In Vitro Permeation 
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DRAFT GUIDANCE 
 
  This guidance document is being distributed for comment purposes only.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publication in the Federal Register of the notice announcing the availability of the draft 
guidance.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 Permeation 
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Draft  Not for Implementation 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.  IVPT METHOD DEVELOPMENT ............................................................................... 3 
A.  IVPT Method Parameters ............................................................................................................. 4 
B.  IVPT Method Considerations ....................................................................................................... 4 
C.  IVPT Method Procedures and Controls ...................................................................................... 5 
D.  IVPT Skin Barrier Integrity Testing: Common Methods .......................................................... 6 
1. Trans-Epidermal Water Loss Skin Barrier Integrity Test ................................................................ 6 
2. Tritiated Water Skin Barrier Integrity Test...................................................................................... 7 
3. Electrical Based Skin Barrier Integrity Tests .................................................................................. 8 
E.  IVPT Skin Barrier Integrity Testing: General Considerations ................................................. 9 
F.  Differences Between IVPT Method Development and Validation .......................................... 10 
1. Optimization of an IVPT Method Prior to Advancing to IVPT Method Validation ....................... 10 
2. Use of a Validated Sample Analytical Method for IVPT Method Validation ................................ 10 
IV.  IVPT METHOD VALIDATION ................................................................................... 11 
A.  Equipment Qualification ............................................................................................................. 12 
B.  Membrane (Skin) Qualification .................................................................................................. 13 
C.  Receptor Solution Qualification ................................................................................................. 13 
D.  Receptor Solution Sampling Qualification ................................................................................ 14 
E.  Environmental Control ............................................................................................................... 14 
F.  Permeation Profile and Range .................................................................................................... 14 
G.  Precision and Reproducibility .................................................................................................... 14 
H.  Dose Depletion .............................................................................................................................. 15 
I.  Discrimination Sensitivity and Selectivity ................................................................................. 15 
1. IVPT Sensitivity .............................................................................................................................. 15 
2. IVPT Selectivity .............................................................................................................................. 18 
J.  Robustness .................................................................................................................................... 18 
V.  SAMPLE ANALYTICAL METHOD VALIDATION ................................................ 19 
VI.  IVPT PIVOTAL STUDY ............................................................................................... 20 
A.  Handling and Retention of Samples ........................................................................................... 20 
B.  Control of Study Procedures ....................................................................................................... 20 
C.  Blinding Procedure ...................................................................................................................... 22 
D.  Randomization ............................................................................................................................. 22 
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E.  Dosing ............................................................................................................................................ 22 
F.  Study Design ................................................................................................................................. 22 
G.  Inclusion Criteria ......................................................................................................................... 23 
H.  Exclusion Criteria ........................................................................................................................ 23 
I.  IVPT Endpoints ........................................................................................................................... 24 
VII. SUBMITTING INFORMATION ON IVPT STUDIES IN AN ANDA ......................... 25 
VIII.  IVPT PIVOTAL STUDY STATISTICAL ANALYSIS .............................................. 25 
APPENDIX I (EXAMPLE SAS CODE) ................................................................................... 29 
APPENDIX II (NUMERICAL EXAMPLES) .......................................................................... 34 
APPENDIX III (EXAMPLE R CODE) .................................................................................... 38 
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1  In Vitro Permeation Test Studies for Topical Drug Products 
2  Submitted 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 permeation test (IVPT) studies comparing a proposed 
24  generic (test) topical product and its reference standard (RS) for the purpose of supporting a 
25  demonstration of bioequivalence (BE) to the reference listed drug (RLD). The reference standard 
26  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, solutions 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 IVPT 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 market place 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 as 
57  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 bioequivalent 
 
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 Release 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. 
7 See 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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59  to the RLD.9 Thus, an ANDA will not be approved if the information submitted in the ANDA is 
60  insufficient to show that the test product is bioequivalent to the RLD.10  
61   
62  An IVPT study may be used to assess the rate and extent to which a drug (i.e., an active 
63  ingredient) from a topical product becomes available at or near a site of action in the skin, and 
64  may be used to characterize and compare the rate and extent of bioavailability for a drug from a 
65  test topical product and RS. The IVPT flux profiles resemble pharmacokinetic profiles and can 
66  be analyzed using unique IVPT endpoints that are somewhat analogous to the pharmacokinetic 
67  endpoints of maximum concentration (C ) and the area under the concentration-time curve 
max
68  (AUC). Yet, IVPT studies characterize the rate and extent of absorption, not the distribution, 
69  metabolism and excretion that occurs in vivo. Therefore, while it is relevant to characterize the 
70  kinetics of topical drug bioavailability monitored by IVPT studies, the use in this guidance of the 
71  term cutaneous pharmacokinetics should not be construed to embody all aspects of 
72  pharmacokineticsonly those related to the absorption component that directly controls the rate 
73  and extent to which a topically applied drug becomes available locally at the site of action. This 
74  guidance focuses on general considerations and recommendations for the method development, 
75  method validation, and conduct of IVPT studies that are submitted in ANDAs and intended to 
76  support a demonstration of BE.11 
77   
78   
79  III.  IVPT METHOD DEVELOPMENT 
80   
81  The development of an IVPT method that is suitable to support a demonstration of BE for a 
82  specific topical product routinely involves a systematic series of exploratory studies. 
83  Inappropriate or insufficient efforts to develop an IVPT method that is suitable for its intended 
84  purpose increases the likelihood that the subsequent IVPT validation, pilot, and pivotal studies 
85  will ultimately be inadequate to support a demonstration of BE. By contrast, appropriate and 
86  systematic IVPT method development studies help to identify IVPT study designs and protocol 
87  (method) parameters which reliably produce flux profiles that can facilitate a comparison of the 
88  cutaneous pharmacokinetics of a drug delivered topically to the skin from test topical products 
89  and RSs.  
90   
91  A detailed and well-organized IVPT method development report should be submitted in an 
92  ANDA to show how the IVPT method was optimized, and to support a demonstration that the 
93  method parameters selected for the IVPT are appropriate or necessary, particularly in situations 
94  where the method parameters are different from the methods recommended in this guidance). 
95  The Agencys interest in reviewing the method development report is to understand why specific 
96  IVPT method parameters were selected and whether the resulting IVPT method is suitably 
97  sensitive and reproducible. This method development report should clearly indicate/distinguish 
 
9 See sections 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 no significant difference in the rate and extent of drug permeation into and through the skin of 
the test topical product and RS using an appropriately validated IVPT 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. 
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98  the method parameters used for each set of data, illustrate the efforts made to optimize the IVPT 
99  method, and demonstrate that the method parameters selected for the IVPT are appropriate.  
100   
101  Applicants are encouraged to use the recommendations in this guidance, and if an applicant 
102  elects to use methods that are different from those recommended in this guidance, the IVPT 
103  method development report should demonstrate why it is scientifically justified to use an 
104  alternative approach than what is recommended in this guidance to optimize the IVPT method.12 
105  Some examples of recommended procedures are described in subsequent sections, to help 
106  applicants identify circumstances when information should be submitted in the ANDA to explain 
107  why a different procedure was utilized.  
108   
109  A.  IVPT Method Parameters 
110   
111  All relevant parameters of the final IVPT method should be summarized (e.g., in a table) and 
112  submitted in the ANDA. Also, information should be provided to briefly explain the choice of 
113  the final IVPT method parameters like the equipment (e.g., a vertical diffusion cell (VDC)), skin 
114  source (e.g., cadaver), skin type (e.g., posterior torso), skin preparation (e.g., dermatomed), skin 
115  barrier integrity test (e.g., trans-epidermal water loss (TEWL) measurement), skin barrier 
116  integrity test acceptance criteria (e.g., < 15 grams/meter2/hour (g/m2/hr)), topical product dose 
117  amount (e.g., 15 milligrams/centimeter2 (mg/cm2)), dose duration (e.g., 6 hours), study duration 
118  (e.g., 24 hours, 48 hours, etc.), receptor solution sampling times (e.g., 1, 2, 4, 6, 8, 12, 16, 20, 
119  and 24 hours), etc.  
120   
121  B.  IVPT Method Considerations 
122   
123  The choice of some IVPT method parameters like the equipment, skin source, skin type, skin 
124  preparation, and skin barrier integrity test procedures may be based upon investigator experience 
125  or convenience, like the availability of specific equipment or instrumentation in a laboratory, 
126  established tissue supply agreements, or other logistical considerations. However, if the chosen 
127  IVPT method parameters do not appear to be well-suited for a specific IVPT method, it is the 
128  applicants responsibility to systematically evaluate alternative method parameters, and 
129  ultimately, to validate that the IVPT method parameters chosen are suitable for the intended 
130  purpose. The recommended procedures for IVPT method validation are detailed in section IV of 
131  this guidance. 
132   
133  The choice of other IVPT method parameters like the topical product dose amount, dose 
134  duration, study duration (which may be longer than the dose duration), sampling schedule, 
135  sampling procedures, receptor solution composition, and sample analytical method may be 
136  different for each IVPT method, and such parameters of IVPT methods should be systematically 
137  developed, optimized, and/or validated for the relevant topical product, as appropriate. The IVPT 
138  method development studies should characterize how differences in these method parameters 
139  influence the resulting IVPT flux profile so that optimal study conditions can be objectively 
140  selected from among those evaluated. 
141   
 
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). 
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142  The selection of the dose amount used in the study should be assessed for each IVPT method 
143  based upon studies performed during IVPT method development. Different dose amounts may 
144  be compared in parallel on replicate skin sections from the same set of donors to optimize the 
145  dose amount for the IVPT study. Considerations for selecting an optimal dose amount may 
146  include (1) the consistency with which the dose can be applied (potentially using different 
147  dispensing and/or spreading techniques), (2) the reproducibility of the flux profiles, (3) the 
148  influence of dose amount and dose duration on the shape of the flux profile, and (4) the 
149  approximate range of drug concentrations in receptor solution samples at different time points 
150  (relative to the sample analytical method limits of quantification).  
151   
152  The selected sampling schedule and study duration should be sufficient to characterize the 
153  cutaneous pharmacokinetics of the drug, which ideally includes a sufficiently complete flux 
154  profile to identify the maximum (peak) flux and a decline in the flux thereafter across multiple 
155  subsequent time points. A dose that remains on the skin for the duration of the study may 
156  continue to deliver the drug for a sustained period and may not necessarily exhibit a suitable 
157  decline in the flux at later time points. In such instances, it may be appropriate to develop an 
158  IVPT method that involves wiping off the applied dose after a suitable duration on the skin and 
159  continuing to monitor the receptor solution for an extended period thereafter, during which the 
160  decline in the flux profile can be characterized. The sampling frequency should be selected to 
161  provide a suitable resolution for the flux profile, and a minimum of eight non-zero sampling time 
162  points is recommended across the study duration (e.g., 48 hours).   
163   
164  C.  IVPT Method Procedures and Controls 
165   
166  Suitable technical procedures and control parameters should be established during method 
167  development. These may include procedures for preparing and mounting the skin on the 
168  diffusion cell in a consistent manner, determining the instrument settings that regulate the skin 
169  surface temperature within the specified range, performing the barrier integrity test 
170  appropriately, controlling the accuracy and precision of the dose amount dispensed on each skin 
171  section.  
172   
173  For example, a dosing procedure may be developed that uses a positive displacement pipette to 
174  dispense a volumetrically controlled amount of a topical product, targeting the deposition on the 
175  skin of a certain mass (e.g., 15 mg/cm2) of topical product. If the inner diameter of the orifice in 
176  the dosing compartment of the diffusion cell is 15 millimeters (mm), and the effective dose area 
177  is ~1.77 cm2, this would indicate a target dose of ~26.5 mg of topical product per diffusion cell. 
178  Experiments during method development may establish that, based upon the density of the 
179  topical product, a specific volumetric setting on a specific model of positive displacement pipette 
180  with a specific pipette tip repeatedly dispenses ~27.5 mg of topical product (e.g., characterized 
181  by multiple replicate pipette dispensations into a weigh boat on a fine balance). This pipette 
182  setting may be optimal for a dosing procedure where the dose spreading instrument, like the flat 
183  bottom of a high performance liquid chromatography (HPLC) glass vial, or the rounded end of a 
184  glass rod or capillary tube, is subsequently used to spread the dispensed dose evenly upon the 
185  skin section mounted in the diffusion cell, and where repeatedly weighing the dose-spreading 
186  instrument before and after the dose spreading indicates that the residual topical product 
187  remaining on the bottom of the glass vial after the dose spreading reproducibly amounts to ~1.0 
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188  mg of topical product (indicating that ~26.5 mg of the topical product would reproducibly be 
189  dosed to each skin section). Such characterizations of the technical procedures and control 
190  parameters for the IVPT method, like the reproducibility of the dosing procedure, should be 
191  established during method development and may not need to be demonstrated thereafter each 
192  time the same procedure is used. 
193   
194  D.  IVPT Skin Barrier Integrity Testing: Common Methods 
195   
196  The technical procedures for the skin barrier integrity test should be established during IVPT 
197  method development. Three types of barrier integrity tests are common, however, there are 
198  currently no applicable compendial standard protocols or acceptance criteria for any of these 
199  three types of human skin barrier integrity tests. Nonetheless, recommended parameters for the 
200  three common types of barrier integrity tests are discussed below.  
201   
202  1.  Trans-Epidermal Water Loss Skin Barrier Integrity Test 
203   
204  A TEWL skin barrier integrity test involves a measurement near the outer surface of the skin of 
205  the rate at which water (vapor) is fluxing through the skin barrier from the underside of the skin 
206  section. For the test, the skin section is mounted in a diffusion cell (e.g., clamped in place 
207  between the donor and receptor compartments), with the underside of the skin in contact with the 
208  receptor solution in the receptor compartment (e.g., phosphate buffered saline, pH 7.4), and 
209  equilibrated to a skin surface temperature of 32C  1C. If skin sections are cut large enough to 
210  cover the flange of the diffusion cell in which they are mounted, then after they have equilibrated 
211  for several hours at a skin surface temperature of 32C  1C, it may be feasible to gently 
212  remove the donor compartment without disrupting a skin sections adherence to the lower flange 
213  of the diffusion cell, thereby allowing the TEWL probe to be placed directly on the skin surface, 
214  instead of being placed atop the donor compartment. Typically, a minimum of three replicate 
215  measurements are made on each skin section, which are recorded after the measurements have 
216  stabilized.  
217   
218  Commercially available devices to measure TEWL may differ in design and operational 
219  principles. The TEWL measured by devices with certain designs (e.g., an open chamber versus a 
220  closed chamber) may be relatively more susceptible to the influence of environmental 
221  conditions. Therefore, environmental temperature and humidity are typically controlled as 
222  precisely as possible (e.g., a temperature range of 21C  2C and a humidity range of 50%  
223  20% relative humidity are ideal, if feasible). More precise control of the relative humidity (e.g., 
224  in the range of 40%  50%) may reduce the variability of TEWL measurements for devices with 
225  certain designs. Certain designs of measurement probes and adapters for in vitro use are 
226  available by the manufacturers of TEWL devices, and may be appropriate to use. Inconsistency 
227  in the diameters for the measurement probe chamber, the measurement probe orifice, the in vitro 
228  adapters, and the skin area being measured, as well as variation in the distance of the probe 
229  sensor(s) from the skin surface, potentially because of the (variable) height of donor 
230  compartments (when applicable), could increase the variability of TEWL measurements. 
231  Inconsistent control of the alignment of the TEWL measurement device in relation to the donor 
232  compartment and/or the skin section may also increase the variability of TEWL measurements. 
233  Also, the TEWL measured by devices with certain designs may be relatively more susceptible to 
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234  the influence of heat transfer from the hand that holds the probe. Applicants should follow 
235  relevant instructions in the manufacturers user manual for the specific TEWL measurement 
236  device used.   
237   
238  No more than approximately 15 grams of water per square meter per hour (i.e.,  15 g/m2/hr) 
239  could be a reasonable skin barrier integrity acceptance (cutoff) criterion for a TEWL barrier 
240  integrity test on human torso or thigh skin; if this was selected as the cutoff criterion, skin 
241  sections with a TEWL > 15 g/m2/hr would fail the test. Skin sections that fail a barrier integrity 
242  test should not be dosed, but may serve as non-dosed control skin sections. A higher cutoff (e.g., 
243   20 g/m2/hr) may also be reasonable if justified by experimental data demonstrating that the 
244  selected acceptance criterion appropriately discriminates skin sections with a compromised 
245  barrier integrity from those with a competent barrier integrity. 
246   
247  However, TEWL measurements for skin sections with a competent barrier integrity can vary 
248  depending upon the TEWL measurement device, the manner in which it is operated, and the 
249  environmental conditions (e.g., higher ambient humidity or greater distance from the skin surface 
250  may decrease the value of the TEWL measurement). Precise control of environmental and 
251  device/operational factors can minimize variability in TEWL measurements. Therefore, the 
252  technical procedures for measuring TEWL should be optimized during IVPT method 
253  development (or based upon prior optimization in the laboratory performing the test). Also, the 
254  TEWL measurement device should be appropriately calibrated (by the manufacturer, and for 
255  some devices, also before each set of tests). Applicants may provide information about the 
256  relevant calibration procedures specified by the manufacturer for the specific TEWL device 
257  used; this can be submitted in the ANDA along with the IVPT method development report, to 
258  support the appropriateness of the technical procedures established by the laboratory for TEWL 
259  measurements. When a TEWL barrier integrity test is used in any study phase (IVPT method 
260  development, pilot study, validation, and/or pivotal study) the ambient laboratory temperature 
261  and humidity during the TEWL barrier integrity test should be monitored and reported.  
262   
263  2.  Tritiated Water Skin Barrier Integrity Test 
264   
265  An example of a recommended approach to a tritiated water skin barrier integrity test would be 
266  to mount the skin in a diffusion cell (e.g., clamped in place between the donor and receptor 
267  compartments) and allow it to equilibrate to a skin surface temperature of 32C  1C with the 
268  stratum corneum exposed to the air in the donor compartment and the underside of the skin in 
269  contact with the receptor solution (e.g., phosphate buffered saline, pH 7.4).  
270   
271  A small amount of tritiated water (sufficient to cover the entire surface of the skin section) would 
272  be briefly dosed on the stratum corneum. This dose of tritiated water would be left on the surface 
273  for a precisely controlled and relatively brief period (e.g., 5 minutes) after which it would be 
274  removed from the skin surface (e.g., using a pipette to remove the bulk volume and then an 
275  absorbent low lint laboratory tissue to gently blot dry). The receptor solution would then be 
276  sampled at a precise duration after the removal of the tritiated water from the skin surface (e.g., 
277  30 minutes after the removal of the 5-minute dose of tritiated water from the skin surface).  
278   
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279  While the entire volume of the receptor compartment may be removed and replenished, typically 
280  only an aliquot of the receptor solution (e.g., phosphate buffered saline, pH 7.4) is transferred to 
281  a suitable volume of scintillation fluid for counting. The volume of the aliquot typically depends 
282  upon the type of scintillation fluid used and the maximum amount of aqueous fluid that is 
283  suitable to mix with the scintillation fluid. A scintillation counter is then used to quantify the 
284  amount of radioactivity in the aliquot sampled, which can be used to calculate the amount of 
285  tritiated water that permeated into the larger (entire) volume of receptor solution; the calculation 
286  is performed using the specific activity of the tritiated water to equate a given amount of 
287  radioactivity to the equivalent volume of tritiated water that permeated per square centimeter of 
288  skin surface area.  
289   
290  Approximately 1.5 equivalent (eq.) microliter (L) of tritiated water per cm2 (i.e., ~1.5 eq. 
291  L/cm2 or ~1.5 eq. mg/cm2) would be a reasonable skin barrier integrity acceptance (cutoff) 
292  criterion for a tritiated water barrier integrity test that involves a 5-minute dose followed by a 30-
293  minute sampling duration (i.e., sampling 30 minutes after dose removal) on human torso or thigh 
294  skin. Skin sections with a tritiated water test result of > 1.5 eq. mg/cm2 would fail the test and be 
295  excluded from the population of skin sections dosed with the topical product; skin sections that 
296  fail a barrier integrity test should not be dosed, but may serve as non-dosed control skin sections. 
297  Other acceptance criteria may also be reasonable if justified by experimental data demonstrating 
298  that the selected acceptance criterion appropriately discriminates skin sections with a 
299  compromised barrier integrity from those with a competent barrier integrity.  
300   
301  When calculating the results for a tritiated water barrier integrity test, it may be important to 
302  account for the surface area dosed. For example, if using an acceptance criterion of 1.5 eq. 
303  mg/cm2 with a diffusion cell that has an orifice diameter of 15 mm and a skin surface area of 
304  1.77 cm2, the mass of tritiated water that would be calculated to have permeated into the receptor 
305  compartment would be ~2.7 eq. mg/cm2 of tritiated water. 
306    
307  3.  Electrical Based Skin Barrier Integrity Tests 
308   
309  There are several variations of electrical based skin barrier integrity tests that report the test 
310  result as a measure of the resistance, conductance, or a related electrical concept that 
311  characterizes the bulk flow of electrical current across the skin. Transepithelial electrical 
312  resistance tests involving the skin may be referred to more specifically as Trans-Epidermal 
313  Electrical Resistance (TEER) skin barrier integrity tests. The test results may be described in 
314  units of conductance, which is the reciprocal of resistance. Electrical based skin barrier integrity 
315  tests often use instruments that are designed to measure the inductance (L), capacitance (C), and 
316  resistance (R) of electronic circuits or electrical components; these instruments are commonly 
317  known as LCR meters and have different settings (test parameters) that can be adjusted.  
318   
319  An example of a recommended approach to a TEER skin barrier integrity test would be to mount 
320  the skin in a diffusion cell (e.g., clamped in place between the donor and receptor compartments) 
321  and allow it to equilibrate to a skin surface temperature of 32C  1C with the stratum corneum 
322  exposed to the air in the donor compartment and the underside of the skin in contact with an 
323  ionic solution (e.g., phosphate buffered saline, pH 7.4).  
324   
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325  A small amount of the ionic solution (sufficient to cover the entire surface of the skin section) 
326  would be briefly dosed on the stratum corneum. Then, one lead/electrode from an LCR meter 
327  would be placed in contact with the solution in the receptor compartment while the other 
328  lead/electrode would be placed in contact with the solution in the donor compartment. After 
329  measuring the resistance across the skin (e.g., in k, normalized for area, noting that resistance 
330  is inversely proportional to area) the solution in the donor compartment would be removed and 
331  the skin surface would be gently blotted dry with an absorbent low lint laboratory tissue. The 
332  skin (still mounted in the diffusion cell) would then be allowed to equilibrate with the dry air 
333  above for a sufficient duration to normalize the hydration state of the stratum corneum before 
334  being dosed with the test topical product or RS. 
335   
336  The results for a TEER skin barrier integrity test can vary substantially depending on the LCR 
337  meter settings (e.g., frequency) and the technical procedures used for the test. The acceptance 
338  criterion for a specific electrical based skin barrier integrity test method may be justified by 
339  experimental data demonstrating that the selected acceptance criterion appropriately 
340  discriminates skin sections with a compromised barrier integrity from those with a competent 
341  barrier integrity.  
342   
343  E.  IVPT Skin Barrier Integrity Testing: General Considerations 
344   
345  There are three general considerations for the development or adoption of technical procedures 
346  for any skin barrier integrity test method during IVPT method development:  
347   
348  i.  The technical procedures should not irreversibly alter the skin barrier. It may be 
349  acceptable to temporarily alter the hydration state of the stratum corneum by briefly 
350  depositing an aqueous solution on the surface of the skin, as long as sufficient time is 
351  afforded for the hydration of the stratum corneum to normalize before dosing of the 
352  topical product. The procedure described above for a brief (e.g., 5-minute) exposure of the 
353  skin surface to tritiated water followed by a 30-minute duration during which the 
354  hydration state of the stratum corneum is re-equilibrating would likely be appropriate. By 
355  contrast, a 30-minute exposure of the skin surface to an aqueous solution for an electrical-
356  based test method, followed within 5 minutes by dosing of the topical product, may not be 
357  appropriate without further characterization of the influence of the hydration state of the 
358  stratum corneum on the discrimination sensitivity of the skin to differences in topical 
359  bioavailability. Similarly, if a portable lamp were placed close to the skin to improve 
360  visibility while study procedures were being performed, the heat from the lamp may alter 
361  the local (micro)environment of the skin in a manner that is not representative of the 
362  ambient environmental conditions in the laboratory; this should be avoided.    
363   
364  ii.  The acceptance criterion should be a cutoff value for the test result, at which a skin section 
365  fails the test. Skin sections that fail a barrier integrity test should not be dosed but may 
366  serve as non-dosed control skin sections. Skin sections with a passing barrier integrity test 
367  result may be considered to have a competent barrier integrity and may be dosed. This 
368  acceptance criterion should be selected based upon an understanding of the distribution of 
369  test results (among multiple replicate skin sections from multiple donors) for the specific 
370  barrier integrity test procedure performed with the specific type and preparation of skin 
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371  under conditions relevant to the IVPT pivotal studies submitted in the ANDA. The 
372  intention of the barrier integrity test is to identify (and exclude) skin sections whose 
373  barrier integrity (intactness) is compromised. The intent is not to reduce the inherent 
374  variability in barrier function (permeability) in human skin that is representative of real 
375  variation in the human population. Also, the relative permeability of the skin to a drug 
376  from a topical product may not necessarily correlate with the permeability of the skin to 
377  water, and therefore, constraining the variability of the skin permeability to water (using a 
378  stricter acceptance criterion that excludes a larger number of skin sections) may not 
379  necessarily reduce the variability in the IVPT study results. 
380   
381  iii.  The acceptance criterion should be able to discriminate skin sections with a compromised 
382  barrier integrity. This may be demonstrated by measuring the barrier integrity of skin 
383  sections mounted and equilibrated in a diffusion cell before and after deliberately 
384  compromising the skin barrier (e.g., by repeatedly using adhesive tape to strip away 
385  increasing amounts of the stratum corneum, piercing the skin several times with a 30 
386  gauge needle, or using other physical or chemical insults to damage the skin barrier). 
387  Based upon the acceptance criterion selected, the test result for skin sections that pass the 
388  test before being damaged should fail the test after the damage. 
389   
390  F.  Differences Between IVPT Method Development and Validation 
391   
392  1.  Optimization of an IVPT Method Prior to Advancing to IVPT Method Validation 
393   
394  Different study designs and method parameters may be evaluated during the IVPT method 
395  development phase. For example, if the selected study parameters initially involve a dose 
396  duration of 48 hours and a study duration of 48 hours, and the flux profile is measurable, but it is 
397  not feasible to identify the maximum (peak) flux and a decline in the flux thereafter across 
398  multiple subsequent time points, then it may be appropriate to evaluate other study parameters as 
399  part of the IVPT method development. For example, a different target dose of the topical product 
400  and/or a longer sampling duration may be evaluated. An alternate study design may involve a 
401  shorter dose duration (e.g., 46 hours) after which the applied dose is removed from the skin, and 
402  the receptor solution continues to be sampled across a study duration that is sufficient to identify 
403  the maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time 
404  points. While shorter dose durations can help to improve the shape of IVPT flux profiles, the 
405  removal of the topical product dose from the skin surface can be challenging and often requires 
406  its own method development and optimization. Also, the design of sensitivity studies for such an 
407  IVPT study design may require a more sophisticated understanding of IVPT studies. While 
408  reasonable efforts should be made to develop an IVPT method that produces a well-defined 
409  maximum (peak) flux and a decline in the flux thereafter across multiple subsequent time points, 
410  this may not be feasible for certain topical products even with study durations of 96 hours, or, at 
411  least, may not be feasible to produce reliably in all donors. In such circumstances, the IVPT 
412  method development report should detail the systematic efforts made to optimize the IVPT 
413  method.  
414   
415  2.  Use of a Validated Sample Analytical Method for IVPT Method Validation 
416   
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417  The IVPT method development studies, being exploratory in nature, are often performed using a 
418  sample analytical method that is not validated (e.g., an HPLC or ultrahigh performance liquid 
419  chromatography (UPLC) method, often involving mass spectrometry (MS)); also, IVPT method 
420  development studies are often conducted in a manner that is not compatible with a quality 
421  management system which would otherwise make the evidence generated suitable to support 
422  valid conclusions. Such method development studies would not be suitable to demonstrate the 
423  validity of an IVPT method, or associated results. Therefore, although it may appear to be 
424  redundant, certain experiments performed during IVRT method development may need to be 
425  repeated during IVPT method validation, using appropriate controls, like a validated analytical 
426  method and procedures that are compatible with a suitable quality management system.  
427   
428  It is important to clearly segregate and consistently identify those experiments and results that 
429  were part of IVPT method development separately from those that were part of IVPT method 
430  validation. It is also important to consistently identify all relevant method parameters and 
431  experimental conditions/controls for each set of IVPT results. Information in the method 
432  development report should clearly identify/distinguish when the results for apparently similar 
433  sets of experiments may have been obtained using different method parameters. Method 
434  development reports should clarify which sets of diffusion cells were run in parallel or separately 
435  (e.g., on separate days). In addition, the sample analytical method parameters used to analyze the 
436  samples from each set of IVPT experiments should be specified, and the report should indicate 
437  whether or not the sample analytical method was validated (either at the time of sample analysis 
438  or subsequently). 
439   
440   
441  IV.  IVPT METHOD VALIDATION  
442   
443  When all the relevant parameters of the IVPT method have been established, a pilot study should 
444  be performed using the final IVPT method and using a validated sample analytical method. The 
445  purpose of the pilot study is to validate the suitability of the selected IVPT method parameters by 
446  demonstrating that the performance characteristics of the IVPT method are appropriate to 
447  compare the cutaneous pharmacokinetics of a drug delivered topically from a test product and 
448  RS. The results from the pilot study, thereby, support the systematic validation of the IVPT 
449  method, which proceeds as a distinct study phase following IVPT method development.  
450   
451  The results from this IVPT pilot study can help to estimate the number of donors that may be 
452  needed to adequately power the IVPT pivotal study. In addition to the test topical product and 
453  RS evaluated in the pilot study, a parallel assessment should be performed with a third topical 
454  product or formulation that is known or designed to be different from the RS, to validate the 
455  selectivity of the IVPT method to discriminate differences in bioavailability. The IVPT pilot 
456  study results should be plotted with error bars, comparing the permeation profiles for the three 
457  treatment groups in the pilot study. Separate plots should be prepared for average flux results and 
458  average cumulative permeation results. These data can be used to support specific IVPT method 
459  validation parameters (e.g., permeation profile and range). 
460   
461  A pilot IVPT study performed with multiple skin donors (e.g., 46 skin donors) and a minimum 
462  of four replicate skin sections per donor per treatment group is recommended. As skin from an 
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463  increasing number of donors is evaluated in the pilot study, the accuracy of the estimated number 
464  of donors needed to adequately power the IVPT pivotal study may improve. While skin from the 
465  same donors evaluated in the pilot study may also be used in the IVPT pivotal study, the results 
466  from the pilot study should not be combined with the results from the IVPT pivotal study for the 
467  purpose of statistical analysis.  
468   
469  The equipment, methodologies, and study conditions used in the IVPT pilot study (and the 
470  eventual IVPT pivotal study) should be appropriately validated or qualified. If an applicant elects 
471  to use equipment, methodologies, or study conditions that are different from those recommended 
472  in this guidance, the applicant should demonstrate why it was necessary and scientifically 
473  justified to do so.  Detailed protocols and well-controlled study procedures are recommended to 
474  ensure the precise control of dosing, sampling, and other IVPT study parameters, as well as 
475  potential sources of experimental bias.  
476   
477  The validation of the IVPT method should incorporate specific qualifications and controls 
478  (described below), performed using a validated sample analytical method, as applicable. The 
479  qualification of an IVPT method parameter refers to the process of defining what attributes make 
480  it suitable to perform its function in the IVPT method. For example, when repeated 
481  measurements of the temperature at the surface of skin mounted in a diffusion cell demonstrate 
482  that an IVPT equipment can maintain the skin surface temperature in the range of 32C  1C, 
483  the results can support a demonstration that the equipment is qualified to perform its function in 
484  an IVPT method for which a method parameter is the control of skin surface temperature in the 
485  range of 32C  1C across the relevant study duration.  
486   
487  A.  Equipment Qualification 
488   
489  Suitable equipment for the IVPT method includes various models of VDCs and flow-through 
490  diffusion cells. The operating principles and specific test procedures differ among the various 
491  equipment; relevant procedures from the manufacturer may be used for installation, operational, 
492  and performance qualifications. The laboratory qualification of each diffusion cell should, at 
493  minimum, include 1) measurements of the diffusional area of the orifices of the donor and 
494  receptor compartments between which the skin is mounted, 2) the empirically measured volume 
495  of the receptor solution compartment in each VDC or the empirically measured outflow tube 
496  length for each flow-through diffusion cell, 3) the stability of the temperature measured at the 
497  skin surface (e.g., 32C  1C) across a relevant duration (e.g., 48 hours), and 4) the rate of 
498  stirring or agitation in VDCs, or the flow rate for flow-through diffusion cells, as applicable. 
499   
500  If information related to the diffusional area of the orifices and the volume of the receptor 
501  solution compartment for each diffusion cell is available from the manufacturer, that information 
502  should be provided for each relevant diffusion cell, in addition to the empirical measurements 
503  made by the laboratory performing the IVPT studies. The equipment should control the diffusion 
504  cell temperature so that the skin surface temperature is verified to be stable (e.g., 32C  1C) for 
505  each diffusion cell before dosing (e.g., measured by a calibrated infrared thermometer), and 
506  monitored periodically throughout the duration of the experiment by repeatedly measuring the 
507  skin surface temperature of a non-dosed control diffusion cell that is run in parallel with the other 
508  replicate dosed diffusion cells and connected to the same water bath or thermoregulation system.  
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509   
510  B.  Membrane (Skin) Qualification 
511   
512  Excised human skin is recommended as the membrane for the IVPT study. The validity of each 
513  skin section dosed in the study should be qualified using an appropriate test procedure to 
514  evaluate the stratum corneum barrier integrity. Acceptable barrier integrity tests may be based 
515  upon tritiated water permeation, TEWL, or electrical impedance/conductance measured across 
516  the skin. The test parameters and acceptance criteria used for the skin barrier integrity test should 
517  be justified for the specific method and instrumentation that is used during the study. The skin 
518  from all donors whose skin is included in the study should be prepared in a consistent manner 
519  and dermatomed to a relatively consistent thickness, within limits specified in the study protocol. 
520  The skin thickness should be measured and reported for each skin section included in the study. 
521  The assignment of replicate skin sections from a donor to each treatment group should be 
522  randomized, as feasible. It is acceptable to balance the distribution of skin thicknesses in each 
523  treatment group (test topical product or RS) by a procedure specified in the study protocol. 
524   
525  C.  Receptor Solution Qualification 
526   
527  The composition and pH of the receptor solution used for the IVPT study should be qualified in 
528  relation to its compatibility with the skin as well as the stability and solubility of the drug in that 
529  receptor solution. The stability of the drug in the receptor solution samples should be validated as 
530  part of the receptor sample analytical method validation. The solubility of the drug in the IVPT 
531  receptor solution should be empirically determined in triplicate, to illustrate that the solubility of 
532  the drug in the receptor solution exceeds the highest sample concentration in the IVPT pivotal 
533  study, ideally by an order of magnitude. The solubility of the drug in the IVPT receptor solution 
534  should be sufficient to characterize the higher amounts of drug permeating from the increased 
535  drug delivery condition evaluated in the IVPT sensitivity assessment during IVPT method 
536  validation. 
537   
538  The inclusion of 0.1% polyoxyethylene[20]oleyl ether (also known as Oleth-20, Volpo-20, or 
539  Brij-20; CAS number 9004-98-2) is recommended to enhance the solubility of physiological 
540  buffer based (aqueous) receptor solutions for hydrophobic drugs. If additional solubility is 
541  needed, small increases in the concentration of polyoxyethylene[20]oleyl ether (e.g., from 0.1% 
542  or 0.2%, which is typically adequate for most hydrophobic drugs, to higher concentrations) are 
543  recommended, but should not exceed 6% polyoxyethylene[20]oleyl ether. Other strategies to 
544  improve the solubility of the drug in the receptor solution that may have the potential to alter the 
545  permeability of the skin (e.g., inclusion of organic solvents and alcohols in the receptor solution) 
546  are not recommended and may invalidate the IVPT method.  
547   
548  The inclusion of an anti-microbial agent in the receptor solution (e.g., ~0.1% sodium azide or ~ 
549  0.01% gentamicin sulfate) is recommended to mitigate potential bacterial decomposition of the 
550  dermis and/or epidermis in the diffusion cell, regardless of the study duration. Other anti-
551  microbial agents may also be acceptable, and if used, information should be included in the 
552  ANDA to explain the reason for their selection (and for the concentration at which they were 
553  used).  
554   
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555  D.  Receptor Solution Sampling Qualification 
556   
557  The accuracy and precision of receptor solution sample collection at each time point should be 
558  appropriately qualified. Evidence to qualify a sampling procedure should illustrate that the 
559  sampling technique can reliably collect a consistent volume of the sample from the well-mixed 
560  volume of the receptor compartment at each sampling event, and that no artifacts are likely to be 
561  created by the sampling technique. Information should be included describing the equipment 
562  manufacturers specification for the accuracy and precision of receptor solution sampling, when 
563  available.  
564   
565  For IVPT studies using a flow-through diffusion cell, it may be appropriate to qualify the lengths 
566  of tubing, and their associated dead volumes, to accurately calculate the lag time before a sample 
567  elutes through the tubing and is collected. For IVPT studies using a VDC, removal of the entire 
568  receptor solution volume and full volume replacement of the receptor solution at each time point 
569  may provide optimal solubility sink conditions. The sampling of small aliquots of the receptor 
570  solution for an IVPT study may introduce anomalous measurements of apparently negative flux 
571  in certain regions of the IVPT study and produce flux profiles that are difficult to interpret.  
572    
573  E.  Environmental Control 
574   
575  Ambient laboratory temperature and humidity during the study should be monitored and 
576  reported. An environmentally controlled temperature range of 21C  2C is recommended, and 
577  a humidity range of 50%  20% relative humidity is recommended, if feasible. 
578   
579  F.  Permeation Profile and Range 
580   
581  The flux profile and cumulative permeation profile for the IVPT pilot study should be plotted 
582  across a range of sampling times, which corresponds to the IVPT pivotal study duration. The 
583  calculation of flux and cumulative total permeation is discussed in more detail below. The results 
584  of the IVPT pilot study should validate that the selected study parameters are suitable to 
585  adequately characterize the permeation profile (the cutaneous pharmacokinetics) of the drug 
586  within the selected study duration (the range of sampling time points).  
587   
588  A sufficiently complete flux profile should be adequate to identify the maximum (peak) flux and 
589  a decline in the flux thereafter across multiple subsequent time points in the IVPT pilot study. 
590  The results of the IVPT pilot study should also validate that the sampling frequency provides 
591  suitable resolution to adequately characterize the permeation profile (particularly the flux 
592  profile).  
593   
594  G.  Precision and Reproducibility 
595   
596  The flux and cumulative permeation results from the IVPT pilot study (and the eventual IVPT 
597  pivotal study) should be calculated, tabulated, and reported for each diffusion cell at each time 
598  point, with summary statistics to describe the intra-donor average, standard deviation, and 
599  percent coefficient of variation (%CV) among replicates, as well as the inter-donor average, 
600  standard error, and %CV. Complete results for all data values used in the calculations should be 
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601  reported in a clear and organized manner, to facilitate the reconstruction of the flux and 
602  cumulative permeation results. The design of the study should be detailed and clear, and data 
603  values should be clearly associated with specific donors, replicates, treatment groups, time 
604  points, etc. 
605   
606  H.  Dose Depletion 
607   
608  The recovery of permeated drug in the receptor solution should be characterized in each 
609  diffusion cell as the cumulative total permeation of the drug in the receptor solution over the 
610  IVPT duration. This may be expressed as a percentage of the nominal amount of drug in the 
611  applied dose (which may be estimated based upon the nominal strength of the drug in the topical 
612  product and the approximate mass of topical product dosed on the skin).  
613   
614  For example, if 10 mg of a topical product containing 5% drug was dosed on the membrane, the 
615  amount of drug in the applied dose may be estimated to be 0.5 mg (or 500 g). If a cumulative 
616  total of 10 g of drug diffused into the receptor solution across a 48-hour duration of the IVPT, it 
617  would be possible to estimate that the 500 g dose would have been depleted by approximately 
618  10 g, amounting to an approximately 2% dose depletion. The average percentage dose 
619  depletion may thereby be estimated (not accounting for skin content) and should be reported.    
620   
621  I.  Discrimination Sensitivity and Selectivity 
622   
623  The discrimination ability of the IVPT method may be described using two concepts: sensitivity 
624  and selectivity. The IVPT sensitivity studies are necessarily performed during IVPT method 
625  development to establish IVPT method parameters like the dose amount, dose duration, study 
626  duration, etc. However, the analysis of the results from these studies is qualitative in nature, and 
627  they need not be repeated during the IVPT method validation phase.  
628   
629  The IVPT sensitivity studies are typically performed toward the end of the IVPT method 
630  development phase, and a key purpose of these studies is to incorporate the final IVPT method 
631  parameters for the target dose and dose duration to be used in the pivotal study so that the IVPT 
632  sensitivity studies can support a demonstration of the validity of the final IVPT method. 
633  Therefore, IVPT sensitivity studies are described within this section of the guidance in the 
634  context of IVPT validation (rather than method development) to avoid dissociating the 
635  discussions of IVPT sensitivity (which is performed to establish the suitability of the final IVPT 
636  method parameters) and IVPT selectivity (which is performed once the final IVPT method 
637  parameters are established, and which is based upon the IVPT pilot study that is performed as 
638  part of the IVPT method validation). With the exception of the alternative dose amounts or dose 
639  durations used in the IVPT sensitivity study, it is important that the IVPT method parameters are 
640  consistent across the IVPT sensitivity, pilot, and pivotal studies (including the anatomical region 
641  specified in the study protocol (e.g., posterior torso), the skin source, and skin preparation).   
642   
643  1.  IVPT Sensitivity  
644   
645  IVPT sensitivity is the ability of the IVPT method to detect changes in the cutaneous 
646  pharmacokinetics of the drug as a function of differences in drug delivery. If the IVPT method 
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647  consistently demonstrates higher and lower flux profiles (i.e., higher and lower values for IVPT 
648  endpoints) in response to increased and decreased drug delivery, respectively (or in response to 
649  other conditions expected to increase and decrease drug delivery, respectively), the IVPT method 
650  may be considered sensitive. 
651   
652  There are a few potential approaches by which to produce the differences in drug delivery that 
653  can be differentiated by a suitably discriminating IVPT method. Regardless of the approach 
654  used, the differences in the IVPT permeation profiles are not necessarily expected to be 
655  specifically proportional to differences in the dose amount, dose duration, or product strength. 
656  For example, three-fold differences in the dose amount (even if outside the recommended target 
657  dose range) may provide distinct flux curves but may not result in three-fold differences in the 
658  IVPT endpoints because the skin barrier may be rate-limiting both in vitro and in vivo.  
659   
660  In other words, if the target dose for the pivotal IVPT study was 10 mg/cm2, a 3-fold lower dose 
661  would be ~3 mg/cm2 and a 3-fold higher dose would be 30 mg/cm2; thus, an IVPT sensitivity 
662  study might compare the flux profiles from 3, 10, and 30 mg/cm2 doses of the topical product. 
663  Similarly, if the target dose for the pivotal IVPT study was 15 mg/cm2, a 3-fold lower dose 
664  would be 5 mg/cm2 and a 3-fold higher dose would be 45 mg/cm2; thus, an IVPT sensitivity 
665  study might compare the flux profiles from 5, 15, and 45 mg/cm2 doses of the topical product. 
666  An IVPT sensitivity study performed with multiple skin donors (e.g., 46 skin donors) and a 
667  minimum of four replicate skin sections per donor per treatment group is recommended. 
668   
669   Modulation of Dose Amount: An IVPT method development study with different dose 
670  amounts may provide supportive evidence that the IVPT methodology is sensitive to 
671  differences in drug delivery.  
672   
673  This approach is well suited to topical products that contain volatile components that 
674  evaporate from the formulation following dose application to the skin. Modulating the 
675  dose amount for such topical products effectively alters the thickness of the applied dose. 
676  The majority of volatile components from a thinner dose will tend to evaporate more 
677  rapidly (compared to a thicker dose), and a thinner dose will tend to deliver less drug into 
678  the skin (and/or for a shorter duration) compared to a thicker dose.  
679   
680  Modulating the dose amount can be an effective technique to modulate differences in 
681  drug delivery for formulations with volatile components, like gels, lotions, and many 
682  creams. However, modulating the dose amount may not necessarily produce perceptible 
683  differences in drug delivery for topical products like petrolatum-based ointments, or other 
684  types of topical products that do not evaporate on the skin, or that may not experience 
685  dose-dependent differences in metamorphosis that can alter the rate and extent of drug 
686  delivery. 
687   
688   Modulation of Dose Duration: For many topical products, it may be more effective to 
689  modulate the dose duration, instead of the dose amount, to produce differences in drug 
690  delivery and associated changes in the cutaneous pharmacokinetics of the drug.  
691   
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692  An IVPT method development study with a controlled dose amount (e.g., 15 mg/cm2) 
693  dosed for different durations (e.g., 2 hours, 6 hours, and 12 hours) may be well suited to 
694  provide supportive evidence that the IVPT methodology is sensitive to differences in 
695  drug delivery from many topical products. The scenario described in this example would 
696  support an IVPT study design where a topical product dose of 15 mg/cm2 is dosed for 6 
697  hours (the target duration for the IVPT study) and then wiped off. The applied dose may 
698  be removed with a series of cotton-tipped swabs, one or more of which may be dry and 
699  one or more of which may be moistened (e.g., with a soap solution or water). The initial 
700  (dry) swab typically removes the bulk of the dose and subsequent swabs are used to 
701  remove the residual dose (i.e., the residue of the topical product which may otherwise 
702  continue to deliver drug into the skin) and/or to rinse the skin.  
703   
704  To support a demonstration of the sensitivity of the IVPT study, the permeation profile 
705  produced by the target dose duration for the IVPT study (e.g., 6 hours) should be 
706  compared with a shorter dose duration (e.g., 2 hours) that is expected to perceptibly 
707  decrease the drug delivery, and also be compared with a longer dose duration (e.g., 12 
708  hours) that is expected to perceptibly increase the drug delivery. Thereby, the three dose 
709  durations compared in the IVPT sensitivity study are designed to produce perceptible 
710  changes in the cutaneous pharmacokinetics of the drug as a function of differences in 
711  drug delivery, and thereby support a demonstration of the sensitivity of the IVPT method. 
712   
713  The specific dose durations may be selected based upon an initial exploratory IVPT study 
714  performed during IVPT method development that characterizes the permeation profile 
715  when the dose is left on the skin for a longer duration (e.g., 24 or 48 hours). An important 
716  feature of the results from such an IVPT study is the duration of the initial phase of the 
717  permeation profile, when the flux is increasing at a relatively rapid rate.  
718   
719  For example, if such an exploratory study indicates that the flux increases on a steep 
720  slope until approximately 12 hours, and then continues to deliver the drug at a gradually 
721  increasing rate thereafter, it may suggest that the permeation profile for a dose duration of 
722  longer than 12 hours (e.g., 24 hours) may not be perceptibly different from that of the 12-
723  hour dose duration, especially when compared in a relatively small number of donors and 
724  replicates (e.g., four donors with four replicates each per dose duration). It may also 
725  suggest that a 12-hour dose duration may be a good choice for the longest of the three 
726  dose durations in the IVPT sensitivity study.  
727   
728  The target dose duration should be selected based upon considerations like the sensitivity 
729  of the sample analytical method, the ability to produce a permeation profile that can be 
730  perceptibly discriminated from that produced by the longer (12 hour) dose duration, 
731  and/or the labeled use of the topical product (which may indicate that the topical product 
732  should be reapplied every 46 hours).  
733   
734  The shortest of the three dose durations in the IVPT sensitivity study should be selected 
735  based upon the sensitivity of the sample analytical method and its ability to produce a 
736  permeation profile that can be perceptibly discriminated from that produced by the target 
737  (6 hour) dose duration. 
  17  Contains Nonbinding Recommendations 
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738   
739   Modulation of Product Strength: To validate the sensitivity, specificity, and selectivity 
740  of an in vitro release test (IVRT) method, altered strength formulations are routinely 
741  prepared. While it may seem convenient to use these altered strength formulations in an 
742  attempt to demonstrate the sensitivity and selectivity of an IVPT method, doing so may 
743  not produce the desired outcomes. There may be circumstances when this strategy may 
744  produce perceptibly different permeation profiles, however, in many instances, the 
745  resulting permeation profiles may not be perceptibly different when compared in a 
746  relatively small number of donors and replicates (e.g., four donors with four replicates 
747  each per topical product strength). In general, the modulation of topical product strength 
748  to support a demonstration of IVPT sensitivity is not recommended because it may not 
749  consistently produce the expected increase or decrease in drug delivery; however, in 
750  certain situations, higher and lower strength formulations (relative to the nominal strength 
751  of the RS) may suitably increase and decrease the drug delivery and cutaneous 
752  pharmacokinetics relative to that from the nominal strength topical product. 
753   
754  2.  IVPT Selectivity  
755   
756  IVPT selectivity is the ability of the IVPT method to discriminate the cutaneous 
757  pharmacokinetics of the drug between the RS and a topical product or formulation that exhibits 
758  differences in drug delivery relative to the RS. The IVPT pilot study with the parallel assessment 
759  of the RS, the test topical product, and a third topical product or formulation that is known or 
760  designed to be different from the RS may provide supportive evidence that the IVPT 
761  methodology is selective for differences in drug delivery. Topical product batch information for 
762  all topical product lots used in IVPT method development, validation and pilot studies, as 
763  applicable, should be submitted in the study reports. The topical product information should 
764  include, but not be limited to, information about the batch formula, manufacturing date, batch 
765  size, altered manufacturing processes (if applicable) and, if available, potency and content 
766  uniformity. The evaluation of inequivalence may be based upon a qualitative or quantitative 
767  comparison of the permeation profiles and/or the IVPT endpoints.  
768   
769  J.  Robustness 
770   
771  A primary assumption related to robustness testing is that the test system performs consistently 
772  when all system variables (e.g., temperature, stirring rate) are at nominal settings. A value of 
773  robustness testing is that it can verify whether the system continues to provide a consistent 
774  output when specific variables are slightly altered, thereby qualifying operational ranges for 
775  those variables. However, the variability inherent in the permeability of human skin, whether in 
776  vitro or in vivo, may not be compatible with the primary assumption related to the consistency of 
777  the test system.  
778   
779  Nonetheless, results from studies during IVPT method development that appear to support the 
780  robustness of the IVPT method or system should be reported, if relevant. For example, an IVPT 
781  method may be robust to substantial variations in the stirring rate of the receptor compartment. 
782  Similarly, the permeation profile of a drug into and through human skin may appear to be robust 
783  to certain differences in the topical product strength. Ultimately, because it may not always be 
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784  feasible to validate the robustness of IVPT method parameters, IVPT study procedures should be 
785  controlled as precisely as possible.  
786   
787   
788  V.  SAMPLE ANALYTICAL METHOD VALIDATION   
789   
790  While exploratory studies performed during IVPT method development may use an unvalidated 
791  sample analytical method, it is essential that all studies conducted as part of the IVPT method 
792  validation use a validated sample analytical method. A validated IVPT method should use a 
793  validated sample analytical method (e.g., HPLC/MS or UPLC/MS). Therefore, a discussion of 
794  the sample analytical method for the IVPT method is included in this guidance under this section 
795  on IVPT method validation.  
796   
797  However, the study protocols and reports related to the IVPT method are distinct from those for 
798  the sample analytical method that is used to quantify drug concentrations in IVPT receptor 
799  solution samples. The validation of a sample analytical method, in and of itself, does not 
800  demonstrate the validity of an IVPT method. Separate and specific reports should be submitted 
801  for the validation of the sample analytical method (e.g., HPLC/MS or UPLC/MS) and for the 
802  validation of the IVPT method.  
803   
804  Any results from studies of the IVPT method that are performed during method development 
805  using a different sample analytical method than that which is ultimately validated, cannot support 
806  a demonstration of the validity of the IVPT method. Information should be provided in the IVPT 
807  method validation report referencing the (separate) sample analytical method validation, and 
808  clearly indicate that all relevant results in the IVPT method validation report were obtained using 
809  a validated sample analytical method (as opposed to a sample analytical method with different 
810  parameters than those which were validated).      
811   
812  The receptor sample analysis procedures (e.g., typically involving an HPLC/MS or UPLC/MS 
813  system) should be performed using chromatography software (e.g., a chromatography data 
814  system) with audit trails, and should include a multi-point (68 concentration) calibration curve 
815  with suitable quality control samples, and should be validated in a manner compatible with the 
816  FDA guidance for industry Bioanalytical Method Validation (May 2018).  
817   
818  The validation of the receptor sample analytical method should include relevant qualifications of 
819  dilution integrity, if applicable, as well as stability assessments with the highest relevant 
820  temperature in the receptor solution for the longest relevant duration; the highest relevant 
821  temperature may be warmer than 32C because the temperature of the receptor solution is often 
822  higher than the temperature at the surface of the skin, and the longest relevant duration may be 
823  the longest interval between sampling time points for methods in which the entire receptor 
824  solution is replaced at each sampling time point, or it could be longer in scenarios with only 
825  partial sampling of the receptor solution (e.g., 34C for 48 hours).  
826   
827  If the samples are processed in specific ways for analysis (e.g., by drying and reconstituting the 
828  receptor samples in a smaller volume to concentrate the sample and increase the effective 
829  analytical sensitivity, or by dilution of receptor solution samples into the validated curve range of 
  19  Contains Nonbinding Recommendations 
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830  the sample analytical method) those procedures should be validated (e.g., by qualifying the 
831  dilution integrity during the sample analytical method validation). The stability of the drug in the 
832  receptor solution sample should be validated in a receptor solution matrix that has been exposed 
833  to the underside of the skin in a diffusion cell under conditions relevant to the IVPT pivotal 
834  study.  
835   
836   
837  VI.  IVPT PIVOTAL STUDY 
838   
839  The IVPT pivotal study protocol should incorporate considerations relevant to BE studies, in 
840  general.  
841   
842  A.  Handling and Retention of Samples  
843   
844  Refer to 21 CFR 320.38, 320.63 and the FDA guidances for industry Handling and Retention of 
845  BA and BE Testing Samples (May 2004) and Compliance Policy for the Quantity of 
846  Bioavailability and Bioequivalence Samples Retained Under 21 CFR 320.38(c) (August 2020), 
847  as applicable, regarding considerations for retention of study drug samples and to 21 CFR 320.36 
848  for requirements for maintenance of records of BE testing. Retention samples should be 
849  randomly selected from the drug supplies received before allocating topical product units for use 
850  in an IVPT study in which the test topical product and RS are compared.  
851   
852  B.  Control of Study Procedures 
853   
854  Study procedures that have the potential to influence the results of the study should be 
855  appropriately controlled. Also, experimental observations that may have the potential to 
856  influence the interpretation of the study results, as well as any protocol or standard operating 
857  procedure (SOP) deviations, should be reported. 
858   
859  Control of procedures related to the skin include the consistent control across the study of the 
860  skin preparation (e.g., dermatoming of skin sections) and the thickness of skin sections mounted 
861  on diffusion cells, as well as the skin storage conditions, including the duration for which the 
862  skin was frozen and the number of freeze-thaw cycles to which the skin was exposed. Skin from 
863  the same anatomical location should be used from all donors, and the demographics (age, race, 
864  sex) should be reported for all donors. Also, the IVPT sensitivity, pilot, and pivotal studies 
865  should use skin from the same anatomical site; otherwise, if skin from different anatomical sites 
866  is used across the different study phases, it may not be possible for the results of the IVPT 
867  sensitivity and pilot studies to support a demonstration of the discrimination ability of the IVPT 
868  method used for the pivotal study because the method parameters would not be aligned across 
869  the respective studies. Similarly, if a non-rate-limiting support membrane is used beneath the 
870  skin section (e.g., a filter membrane used in a validated IVRT method for the same topical 
871  product) then it should be used in a consistent manner for the IVPT sensitivity, pilot, and pivotal 
872  studies. 
873   
874  Control of procedures related to the dose include the control of the area of dose application, the 
875  dose amount, the dosing technique, the dose duration, and the blinding and randomization 
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876  procedures for dosing. The test topical product and RS should be dosed in an identical and 
877  consistent manner for all diffusion cells in the study. Differences in dosing technique may alter 
878  the metamorphosis of the dosage form on the skin, and inconsistencies in the diameter of the area 
879  dosed on each diffusion cell may significantly influence the dosed area and contribute to errors 
880  in the calculation of flux.  
881    
882  Control of procedures related to sampling include the control of sampling time precision, the 
883  sampling technique, the duration of sampling and replacement of receptor solution, the sample 
884  volume or flow rate, and sample handling and storage. 
885   
886  Control of procedures related to the pivotal study should include a non-dosed control skin section 
887  from each skin donor, which should be mounted in a diffusion cell and otherwise treated 
888  identically to the dosed skin sections, including sampling of the receptor solution at all time 
889  points to ensure that drug concentrations monitored in the receptor solution are associated with 
890  the dose applied in the IVPT pivotal study, and not drug contamination in the skin from that 
891  donor that might permeate into the receptor solution across the duration of the study. A pre-dose 
892  zero sample collected from each diffusion cell is also recommended, which may identify 
893  potential contamination associated with each skin section and/or each diffusion cell. 
894   
895  In addition, investigators should perform the IVPT validation and pivotal studies within a quality 
896  management system that includes, but is not limited to, documented procedures for: 
897   
898    Study personnel identification, training, qualification, and responsibilities 
899   
900    Study management and study management personnel responsibilities 
901   
902    Quality control (QC) and QC personnel responsibilities 
903   
904    Quality assurance (QA) and QA personnel responsibilities 
905   
906    Use of SOPs 
907   
908    Use of study protocols 
909   
910    Use of study reports 
911   
912    Maintenance and control of the study facility environment and systems 
913   
914    Qualification and calibration of instruments and computerized systems 
915   
916    Good documentation practices including, but not limited to, contemporaneous 
917  documentation of study procedures and recording of experimental observations or 
918  deviations from procedures specified in the study protocol or in relevant SOPs  
919   
920    Maintenance of suitable records that facilitate the reconstruction of study events and 
921  procedures, including study sample handling and storage records (e.g., sample tracking 
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Draft  Not for Implementation 
922  logs), audit trails for sample analysis procedures, control of study materials and reagents, 
923  and electronic data control 
924   
925    Archival of study records 
926   
927  C.  Blinding Procedure 
928   
929  A detailed description of the blinding procedure should be provided in the study protocol and 
930  final report. The packaging of the test topical product and RS should be similar in appearance to 
931  maintain adequate blinding of the investigator and any experimental operators.  
932   
933  D.  Randomization 
934   
935  The method of randomization should be described in the protocol of the IVPT study and the 
936  randomization schedule provided, preferably in a SAS data set in .xpt format (created using the 
937  SAS XPORT procedure). It is recommended that an independent third party generate and hold 
938  the randomization code throughout the conduct of the study to minimize bias. The applicant may 
939  generate the randomization code if not involved in the packaging and labeling of the test topical 
940  product and RS dosed in the study. A sealed copy of the randomization scheme should be 
941  retained at the study site and should be available to FDA investigators at the time of site 
942  inspection to allow for verification of the treatment identity of each skin section. 
943   
944  E.  Dosing 
945   
946  In the IVPT pivotal study, the test topical product and RS should be dosed in an alternating 
947  pattern on successive diffusion cells (skin sections) from each donor. One of two dosing 
948  sequences (illustrated below) may be randomly assigned for each donor: 
949   
950  a.  ABABAB... 
951  b.  BABABA... 
952   
953  F.  Study Design 
954   
955  The IVPT pivotal study should compare the cutaneous pharmacokinetics of the drug from the 
956  test topical product versus that from the RS using excised human skin with a competent skin 
957  barrier mounted on a qualified diffusion cell system. The IVPT pivotal study should use a design 
958  that directly compares the test topical product and RS on skin from the same set of donors, each 
959  with the same number of replicate skin sections per donor per treatment group (dosed with either 
960  test topical product or RS topical), using the same IVPT method parameters.  
961   
962  The IVPT pivotal study design, methodology, and diffusion cell equipment considerations 
963  relating to sampling precision should be controlled as precisely as possible. For example, it may 
964  be appropriate to stagger the dose application on successive diffusion cells and to synchronize 
965  the sampling time points with the dosing time for that diffusion cell, to ensure consistent 
966  durations between dosing and sampling of all diffusion cells.  
967   
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968  G.  Inclusion Criteria  
969   
970  In general, the following inclusion criteria should apply: healthy, normal, barrier-competent skin 
971  from male and/or female donors of at least 18 years of age. Inclusion criteria related to donor 
972  demographics (e.g., age, race, sex) should be specified in the study protocol and demographic 
973  information should be reported for each donor. Additional criteria may be added by the 
974  applicant. 
975   
976  The skin may be harvested following excision from patients undergoing a surgical procedure or 
977  excised from cadavers. A consistent source is recommended for all the skin used. The anatomical 
978  region specified in the study protocol (e.g., posterior torso) should be consistent for all donors 
979  whose skin is included in the study.   
980   
981  The study protocol should specify the inclusion (acceptance) criteria for skin sections based upon 
982  the barrier integrity test result, which should be reported for each skin section. 
983   
984  The study protocol should specify inclusion criteria related to the temperature and duration of 
985  skin storage as well as the number of freeze-thaw cycles, all of which should be reported for each 
986  donors skin. 
987   
988  The study protocol should specify the inclusion criteria related to the skin harvesting/processing 
989  procedures and skin thickness (e.g., dermatomed skin of 500 m  250 m thickness) used in the 
990  IVPT study.  
991   
992  H.  Exclusion Criteria  
993   
994  In general, the following exclusion criteria should apply. Skin from subjects with a known 
995  (history of) dermatological disease should be excluded from the study. Skin with tattoos, stretch 
996  marks, or any visible sign of abnormality should be excluded from the study. Skin exhibiting a 
997  significant density of terminal hair is not recommended and should be excluded from the study.  
998  Additional criteria may be added by the applicant. 
999   
1000  While gentle washing or rinsing of the skin surface is appropriate, submerging the skin in an 
1001  aqueous solution for more than a few minutes may damage the skin barrier and should be 
1002  avoided; such skin sections should be excluded from the study. Also, skin that has been 
1003  subjected to shaving with a blade; abrasive polishing; tape-stripping; or cleansing with alcohols, 
1004  solvents, or other strong solutions that could damage the skin barrier should be excluded from 
1005  the study.  
1006   
1007  Skin from donors with significant background levels of the drug or other compounds that may 
1008  interfere with the quantification of the drug in receptor solution samples should be excluded from 
1009  the study. 
1010   
1011  Skin from donors exhibiting a high barrier integrity test failure rate among replicate skin sections 
1012  may be excluded from the study, and skin from an alternative donor may be used instead.  
1013   
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Draft  Not for Implementation 
1014  I.  IVPT Endpoints  
1015   
1016  The endpoints for the IVPT pivotal study are based upon parameters that characterize the rate 
1017  and extent to which the drug permeates into and through the skin and becomes available in the 
1018  receptor solution. Specifically, the rate of drug permeation is characterized by the flux (J) and the 
1019  extent of drug permeation is characterized by the total cumulative amount (AMT) of drug 
1020  permeated into the receptor solution across the study duration. 
1021    
1022  The flux (rate of drug permeation) should be plotted as J on the Y-axis in units of mass/area/time 
1023  (e.g., nanograms (ng)/cm2/hr) versus time on the X-axis. Flux profiles commonly resemble 
1024  plasma pharmacokinetic profiles, however, it is important to distinguish that the flux is a rate, 
1025  rather than a concentration. The extent of drug permeation should also be plotted, as the total 
1026  cumulative amount (AMT) of drug permeated on the Y-axis in units of mass/area (e.g., ng/cm2) 
1027  versus time on the X-axis. 
1028   
1029  The flux should be calculated based upon: the receptor sample concentration (e.g., 2.0 ng/mL) at 
1030  each time point; the precise, empirically measured volume of that specific diffusion cell (e.g., 6.0 
1031  mL) which may vary between individual cells; the area of dose application (e.g., 1 cm2); and the 
1032  duration for which the receptor volume was accepting the drug.  For example, if the sample 
1033  exemplified here represented a 2-hour period following dosing, then J would be calculated based 
1034  upon the values above as: 
1035   
1036                   J = [(2.0 ng/mL) x (6.0 mL)]/(1 cm2)/(2 hrs) = 6 ng/cm2/hr 
1037   
1038  This flux should be calculated and reported for each diffusion cell for each sampling interval and 
1039  plotted across the entire study duration to generate the flux profile for each diffusion cell.  The 
1040  rate calculated above may be plotted at the 2-hour time point, or at the midpoint between 0 and 2 
1041  hours (i.e., 1 hour).  
1042   
1043  In addition, the AMT should be calculated and reported for each diffusion cell. This cumulative 
1044  amount of drug that has permeated (in total across the entire study) should be reported as the 
1045  AMT endpoint, rather than using a trapezoid rule to calculate the area under the flux curve. 
1046   
1047  The maximum flux (J ) at the peak of the drug flux profile and the AMT should both be 
max
1048  compared for locally-acting test topical products and RSs. This is somewhat analogous to the 
1049  comparison of the C  and AUC for systemically-acting test products and RSs, inasmuch as the 
max
1050  pair of endpoints in each case facilitates a comparison of the rate and extent to which the drug 
1051  from each type of product (locally-acting or systemically-acting) becomes available at the site of 
1052  action.    
1053   
1054  A confidence interval (CI) should be calculated for each IVPT endpoint: 
1055   
1056  a.  the natural log-transformed maximum flux (J ) 
max
1057  b.  the natural log-transformed total cumulative amount (AMT) permeated  
1058   
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Draft  Not for Implementation 
1059  It is the responsibility of the applicant to determine the number of donors required to adequately 
1060  power the IVPT pivotal study, however, a minimum of four dosed replicates per donor per 
1061  treatment group (test product or RS) is recommended.  
1062   
1063  At the completion of the study, if the number of skin replicates is the same for all donors in the 
1064  test topical product and RS treatment groups in the IVPT study, a statistical analysis for a 
1065  balanced design is recommended. If skin sections or diffusion cells are excluded from the final 
1066  statistical analysis because of experimental loss/issues, and the resulting data set is unbalanced, a 
1067  statistical analysis for an unbalanced design is recommended.  
1068   
1069  Approaches to statistical analysis of the pivotal study are described in section VIII of this 
1070  guidance.  Appendix I provides example SAS code for determining BE with both a balanced 
1071  dataset and an unbalanced dataset. Appendix II provides numerical examples with simulated data 
1072  sets. Appendix III provides example R code for determining BE. 
1073   
1074   
1075  VII. SUBMITTING INFORMATION ON IVPT STUDIES IN AN ANDA 
1076   
1077  For IVPT studies with topical products submitted in ANDAs that are intended to support a 
1078  demonstration of BE, detailed study protocols, relevant SOPs, and detailed reports should be 
1079  submitted for the IVPT method validation (including the IVPT pilot study) and the IVPT pivotal 
1080  study. In addition, a detailed report describing the IVPT method development should be 
1081  submitted. These protocols, SOPs, and reports should be submitted in module 5.3.1.2 of the 
1082  electronic Common Technical Document (eCTD) and should describe experimental procedures, 
1083  study controls, quality management procedures, and data analyses.  
1084   
1085  Note that the study protocols, SOPs, and reports related to the IVPT method are distinct from 
1086  those for the sample analytical method that is used to quantify drug concentrations in IVPT 
1087  receptor solution samples (e.g., an HPLC/MS or UPLC/MS method). Separate protocols and 
1088  SOPs should be submitted for the sample analytical method validation. Sample analytical 
1089  method development and validation reports, pilot and pivotal IVPT study sample analysis 
1090  reports, as well as associated SOPs and protocols relevant to the sample analysis of an IVPT 
1091  study with human skin should be submitted in Module 5.3.1.4 of the eCTD. 
1092   
1093   
1094  VIII.  IVPT PIVOTAL STUDY STATISTICAL ANALYSIS  
1095    
1096  The two treatment groups would correspond to the test topical product (T) and the RS (R). The 
1097  statistical analysis should consider a sample of   donors, for which   replicate skin sections 
1098  from the jth donor ( ) are available for the T group and   rTTeplicate skin sections from 
nn rrjj
1099  the jth donor ( ) are available for the R group. Each replicRRate (i) from each donor (j) 
jj = 1,,nn rrjj
1100  should have been randomly assigned to each product.   
1101    jj = 1,,nn
1102  Define the following quantities: 
1103   
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Draft  Not for Implementation 
1104     = the natural log-transformed IVPT endpoint (J  or AMT) dosed with the test 
max
1105  topical product for the ith skin replicate from the jth donor ( ) 
TTiijj
TT
1106     = the natural log-transformed IVPT endpoint (J  or AiiM=T1) ,d2o,sed, wrrjjit,hjj th=e 1R,S2 ,for ,tnnhe 
max
1107  ith skin replicate from the jth donor ( ) 
RRiijj
RR
1108     = the number of skin replicates friio=m 1th,2e ,jth d,orrjjno,rjj d=os1ed,2 w,ith, tnnhe test topical product 
1109  (TT ) 
rrjj
1110     jj == t1h,e2 n,um,bnner of skin replicates from the jth donor dosed with the RS ( ) 
RR
1111    rrjj  = the total number of skin replicates in the R gro jjup=  1,2,,nn
 RR RR RR
1112    rr == thrre1 n+umrr2be+r of d+onrronnrs 
1113  If the nunnmbers of skin replicates available for the final statistical analysis are the same for the   
1114  donors for the T group and the R group, the resulting data set is balanced. For simplicity of 
1115  notation, the common number of skin replicates for one donor for one treatment group in a  nn
1116  balanced data set is denoted as  .  
1117    TT TT TT RR RR RR
1118  A diffusion cell may be excluderrd= frorr1m =amrro2n=g the =reprrnnlic=aterrs1 in= a rrd2at=a set w=hrrennn there is a 
1119  documented observation of a failure (e.g., visual observation that a skin section tears and leaks 
1120  during the study) or a protocol deviation (e.g., the receptor compartment in a diffusion cell is 
1121  discovered to be empty at the first sampling time point). In such instances, if sufficient skin 
1122  remains from the same donor, and no samples from that diffusion cell have been analyzed, a 
1123  replacement diffusion cell can be set up and studied. Otherwise (if the diffusion cell cannot be 
1124  replaced) the resulting data set becomes unbalanced. 
1125   
1126  The statistical analysis methods for assessing BE in the cases of a balanced data set and an 
1127  unbalanced data set are described below. For a donor to be included in the statistical analysis, 
1128  there should be at least 3 replicate skin sections from the donor for each (T and R) treatment 
1129  group. 
1130   
1131  Step 1. 
1132  Determine  , the estimated within-donor standard deviation of the RS, for each of 
1133  the natural log-transformed IVPT endpoints J  and AMT: 
max
1134    SSWWRR
RR 1/2
1135  rrjj 2  
nn
jj=1ii=1RRiijj RR.jj
SSWWRR =   
1136   
rr nn
1137  where   is the average of log-transformed observations across all   
RR
1 rrjj RR
1138  replicaRRte.sjj f=rorrmjjRR doii=n1oRRr jiijj dosed with the RS.  rrjj
1139   
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Draft  Not for Implementation 
1140  (a) If  , use the scaled average BE (SABE) approach to determine BE 
1141  for the individual IVPT endpoint(s) in Steps 2, 3.1, and 4.1 
SSWWRR  0.294
1142  (b) If  , use the regular average BE (ABE) approach through the two 
1143  one-sided tests (TOST) procedure (Schuirmann, 1987) to determine BE for 
SSWWRR < 0.294
1144  the individual  IVPT endpoint(s) in Steps 2, 3.2, and 4.2 
1145  Step 2. 
1146  Determine the point estimate for the mean difference of T and R products ( ), its 
1147  standard error ( ), and the corresponding degrees of freedom ( ). 
1148     II
1149  For a balanced ssdssa(tIIa) set, determine  ,  , and   by the followddinddg: 
1150    
II ssss(II) dddd
1151      where   
1 nn 1 rr
II = II. = nnjj=1IIjj IIjj = rrii=1TTiijj RRiijj
1152     (estimate of inter-donor variability) 
2 1 nn 2
SSII = (nn1)jj=1IIjj II.
1153     
2
1154    ssssII = SSII nn

1155  For an uddnddba=lannnced1 data set, approximate  ,  , and   by using PROC MIXED 
1156  (or PROC GLM) in SAS.  The example code is provided in Appendix I.  
1157    II ssss(II) dddd
1158   
1159  Step 3.1.   Scaled Average BE (SABE) Approach 
1160   
1161  In the SABE approach, the hypotheses to be tested are: 
1162   
1163   
2
(TT RR)
HH0: 2   
1164  WWRR  
2
(TT RR)
1165    HHaa: 2 <  
1166  where:  WWRR
1167   
1168     = mean difference of T and R products 
1169    TT  = wRRithin-donor variance of R product 
2
1170    WWRR ,   (BE limit), and   (regulatory constant) 
2
(ln (mm))
1171  Rejection =of t(hWWe 0n)u2ll hmmyp=ot1h.e2s5is0 s0upports the concluWWsi0on= o0f .e2q5uivalence of the two 
1172  products. 
1173   
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1174  Determine (1-)*100% upper confidence bound for   based on 
1175  Howes Approximation (Howe, 1974) ( = 0.05):  2 2
1176    (TT RR) WWRR
1177   
1178    1/2
1179  where:  XX +YY+ssiissnn(VV)|VV|
1180   
1181     
2 2
1182    XX = II ssss (II)
2
1183    YY = SSWWRR  
2
 
XX = II+tt(1),dddd ssss(II)
1184     
 2
 (rr nn)SSWWRR
YY = (21),(rrnn)
1185     
   
1186    VV = XX XXXX XX+ YY YYYY YY 
1187  Note thasstii ssnn(VV) = 1is i f VV > 0; 0 iidd VV = pe0r;c e nt1il eif o VVf t<he0 Students t-distribution with 
1188   degrees of freedom and  th  is   percentile of the Chi-
tt(1),dddd (1)100
1189  square distribution with  2  degrees of freedom.  th
dddd (1),(rr nn) (1)100
1190    
1191    (rr nn)
1192  Step 3.2.   Regular Average BE (ABE) Approach 
1193   
1194  In the ABE approach, the hypotheses to be tested are: 
1195   
1196   
1197   
1198  where:  HH0: TT RR  ln(mm)or TT RR  ln(mm)
1199    HHaa: ln (mm) < TT RR < ln(mm)
1200     = mean difference of T and R products 
1201    TT RR  (BE limit) 
1202    mm = 1. 2is5 t0h0e natural logarithm of the BE limit  
1203  Rejectiolnn  (ommf t)he null hypothesis supports the conclusion of equivalence of the two 
1204  products. 
1205   
1206  Determine the (1  2)*100% confidence interval for   ( = 0.05): 
1207   
1208   TT RR
1209    
IItt(1),dddd ssss(II)
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1210  where   is   percentile of the Students t-distribution with   
1211  degrees of freedom.  th 
tt(1),dddd (1)100 dddd
1212   
1213   
1214  Step 4.1.   BE Determination with SABE Approach 
1215   
1216  For the test product to be bioequivalent to the reference standard, both of the 
1217  following conditions must be satisfied for each IVPT endpoint tested: 
1218  a.  the 95% upper confidence bound for   must be less than or 
1219  equal to zero (numbers should be kept to a mini2mum of2 four significant 
(TT RR) WWRR
1220  figures for comparison). 
1221  b.  the point estimate of the T/R geometric mean ratio must fall within the pre-
1222  specified limits  , where m = 1.2500. 
1
1223    mm, mm
1224  Step 4.2.   BE Determination with ABE Approach 
1225   
1226   For the test product to be bioequivalent to the reference standard, the 90% 
1227  confidence interval for   must be contained within the limits   in the 
1
1228  original scale for each IVPT endpoint tested, where m = 1.2500. 
TT RR mm, mm
1229   
1230  APPENDIX I (EXAMPLE SAS CODE)  
1231   
1232  This section provides an example SAS code for use in determining BE in an in vitro permeation 
1233  test (IVPT) study with either a balanced data set or an unbalanced data set. The example data 
1234  sets, Data-Balanced.csv and Data-Unbalanced.csv, are provided in Appendix II. 
1235   
1236   
/* 
INPUT 
* dat = name of the data set 
* don = column name of donor variable in dat 
* reps = column name of replicate variable in dat 
* trt = column name of treatment variable in dat (treatment variable: 'T', 
'R') 
* ly = column name of log-transformed endpoint in dat 
 
OUTPUT: result 
* Swr = estimated within-donor standard deviation of reference 
* lpointest = point estimate for mean difference in log scale 
* testmean = test mean estimate in original scale 
* refmean = reference mean estimate in original scale 
* pointest = point estimate transformed back to original scale 
* ub = SABE 95% upper confidence bound 
* (l, u) = ABE 90% CI for mean difference transformed back to original scale 
*/ 
 
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%MACRO ivpt(dat=, don=, reps=, trt=, ly=); 
 
  * Remove missing values before analysis; 
  DATA wdat; 
    SET &dat; 
    IF &ly = . THEN DELETE; 
  RUN; 
 
  * Create the data sets for test & reference; 
  DATA tdat; 
    SET wdat; 
    IF &trt = 'T'; 
  RUN; 
 
  DATA rdat; 
    SET wdat; 
    if &trt = 'R'; 
  RUN; 
 
  * Sort tdat and rdat by donor id and reps id; 
  PROC SORT DATA=tdat; 
    BY &don &reps; 
  RUN; 
 
  PROC SORT DATA=rdat; 
    BY &don &reps; 
  RUN; 
 
  * Determine if the data is balanced or unbalanced; 
  PROC SQL; 
    CREATE TABLE num as 
    SELECT &don, &trt, n(&don) as nrep 
    FROM wdat 
    GROUP BY &don, &trt; 
 
    CREATE TABLE unum as 
    SELECT DISTINCT(nrep) as nr 
    FROM num; 
 
    CREATE TABLE rcount as 
    SELECT COUNT(*) as nnr 
    FROM unum; 
 
    DROP TABLE num, unum; 
  QUIT; 
 
  DATA _NULL_; 
    SET rcount; 
    CALL SYMPUT("nnr", nnr); 
  RUN; 
 
  %IF &nnr=1 %THEN %DO;   * if the data is balanced;     
 
    * Calculate the necessary quantities; 
    PROC SQL; 
      CREATE TABLE tmp1 as 
    SELECT &don, mean(&ly) as mtest, var(&ly) as vtest, n(&ly) as rt 
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    FROM tdat GROUP BY &don; 
 
    CREATE TABLE tmp2 as 
    SELECT &don, mean(&ly) as mref, var(&ly) as vref, n(&ly) as rr 
    FROM rdat GROUP BY &don; 
 
    CREATE TABLE mergetmp0 as 
    SELECT * FROM tmp1 as tmp1 
    FULL JOIN tmp2 as tmp2 
    on tmp1.&don = tmp2.&don; 
 
    CREATE TABLE mergetmp as 
    SELECT *, mtest-mref as Ij 
    FROM mergetmp0; 
 
    DROP TABLE tmp1, tmp2, mergetmp0; 
    QUIT; 
 
    PROC IML; 
      USE mergetmp; 
    READ ALL VAR {&don "vref" "rr" "Ij" "mtest" "mref"}; 
    m = 1.2500; 
    alpha = 0.05; 
 
    * Determine Swr; 
    Swr2 = mean(vref); 
    Swr = sqrt(Swr2); 
    Ihat = mean(Ij); 
    SI2 = var(Ij); 
    nd = nrow(&don); 
    nr = rr[1,1]; 
    df = (nr-1)*nd; 
 
    * Treatment means; 
    testmean = exp(mean(mtest)); 
    refmean = exp(mean(mref)); 
 
    * SABE for balanced data; 
    theta = (log(m)/0.25)**2; 
    qchi = quantile('chisq', 1-alpha, df); 
    qt = quantile('t', 1-alpha, nd-1); 
    x = Ihat**2 - SI2/nd; 
    y = - theta * Swr2; 
    xp = ( abs(Ihat) + qt * sqrt(SI2/nd) )**2; 
    yp = - theta * df * Swr2 / qchi;   
    v = sign(xp-x) * (xp-x)**2 + sign(yp-y) * (yp-y)**2; 
    ub = x + y + sign(v)*sqrt(abs(v)); 
 
    * ABE  for balanced data; 
    se = sqrt(SI2/nd); 
    logl = Ihat - qt * se; 
    logu = Ihat + qt * se; 
 
    l = exp(logl); 
    u = exp(logu); 
 
    * Rename the point estimate; 
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    lpointest = Ihat; 
    pointest = exp(Ihat); 
 
    CREATE result var {Swr lpointest  
              testmean refmean pointest ub l u}; 
    APPEND; 
    CLOSE result; 
    QUIT; 
 
    PROC PRINT DATA = result noobs; 
      TITLE "IVPT Study Data Analysis Results for &ly: Balanced Data"; 
    RUN; 
 
  %END; 
 
  %ELSE %DO;   * if the data is unbalanced; 
 
    * Estimate the mean difference; 
    PROC MIXED DATA = wdat; 
      CLASS &don &trt; 
    MODEL &ly = &don &trt; 
    ESTIMATE "&ly Test-Ref" &trt -1 1/cl alpha=0.1; 
    LSMEANS &trt; 
    ODS OUTPUT ESTIMATES = iout; 
    ODS OUTPUT LSMEANS = mout; 
    RUN; QUIT; 
 
    * Calculate the necessary quantities; 
    PROC SQL; 
      CREATE TABLE tmp1 as 
    SELECT &don, mean(&ly) as mref, n(&ly) as rr 
    FROM rdat GROUP BY &don; 
 
    CREATE TABLE tmp2 as 
    SELECT count(*) as nd, sum(rr) as rstar 
    FROM tmp1; 
    QUIT; 
 
    PROC IML; 
      USE rdat; 
    READ ALL VAR {&ly}; 
 
    USE tmp1; 
    READ ALL VAR {&don "mref" "rr"}; 
 
    USE tmp2; 
    READ ALL VAR {"nd" "rstar"}; 
 
    USE iout; 
    READ ALL VAR {"estimate" "stderr" "df"}; 
 
    USE mout; 
    READ ALL VAR {"estimate"} into lsmean; 
 
    m = 1.2500; 
    alpha = 0.05; 
 
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    * Determine Swr; 
    mref2 = repeat(mref, rr); 
    mref2 = shape(mref2, rstar, 1); 
 
    Swr2 = sum( (&ly - mref2)##2 ) / (rstar - nd); 
    Swr = sqrt(Swr2); 
 
    * Treatment means; 
    testmean = exp(lsmean[2,1]); 
    refmean = exp(lsmean[1,1]); 
 
    * SABE for unbalanced data; 
    theta =  (log(m)/0.25)**2; 
    qchi = quantile('chisq', 1-alpha, rstar-nd); 
    qt = quantile('t', 1-alpha, df); 
 
    estimate = estimate[1,1]; 
    stderr = stderr[1,1]; 
 
    x = estimate**2 - stderr**2; 
    y = - theta * Swr2; 
 
    xp = (abs(estimate) + qt * stderr)** 2; 
    yp = - theta * (rstar - nd) * Swr2 / qchi; 
 
    v = sign(xp-x) * (xp-x)**2 + sign(yp-y) * (yp-y)**2; 
    ub = x + y + sign(v)*sqrt(abs(v)); 
   
    * ABE for unbalanced data; 
    logl = estimate - qt*stderr; 
    logu = estimate + qt*stderr; 
 
    l = exp(logl); 
    u = exp(logu); 
 
    * Rename the point estimate; 
    lpointest = estimate; 
    pointest = exp(estimate); 
 
    CREATE result var {Swr lpointest  
              testmean refmean pointest ub l u}; 
    APPEND; 
    CLOSE result; 
    QUIT; 
 
    PROC PRINT DATA = result noobs; 
      TITLE "IVPT Study Data Analysis Results for &ly: Unbalanced Data"; 
    RUN; 
 
  %END; 
 
%MEND ivpt; 
 
proc import datafile = "Data-Balanced.csv" 
  out = bdat 
  dbms = csv 
  replace; 
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run; 
 
ivpt
% (dat=bdat, don=donor, reps=replicate, trt=treat, ly=LAMT) 
 
 
proc import datafile = "Data-Unbalanced.csv" 
  out = udat 
  dbms = csv 
  replace; 
run; 
 
ivpt
% (dat=udat, don=donor, reps=replicate, trt=treat, ly=LAMT) 
 
1237   
1238   
1239  APPENDIX II (NUMERICAL EXAMPLES)  
1240   
1241  This section provides numerical examples using simulated data sets illustrating the determination 
1242  of BE. 
1243   
1244  (a) Balanced Data 
1245   
1246  The simulated data set Data-Balanced.csv shown below provides an example of in vitro 
1247  permeation test (IVPT) study outcomes when the data is balanced. The SAS output and the 
1248  determination of BE for LAMT in this data set follows. 
1249   
1250  Data-Balanced.csv 
donor  replicate  treat  AMT  Jmax  LAMT  LJmax 
1  1  T  2.361749  0.081326  0.859402  -2.50929 
1  2  T  0.916571  0.041008  -0.08712  -3.19398 
1  3  T  1.246243  0.038537  0.220133  -3.25613 
1  4  T  0.890018  0.04296  -0.11651  -3.14747 
1  5  T  0.663551  0.031219  -0.41015  -3.46674 
1  6  T  0.479143  0.015747  -0.73576  -4.15108 
2  1  T  0.998845  0.030073  -0.00116  -3.50412 
2  2  T  0.814457  0.061644  -0.20523  -2.78637 
2  3  T  0.648741  0.019984  -0.43272  -3.91283 
2  4  T  1.142716  0.044332  0.133408  -3.11604 
2  5  T  0.767291  0.028453  -0.26489  -3.55951 
2  6  T  1.392406  0.166782  0.331033  -1.79107 
3  1  T  1.388867  0.096822  0.328488  -2.33488 
3  2  T  0.45757  0.02184  -0.78182  -3.82402 
3  3  T  1.377438  0.0651  0.320226  -2.73182 
3  4  T  0.870988  0.073199  -0.13813  -2.61457 
3  5  T  1.753523  0.067281  0.561627  -2.69888 
3  6  T  0.995674  0.116414  -0.00434  -2.15061 
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4  1  T  0.811458  0.053465  -0.20892  -2.92872 
4  2  T  0.913538  0.060217  -0.09043  -2.8098 
4  3  T  2.251438  0.083596  0.811569  -2.48176 
4  4  T  1.163818  0.054213  0.151706  -2.91483 
4  5  T  1.027813  0.065446  0.027433  -2.72653 
4  6  T  1.081988  0.062279  0.078801  -2.77614 
5  1  T  1.275517  0.069859  0.243352  -2.66128 
5  2  T  1.231649  0.051342  0.208354  -2.96924 
5  3  T  1.454325  0.161016  0.374542  -1.82625 
5  4  T  1.195989  0.064734  0.178973  -2.73746 
5  5  T  2.07678  0.088355  0.730819  -2.42639 
5  6  T  1.893399  0.093223  0.638374  -2.37276 
6  1  T  1.564164  0.137378  0.447352  -1.98502 
6  2  T  1.504557  0.0728  0.408499  -2.62004 
6  3  T  1.049724  0.064531  0.048527  -2.74061 
6  4  T  1.047633  0.043859  0.046533  -3.12676 
6  5  T  1.159634  0.09236  0.148105  -2.38206 
6  6  T  1.129313  0.06546  0.12161  -2.72632 
1  1  R  1.598636  0.04239  0.469151  -3.16084 
1  2  R  2.24476  0.117486  0.808599  -2.14143 
1  3  R  1.60912  0.044199  0.475687  -3.11906 
1  4  R  1.8834  0.066452  0.633079  -2.71127 
1  5  R  1.101948  0.031705  0.097079  -3.45129 
1  6  R  1.165342  0.034002  0.153015  -3.38133 
2  1  R  0.622369  0.052794  -0.47422  -2.94135 
2  2  R  0.833337  0.033419  -0.18232  -3.39863 
2  3  R  0.386763  0.029507  -0.94994  -3.52311 
2  4  R  0.294178  0.02005  -1.22357  -3.9095 
2  5  R  0.851759  0.03968  -0.16045  -3.2269 
2  6  R  0.677715  0.032379  -0.38903  -3.43024 
3  1  R  0.96461  0.042626  -0.03603  -3.15528 
3  2  R  0.838346  0.101628  -0.17632  -2.28643 
3  3  R  0.130884  0.008774  -2.03344  -4.73601 
3  4  R  0.635926  0.039118  -0.45267  -3.24118 
3  5  R  0.804131  0.114582  -0.21799  -2.16646 
3  6  R  2.324877  0.229704  0.843667  -1.47096 
4  1  R  1.694799  0.088825  0.527564  -2.42109 
4  2  R  0.977661  0.030392  -0.02259  -3.49356 
4  3  R  3.13529  0.217896  1.142722  -1.52374 
4  4  R  0.922805  0.040161  -0.08034  -3.21485 
4  5  R  1.504834  0.082443  0.408683  -2.49565 
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4  6  R  1.330167  0.055237  0.285305  -2.89612 
5  1  R  2.104036  0.101673  0.743858  -2.28599 
5  2  R  0.842231  0.094771  -0.1717  -2.35629 
5  3  R  0.985656  0.081963  -0.01445  -2.50148 
5  4  R  0.931461  0.069496  -0.071  -2.66648 
5  5  R  1.580578  0.059193  0.45779  -2.82695 
5  6  R  1.209059  0.067989  0.189842  -2.68841 
6  1  R  1.038591  0.037859  0.037865  -3.27389 
6  2  R  1.064539  0.049079  0.062542  -3.01433 
6  3  R  0.795337  0.028705  -0.22899  -3.55068 
6  4  R  0.922567  0.035194  -0.0806  -3.34689 
6  5  R  0.780047  0.034144  -0.2484  -3.37716 
6  6  R  1.415222  0.066506  0.347286  -2.71046 
1251   
1252   
1253  SAS Output 
1254    
SWR  LPOINTEST TESTMEAN REFMEAN POINTEST  UB  L  U 
0.50242  0.096445  1.11571  1.01313  1.10125  -0.022242 0.80470 1.50708 
1255   
1256  The estimated within-donor standard deviation of the RS is 0.5024, which is greater than 0.294. 
1257  Using the SABE approach, the 95% upper confidence bound is -0.0222 < 0 and the point 
1258  estimate of 1.1013 is within the BE limit of [0.8000, 1.2500]. Thus, BE for AMT can be 
1259  concluded. The BE test for J  can be performed similarly. 
max
1260   
1261  (b) Unbalanced Data 
1262   
1263  The simulated data set Data-Unbalanced.csv shown below provides an example of IVPT study 
1264  outcomes when the data is unbalanced. The SAS output and the determination of BE for LAMT 
1265  in this data set follows. 
1266   
1267  Data-Unbalanced.csv 
donor  replicate  treat  AMT  Jmax  LAMT  LJmax 
1  1  T  2.361749  0.081326  0.859402  -2.50929 
1  2  T  0.916571  0.041008  -0.08712  -3.19398 
1  3  T  1.246243  0.038537  0.220133  -3.25613 
1  4  T  0.890018  0.04296  -0.11651  -3.14747 
1  5  T  0.663551  0.031219  -0.41015  -3.46674 
1  6  T  0.479143  0.015747  -0.73576  -4.15108 
2  1  T  0.998845  0.030073  -0.00116  -3.50412 
2  2  T  0.814457  0.061644  -0.20523  -2.78637 
2  3  T  0.648741  0.019984  -0.43272  -3.91283 
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2  4  T  0.767291  0.028453  -0.26489  -3.55951 
2  5  T  1.392406  0.166782  0.331033  -1.79107 
3  1  T  0.45757  0.02184  -0.78182  -3.82402 
3  2  T  1.377438  0.0651  0.320226  -2.73182 
3  3  T  2.170988  0.073199  0.775183  -2.61457 
3  4  T  1.753523  0.067281  0.561627  -2.69888 
3  5  T  0.995674  0.116414  -0.00434  -2.15061 
4  1  T  0.811458  0.053465  -0.20892  -2.92872 
4  2  T  0.913538  0.060217  -0.09043  -2.8098 
4  3  T  1.251438  0.083596  0.224293  -2.48176 
4  4  T  1.163818  0.054213  0.151706  -2.91483 
4  5  T  1.027813  0.065446  0.027433  -2.72653 
4  6  T  1.081988  0.062279  0.078801  -2.77614 
5  1  T  1.275517  0.069859  0.243352  -2.66128 
5  2  T  1.231649  0.051342  0.208354  -2.96924 
5  3  T  2.454324  0.161016  0.897852  -1.82625 
5  4  T  1.195989  0.064734  0.178973  -2.73746 
5  5  T  2.07678  0.088355  0.730819  -2.42639 
5  6  T  1.893399  0.093223  0.638374  -2.37276 
6  1  T  1.564164  0.137378  0.447352  -1.98502 
6  2  T  1.049724  0.064531  0.048527  -2.74061 
6  3  T  1.047633  0.043859  0.046533  -3.12676 
6  4  T  1.159634  0.09236  0.148105  -2.38206 
1  1  R  1.598636  0.04239  0.469151  -3.16084 
1  2  R  2.24476  0.117486  0.808599  -2.14143 
1  3  R  1.60912  0.044199  0.475687  -3.11906 
1  4  R  1.8834  0.066452  0.633079  -2.71127 
1  5  R  1.101948  0.031705  0.097079  -3.45129 
1  6  R  1.165342  0.034002  0.153015  -3.38133 
2  1  R  0.622369  0.052794  -0.47422  -2.94135 
2  2  R  0.833337  0.033419  -0.18232  -3.39863 
2  3  R  0.386763  0.029507  -0.94994  -3.52311 
2  4  R  0.851759  0.03968  -0.16045  -3.2269 
2  5  R  0.677715  0.032379  -0.38903  -3.43024 
3  1  R  0.838346  0.101628  -0.17632  -2.28643 
3  2  R  0.130884  0.008774  -2.03344  -4.73601 
3  3  R  0.635926  0.039118  -0.45267  -3.24118 
3  4  R  0.804131  0.114582  -0.21799  -2.16646 
3  5  R  2.324877  0.229704  0.843667  -1.47096 
4  1  R  1.694799  0.088825  0.527564  -2.42109 
4  2  R  0.977661  0.030392  -0.02259  -3.49356 
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4  3  R  3.13529  0.217896  1.142722  -1.52374 
4  4  R  0.922805  0.040161  -0.08034  -3.21485 
4  5  R  1.504834  0.082443  0.408683  -2.49565 
4  6  R  1.330167  0.055237  0.285305  -2.89612 
5  1  R  2.104036  0.101673  0.743858  -2.28599 
5  2  R  0.842231  0.094771  -0.1717  -2.35629 
5  3  R  0.985656  0.081963  -0.01445  -2.50148 
5  4  R  0.931461  0.069496  -0.071  -2.66648 
5  5  R  1.580578  0.059193  0.45779  -2.82695 
5  6  R  1.209059  0.067989  0.189842  -2.68841 
6  1  R  1.038591  0.037859  0.037865  -3.27389 
6  2  R  0.795337  0.028705  -0.22899  -3.55068 
6  3  R  0.922567  0.035194  -0.0806  -3.34689 
6  4  R  0.780047  0.034144  -0.2484  -3.37716 
6  5  R  1.415222  0.066506  0.347286  -2.71046 
1268   
1269   
1270  SAS Output 
1271   
SWR  LPOINTEST TESTMEAN REFMEAN POINTEST  UB  L  U 
0.50651  0.067494  1.10723  1.03497  1.06982  -0.10907 0.87627 1.30613 
1272   
1273  The estimated within-donor standard deviation of the RS is 0.5065, which is greater than 0.294. 
1274  Using the SABE approach, the 95% upper confidence bound is -0.1091 < 0 and the point 
1275  estimate of 1.0698 is within the BE limit of [0.8000, 1.2500]. Thus, BE for AMT can be 
1276  concluded. The BE test for J  can be performed similarly. 
max
1277   
1278   
1279  APPENDIX III (EXAMPLE R CODE)  
1280   
1281  This section provides an example of R code that performs the same calculations as the SAS code 
1282  in Appendix I. 
1283   
 
## INPUT 
 
# DAT = a data frame 
# DON = column name of donor variable in DAT (donor variable: numeric) 
# REPS = column name of replicate variable in DAT 
#        (replicate variable: numeric) 
# TRT = column name of treatment variable in DAT 
#        (treatment variable: "T", "R") 
# LY = column name of log-transformed endpoint in DAT 
 
 
## OUTPUT 
  38  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
# BU = balanced data or unbalanced data 
# Swr = estimated within-donor standard deviation of reference 
# Ihat = point estimate for mean difference in log scale 
# testMean = test mean estimate in original scale 
# refMean = reference mean estimate in original scale 
# pointest = point estimate transformed back to original scale 
# UB = SABE 95% upper confidence bound 
# CI = ABE 90% CI for mean difference transformed back to original scale 
 
ivpt <- function(DAT, DON, REPS, TRT, LY){ 
   
  # Remove missing values before analysis 
  DAT <- DAT[!is.na(DAT[[LY]]),] 
   
  # If zero values in AMT or Jmax are not imputed, 
  # remove them to avoid a computational burden 
  DAT <- DAT[!(DAT[[LY]]==-Inf),] 
   
  # Create the data sets for test & reference 
  tdat <- DAT[DAT[[TRT]]=="T",] 
  rdat <- DAT[DAT[[TRT]]=="R",] 
   
  # Sort tdat and rdat by don and reps 
  ii1 <- order(tdat[[DON]], tdat[[REPS]]) 
  tdat <- tdat[ii1,] 
  ii2 <- order(rdat[[DON]], rdat[[REPS]]) 
  rdat <- rdat[ii2,] 
   
  # Determine the numbers of replicates from each donor for T & R 
  rT <- as.vector(table(tdat[[DON]])) 
  rR <- as.vector(table(rdat[[DON]])) 
   
  nd <- length(unique(tdat[[DON]])) # the number of donors 
  rstar <- sum(rR)        # the total number of replicates in R group 
  nr <- unique(c(rT, rR)) # This is length of 1 when data is balanced 
   
  # Set m and alpha 
  m <- 1.2500 
  alpha <- 0.05 
  theta <- (log(m)/0.25)^2 
   
  if(length(nr)==1){   # if the data is balanced 
     
    BU <- "Balanced" 
     
    # Determine SWR 
    mRef <- tapply(rdat[[LY]], rdat[[DON]], mean, na.rm=TRUE) 
    vv <- tapply(rdat[[LY]], rdat[[DON]], var, na.rm=TRUE) 
    Swr2 <- sum(vv) / nd 
    Swr <- sqrt(Swr2) 
     
    mTest <- tapply(tdat[[LY]], tdat[[DON]], mean, na.rm=TRUE) 
    Ij <- mTest - mRef 
    Ihat <- mean(Ij) 
    SI2 <- var(Ij, na.rm=TRUE) 
     
  39  Contains Nonbinding Recommendations 
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    # Treatment means 
    testMean <- exp(mean(mTest)) 
    refMean <- exp(mean(mRef)) 
     
    # SABE for balanced data 
    X <- Ihat^2 - SI2 / nd 
    Y <- - theta * Swr2 
    Xp <- ( abs(Ihat) + qt(1-alpha, nd-1) * sqrt(SI2/nd) )^2 
    Yp <- - theta*(nr-1)*nd*Swr2 / qchisq(1-alpha, (nr-1)*nd) 
    V <- sign(Xp-X)*(Xp-X)^2 + sign(Yp-Y)*(Yp-Y)^2 
    UB <- X + Y + sign(V)*sqrt(abs(V)) 
     
    # ABE for balanced data 
    se <- sqrt(SI2/nd) 
    L <- Ihat - qt(1-alpha, nd-1)*se 
    U <- Ihat + qt(1-alpha, nd-1)*se 
     
  }else{              # if the data is unbalanced 
     
    BU <- "Unbalanced" 
     
    # Determine SWR 
    mRef <- tapply(rdat[[LY]], rdat[[DON]], mean, na.rm=TRUE) 
    vv <- sum( (rdat[[LY]] - rep(mRef, times=rR))^2 ) 
    Swr2 <- vv / (rstar - nd) 
    Swr <- sqrt(Swr2) 
     
    # Estimate the mean difference 
    DAT[[DON]] <- factor(DAT[[DON]]) 
    f <- as.formula(paste(LY, "~", DON, "+", TRT)) 
    obj <- lm(f, data = DAT) 
    tname <- paste0(TRT, "T") 
    tcoef <- summary(obj)$coef[rownames(summary(obj)$coef)==tname,] 
    Ihat <- as.numeric(tcoef[1]) 
    se <- as.numeric(tcoef[2]) 
    dfstar <- summary(obj)$df[2] 
     
    # Treatment means 
    udon <- unique(DAT[[DON]]) 
    newdat1 <- data.frame(DON = udon, TRT = rep("T", length(udon))) 
    newdat2 <- data.frame(DON = udon, TRT = rep("R", length(udon))) 
    colnames(newdat1) <- c(DON, TRT) 
    colnames(newdat2) <- c(DON, TRT) 
    testMean <- exp(mean(predict(obj, newdata = newdat1))) 
    refMean <- exp(mean(predict(obj, newdata = newdat2))) 
     
    # SABE for unbalanced data 
    X <- Ihat^2 - se^2 
    Y <- - theta * Swr2 
    Xp <- ( abs(Ihat) + qt(1-alpha, dfstar)*se )^2 
    Yp <- - theta*(rstar-nd)*Swr2 / qchisq(1-alpha, rstar-nd) 
    V <- sign(Xp-X)*(Xp-X)^2 + sign(Yp-Y)*(Yp-Y)^2 
    UB <- X + Y + sign(V)*sqrt(abs(V)) 
     
    # ABE for unbalanced data 
    L <- Ihat - qt(1-alpha, dfstar)*se 
    U <- Ihat + qt(1-alpha, dfstar)*se 
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  } 
   
  result <- list(BU=BU, Swr=Swr, Ihat=Ihat,  
                 testMean=testMean, refMean=refMean, pointest=exp(Ihat),  
                 UB=UB, CI=exp(c(L, U))) 
   
  return(result) 
} 
 
bdat <- read.csv("Data-Balanced.csv") 
res1 <- ivpt(bdat, "donor", "replicate", "treat", "LAMT") 
 
udat <- read.csv("Data-Unbalanced.csv") 
res2 <- ivpt(udat, "donor", "replicate", "treat", "LAMT") 
 
1284   
1285   
  41