Topical Dermatologic 
Corticosteroids:  
In Vivo Bioequivalence 
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DRAFT GUIDANCE 
 
  This guidance document is being distributed for comment purposes only.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publication in the Federal Register of the notice announcing the availability of the draft 
guidance.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 240-402-7936.  
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
October 2023 
Generic Drugs 
Revision 1 
 
  Topical Dermatologic 
Corticosteroids:  
In Vivo Bioequivalence 
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/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
 October 2023 
Generic Drugs 
Revision 1 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
 
II.  BACKGROUND ............................................................................................................... 2 
III.  PHARMACODYNAMIC VASOCONSTRICTOR STUDIES ..................................... 3 
A.  Vasoconstrictor Method Qualification ......................................................................................... 3 
1. Chromameter Qualification ............................................................................................................. 4 
2. Operator Qualification .................................................................................................................... 4 
B.  Dose Duration-Response Model .................................................................................................... 4 
C.  Study Design ................................................................................................................................... 6 
1. Pilot Study ........................................................................................................................................ 6 
2. Pivotal Study .................................................................................................................................... 6 
D.  Subject Inclusion Criteria ............................................................................................................. 7 
E.  Subject Exclusion Criteria ............................................................................................................ 7 
F.  Subject Screening for Response .................................................................................................... 8 
G.  Occlusion Versus Nonocclusion .................................................................................................... 8 
H.  Methods of Application and Removal .......................................................................................... 9 
I.  Study Day Activities and Restrictions .......................................................................................... 9 
1. Pilot Study ........................................................................................................................................ 9 
2. Pivotal Study .................................................................................................................................. 11 
J.  Data Analyses and Pharmacodynamic Modeling ..................................................................... 12 
1. AUEC Calculation for the Pilot and Pivotal Studies ..................................................................... 12 
2. Pharmacodynamic Modeling for the Pilot Study ........................................................................... 12 
3. Data Analysis for the Pivotal Study ............................................................................................... 13 
4. Formatted Data Submission .......................................................................................................... 14 
APPENDIX I: SCHEMATIC FOR STAGGERED APPLICATION WITH 
SYNCHRONIZED REMOVAL FOR PILOT STUDY PROTOCOLS ................................ 15 
APPENDIX II: EXAMPLE FOR SKIN BLANCHING STUDY DESIGN FOR 
PILOT DOSE-DURATION RESPONSE STUDY .......................................................... 16 
APPENDIX III: CALCULATION OF AUEC ......................................................................... 17 
APPENDIX IV: EMAX MODELS ............................................................................................ 18 
APPENDIX V: LOCKE METHOD FOR BIOEQUIVALENCE ASSESSMENT AND 
A WORKED EXAMPLE ......................................................................................................... 19 
 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1  Topical Dermatologic Corticosteroids: In Vivo Bioequivalence 
2  Guidance for Industry1 
3   
4  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
5  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
6  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
7  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
8  for this guidance as listed on the title page.   
9   
10   
11  I.  INTRODUCTION  
12   
13  This guidance is intended to assist applicants who submit abbreviated new drug applications 
14  (ANDAs) for topical dermatologic corticosteroid products of all potency groups2, hereinafter 
15  referred to as topical corticosteroids. This guidance describes recommendations for in vivo 
16  studies to demonstrate the bioequivalence of topical corticosteroids.  
17   
18  When finalized, this guidance will replace the guidance for industry Topical Dermatologic 
19  Corticosteroids: In Vivo Bioequivalence that was issued in June 1995.3  Revising this guidance 
20  will provide clarity for potential ANDA applicants on the appropriate pilot and pivotal studies 
21  and other recommendations for pharmacodynamic approach to assess the bioequivalence of 
22  topical dermatologic corticosteroids.  These recommendations have evolved since the original 
23  guidance was issued in 1995.    
24   
25  This guidance provides recommendations for the study design, method qualification, data 
26  analysis, and data reporting for the pilot dose-duration vasoconstrictor response study and pivotal 
27  vasoconstrictor bioequivalence study used to demonstrate bioequivalence of topical 
28  corticosteroids. The guidance also discusses considerations and approaches for estimating key 
29  study parameters (e.g., dose corresponding to half the maximal vasoconstrictor response (ED50)) 
30  and sample size for the pivotal vasoconstrictor bioequivalence study). 
31   
32  In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
33  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
34  as recommendations, unless specific regulatory or statutory requirements are cited. The use of 
 
1 This guidance has been prepared by the Office of Generic Drugs in consultation with the Office of Clinical 
Pharmacology in the Center for Drug Evaluation and Research at the U.S. Food and Drug Administration.  You may 
submit comments on this guidance at any time.  Submit comments to Docket No. FDA-2017-D-6821 (available at 
https://www.regulations.gov/docket?D=FDA-2017-D-6821).  See the instructions in that docket for submitting 
comments on this and other Level 2 guidances. 
 
2 The potency of topical corticosteroids is the amount of drug needed to produce a desired therapeutic effect. The 
vasoconstrictor assay could be used to determine potency. 
 
3 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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35  the word should in Agency guidances means that something is suggested or recommended, 
36  but not required. 
37   
38  II.  BACKGROUND 
39   
40  The Federal Food, Drug, and Cosmetic Act (FD&C Act) generally requires an ANDA to contain, 
41  among other things, information to show that the proposed generic drug product (test product) is 
42  bioequivalent to its reference listed drug (RLD).4 Bioequivalence is the absence of a significant 
43  difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical 
44  equivalents or pharmaceutical alternatives becomes available at the site of drug action when 
45  administered at the same molar dose under similar conditions in an appropriately designed 
46  study.5 
47   
48  This guidance describes an in vivo pharmacodynamic approach to demonstrate the 
49  bioequivalence of topical corticosteroids. Topical corticosteroids are known to cause 
50  vasoconstriction of the dermal vasculature that produces the pharmacodynamic effect of skin 
51  blanching. The magnitude of blanching (change in skin color) depends upon the potency of the 
52  corticosteroid, and it increases relative to the amount of the corticosteroid permeating into the 
53  skin, when study parameters are suitably controlled. Thus, the pharmacodynamic vasoconstrictor 
54  response can be a surrogate measure of the rate and extent to which a topical corticosteroid 
55  becomes available at the site of action in the skin.  
56   
57  A pilot vasoconstrictor study is routinely performed to define appropriate parameters for a 
58  pivotal vasoconstrictor study used to support a demonstration of bioequivalence between a test 
59  topical corticosteroid and its reference standard, which ordinarily is the RLD. Therefore, this 
60  guidance recommends that ANDA applicants who propose to use an in vivo pharmacodynamic 
61  approach to demonstrate bioequivalence between a test topical corticosteroid and its reference 
62  standard conduct two in vivo vasoconstrictor studies: (1) a pilot dose-duration vasoconstrictor 
63  response study, using the reference standard; and (2) a pivotal vasoconstrictor bioequivalence 
64  study, comparing the test topical corticosteroid and reference standard. The proposed 
65  methodology, including the study design, model selection, and model optimization for the pilot 
66  dose-duration vasoconstrictor response study, and the statistical method for the pivotal 
67  vasoconstrictor bioequivalence study are discussed in more detail in subsequent sections of this 
68  guidance.  
69   
70  The purpose of the pilot dose duration vasoconstrictor response study (or pilot vasoconstrictor 
71  study or pilot study) is to determine the dose duration-response relationship of the topical 
72  corticosteroid to be studied in the pivotal vasoconstrictor bioequivalence study. The results of the 
73  pilot vasoconstrictor study provide the dose duration-response information necessary to 
 
4 See section 505(j)(2)(A), (j)(2)(C), and (j)(4) of the FD&C Act (21 U.S.C. 355(j)(2)(A), (j)(2)(C), and (j)(4)); see 
also 21 CFR 314.94. Bioequivalence to the RLD may be demonstrated via comparative assessments of the test 
product to the designated reference standard (RS). See, e.g.,  314.3(b) (21 CFR 314.3(b)) (defining reference 
standard). 
5  314.3(b) (defining bioequivalence); see also section 505(j)(8)(B) of the FD&C Act (describing when a drug shall 
be considered to be bioequivalent to a listed drug); see also 21 CFR 320.23(b). 
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74  determine the parameters ED , D , and D 6 to be used in the prospective applicants pivotal 
50 1 2
75  vasoconstrictor bioequivalence study. Development and validation of a suitably sensitive and 
76  discriminating region of a dose duration-response standard curve is essential to estimate ED , 
50
77  D , and D  for a vasoconstrictor response. This approach is analogous to using a standard curve 
1 2
78  to characterize the linearity, range, and limits of quantification for a bioanalytical method for a 
79  drug in a biological fluid. The pivotal study should be performed under the same conditions as 
80  the pilot study for each topical corticosteroid under investigation.     
81   
82  The purpose of the pivotal vasoconstrictor study is to demonstrate bioequivalence of the test 
83  product to the RLD using an in vivo approach. Alternatively, an in vitro characterization-based 
84  approach to establish the bioequivalence of a topical corticosteroid product may be acceptable 
85  when the proposed generic formulation contains no difference in inactive ingredients or in other 
86  aspects of the formulation relative to the RLD that may significantly affect the local or systemic 
87  availability of the active ingredient(s). Prospective applicants are encouraged to submit a 
88  controlled correspondence, if appropriate, or to request a product development meeting for 
89  relevant complex products that may be submitted in an ANDA to discuss specific scientific 
90  issues or questions (e.g., a proposed study design or issues related to method qualification, dose 
91  duration-response, or other aspects of a pilot dose duration-response study before conducting the 
92  pivotal vasoconstrictor study), or to discuss an alternative bioequivalence approach (e.g., a 
93  characterization-based approach).7,8 An applicant must submit with their ANDA a complete 
94  study report for the bioequivalence study upon which the ANDA relies for approval.9  
95   
96  III.  PHARMACODYNAMIC VASOCONSTRICTOR STUDIES  
97   
98  A.  Vasoconstrictor Method Qualification 
99   
100  The chromameter is the apparatus most commonly used to measure the pharmacodynamic skin 
101  blanching response induced following the application of topical corticosteroids. Prior to 
102  collecting data for vasoconstrictor studies, the chromameter should be calibrated and qualified 
103  for its intended use. In addition, the repeatability and ruggedness10 of chromameter 
104  measurements by different operators should be qualified. These qualifications should be 
 
6 ED : half of the maximal vasoconstrictor response; D : the dose duration equal to approximately 0.5 times the 
50 1
population ED ; and D the dose duration equal to approximately 2 times the population ED for the simple E  
50 2:  50  max
model used.  
7 See the guidance for industry Formal Meetings Between FDA and ANDA Applicants of Complex Products Under 
GDUFA (November 2020) for more information on product development meetings.  
8 See also the draft guidances for industry Physicochemical and Structural (Q3) Characterization of Topical Drug 
Products Submitted in ANDAs (October 2022), In Vitro Release Test Studies for Topical Drug Products Submitted 
in ANDAs (October 2022), and In Vitro Permeation Test Studies for Topical Drug Products Submitted in ANDAs 
(October 2022). When final, these guidances will represent FDAs current thinking on these topics. 
9 21 CFR 314.94(a)(7). 
10 Repeatability expresses the precision under the same operating conditions over a short interval of time. 
Ruggedness is the reproducibility of the method under a variety of normal, but variable, test conditions. Variable 
conditions might include different machines, operators, and reagent lots. Ruggedness provides an estimate of 
experimental reproducibility with unavoidable error.    
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105  completed before the start of a study. If studies have multiple groups, method qualifications 
106  should be performed, at a minimum, before the start date of the first group. 
107   
108  1.  Chromameter Qualification 
109   
110  Chromameter qualification is conducted with calibrated chromameters to support a 
111  demonstration of the ruggedness of the chromameter measurements across multiple chromameter 
112  units. Multiple chromameter units can be set up to measure the vasoconstrictor response in both 
113  the pilot dose-duration vasoconstrictor response study and the pivotal vasoconstrictor 
114  bioequivalence study. All chromameters used in these pilot and pivotal vasoconstrictor studies 
115  should be reported with their specific identification numbers and qualified to ensure consistent 
116  performance in study data collection. Chromameter qualification should be performed on all 
117  chromameters planned to be used in pilot and pivotal vasoconstrictor studies using one operator, 
118  one subject, and, with at least four readings each at one designated skin site. Intra-chromameter 
119  variability is calculated as the variability within multiple readings at one skin site by one 
120  operator using one chromameter. Inter-chromameter variability is calculated as the variability in 
121  readings between different chromameters, with the mean value of multiple readings from each 
122  chromameter at one skin site by one operator. The chromameter qualification should be repeated 
123  with at least four study subjects, using at least four skin sites in each study subject to demonstrate 
124  the reproducibility of the chromameter measurements. To determine procedure consistency 
125  between and within chromameters, the variability (% coefficient of variation (CV)) for the intra-
126  chromameter and the inter-chromameter measurements should be not more than 15% in each and 
127  every subject. 
128   
129  2.  Operator Qualification 
130   
131  Operator qualification is conducted to support a demonstration of the ruggedness of the 
132  chromameter measurements across multiple operators. The operators who conduct pilot and 
133  pivotal vasoconstrictor studies should be reported with their specific identification numbers or 
134  names and qualified to ensure that each one is operating the chromameters and measuring the 
135  skin response consistently. Operator qualification should be performed by multiple operators 
136  using one chromameter on one subject with at least four readings each at one designated skin 
137  site. Intra-operator variability is calculated as the variability within multiple readings by one 
138  operator using one chromameter at one skin site. Inter-operator variability is calculated as the 
139  variability between different operators, with the mean value of multiple readings from each 
140  operator, using one chromameter at one skin site of the same subject. The operator qualification 
141  should be repeated with at least four study subjects, with at least four skin sites in each study 
142  subject, to support a demonstration of method reproducibility. To determine procedure 
143  consistency between and within operators, the variability (CV) for the intra-operator and the 
144  inter-operator measurements should be not more than 15% in each and every subject. 
145   
146  B.  Dose Duration-Response Model 
147   
148  The conditions under which the pivotal vasoconstrictor bioequivalence study is performed 
149  should be optimized to assure that the test topical corticosteroid and reference standard are 
150  compared in the sensitive (steep) portion of the response curve, where the vasoconstrictor 
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151  response would be sensitive and discriminating to differences in the bioavailability of the 
152  corticosteroid between the test and reference standard. Development of a dose duration- response 
153  relationship for a topical corticosteroid relies on consistent administration of a predetermined 
154  dose of the drug product to the skin. Development of a dose duration-response relationship for a 
155  topical corticosteroid will identify the sensitive dose duration-response region to support pivotal 
156  study design. The time course of the response should be measured until it returns to baseline to 
157  ensure that at each dose duration, the maximal pharmacodynamic response is observed.  
158   
159  To identify the sensitive and discriminating region of the dose duration-response curve for the 
160  pharmacodynamic skin blanching effect, it is useful to (1) produce conditions that are expected 
161  to deliver increasing amounts of a corticosteroid drug into the skin (a practical way to modulate 
162  the amount of drug (corticosteroid) delivered into the skin is to dose the fixed amount of topical 
163  corticosteroid product on the skin for progressively increasing dose durations), and (2) measure 
164  the resulting skin blanching effect caused by dermal vasoconstriction. 
165   
166  Although various models are available to express a relationship between drug dose and 
167  pharmacodynamic effect, the Agency recommends use of the E  model below to describe the 
max
168  dose duration-response of topical corticosteroids, which describes the measure of effect (E) in 
169  terms of a baseline effect (E ), a maximal effect (E ) and a dose duration (D) at ED : 
0 max 50
170   
171   
EEmmmmmm DD
172     EE = EE0 +
173  Alternative models can be used, with justificationEEs DDa5n0d +apDDpropriate model selection procedures, 
174  if a prospective applicant finds the above E  model is not appropriate (see Appendix IV). 
max
175  Prospective applicants should justify their selected E  model and are encouraged to use the 
max
176  pharmacodynamic vasoconstrictor study data to support the dose duration selection from a dose 
177  duration-response model for population estimation. In the population dose duration-response 
178  model, both fixed effect and/or random effect for E  and ED  can be considered. The type of 
max 50
179  model parameter distribution assumption (normal or log-normal) for E  and ED  parameters 
max 50
180  within the population analysis should be specified. Prospective applicants should describe their 
181  model optimization procedures and provide the rationale for ED  selection in the pre-ANDA 
50
182  meeting request or ANDA submission. Some aspects of model optimization that are 
183  recommended to be included are provided below: 
184    
185    E  model selection 
max
186    Estimation methods comparison 
187    Model parameter selection 
188    Error models selection 
189    Initial estimates procedure11 
190   
191  The in vivo vasoconstrictor response (detected as skin blanching) generally approaches a 
192  maximum when the dermal vasculature is not able to vasoconstrict further. At relatively high 
 
11 For detailed modeling procedures, refer to the guidance for industry Population Pharmacokinetics (February 
2022).  
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193  strengths for highly potent topical corticosteroids, there may be a diminishing change in the 
194  vasoconstrictor response to increases in dose duration (flattening the response curve at the upper 
195  end). Conversely, at relatively low strengths for low potency topical corticosteroids, it may be 
196  challenging to elicit a vasoconstrictor response despite increases in dose duration (flattening the 
197  response curve at the lower end). Therefore, a prospective applicant should design the pilot 
198  vasoconstrictor study to cover a full dose duration-response curve appropriately according to the 
199  potency of topical corticosteroids, and hence improve the dose duration-response model.  
200   
201  C.  Study Design  
202   
203  1.  Pilot Study 
204   
205    This dose duration-response study should be based on the reference standard only, with 
206  randomization of dose-duration skin sites. 
207   
208    Untreated control sites on each arm should be used to enable correction of active drug 
209  skin sites for color changes during the study unrelated to drug exposure. Because the 
210  vehicle corresponding to the reference standard is not generally available, untreated 
211  control sites refer to untreated areas of skin, not to areas of skin to which vehicle has been 
212  applied. 
213   
214    Dose durations (e.g., from 0.25 to 6.0 hours) should be designed properly to explore the 
215  dose duration-response relationship and to determine the appropriate dose duration for 
216  the pivotal study. Pharmacodynamic responses are measured in terms of area under the 
217  effect curve (AUEC) by readings of a chromameter at the end of each dose duration after 
218  the removal of residual topical corticosteroid. 
219   
220    Dose duration-response data should be modeled using a nonlinear mixed effect modeling 
221  method to determine the population ED  value, which will serve as the approximate dose 
50
222  duration for the pivotal vasoconstrictor study. 
223   
224    A minimum of twelve subjects is recommended.  
225   
226  2.  Pivotal Study 
227   
228    This pharmacodynamic bioequivalence study uses replicates of single dose duration of 
229  test topical corticosteroid and reference standard based on the population ED  identified 
50
230  in the pilot study. Also, the replicates of each of the dose durations (D  and D ) of the 
1 2
231  reference standard should be included in the pivotal study. 
232   
233    For a bioequivalence analysis, selection of an individual subject is based upon an 
234  acceptable ratio of mean reference AUEC at D  over mean reference AUEC at D  for 
2 1
235  each subject. The minimum value of the ratio should be 1.25 and both mean AUEC 
236  values at D and D are negative,12 if simple E  model is proposed. However, other 
1  2  max
 
12 Refer to section J.1.(b) for AUEC calculation 
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237  values for the ratio can be used with justification depending on the selected dose 
238  duration-response model. The individual subject who meets this dose duration-response 
239  criterion (under conditions when both mean D and D values are negative) is defined as a 
1  2 
240  detector (i.e., evaluable subject). 
241   
242    It is the applicants responsibility to design an adequately powered pivotal 
243  bioequivalence study. It is recommended that applicants enroll a sufficient number of 
244  subjects to yield a number of detectors sufficient to power the study. When determining 
245  the sample size of enrolled subjects, dropouts and estimated required number of detectors 
246  should be taken into consideration. Based on observations from studies submitted in 
247  ANDAs, forty or more detectors are generally used for the pivotal study. The sample size 
248  determination for the pivotal study should be prespecified in the protocol and justified. 
249  Sufficient subjects should be recruited, randomized with respect to dose duration skin 
250  site, and dosed at the beginning of the study to ensure that the desired number of 
251  detectors will be available for analysis. All detectors should be included in the analysis. 
252   
253  D.  Subject Inclusion Criteria 
254   
255    Males and non-pregnant, non-lactating females, general population. 
256   
257    Subjects demonstrating adequate vasoconstrictor response to the reference standard. 
258  (Refer to section F for subject screening for response). 
259   
260    Willing to shower using the same soap/cleansers throughout the study (Screening Visit 
261  through study completion). 
262   
263    Willing to follow study restrictions. (Refer to section I.1.(c)-(f)). 
264   
265  E.  Subject Exclusion Criteria  
266   
267    Clinically significant hypertension or circulatory disease.  
268   
269    Smoking within one week of study. 
270   
271    Caffeine intake greater than 500 mg per day prior to or during the study.  Coffee, tea, and 
272  energy drinks should all be considered as important caffeine sources. 
273   
274    Clinically significant history of alcoholism or drug abuse. 
275   
276    Use of topical dermatologic drug therapy (either as therapy or participation in the clinical 
277  study) on ventral forearms within one month prior to the study. 
278   
279    Adverse reactions to topical or systemic corticosteroids. 
280   
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281    Any current or past medical condition, including active dermatitis or any other 
282  dermatologic condition, which might significantly affect the pharmacodynamic response 
283  to the administered drug. 
284   
285    Would require shaving ventral forearms to ensure consistent dosing on the skin surface.  
286    Use of any vasoactive (constrictor or dilator) medication (prescription or over-the-
287  counter) that could modulate blood flow. Examples of such drugs include nitroglycerin, 
288  antihypertensives, antihistamines, nonsteroidal anti-inflammatory drugs, aspirin, and 
289  over-the-counter cough/cold products containing antihistamines and/or either 
290  phenylpropanolamine or phentolamine. 
291   
292    Any obvious difference in skin color between arms. 
293   
294  F.  Subject Screening for Response  
295   
296  In this guidance, a responder is defined as a subject who shows the skin blanching 
297  vasoconstriction response to a single-dose duration of the corresponding reference standard 
298  under the same occlusive or non-occlusive conditions used in the pilot and pivotal 
299  vasoconstrictor studies. Quantification of skin blanching in the pilot and pivotal vasoconstrictor 
300  studies by a chromameter is considered to be the most satisfactory response measurement. 
301  However, responder status may be based on visual readings with the discrete multiple unit scale 
302  (0 - 3 or 0  4). A dose duration of 4 hours or 6 hours is suggested, with skin blanching 
303  assessment 2 hours following drug product removal. A responder shows a visual reading of at 
304  least one unit. 
305   
306  Inclusion of nonresponders reduces the ability of a study to detect true differences between the 
307  test topical corticosteroid and reference standard, should they exist. Therefore, for both the pilot 
308  dose duration-response study and the pivotal bioequivalence study, only responders should be 
309  included for the enrollment. 
310   
311  To conserve skin sites on the ventral forearm for use in the dose duration-response study or 
312  bioequivalence study, responder status may be based on studies conducted at sites other than the 
313  forearm (e.g., upper arm). 
314   
315  Criteria for identification of responders, including dose duration, magnitude of response, and 
316  skin site tested, should be included in the study report. 
317   
318  G.  Occlusion Versus Nonocclusion  
319   
320  When use of occlusion is allowed in the label of the specific reference standard, the pilot dose 
321  duration-response vasoconstrictor study and pivotal vasoconstrictor (bioequivalence) study may 
322  be conducted using a non-absorbent occlusive film. Occlusion may be appropriate only for the 
323  lower potency products in the vasoconstrictor study. Caution is recommended, as observations 
324  from pilot studies data suggest that the ED (the dose duration to be used in the pivotal study) 
50 
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325  decreases with increasing topical corticosteroid product potency.13 Evaluation of dose duration-
326  response requires dose duration data at some time (i.e., D ) less than the ED . Very short dose 
1 50
327  durations are difficult to conduct experimentally and tend to produce high variability in response. 
328  If occlusion is used for the pilot vasoconstrictor study, it should also be used for the pivotal 
329  vasoconstrictor study. 
330   
331  H.  Methods of Application and Removal  
332   
333  Staggered application with synchronized removal (i.e., the topical corticosteroid is applied to 
334  skin sites at different times, and removed at the same time) could be utilized in the pilot and 
335  pivotal vasoconstrictor studies (see Appendix I).  
336   
337  I.  Study Day Activities and Restrictions 
338   
339  1.  Pilot Study 
340   
341  a)  Subjects should begin the study sessions at approximately the same time (within 
342  one hour) each study day. 
343   
344  b)  Verification by history of adequate washout of excluded drugs that could 
345  modulate blood flow (constrictor or dilator). 
346   
347  c)  No exercise with either arm, and no strenuous exercise overall, for duration of 
348  study session. 
349   
350  d)  No bathing or showering during the periods of drug application and assessment of 
351  skin blanching. 
352   
353  e)  No use of creams, emollients, or similar products on forearms for 24 hours prior 
354  to, and throughout, the study. 
355   
356  f)  The forearms should be free of any dirt or particulate matter that would interfere 
357  with proper drug application or the assessment of a pharmacodynamic response. 
358  Cleansing of the skin is not encouraged because of the possible effects on drug uptake 
359  and the pharmacodynamic response to the drug product. If necessary, cleansing 
360  should be performed not less than 2 hours before drug product application. If 
361  cleansing is performed, this should be noted in the study report. 
362   
363  g)  Whether the study is conducted using occlusion or under non-occlusive 
364  conditions, the use of a protective, non-occlusive guard is recommended to prevent 
365  smearing or removal of the topical corticosteroid from the skin site. Care should be 
366  taken to avoid contact between the guard and the topical corticosteroid to prevent 
367  inadvertent contamination of untreated control sites or other test sites. 
368   
 
13Singh GJP, W P Adams, Lesko LJ, Shah VP, et al. Development of in vivo bioequivalence methodology for 
dermatologic corticosteroids based on pharmacodynamic modeling; Clin Pharmacol Ther 1999 Oct, 66(4): 346-57. 
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369  h)  Skin sites should be no closer than 34 cm to the antecubital fossa or to the wrist. 
370   
371  i)  The reference standard should be applied to skin sites of identical surface area on 
372  the ventral forearms. Suggested dose durations for the pilot study are 0.25, 0.5, 0.75, 
373  1, 1.5, 2, 4 and 6 hours, but may vary depending on the topical corticosteroid under 
374  investigation. 
375   
376  j)  Eight dose durations, i.e., active drug sites, should be equally divided between the 
377  two arms. 
378   
379  k)  Amount of drug product, skin site size, and spacing between sites should be 
380  determined prior to the initiation of the study. For example, investigators may use 
381  doses of 5-12 microliters (L) of formulation per centimeter (cm)2 of skin surface 
382  area, and 1.6 cm diameter sites. Sites may be spaced as close as 2.5 cm center-to-
383  center and may be in a straight line or staggered pattern, depending on skin surface 
384  suitability (e.g., vascularity, nevi, etc.) and arm length. If vasoconstrictor effects of 
385  two adjacent test sites overlap and the investigator cannot discern between the 
386  vasoconstrictor effect at each test site, the subject should be excluded from the data 
387  analysis. 
388   
389  l)  Application to each subject of eight dose durations (in duplicate; see Appendix II) 
390  and four untreated control sites should be randomly assigned among the 20 sites, 
391  maintaining two untreated control sites, eight dosed sites on each arm (ten sites per 
392  arm), and duplicate measurements for each duration.  
393   
394  m) Prior to measurement of the pharmacodynamic skin blanching (vasoconstrictor) 
395  response at the end of the application period, remaining topical corticosteroid should 
396  be gently removed from the skin. This may be accomplished by either of the methods 
397  below: 
398   
399    Three consecutive swabbings with dry cotton swabs. 
400   
401    Washing all skin sites with mild skin cleanser and water, blotting the sites 
402  dry with a nonabrasive towel, and allowing to air-dry for at least 5 minutes prior 
403  to evaluation. Cleanse arm surfaces with a minimum amount of mild liquid skin 
404  cleanser, for example one drop of a liquid cleanser worked to a lather in wetted 
405  hands, followed by rinsing. If after 5 minutes the subject has any visible 
406  cutaneous effects related to washing, a longer waiting period may be necessary. 
407  This method is suitable for the staggered application with synchronized removal 
408  method. 
409   
410  n) Assessment of baseline skin color and skin blanching at each site.  
411  Examples of assessment time periods for staggered application with synchronized 
412  removal are: 
413   
10 
  Contains Nonbinding Recommendations 
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414    For all dose durations and untreated control sites, baseline readings within 
415  1 hour prior to drug application of the longest dose duration, and at 0, 2, 4, 6, 8, 
416  10, 12, 20, and 24 hours or longer until the response returns to baseline after drug 
417  product removal (see Appendix I).  Dose duration will depend upon the topical 
418  corticosteroid being studied. 
419   
420    Time zero (0) is defined as within 15 minutes after drug product removal. 
421   
422  2.  Pivotal Study 
423   
424  a)  Follow the recommendation listed in the section III.I.1 above where applicable. 
425  To remove potential operator bias, the analyst (e.g., chromameter operator) should be 
426  blinded to the product treatment assignments.   
427   
428  b)  Application of dose durations to skin sites on the ventral forearms of each subject 
429  should be randomly assigned, maintaining the recommendations described below.  
430  Sites may be occluded or nonoccluded, based on the considerations of section III.G 
431  above and the study design used in the pilot study. Untreated control skin sites should 
432  also be included. Dose durations and control sites on each arm should include: 
433   
434  R: the reference standard at the dose duration corresponding approximately to 
435  ED , as determined with the reference standard in the pilot study (e.g., two sites 
50
436  per arm) 
437   
438  T: the test topical corticosteroid at the same dose duration corresponding 
439  approximately to ED  as for the reference standard (e.g., two sites per arm) 
50
440   
441  D : the shorter dose duration reference standard calibrator (e.g., two sites per arm) 
1
442   
443  D : the longer dose duration reference standard calibrator (e.g., two sites per 
2
444  arm);and  
445   
446  UNT: the untreated control (e.g., two sites per arm) 
447   
448  The total number of treated sites is 16 (i.e., eight sites per arm). The eight treatments 
449  and two UNTs each arm should be randomized, as noted above. Application patterns 
450  on each arm should be complementary, i.e., D  is complementary to D , R is 
2 1
451  complementary to T, and UNT is complementary to UNT. As examples, where T is 
452  assigned a specific skin site location on one arm, R should be assigned to the 
453  corresponding skin site on the other arm. Where UNT is assigned a specific skin site 
454  location on one arm, UNT should be assigned to the corresponding skin site on the 
455  other arm. 
456   
457  A representative application sequence for a particular subject might be: 
458   
ANTECUBITAL FOSSA 
11 
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Left Arm  Right Arm 
D1  D2 
T  R 
UNT  UNT 
R  T 
D1  D2 
UNT  UNT 
T  R 
D2  D1 
R  T 
D2  D1 
WRIST 
459   
460  The specific pattern of skin sites, i.e., medial (ulnar) to lateral (radial), and superior to 
461  inferior, should be described in the study report/study protocol. 
462   
463  c)  The staggered application with synchronized removal method consistent with the 
464  methodology used in the pilot study should be used for D , D , and ED  dose 
1 2 50
465  durations. 
466   
467  d)  Refer to section III.I.1(n) Assessment of baseline skin color and skin blanching at 
468  each site. 
469   
470  J.  Data Analyses and Pharmacodynamic Modeling 
471   
472  1.  AUEC Calculation for the Pilot and Pivotal Studies   
473   
474  a)  Adjust (by subtraction) the chromameter raw data of each skin blanching response 
475  versus time profile (both active drug sites and untreated control sites) for the 
476  baseline value at that site. Correct each baseline-adjusted active drug site for the 
477  mean of the two baseline-adjusted untreated control sites on the same arm. 
478   
479  b)  Using the trapezoidal rule, compute the AUEC for each baseline-adjusted, 
480  untreated control site -corrected dose duration (see Appendix III): 
481   
482  AUEC  for the staggered application with synchronized removal method 
(0-t)
483  0: within 15 minutes after drug removal 
484  t: at least 24 hours after drug removal 
485   
486  2.  Pharmacodynamic Modeling for the Pilot Study 
487   
488  a)  Fitting dose duration-response data by averaging across subjects at each dose 
489  duration is not recommended. Rather, the data should be fitted by using all 
490  observations of all individual subjects simultaneously using nonlinear mixed 
491  effects modeling. The modeling software should provide population estimation 
492  for ED  and E  parameters for the data from at least 12 subjects. 
50 max
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493   
494  b)  Determine the ED  (the dose duration corresponding to half-maximal response). 
50
495   
496  c)  Determine D  and D  corresponding to approximately one-half ED  and two 
1 2 50
497  times ED  (for simple E  model used), respectively, for use in the pivotal 
50 max
498  study.14 These values bracket ED , correspond to approximately 33% and 67% 
50
499  respectively of the maximal response, and represent the sensitive portion of the 
500  dose duration-response curve.  
501   
502  3.  Data Analysis for the Pivotal Study 
503   
504  a)  Only the data of detectors should be included in the data analysis. The dose 
505  duration-response criterion to define detector is: 
506   
507   
AAAAEEAA aaaa DD2
508   
  1.25
509  AUEC at D =average of AAAAAUEEEAAC saa aaat  DDD1  from both left arm and right arm 
2 2
510  AUEC at D =average of AUECs at D  from both left arm and right arm 
1 1
511   
512  b)  The bioequivalence comparison should be based on AUEC values computed 
513  according to Appendix III at the dose duration corresponding approximately to ED  
50
514  (treatments T and R). 
515  i.  The statistical analysis requires the use of untransformed data because 
516  AUEC values of treatments T and R, calculated from baseline-adjusted, 
517  untreated control site-corrected data, are generally negative, although 
518  sometimes positive. The presence of both positive and negative data 
519  prevents the use of conventional statistical transformations. Locke's 
520  method15 provides an exact confidence interval from untransformed data. 
521   
522  ii.  Using data from the detectors, the 90% confidence interval should be 
523  calculated for the ratio of the average AUEC (e.g., AUEC ) response 
0-24hr
524  due to the test product (average of four replicates) to the average AUEC 
525  (e.g., AUEC ) response due to the reference product (average of four 
0-24hr
526  replicates) should be calculated using Locke's method. The formulae and a 
527  worked example based on the data are given in Appendix V. 
528   
529             The 90% confidence interval for the test to reference AUEC ratio should  
530  be within the 80.00-125.00% interval. 
531   
 
14 The estimated ED value may be rounded by up to 15 minutes to obtain the ED  value used in the pivotal study.  
50  50
For potent corticosteroids with short ED  values, these recommendations may require adjustment. If so, FDA may 
50
be consulted via a controlled correspondence or, for relevant complex products, via a pre-ANDA meeting. 
15 Locke CS. An exact confidence interval from untransformed data for the ratio of two formulation means. J 
Pharmacokinet Biopharm 1984;12:649-55. 
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  Contains Nonbinding Recommendations 
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532  4.  Formatted Data Submission 
533   
534  The study data for the pilot and pivotal studies should be submitted, as recommended by 
535  the Agency, in the following format: https://www.fda.gov/media/87599/download. 
536  Chromameter raw data; baseline-adjusted data; baseline-adjusted, untreated control site-
537  corrected data; and AUEC data should be arranged in separate files.   
538   
539  All study data, including the data of nondetectors, should be submitted. An explanation 
540  (e.g., nondetector, overlap of vasoconstrictor effect due to an adjacent site, etc.) should 
541  accompany any data not used in the vasoconstrictor study evaluation. The randomization 
542  code, indicating the specific skin sites to which each dose duration and control site was 
543  assigned, should be submitted with the study report. 
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  Contains Nonbinding Recommendations 
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544  APPENDIX I: SCHEMATIC FOR STAGGERED APPLICATION WITH 
545  SYNCHRONIZED REMOVAL FOR PILOT STUDY PROTOCOLS  
546   
547  Figure A1: Example of Baseline (BL) Measurement, Drug Application and Drug Removal 
548   
BL 
 
BL 
 
BL 
 
BL 
 
BL 
 
  BL  Application Time 
BL 
 
Removal Time 
BL 
 
-7 -6 -5 -4 -3 -2 -1 0
Hours before Drug Removal
DD8 (6 hr) DD7 (4  hr) DD6 (2 hr) DD5 (1.5 hr)
DD4 (1 hr) DD3 (0.75 hr) DD2 (0.5 hr) DD1 (0.25 hr)
549 
550  DD: Dose Duration 
551   
552  Figure A2: Skin Blanching Measurements 
Measurement 
Removal Time 
0 2 4 6 8 10 12 14 16 18 20 22 24
Hours After Drug Removal
553   
 
554  Note: Time zero (0) is defined as the within 15 minutes after drug product removal.
15 
   
555  APPENDIX II: EXAMPLE FOR SKIN BLANCHING STUDY DESIGN FOR 
556  PILOT DOSE-DURATION RESPONSE STUDY 
557   
558   
559    RIGHT ARM  LEFT ARM 
560   
561    DOSE  ANTECUBITAL  ANTECUBITAL  DOSE 
562    DURATION   FOSSA  FOSSA  DURATION 
563   
564    (hours)  (hours) 
565   
566   
567   
568  6.0  1.5  1  2    11  12  4.0  1.0 
569   
570   
571   
557723   0.25  1.0  3  4     13  14  UNT  1.5 
574   
575   
576  4.0  0.75  5  6    15  16  6.0  2.0 
577   
0.5  UNT  7  8  17  18  0.75  UNT 
578   
579   
UNT  2.0  9  10  19  20  0.25  0.5 
580   
581   
WRIST  WRIST 
582   
583  Light circle: untreated site; Dark circle: treated site with different dose-duration  
584  Dose duration of 0.25 to 6.0 hours represent times for exposure of skin to the reference standard. 
585   
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  Contains Nonbinding Recommendations 
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586  APPENDIX III: CALCULATION OF AUEC  
587   
588  Step 1. Calculate baseline-adjusted, untreated control site-corrected a-scale data (C ) for 
i,j 
589  each corresponding treated site: 
590             
591           C = A  A   A   
i,j  i,j  0,j i,0 
592   
593  where i is i th measurement after drug removal (hours): e.g., from 0 hr to t (at least 24 hr); 
594  j is the j th dose duration: from dose duration DD  to last dose duration DD ; 
1 n
595  A  is the raw a-scale data site reading for each corresponding treated site for j th dose 
i,j
596  duration at time i after drug removal;   
597  A is baseline (pre-dose) reading within one hour prior to drug application of the longest 
0,j 
598  dose duration; 
599  and  A is mean of untreated control site reading at time i after drug removal of the same 
  i,0 
600  arm.  
601   
602  Step 2. AUEC calculation from the baseline-adjusted and untreated control site-corrected a-
603  scale data (C ) for the test topical corticosteroid and reference standard for all subjects.  
i,j
604  nn  
ttlllllltt AAii +AAii+11
605    AAAAAAAAtt00 =  ttii
ii=11 22
606  where t  denotes the time of first measured pharmacodynamic response, e.g., 0.25 hr after 
0
607  drug removal;  
608  t = t -t and t  denotes the time of the last measured pharmacodynamic response 
i i+1  i last
609   
  17  Contains Nonbinding Recommendations 
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610  APPENDIX IV: E  MODELS  
MAX
611   
612  A population modeling approach should be used to develop a simple E  model as shown 
max
613  below, because the E  model needs to account for between-subject variability. Naive pools (all 
max
614  subjects pooled as one) are no longer recommended by FDA.  
615   
EEmmmmmm DD
616  E is the response (baseline-adjusted, untreated control site-corrected AUEC) at the dose duration 
EE =
617  of application (D), E  is the maximal responEEseDD, 5a0nd+  DD  is the duration at which half-maximal 
max
618  response occurs.  
619    EEDD50
620  Alternative sigmoidal models can be used with justifications and appropriate model selection 
621  procedures if the above E  model cannot fit dose duration-response response data well.  
max
622  Potential alternative models are provided below: 
623   
624  Sigmoid E  model which incorporates a Hill coefficient  :  
max
625   


EEmmmmmm DD
626  Note:   and   should be adjusted usinEEg t=heEE fDDol5l0ow+inDDg equations:  1 ; 
 1
627  DD1 DD2 .   DD1 = (ff1) EEDD50,ff1  2
1
628    
629  DDO2th=er (affl2te)rnatiEEvDDe 5m0,offd2els m2ay be acceptable with sufficient justification. For detailed information 
630  about population modeling, model verification/validation and E  models, refer to the following 
max
631  guidance and publications:  
632    Guidance for industry Population Pharmacokinetics (February 2022) 
633    Guidance for industry Exposure-Response Relationships  Study Design, Data Analysis, 
634  and Regulatory Applications (April 2003)   
635    Deniz Ozdin, Naveen Sharma, Jorge Lujan-Zilbermann, Philippe Colucci, Isadore 
636  Kanfer, Murray P Ducharme, Revisiting FDA's 1995 Guidance on Bioequivalence 
637  Establishment of Topical Dermatologic Corticosteroids: New Research Based 
638  Recommendations, J PharmSci. 2018;21(1):413-28. 
639    RN Upton and DR Mould. Basic Concepts in Population Modeling, Simulation, and 
640  Model-Based Drug Development: Part 3Introduction to Pharmacodynamic Modeling 
641  Methods. CPT Pharmacometrics Syst Pharmacol. 2014 Jan; 3(1): e88. 
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642  APPENDIX V: LOCKE METHOD FOR BIOEQUIVALENCE ASSESSMENT AND 
643  A WORKED EXAMPLE 
644   
645  The calculation of the 90% confidence interval for the pivotal bioequivalence data set of Table 1 
646  (Mean AUEC Values of Subjects in the Pivotal Study) is given below. The data used to calculate 
647  the confidence interval are the average baseline-adjusted and untreated control site-corrected 
648  AUEC values of detectors.  
649   
650  The calculation of the confidence interval is facilitated by the calculation of the following 
651  intermediate quantities:  
652  nn  
11
653    XXTT = XXTTii
nnii=11
654  nn  
11
655    XXRR = XXRRii
nnii=11
nn
22
656    
 XXTTii  XXTT  
ii=11
657   
TTTT =
nn11
nn
22
658    
 XXRRii  XXRR  
ii=11
659   
RRRR =
nn11
nn
660     
 XXTTii  XXTTXXRRii  XXRR 
ii=11
661   
TTRR =
662  where n is the number of evaluable subjects,  nn11
663   
664  And define t as the 95th percentile of the t-distribution for n-1 degrees of freedom, then define: 
665   
666   
22
tt  RRRR
667    GG = 22
668  G < 1 is required to have a proper confidence intennr vXXaRRl. If G  1, the study does not meet the in 
669  vivo bioequivalence requirements. 
670   
671  Under the assumption that G < 1, calculate: 
672   
673   
22
XXTT TTTT TTRR TTRR XXTT
674    KK =   + (11GG)+ GG 22 
675  The confidence interval limitXXs mRR ay nowRR RRbe calculated: RRRR RRRR XXRR
  19  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
676   
677  XXTT TTRR tt RRRR  
 GG   KK 
678    XXRR RRRR XXRR  nn
679  Table 1. Mean AUEC Value11s of GGSubjects in the Pivotal Study 
680   
Subject  AUEC(0-t)  AUEC(0-t) 
Test Product  Reference Product 
  (Average)  (Average) 
2  -48.52  -22.20 
3  -38.99  -18.65 
4  -7.62  -22.42 
7  0.98  -10.96 
9  -32.05  -37.40 
11  -26.18  -26.73 
12  -11.62  -12.56 
681   
682   
683  For the example, these are    =  -23.43,    =  -21.56,   = 323.13,   = 80.10, and 
684   = 78.83. 
685  In the example, for n = 7, t XX(6TT degrees of fXXreRRedom) is 1.94TT3TT2.   G = 0.093RR0RR <1, then K = 
686  2.TT7RR91.  
687   
688  Based on the data of evaluable subjects, the 90% confidence interval limits are 53.6% and 
689  165.9%, which are not within the acceptable limits of 80.00- 125.00%. . 
  20