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Irritative Activity of Topical Lotions With Minoxidil Content Assessment in a HET-CAM Test

Organized by Faculty of Pharmacy, Lithuanian University of Health Sciences
OCTOBER 22ND, 2021
The Joint International Pharmacy Symposium “Contemporary Pharmacy: Issues Challenges
and Expectations 2021“ and “11th Conference: Pharmacy Science and Practice“
The Conference is dedicated to Pharmacist Role during COVID-19 pandemic situation.
Organizing Committee
Prof. dr. Ramune Morkuniene
Prof. dr. Nijole Savickiene
Prof. dr. Jurga Bernatoniene
Prof. dr. Liudas Ivanauskas
Prof. dr. Valdas Jakstas
Prof. dr. Lina Raudone
Assoc. Prof. dr. Vilma Gudiene
Assoc. Prof. dr. Tauras Antanas Mekas
Lect. dr. Agne Mazurkeviciute
Dr. Lauryna Pudziuvelyte
PhD student Justina Kamarauskaite
PhD student Jurga Andreja Kazlauskaite
PhD student Inga Matulyte
PhD student Gabriele Vilkickyte
Scientific Committee
Scientists section: Prof. dr. Lina Raudone and
PhD student Gabriele Vilkickyte
History of pharmacy section: Assoc.
Prof. dr. Vilma Gudiene and Dr. Ramunas
Students and PhD students section: PhD
student Jurga Andreja Kazlauskaite and PhD
student Justina Kamarauskaite
PhD student Inga Matulyte
Assoc. Prof. dr. Raimondas Benetis
Dr. Aiste Jekabsone
Assoc. Prof. dr. Giedre Kasparaviciene
Assoc. Prof. dr. Daiva Kazlauskiene
Lect. dr. Mindaugas Liaudanskas
Assoc. Prof. dr. Augusta Zevzikoviene
Lect. dr. Modestas Zilius
ISBN 978-9955-15-711-3
2021, Kaunas
Language of abstracts was not corrected.
Irritative Activity of Topical Lotions With Minoxidil Content Assessment
in a HET-CAM Test
Vira Turkina, Natalia Chemodurova
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
*Corresponding author’s e-mail:
Introduction: The use of lotions containing minoxidil for the various alopecia forms correction and
prevention has become increasingly common in trichological practice throughout the last decade [1, 2, 3].
Since these lotions are mostly positioned as cosmetics with a therapeutic effect and are often used without
consulting a dermatologist, the probable side effects assessment of these products is particularly important.
The aim of the present study was to assess the possibility of using the HET-CAM test as an alternative test in
rabbits for the irritant potential of lotions containing 5% and 10% minoxidil.
Materials and methods: According to the test protocol, the lotions containing 5% and 10% minoxidil were
applied to the chorioallantois membrane (CAM) of a chicken embryo in an amount of at least 3 pieces [4]. The
reaction of chorioallantoic membrane blood vessels was assessed in points by signs of hemorrhage,
vascular lysis and coagulation. We used 0.9% NaCl solution as a negative control and 1% sodium
dodecyl sulfate solution as a positive control.
CAM reactions were monitored for 300 seconds; photo fixation of changes was performed using an
endoscope microcamera. The overall subdivision was assessed by the irritant index classification of the tested
substance, based on the calculated mean score.
Results: Application of 0.3 ml 0.9% saline to healthy membranes caused no visual effect for 300 seconds. In
contrast, application of 0.3 ml of NaOH caused a severe, momentary hemorrhage, which increased for 300
seconds. When lotion containing 5% minoxidil was studied, hemorrhage and vascular lysis were recorded
starting from the 30th second. Application of lotion containing 10% minoxidil caused coagulation
and hemorrhage the 30th second, vascular lysis from the 120th second of observation.
Conclusions: It was found that the use of the HET-CAM test to test lotions containing 5% and
10% minoxidil revealed a pronounced irritant effect on the mucous membranes.
1. Tr̈eb, R., n.d. The difficult hair loss patient.
2. Rossi, A., Cantisani, C., Melis, L., Iorio, A., Scali, E. and Calvieri, S., 2012. Minoxidil Use in Dermatology, Side
Effects and Recent Patents. Recent Patents on Inflammation & Allergy Drug Discovery, 6(2), pp.130-136.
3. Spielmann, H., Kalweit, S., Liebsch, M., Wirnsberger, T., Gerner, I., Bertram-Neis, E., Krauser, K., Kreiling,
R., Miltenburger, H., Pape, W. and Steiling, W., 1993. Validation study of alternatives to the Draize eye irritation test in
Germany: Cytotoxicity testing and HET-CAM test with 136 industrial chemicals. Toxicology in Vitro, 7(4), pp.505-510.
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Full-text available
Minoxidil, a vasodilator medication known for its ability to slow or stop hair loss and promote hair regrowth, was first introduced, exclusively as an oral drug, to treat high blood pressure. It was however discovered to have the important side-effect of increasing growth or darkening of fine body hairs; this led to the development of a topical formulation as a 2% concentration solution for the treatment of female androgenic alopecia or 5% for treating male androgenic alopecia. Measurable changes disappear within months after discontinuation of treatment. The mechanism by which it promotes hair growth is not fully understood. Minoxidil is a potassium channel opener, causing hyperpolarization of cell membranes and it is also a vasodilator, it is speculated that, by widening blood vessels and opening potassium channels, it allows more oxygen, blood and nutrients to the follicle. This can also cause follicles in the telogen phase to shed, usually soon to be replaced by new, thicker hairs in a new anagen phase. It needs to be applied regularly, once or twice daily, for hair gained to be maintained, and side effects are common. The most common adverse reactions of the topical formulation are limited to irritant and allergic contact dermatitis on the scalp. There have been cases of allergic reactions to the nonactive ingredient propylene glycol, which is found in some topical solution especially if they are galenic. Increased hair loss which can occur during Minoxidil use, is due to the synchronization of the hair cycle that the treatment induces. In this review, we described its mechanism of action, use in dermatology and some patents related to alternative treatment of allergic reactions due to its use.
According to OECD guideline 405 revised in 1987 Draize eye tests need not be performed for severely irritating and corrosive chemicals if results from 'well-validated alternative studies' are presented. In 1988 a validation study on alternatives to the Draize eye test was started in Germany to establish 'well-validated alternative methods' for this purpose. During database development, the last stage of the validation programme, 136 chemicals from the German chemical industry were classified in a blind trial with the 3T3 cell neutral red/kenacid blue cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test using fertile chicken eggs. The major goal of this stage of validation was to demonstrate the feasibility and limitations of the two alternative methods. Chemicals were, therefore, selected as representatives of chemical structural groups as well as of physicochemical and toxicological properties. In addition, some of the chemicals were chosen because they were of interest to the cosmetic and detergent industries. Draize eye testing data in vivo were provided by industry. In contrast to data from a previous interlaboratory assessment trial, it was impossible to correlate cytotoxicity data to the EEC classification for in vivo eye irritation. However, seven of 10 severely irritating chemicals (EEC labelling R-41) could be identified correctly in the HET-CAM assay, whereas test conditions of the study described here did not allow identification of irritating chemicals (EEC labelling R-36). The HET-CAM test is, therefore, fulfilling the criteria of a 'well-validated alternative method' according to OECD guideline 405 and should be incorporated into eye irritation testing at the earliest possible stage to reduce effectively the suffering of rabbits in the Draize eye test. Although an 80% correct prediction of 'non-labelled' chemicals in the HET-CAM test is encouraging, for safety assessment of non-irritant chemicals, for use as cosmetic formulations, for example, both government and industry will accept an in vitro assay only if its prediction of the absence of irritant properties is 100% correct.