In Vitro Release Test 
Studies for Topical 
Drug Products 
Submitted in ANDAs 
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DRAFT GUIDANCE 
 
  This guidance document is being distributed for comment purposes only.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publication in the Federal Register of the notice announcing the availability of the draft 
guidance.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 https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (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) Susan Levine at 240-402-7936.  
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
October 2022 
Generic Drugs 
 
   
In Vitro Release Test 
Studies for Topical 
Drug Products 
Submitted in ANDAs 
Guidance for Industry 
 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs  
   
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
October 2022 
Generic Drugs 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
TABLE OF CONTENTS 
 
 
I.  INTRODUCTION ...................................................................................................... 1 
II.  BACKGROUND ........................................................................................................ 2 
III.  IVRT METHOD DEVELOPMENT .......................................................................... 3 
A.  IVRT Method Parameters ............................................................................................. 4 
B.  IVRT Receptor Solution ................................................................................................ 4 
C.  IVRT Membrane .......................................................................................................... 5 
IV.  IVRT METHOD VALIDATION ............................................................................... 5 
A.  Equipment Qualification ............................................................................................... 6 
B.  Membrane Qualification ............................................................................................... 7 
C.  Receptor Solution Qualification ..................................................................................... 7 
D.  Receptor Solution Sampling Qualification ...................................................................... 7 
E.  Environmental Control ................................................................................................. 7 
F.  Linearity and Range...................................................................................................... 7 
G.  Precision and Reproducibility ........................................................................................ 8 
H.  Dose Depletion .............................................................................................................. 8 
I.  Discrimination Sensitivity, Specificity, and Selectivity...................................................... 8 
1. IVRT Sensitivity.............................................................................................................. 9 
2. IVRT Specificity ............................................................................................................. 9 
3. IVRT Selectivity.............................................................................................................10 
4. IVRT Supplemental Selectivity .........................................................................................10 
J.  Robustness ..................................................................................................................11 
V.  SAMPLE ANALYTICAL METHOD VALIDATION ............................................. 12 
VI.  IVRT PIVOTAL STUDY ......................................................................................... 12 
A.  Handling and Retention of Samples...............................................................................13 
B.  Control of Study Procedures .........................................................................................13 
C.  Blinding Procedure ......................................................................................................14 
D.  Dosing .........................................................................................................................14 
VII.  SUBMITTING INFORMATION ON IVRT STUDIES IN AN ANDA.................... 14 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
In Vitro Release Test Studies for Topical Drug Products Submitted 
1 
in ANDAs 
2 
Guidance for Industry1 
3 
4   
5   
6  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
7  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
8  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
9  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
10  for this guidance as listed on the title page.   
11   
12   
13   
14   
15  I.  INTRODUCTION  
16   
17  This guidance is intended to assist applicants who are submitting abbreviated new drug 
18  applications (ANDAs) for liquid-based and/or other semisolid products applied to the skin, 
19  including integumentary and mucosal (e.g., vaginal) membranes, which are hereinafter called 
20  topical products.2 Because of the complex route of delivery associated with these products, 
21  which are typically locally acting, and the potential complexity of certain formulations, topical 
22  products (other than topical solutions) are classified as complex products.3 This guidance 
23  provides recommendations for in vitro release test (IVRT) studies that can be used to compare a 
24  proposed generic (test) topical product and its reference standard (RS) for the purpose of 
25  supporting a demonstration of bioequivalence (BE) to the reference listed drug (RLD). The 
26  reference standard ordinarily is the RLD.4  
27   
 
1 This guidance has been prepared by the Office of Generic Drugs in the Center for Drug Evaluation and Research at 
the Food and Drug Administration.  
2 Topical products in ANDAs within the scope of this guidance include ointments, creams, lotions, emulsions, 
pastes, shampoos, gels, suspensions, sprays, aerosols, foams, and other semisolid and/or liquid-based dosage forms 
dispensed with a structured arrangement of matter (which may include more than one phase state). 
3 A complex product, as defined in the GDUFA Reauthorization Performance Goals and Program Enhancements 
Fiscal Years 20232027 (GDUFA III Commitment Letter) (accessible at 
https://www.fda.gov/media/153631/download), includes, among others, products with complex formulations (e.g., 
colloids) and complex routes of delivery (e.g., locally acting drugs such as dermatological products).  
4 A reference listed drug is the listed drug identified by FDA as the drug product upon which an applicant relies in 
seeking approval of its ANDA (21 CFR 314.3(b)).  A reference standard, which is selected by FDA, is the specific 
drug product that the ANDA applicant must use in conducting any in vivo bioequivalence testing required to support 
approval of its ANDA (see  314.3(b)). We recommend that the reference standard also be used for in vitro testing. 
There may be circumstances (e.g., when the RLD is no longer marketed) in which the reference standard is a drug 
product other than the RLD.  For more information on RLD and reference standard products, see the guidance for 
industry Referencing Approved Drug Products in ANDA Submissions (October 2020). We update guidances 
periodically. For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
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28  This guidance does not address drug products that are administered via ophthalmic, otic, nasal, 
29  inhalation, oral, or injection-based routes, or that are transdermal or topical delivery systems 
30  (including products known as patches, topical patches, or extended-release films).  
31   
32  It is beyond the scope of this guidance to discuss specific topical products to which this guidance 
33  applies. FDA recommends that applicants consult this guidance and any relevant product-
34  specific guidances (PSGs)5 and any other relevant guidances for industry,6 when considering the 
35  design and conduct of IVRT studies that, in conjunction with other studies, as deemed necessary, 
36  may be appropriate to support a demonstration that a proposed generic topical product and its 
37  RLD are bioequivalent. FDA also recommends that applicants routinely refer to FDAs guidance 
38  web pages, because additional guidances may become available that could assist in the 
39  development of a generic topical product. 
40   
41  In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
42  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
43  as recommendations, unless specific regulatory or statutory requirements are cited. The use of 
44  the word should in Agency guidance means that something is suggested or recommended, but 
45  not required.  
46   
47   
48  II.  BACKGROUND 
49   
50  This guidance has been developed as part of FDAs Drug Competition Action Plan,7 which, in 
51  coordination with the Generic Drug User Fee Amendments (GDUFA)8 program and other FDA 
52  activities, is intended to increase competition in the marketplace for prescription drugs, facilitate 
53  the entry of high-quality and affordable generic drugs, and improve public health. 
54   
55  The Federal Food, Drug, and Cosmetic Act (FD&C Act) generally requires an ANDA to contain, 
56  among other things, information to show that the proposed generic drug product (1) is the same 
57  as the RLD with respect to the active ingredient(s), conditions of use, route of administration, 
58  dosage form, strength, and labeling (with certain permissible differences); and (2) is 
 
5 Generic drug product-specific guidances are available at FDAs Product-Specific Guidances for Generic Drug 
Development web page at https://www.fda.gov/drugs/guidances-drugs/product-specific-guidances-generic-drug-
development. 
6 Other relevant guidances include the draft guidances for industry In Vitro Permeation Test Studies for Topical 
Drug Products Submitted in ANDAs (October 2022) and Physicochemical and Structural (Q3) Characterization of 
Topical Drug Products Submitted in ANDAs (October 2022). When final, these guidances will represent the FDAs 
current thinking on these topics. For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
7 See the FDA Drug Competition Action Plan (describing the FDAs Drug Competition Action Plan, implemented in 
2017 and designed to, among other things, further encourage robust and timely market competition for generic 
drugs), available at https://www.fda.gov/drugs/guidance-compliance-regulatory-information/fda-drug-competition-
action-plan.  
8 In this guidance, GDUFA refers to the generic drug user fee program codified in the Generic Drug User Fee 
Amendments of 2012, Title III, Food and Drug Administration Safety and Innovation Act (Public Law 112-144), the 
Generic Drug User Fee Amendments of 2017, Title III, FDA Reauthorization Act of 2017 (Public Law 115-52), and 
the Generic Drug User Fee Amendments of 2022, Title III of Division F (the FDA User Fee Reauthorization Act of 
2022) of the Continuing Appropriations and Ukraine Supplemental Appropriations Act, 2023 (Public Law 117-180). 
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Draft  Not for Implementation 
59  bioequivalent to the RLD.9 Thus, an ANDA will not be approved if the information submitted in 
60  the ANDA is insufficient to show that the test product is bioequivalent to the RLD.10  
61   
62  An IVRT study may be used to assess the rate of drug release (i.e., release of an active 
63  ingredient) from a topical product. Once validated, an IVRT study may also be useful in 
64  controlling product quality and/or establishing the acceptability of post-approval manufacturing 
65  changes. This guidance focuses on general considerations and recommendations for the method 
66  development, method validation, and conduct of IVRT studies that are submitted in ANDAs and 
67  intended to support a demonstration of BE.11  
68   
69   
70  III.  IVRT METHOD DEVELOPMENT  
71   
72  If an IVRT study is intended to support a demonstration of BE, the IVRT method development 
73  report should be submitted in the ANDA to show how the IVRT method was optimized, and to 
74  support a demonstration that the method parameters selected for the IVRT are appropriate or 
75  necessary, particularly in situations where the method parameters are different from the methods 
76  recommended in this guidance and described in the United States Pharmacopeia (USP) General 
77  Chapter <1724>.12 The Agencys interest in reviewing the method development report is to 
78  understand why specific IVRT method parameters were selected and whether they are suitably 
79  sensitive and reproducible. This method development report should clearly indicate/distinguish 
80  the method parameters used for each set of data, illustrate the efforts made to optimize the IVRT 
81  method, and demonstrate that the method parameters selected for the IVRT are appropriate. 
82   
83  Applicants are encouraged to use the recommendations in this guidance, and if an applicant 
84  elects to use methods that are different from those recommended in this guidance, the IVRT 
85  method development report should demonstrate why it is scientifically justified to use an 
86  alternative approach than what is recommended in this guidance or USP <1724> to optimize the 
87  IVRT method.13 Specific examples of procedures are described in subsequent sections, to help 
88  applicants identify circumstances when information should be submitted in the ANDA to explain 
89  why an alternative procedure was utilized. 
90   
91  The IVRT method development studies, being exploratory in nature, are often performed using a 
92  sample analytical method that is not validated (e.g., a high-performance liquid chromatography 
93  (HPLC) or ultrahigh performance liquid chromatography (UPLC) method); also, IVRT method 
94  development studies are often conducted in a manner that is not compatible with a quality 
95  management system which would otherwise make the evidence generated suitable to support 
 
9 See section 505(j)(2)(A), (j)(2)(C), and (j)(4) of the FD&C Act (21 U.S.C. 355(j)(2)(A), (j)(2)(C), (j)(4)); see also 
21 CFR 314.94.  
10 21 CFR 314.127(a)(4), (6). 
11 A demonstration of equivalent drug release rates for the test topical product and RS using an appropriately 
validated IVRT method can be used to support a demonstration of BE along with other data in the application 
(which may be specified in a PSG), as part of a comparative product characterization-based approach. 
12 Applicants may choose to use an approach different from the approach recommended in this guidance.  However, 
the alternative approach must comply with relevant statutes and regulations (see 21 CFR 10.115(d)). 
1313 Ibid.   
  3  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
96  valid conclusions. Such method development studies would not be suitable to demonstrate the 
97  validity of an IVRT method, or the associated results. Therefore, although it may appear to be 
98  redundant, certain experiments performed during IVRT method development may need to be 
99  repeated during IVRT method validation, using appropriate controls, like a validated analytical 
100  method and procedures that are compatible with a suitable quality management system.  
101   
102  It is important to clearly segregate and consistently identify those experiments and results that 
103  were part of IVRT method development separately from those that were part of IVRT method 
104  validation. It is also important to consistently identify all relevant method parameters and 
105  experimental conditions/controls for each set of IVRT results. Information in the method 
106  development report should clearly identify/distinguish when the results for apparently similar 
107  sets of experiments may have been obtained using different method parameters. Method 
108  development reports should clarify which sets of diffusion cells were run in parallel or separately 
109  (e.g., on separate days). In addition, the sample analytical method (e.g., a HPLC or UPLC 
110  method) used to analyze the samples from each set of IVRT experiments should be specified, 
111  and the reports should indicate whether or not the sample analytical method was validated (either 
112  at the time of sample analysis or subsequently).  
113   
114  A.  IVRT Method Parameters 
115   
116  Theoretical or empirical information should be provided to explain the selection of IVRT method 
117  parameters such as the equipment, product dose amount, sampling times, stirring/agitation rate, 
118  etc. When the equipment selected is among the models of equipment in the USP <1724>, 
119  Semisolid Drug Products  Performance Tests, and when the product dose amount or stirring rate 
120  is a parameter that is fixed (not adjustable) with the selected equipment, it may be sufficient to 
121  explain these facts.  
122   
123  It is unconventional for IVRT sampling times to be selected within a study duration of less than 
124  4 hours. This may occur in situations where the fixed product dose was depleted to such a great 
125  extent by 4 hours that the release kinetics were no longer linear thereafter (when plotted vs. the 
126  square root of time). In such instances, it would be appropriate to explain the efforts that were 
127  made to optimize the IVRT method (e.g., using a different diffusion cell equipment that allowed 
128  for a larger product dose to be used) so that the sustained steady state release kinetics could 
129  potentially be characterized over a conventional IVRT duration of 4 to 6 hours. 
130   
131  B.  IVRT Receptor Solution 
132   
133  It is conventional to evaluate different receptor solutions during IVRT method development (all 
134  using the same membrane that has broad chemical compatibility with the receptor solutions 
135  evaluated); these receptor solutions are frequently binary hydro-alcoholic mixtures selected 
136  based upon the solubility and stability of the (frequently hydrophobic) drug in the receptor 
137  solution. The receptor solutions are conventionally sampled at least hourly across a 6-hour 
138  duration.  
139   
140  Information on the empirical solubility and stability of the drug in the receptor solution, as well 
141  as information on the linearity and precision of the resulting drug release rate in an IVRT should 
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142  be provided to help explain the selection of a receptor solution for the test method. The linearity 
143  of the drug release rate (slope) across all time points should ideally have an r2 value of  0.97. In 
144  situations where the solubility of the drug in the receptor solution limits the release kinetics, 
145  causing a reduction in the release rate at the last time point(s), it may be appropriate to evaluate 
146  different receptor solutions. It may be appropriate to truncate the IVRT method to a 4- or 5-hour 
147  sampling duration if the linearity of the release rate in that truncated duration is improved 
148  (exhibiting a higher r2 value), and if other aspects of the release kinetics (e.g., precision) in that 
149  receptor solution are optimized compared to other receptor solutions evaluated.  
150   
151  One advantage of selecting an optimal receptor solution as an initial step in IVRT method 
152  development is that it allows for the sample analysis method to be optimized for the selected 
153  receptor solution sample matrix before proceeding to an evaluation of different membranes using 
154  that receptor solution.  
155   
156  C.  IVRT Membrane 
157   
158  It is conventional to evaluate different membranes during IVRT method development (all using 
159  the same receptor solution); these membranes are frequently synthetic membranes used for the 
160  filtration of particulate matter in solutions. IVRT membranes are selected based upon their 
161  effective pore size (e.g., 0.45 micrometers (m)), as well as their expected inertness to binding 
162  the drug. Information should be provided in the IVRT method development report on each 
163  membranes binding to the drug and its chemical compatibility with relevant receptor solution(s) 
164  selected for the IVRT method (based on the preceding phase of IVRT method development), as 
165  well as information on the linearity and precision of the resulting release rate when each 
166  membrane is used in an IVRT, as this information can help to explain why a specific membrane 
167  is optimal for the IVRT method.  
168   
169   
170  IV.  IVRT METHOD VALIDATION  
171   
172  The equipment, methodologies, and study conditions used in the IVRT pivotal study should be 
173  appropriately validated or qualified. It is conventional to initiate the validation of the sample 
174  analytical method (e.g., a HPLC or UPLC method) for the IVRT before initiating the IVRT 
175  method validation itself, although certain components of the sample analysis method validation 
176  (e.g., stability) often proceed in parallel with the IVRT method validation. If an applicant elects 
177  to use equipment, methodologies, or study conditions that are different from those recommended 
178  in this guidance or in USP <1724>, the applicant should demonstrate why the differences are 
179  scientifically justified.14 It is important to consistently identify all relevant method parameters for 
180  each set of IVRT results, making it clear that the results were obtained using the same IVRT 
181  method parameters, and clarifying which sets of diffusion cells were run in parallel or separately. 
182  Detailed protocols and well-controlled test procedures are recommended to ensure the precise 
183  control of dosing, sampling, and other IVRT study parameters, and of potential sources of 
184  experimental bias.  
 
14 Applicants may choose to use an approach different from the approach recommended in this guidance.  However, 
the alternative approach must comply with relevant statutes and regulations (see 21 CFR 10.115(d)). 
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185   
186  The qualification of an IVRT method parameter refers to the process of defining what attributes 
187  make it suitable to perform its function in the IVRT method. For example, when hourly 
188  measurements of the temperature at the membrane surface (when mounted in a diffusion cell) 
189  demonstrate that an IVRT equipment can maintain a membrane surface temperature in the range 
190  of 32C  1C across 6 hours, the results can support a demonstration that the equipment is 
191  qualified to perform its function in an IVRT method for which a method parameter is the control 
192  of membrane surface temperature in the range of 32C  1C across 6 hours. While an IVRT 
193  membrane surface temperature in the range of range of 32C  1C is appropriate for topical 
194  products applied on the skin, for topical products applied on mucosal membranes (e.g., a vaginal 
195  gel) the relevant IVRT membrane surface temperature would be 37C  1C. The validation of 
196  the IVRT method should incorporate the following qualifications and controls, performed using 
197  validated sample analytical procedures, as applicable.  
198   
199  A.  Equipment Qualification 
200   
201  Suitable equipment for the IVRT method are described in USP General Chapter <1724>. These 
202  include different models of a vertical diffusion cell and an immersion cell. Other models of 
203  vertical diffusion cells and immersion cells that are essentially the same in design and/or 
204  operational principles as those described in USP General Chapter <1724> may also be suitable.  
205   
206  The operating principles and specific test procedures differ among the various equipment; 
207  relevant procedures from the manufacturer may be used for installation, operation, and 
208  performance qualifications. The laboratory qualification of each diffusion cell should, at 
209  minimum, include: (1) measurements of the diffusional area of the orifices of the donor and 
210  receptor compartments between which the membrane is mounte; (2) the empirically measured 
211  volume of the receptor solution compartment/vessel for each diffusion cell; (3) the stability of 
212  the temperature measured at the membrane surface (e.g., at 32C  1C), or just below the 
213  membrane, across a relevant duration (e.g., 6 hours); and (4) the rate of stirring or agitation, as 
214  applicable.  
215   
216  If information related to the diffusional area of the orifice and the volume of the receptor solution 
217  compartment for each diffusion cell is available from the manufacturer, that information should 
218  be provided for each relevant diffusion cell, in addition to the empirical measurements made by 
219  the laboratory. The equipment should control the diffusion cell thermoregulation so that the 
220  membrane surface temperature is verified to be stable (e.g., at 32C  1C) for each diffusion 
221  cell (e.g., measured by a calibrated infrared thermometer) before dosing. If it is not feasible to 
222  verify that the membrane surface temperature of a diffusion cell has equilibrated and stabilized 
223  (e.g., at 32C  1C) before dosing because of design and operating principles of a specific 
224  equipment, the qualification of that equipment should demonstrate that, under the specific 
225  conditions used for the IVRT method, the membrane surface temperature can be expected to be 
226  stable (e.g., at 32C  1C) for each diffusion cell throughout the test. 
227     
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228   
229  B.  Membrane Qualification 
230   
231  Membrane inertness should be evaluated in relation to membrane binding of the drug in the 
232  receptor solution at a concentration relevant to the range of drug concentrations in the receptor 
233  solution during the test. Determinations should be based upon a minimum of three replicate 
234  membrane incubations for the IVRT duration at the relevant temperature (e.g., 6 hours at 32C  
235  1C). Three replicate control incubations should be performed in parallel, without membranes, to 
236  monitor for drug loss that is not associated with membrane binding. Aliquots of these solutions 
237  should be collected before and after the duration of incubation, to assess any decrease in the 
238  amount of drug in solution. The recovery of drug in solution is recommended to be within the 
239  range of 100%  5% at the end of the test duration to qualify the inertness of the membrane. 
240   
241  C.  Receptor Solution Qualification 
242   
243  The reason for selecting the composition of the receptor solution used for the IVRT study should 
244  be explained. The solubility of the drug in the IVRT receptor solution should be empirically 
245  determined in triplicate, to illustrate that the solubility of the drug in the receptor solution 
246  exceeds the highest sample concentration in the IVRT pivotal study, ideally by an order of 
247  magnitude, but demonstrably sufficient to facilitate a linear (steady state) release rate for the 
248  duration of the study (even when evaluating the relatively higher release rate of a formulation 
249  that is 150% of the nominal strength of the RS during the IVRT method validation).  
250   
251  D.  Receptor Solution Sampling Qualification 
252   
253  The accuracy and precision of receptor solution sample collection at each time point should be 
254  appropriately qualified. Evidence to qualify a sampling procedure should illustrate that the 
255  sampling technique can reliably collect a consistent volume of the sample from the well-mixed 
256  volume of the receptor compartment at each sampling event, and that no artifacts are likely to be 
257  created by the sampling technique (e.g., because of carryover between samples in automated 
258  sampling systems or because of sampling from an unmixed volume in the sampling arm of a 
259  vertical diffusion cell). Information should be included describing the equipment manufacturers 
260  specification for the accuracy and precision of receptor solution sampling, when available. 
261   
262  E.  Environmental Control 
263   
264  Ambient laboratory temperature and humidity during the study should be monitored and 
265  reported. An environmentally controlled temperature range of 21C  2C is recommended, and, 
266  if feasible, a humidity range of 50%  20% relative humidity is recommended.   
267   
268  F.  Linearity and Range 
269   
270  The linearity (r2 value) of the release rate (slope) should be plotted across the range of the 
271  sampling times, which corresponds to the IVRT study duration. The linearity of drug release 
272  should be calculated and reported for each diffusion cell and compared within and across all 
273  IVRT runs. For the release rate to be considered suitably linear, it should have an r2 value  0.97 
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274  across the recommended IVRT study duration of 46 hours. An IVRT study duration of less than 
275  4 hours may be insufficient to assess whether the release rates being compared for the test topical 
276  product and RS represent their steady state drug release kinetics, but an IVRT study duration of 
277  less than 4 hours (which is not recommended) may be justified if supported by compelling 
278  experimental data within the method development report to illustrate that reasonable and 
279  scientifically appropriate efforts were made to optimize the IVRT method. The IVRT method 
280  linearity and range should be established based upon the results of the precision and 
281  reproducibility runs, described further below. 
282   
283  G.  Precision and Reproducibility 
284   
285  The intra-run and inter-run precision and reproducibility may be compared for the release rate 
286  (slopes) calculated for each diffusion cell. The mean, standard deviation, and percent coefficient 
287  of variation (%CV) among slopes may be calculated within and across all runs, and a minimum 
288  intra-run and inter-run %CV  15% is recommended. Runs may be organized to facilitate a 
289  simultaneous evaluation of intra/inter-instrumentation and/or intra/inter-operator precision and 
290  reproducibility. A minimum of three independent precision and reproducibility runs is 
291  recommended.  
292   
293  H.  Dose Depletion 
294   
295  The recovery of released drug in the receptor solution should be characterized in each diffusion 
296  cell as the cumulative amount of drug released into the receptor solution over the IVRT study 
297  duration. This may be expressed as a percentage of the amount of drug in the applied dose 
298  (which may be estimated based upon the nominal strength of the drug in the topical product and 
299  the approximate mass of topical product dosed on the membrane). For example, if 1 gram (g) of 
300  a topical product containing 5% drug was dosed on the membrane of each diffusion cell, the 
301  amount of drug in the applied dose may be estimated to be 50 mg. If a total of 10 mg of drug 
302  diffused into the receptor solution of each diffusion cell across the 6-hour duration of the IVRT, 
303  it would be possible to estimate that the 50 mg dose would have been depleted by 10 mg, 
304  amounting to a 20% dose depletion. The average percentage dose depletion may thereby be 
305  estimated and should be reported. While steady state release kinetics can typically be assumed 
306  under conditions when the dose depletion is less than 30%, for some topical products, steady 
307  state release kinetics may continue to be observed at higher percentage dose depletions. The 
308  IVRT method may be considered adequate despite a dose depletion of greater than 30% when 
309  experimental evidence illustrates that the release rate (slope) remains suitably linear for each 
310  diffusion cell when plotted versus the square root of time. 
311   
312  I.  Discrimination Sensitivity, Specificity, and Selectivity 
313   
314  The IVRT method should be able to discriminate drug release rates from similar formulations. 
315  This should be evaluated by comparing the release rate from the test formulation with that from 
316  two comparable formulations in which the concentration of drug has been altered  one with a 
317  higher strength (150% of the nominal concentration of the RS) and one with a lower strength 
318  (50% of the nominal concentration of the RS). If precipitation of the active ingredient is 
319  observed when formulating a topical product at 150% compared to the nominal strength, it may 
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320  be necessary to use different strategies, which may be discussed with the Agency before the 
321  submission of an ANDA during a pre-ANDA product development meeting15 or via a controlled 
322  correspondence.16 The composition and procedures for preparation of these higher and lower 
323  strength formulations should be reported, although these formulations need not be prepared in a 
324  manner compatible with current Good Manufacturing Practices. The discrimination ability of the 
325  IVRT method should be described using three concepts of discrimination ability: sensitivity, 
326  specificity, and selectivity. 
327   
328  1.  IVRT Sensitivity  
329   
330  IVRT sensitivity is the ability to detect changes in the release rate, as a function of drug 
331  concentration in the formulation. If the IVRT method consistently identifies higher or lower rates 
332  of release for test formulations with increased or decreased drug concentrations, respectively, 
333  relative to the formulation at the nominal strength of the RS run in parallel on the same day, the 
334  IVRT method would generally be considered sensitive.  
335   
336  2.  IVRT Specificity  
337   
338  IVRT specificity is the ability to accurately monitor the proportionality of changes in the release 
339  rate as a function of drug concentration in the formulation. This proportionality may be 
340  illustrated in a plot of the relationship between the formulation concentration and the average 
341  IVRT release rate (slope). The specificity of the IVRT method should be calculated, plotted with 
342  a linear trendline, and the linearity quantified and reported as an r2 value. To be considered 
343  suitably specific, an IVRT method should be proportionally linear in its response to differences 
344  in release rates, with a minimum r2 value  0.95 for the correlation of the formulation 
345  concentration to the average IVRT release rate (slope).  
346   
347  IVRT specificity is a function of the proportionality of release rates across different strengths of 
348  the product, some, or all of which may be formulated as small-scale laboratory batches, with 
349  each strength having a slightly different formulation composition to accommodate for the 
350  different amount of the active ingredient in that strength of the product. These slight formulation 
351  differences across the different strengths of the product may impact the ideal proportionality of 
352  release rates across the different strengths of the product.  
353   
354  Thus, the proportional linearity of release rates across different strengths of the product may be 
355  impacted by formulation differences across the strengths that are independent of the proportional 
356  responsiveness of the IVRT method. The minimum r2 value  0.95 for the correlation of the 
357  formulation concentration to the average IVRT release rate (slope) takes into account that the 
358  IVRT methods response to differences in release rates may not appear to be perfectly 
359  proportional because of formulation differences that are independent of the IVRT method. 
 
15 See the guidance for industry Formal Meetings Between FDA and ANDA Applicants of Complex Products Under 
GDUFA (November 2020) for information on the enhanced pathway for discussions between FDA and a 
prospective applicant preparing to submit an ANDA for a complex product as defined in that guidance.   
16 See the guidance for industry Controlled Correspondence Related to Generic Drug Development (December 
2020) for information on the types of inquiries accepted as controlled correspondence and on how to submit 
controlled correspondence to OGD.   
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360   
361  Note that the linearity of release rates across different strengths of the product (which assesses 
362  the specificity of the IVRT method, with a minimum r2 value  0.95) is fundamentally different 
363  and has different scientific considerations than the linearity of the release rate for a single 
364  strength of the product across the range of the sampling times (which assesses the IVRT 
365  methods ability to monitor the steady state release kinetics of the active ingredient, with a 
366  minimum r2 value  0.97). Despite the potential for different scientific considerations to impact 
367  the linearity of the IVRT results in each context, for well-developed and suitably controlled 
368  IVRT methods, the r2 value  0.99 is routinely observed in both contexts. 
369   
370  3.  IVRT Selectivity  
371   
372  IVRT selectivity is the ability of the IVRT method to discriminate the drug release rates between 
373  the reference topical product and the altered (50% and 150% nominal strength) concentration test 
374  formulations such that their release rates are determined to be statistically inequivalent compared 
375  to that from the nominal reference strength formulation. Determination of inequivalence between 
376  release rates should be evaluated using the statistical approach described in USP General Chapter 
377  <1724>.  
378   
379  Specifically, the release rates from six cells dosed with the nominal reference strength 
380  formulation should be compared with the release rates from 6 cells dosed with the formulation at 
381  150% the nominal reference strength, using the statistical approach described in USP General 
382  Chapter <1724>. All 12 cells being compared should have been run in parallel on the same day, 
383  and the release rate from the formulation at 150% the nominal reference strength should fail to 
384  show equivalence to the release rate from the nominal reference strength formulation.  
385   
386  The release rates from 6 cells dosed with the nominal reference strength formulation should also 
387  be compared with the release rates from 6 cells dosed with the formulation at 50% the nominal 
388  reference strength, using the statistical approach described in USP General Chapter <1724>. All 
389  12 cells being compared should have been run in parallel on the same day, and the release rate 
390  from the formulation at 50% the nominal reference strength should fail to show equivalence to 
391  the release rate from the nominal reference strength formulation. 
392   
393  4.  IVRT Supplemental Selectivity  
394   
395  IVRT supplemental selectivity is the ability of the IVRT method to discriminate the drug release 
396  rates between the reference topical product and an altered formulation with the same nominal 
397  reference strength, such that their release rates are determined to be statistically inequivalent.  
398   
399  The demonstration of IVRT selectivity (distinct from supplemental selectivity) validates the 
400  ability of the IVRT method to discriminate differences in release rates under conditions when the 
401  release rate is expected to differ in a predictable manner (i.e., when there are different 
402  concentrations of drug in the formulation).  
403   
404  A separate and supplemental demonstration of the selectivity of an IVRT method, when feasible, 
405  independently validates the ability of the IVRT method to discriminate differences in release 
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406  rates under the conditions of the pivotal IVRT study, in which the test and reference topical 
407  products are compared at the same strength. Thus, the supplemental demonstration of the 
408  selectivity of the IVRT method validates that it can detect differences in the release rate that are 
409  associated with aspects of the formulation other than the strength, and this is ideal, when 
410  feasible. 
411   
412  Determination of inequivalence between release rates should be evaluated using the statistical 
413  approach described in USP General Chapter <1724>. Specifically, the release rates from 6 cells 
414  dosed with the nominal reference strength formulation should be compared with the release rates 
415  from 6 cells dosed with an altered formulation, also at the nominal reference strength, using the 
416  statistical approach described in USP General Chapter <1724>. All 12 cells being compared 
417  should have been run in parallel on the same day, and the release rate from the altered 
418  formulation at the same nominal reference strength should fail to show equivalence to the release 
419  rate from the nominal reference strength formulation.  
420   
421  The altered formulation used in the assessment of supplemental selectivity should have the same 
422  nominal strength as the reference topical product, and may include changes in inactive 
423  ingredients, changes in inactive ingredient concentration(s), changes in the manufacturing 
424  processes, or combinations thereof. However, not all variations in a formulation will necessarily 
425  produce a difference in the release rate compared to the reference formulation, and if two similar 
426  formulations are found to have equivalent release rates, the demonstration of supplemental 
427  selectivity may be inconclusive. Therefore, applicants are encouraged to develop or select an 
428  altered formulation for the demonstration of supplemental selectivity based on differences in 
429  physicochemical and structural properties of the formulation (relative to the reference 
430  formulation) that are likely to alter the release rate of the active ingredient from the formulation. 
431  The altered formulation may be a marketed topical product, such as a different dosage form at 
432  the same strength of the same drug (e.g., a 5% gel versus a 5% ointment). Product batch 
433  information for all topical product lots used in IVRT method development, and validation 
434  studies, as applicable, should be submitted in the study reports. The topical product information 
435  should include, but not be limited to, information about the batch formula, manufacturing date, 
436  batch size, altered manufacturing processes (if applicable) and, if available, potency and content 
437  uniformity. 
438   
439  J.  Robustness 
440   
441  The IVRT method may be considered robust to a variation in the test method if the average slope 
442  of an IVRT run under the altered IVRT method parameters is within  15% of the average slope 
443  of the precision and reproducibility IVRT runs. Robustness testing may encompass variations in 
444  the IVRT method that are relevant to the equipment and test method, for example: 
445   
446    Temperature variations (e.g., - 1C and +1C relative to 32C  1C) 
447    Dose volume variations (e.g., +10% and -10% in the dose volume) 
448    Receptor solution variations (e.g., slight change in composition and/or pH) 
449    Mixing rate variation (e.g., slight change in stirring speed, as applicable) 
450   
451   
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452  V.  SAMPLE ANALYTICAL METHOD VALIDATION 
453   
454  While exploratory studies performed during IVRT method development may use an unvalidated 
455  sample analytical method, it is essential that all studies conducted as part of the IVRT method 
456  validation use a validated sample analytical method. A validated IVRT method should use a 
457  validated receptor solution sample analytical method. Therefore, a discussion of the sample 
458  analytical method for the IVRT method is included in this guidance under this section.  
459   
460  It is important to note that the study protocols and reports related to the IVRT method are distinct 
461  from those for the sample analytical method that is used to quantify drug concentrations in IVRT 
462  receptor solution samples. The validation of a sample analytical method, in and of itself, does not 
463  demonstrate the validity of an IVRT method. Separate and specific reports should be submitted 
464  for the validation of the sample analysis (e.g., HPLC or UPLC) method and for the validation of 
465  the IVRT method.  
466   
467  Any results from studies of the IVRT method that are performed (during method development) 
468  using a different sample analytical method than that which is ultimately validated, cannot support 
469  a demonstration of the validity of the IVRT method. Information should be provided in the IVRT 
470  method validation report referencing the (separate) sample analytical method validation, and 
471  clearly indicate that all relevant results in the IVRT method validation report were obtained using 
472  a validated sample analytical method (as opposed to an analytical method with different 
473  parameters than those which were validated). 
474   
475  The receptor sample analysis procedures, typically involving HPLC or UPLC, should be 
476  performed using chromatography software (e.g., a chromatography data system) with audit trails, 
477  and should include a multi-point (68 concentration) calibration curve with suitable quality 
478  control samples, and should be validated in a manner compatible with the FDA guidance for 
479  industry Bioanalytical Method Validation (May 2018).  
480   
481  The validation of the receptor sample analytical method should include relevant qualifications of 
482  dilution integrity, if applicable, as well as stability assessments with the highest relevant 
483  temperature in the receptor solution for the longest relevant duration; the highest relevant 
484  temperature may be warmer than the IVRT membrane surface temperature because the 
485  temperature of the receptor solution is often higher than the temperature at the surface of the 
486  membrane (e.g., the temperature of the receptor solution may be 34C when the temperature of 
487  membrane surface is 32C, so stability assessments with the IVRT receptor solution may be 
488  performed at 34C for 6 hours; the temperature would be higher for an IVRT with a vaginal gel, 
489  for example).  
490   
491   
492  VI.  IVRT PIVOTAL STUDY  
493   
494  The IVRT pivotal study comparing the drug release rates between the test and reference topical 
495  products should be performed in a manner compatible with the general procedures and statistical 
496  analysis method specified in USP General Chapter <1724>. The cumulative amount of drug 
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497  released at each sampling time point should be reported for each diffusion cell. Relevant 
498  summary statistics for the IVRT study should also be reported.  
499   
500  A.  Handling and Retention of Samples 
501   
502  Refer to 21 CFR 320.38, 320.63 and the guidances for industry Handling and Retention of BA 
503  and BE Testing Samples (May 2004) and Compliance Policy for the Quantity of Bioavailability 
504  and Bioequivalence Samples Retained Under 21 CFR 320.38(c) (August 2020), as applicable, 
505  regarding considerations for retention of study drug samples and to 21 CFR 320.36 for 
506  requirements for maintenance of records of BE testing. Retention samples should be randomly 
507  selected from the drug supplies received before dispensing during the IVRT study in which the 
508  test topical product and RS are compared. Experimental observations that may have the potential 
509  to influence the interpretation of the study results, as well as any protocol deviations, should be 
510  reported. 
511   
512  B.  Control of Study Procedures 
513   
514  Study procedures that have the potential to influence the results of the study should be 
515  appropriately controlled. Also, experimental observations that may have the potential to 
516  influence the interpretation of the study results, as well as any protocol or standard operating 
517  procedure (SOP) deviations, should be reported. 
518   
519  In addition, investigators should perform the IVRT validation and pivotal studies within a quality 
520  management system that includes, but is not limited to, documented procedures for:  
521   
522    Study personnel identification, training, qualification, and responsibilities 
523   
524    Study management and study management personnel responsibilities 
525   
526    Quality control (QC) and QC personnel responsibilities 
527   
528    Quality assurance (QA) and QA personnel responsibilities 
529   
530    Use of SOPs 
531   
532    Use of study protocols 
533   
534    Use of study reports 
535   
536    Maintenance and control of the study facility environment and systems 
537   
538    Qualification and calibration of instruments and computerized systems 
539   
540    Good documentation practices including, but not limited to, contemporaneous 
541  documentation of study procedures and recording of experimental observations or 
542  deviations from procedures specified in the study protocol or in relevant SOPs 
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543   
544    Maintenance of suitable records that facilitate the reconstruction of study events and 
545  procedures, including study sample handling and storage records (e.g., sample tracking 
546  logs), audit trails for sample analysis procedures, control of study materials and reagents, 
547  and electronic data control 
548   
549    Archival of study records 
550   
551  C.  Blinding Procedure 
552   
553  A detailed description of the blinding procedure should be provided in the study protocol and 
554  final report for the IVRT pivotal study. The packaging of the test topical product and RS should 
555  be similar in appearance to maintain adequate blinding of the investigator and any experimental 
556  operators. Once blinded, the test topical product and RS should be identified by a random 
557  designation, e.g., A or B.  
558   
559  D.  Dosing 
560   
561  In the IVRT pivotal study, the test topical product and RS should be dosed in an alternating 
562  pattern on successive diffusion cells. There are two possible sequences for the alternating pattern 
563  (either ABABAB or BABABA). One of these two dosing sequences should be randomly 
564  selected. 
565   
566   
567   
568  VII.  SUBMITTING INFORMATION ON IVRT STUDIES IN AN ANDA 
569   
570  For IVRT studies with topical products submitted in ANDAs that are intended to support a 
571  demonstration of BE, detailed study protocols, relevant SOPs, and detailed reports should be 
572  submitted for the IVRT method validation and the IVRT pivotal study. In addition, a detailed 
573  report describing the IVRT method development should be submitted. These protocols, SOPs, 
574  and reports should be submitted in module 5.3.1.2 of the electronic Common Technical 
575  Document (eCTD) and should describe experimental procedures, study controls, quality 
576  management procedures, and data analyses.  
577   
578  Note that the study protocols, SOPs, and reports related to the IVRT method are distinct from 
579  those for the sample analytical method that is used to quantify drug concentrations in IVRT 
580  receptor solution samples (e.g., a HPLC or UPLC method). Separate protocols and SOPs should 
581  be submitted for the sample analytical method validation. Sample analytical method 
582  development and validation reports, pivotal IVRT study sample analysis reports, as well as 
583  associated SOPs and protocols relevant to the sample analysis for an IVRT study should be 
584  submitted in Module 5.3.1.4 of the eCTD. 
585   
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