Bioequivalence Studies With 
Pharmacokinetic Endpoints for 
Drugs Submitted Under 
an ANDA 
Guidance for Industry 
 
 
 
DRAFT GUIDANCE
 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to http://www.regulations.gov.  Submit written 
comments to the Division 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 listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact (CDER) David Coppersmith at 301-796-
9193. 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
August 2021 
Generics   
Bioequivalence Studies With 
Pharmacokinetic Endpoints 
for Drugs Submitted Under 
an ANDA 
 
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  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
August 2021 
Generics    Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
TABLE OF CONTENTS 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  ESTABLISHING BIOEQUIVALENCE ........................................................................ 2 
A.  Pharmacokinetic Studies ........................................................................................................ 3 
1.  General Considerations ...................................................................................................... 3 
2.  Pilot Studies ........................................................................................................................ 3 
3.  Pivotal Bioequivalence Studies ........................................................................................... 3 
4.  Study Designs ...................................................................................................................... 3 
5.  Study Population ................................................................................................................. 4 
6.  Single-Dose Studies ............................................................................................................ 5 
7.  Steady-State Studies ............................................................................................................ 6 
8.  Bioanalytical Methodology ................................................................................................. 6 
9.  Pharmacokinetic Measures of Rate and Extent of Absorption ........................................... 6 
10. Fed Bioequivalence Studies ................................................................................................. 7 
11. Sprinkle Bioequivalence Studies .......................................................................................... 8 
12. Bioequivalence Studies of Products Administered in Specific Beverages ........................... 8 
B.  General Considerations on Other Bioequivalence Studies .................................................. 8 
1.  In Vitro Studies ................................................................................................................... 8 
2.  In Vitro Tests Predictive of Human In Vivo Bioavailability (In Vitro-In Vivo    
Correlation Studies or IVIVC)........................................................................................ 9 
3.  Pharmacodynamic Studies .................................................................................................. 9 
4.  Comparative Clinical Endpoint Studies .............................................................................. 9 
IV.  ESTABLISHING BIOEQUIVALENCE FOR DIFFERENT DOSAGE FORMS ..... 9 
A.  Oral Solutions ........................................................................................................................ 10 
B.  Immediate-Release Products:  Capsules and Tablets ........................................................ 10 
1.  Bioequivalence Study Designs and Dose .......................................................................... 10 
2.  Demonstration of Bioequivalence:  Additional Strengths ................................................. 10 
3.  Post-Approval Changes .................................................................................................... 11 
C.  Suspensions ............................................................................................................................ 11 
D.  Modified-Release Products ................................................................................................... 12 
1. Delayed-Release Products ................................................................................................ 12 
2. Extended-Release Products .............................................................................................. 12 
3. Bioequivalence Study Designs and Dose .......................................................................... 12 
4. Demonstration of Bioequivalence:  Additional Strengths ................................................. 13 
5. Post-Approval Changes .................................................................................................... 13 
E.  Chewable Tablets .................................................................................................................. 14 
F.  Orally Disintegrating Tablets ............................................................................................... 14 
G.  Sublingual .............................................................................................................................. 14  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
H.  Transdermal........................................................................................................................... 14 
V.  SPECIAL TOPICS ......................................................................................................... 15 
A.  Moieties To Be Measured ..................................................................................................... 15 
1.  Parent Drug Versus Metabolites ...................................................................................... 15 
2.  Enantiomers Versus Racemates ........................................................................................ 16 
3.  Drug Products with Complex Mixtures as the Active Ingredients .................................... 16 
B.  Long Half-Life Drugs ............................................................................................................ 16 
C.  First Point C  ...................................................................................................................... 16 
max
D.  Alcoholic Beverage Effects on Modified-Release Drug Products...................................... 17 
E.  Endogenous Compounds ...................................................................................................... 17 
F.  In Vitro Dissolution Testing ................................................................................................. 18 
1.  Immediate-Release Products ............................................................................................ 18 
2.  Modified-Release Products ............................................................................................... 18 
G.  Enteral Feeding Tube ............................................................................................................ 19 
APPENDIX A:  GENERAL DESIGN AND DATA HANDLING OF BIOEQUIVALENCE 
STUDIES WITH PHARMACOKINETIC ENDPOINTS ...................................................... 20 
APPENDIX B:  METHOD FOR STATISTICAL ANALYSIS USING THE REFERENCE-
SCALED AVERAGE BIOEQUIVALENCE APPROACH:  HIGHLY VARIABLE 
DRUGS......................................................................................................................................... 25 
APPENDIX C:  METHOD FOR STATISTICAL ANALYSIS USING THE REFERENCE-
SCALED AVERAGE BIOEQUIVALENCE APPROACH:  NARROW THERAPEUTIC 
INDEX DRUGS ........................................................................................................................... 33 
GLOSSARY................................................................................................................................. 39  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1  Bioequivalence Studies With Pharmacokinetic Endpoints for Drugs 
2  Submitted Under an ANDA 
3   
4  Guidance for Industry1 
5   
6   
7  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
8  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
9  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
10  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
11  for this guidance as listed on the title page.   
12   
13   
14   
15  I.  INTRODUCTION  
16   
17  This guidance provides recommendations to applicants planning to include bioequivalence (BE) 
18  information in abbreviated new drug applications (ANDAs) and ANDA supplements.  In 
19  addition, this guidance describes how to meet the BE requirements set forth in the Federal Food, 
20  Drug, and Cosmetic Act (FD&C Act) and FDA regulations.  This guidance is generally 
21  applicable to dosage forms intended for oral administration and to non-orally administered drug 
22  products in which reliance on systemic exposure measures is suitable for establishing BE (e.g., 
23  transdermal delivery systems and certain rectal and nasal drug products).  This guidance will also 
24  be useful to applicants planning BE studies intended to be conducted during the post-approval 
25  period for changes to a drug product approved under an ANDA. 
26   
27  This guidance revises the draft guidance for industry Bioequivalence Studies with 
28  Pharmacokinetic Endpoints for Drugs Submitted Under an ANDA that was issued in December 
29  2013.2 
30   
31  The contents of this document do not have the force and effect of law and are not meant to bind 
32  the public in any way, unless specifically incorporated into a contract.  This document is 
33  intended only to provide clarity to the public regarding existing requirements under the law.  
 
1 This guidance has been prepared by the Office of Generic Drugs in the Center for Drug Evaluation and Research 
(CDER) in cooperation with CDERs Office of Translational Sciences and the Office of Pharmaceutical Quality at 
the Food and Drug Administration.  
2 FDA recommends that applicants for investigational new drug applications, new drug applications, and new drug 
application supplements consult the draft guidance for industry Bioavailability Studies Submitted in NDAs or INDs 
 General Considerations (February 2019), which addresses bioavailability studies for these submission types.  
When final, this guidance will represent the FDAs current thinking on this topic.  For the most recent version of a 
guidance, check the FDA guidance web page at https://www.fda.gov/RegulatoryInformation/Guidances/default.htm. 
FDA also recommends that ANDA applicants consult routinely published product-specific guidances (PSGs) when 
considering the appropriate BE study and/or other studies for a proposed drug product.  For more information about 
FDAs PSG publications and to search for the most recent version of a PSG, see the Product-Specific Guidances for 
Generic Drug Development web page at  https://www.fda.gov/drugs/guidances-drugs/product-specific-guidances-
generic-drug-development. 
  1  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
34  FDA guidance documents, including this guidance, show be viewed only as recommendations, 
35  unless specific regulatory or statutory requirements are cited.  The use of the word should in 
36  Agency guidance means that something is suggested or recommended, but not required.   
37   
38   
39  II.  BACKGROUND 
40   
41  To receive approval for an ANDA, an applicant generally must demonstrate among other things, 
42  that its proposed drug product is bioequivalent to the reference listed drug (RLD).3  The FD&C 
43  Act provides that a generic drug is bioequivalent to the listed drug if: 
44   
45  The rate and extent of absorption of the drug do not show a significant difference 
46  from the rate and extent of absorption of the listed drug when administered at the 
47  same molar dose of the therapeutic ingredient under similar experimental 
48  conditions in either a single dose or multiple doses.4 
49   
50  For most products, the focus of BE studies is on the release of the drug substance from the drug 
51  product into the systemic circulation.  During such BE studies, an applicant compares the 
52  systemic exposure profile of a test drug product to that of the RLD designated in FDAs 
53  Approved Drug Products with Therapeutic Evaluations (the Orange Book).5, 6 
54   
55   
56  III.  ESTABLISHING BIOEQUIVALENCE 
57   
58  Under FDA regulations, an applicant must use the most accurate, sensitive, and reproducible 
59  approach available among those set forth in 21 CFR 320.24(b) to demonstrate BE.7  As noted in 
60  21 CFR 320.24, in vivo and/or in vitro methods can be used to establish BE.  These methods 
61  include comparative pharmacokinetic (PK), in vitro tests predictive of human in vivo 
62  bioavailability (BA) (in vitro-in vivo correlation (IVIVC)), pharmacodynamic (PD), clinical 
63  endpoint, and in vitro studies.8 
64   
 
3 See section 505(j)(2)(A)(iv) of the FD&C Act (21 U.S.C. 355(j)(2)(A)(iv)) and 21 CFR 314.94(a)(7).   In general, 
to obtain approval of an ANDA for a generic drug, an ANDA applicant first must identify the previously approved 
drug product it seeks to duplicate, i.e., the RLD, and must show, among other things, that the generic drug is 
bioequivalent to the RLD. A reference standard (RS) selected by FDA is the specific drug product that the ANDA 
applicant must use in conducting any in vivo BE testing required to support approval of its ANDA. The RS, selected 
by FDA, is ordinarily the RLD.  For ease of the reader, this guidance document will only use the terms RLD or 
reference product when describing regulatory requirements and recommendations relating to BE.  For more 
information regarding the distinction between an RLD and RS, refer to FDAs guidance for industry Referencing 
Approved Drug Products in ANDA Submissions (October 2020).   
4 Section 505(j)(8)(B)(i) of the FD&C Act.  See also section 505(j)(8)(B)(ii) and (C) of the FD&C Act; 21 CFR 
320.1(e); and 21 CFR 320.23(b).   
5 The Orange Book is available at https://www.accessdata.fda.gov/scripts/cder/ob/. 
6 21 CFR 314.3(b) and FDAs guidance for industry Referencing Approved Drug Products in ANDA Submissions 
(October 2020).   
7 See 21 CFR 320.24(a). 
8 See 21 CFR 320.24(b). 
  2  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
65  A.  Pharmacokinetic Studies 
66   
67  1.  General Considerations 
68   
69  As provided above, the statutory definition of BE, expressed in terms of rate and extent of 
70  absorption of the active ingredient or moiety, emphasizes the use of PK endpoints in an 
71  accessible biological matrix (such as whole blood, plasma, and/or serum) to indicate release of 
72  the drug substance from the drug product into the systemic circulation.9  BE frequently relies on 
73  PK endpoints such as C 10 and AUC that are reflective of the rate and extent of absorption, 
max
74  respectively. 
75   
76  If serial measurements of the drug and/or its metabolites in plasma, serum, or whole blood 
77  cannot be accomplished, measurement of urinary excretion can be used to demonstrate BE. 
78   
79  2.  Pilot Studies 
80   
81  If the applicant chooses, a pilot study in a small number of subjects can be carried out before 
82  proceeding with a pivotal BE study.  This pilot study can be used to validate analytical 
83  methodology, assess PK variability, estimate sample size to achieve adequate power, optimize 
84  sample collection time intervals, and provide other information.  ANDA applicants are required 
85  to submit information from all BE studies conducted with the same formulation of the proposed 
86  drug product.11 
87    
88  3.  Pivotal Bioequivalence Studies 
89   
90  General recommendations for a standard BE study based on PK endpoints are provided in 
91  Appendix A. 
92   
93  4.  Study Designs  
94   
95  FDA recommends that applicants use (1) a two-period, two-sequence, two-treatment, single-dose 
96  crossover study design, (2) a single-dose parallel study design, or (3) a single-dose replicate 
97  study design for BE studies.  The BE studies generally should be conducted on the highest 
98  strength of the drug product, unless safety considerations preclude the use of that dose in study 
99  subjects.  The general recommendations for study designs provided in Appendix A should be 
100  considered in designing studies.  FDA recommends that applicants use the average BE method of 
101  analysis with these study designs.  
102   
103  For most dosage forms that release a drug intended to be systemically available, FDA 
104  recommends that applicants perform a two-period, two-sequence, two-treatment, single-dose, 
 
9 See section 505(j)(8)(B) of the FD&C Act. 
10 Terms that appear in bold type are defined in the glossary at the end of this guidance. 
11 See 21 CFR 314.94(a)(7) and FDAs guidance for industry Submission of Summary Bioequivalence Data for 
ANDAs (May 2011).  
 
  3  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
105  crossover study using either healthy subjects or other populations, as appropriate.  In this design, 
106  each subject should receive each treatment (the test and the reference product) in a random order. 
107   
108  A replicate crossover study design (either partial or fully replicate) is appropriate for drugs 
109  whether the reference product is a highly variable drug or not.  A replicate design can have the 
110  advantage of using fewer subjects compared to a non-replicate design, although each subject in a 
111  replicate design study would receive more treatments. 
112   
113  Further, a replicate design is recommended to be used under the following scenarios: 
114   
115    A replicate design is advantageous over a non-replicate design for non-narrow 
116  therapeutic index (NTI) drugs with a high intrasubject variability.12  Either a partial or 
117  fully replicate design may be used, but a reference-scaled BE analysis approach should 
118  only be applied to specific PK metrics that exhibit a high within-subject variability for the 
119  reference product in the pivotal BE study.  Refer to Appendix B for the method of 
120  statistical analysis for the reference-scaled average BE analysis approach for highly 
121  variable drugs and to product-specific guidances (PSGs)13 for detailed recommendations 
122  for particular highly variable drugs. 
123   
124    A fully replicate design is recommended for NTI drugs,14 where within-subject 
125  variability for both the reference and test products can be computed and a reference 
126  scaled-average BE analysis can be conducted to properly adjust the BE acceptance 
127  criteria.  Refer to Appendix C for the method of statistical analysis for the reference-
128  scaled average BE analysis approach for NTI drugs and to PSGs for detailed 
129  recommendations for particular NTI drugs.   
130   
131  FDAs recommendations for replicate study designs and the average BE approach method can 
132  also be found in the guidance for industry Statistical Approaches to Establishing Bioequivalence 
133  (February 2001).  
134   
135  Applicants wishing to use variations of these study designs or analysis methods (e.g., a 
136  sequential design) may submit a controlled correspondence15 with specific questions about their 
137  approach before starting the study. 
138   
139  5.  Study Population 
140   
141  In general, unless otherwise recommended in a PSG: 
142   
143    Healthy subjects or other populations as appropriate are recruited. 
 
12 See Appendix B. 
13 See footnote 2. 
14 See e.g., the draft PSG on Warfarin Sodium tablets (December 2012), which is available on the Product-Specific 
Guidances for Generic Drug Development web page at https://www.fda.gov/drugs/guidances-drugs/product-
specific-guidances-generic-drug-development.  When final, this guidance will represent the FDAs current thinking 
on this topic. 
15 See FDAs guidance for industry Controlled Correspondence Related to Generic Drug Development (December 
2020).  
  4  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
144   
145    Subjects recruited for in vivo BE studies should be 18 years of age or older. 
146   
147    If a drug product is intended for use in both sexes, the applicant should include similar 
148  proportions of males and females in the study or provide a justification supporting the use 
149  of a single-sex population.  Likewise, if a drug product is intended for use in a single sex, 
150  then the applicant should only include subjects of that sex.  Females should not be 
151  pregnant or lactating, and, if applicable, should practice abstention or contraception. 
152   
153    If the drug product is predominantly intended for use in the elderly, the applicant should 
154  include as many subjects as possible at or above age 60 or provide a justification if no 
155  subject at or above age 60 is included in the study. 
156   
157    In general, a BE assessment in adults between two products can be used to support a BE 
158  assessment in pediatric patients.  If the drug product is predominantly intended for use in 
159  pediatric patients younger than 6 years, the applicant should justify that the BE study 
160  results obtained from adult subjects are relevant to the pediatric population.  FDA 
161  recommends that this justification include information supporting that the inactive 
162  ingredients in the proposed products are appropriate for use in the pediatric population. 
163   
164    The total number of subjects in a study should be sufficient to provide adequate statistical 
165  power for a BE demonstration in the proposed study design.   
166   
167  We also recommend that any restrictions on admission into a study be primarily based on safety 
168  considerations.  Sometimes, safety considerations preclude the use of either healthy subjects or 
169  the general population.16  In such situations, applicants should attempt to enroll patients for 
170  whom the drug is intended to treat and whose disease process and treatments are stable for the 
171  duration of the BE study.  An investigational new drug application may be required for certain 
172  products (such as cytotoxic products).17  
173   
174  6.  Single-Dose Studies 
175   
176  We usually recommend single-dose PK studies for both immediate- and modified-release drug 
177  products to demonstrate BE because these studies are generally more sensitive than steady-state 
178  studies in assessing differences in the release of the drug substance from the drug product into 
179  the systemic circulation.  
180   
 
16 Healthy subjects are in general non-smoking adults 18 years of age or older without existing medical conditions or 
required medications that exert physiological effects.  However, general population is a broad collection of adults 
18 years of age or older with or without stable, chronic medical conditions, who may or may not be treated with 
therapeutic drugs that will not interfere with the test medication or bioassay.  Individuals in the general population 
may be enrolled in BE studies if they are in relatively stable condition and their medications are not considered to 
interfere with the test medication or bioassay.  The inclusion criteria for healthy subjects are more restrictive than 
the criteria for the general population, and healthy subjects is a subset of general population.  These two terms are 
not used interchangeably. 
17 See 21 CFR 312.2(c) and 320.31. 
  5  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
181  7.  Steady-State Studies 
182   
183  When safety considerations suggest using patients who are already receiving a medication, often 
184  the only approach to establish BE without disrupting a patients ongoing treatment is in a steady-
185  state study.  If a steady-state study is used, we recommend that applicants carry out appropriate 
186  dosage administration and sampling to demonstrate the attainment of steady state.   
187   
188  8.  Bioanalytical Methodology 
189   
190  We recommend applicants ensure that their bioanalytical methods for BE studies are accurate, 
191  precise, selective, sensitive, and reproducible.  The guidance for industry Bioanalytical Method 
192  Validation (May 2018) is available to assist applicants in validating bioanalytical methods. 
193   
194  9.  Pharmacokinetic Measures of Rate and Extent of Absorption  
195   
196  a.  Rate of absorption (peak exposure) 
197   
198  For both single-dose and steady-state studies, FDA recommends that applicants assess the rate of 
199  absorption by measuring the C  obtained directly from the data (i.e., without interpolation).  
max
200  T  can also provide important information regarding the rate of absorption.  Applicants should 
max
201  evaluate T  differences between their product and the reference product for any clinical 
max
202  implications.   
203   
204  b.  Extent of absorption (total exposure)  
205   
206  For single-dose studies, FDA recommends that the indicators for the extent of absorption be both 
207  of the following: 
208   
209    Area under the plasma, serum, or blood concentration-time curve from time zero to 
210  time t (AUC ), where t is the last time point with a measurable concentration 
0-t
211   
212    Area under the plasma, serum, or blood concentration-time curve from time zero to 
213  time infinity (AUC ), where: 
0-inf
214   
215  AUC  = AUC + C/
0-inf 0-t  t z 
216   
217    C is the last measurable drug concentration 
t
218   
219      is the terminal or elimination rate constant calculated according to an 
z
220  appropriate method 
221   
222  For steady-state studies, FDA recommends that the indicator for the extent of absorption be the 
223  area under the plasma, serum, or blood concentration-time curve over a dosing interval at steady- 
224  state (AUC , where tau is the length of the dosing interval). 
0-tau
225   
  6  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
226  c.  Partial exposure 
227   
228  Although BE generally can be demonstrated by measurements of C  and AUC, FDA 
max
229  recommends applicants use a partial AUC (pAUC) as an exposure measure if specified in the 
230  applicable PSG.  For instance, pAUC may be used for certain modified-release products in which 
231  the different phases of release correspond to a clinical effect.  The beginning and ending times 
232  for the pAUC should relate to a clinically relevant measure.  FDA recommends that sufficient 
233  quantifiable samples be collected to allow adequate estimation of the pAUC.  As mentioned in 
234  section I of this guidance, for further information on specific products, applicants should consult 
235  the FDAs website to determine whether a PSG for the proposed product is available.18 
236   
237  For drugs with a long elimination half-life, a truncated AUC can be used,19 provided that the 
238  truncated AUC covers the complete absorption phase.  
239   
240   
241  10.  Fed Bioequivalence Studies 
242   
243  Co-administration of food with oral drug products can impact BA.  Therefore, fed BE studies can 
244  determine whether test and RLD products are bioequivalent when co-administered with meals.  
245  The design of the fed BE study should generally be one of the designs described in section 
246  III.A.4 of this guidance.  The fed BE study for products where variability is different (i.e., when 
247  compared to fasting conditions) may use a different design from the fasting BE study based on 
248  the considerations in section III.A.4 of this guidance.  Refer to Appendix A for details on study 
249  design. 
250   
251  For an orally administered immediate-release product, FDA recommends that applicants conduct 
252  a fed BE study, in addition to a fasting BE study, except when the RLD labeling states that the 
253  product should be taken on an empty stomach or when serious adverse events are anticipated 
254  with administration of the drug product under fed conditions.  Similarly, both fasting and fed BE 
255  studies are recommended for products even when the RLD labeling states that the product should 
256  be taken with food, except when serious adverse events are anticipated with fasting 
257  administration, we recommend that applicants conduct only a fed study; a fasting study is not 
258  recommended. 
259   
260  For all orally administered modified-release drug products, FDA recommends that applicants 
261  conduct a fed BE study, in addition to a fasting BE study, irrespective of dosing instructions in 
262  the RLD labeling.  However, a fed study is not recommended when serious adverse events are 
263  anticipated with administration of the drug product under fed conditions.  Similarly, when 
264  serious adverse events are anticipated with fasting administration, we recommend that applicants 
265  conduct only a fed study; a fasting study is not recommended.   
266   
267  If neither a fasting nor fed BE study can be safely conducted in healthy subjects or the general 
268  population, then a BE study in patients is recommended.  
269   
 
18 See footnote 2. 
19 See section V.B of this guidance. 
  7  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
270  11.  Sprinkle Bioequivalence Studies 
271   
272  If the labeling of a modified-release RLD product states that the product can be administered 
273  sprinkled in soft foods, FDA recommends that applicants conduct a sprinkle BE study.  For each 
274  treatment arm of a sprinkle BE study, the product should be sprinkled on one of the soft foods 
275  mentioned in the labeling of the RLD, normally applesauce.  Aside from administration in the 
276  soft food, a sprinkle BE study should follow the recommendations for the fasting BE study 
277  described in Appendix A.  When serious adverse events are anticipated with fasting 
278  administration, a sprinkle BE study should follow the recommendations for the fed BE study 
279  described in Appendix A. 
280   
281  12.  Bioequivalence Studies of Products Administered in Specific Beverages 
282   
283  If the labeling specifies that the product must be administered in a specific beverage or 
284  beverages, applicants should administer the product mixed with one of the beverages mentioned 
285  in the labeling for BE studies.  If additional beverages are listed in the labeling, applicants should 
286  provide evidence that the use of these additional beverages would not result in BE differences.  
287   
288  If applicants have questions not addressed in the applicable PSG about the use of other vehicles 
289  or about the design or analysis of such BE studies, they should contact the Office of Generic 
290  Drugs via a controlled correspondence. 
291   
292  B.  General Considerations on Other Bioequivalence Studies 
293   
294  In certain circumstances, other types of approaches are recommended to support BE.  Some 
295  general considerations regarding these approaches are described in the following sections.  
296  Applicants should consult FDAs guidances for industry for additional information on these 
297  methods as well.20 
298   
299  1.  In Vitro Studies 
300   
301  In general, FDA does not recommend in vitro approaches for drug products that are intended to 
302  be systemically absorbed.  However, under certain circumstances, BE can be evaluated using in 
303  vitro approaches (e.g., dissolution/drug-release testing).21 
304   
305  For highly soluble and rapidly dissolving, orally administered immediate-release drug products, 
306  in vitro data may be acceptable to demonstrate BE based on the biopharmaceutics classification 
307  system as described in the guidance for industry M9 Biopharmaceutics Classification System-
308  Based Biowaivers (May 2021).  
309   
310  The following FDA guidances for industry provide recommendations on developing dissolution 
311  methodology, setting specifications, and the regulatory applications of dissolution testing for 
312  immediate-release drug products: 
313   
 
20 See footnote 2. 
21 See 21 CFR 320.24(b)(5) and (6). 
  8  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
314    Dissolution Testing of Immediate Release Solid Oral Dosage Forms (August 1997) 
315   
316    Dissolution Testing and Acceptance Criteria for Immediate-Release Solid Oral Dosage 
317  Form Drug Products Containing High Solubility Drug Substances (August 2018) 
318   
319  2.  In Vitro Tests Predictive of Human In Vivo Bioavailability (In Vitro-In Vivo  
320      Correlation Studies or IVIVC) 
321   
322  IVIVC is a scientific approach to describe the relationship between an in vitro attribute of a 
323  dosage form (e.g., the rate or extent of drug release) and a relevant in vivo response (e.g., plasma 
324  drug concentration or amount of drug absorbed).  This model relationship facilitates the rational 
325  development and evaluation of modified-release dosage forms and, less commonly, other dosage 
326  forms.  Once an IVIVC is validated, the in vitro test serves as a surrogate for in vivo BA and/or 
327  BE testing, as well as a tool for formulation screening and for setting the dissolution/drug release 
328  acceptance criteria. 
329   
330  Additional information on the development and validation of an IVIVC can be found in the 
331  guidance for industry Extended Release Oral Dosage Forms:  Development, Evaluation, and 
332  Application of In Vitro/In Vivo Correlations (September 1997).  
333   
334  3.  Pharmacodynamic Studies 
335   
336  A validated PD method can be used to demonstrate BE.  However, FDA does not recommend 
337  PD studies for drug products that are intended to be absorbed into the systemic circulation and 
338  for which a PK approach can be used to establish BE. 
339   
340  4.  Comparative Clinical Endpoint Studies 
341   
342  When it is not possible to use the previously described methods, well-controlled BE studies with 
343  comparative clinical endpoints in patients can be used to establish BE. 
344   
345   
346  IV.  ESTABLISHING BIOEQUIVALENCE FOR DIFFERENT DOSAGE FORMS 
347   
348  The following subsections provide recommendations for establishing BE for specific dosage 
349  forms.  As explained below, in certain cases, a requirement for in vivo BE testing may be 
350  waived22 or an alternative approach may be more accurate, sensitive, and reproducible.23 
351   
352   
353   
 
22 See 21 CFR 320.22.   
23 In addition to waiver of an in vivo BE requirement under 21 CFR 320.22, there are certain circumstances in which 
BE can be evaluated using in vitro approaches under 21 CFR 320.24(b)(6). In such circumstances, an in vivo data 
requirement is not waived, but rather, FDA has determined that in vitro data are the most accurate, sensitive, and 
reproducible for a product, as required under 21 CFR 320.24(a).  
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354  A.  Oral Solutions 
355   
356  For oral solutions, elixirs, syrups, tinctures, or other solubilized forms, the in vivo BE testing 
357  requirement may be waived if in vivo BE is self-evident.24  In such instances, the applicant 
358  would be deemed to have complied with and fulfilled any requirement for in vivo BE data.25  For 
359  example, an in vivo BE data requirement can be waived for an oral solution if the formulation 
360  has the same active ingredient in the same concentration and dosage form as the RLD and does 
361  not contain any excipient that significantly affects drug absorption or availability.26 
362   
363  B.  Immediate-Release Products:  Capsules and Tablets 
364   
365  1.  Bioequivalence Study Designs and Dose 
366   
367  For immediate-release capsule and tablet products, FDA generally recommends that applicants 
368  conduct the following studies:  (1) a single-dose, fasting BE study comparing the highest 
369  strength of the test and reference products and (2) a single-dose, fed BE study comparing the 
370  highest strength of the test and reference products.27 If an applicant does not intend to submit an 
371  ANDA for the highest strength of the reference product, then FDA generally recommends using 
372  the highest strength included in the ANDA for BE studies. 
373   
374  Conducting an in vivo BE study on a strength other than the highest may be appropriate for 
375  reasons of safety.  Use of a lower strength for reasons of safety is generally acceptable if the 
376  following conditions are met: 
377   
378    Linear elimination has been documented over the therapeutic dose range. 
379    The recommendations in section IV.B.2 in this guidance are followed. 
380   
381  In other cases (such as non-linear elimination), applicants may contact the Office of Generic 
382  Drugs via a controlled correspondence if there is no applicable PSG or if the proposed strength 
383  differs from what is recommended in the applicable PSG. 
384   
385  2.  Demonstration of Bioequivalence:  Additional Strengths 
386   
387  An in vivo BE requirement for one or more strength(s) can be waived based on (1) acceptable 
388  BE study(ies) on the designated strength, (2) acceptable in vitro dissolution testing of all the 
389  strengths, and (3) proportional similarity of the formulations across all strengths.28 
390   
391  In this guidance, proportionally similar means any of the following:  
392   
393    All active and inactive ingredients are in similar proportion between different strengths 
394  (e.g., a tablet of 50-milligram (mg) strength has all the inactive ingredientsalmost 
 
24 See 21 CFR 320.22(b)(3). 
25 Ibid. 
26 Ibid. 
27 See section III.A.10 of this guidance for more information on fed BE studies. 
28 See 21 CFR 320.22(d)(2).  
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395  exactly half that of a tablet of 100-mg strength, and almost twice that of a tablet of 25-mg 
396  strength). 
397   
398    For drug products that meet the following criteria:  (1) the total weight of the dosage form 
399  remains nearly the same for all strengths (within +/- 10 percent of the total weight of the 
400  strength on which a biostudy was performed), (2) the same inactive ingredients are used 
401  for all strengths, and (3) the change in any strength is obtained by altering the amount of 
402  the active ingredients and one or more of the inactive ingredients.   
403   
404    Active and inactive ingredients that are not in similar proportion between different 
405  strengths can be considered proportionally similar with adequate justification.  FDAs 
406  determination of proportionality will be assessed during the ANDA assessment. 
407   
408  Under any of these scenarios, we recommend that in vivo BE studies be accompanied by in vitro 
409  dissolution profiles on all strengths of each product with the method set forth in the U. S. 
410  Pharmacopeia (USP) drug product monograph or FDAs dissolution database method.29  We also 
411  recommend that applicants conduct a BE study using the strength(s) recommended in the 
412  applicable PSG.30 
413   
414  For additional information on the BE study design for a specific product, we recommend that 
415  applicants consult FDAs Product-Specific Guidances for Generic Drug Development web 
416  page31 to determine whether a PSG for the proposed product is available.  
417   
418  3.  Post-Approval Changes 
419   
420  Refer to the guidance for industry Immediate Release Solid Oral Dosage Forms:  Scale-Up and 
421  Postapproval Changes:  Chemistry, Manufacturing, and Controls, In Vitro Dissolution Testing, 
422  and In Vivo Bioequivalence Documentation (November 1995) for information regarding the BE 
423  testing recommended for specified types of post-approval changes. 
424   
425  For post-approval changes generally, we recommend that applicants make the in vitro 
426  comparison between the pre-change and post-change products.  When in vivo BE studies are 
427  recommended to support a post-approval change for an ANDA product, FDA recommends that 
428  applicants compare the post-change ANDA product to the RLD and not to the pre-change 
429  ANDA product. 
430   
431  C.  Suspensions 
432   
433  FDA generally recommends that applicants establish BE for a suspension in the same manner as 
434  for other solid oral dosage forms.  In vivo studies and dissolution testing should be performed as 
435  described in section IV.B of this guidance on immediate-release products or in section IV.D of 
436  this guidance on modified-release products.  
437   
 
29 See section V.F of this guidance for more information on this method. 
30 See footnote 2. 
31 Ibid. 
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438  D.  Modified-Release Products 
439   
440  Modified-release products include delayed-release products and extended-release (controlled-
441  release or sustained-release) products. 
442   
443  1.  Delayed-Release Products 
444   
445  A delayed-release drug product is a dosage form that releases the active pharmaceutical 
446  ingredient or active moiety at a time later than immediately after administration (e.g., the drug 
447  product exhibits a lag time in quantifiable plasma concentrations).  Typically, the coatings (e.g., 
448  enteric coatings) of delayed-release products have been designed to delay the release of the 
449  medication until the dosage form has passed through the acidic medium of the stomach.  In vivo 
450  tests for delayed-release drug products are similar to those for extended-release drug products, 
451  described below.  We recommend that in vitro dissolution tests for these products document 
452  that they are stable under acidic conditions and that they release the drug only in a neutral 
453  medium (e.g., a pH of 6.8).  For certain delayed-release products, differences in the delayed-
454  release coating polymer(s) between the test and reference product can impact the PK profiles at 
455  a pH between acidic and neutral which may be clinically undesirable, thus dissolution testing in 
456  additional pH/media may be warranted.  FDA recommends that applicants consult this guidance 
457  in conjunction with any relevant PSGs that contain product specific recommendations for a 
458  need to conduct dissolution testing in additional pH/media.32 
459   
460  2.  Extended-Release Products 
461   
462  An extended-release drug product is a dosage form that both allows a reduction in the dosing 
463  frequency and reduces fluctuations in plasma concentrations when compared to an immediate-
464  release dosage form.  Extended-release products can be formulated as capsules, tablets, granules, 
465  pellets, or suspensions.  If any part of a drug product includes an extended-release component, 
466  the product should be treated as a modified-release dosage form to establish BE, as specified in 
467  sections IV.D.3 and IV.D.4 of this guidance. 
468   
469  3.  Bioequivalence Study Designs and Dose 
470   
471  For modified-release products, we generally recommend the following studies:  (1) a single-dose, 
472  fasting BE study comparing the highest strength of the test with the reference product and (2) a 
473  single-dose, fed BE study comparing the highest strength of the test with the reference product.  
474  Because single-dose studies are considered more sensitive in addressing the primary question of 
475  BE (e.g., release of the drug substance from the drug product into the systemic circulation), 
476  multiple-dose studies are generally not recommended. 
477   
478  Conducting an in vivo BE study on a strength other than the highest may be appropriate for 
479  reasons of safety.  Use of a lower strength for reasons of safety is generally acceptable if the 
480  following conditions are met: 
481   
 
32 See footnote 2. 
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482    Linear elimination has been documented over the therapeutic dose range. 
483    The recommendations in section IV.D.4 of this guidance are followed. 
484   
485  In other cases (such as non-linear elimination), applicants may contact the Office of Generic 
486  Drugs via a controlled correspondence if there is no applicable PSG or the proposed strength 
487  differs from what is recommended in the applicable PSG. 
488   
489  4.  Demonstration of Bioequivalence:  Additional Strengths 
490   
491  Additional strengths of modified-release products may be demonstrated to be bioequivalent to 
492  the corresponding reference product strengths under 21 CFR 320.24(b)(6) if all the following 
493  conditions have been met: 
494   
495    The reference product demonstrates dosage form equivalence among different strengths 
496  and demonstrates similar dissolution performance across different strengths. 
497   
498    The test product includes the same excipients for different strengths and the ratios of drug 
499  and excipients among different strengths of the test product is justified and appropriate 
500  for the drug release mechanism of the test product (e.g., drug and excipients of different 
501  strengths can be either proportional or not proportional in quantity).  
502   
503    The additional strength of the test product has the same drug release mechanism as the 
504  strength of the test product that underwent an acceptable in vivo BE study compared to 
505  the reference product. 
506   
507    Dissolution testing of all strengths is acceptable.  The drug products should exhibit 
508  similar dissolution profiles between the strength on which the BE testing was conducted 
509  and other strengths, based on the similarity factor (f ) test or other appropriate statistical 
2
510  approaches (e.g., a multivariate model independent approach or a model dependent 
511  approach) in at least three dissolution media (e.g., a pH of 1.2, 4.5, and 6.8).33 
512   
513  We recommend that applicants generate dissolution profiles on the test and reference products of 
514  all strengths.  To note, there may be instances in which an in vivo BE study for non-
515  proportionally formulated strengths may be necessary to demonstrate bioequivalence.  The 
516  decision of the acceptability of the approach will be made during ANDA assessment based on 
517  the totality-of-evidence (in addition to the dissolution data).  
518   
519  5.  Post-Approval Changes 
520   
521  Refer to FDAs guidance for industry SUPAC-MR:  Modified-Release Solid Oral Dosage Forms:  
522  Scale-Up and Postapproval Changes:  Chemistry, Manufacturing, and Controls; In Vitro 
523  Dissolution Testing and In Vivo Bioequivalence Documentation (October 1997) for information 
 
33 In such instances, we anticipate that such approach will be adequate to demonstrate BE.  See 21 CFR 
320.24(b)(6). 
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524  regarding BE testing recommended for specified types of post-approval changes for modified-
525  release dosage forms. 
526   
527  For post-approval changes, we recommend that applicants perform an in vitro comparison 
528  between the approved (pre-change) product and the test (post-change) product.  If appropriate, 
529  we recommend that the f test be used to compare dissolution profiles.  If the f2 test requirements 
2 
530  are not met, for the comparison of the dissolution profiles, applicants should use another 
531  appropriate statistical approach (e.g., a multivariate model independent approach or a model 
532  dependent approach).  An in vivo BE study may be needed if dissolution profiles are not shown 
533  to be similar.  When an in vivo BE study is recommended to support a post-approval change for 
534  an ANDA product, FDA recommends that applicants compare the post-change ANDA drug 
535  product to the RLD product and not to the pre-change ANDA product. 
536   
537  E.  Chewable Tablets 
538   
539  Applicants should administer chewable tablets according to the directions in the RLD labeling.  
540  If the labeling states that the tablet must be chewed before swallowing, the product should be 
541  chewed when administered in BE studies.  If the labeling gives the option of either chewing the 
542  product or swallowing it whole, the product should be swallowed whole, with 240 milliliters of 
543  water, when administered in BE studies.  We also recommend that applicants conduct in vitro 
544  dissolution testing on intact, whole tablets of the chewable drug product. 
545   
546  F.  Orally Disintegrating Tablets 
547   
548  Applicants should administer orally disintegrating tablets according to the directions in the RLD 
549  labeling.  If the labeling states that the tablet may be administered with or without water, BE 
550  studies should be conducted without water. 
551   
552  G.  Sublingual 
553   
554  Sublingual tablets should not be swallowed.  The tablets should be placed under the tongue until 
555  they are dissolved.  Follow the labeling instruction or the applicable PSG for additional 
556  information on the method of administration. 
557   
558  H.  Transdermal 
559   
560  Transdermal drug products are administered to the skin and designed to deliver the drug through 
561  (rather than to) the skin.  Most transdermal products are extended-release film dosage forms, 
562  more commonly known as transdermal delivery systems.  These deliver drugs into the systemic 
563  circulation at a controlled rate for a specified duration.  To demonstrate the BE of transdermal 
564  delivery system, an in vivo single-dose, two-treatment, two-period crossover BE study with PK 
565  endpoints is recommended.  Studies on adhesion and skin irritation/sensitization are 
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566  recommended as well to assess the noninferiority of the generic product to the reference 
567  product.34 
568   
569  Administration of TDS products should be to the intact skin unless the labeling indicates 
570  otherwise.  Transdermal delivery systems should be applied as directed unless recommended 
571  otherwise in the relevant PSGs.  Reservoir transdermal delivery systems should not be cut or 
572  otherwise altered before application.  Topically applied creams, gels, ointments, lotions, or other 
573  formulations intended for a systemic effect should be applied as directed over a body surface 
574  area consistent with the labeled use.  Systemic BE assessments can be made for transdermal 
575  delivery systems and topical formulations.  If a product can be administered interchangeably to 
576  multiple body sites, it is generally suggested that applicants use a single administration site to 
577  demonstrate BE or refer to recommendations in the applicable PSGs. 
578   
579   
580  V.  SPECIAL TOPICS 
581   
582  A number of topics that may warrant special consideration are addressed in the following 
583  subsections.  If a PSG is available on FDAs Product-Specific Guidances for Generic Drug 
584  Development web page,35 the recommendations in that PSG generally supersede those described 
585  within this section. 
586   
587  A.  Moieties To Be Measured 
588   
589  1.  Parent Drug Versus Metabolites  
590   
591  The parent drug in the dosage form should always be measured in the biological fluids collected 
592  in BE studies, unless accurate assay quantitation is not possible using state-of-the-art-technology.  
593  We generally recommend that applicants measure only the parent drug, rather than metabolites, 
594  because the concentration-time profile of the parent drug is more sensitive to changes in 
595  formulation performance than a metabolite, which is more reflective of metabolite formation, 
596  distribution, and elimination.  Primary metabolite(s), formed directly from the parent compound, 
597  should be measured if they (1) are formed substantially through presystemic metabolism (gut 
598  wall or gut lumen metabolism) and (2) contribute significantly to the safety and/or efficacy of the 
599  product.  This approach should be used for all drug products, including prodrugs.  We 
600  recommend that applicants analyze the parent drug measured in these BE studies using a 
601  confidence interval approach.  Applicants can use the metabolite data to provide supportive 
602  evidence of a comparable therapeutic outcome. 
603   
604  If the parent drug concentrations are too low to allow reliable analytical measurement in blood, 
605  plasma, or serum for an adequate length of time, the metabolite data obtained from these studies 
606  should be subject to the confidence interval approach for BE demonstration. 
 
34 Refer to the draft guidances for industry Assessing Adhesion with Transdermal and Topical Delivery Systems for 
ANDAs (October 2018) and Assessing the Irritation and Sensitization Potential of Transdermal and Topical 
Delivery Systems for ANDAs (October 2018) for details.  When final, these guidances will represent the FDAs 
current thinking on these topics. 
35 See footnote 2. 
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607   
608  2.  Enantiomers Versus Racemates  
609   
610  For BE studies, we recommend using an achiral assay to measure the racemate.  We recommend 
611  measuring individual enantiomers in BE studies only when all the following conditions have 
612  been met:  (1) the enantiomers exhibit different PD characteristics, (2) the enantiomers exhibit 
613  different PK characteristics, (3) the primary efficacy and safety activity reside with the minor 
614  enantiomer, and (4) nonlinear absorption is present (as expressed by a change in the enantiomer 
615  concentration ratio with change in the input rate of the drug) for at least one of the enantiomers.  
616  When all these conditions are met, we recommend that applicants separately apply their BE 
617  analysis to the enantiomers.  
618   
619  3.  Drug Products with Complex Mixtures as the Active Ingredients 
620   
621  Certain drug products contain complex drug substances (e.g., active moieties or active 
622  ingredients that are mixtures of multiple synthetic and/or natural source components).  Some or 
623  all the components of these complex drug substances cannot be fully characterized with regard to 
624  chemical structure and/or biological activity.  For these complex drug products, we do not 
625  encourage quantification of all active or potentially active components in PK studies.  Rather, we 
626  recommend that applicants base BE studies on a small number of markers of rate and extent of 
627  absorption.  Selection of the markers should be based on the characteristics and mechanism of 
628  action of the drug product.  Criteria for marker selection can include the biopharmaceutics of the 
629  dosage form; the amount of the moiety in the dosage form; the plasma or blood concentrations of 
630  the moiety; and the biological activity of the moiety relative to other moieties in the complex 
631  mixture. 
632   
633  B.  Long Half-Life Drugs 
634   
635  For an oral immediate-release product with a long elimination half-life drug (> 24 hours), 
636  applicants can conduct a single-dose, crossover study, provided an adequate washout period is 
637  used.  If the crossover study is problematic, applicants should conduct a BE study with a parallel 
638  design.  For either a crossover or parallel study, sample collection times should be adequate to 
639  ensure completion of gastrointestinal transit of the drug product and absorption of the drug 
640  substance (which usually occurs within approximately 2 to 3 days).  Applicants can use C  and 
max
641  a suitably truncated AUC (for instance, an AUC truncated at 72 hours (AUC )) to 
0-72 hr
642  characterize peak and total drug exposure, respectively.  However, sampling should ensure that 
643  the complete drug absorption phase is covered and characterized.  For drugs exhibiting flip-flop 
644  kinetics with reported t > 24 hours, truncation of AUC may not be appropriate. 
1/2 
645   
646  C.  First Point C  
max
647   
648  The first point of a concentration-time curve in a BE study, based on blood or plasma 
649  measurements, is sometimes the highest point, which raises questions of bias in the estimation of 
650  C  because of insufficient early sampling times.  A carefully conducted pilot study can enable 
max
651  an applicant to avoid this problem. 
652   
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653  In the BE study, collection of blood samples at an early time point, between 5 and 15 minutes 
654  after dosing, followed by additional sample collections (e.g., two to five) in the first hour after 
655  dosing is usually sufficient to assess peak drug concentrations.  Failure to include early (5- to 15-
656  minute) sampling times leading to first time point C  values may result in FDA excluding the 
max
657  data from affected subjects from the BE analysis.  
658   
659  D.  Alcoholic Beverage Effects on Modified-Release Drug Products 
660   
661  The consumption of alcoholic beverages can affect the release of a drug substance from a 
662  modified-release formulation.  The formulation can lose its modified-release characteristics, 
663  leading to a more rapid drug release and an altered systemic exposure, which can have 
664  deleterious effects on the drug's safety and/or efficacy. 
665   
666  FDA recommends that applicants developing certain modified-release solid oral dosage forms 
667  conduct in vitro studies to determine the potential for dose dumping in alcohol which may occur 
668  in vivo.  In vitro assessments of the drug release from the drug product using media with various 
669  alcohol concentrations may be recommended.  An in vivo BE study of the drug product when 
670  administered with alcohol may be appropriate in some cases.  For information on specific 
671  products, we recommend that applicants consult any relevant PSG.36 
672   
673  E.  Endogenous Compounds 
674   
675  Endogenous compounds are already present in the body either because the body produces them 
676  or because they are present in a normal diet.  Because these compounds are identical to the drug 
677  that is being administered, determining the amount of drug released from the dosage form and 
678  absorbed by each subject can be difficult.  We recommend that applicants measure and 
679  approximate the baseline endogenous concentrations in blood (plasma) or urine and subtract 
680  these concentrations from the total concentrations measured from each subject after the drug 
681  product is administered to achieve an estimate of the actual drug availability from the drug 
682  product.  Depending on whether the endogenous compound is naturally produced by the body or 
683  is present in the diet, the recommended approaches for determining BE differ as follows:  
684   
685    When the body produces the compound, we recommend that applicants measure multiple 
686  baseline concentrations from each individual subject in the time period before 
687  administration of the study drug and subtract the time-averaged baseline or time-matched 
688  baseline from post-dose concentrations for those subjects in an appropriate manner 
689  consistent with the PK properties of the drug. 
690   
691    When there is a dietary intake of the compound, we recommend that applicants strictly 
692  control the intake both before and during the study.  Subjects should be housed at a clinic 
693  before the study and served standardized meals containing an amount of the compound 
694  similar to that in the meals to be served on the PK sampling day. 
695   
 
36 See footnote 2. 
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696  For both approaches above, we recommend that applicants determine baseline concentrations for 
697  each dosing period and perform baseline corrections that are period specific.  If a baseline 
698  correction results in a negative plasma concentration value, the value should be set equal to 0 
699  before calculating the baseline-corrected AUC.  PK and statistical analyses should be performed 
700  on both uncorrected and corrected data.  Determination of BE should be based on the baseline-
701  corrected data. 
702   
703  F.  In Vitro Dissolution Testing  
704   
705  The following guidances for industry provide recommendations for developing a dissolution 
706  methodology, setting acceptance criteria/criterion, and applying the regulatory applications of 
707  dissolution testing:  
708   
709    Dissolution Testing of Immediate Release Solid Oral Dosage Forms (August 1997) 
710   
711    Dissolution Testing and Acceptance Criteria for Immediate-Release Solid Oral Dosage 
712  Form Drug Products Containing High Solubility Drug Substances (August 2018) 
713   
714    Extended Release Oral Dosage Forms:  Development, Evaluation, and Application of In 
715  Vitro/In Vivo Correlations (September 1997) 
716   
717    Tablet Scoring:  Nomenclature, Labeling, and Data for Evaluation (March 2013) 
718   
719  1.  Immediate-Release Products 
720   
721  The dissolution of a drug is product specific; FDA recommends that for immediate-release drug 
722  products, applicants develop optimal discriminating dissolution methods.  Applicants may also 
723  use the dissolution method set forth in any related official USP drug product monograph or in 
724  FDAs dissolution database,37 provided that applicants submit adequate dissolution 
725  data/information supporting the discriminating ability of the USP or FDA database method being 
726  proposed for the proposed immediate-release product.   
727   
728  If a new dissolution method is developed, FDA recommends that the submission include the 
729  dissolution method development and validation report with the complete information/data 
730  supporting the proposed method. 
731   
732  2.  Modified-Release Products 
733   
734  For modified-release drug products, FDA recommends that applicants develop specific 
735  discriminating dissolution methods.  Applicants may also use the dissolution method set forth in 
736  any related official USP drug product monograph or in FDAs dissolution database,38 provided 
737  that applicants submit adequate dissolution data supporting the discriminating ability of the USP 
738  or FDA database method being proposed. 
 
37 FDAs dissolution database, which describes FDAs dissolution methods, is available at 
http://www.accessdata.fda.gov/scripts/cder/dissolution/index.cfm. 
38 Ibid. 
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739   
740  If a new dissolution method is developed for the modified-release drug product, FDA 
741  recommends that the submission includes the dissolution method development and validation 
742  report with the complete information/data supporting the proposed method. 
743   
744  Overall, the selected dissolution method and acceptance criteria should be discriminating and 
745  sensitive enough to reject batches/lots that would perform differently from the batches/lots used 
746  in the pivotal BE studies.  
747   
748  If applicants propose a method different from the dissolution method described in the FDA 
749  dissolution database or USP, FDA recommends that they submit data using the dissolution 
750  method described in the FDA dissolution database or USP, in addition to their proposed method, 
751  for comparison. 
752   
753  G.   Enteral Feeding Tube 
754   
755  If the approved labeling for the RLD states that the product may be administered by an enteral 
756  feeding tube (e.g., a nasogastric or a gastric tube), the applicant should conduct in vitro 
757  comparative testing to compare the performance of the test product to that of the reference 
758  product; this comparative testing supports the administration of drugs via enteral feeding tubes.  
759  Refer to PSGs for individual product recommendations.39 
 
39 See footnote 2. 
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760  APPENDIX A:  GENERAL DESIGN AND DATA HANDLING OF BIOEQUIVALENCE 
761  STUDIES WITH PHARMACOKINETIC ENDPOINTS 
762   
763  For both replicate and non-replicate in vivo bioequivalence (BE) studies with pharmacokinetic 
764  (PK) endpoints, the Food and Drug Administration (FDA) recommends the following general 
765  approaches.  However, elements can be adjusted for certain drug substances and drug products. 
766   
767  Study conduct: 
768   
769    Fasting Study:  The test or reference product should be administered with about 8 ounces 
770  (240 milliliters) of water to subjects under fasting conditions (i.e., after an overnight fast 
771  of at least 10 hours). 
772   
773    Fed Study: We recommend that subjects start the recommended meal 30 minutes before 
774  administration of the test or refrence product following an overnight fast of at least 10 
775  hours.  Study subjects should finish eating this meal in 30 minutes or less, and the drug 
776  product should be administered 30 minutes after start of the meal.  The drug product 
777  should be administered with about 8 fluid ounces (240 milliliters) of water.  
778    In general, we recommend that applicants conduct fed BE studies using meals that 
779  provide the greatest effects on gastrointestinal physiology and systemic drug availability.  
780  We recommend a high-fat (approximately 50 percent of total caloric content of the meal), 
781  high-calorie (approximately 800 to 1000 kilocalories) test meal for fed BE studies.  This 
782  test meal should derive approximately 150, 250, and 500 to 600 kilocalories from protein, 
783  carbohydrate, and fat, respectively.40  The caloric breakdown of the test meal should be 
784  provided in the study report. No food should be allowed for at least 4 hours post-dose.  
785  Water may be allowed as desired except for 1 hour before to 1 hour after drug 
786  administration.  Subjects should receive standardized meals scheduled at the same time in 
787  each period of the study. 
788   
789    Before and during each study phase, we recommend that subjects abstain from alcohol 
790  for at least 24 hours before each study period and until after the last sample from each 
791  period has been collected. 
792   
793    Generally, the highest-marketed strength can be administered as a single unit.  If the 
794  highest strength is not deemed safe for healthy subjects or the general population, then 
795  the study can be performed with individuals already prescribed and taking the drug at the 
796  highest marketed strength, or alternatively, in healthy subjects or the general population 
797  using a lower strength, where appropriate.  If warranted to achieve sufficient 
798  bioanalytical sensitivity, multiple units of the highest strength can be administered, 
799  provided that the total single dose remains within the labeled dose range and the total 
800  dose is safe for administration to the study subjects. 
 
40 An example test meal would be two eggs fried in butter, two strips of bacon, two slices of toast with butter, four 
ounces of hash brown potatoes, and eight ounces of whole milk.  Substitutions in this test meal (e.g., beef or chicken 
instead of bacon) can be made as long as the meal provides a similar amount of calories from proteins, 
carbohydrates, and fat and has a comparable meal volume, density, and viscosity. 
  20  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
801   
802    An adequate washout period (e.g., more than five half-lives of the moieties to be 
803  measured) should separate each treatment.  
804   
805    The lot numbers of both test and reference products and the expiration date for the 
806  reference product used in the study should be stated in the study report and the applicable 
807  Bioequivalence Summary Tables.  We recommend that the assayed drug content of the 
808  test product batch not differ from the reference product by more than +/- 5 percent.  The 
809  applicant should include a statement of the composition of the test product and, if 
810  possible, a side-by-side comparison of the compositions of the test and reference 
811  products.  Under 21 CFR 320.63, the study drug test article of the test and reference 
812  products must be retained for 5 years.  For additional information, refer to the guidance 
813  for industry Handling and Retention of BA and BE Testing Samples (May 2004). 
814   
815  Sample collection and sampling times: 
816   
817  We recommend that under normal circumstances, applicants sample blood, rather than urine or 
818  tissue.  In most cases, drug or metabolites are measured in serum or plasma.  However, in certain 
819  cases, whole blood may be more appropriate for analysis.  We recommend drawing blood 
820  samples at appropriate times to describe the absorption, distribution, and elimination phases of 
821  the drug.  For most drugs, we recommend collecting 12 to 18 samples, including a predose 
822  sample, per subject, per dose.  This sampling should continue for at least three or more terminal 
823  elimination half-lives of the drug.  The exact timing for sample collection depends on factors 
824  such as the nature of the drug and the rate of input from the administered dosage form.  The 
825  sample collection should be spaced in such a way that the C 41 and z can be estimated 
max
826  accurately.  At least three samples should be obtained during the terminal log-linear phase to 
827  obtain an accurate estimate of z from linear regression.  We recommend recording the actual 
828  clock time when samples are drawn as well as the elapsed time related to drug administration. 
829   
830  Subjects with pre-dose plasma drug concentrations: 
831   
832  If the pre-dose concentration is  5 percent of the C  value in a subject with a pre-dose plasma 
max
833  concentration, applicants can include the subjects data without any adjustments in all PK 
834  measurements and calculations.  We recommend that if the pre-dose value is > 5 percent of the 
835  C , applicants drop the subject from all BE study evaluations. 
max
836   
 
41 Terms that appear in bold type are defined in the glossary at the end of this guidance. 
  21  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
837  Data deletion because of vomiting: 
838   
839  We recommend that data from subjects who experience vomiting during a BE study for 
840  immediate-release products be deleted from statistical analysis if that vomiting occurred at or 
841  before 2 times median T .  For modified-release products, we recommend deleting data from 
max
842  the analysis if a subject vomits during a period of time less than or equal to the dosing interval 
843  stated in the labeling of the product.  The concentration data for the subject who vomited should 
844  be reported. 
845   
846  Handling of outliers: 
847   
848  Applicants should not remove data from the statistical analysis of BE studies solely because that 
849  data are identified as statistical outliers.  Outlier data may only be removed from the BE 
850  statistical analysis if there is a real-time documentation demonstrating a protocol violation during 
851  the clinical and/or analytical phase of the BE study.  Applicants should include a prospective 
852  plan in the BE study protocol for removing subjects from the BE statistical analysis (e.g., a 
853  clinician documents in a case report form that the subject did not swallow the tablet, based on a 
854  mouth check of the subject).  Data from redosing studies are not considered as evidence to 
855  support removal of outlier data from the statistical analysis.  Note that all subject data should be 
856  submitted and potential outliers flagged with appropriate documentation as part of the 
857  submission. 
858   
859  Pharmacokinetic information in submissions: 
860   
861  We recommend that applicants provide the following PK information in their submissions: 
862   
863    Plasma or other acceptable matrix concentrations and time points (both actual and 
864  nominal sampling time points). 
865   
866    Subject, period, sequence, treatment. 
867   
868    Intersubject, intrasubject, and/or total variability, if available. 
869   
870    For single-dose BE studies:  AUC , AUC , AUC truncated or partial AUCs if 
0-t 0-inf
871  applicable, and C .  In addition, report the following supportive information:  T , K  
max max el
872  and t .  
1/2
873   
874    For steady-state BE studies:  AUC  and C .  In addition, report C (lowest 
0-tau maxSS minSS 
875  concentration in a dosing interval), C (average concentration during a dosing 
avSS 
876  interval), degree of fluctuation [(C -C )/C ], swing [(C -C )/C ], and 
maxSS minSS avSS maxSS minSS minSS
877  T .  
max
878   
879    Additional analysis may be needed in certain cases to ensure that the two products are 
880  bioequivalent.  
  22  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
881   
882  Submission of data from in vivo bioequivalence studies: 
883   
884    For information about submitting electronic datasets, including plasma concentration data 
885  (under PC domain), PK parameter data (under PP domain), and other applicable data 
886  domains for ANDA submissions, refer to the Study Data Tabulation Model 
887  Implementation Guide web page.42  
888   
889    For the most recent version of FDAs study data guidance and technical specifications, 
890  check FDAs Study Data Standards Resources web page.43  This page includes links to 
891  the following:  
892   
893    The guidance for industry on study data standards entitled Providing Regulatory 
894  Submissions in Electronic FormatStandardized Study Data (October 2020) 
895   
896    Relevant technical specifications found in the FDA Data Standards Catalog and the 
897  Study Data Technical Conformance Guide 
898   
899  Statistical information for AUC , AUC , and C : 
0-t 0-inf max
900   
901  We recommend that applicants provide the following statistical information for AUC ,  
0-t
902  AUC , and C : 
0-inf max
903   
904    Geometric means 
905   
906    Arithmetic means 
907   
908    Geometric mean ratios and their corresponding 90 percent confidence intervals and/or 95 
909  percent upper confidence bound, as applicable 
910   
911  We also recommend that applicants provide logarithmic transformation for measures used for BE 
912  demonstration and consult the guidance for industry Statistical Approaches to Establishing 
913  Bioequivalence (February 2001).  
914   
915  Confidence interval values for unscaled average bioequivalence anaylses: 
916   
917  For unscaled average bioequivalence analyses, to pass a confidence interval limit of 80 to 125 
918  percent, the rounded confidence interval value should be at least 80.00 percent and not more than 
919  125.00.  We thus recommend that when applicants evaluate the confidence interval to assess 
920  bioequivalence using an unscaled average bioequivalence analysis during the development 
921  program, applicants round confidence interval values to two digits after the decimal point.  
922   
 
42 The Study Data Tabulation Model Implementation Guide web page is available on the Clinical Interchange 
Standards Consortiums website at https://www.cdisc.org/standards/foundational/sdtmig. 
43 FDAs Study Data Standards Resources web page is available at 
http://www.fda.gov/ForIndustry/DataStandards/StudyDataStandards/default.htm. 
  23  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
923  Highly variable drugs: 
924   
925  For non-narrow therapeutic index (non-NTI) drugs exhibiting high intra-subject variability, 
926  applicants may consider using a reference-scaled average BE approach.  If using this approach, 
927  the applicant should provide evidence of high variability in the PK parameters including AUC 
928  and/or C  for BE assessment.  For the method of statistical analysis using the reference-scaled 
max
929  average BE approach for highly variable drugs, refer to Appendix B and product-specific 
930  guidances for individual product recommendations.44 
931   
932  Narrow therapeutic index drugs: 
933   
934  Narrow therapeutic index (NTI) drugs are defined as those drugs where small differences in dose 
935  or blood concentration may lead to serious therapeutic failures and/or adverse drug reactions that 
936  are life-threatening or result in persistent or significant disability or incapacity. For BE 
937  assessment for NTI drugs, we recommend a reference-scaled average BE approach with a four-
938  way, fully replicated, crossover design study that permits the simultaneous equivalence 
939  comparison of the mean and within-subject variability of the test and reference products.45 For 
940  the method for statistical analysis using the reference-scaled average BE approach for NTI drugs, 
941  refer to Appendix C and product-specific guidances for individual product recommendations.46 
942    
943   
 
44 See the Product-Specific Guidances for Generic Drug Development web page at 
https://www.fda.gov/drugs/guidances-drugs/product-specific-guidances-generic-drug-development to search for 
published product-specific guidances. 
45 Yu L, et. al., Novel Bioequivalence Approach for Narrow Therapeutic Index Drugs. Clin Pharm & Ther, 97(3), 
286-291, 2015. 
46 See footnote 2. 
  24  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
944   
945  APPENDIX B:  METHOD FOR STATISTICAL ANALYSIS USING THE REFERENCE-
946  SCALED AVERAGE BIOEQUIVALENCE APPROACH:  HIGHLY VARIABLE DRUGS 
947   
948  For highly variable drugs, a mixed scaling approach is used. Namely, the reference-scaled 
949  procedure is used for specific PK parameters that have a within subject variability of the 
950  reference product (s )  0.294, and the two one-sided tests procedure is used for PK parameters 
WR
951  with s  < 0.294. In other words, if AUC (AUC0-t47 and AUC0-inf, as applicable) and Cmax have 
WR
952  different s  values, different BE analysis should be conducted. 
WR
953   
954  The following are the steps that can be followed to carry out the statistical analysis for the 
955  reference-scaled average bioequivalence assessment for highly variable drugs: 
956   
957  Step 1.  Determine s , the within-subject standard deviation of the reference product, for the 
WR
958  pharmacokinetic (PK) parameters including AUC and C . 
max
959   
960  a.  If s  < 0.294, use the two one-sided tests procedure to determine bioequivalence 
WR
961  (BE) for the individual PK parameter(s). 
962   
963  b.  If s   0.294, use the reference-scaled procedure to determine BE for the 
WR
964  individual PK parameter(s). 
965   
966  Calculation for s  can be conducted as follows:  
WR
m ni (  )2
Dij Di
967  s2   =  i=1 j=1  
WR 2(nm)
968   
969  Where: 
970   
971    i = number of sequences m used in the study 
972   
973  [m=3 for partially replicate design:  TRR, RTR, and RRT; 
974  m=2 for fully replicate design:  TRTR and RTRT]  
975   
976    j = number of subjects within each sequence 
977   
978    T = Test product 
979   
980    R = Reference product 
981   
982    D  = R   R  (where 1 and 2 represent replicate reference treatments) 
ij ij1 ij2
 
47 Terms that appear in bold type are defined in the glossary at the end of this guidance. 
  25  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
n
i

D
ij
= j=1
D
i
983    ni  
984   
m
985    n = n  (i.e., total number of subjects used in the study, while n is number 
i i
i=1
986  of subjects used in sequence i) 
987   
988  Continue with steps 2 and 3 for PK parameters that have a s   0.294. 
WR
989   
990  Step 2.  Determine the 95% upper confidence bound48 for: 
991   
2
 _ _ 
992  Y Y  s2  
T R WR
 
993  Where:  
_ _
994    Y and Y  are the means of the ln-transformed PK endpoint (AUC and/or 
T R
995  C ) obtained from the BE study for the test and reference products, 
max
996  respectively. 
997   
ln(1.25)2
998       (scaled average BE limit). 

 
W0
999      = 0.25 (regulatory constant). 
W0
1000   
1001  Step 3.  For the test product to be bioequivalent to the reference product, both of the following 
1002  conditions must be satisfied for each PK parameter tested:  
2
 _ _ 
1003  a.  The 95% upper confidence bound for Y Y  s2  must be  0 (numbers 
T R WR
 
1004  should be kept to a minimum of four significant figures for comparsion).  
1005  b.  The point estimate of the Test/Reference geometric mean ratio must fall within 
1006  [0.8000, 1.2500]. 
1007   
1008   
1009  Example SAS codes are presented below.  It is not necessary to use SAS if other software 
1010  accomplish the same objectives. 
1011   
1012  If SAS is used for statistical analysis, note the following: 
 
48 The method for obtaining the upper confidence bound is based on Howes Approximation I, which is described in 
the following paper:  WG Howe, 1974, Approximate Confidence Limits on the Mean of X+Y Where X and Y are 
Two Tabled Independent Random Variables, J Am Stat Assoc, 69(347):789794. 
 
  26  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1013   
1014    PROC GLM should be used for partially replicate (3-way) BE studies 
1015    PROC MIXED should be used for fully replicate (4-way) BE studies 
1016   
1017   
1018    Example SAS Codes:  Partial reference-replicate 3-way design 
1019   
1020  For a BE study with the following sequence assignments in a partial reference-replicate 3-way 
1021  crossover design: 
1022   
  Period 1  Period 2  Period 3 
Sequence 1  T  R  R 
Sequence 2  R  T  R 
Sequence 3  R  R  T 
1023   
1024  1.  For PK parameters with a s   0.294,  use the reference-scaled procedure to determine 
WR
1025  BE. 
1026   
1027  The following codes are an example of the determination of reference-scaled average BE for 
1028  LAUCT with a partially replicate 3-way BE design: 
1029   
1030  Dataset containing TEST observations: 
1031 
data test; 
1032 
set pk; 
1033 
if trt='T'; 
1034 
latt=lauct; 
1035 
run; 
1036 
 
1037  Dataset containing REFERENCE 1 observations: 
1038 
data ref1; 
1039 
set ref; 
1040 
if (seq=1 and per=2) or (seq=2 and per=1) or (seq=3 and per=1); 
1041 
lat1r=lauct; 
1042 
run; 
1043 
 
1044  Dataset containing REFERENCE 2 observations: 
1045 
data ref2;  
1046 
set ref; 
1047 
if (seq=1 and per=3) or (seq=2 and per=3) or (seq=3 and per=2); 
1048 
lat2r=lauct; 
1049 
run; 
1050   
1051  Define the following quantities: 
1052   
T =  the observation on T for subject j within sequence i 
ij 
  27  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
R =  kth observation (k = 1 or 2) on R for subject j within sequence i 
ijk 
R +R
1053  I   =         T  ij1 ij2  
ij ij
2
1054  Dij  =  Rij1  Rij2 
1055  I is the difference between a subjects (specifically, subject j within sequence i) 
ij 
1056  observation on T and the mean of the subjects two observations on R, while D is the 
ij 
1057  difference between a subjects two observations on R. 
1058   
1059  Determine Iij and Dij 
1060 
data scavbe; 
1061 
merge test ref1 ref2;  
1062 
by seq subj; 
1063 
ilat=latt - 0.5*(lat1r+lat2r)); 
1064 
dlat=lat1r-lat2r; 
1065 
run; 
1066   
1067   Intermediate analysis - ilat 
1068 
proc glm data=scavbe; 
1069 
class seq; 
1070 
model ilat=seq/clparm alpha=0.1; 
1071 
estimate 'average' intercept 1 seq 0.3333333333 0.3333333333 
1072 
0.3333333333; 
1073 
ods output overallanova=iglm1; 
1074 
ods output Estimates=iglm2; 
1075 
ods output NObs=iglm3; 
1076 
title1 'scaled average BE'; 
1077 
run; 
1078   
1079  From the dataset IGLM2, calculate the following: 
1080   
1081  IGLM2:   
1082   
pointest=exp(estimate); 
1083 
x=estimate**2stderr**2; 
1084 
boundx=(max((abs(LowerCL)),(abs(UpperCL))))**2; 
1085   
1086  Intermediate analysis - dlat 
1087 
proc glm data=scavbe;  
1088 
class seq; 
1089 
model dlat=seq; 
1090 
ods output overallanova=dglm1;  
1091 
ods output NObs=dglm3; 
1092 
title1 'scaled average BE'; 
1093 
run; 
1094   
1095  From the dataset DGLM1, calculate the following:  
1096   
1097  DGLM1: 
  28  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1098         
dfd=df; 
1099 
  s2wr=ms/2; 
1100   
1101  From the above parameters, calculate the final 95% upper confidence bound: 
1102   
1103 
theta=((log(1.25))/0.25)**2; 
1104 
y=-theta*s2wr;  
1105 
boundy=y*dfd/cinv(0.95,dfd); 
1106 
sWR=sqrt(s2wr); 
1107 
      critbound=(x+y)+sqrt(((boundx-x)**2)+((boundy-y)**2)); 
1108 
 
1109 
 
1110  2.  For PK parameters with a s  < 0.294, use the unscaled average BE approach.  
WR
1111   
1112  The following codes are an example of the determination of unscaled average BE for LAUCT 
1113  with a partially replicate 3-way BE design: 
1114   
1115 
PROC MIXED  
1116 
  data=pk; 
1117 
  CLASSES SEQ SUBJ PER TRT; 
1118 
  MODEL LAUCT = SEQ PER TRT/ DDFM=SATTERTH; 
1119 
  RANDOM TRT/TYPE=FA0(2) SUB=SUBJ G; 
1120 
  REPEATED/GRP=TRT SUB=SUBJ; 
1121 
  ESTIMATE 'T vs. R' TRT 1 -1/CL ALPHA=0.1; 
1122 
  ods output Estimates=unsc1; 
1123 
  title1 'unscaled BE 90% CI - guidance version'; 
1124 
  title2 'AUCt'; 
1125 
run; 
1126 
 
1127 
data unsc1;  
1128 
set unsc1;  
1129 
unscabe_lower=exp(lower);  
1130 
unscabe_upper=exp(upper); 
1131 
run; 
1132   
1133 
 
1134    Example SAS Codes:  Fully replicate 4-period, 2-sequence, 4-way crossover design 
1135   
1136  For a BE study with the following sequence assignments in a fully replicate 4-way crossover 
1137  design: 
1138   
  Period 1  Period 2  Period 3  Period 4 
Sequence 1  T  R  T  R 
Sequence 2  R  T  R  T 
1139   
1140  1.  For PK parameters with a s   0.294,  use the reference-scaled procedure to determine 
WR
1141  BE. 
1142   
1143  The following codes are an example of the determination of reference-scaled average BE for 
1144  LAUCT with a fully replicate 4-way BE design: 
  29  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1145   
1146    Dataset containing TEST 1 observations: 
1147   
1148 
data test1; 
1149 
  set test; 
1150 
  if (seq=1 and per=1) or (seq=2 and per=2);  
1151 
  lat1t=lauct; 
1152 
run; 
1153   
1154    Dataset containing TEST 2 observations: 
1155   
1156 
data test2; 
1157 
set test; 
1158 
if (seq=1 and per=3) or (seq=2 and per=4);  
1159 
  lat2t=lauct; 
1160 
run; 
1161   
1162    Dataset containing REFERENCE 1 observations: 
1163   
1164 
data ref1;  
1165 
  set ref;  
1166 
  if (seq=1 and per=2) or (seq=2 and per=1);  
1167 
  lat1r=lauct; 
1168 
run; 
1169   
1170    Dataset containing REFERENCE 2 observations: 
1171   
1172 
data ref2; 
1173 
  set ref; 
1174 
  if (seq=1 and per=4) or (seq=2 and per=3);  
1175 
  lat2r=lauct; 
1176 
run; 
1177   
1178  The number of subjects in each sequence is n  and n  for sequences 1 and 2, respectively. 
1 2
1179   
1180  Define the following quantities: 
1181   
1182    T   =  kth observation (k = 1 or 2) on T for subject j within sequence i 
ijk
1183   
1184    R   =  kth observation (k = 1 or 2) on R for subject j within sequence i 
ijk
1185   
T +T R +R
1186    I   =  ij1 ij2  ij1 ij2  
ij
2 2
1187    D   =  R   R  
ij ij1 ij2
1188   
1189  I  is the difference between the mean of two observations of a subject (specifically, subject j 
ij
1190  within sequence i) on T and the mean of the subjects two observations on R, while D  is the 
ij
1191  difference between a subjects two observations on R. 
  30  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1192   
1193  Determine I andD
ij    ij 
1194   
1195 
data scavbe; 
1196 
  merge test1 test2 ref1 ref2;  
1197 
  by seq subj; 
1198 
  ilat=0.5*(lat1t+lat2t-lat1r-lat2r); 
1199 
  dlat=lat1r-lat2r; 
1200 
run; 
1201   
1202  Intermediate analysis  ilat 
1203   
1204 
proc mixed data=scavbe; 
1205 
  class seq; 
1206 
  model ilat =seq/ddfm=satterth; 
1207 
  estimate 'average' intercept 1 seq 0.5 0.5/e cl alpha=0.1; 
1208 
  ods output CovParms=iout1; 
1209 
  ods output Estimates=iout2; 
1210 
  ods output NObs=iout3; 
1211 
  title1 'scaled average BE'; 
1212 
  title2 'intermediate analysis - ilat, mixed'; 
1213 
run; 
1214   
1215  From the dataset IOUT2, calculate the following: 
1216   
1217  IOUT2:   
1218 
pointest=exp(estimate); 
1219 
    x=estimate**2stderr**2; 
1220 
    boundx=(max((abs(lower)),(abs(upper))))**2; 
1221   
1222  Intermediate analysis  dlat 
1223   
1224 
proc mixed data=scavbe; 
1225 
  class seq; 
1226 
  model dlat=seq/ddfm=satterth; 
1227 
  estimate 'average' intercept 1 seq 0.5 0.5/e cl alpha=0.1; 
1228 
  ods output CovParms=dout1; 
1229 
  ods output Estimates=dout2; 
1230 
  ods output NObs=dout3; 
1231 
  title1 'scaled average BE'; 
1232 
  title2 'intermediate analysis - dlat, mixed'; 
1233 
run; 
1234   
1235  From the dataset DOUT1, calculate the following: 
1236   
1237  DOUT1:   
1238 
s2wr=estimate/2; 
1239   
1240  From the dataset DOUT2, calculate the following: 
1241   
1242  DOUT2:   
1243   
dfd=df;
  31  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1244   
1245  From the above parameters, calculate the final 95% upper confidence bound:  
1246   
1247 
theta=((log(1.25))/0.25)**2; 
1248 
y=-theta*s2wr;  
1249 
boundy=y*dfd/cinv(0.95,dfd); 
1250 
sWR=sqrt(s2wr); 
1251 
critbound=(x+y)+sqrt(((boundx-x)**2)+((boundy-y)**2)); 
1252 
 
1253   
1254  2.  For PK parameters with a  < 0.294, use the unscaled average BE approach.  
1255  The following codes are an example of the determination of unscaled average BE for LAUCT 
1256  with a fully replicate 4-way BE design: 
1257   
1258 
PROC MIXED  
1259 
  data=pk; 
1260 
  CLASSES SEQ SUBJ PER TRT; 
1261 
  MODEL LAUCT = SEQ PER TRT/ DDFM=SATTERTH; 
1262 
  RANDOM TRT/TYPE=FA0(2) SUB=SUBJ G; 
1263 
  REPEATED/GRP=TRT SUB=SUBJ; 
1264 
  ESTIMATE 'T vs. R' TRT 1 -1/CL ALPHA=0.1; 
1265 
  ods output Estimates=unsc1; 
1266 
  title1 'unscaled BE 90% CI - guidance version'; 
1267 
  title2 'AUCt'; 
1268 
run; 
1269 
 
1270 
data unsc1;  
1271 
set unsc1;  
1272 
unscabe_lower=exp(lower);  
1273 
unscabe_upper=exp(upper); 
1274 
run; 
1275 
 
1276   
  32  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1277   
1278  APPENDIX C:  METHOD FOR STATISTICAL ANALYSIS USING THE REFERENCE-
1279  SCALED AVERAGE BIOEQUIVALENCE APPROACH:  NARROW THERAPEUTIC 
1280  INDEX DRUGS 
1281   
1282  For narrow therapeutic index (NTI) drugs, the study should be a fully replicated, 4-way 
1283  crossover design to scale the bioequivalence limit to the variability of the reference product and 
1284  to simultaneously compare the mean and within-subject variability of the test and reference 
1285  products. The procedure described below includes both reference scaling and unscaled analysis 
1286  and they are combined to ensure that for NTI drugs the BE limits do not exceed 80.00%-
1287  125.00%. 
1288   
1289  The following are the steps that can be followed to carry out the statistical analysis for the 
1290  reference scaled average bioequivalence for narrow therapeutic index drugs: 
1291   
1292   
1293  Step 1.  Determine s , the estimate of within-subject standard deviation of the reference 
WR
1294  product, for the PK parameters including AUC49 and C . 
max
1295   
1296  Calculation for s  can be conducted as follows:  
WR
m ni (  )2
Dij Di
1297  s2 =  i=1 j=1  
WR 2(nm)
1298   
1299  Where: 
1300   
1301    i = number of sequences m used in the study 
1302   
1303  [m=2 for fully replicate design:  TRTR and RTRT]  
1304   
1305    j = number of subjects within each sequence 
1306   
1307    T = Test product 
1308   
1309    R = Reference product 
1310   
1311    D  = R   R  (where 1 and 2 represent replicate reference treatments) 
ij ij1 ij2
n
i

D
ij
= j=1
D
i
1312    ni  
1313   
 
49 Terms that appear in bold type are defined in the glossary at the end of this guidance. 
  33  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
m
1314    n = n  (i.e., total number of subjects used in the study, while n is number 
i i
i=1
1315  of subjects used in sequence i) 
1316   
1317  Step 2.  Use the referenced-scaled procedure to determine BE for individual PK 
1318  parameter(s).   
1319   
1320  Determine the 95% upper confidence bound50 for: 
( )2
1321        YT YR sW2R 
1322  Where: 
   
1323    Y  and Y  are the means of the ln-transformed PK endpoint (AUC and/or 
T R
1324  C ) obtained from the BE study for the test and reference products, 
max
1325  respectively 
2
ln()
1326           (scaled average BE limit) 

 
W0
1327    and  =0.10 (regulatory constant),   (approximately=1.11111, 
W0
1328  the upper BE limit) 
 = 1./0.9
1329   
1330  Step 3.  Use the unscaled average bioequivalence procedure to determine BE for 
1331  individual PK parameter(s).  
1332   
1333  Step 4.  Calculate the 90% confidence interval of the ratio of the within subject standard 
1334  deviation of test product to reference product   . The upper limit of the 
WT WR
1335  90% confidence interval for    will be evaluated to determine if  and 
WT WR WT 
1336  WR are comparable.  
1337 
 

1338  The (1)100%CI for  WT is given by 

WR
1339   
1340   
1341  Where: 
1342   
 
50 The method of obtaining the upper confidence bound is based on Howes Approximation I, which is described in 
the following paper: WG Howe, 1974, Approximate Confidence Limits on the Mean of X+Y Where X and Y  are 
Two Tabled Independent Random Variables, J Am Stat Assoc, 69 (347):789794. 
 
  34  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1343    s is the estimate of  with v as the degree of freedom 
WT WT 1
1344    s is the estimate of   with v as the degree of freedom 
WR WR 2
1345    F is the value of the F-distribution with   (numerator) and 
/2,1,2 1 2
1346  (denominator) degrees of freedom that has probability of /2to its right.  
1347    F  is the value of the F-distribution with   (numerator) and 
1/2,1,2 1 2
1348  (denominator) degrees of freedom that has probability of 1-/2to its 
1349  right.  
1350    here =0.1. 
1351   
1352  Step 5.  For the test product to be bioequivalent to the reference product, the following 
1353  conditions must be satisfied for each PK parameter tested:  
2
 _ _ 
1354  a.  The 95% upper confidence bound for Y Y  s2  must be  0 
T R WR
 
1355  (numbers should be kept to a minimum of four significant figures for 
1356  comparison). 
1357   
1358  b.  Regular unscaled bioequivalence limits of 80.00%-125.00% should be 
1359  passed. 
1360   
1361  c.  The proposed requirement for the upper limit of the 90% equal-tails 
1362  confidence interval for  /  is less than or equal to 2.500. 
WT WR
1363   
1364  Example SAS codes are presented below.  It is not necessary to use SAS if other software 
1365  accomplish the same objectives. 
1366   
1367  If SAS is used for statistical analysis, PROC MIXED should be used for fully replicate 4-way 
1368  crossover BE studies. 
1369   
1370    Example SAS Codes: Fully replicate 4-period, 2-sequence, 4-way crossover design  
1371   
1372  For a BE study with the following sequence assignments in a fully replicate 4-way crossover design:  
1373   
  Period 1  Period 2   Period 3  Period 4 
Sequence 1   T   R   T   R  
Sequence 2   R   T   R   T  
1374   
1375   
1376  The following codes are an example of the determination of reference-scaled average BE for 
1377  LAUCT. Assume that the datasets TEST and REF, have already been created, with TEST having all 
1378  the test observations and REF having all the reference observations. 
1379   
1380  Dataset containing TEST 1 observations:  
1381 
 
  35  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1382 
data test1; 
1383 
 set test; 
1384 
 if (seq=1 and per=1) or (seq=2 and per=2); 
1385 
 lat1t=lauct;  
1386 
run;  
1387   
1388  Dataset containing TEST 2 observations:  
1389   
1390 
data test2;  
1391 
  set test; 
1392 
  if (seq=1 and per=3) or (seq=2 and per=4); 
1393 
  lat2t=lauct;  
1394 
run;  
1395   
1396  Dataset containing REFERENCE 1 observations:  
1397   
1398 
data ref1; 
1399 
  set ref;  
1400 
  if (seq=1 and per=2) or (seq=2 and per=1); 
1401 
  lat1r=lauct;  
1402 
run; 
1403 
 
1404  Dataset containing REFERENCE 2 observations:  
1405   
1406 
data ref2; 
1407 
  set ref; 
1408 
  if (seq=1 and per=4) or (seq=2 and per=3); 
1409 
  lat2r=lauct;  
1410 
run;  
1411 
 
1412  The number of subjects in each sequence is n1 and n2 for sequences 1 and 2, respectively.  
1413   
1414  Define the following quantities: 
1415   
1416  T =kthobservation (k = 1 or 2) on T for subject  j within sequence i 
ijk
1417   
1418  R =kthobservation (k = 1 or 2) on Rfor subject  j within sequence i 
ijk
T +T R +R
1419  I = ij1 ij2  ij1 ij2  
ij
2 2
1420   
1421  and  
1422  D = R R  
ij ij1 ij2
1423   
1424  Iij is the difference between the mean of a subjects (specifically subject j within sequence i) two 
1425  observations on T and the mean of the subjects two observations on R, while Dij is the difference 
1426  between a subjects two observations on R. 
1427   
1428  Determine Iij and Dij 
1429   
  36  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1430 
data scavbe; 
1431 
  merge test1 test2 ref1 ref2; 
1432 
  by seq subj; 
1433 
  ilat=0.5*(lat1t+lat2t-lat1r-lat2r); 
1434 
  dlat=lat1r-lat2r;  
1435 
run;  
1436   
1437  Intermediate analysis - ilat  
1438   
1439 
proc mixed data=scavbe; 
1440 
  class seq; 
1441 
  model ilat =seq/ddfm=satterth; 
1442 
  estimate 'average' intercept 1 seq 0.5 0.5/e cl alpha=0.1; 
1443 
  ods output CovParms=iout1; 
1444 
  ods output Estimates=iout2; 
1445 
  ods output NObs=iout3; 
1446 
  title1 'scaled average BE'; 
1447 
  title2 'intermediate analysis - ilat, mixed'; 
1448 
run; 
1449 
 
1450  From the dataset IOUT2, calculate the following:  
1451  IOUT2:    
1452 
pointest=exp(estimate); 
1453 
x=estimate**2stderr**2; 
1454  boundx=(max((abs(lower)),(abs(upper))))**2; 
1455   
1456  Intermediate analysis - dlat  
1457   
1458 
proc mixed data=scavbe; 
1459 
  class seq; 
1460 
  model dlat=seq/ddfm=satterth; 
1461 
  estimate 'average' intercept 1 seq 0.5 0.5/e cl alpha=0.1; 
1462 
  ods output CovParms=dout1; 
1463 
  ods output Estimates=dout2; 
1464 
  ods output NObs=dout3; 
1465 
  title1 'scaled average BE'; 
1466 
  title2 'intermediate analysis - dlat, mixed'; 
1467 
run;  
1468   
1469  From the dataset DOUT1, calculate the following:  
1470  DOUT1:  
1471 
s2wr=estimate/2;  
1472   
1473  From the dataset DOUT2, calculate the following:  
1474  DOUT2:  
1475 
dfd=df;  
1476   
1477  From the above parameters, calculate the final 95% upper confidence bound:  
1478 
 
1479 
theta=((log(1.11111))/0.1)**2; 
1480 
y=-theta*s2wr; 
1481 
boundy=y*dfd/cinv(0.95,dfd); 
1482 
sWR=sqrt(s2wr); 
  37  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1483  critbound=(x+y)+sqrt(((boundx-x)**2)+((boundy-y)**2)); 
1484   
1485  Calculation of unscaled 90% bioequivalence confidence intervals: 
1486   
1487 
PROC MIXED data=pk; 
1488 
CLASSES SEQ SUBJ PER TRT; 
1489 
MODEL LAUCT = SEQ PER TRT/ DDFM=SATTERTH; 
1490 
RANDOM TRT/TYPE=FA0(2) SUB=SUBJ G; 
1491 
REPEATED/GRP=TRT SUB=SUBJ; 
1492 
ESTIMATE 'T vs. R' TRT 1 -1/CL ALPHA=0.1; 
1493 
ods output Estimates=unsc1; 
1494 
title1 'unscaled BE 90% CI - guidance version';  
1495 
title2 'AUCt';  
1496 
run;  
1497 
 
1498 
data unsc1; 
1499 
  set unsc1; 
1500 
  unscabe_lower=exp(lower); 
1501 
  unscabe_upper=exp(upper); 
1502 
run; 
1503     
  38  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1504  GLOSSARY 
1505   
AUC  Area under the curve 
AUC   Area under the curve extrapolated to infinity 
0-inf
AUC   Area under the curve from time zero to the last measurable time 
0-t
point 
AUC   Area under the curve for one dosing interval at steady state 
0-tau
C   Average plasma concentration at steady state 
avSS
C   Maximum plasma concentration 
max
C   Maximum plasma concentrations during the dosing interval at 
maxSS
steady state 
C   Minimum plasma concentrations at steady state 
minSS
Enantiomers  Two stereoisomers (molecules that are identical in atomic 
constitution and bonding but different in the three-dimensional 
arrangement of the atoms) that are related to each other by a 
reflection; they are mirror images of each other, which are 
nonsuperimposable.  Every stereocenter in one has the opposite 
configuration in the other.  Two compounds that are enantiomers 
of each other have the same physical properties, except for the 
direction in which they rotate the polarized light and how they 
interact with different optical isomers of other compounds. 
pAUC  Area under the curve between two specific time points 
   Terminal or elimination rate constant 
z
Racemate  A racemate is optically inactive.  Because the two isomers rotate 
plane-polarized light in opposite directions, they cancel out; 
therefore, a racemic mixture does not rotate plane-polarized light.  
In contrast to two separate enantiomers, which generally have 
identical physical properties, a racemate often has different 
properties compared to either one of the pure enantiomers.  
Different melting points and solubilities are very common, but 
differing boiling points are also possible.  Pharmaceuticals can be 
available as a racemate or as a pure enantiomer, which might have 
different potencies. 
T   Time to maximum observed plasma concentration 
max
t   Half-life 
1/2
1506   
  39