For most drugs, we recommend collecting 12 to 18 samples (including a pre-dose sample) per 
subject, per dose.  This sampling should continue for at least three terminal elimination half-
lives.  For multiple-dose studies, sampling must occur at steady-state across the dose interval and 
include the beginning and the end of the interval.66  The exact timing for sample collection 
depends on the nature of the drug and the rate of input from the administered dosage form.  The 
sample collection should be spaced in such a way that the C  of the drug in the blood and 
max
terminal elimination rate constant ( ) can be estimated accurately.  The sponsor should collect 
z
three or more samples during the terminal log-linear phase to obtain an accurate estimate of   
z
from linear regression.  We recommend recording the actual clock time when the samples are 
drawn as well as the elapsed time after drug administration. 
 
C.  Subjects With Pre-Dose Plasma Concentrations 
 
If the pre-dose concentration is less than or equal to 5 percent of the C  value in that subject, 
max
the subjects data without any adjustments can be included in all PK measurements and 
calculations.  We recommend that if the pre-dose value is greater than 5 percent of the C , the 
max
subject should be dropped from all PK evaluations.  However, this subjects data should be 
flagged and reported, and the subject should be included in the safety evaluations. 
 
D.  Handling Outliers 
 
If any data are identified as statistical outliers, sponsors should not remove the data from the 
statistical analysis of BA studies solely based on this fact.  The only instance where outlier data 
should be removed from the statistical analysis of a BA study is when there is coinciding 
documentation demonstrating a protocol violation (e.g., real-time documentation of a sample 
processing error as opposed to a retrospective investigation based on the analytical results).  Data 
from re-dosing studies should not be considered as evidence to support the removal of outlier 
data from the statistical analysis.  Data from all subjects should be submitted, and potential 
outliers should be flagged with appropriate documentation as part of the submission.  The 
protocol should specify how outliers will be defined and handled.  
 
E.  Data Deletion Because of Vomiting 
  
 
66 21 CFR 320.27(d)(1). 
  26  Contains Nonbinding Recommendations 
 
  We recommend that data from subjects who experience emesis during a study for IR 
products be deleted from statistical analysis if vomiting occurs at, or before, two times 
the median T .   
max
 
  For MR products, subjects who experience emesis at any time during the labeled dosing 
interval should not be included in the statistical analysis. 
 
  Plasma concentration data from subjects who experience emesis during the study should 
be flagged and reported even though they were excluded from the statistical analysis. 
 
F.  Data Submission and Analysis 
 
The sponsor should submit the following PK information: 
 
  Drug concentrations in plasma/other acceptable matrices and their corresponding 
sampling time points 
 
  Study design elements:  Subject, period, sequence of drug administration, and treatment 
 
  Measures of variability:  Intersubject, intrasubject, and total variability, if available 
 
  PK parameters for single-dose studies:  AUC , AUC , truncated or partial AUC and 
0-t 0-INF
t , if applicable, C , T , z, t apparentclearance, and volume of distribution 
lag max max 1/2,   
 
  Steady-state PK parameters for multiple-dose studies:  AUC , C , T , the lowest 
0-TAU max max
concentration in a dosing interval (C ), the concentration at the end of the dosing 
min
interval (C ), the average concentration during a dosing interval (C ), the degree of 
trough av
fluctuation [(C -C )/C ], and the swing [(C -C )/C ].  C  should be 
max min av max min min trough
measured for at least two dosing intervals to assess whether steady-state was achieved.  
The drugs clearance and volume of distribution should also be reported. 
 
  Statistical information for AUC  AUC  and C :  Geometric means, arithmetic 
0-t, 0-INF, max
means, as well as geometric mean ratios and their corresponding 90 percent CIs 
 
For details pertaining to data analysis, consult the FDA guidance entitled Statistical Approaches 
to Establishing Bioequivalence (February 2001). 
 
G.  Confidence Interval Values for Unscaled Average Bioequivalence Analyses 
 
Sponsors should round off CI values to two decimal places.  For unscaled average 
bioequivalence analyses, to pass a confidence interval limit of 80 to 125 percent, the rounded 
confidence interval value should be at least 80.00 percent and not more than 125.00.  We thus 
recommend that when applicants evaluate the confidence interval to assess bioequivalence using 
an unscaled average bioequivalence analysis during the development program, applicants round 
confidence interval values to two digits after the decimal point. 
 
  27  Contains Nonbinding Recommendations 
 
APPENDIX B:  GUIDELINES FOR CONDUCTING FED OR FASTED STUDIES 
 
For new IR drug products developed via the pathway under section 505(b)(1) of the FD&C Act 
for which BA is determined using a solution, IV, or a previously developed formulation as a 
reference, the BA study should be conducted under fasted conditions except when tolerability 
issues are anticipated in the fasted state.  Additionally, the effect of food on the BA of the new 
drug product should be evaluated using a high-fat and high-calorie meal.  If the objective is to 
evaluate the effect of other meal types, then other meals with different compositions can also be 
assessed in addition to the high-fat and high-calorie meal.67 
 
For new IR drug products developed under either section 505(b)(1) or 505(b)(2) of the FD&C 
Act for which relative BA is determined using an approved product as a reference: 
 
  If the reference drug product is labeled to be taken under fasted conditions, then the test 
drug product should be compared under fasted conditions to the reference drug product 
for the relative BA comparison.  In addition, evaluation of the effect of a high-fat meal on 
the new drug product can be useful to support labeling of the test product.  A three-way 
crossover study can be considered because it allows for the relevant comparisons (e.g., 
test fasted vs reference fasted and food-effect assessment) to be made directly. 
 
  If the reference drug product is labeled to be taken without regard to meals, then the test 
and reference drug product should be compared under fasted conditions.  In addition, the 
effect of a high-fat meal on the new drug product should be evaluated.  Alternatively, the 
BA of the new drug product under fed conditions can be established by comparing the 
test product to the reference drug product both administered with a high-fat meal. 
 
  If the reference drug product is labeled to be taken with food, then the test drug product 
should be compared under fed conditions.  The fed conditions in this study should be the 
same as described in the labeling for the reference product.  However, if no specific meal 
type is described in the reference product labeling, then the high-fat meal should be used 
for the comparison for the fed condition.  In addition, the evaluation of the effect of a 
high-fat meal on the new drug product (test fed versus test fasted) can be useful to inform 
and support labeling of the test product.  A three-way crossover study can be considered 
because it allows for the relevant comparisons to be made directly (e.g., test fed vs 
reference fed and food-effect assessment).  
 
  If the reference drug product is labeled to be taken with food to avoid tolerability issues 
in the fasted state, then the BA for the test drug product should be evaluated under fed 
conditions according to the labeling instructions for the reference product.   
 
The above principles can be adopted for post-approval changes to formulations to determine the 
appropriate studies needed to address issues related to BA and the impact of food intake on the 
 
67 For more information, consult the FDA draft guidance entitled Assessing the Effects of Food on Drugs in INDs 
and NDAs  Clinical Pharmacology Considerations (February 2019).  When final, this guidance will represent the 
FDAs current thinking on this topic. 
  28  Contains Nonbinding Recommendations 
 
BA.  For most post-approval scenarios where the changes to the formulation are not significant, 
food-effect evaluations will likely not be recommended.68  
 
 
 
68 For more information, consult the FDA draft guidance entitled Assessing the Effects of Food on Drugs in INDs 
and NDAs  Clinical Pharmacology Considerations (February 2019).  When final, this guidance will represent the 
Agencys current thinking on this topic. 
  29  Contains Nonbinding Recommendations 
 
APPENDIX C:  GUIDELINES FOR CONDUCTING AN IN VITRO ALCOHOL DOSE-
DUMPING STUDY  
 
The sponsor should conduct in vitro assessments of the drug release from the drug product using 
media with various alcohol concentrations on the lowest and highest strengths of the MR drug 
product.  The following points should be considered during the evaluation of the in vitro, 
alcohol-induced, dose dumping of MR drug products: 
 
  Dissolution testing should be conducted using the optimal apparatus and agitation speed.  
Dissolution data should be generated from twelve dosage units (n=12) at multiple time 
points to obtain a complete dissolution profile. 
 
  The following alcohol concentrations are recommended for the in vitro dissolution 
studies: 0, 5, 20, and 40 percent. 
 
  The general considerations for selecting the media are as follows: 
 
-If the optimal dissolution medium is 0.1N HCl:  Dissolution profiles in 0.1 N HCl 
(pH 1.2) containing the above range of alcohol concentrations are recommended. 
 
-If the optimal dissolution medium is not 0.1N HCl:  Dissolution profiles using the 
above range of alcohol concentrations in 0.1N HCl and in the optimal, proposed 
regulatory dissolution medium are recommended. 
 
  The shape of the dissolution profiles should be compared to determine if the MR 
characteristics are maintained, especially in the first 2 hours. 
 
  The f  values assessing the similarity (or lack thereof) between the dissolution profiles 
2
should be estimated (using 0 percent alcohol as the reference).   
 
  The report should include complete data (e.g., individual, mean, standard deviation, 
comparison plots, f  values) collected during the evaluation of the in vitro, alcohol-
2
induced, dose-dumping study.  
 
Based on the results of the in vitro assessments, an in vivo BA study of the drug product when 
administered with alcohol could be needed.69  Sponsors should consult the appropriate review 
division for need and design of in vivo study or appropriate labeling.  
 
 
 
 
 
69 21 CFR 320.25(f)(1)(ii). 
  30