In Vitro Release Tests as a Critical Quality Attribute in Topical Product Development

  • Lakshmi RaghavanEmail author
  • Marc Brown
  • Bozena Michniak-Kohn
  • Stephanie Ng
  • Srinivasa Sammeta
Part of the AAPS Advances in the Pharmaceutical Sciences Series book series (AAPS, volume 36)


Critical quality attributes (CQAs) play an important role in demonstrating Q1, Q2, and Q3 equivalence of topical products between the test and reference products. In vitro testing is one of the CQAs that is predominantly used in the correlation of Q3 microstructure to product performance. Two types of in vitro tests are typically employed to understand the microstructure, namely, in vitro release tests (IVRT) and in vitro permeation tests (IVPT). IVRT provides information on the release of the drug from a formulation and IVPT provides information on the permeation of the drug through the skin. Both can be influenced by the type of dosage form that includes gels, creams, ointments, and lotions. Each of the dosage forms has a different matrix that varies in complexity and affects the IVRT/IVPT differently. Different microstructure parameters influence release rates and some of these include: viscosity and rheological properties, globule and particle sizes, pH, phase homogeneity, polymorphism, etc. Excipients have an influence on microstructure properties: for example, the type and grade of the excipient, different solvents, co-solvents, penetration enhancers, preservatives, coloring agents, and fragrances. Microstructure properties are also greatly influenced by the critical processing parameters (CPPs). Examples are mixing time, cooling/heating, mixer type, mixing temperature, speed and duration, and hold times at various process stops. IVRT plays a very important role in evaluating any post-approval change in process that can impact product quality and performance. FDA has issued the SUPAC-SS guidance to identify the different levels of process changes and how IVRT should be conducted to evaluate the impact. The various process level changes and what methodology should be adopted are discussed. In summary, this chapter provides an introduction to in vitro release and permeation testing and how this test can be a tool in evaluating Q3 microstructure.


Topicals Semisolids Product performance Critical quality attributes Microstructure In vitro permeability testing In vitro release testing Excipients Critical process parameters 


  1. M. Alberti, Y. Dancik, G. Sriram, B. Wu, Y.L. Teo, Z. Feng, M. Bigliardi-Qi, R.G. Wu, Z.P. Wang, P.L. Bigliardi, Multi-chamber microfluidic platform for high-precision skin permeation testing. Lab Chip 17, 1625–1634 (2017)PubMedCrossRefGoogle Scholar
  2. W.L. Au, M. Skinner, I. Kanfer, Comparison of tape stripping with the human skin blanching assay for the bioequivalence assessment of topical clobetasol propionate formulations. J. Pharm. Pharma. Sci. 13(1), 11–20 (2010)CrossRefGoogle Scholar
  3. B. Balazs, G. Vizseralek, S. Berko, M. Budai-Szucs, A. Kelemen, B. Sinko, K. Takacs-Novak, P. Szabo-Revesz, E. Csanyi, Investigation of the efficacy of transdermal penetration enhancers through the use of human skin and a skin mimic artificial membrane. J. Pharm. Sci. 105, 1134–1140 (2016)PubMedCrossRefGoogle Scholar
  4. R. Baynes, J. Riviere, T. Franz, N. Monteiro-Riviere, P. Lehman, M. Peyrou, P.L. Toutain, Challenges obtaining a biowaiver for topical veterinary dosage forms. J. Vet. Pharmacol. Ther. 35(Suppl 1), 103–114 (2012)PubMedCrossRefGoogle Scholar
  5. M. Bodenlenz, C. Ho¨fferer, C. Magnes, et al., Dermal PK/PD of a lipophilic topical drug in psoriatic patients by continuous intradermal membrane-free sampling. Eur. J. Pharm. Biopharm 81(3), 635–641 (2012)PubMedCrossRefGoogle Scholar
  6. M. Bodenlenz, B. Aigner, C. Dragatin, et al., Clinical applicability of dOFM devices for dermal sampling. Skin Res. Technol. 19(4), 474–483 (2013)PubMedGoogle Scholar
  7. M. Bodenlenz, K.I. Tiffner, R. Raml, T. Augustin, C. Dragatin, T. Birngruber, D. Schimek, G. Schwagerlez, T.R. Pieber, S.G. Raney, I. Kanfer, F. Sinner, Open flow microperfusion as a dermal pharmacokinetic approach to evaluate topical bioequivalence. Clin Pharmacokinetics 56, 91–98 (2017)CrossRefGoogle Scholar
  8. F. Bonina, C. Puglia, D. Trombetta, M.C. Dragani, M.M. Gentile, G. Clavenna, Vehicle effects on in vitro skin permeation of thiocolchicoside. Pharmazie 57, 750–752 (2002)PubMedGoogle Scholar
  9. R.-K. Chang, A. Raw, R. Lionberger, L. Yu, Generic development of topical dermatologic products: Formulation development, process development, and testing of topical dermatologic products. AAPS J. 15(1), 41–52 (2013a)PubMedCrossRefGoogle Scholar
  10. R.-K. Chang, A. Raw, R. Lionberger, L. Yu, Generic development of topical dermatologic products, part II: Quality by Design for Topical Semisolid Products. AAPS J. 15(3), 674–683 (2013b)PubMedPubMedCentralCrossRefGoogle Scholar
  11. H.V. Chavda, Qbd in developing topical dosage forms. Ely. J. Pharm. Res. 2(1), 1–2 (2016)Google Scholar
  12. Y.W. Chien, Chapter 2: Developmental Concepts & Practice in Transdermal Therapeutic Systems, in Transdermal Controlled Systemic Medications, (Marcel Dekker, New York, 1987), pp. 44–47Google Scholar
  13. J.M. Christensen, M.C. Chuong, H. Le, L. Pham, E. Bendas, Hydrocortisone diffusion through synthetic membrane, mouse skin, and Epiderm cultured skin. Arch. Drug Inform. 4, 10–21 (2011)CrossRefGoogle Scholar
  14. S.E. Cross, M.S. Roberts, Use of in vitro human skin to model and predict the effect of changing blood flow on the flux and retention of topically applied solutes. J. Pharm. Sci. 97(8), 3442–3450 (2008)PubMedCrossRefGoogle Scholar
  15. C. Dragatin, F. Polus, M. Bodenlenz, et al., Secukinumab distributes into dermal interstitial fluid of psoriasis patients as demonstrated by open flow microperfusion. Exp Dermatol 25(2), 157–159 (2016)PubMedCrossRefGoogle Scholar
  16. D. Dupuis, R. Rougier, R. Roguet, C. Lotte, The measurement of the stratum corneum reservoir: A simple method to predict the influence of vehicles on in vivo percutaneous absorption. Br. J. Dermatol. 115, 233–238 (1986)PubMedCrossRefGoogle Scholar
  17. B.A. Elewski, Percutaneous absorption kinetics of topical metronidazole formulations in vitro in the human cadaver skin model. Adv Ther 24(2), 239–246 (2007)PubMedCrossRefGoogle Scholar
  18. Food & Drug Administration, Product Specific Guidance for Generic Drug Development (2016).
  19. Food and Drug Administration, Guidance for Industry: Topical Dermatologic Drug Product NDAs and ANDAs In Vivo Bioavailability, Bioequivalence, in Vitro Release, and Associated Studies (1998)Google Scholar
  20. Food and Drug Administration (FDA), Guidance for Industry: PAT — A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance (Food and Drug Administration (FDA), Rockville, MD, 2004)Google Scholar
  21. Food and Drug Administration (FDA), Role of Models in the Quality by Design (QbD) Paradigm: Regulatory Perspective (2011).
  22. Fowler M, Quality by Design (QbD) Approach to Generic Transdermal or Topical Product Development (2015). Available at:
  23. T.J. Franz, On the relevance of in vitro data. J. Invest. Dermatol. 64, 190–195 (1975)PubMedCrossRefGoogle Scholar
  24. P.O. Fritsch, G. Gschnait, G. Kaaserer, et al., PUVA suppresses the proliferative stimulus produced by stripping on hairless mice. J. Invest. Dermatol. 73(2), 188–190 (1979)PubMedCrossRefGoogle Scholar
  25. S.J. Gallagher, C.M. Heard, Solvent content and macroviscosity effects on the in vitro transcutaneous delivery and skin distribution of ketoprofen from simple gel formulations. Skin Pharmacol. Physiol. 18, 186–194 (2005)PubMedCrossRefGoogle Scholar
  26. S.J. Gallagher, L. Trottet, C.M. Heard, Ketoprofen: Release from, permeation across and rheology of simple gel formulations that simulate increasing dryness. Int. J. Pharm. 268, 37–45 (2003)PubMedCrossRefGoogle Scholar
  27. K. Goebel, M.E.O. Sato, D.F. Souza, S. Murakami, I.F. Andreazza, In vitro release of diclofenac diethylamine from gels: Evaluation of generic semisolid drug products in Brazil. Braz. J. Pharm. Sci. 49(2), 211–220 (2013)CrossRefGoogle Scholar
  28. Z. Guerol, S. Hekimoglu, R. Demirdamar, M. Sumnu, Percutaneous absorption of ketoprofen. I. In vitro release and percutaneous absorption of ketoprofen from different ointment bases. Pharm. Acta Helv. 71, 205–212 (1996)CrossRefGoogle Scholar
  29. J. Hadgraft, R.H. Guy, Feasibility Assessment in Topical and Transdermal Drug Delivery: Mathematical Models and In Vitro Studies, in Transdermal Drug Delivery, ed. by R. Guy, J. Hadgraft, 2nd edn., (Marcel Dekker, New York, 2003), pp. 1–23Google Scholar
  30. J. Hadgraft, M. Whitefield, P.H. Rosher, Skin penetration of topical formulations of ibuprofen 5%: An in vitro comparative study. Skin Pharmacol. Appl. Ski. Physiol. 16, 142 (2003)Google Scholar
  31. C. Herkenne, A. Naik, Y.N. Kalia, J. Hadgraft, R.H. Guy, Ibuprofen transport into and through skin from topical formulations: In vitro-in vivo comparison. J. Invest. Dermatol. 127, 135–142 (2007)PubMedCrossRefGoogle Scholar
  32. T. Higuchi, Rate of release of medicaments from ointment bases containing drugs in suspension. J. Pharm. Sci. 50, 874–875 (1961)PubMedCrossRefGoogle Scholar
  33. (2009)Google Scholar
  34. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline, Quality Risk Management– Q9 (November 2005). Available at:
  35. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline, Pharmaceutical Quality Systems–Q10 (June 2008), Available at: Accessed 21 Nov 2014
  36. International Conference on Harmonization (ICH): Implementation of ICH Q8, Q9, Q10 (2010). (Accessed June 2016)
  37. International Conference on Harmonization (ICH) Q8(R2): Pharmaceutical Development (2009).
  38. J. Jain, Bioequivalence of topical dosage forms. J. Pharm. 2(2), 1–2 (2014)Google Scholar
  39. A. Jankowski, R. Dyja, B. Sarecka-Hujar, Dermal and transdermal delivery of active substances from semisolid bases. Indian J. Pharm. Sci. 79(4), 488–500 (2017)CrossRefGoogle Scholar
  40. J.M. Juran, The Quality Trilogy: A Universal Approach to Managing for Quality. Quality Progress, 19(8), 19–24 (1986)Google Scholar
  41. J.M. Juran, Juran on Quality by Design: The New Steps for Planning Quality into Goods and Services (Free Press, New York, 1992)Google Scholar
  42. M. Kietzmann, B. Blume, Percutaneous absorption of betamethasone from different formulations using the isolated perfused bovine udder. In Vitro Toxicol 10, 11–15 (1997)Google Scholar
  43. R.R. Klein, J.Q. Tao, S. Wilder, K. Burchett, Q. Bui, K.D. Thakker, Development of an in vitro release test (IVRT) for a vaginal microbicide gel. Dissolut. Technol. 17(4), 6–10 (2010)CrossRefGoogle Scholar
  44. Y.S. Krishnaiah, X. Xu, Z. Rahman, Y. Yang, U. Katragadda, R. Lionberger, J.R. Peters, K. Uhl, M.A. Khan, Development of performance matrix for generic product equivalence of acyclovir topical creams. Int. J. Pharm. 475(1–2), 110–122 (2014a)CrossRefPubMedGoogle Scholar
  45. Y.S. Krishnaiah, N. Kamal, N. Pavurala, X. Xu, M.A. Khan, Effect of Aqueous Phase pH on the In Vitro Performance of Acyclovir Topical Creams AAPS poster, AAPS Annual Meeting and Exposition, San Diego, CA (2014b)Google Scholar
  46. Y.S. Krishnaiah, N. Kamal, N. Pavurala, X. Xu, M.A. Khan, Development of Discriminatory In Vitro Release Test (IVRT) and In Vitro Permeation Test (IVPT) Methods for Acyclovir Topical Cream Using Quality by Design Approach (AAPS poster, San Diego, CA, 2016)Google Scholar
  47. P.A. Lehman, S.G. Raney, T.J. Franz, Percutaneous absorption in man: in vitro-in vivo correlation. Skin Pharmacol. Physiol. 24(4), 224–230 (2011)PubMedCrossRefGoogle Scholar
  48. R. Lionberger, S. Lee, A. Raw, L. Yu, Quality by design: Concepts for ANDAs. AAPS J. 10(2), 268–276 (2008)PubMedPubMedCentralCrossRefGoogle Scholar
  49. N.A. Megrab, A.C. Williams, B.W. Barry, Oestradiol permeation through human skin and silastic membrane: effects of propylene glycol and supersaturation. J. Control. Release 36, 277–294 (1995)CrossRefGoogle Scholar
  50. P. Minghetti, A. Casiraghi, F. Cilurzo, L. Montanari, M. Marazzi, L. Falcone, V. Donati, Comparison of different membranes with cultures of keratinocytes from man for percutaneous absorption of nitroglycerine. J. Pharm. Pharmacol. 51, 673–678 (1999)PubMedCrossRefGoogle Scholar
  51. S.N. Murthy, Characterizing the critical quality attributes and in vitro bioavailability of acyclovir and metronidazole topical products. Perspectives in Percutaneous Penetration Conference 2016, 2014Google Scholar
  52. C. Nagelreiter, S. Raffeiner, C. Geverhofer, V. Klang, C. Valenta, Influence of drug content, type of semi-solid vehicle and rheological properties on the skin penetration of the model drug fludrocortisone acetate. Int. J. Pharm. 448(1), 305–312 (2013)PubMedCrossRefGoogle Scholar
  53. G.M. Nemecek, A.D. Dayan, Safety evaluation of human living skin equivalents. Toxicol. Pathol. 27, 101–103 (1999)PubMedCrossRefGoogle Scholar
  54. V.E. Nwoko, Semi Solid dosage Forms Manufacturing: Tools, Critical Process Parameters, Strategies, Optimization and Validation. Sch. Acad. J. Pharm. 3, 153–161 (2014)Google Scholar
  55. D.W. Osborne Compositions for topical application of therapeutic agents. US patent 6,620,435 B1, assignee ViroTex Coroporation (2003)Google Scholar
  56. D.W. Osborne, Impact of quality by design on topical product excipient suppliers, part I: A drug Manufacturer’s perspective. Pharm. Technol. 40(10), 38–43 (2015)Google Scholar
  57. C. Puglia, P. Blasi, A. Rizza, F. Schoubben, F. Bonina, C. Rossi, M. Ricci, Lipid nanoparticles for prolonged topical delivery: An in vitro and in vivo investigation. Int. J. Pharm. 357, 295–304 (2008)PubMedCrossRefGoogle Scholar
  58. S.A. Rabbani, Approaches for bioequivalence assessment of topical dermatological formulations. Adv. Bioequival. Bioavail. 1(1), 1–3 (2018)Google Scholar
  59. F.S. Radulescu, D.S. Miron, Physicochemical characterization of acyclovir topical semisolid dosage forms towards TCS validation. 3rd FDA/PQRI Conference on Advancing Product Quality, 22-24 Mar2017Google Scholar
  60. V. Rai, J. Terebetski, S. Silva, B. Michniak-Kohn, Human Skin Equivalents (HSEs) as an alternative for transdermal permeation, phototoxicity and cytotoxicity studies. Transdermal 2(3), 5–8 (2010)Google Scholar
  61. S. Raney, Strategies to improve patient access to high quality topical products AAPS workshop dermatological drug products: developmental & regulatory considerations, 12 Nov 2017aGoogle Scholar
  62. S. Raney, In vitro characterization of topical semisolid dosage forms. Presented at the 3rd PQRI/FDA Conference on Advancing Product Quality (2017b)Google Scholar
  63. Roberts MS, Mohammed Y, Namjoshi S, Jung N, Chaitanya K, Cheruvu S, Windbergs M, Liu X, Benson HAE, Naegel A, Wittum R, Stokes J, Shewan H, Ghosh P, Ramezanli T, Raney S, Grice JE, Correlation of physicochemical characteristics and in vitro permeation test (IVPT) results for acyclovir and metronidazole topical products. FDA Workshop on Bioequivalence Testing of Topical Drug Products, 2017Google Scholar
  64. F.P. Schmook, J.G. Meingasser, A. Billich, Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption. Int. J. Pharm. 215, 51–56 (2001)PubMedCrossRefGoogle Scholar
  65. V.P. Shah, J.S. Elkins, R.L. Williams, Evaluation of the test system used for in vitro release of drugs for topical dermatological drug products. Pharm. Dev. Technol. 4, 377–385 (1999)PubMedCrossRefGoogle Scholar
  66. A.H. Shojaei, B. Berner, X.L. Li, Transbuccal delivery of acyclovir: I in vitro determination of routes of buccal transport. Pharm. Res. 15, 1182–1118 (1998)PubMedCrossRefGoogle Scholar
  67. C. Shukla, E.D. Bashaw, G. Stagni, E. Benfeldt, Applications of dermal microdialysis: A review. J. Drug Deliv. Sci. Technol. 24(3), 259–269 (2014)CrossRefGoogle Scholar
  68. O. Siddiqui, M.S. Roberts, A.E. Polack, The effect of iontophoresis and vehicle pH on the in-vitro permeation of lignocaine through human stratum corneum. J. Pharm. Pharmacol. 37(10), 732–735 (1985)PubMedCrossRefGoogle Scholar
  69. P.J. Sinko, Diffusion, in Martin’s Physical Pharmacy and Pharmaceutical Sciences, ed. by P. J. Sinko, 5th edn., (Lippincott Williams and Wilkins, Philadelphia, PA, 2006), pp. 301–335Google Scholar
  70. A. Sivaraman, A.K. Banga, Quality by design approaches for topical dermatological dosage forms. Res. Rep. Transdermal Drug Deliv. 4, 9–21 (2015)Google Scholar
  71. R.K. Subedi, S.Y. Oh, M.K. Chun, H.K. Choi, Recent advances in transdermal drug delivery. Arch. Pharm. Res. 33, 339–351 (2010)PubMedCrossRefGoogle Scholar
  72. K.D. Thakker, W.H. Chern, Development and validation of in vitro release tests for semisolid dosage forms – Case study. Dissolut. Technol. 10(2), 10–15 (2003)CrossRefGoogle Scholar
  73. K.I. Tifffner, I. Kanfer, T. Augustin, R. Raml, S.G. Raney, A comprehensive approach to quality and validate the essential parameters of an in vitro release test (IVRT) method for acyclovir cream, 5%. Int. J. Pharm. 535, 217–227 (2018)CrossRefGoogle Scholar
  74. K. Tojo, P.R. Keshary, Y.W. Chien, Drug permeation through skin from matrix-type drug delivery systems. Chem. Engineer. J. 32(3), B57–B64 (1986)CrossRefGoogle Scholar
  75. J.C. Tsia, S.A. Chuang, M.Y. Hsu, H.M. Sheu, Distribution of salicylic acid in human stratum corneum following topical application in vivo: A comparison of six different formulations. Int. J. Pharm. 188, 145–153 (1999)CrossRefGoogle Scholar
  76. U.S. USP chapter <1724> Semisolid drug products – performance tests, in: USP 40, pp 2055–2067 (2017)Google Scholar
  77. U.S. FDA, Guidance for industry nonsterile semisolid dosage forms, scale-up and Post-approval changes: chemistry, manufacturing, and controls; in vitro release testing and in vivo bioequivalence documentation (SUPAC-SS). Guidance for Industry. US. (1997)Google Scholar
  78. U.S. FDA. Formal Meetings Between FDA and ANDA Applicants of Complex Products Under GDUFA, Guidance for Industry, draft guidance (2017)Google Scholar
  79. U.S. FDA, Product-Specific Guidances for Generic Product Development (2019)Google Scholar
  80. U.S. Food and Drug Administration Center for Drug Evaluation and Research (CDER), Manual of Policies and Procedures MAPP 5016.1. Applying ICH Q8(R2), Q9, and Q10 Principles to CMC Review. February 2011. Available at: Accessed 21 Nov 2014.
  81. J. Vonguru, R. Gilman, R. Klein, D.M. Mattocks, K.D. Thakker, Developing in vitro release testing (IVRT) methods for petrolatum based semi-solid dosage forms. Abstract submission, AAPS (2015)Google Scholar
  82. C.K. Wang, C.F. Nelson, A.M. Brinkman, A.C. Miller, W.K. Hoffler, Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent. J Invest Dermatol 114, 674–680 (2000)PubMedCrossRefGoogle Scholar
  83. J. Wolf, Die innere Struktur der Zellen des Straum desquamans der mensschlichen Epidermis. Z mikranat Forsch 46, 170–202 (1939)Google Scholar
  84. X. Xu, M. Al-Ghabeish, Y.S. Krishnaiah, Z. Rahman, M.A. Khan, Kinetics of drug release from ointments: Role of transient-boundary layer. Int J Pharm. 494, 31–39 (2015)PubMedCrossRefGoogle Scholar
  85. L.X. Yu, G. Amidon, M.A. Khan, S.W. Hoag, J. Polli, G.K. Raju, J. Woodcock, Understanding pharmaceutical quality by design. APPS J 16(4), 771–783 (2014)Google Scholar
  86. Z. Zheng, B. Michniak-Kohn, Tissue engineered human skin equivalents. Pharmaceutics ISSN 1999–4923 (2012).;, 4: 26–41.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© AAPS (American Association of Pharmaceutical Scientists) 2019

Authors and Affiliations

  • Lakshmi Raghavan
    • 1
    Email author
  • Marc Brown
    • 2
  • Bozena Michniak-Kohn
    • 3
  • Stephanie Ng
    • 4
  • Srinivasa Sammeta
    • 5
  1. 1.Solaris Pharma CorporationBridgewaterUSA
  2. 2.MedPharm Ltd.SurreyUK
  3. 3.RutgersThe State University of New JerseyPiscatawayUSA
  4. 4.Acrux DDS Pty Ltd.West MelbourneAustralia
  5. 5.Teva PharmaceuticalsSalt Lake CityUSA

Personalised recommendations