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DRAFT GUIDANCE
This guidance document is being distributed for comment purposes only.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publication in the Federal Register of the notice announcing the availability of the draft
guidance.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., 4 th Floor
Silver Spring, MD 20993-0002
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353
Email: [email protected]
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
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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
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1 In Vitro Release Test Studies for Topical Drug Products Submitted

2 in ANDAs
3 Guidance for Industry1
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 2023–2027 (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 f or
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 FDA’s 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, FDA’s guidance documents do not establish legally enforceable responsibilities.
42 Instead, guidances describe the Agency’s 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 FDA’s “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 FDA’s 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 FDA’s
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 FDA’s 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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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 Agency’s 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.

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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 (µm)), as well as their expected inertness to binding
162 the drug. Information should be provided in the IVRT method development report on each
163 membrane’s 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 32°C ± 1°C 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 32°C ± 1°C across 6 hours. While an IVRT
193 membrane surface temperature in the range of range of 32°C ± 1°C 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 37°C ± 1°C. 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 32°C ± 1°C), 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 32°C ± 1°C) 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 32°C ± 1°C) 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 32°C ± 1°C) 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 32°C ±
235 1°C). 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 manufacturer’s
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 21°C ± 2°C 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 4–6 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 method’s 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 method’s 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., - 1°C and +1°C relative to 32°C ± 1°C)
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 (6–8 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 34°C when the temperature of
487 membrane surface is 32°C, so stability assessments with the IVRT receptor solution may be
488 performed at 34°C 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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