Universidade de Lisboa
Faculdade de Farmácia
Evaluation of Hair Dyes Influence on the
Epidermal Barrier Using an In Vitro Test
Joana Rita Fernandes Castilho de Brito
Mestrado Integrado em Ciências Farmacêuticas
Universidade de Lisboa
Faculdade de Farmácia
Evaluation of Hair Dyes Influence on the
Epidermal Barrier Using an In Vitro Test
Joana Rita Fernandes Castilho de Brito
Monografia de Mestrado Integrado em Ciências Farmacêuticas apresentada
à Universidade de Lisboa através da Faculdade de Farmácia
Orientador: Professora Associada, Doutora Helena Margarida
Ribeiro
This work was developed through the Erasmus+ Programme, with the practical part being developed in the Faculty of Pharmacy, University of Ljubljana. The development of the practical work had Prof. Mirjam Gonseča as supervisor. All this work could also count on the help of Assistant Prof. Maja Bjelošević.
“The greatness of a nation and its moral progress can be judged by the way its animals are treated.”
5 Resumo
O ser humano possui o desejo intrínseco de melhorar a sua aparência. As tintas para o cabelo tornam-se assim um produto cosmético muito popular, que não conhece barreiras de género ou etárias.
A utilização de tintas para o cabelo remonta à época dos egípcios, há cerca de 4000 anos, onde as mulheres utilizavam um pó, feito a partir de folhas de henna, para pintar o cabelo, as unhas, ou até partes do corpo. Hoje em dia, podemos optar por pintar o cabelo em casa ou fazê-lo com um profissional num salão de cabeleireiros. Encontramos também ao nosso dispor uma grande variedade de cores, marcas e diferentes formas de colorir os nossos cabelos. Dentro desta categoria, as tintas permanentes possuem a maior quota de mercado, 80%.
Existem inúmeras razões para a utilização de tintas para o cabelo, sendo a cobertura dos cabelos brancos apontada como a principal razão. Os aspetos psicológicos da transformação da cor do cabelo, relacionados muitas vezes com uma aparência mais jovem, são o principal impulsionador da utilização destes produtos cosméticos.
De acordo com as estatísticas, mais de 70% das mulheres no mundo desenvolvido admitem pintar o seu cabelo pelo menos uma vez na vida e grande parte fá-lo de uma forma regular. Devido à extensa utilização deste tipo de produtos e à sua elevada complexidade química, a segurança e regulamentação são aspetos muito importantes para garantir elevados padrões de segurança para os consumidores.
Em todo o mundo, a legislação de produtos cosméticos tem vindo a sofrer inúmeras mudança e a preocupação com os testes realizados em animais encontra-se no topo das prioridades. Na Europa, em 2003, foi publicada a 7ª Alteração à Diretiva de Cosméticos que estabeleceu a proibição de testes realizados em animais para ingredientes e produtos cosméticos acabados, bem como a proibição da sua comercialização. Em 2013, após um período de phasing out, esta lei entrou em vigor.
Pelo contrário, na China, estima-se que todos os anos são usados entre 100.000 a 300.000 animais para testar produtos cosméticos. Em 2014, o governo chinês procedeu a algumas alterações e estabeleceu que para cosméticos comuns produzidos no próprio país, os testes em animais deixariam de ser um requisito obrigatório para a sua comercialização. Segundo as estimativas, a China tornar-se-á a maior potência mundial no que respeita ao mercado dos cosméticos, tornando-se preponderante terminar com os testes realizados em animais, sobretudo neste mercado.
6 Muitos utilizadores destes produtos cosméticos não se encontram cientes dos efeitos que estes podem provocar, a curto e a longo prazo. Torna-se assim importante estudar a influência que estes produtos podem ter na pele, especialmente na sensibilização ou irritação da pele.
Este trabalho teve como objetivo estudar a influência das tintas para o cabelo na barreira epidérmica da pele. A avaliação realizou-se indiretamente recorrendo a um modelo in vitro. Para a realização destes testes foi utilizada pele de orelha de porco, colocada em células de difusão de Franz. As alterações na barreira epidérmica foram monitorizadas através da medição da perda de água transepidérmica (TEWL), que é considerado um critério para a avaliação da função de barreira da pele.
Os primeiros testes foram realizados utilizando dois controlos: a água como controlo negativo e uma solução de SDS com uma concentração de 20% como controlo positivo. Posteriormente, foram testadas 10 combinações diferentes de 8 cremes e quatro reveladores para avaliar a mudança produzida por estes nos valores de TEWL, após a sua aplicação, em relação aos valores basais, previamente medidos.
Pode concluir-se que os todos os produtos cosméticos testados têm um efeito nocivo ao nível da barreira epidérmica da pele, uma vez que todos produziram um aumento nos valores de TEWL após a sua aplicação na pele. Segundo os resultados produzidos, parecem também não existir diferenças significativas entre as duas marcas testadas e os efeitos por estas produzidos ao nível da barreira cutânea. Através deste trabalho não foi possível confirmar a participação do H2O2 na perturbação da barreira
da pele, nem relacionar a percentagem e a quantidade em que se encontra na formulação e a magnitude dos efeitos produzidos.
Os testes realizados foram considerados insuficientes para averiguar a relação entre os compostos testados e os efeitos produzidos ao nível da pele, não permitindo também retirar elações acerca da relação entre um composto em particular e as mudanças verificadas na barreira epidérmica. Por outro lado, os testes foram realizados com recurso a pele de orelha de porco, o que não permite extrapolar os resultados obtidos para a pele humana.
Palavras-chave: sensibilização da pele, tintas para o cabelo, perda de água transepidérmica, células Franz
7 Abstract
The human being has the intrinsic desire to improve his appearance. Therefore, hair dyes have become a very popular product, which knows no gender or age barriers. According to statistics, more than 70% of women in developed world admit dyeing their hair at least once in their lifetime and most do so on a regular basis. Due to the extensive use of this type of products and their high chemical complexity, safety and legislation are very important aspects to ensure high standards of safety to consumers.
Many users of these cosmetic products are not aware of the short-term and long-term effects they may cause. It is therefore important to study the influence these products can have on the skin, especially in the sensitization or irritation of the skin.
This study aimed to study the influence of hair dyes on the epidermal barrier of the skin. The evaluation was performed indirectly using an in vitro model. Changes in the epidermal barrier were monitored through the measurement of TEWL, which is considered a criterion for the evaluation of skin barrier function.
The first tests were performed using two controls: water as a negative control and a 20% SDS solution as a positive control. Subsequently, 10 different combinations were tested to evaluate the changes they can produced in the TEWL values, after their application, in relation to the basal values.
It can be concluded that all the cosmetic products tested have a deleterious effect on the epidermal barrier of the skin, since all produced an increase in TEWL values. According to the results, there also appear to be no significant differences between the two brands tested and the effects produced on the epidermal barrier. Through this work, it was not possible to confirm the of involvement H2O2 in skin barrier disturbance, nor to
relate the percentage and amount of H2O2 present in the formulation and the magnitude
of the effects produced.
The tests performed were considered insufficient to ascertain the relationship between the compounds tested and the effects produced on the skin, also not allowing elicit associations regarding a particular compound. On the other hand, the tests were performed using pig ear skin, which does not allow extrapolating results obtained for human skin.
9 Acknowledgments
This dissertation arises not only due to the long and hard work developed and to all the dedication and commitment but also due to the support, friendship and affection of people who were an added value to make this work possible.
I would like to thank to Prof. Dr. Helena Margarida Ribeiro for all academic support, guidance and supervision.
I also would like to thank to Professor Mirjam Gonseča and Assistant Prof. Maja
Bjelošević for all the dedication, support and help during my experience abroad.
I would like to thank to Prof. Graça Bernardino, who is also a friend, for all the support that contributed to the conclusion of this work.
I would like to extend my sincere gratitude and appreciation to my colleagues and friends Inês Beato, Inês Correia, Inês dos Reis Correia, Joana Godinho, Joana Lima, Joana Maia, Patrícia Gomes, João Filipe Gonçalves, Guilherme Lopes, Miguel Teixeira, Mariana Correia, Maria Campos, Catarina Gutierres and Cristina Ramalho for all the help, true friendship and support throughout my academic journey. I would not have accomplished these years without all of you.
I leave words of appreciation to Filipe Loureiro for all the love, support and especially for all the patience and dedication, that were remarkable, without you this work would never have been accomplished.
A special word to express my profound gratitude towards my lovely parents, Luísa and Raúl Brito, my dear grandmother Rosária António, my brother Ricardo Brito, my cousin Ivo Fernandes and to all my family for all their unconditional love and encouragement, which were always present, even being thousands of miles away.
11 Abbreviations List
ADC Allergic Contact Dermatitis AOP Adverse Outcome Pathway APC Antigen Presenting Cells DPRA Direct Peptide Reactivity Assay
EU European Union
EURL ECVAM European Union Reference Laboratory for Alternatives to Animal Testing GPMT Guinea Pig Maximization Test
HMT Human Maximization Test
HRIPT Human Repeated Insult Patch Test LLNA Local Lymph Node Assay
RH Relative Humidity SC Stratum Corneum SD Standard Deviation SDS Sodium Dodecyl Sulfate
SP Subrina Professional
T Temperature
13 Contents
1. Introduction ... 17
2. State of Art ... 19
2.1. History of Hair Dyes ... 19
2.2. Plant Colours ... 19
2.3. Types of Hair Dyes ... 20
2.3.1. Temporary Hair Dyes... 20
2.3.2. Semi-permanent Hair Dyes... 21
2.3.3. Demi-permanent and Permanent Hair Dyes ... 21
2.3.3.1. Oxidative Dye Formation ... 22
2.4. Skin ... 23
2.4.1. Epidermal Barrier ... 24
2.4.2. Hair and Scalp ... 24
2.5. Methods for Assessing Skin Irritation and Sensitization ... 25
2.5.1. Irritation Tests ... 26
2.5.2. Sensitization Tests ... 27
2.6. Transepidermal Water Loss (TEWL) ... 30
2.6.1. Equipment ... 30
2.6.2. The Measuring Principle of Tewameter® TM 3000 ... 32
2.7. Legislation ... 32
2.7.1. European Union (EU) Cosmetics Legislation ... 33
2.7.2. Chinese Cosmetics Legislation ... 34
3. Purpose of Work ... 35
4. Materials and Methods ... 37
4.1. Materials ... 37
4.1.1. Hair Dyes and Colour Developers ... 37
4.1.2. Reagents and Solvents ... 39
4.1.3. Equipment ... 39
4.2. Methods ... 40
4.2.1. Skin Preparation ... 40
4.2.2. Preparation of Receptor Medium ... 40
14
4.2.4. Sample Preparation ... 41
4.2.5. Execution of the Measurements ... 42
5. Results and Discussion ... 45
5.1. Preliminary Testing ... 45
5.2. Hair Dyes Testing ... 47
6. Conclusion ... 53
Bibliography ... 55
Appendix ... 59
A1. Colour Creams Composition ... 59
15 List of Figures
Figure 2.1 - The three main steps in oxidative dye formation, available from (1)
... 23
Figure 2.2 - Schematic illustration of a condenser-chamber measurement head, available from (29) ... 31
Figure 2.3 - Schematic illustration of an open-chamber measurement head, available from (29) ... 32
Figure 4.1 - Ingredients of Subrina Professional Hair Colour Cream ECHOES 37 Figure 4.2 - Ingredients of Schwarzkopf Professional Hair Colour Cream Essensity ... 38
Figure 4.3 - Complete Franz Cells ... 41
Figure 5.1 - TEWL measurements for negative control (H2O) ... 46
Figure 5.2 - TEWL measurements for positive control (20% SDS) ... 47
Figure 5.3 – Combinations grouped in 2 groups ... 47
Figure 5.4 – Comparison between Combinations 3, 6 and 7 ... 49
Figure 5.5 – Comparison between Combinations 6 and 10... 49
Figure 5.6 - Comparison between Combinations 5 and 8 ... 50
Figure 5.7 – Comparison between Combinations 3 and 4 ... 51
Figure 5.8 – Comparison between Combinations 4 and 5 ... 51
List of Tables Table 4.1 - Subrina Professional Echoes Colour Developers Ingredients ... 39
Table 4.2 - Schwarzkopf Professional Colour Developer Essensity Oil Ingredients ... 39
Table 5.1 - Interpretation of the measured values of TEWL in healthy skin (27) ... 45
Table A1. 1 - Colour Creams Constituents ... 59
Table A1. 2 - Colour Creams Composition ... 59
Table A2. 1 - TEWL measurements for combination 1 ... 61
Table A2. 2 - TEWL measurements for combination 2 ... 61
Table A2. 3 - TEWL measurements for combination 3 ... 62
Table A2. 4 - TEWL measurements for combination 4 ... 62
Table A2. 5 - TEWL measurements for combination 5 ... 63
Table A2. 6 - TEWL measurements for combination 6 ... 63
16 Table A2. 8 - TEWL measurements for combination 8 ... 64 Table A2. 9 - TEWL measurements for combination 9 ... 65 Table A2. 10 - TEWL measurements for combination 10 ... 65
17 1. Introduction
Cosmetic products are present in our everyday life, since they are crucial for our daily hygiene and well-being. Hair dyes are no exception in this field, owning to the fact that hairstyle, including hair colour, play an important role on the overall appearance, which we present to the outside world.
The use of hair dyes evolved through the centuries: vegetable preparations were initially used, such as henna, nowadays we can choose between dyeing our hair at home or doing it with a help of a professional at a salon. We also have a wide range of different products in the market for different results.
The development of electronic technology, especially colour television, has had a major influence on the use of these products since it started to show a more clear and real image of the people and the world. Therefore, hair colours eventually gained a new meaning.
Over the years, the technology has brought more changes in the world of cosmetics and more specifically for hairstyling. A wide range of different colours, shades, brands, and different ways to dye our hair have invaded the markets all over the world. Within this category, permanent or oxidative hair dyes hold the largest market share with 80%.
According to statistics, more than 70% of women in the developed world dye their hair at least once in a lifetime and a large proportion of them do it regularly. Due to their great chemical complexity, safety and regulation are two key aspects of modern hair dyes. (1,2)
Many people use this type of products without being aware of their side effects, in short and long term. It has thus become important to study the influence that these products have on skin problems, especially on skin sensitization.
19 2. State of Art
2.1. History of Hair Dyes
Humans have an intrinsic desire to improve their appearance, therefore hair dyes are a popular cosmetic product among users of all age groups and gender, as they allow the change of the natural colour. There are a greater number of reasons for people to use hair dyes: to hide gray hair, to lighten hair colour or add an additional highlight, to remove the yellow look from gray hair, or to enhance the natural hair colour but the number one reason is to cover their gray hair. The psychological aspects of hair transformation associated with hair dyes are a key decision driver for the use of these products.(1)
The use of hair dyes can be traced back at least 4000 years, to the Egyptians. Powders made from henna leaves, used by women to dye their hair and nails, were discovered in the tombs of the Pharaohs. Also in the Roman era, there are records of the use of hair dyes, lead combs and dyes made from various lead compounds were in common use at that time. It was common fashion for the ladies of the Roman aristocracy to have blonde hair and for that purpose they wore blonde wigs or bleached their hair.
Since the late 19th century, some synthetic substances, like
paraphenylenediamine, which were already used to dye animal fibers, began to be also used for colouring human hair.
After the mid-1920s the world of hair dyes had a great boost: the use of hair dyes has become a fashion phenomenon. Many people began to use this type of products as a fashion accessory since they allowed them to meet the desire of changing their appearance. The oxidative dyes have experienced some changes, which have revealed significant improvements for this cosmetic sector and have allowed some preconceived ideas to fade away.
In 1945, after the end of World War II, a lot of new colorants were developed and added to the great number already existing in the market. Nowadays, the hair dyes’ market has evolved greatly and has expanded beyond borders or religions, being now a very lucrative worldwide business. (1,2)
2.2. Plant Colours
Henna, Lawsonia inermis, is one of the most used vegetable dyes and it is used since thousands of years to dye hair, nails, and even different parts of the body, for example men used to dye their beards with this plant. Nowadays it continues to be used for this purpose in Arab countries.
20 This plant is a shrub that grows mainly in India, Tunisia, Arabia and Iran and it has different denominations for all these different parts of the globe. Its Egyptian name is Khenna, the Arab name is Al Khanna and its Indian name is Mendee. It can also be called red henna because it stains the skin reddish-brown. Its white flowers have a strong smell, so they are used to produce fragrances, while the leaves are dried and crushed to produce a greenish-yellow powder, which must be mixed with hot water before use. The paste produced from this mixture is applied on the hair and left on for 30 to 40 minutes before rinsing. The result of this colouring varies depends on the colour of the hair where the paste was applied. When henna is applied on dark hair it produces an auburn colour or a brown-orange colour but on white and light-coloured hair it produces a strong “carrot” shade.
Henna’s active ingredient is 2-hydroxy-1,4-naphtoquinone, a substance known as lawsone or hennotannic acid (C.I. 75480). This substance can be found as a glucoside in the leaves in an approximate amount of 1%. Lawsone reacts chemically with keratin, a protein that exists on skin and hair, in a process known as Michael addition, resulting in a strong permanent colour that lasts until the next wash of the skin or hair. (2,3)
A considerable number of studies have been devoted to henna and its use as a vegetable dye since some cases of allergic contact dermatitis have been reported. (4) Over the years and due to the evolution that has arisen in the cosmetic world, henna has been replaced by semi-permanent dye products. (2)
2.3. Types of Hair Dyes
According to the book Cosmetic Dermatology: Products and Procedures, hair dyes can be defined as “products that alter the colour appearance of hair temporarily or permanently, by removing some of the existing colour and/or adding new colour”. There are different types of hair dyes that can be classified according to their colour resistance and according to their capacity to reach the cortex or to not reach the cortex. (1,4)
2.3.1. Temporary Hair Dyes
Temporary hair dyes are generally formulated with high molecular acids or dispersed dyes, which in turn have little affinity for the hair and are quite soluble in the dye base. These are further complexed with a cationic polymer in order to decrease its solubility and increase its affinity for the hair. In turn, this complex is dispersed in the dye base through surfactants. This complex will form a film that will coat the outside of the hair shaft and its excess can be rinsed off. The reason for its temporary effect is that the dye will only deposit on the cuticle and so the bonding forces established between the
21 hair and the dye are weak. Therefore, they can be easily washed away with the next shampoo. (1)
Thereby, the purpose of this type of hair dyes is to allow a slight change or modify the natural hair colour, quickly and simply. The duration of these changes can vary from a few hours to some days and it should be easily washed off with the next use of shampoo. (1) The best way to describe temporary hair dyes is: “a rinse brings it, and a rinse takes it away”. (2) This type of formulations is typically presented in the form of shampoos and sprays.
2.3.2. Semi-permanent Hair Dyes
The main characteristic of these dyes is their low molecular weight, which allows them to diffuse into the outer layers of the cuticle, without having to form a firm bond with the hair protein. Nitro-dyes are the most important and most used type of dyes for the productions of semi-permanent dyes since they are able to penetrate into the cuticle of the hair. This is due not only to their small size but also to the fact that the negative charges of the hair surface do not influence them since they are non-ionic (uncharged). Because of their solubility in water, eventually, it will vanish completely with each shampoo since washing the hair opens the cuticle allowing the colour molecules to be washed off.
Semi-permanent hair dyes are considered to be the “direct” hair colouring since they are a mixture of preformed dyes and are ready to use without any kind of mixing prior to their application. This mixture is usually left on the hair for 20 to 30 minutes to achieve a meaningful colour change, although the results are very limited: no lightning and subtly blending gray hair. (2)
2.3.3. Demi-permanent and Permanent Hair Dyes
Demi-permanent and permanent hair dyes have in common the fact that they involve oxidative chemistry, just require different components to be mixed before their application. One component contains the dye precursors, the couplers and the alkalizing agent in a surfactant base and the other is a stabilized solution of hydrogen peroxide (developer). The major differences between these two groups are the type and level of alkalizing agent and the concentration of peroxide, which determines the effectiveness of the colouring. (1,2)
Demi-permanent colours typically use 2% hydrogen peroxide and low levels of alkalizing agent (usually monoethanolamine, not ammonia) resulting in a less efficient dye penetration but in a gentler hair dye than permanent dyes. Thereby, it will wash out
22 after approximately 24 shampoos. As a result, they can enhance and bright the natural hair colour and blend or cover up gray hair up to 50% but its potential to lighten the hair is practically null.
Permanent colours use up to 6% peroxide and typically contain ammonia as an alkalizer agent to increase the pH of the final product to values between 9.0 and 10.5, which allows total penetration across the cortex. The use of these hair dyes can lighten the hair significantly, make a real colour change in subtle or dramatic ways, and provide 100% gray coverage. Reapplication is only dependent on the rate of hair growth, which is usually required after 4 to 6 weeks, when gray hair starts to appear in the root line because of new hair growth. These are the most versatile and long-lasting hair dye products and are available in the great variety of shades. (1)
2.3.3.1. Oxidative Dye Formation
The reactions responsible for the colouring process of the demi and permanent hair dyes are redox types and require four major components: the aromatic amine with substitutions at positions ortho or para as the coupling bases, the reaction modifiers and alkalizing and oxidizing agents. (5) The formation of colour happens after mixing of two components, where the precursors undergo oxidation by hydrogen peroxide in an alkaline medium. This medium, with highly alkaline pH, will swell the hair fiber and consequently promote the opening of the cuticles allowing the small dye molecules to penetrate the cortex. (1,5) The most commonly alkalizing compounds used are ammonium hydroxide and monoethanolamine, when the formulations contain water, or sodium salicylate when it is in the solid form (powder). (5)
The first step of this process is the oxidation of the primary intermediates or oxidation basis to highly active p-benzoquinone imines. In the presence of couplers, or colour modifiers, the nitrogen atom of imines, preferentially, reacts with the most nucleophilic carbon atom of the couplers molecule. In the third step, the previously formed product is oxidized to form wash-resistant indo dyes.
As constituents they also have reducing agents and oxidants. Reducing agents are added to oxidative formulations to prevent the auto-oxidation of the dyes during their shelf life while the antioxidants are added to avoid the onset of the reaction from occurring without the prior addition of the oxidant itself. The oxidants used are hydrogen peroxide, when the vehicle is water, and sodium persulfate, when it is a powder. The hydrogen peroxide of the formulation also acts as a bleaching agent on the natural pigment of the hair.
23 The final result is the formation of a colour complex with high molecular weight, which makes it difficult to escape from the hair structure and consequently, resistant to hair washes. (1,5)
The colour result depends on factors such as the precursors and the direct dyes present in the dyeing solution, the pH and the contact time between the hair and the dyeing solution.
Figure 2.1 - The three main steps in oxidative dye formation, available from (1)
Oxidative dyes have been shown to cause several changes to the hair structure. This is due not only to its mechanism of action but also to the ingredients involved in the reactions that cause transformations at the epidermal barrier, that ultimately lead to disturb and disrupt it, contributing to increase the values of transepidermal water loss. Furthermore, their frequent use and re-application also contribute to these compounds staying in contact with the hair and the scalp more often, increasing the probability and severity of their side effects. For example, the outer hydrophobic surface barrier of hair, made of 18-methyl eicosanoic acid, is partially removed by hydrogen peroxide. Its destructions cause significant changes in important hair properties, since this layer works as a natural protection or conditioning system of the hair. The resulting physiochemical changes of the hair surface can be irreversible. (1)
2.4. Skin
The skin is considered to be the body’s largest organ. It is the interface between the body and the environment and plays an important role in multiple defensive and regulatory functions in the body. (1) It consists of several layers that differ from each other in their composition and structure. There are two main layers of tissue: epidermis and dermis and another compartment which is hypodermis, the deepest layer. Epidermis
24 is the outermost layer of the skin, stratified with several layers of epithelial tissue and the stratum corneum is its outermost layer. This hydrophobic layer is the main component of the epidermal barrier, made from dead cells. Dermis is a hydrophilic layer composed of connective tissue providing mechanical strength to the skin.
It is a barrier against many external factors, including physical, chemical, and biological. The skin also acts as a protective barrier since it ensures the integrity of the body by preventing the loss of important substances from the organism, such as ions, water, and serum proteins. (6–10)
2.4.1. Epidermal Barrier
Nowadays, it is considered that more than 90% of the protective barrier function, attributed to the skin, resides in its outermost layer, the epidermis, more particularly in the stratum corneum. The stratum corneum is composed of anucleated cells, called corneocytes, that are formed by differentiation of keratinocytes from the granular layer of epidermis. It limits the transcutaneous movements of water through the skin, allowing it to remain in the body in sufficient quantities to survival. The movement of water depends on several factors, among them: the integrity of the SC and the size of its cells, the degree of hydration of the skin and the state of the intercellular lipid matrix. (11)
The epidermal barrier is not completely impermeable to chemical substances directly applied to the skin surface. This is the underlying principle of topical dermatology treatments as well as the application of transdermal systems. Frequent, or even daily, exposure of epidermal barrier to hair chemicals, like hair dyes, can compromise its properties and cause unhealthy conditions for the skin. Some of the problems that may arise are the allergic reactions to these topically applied products, resulting in redness and itching and leading to disturbances in the skin barrier functions. These disturbances lead to increased transepidermal water loss (TEWL). (1,8)
2.4.2. Hair and Scalp
The scalp is composed of five layers: the skin, connective tissue, epicranial aponeurosis, loose areolar tissue, and pericranium. The skin, the outermost layer, is thick and composed of numerous sebaceous glands and hair follicles. The connective tissue is a fibrofatty layer that is localized beneath the skin and contains the nerves and blood vessels that irrigate the skin. The epicranial aponeurosis is a thin layer consisting of dense fibers that form a tendinous structure. These three layers function as a single layer since they are bound together. The loose areolar tissue is made up collagen I and III and is also rich in glycosaminoglycans. This is considered a danger zone since infectious
25 agents can easily spread, through emissary veins, into the cranium. The pericranium is the periosteum of the skull bones. (12)
As mentioned, the scalp is composed of an abundance of large hair follicles that produce long hair fibers with large associated sebaceous glands, thus differing from the skin covering the rest of the body. From the hair follicle is generated a hair fiber whose main constituents are sulphur-rich proteins, lipids, melanin, and water. Hair is composed of dead keratinized cells, being keratin its main protein.
The hair shaft is divided into three main components, from the outside to the inside: cuticle, cortex, and medulla. The cuticle is also composed of keratin and it contains 6 to 8 layers of overlapping cells. The cuticle is divided in two parts: endocuticle and exocuticle. The exocuticle is composed of three important layers: b-layer, a-layer and epicuticle. The epicuticle or f-layer is a hydrophobic lipid layer with 18-methyl eicosanoic acid being the main constituent. It acts as a defense barrier for the hair fiber. The cortex is the principal component of the hair shaft and it consists of cylindrical cells rich in keratin filaments and an amorphous matrix of sulfur proteins. Cysteine residues in adjacent keratin filaments form covalent disulphide bonds, which confer stability and shape to the hair shaft. The matrix contains melanin granules which are responsible for the hair natural colour and its photo protection. The medulla is the innermost region of the hair shaft and its presence along the hair can be continuous, discontinuous, or even absent. It can be empty or filled with keratin, can serve as a pigment reservoir, and can contribute to the brightness of the hair. (1,5)
Humans have between 90 and 150 thousand of hair fibers, however the normal daily loss of hair is about 50 to 100 hairs. The hair follicle is a complex structure, consisting of several layers, with the ability to renew itself, due to the life cycle of hair fibers, that allows its growth, its fall out and the subsequent renewal. Its complex structure is formed to provide resistance to multiple environmental aggressions, such as UV radiation, and to some chemicals.
Virgin hair is the healthiest and strongest but simple gestures that are practiced daily, e.g. brushing and shampooing, can inflict damage on the hair. The cosmetic manipulation can also cause some disturbances on the hair. (1)
2.5. Methods for Assessing Skin Irritation and Sensitization
The roots of animal testing date back to early Greece, where physician-scientists developed and performed animal experiments to promote the evolution of, for example, anatomy, physiology, and pharmacology. Over the years, animal testing has become
26 quite popular and transversal to many industries (pharmaceutical, cosmetic, among others), revolutionizing the studies of products that would later be used in humans.
In the 20th century, the growing concern with animal tests, especially due to the
suffering caused to these beings, gained strength and the abolition of animal testing began to be idealized. New concepts ad approaches have emerged for the discovery and validation of methods that can replace animal tests. The concept of 3Rs was established to Replace animal tests by non-animal methods, to Refine the animal tests in order to reduce the stress and suffering of these beings and to Reduce the number of animals used for the tests. (13) The term “alternatives” was first described by David Smyth in his book “Alternatives to Animal Experiments” in 1978 and has since been used to describe the replacement of animals which may occur due to any change to established procedures. (14)
Nowadays, some of these methods are no longer considered the alternative methods since some, which have already been approved and are already in use, are the standard methods.
2.5.1. Irritation Tests
Adverse skin effects, such as irritation, may occur due to topical exposure of irritants. These substances can be classified as acute irritants, when only through a one-time exposure they cause irritation, and cumulative irritants, when they trigger an irritant response only after repeated exposures to the same substance in the same skin area. The irritant substances lead to a reversible local inflammatory reaction, which is manifested by redness (erythema), swelling (edema), and itching. This is the result of a cascade of events that begins with the penetration of an irritant substance into the stratum corneum, which will also cause damages in the adjacent layers, for example in keratinocytes. (15,16)
These substances must undergo some tests before they can be used in products for human use, so that only substances with a risk/benefit ratio as lower as possible can be used to formulate these products. Currently, officially accepted tests for assessing skin irritation include the traditional in vivo test (Draize rabbit test) and some in vitro tests which are based on RhE technology (Reconstructed human Epidermis). This technology is a three-dimensional model based on reconstructed human epidermis made from non-transformed keratinocytes, derived from the human epidermis, which are cultured to form a multi-layered highly differentiated model of in vitro human epidermis. This model mimics the biochemical and physiological properties of the most superficial parts of human skin since the multilayers are constituted by the basal, spinous and granular
27 layers and by a multi-layered stratum corneum. There are three in vitro test methods validated by ECVAM, so far: EpiSkin™, (modified) EpiDerm™ SIT, and SkinEthic™ RHE. These in vitro tests have been replacing tests requiring the use of animals since there is a growing concern for animal welfare. (15,16)
The cumulative irritation test (CIT) is an in vivo method used to evaluate the cumulative skin irritation potential of leave-on substances. It is used to test skin care and cosmetic products and compounds. The samples are applied directly to the skin using clinical patches, for a pre-defined period of time. These tests are performed in human volunteers. These volunteers are left with the patch placed on their body, for an established period, and after each patch removal a score is given according to the effects observed on the skin. (17)
The Draize rabbit test is an increasingly disused model, since the substances are tested on the skin of a living animal. Healthy young adults of albino rabbits are the preferred animals for this type of testing. The selected samples are tested by applying 0.5 ml, in the case of a liquid, or 0.5 g, in the case of a solid or semi-solid, on the experimental animal’s skin. Skin areas that are not treated with any kind of substance are used as controls. The degree of irritation may be observed after defined time intervals and the registration of this degree is done by assigning a score. The duration of this type of study should be sufficient to assess the reversibility or irreversibility of the observed effects. (18)
2.5.2. Sensitization Tests
Skin sensitizers are substances capable to elicit an allergic response after a first contact between the skin and an allergen, in sensitized individuals. This type of allergy is called allergic contact dermatitis (ACD), when it happens in humans. Skin sensitization is a process that involves two steps: the development of a specialized immunological memory, after the first contact with an hapten, and the production of a specific immune response, after a new contact with the same allergen. Contact allergens, designated haptens, are low molecular weight compounds that bind to skin components, such as proteins, and form conjugates that function as antigens. These antigens are presented to antigen presenting cells (APC) and recognized by T-cells. After these phenomena, the sensitized individual can trigger an allergic contact dermatitis when a new contact with the same allergen occurs. (19,20)
The ability of a substance to be a skin sensitizer can be studied using standardised methods in vivo, in animals and humans, and in vitro. Currently, internationally accepted animal tests to assess the skin sensitization potential include
28 the Mouse Local Lymph Node Assay (LLNA) and its non-radioactive modifications (LLNA-DA and LLNA-BrdU Elisa), the Guinea Pig Maximisation Test (GPMT) and the Buehler occluded patch test in the guinea pig. (19)
The GPMT is a highly sensitive method and it includes topical and intradermal induction and closed patch for 48 hours. Initially, the guinea pigs are exposed to the test substance by three pairs of intradermal injections given in the shoulder region. Between day 6 and day 8, a filter paper, containing the test substance in an appropriate vehicle, is applied to the test zone by occlusion for 48 hours. Animals belonging to the control group receive the same filter paper containing only the vehicle previously used. After a rest period, between the 20th and the 22nd day, two patches are placed on their flanks:
one containing the test substance and another containing only the vehicle. The patches are occluded for 24 hours and the skin reaction, erythema and oedema, is evaluated 24 and 48 hours after removal of the patch. The Buehler test is similar to the test described above. It consists of three phases of occlusive induction during 6h. The second phase occurs between days 6 and 8, and the third phase between days 13 and 15. Between days 27 and 29, an occlusive patch test, containing the maximum non-irritating concentration of the test substance, is performed. These are applied for 6 hours and the skin reaction is evaluated 24 and 48 hours after removal of the patches. In both guinea pig tests, the evaluation of coloured substances, such as pigments, is often impossible due to the staining that they cause on the skin. (21–23)
In the LLNA method, mice are divided into 4 groups, including a control group, and are exposed to the test substance by application to the back of the ears for 3 consecutive days. On day 6, lymphocyte proliferation in the lymph node, caused by the primary immune response, is measured by the incorporation of 3H-methyl thymidine into
the DNA of proliferating lymphocytes. Subsequently, the animals are sacrificed and the contents of the lymph nodes that drain the ears are removed so that the DNA can be extracted from lymphocytes. The proliferation of lymphocytes is proportional to the dose and to the potency of the applied substance as an allergen. The endpoint of this method is the stimulation index, calculated from the ratio of thymidine incorporation in lymphocytes of mice treated with the test substance to those in the control group. (21,22) The differences between the method performed on mice or the methods performed on guinea pigs are mainly in the endpoint: LLNA measures the response induced by sensitization in animals, while the other two measure challenge induced elicitation reactions in previously sensitised animals. The LLNA is considered a reduction and refinement method, when compared to the tests performed on guinea pigs, since it requires a smaller number of animals and reduces the pain and distress caused. These
29 animal test methods have been an added value to assess the safety of cosmetic ingredients. (19,20,24,25)
As mentioned before, skin sensitisation tests can also be performed using human volunteers. Some of these tests include Schwarz-Peck Test, Human Repeated Insult Patch Tests (HRIPT) and Human Maximisation Test (HMT). The Schwarz-Peck Test detects only potent sensitizing substances and is considered obsolete when compared to other human tests. It consists of the topical application of different doses of the same substance for 24, 72 or 96 hours, in the induction phase, followed by a rest period of 10 to 14 days. Subsequently, the challenge phase occurs, in which a patch is applied for 48 hours. After its removal, the skin reaction is scored.
Nowadays, HRIPT is only used to ensure the safe use of a potentially sensitizing substance in consumer products, such as cosmetics. The HRIPT can be performed at least through four different ways. It is also common practice to use one test to test multiple substances at the same time, as it saves time and costs. The test includes an induction phase, where volunteers are exposed to a substance 9 x 24 or 48 hours for 3 weeks. After a rest period of 2 weeks, the challenge phase is then performed, where patch tests are applied to two different locations: one previously exposed to the substance and the other that has not been exposed, for 24 or 48 hours. These tests are usually performed in groups of, approximately, 100 volunteers. (21,22)
The HMT includes an induction period consisting of five consecutive occlusive patch tests, always applied to the same place of the skin, with a rest period of 24 hours between their removal and reapplication. Substances with an irritant potential are applied at a concentration that generates moderate erythema, while non-irritating substances are applied to a skin site previously treated with 5% SDS for 24 hours. After a 2-week rest period, the extent of skin sensitization is assessed through a 24-hour occlusive patch test at a previously irritated skin site and using the maximum non-irritating concentration of the test substance. The effects verified on the skin are evaluated by assigning a score, after 24 and 48 hours after removal of the patch. (22)
Some methods have not yet been validated by EURL ECVAM, i.e. they were considered to be mechanistically relevant to contribute to reduce or replace animal tests but are not internationally accepted. These methods include the Direct Peptide Reactivity Assay (DPRA) and the KeratinoSens™. (19)
Currently, there are no formally validated or regulatory adopted alternative tests and it was proposed that non-animal tests would not completely replace animal testing,
30 but a combination of methods that address the key mechanisms of the sensitization adverse outcome pathway (AOP) will be required to achieve full replacement. (19)
2.6. Transepidermal Water Loss (TEWL)
As part of the normal metabolism of the skin, a certain amount of water evaporates from the skin. Transepidermal water loss (TEWL) represents the amount of condensed water that is diffused from the skin through the stratum corneum (SC), to the atmosphere, excluding perspiration. The measurements of the TEWL have been widely used to assess the skin barrier function since skin health influences the values of TEWL. A low TEWL value is generally associated with healthy skin conditions. On the contrary, when the skin is damaged or irritated or has its barrier function curtailed, consequently there is a greater amount of water evaporating from the skin, resulting in higher measured TEWL values. Elevated values of TEWL have been associated with an altered skin barrier function, such as in several skin diseases (e.g. atopic dermatitis). Therefore, TEWL measurements can evaluate the skin barrier properties and indirectly its integrity. They can also be used to assess the influence of a topically applied substance to the skin. However, it only reflects the inside-out barrier function. Some evidence lead us to suggest that when the skin barrier is damaged or disturbed, the absorption of chemical substances, that previously could not penetrate the skin, increases.
TEWL values can be influenced by some endogenous, exogenous and environmental factors. The endogenous factors that may influence TEWL are, for example, age, ethnicity, anatomical position, sweating and skin temperature. Air convection or air movement, relative humidity and ambient temperature are some of the environmental factors that may influence TEWL measurements. Exogenous factors such as the use of solvents and surfactants and even the simple gesture of hand-washing can also influence the values of TEWL. Some experimental and instrumental related factors may also influence TEWL measurements but numerous of these factors can be controlled or minimized. (1,7,26–28)
2.6.1. Equipment
Different methodology settings are used in TEWL measuring devices: closed and open chamber. The closed-chamber method measures the increase of relative humidity (RH) in a chamber that is in the form of a cylinder with one end closed and the other end is placed in contact with the skin. The main problem with this method is its inability to perform continuous measurements because the water vapor accumulated inside the chamber needs to be removed so that it does not interfere with the following measurements. In order to solve this problem, a closed-chamber with a condenser, on
31 its upper end, was created to remove the water vapor from the chamber, allowing the measurements to be made continuously without needing to stop between them. The lower end of the chamber is open and is the end that is placed in contact with the skin surface. All these characteristics are illustrated in Figure 2.2. The condenser closes the measurement chamber protecting the diffusion zone from disturbance by ambient air movements and it controls the humidity inside the chamber despite the humidity of the surrounding area. (7,29,30)
Figure 2.2 - Schematic illustration of a condenser-chamber measurement head, available from (29) The open-chamber method is the only one that can assess the TEWL continuously without influencing its micro environment. This method uses a chamber with cylindrical design with both its lower and upper ends open. The main features of this chamber are illustrated in Figure 2.3, where the location of temperature (T) and relative humidity (RH) sensors can be observed. The humidity gradient generated by its both open ends allows the measurement of the density gradient of the water evaporation to be performed, since the water vapor migrates from the skin surface through the chamber to the surrounding environment, allowing a density gradient to be formed between two distinct points on the chamber. However, the open chamber method contains some limitations being the major one its vulnerability to disturbance from ambient air movements. (7,27,29,30)
32 Figure 2.3 - Schematic illustration of an open-chamber measurement head, available from (29)
2.6.2. The Measuring Principle of Tewameter® TM 3000
The open chamber instruments, for example Tewameter® TM 3000 (Courage & Khazaka GmbH, Köln, Germany) used in this work, are based on Adolf Fick’s Diffusion Law from 1855: 𝑑𝑚 𝑑𝑡 = −𝐷. 𝐴. 𝑑𝑝 𝑑𝑥 (2.1) where: A = surface (m2) m = water transported (g) t = time (h) D = diffusion constant (=0,0877 g7m.h.mm Hg) p = vapor pressure of the atmosphere (mm Hg)
x = distance from the skin surface to point of measurement (m) The diffusion law indicates the mass per m2 being transported in a period of time.
The probe measures the density gradient of the water evaporation from the skin indirectly by two pairs of sensors (temperature and relative humidity). The data obtained is processed by a microprocessor and the numerical values of TEWL are usually shown in g/h/m2. (7,27)
2.7. Legislation
All over the world the cosmetic legislation has suffered many changes and the concern regarding the animal tests has become a priority, although this subject is still highly sensitive, especially when it comes to cosmetics. The cosmetics industry believes that: “Ending animal testing is a benefit for all: consumers, animal welfare and industry”. (31) The Cosmetics Europe, the European trade association for the cosmetics and personal industry, (32) has been working at an international level to promote an increase in validation and acceptance of alternative methods, via collaboration with cosmetic
33 associations, regulatory bodies as well as academic bodies, industry regulators, such as the European Commission, and other industry sectors. Many of these alternative methods driven by the cosmetics industry, which is the pioneer in researching alternative methods to animal testing, have been adopted by other sectors, such as pharmaceuticals and chemicals industries. (31)
2.7.1. European Union (EU) Cosmetics Legislation
Every year, over five billion units of cosmetic products are sold in the EU and the predictions show that these numbers will continue to rise. For the cosmetics industry, the number one priority is the safety of consumers, therefore the EU cosmetics legislation has become increasingly restricted. The EU cosmetics legislation has a history with more than 40 years. Since 1976, when the Directive 76/768/EEC was published, where the member states claim their intention to harmonize their cosmetic regulations, at national level, with the purpose of ensuring a high level of consumer protection and allow the free circulation of these products within all the EU countries. (14)
Over the years, the concerns of the EU regarding this subject have grown and many new directives have been published and many decisions have been taken, always to ensure the possible higher standards for cosmetics sold within the community. In 1979, the Scientific Committee on Cosmetology, constituted by an independent group of experts, was established to advise on various matters related to cosmetics, e.g. safety of cosmetics ingredients, for regulatory decisions. (14)
The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 and since then has been validating methods which can reduce, refine or replace use of animals for safety and efficacy testing for chemicals, biologicals and vaccines. The first alternative method to assess skin irritation was accepted for cosmetics in 2000, after its prior validation by ECVAM and adopted by the EU and OECD. (14,25)
The 7th Amendment to the Cosmetics Directive was published in 2003 and
instated a test and a marketing ban, for animal tests performed in ingredients and finished cosmetic products, in the European Community. Thus, it was imposed a deadline for phasing out animal testing.
Recently, in 2013, after a phasing out period of ten years the animal testing ban of cosmetic products and ingredients entered into full force. The marketing ban was put into practice and it became prohibited to market cosmetic products that included ingredients tested on animals. (14,33–35)
34 2.7.2. Chinese Cosmetics Legislation
Until now, it was estimated that between 100.000 and 300.000 animals, e.g. rabbits, guinea-pigs, and mice, have been used to test cosmetics in China every year, since it was compulsory to conduct animal tests for any company that sold cosmetic products in the Chinese market. This causes an ethical dilemma for companies that wish to be part of China’s $26.3 bn cosmetics market. These companies had to decide if they go against the cruelty free company policy or miss this profitable market. Some cosmetic brands cannot afford to miss out this market so they ended up performing animal tests just because of Chinese laws. (35,36)
In 2014, the Chinese Government made same changes in their laws regarding to cosmetic products and it became no longer legally mandate for domestically produced ordinary and normal cosmetics to conduct animal tests. With this important step, China is revealing awareness of the global trend towards non-animal testing cosmetics. Although some products, e.g. foreign imported ordinary cosmetics and both foreign imported and domestically produced special use cosmetics, such as hair dyes, perms and hair growth products, deodorants, sunscreens, skin-whitening creams, and products that have functional claims on product labels, still require animal testing. Ending complete animal testing in China will be a long and lasting process and it will require support from other countries and international institutions. (35)
It is estimated that the consumption of cosmetic products in China reached a growth of 15% between 2014 and 2015, which includes domestic and online shopping and purchases made while travelling abroad. These numbers will continue to increase and China will become the world’s largest beauty market. The power of Chinese consumers will remain an economic concern, as they become the largest force driving the global beauty market. (37)
35 3. Purpose of Work
The purpose of this master thesis was to evaluate the influence of different hair dyes on skin irritation and sensitization using an in vitro test.
The evaluation will be carried out indirectly on a pig ear skin model, placed in static Franz diffusion cells. In doing so, we will monitor the change in transepidermal water loss (TEWL) values, which is considered a criterion for the assessment of the skin’s barrier function.
The first tests will be carried out to determine the two work controls: water, as a negative control, and a solution of SDS with a concentration of 20%, as a positive control. Posteriorly, we will test ten different combinations of eight colour creams and four developers to evaluate the change in TEWL values after the application of each combination regarding the basal values, previously measured.
37
4. Materials and Methods
4.1. Materials
Pig ear skin (ears acquired in January 2017 adequately stored in the freezer immediately after withdrawal).
4.1.1. Hair Dyes and Colour Developers
• Subrina Professional (SP) hair colour cream Echoes shades of 8/04, light brown (5/0), extra light blonde - ash (10/1), dark blonde – intensive brown (6/77), medium blonde – red (7/5) (Ilirija d.d., Slovenia)
• Subrina Professional hair colour cream Senseo 6/7
• Schwarzkopf Professional, hair colour cream Essensity of ultra blonde (10-19), dark blonde (6-0)
• Subrina Professional, colour developer 5.5% and 11.5% hydrogen peroxide (Ilirija d.d., Slovenia)
• Schwarzkopf Professional, colour developer Essensity Oil 5.5% and 11.5% hydrogen peroxide
Regarding the composition of the colour creams from different brands used to perform this work, when analysing the Figure 4.1 and Figure 4.2, we can verify that the colour creams Echoes have the same basic constitution that the colour creams Essensity. Both are constituted by a solvent, water, by a buffering, ethanolamine, which is in this case an alkalizing agent, by an emulsion stabilizer, cetearyl alcohol, and by a surfactant, which in the case of the Echoes brand is ceteareth-25 while in the Essensity brand is the glyceryl stearate. However, differences can be highlighted. The colour creams of Echoes brand contain some natural oils and butters, oleic acid which is a skin enhancer, polyquaternium-22 which is an antistatic agent and lauryl glucoside which is a surfactant that does not contain sulfates and which in this case contrasts with the use of betaine in Essensity formulation, which is an amphoteric surfactant.
38 Figure 4.2 - Ingredients of Schwarzkopf Professional Hair Colour Cream Essensity The pigments that constitute each of the colour creams are described in the Table A1. 2 in the appendix.
39 Regarding the developers’ composition used to perform this work, two different brands were used. However, we can see from Table 4.1 and Table 4.2 that the base composition of the developers is the same, as it happened in the colour creams. They consist of a solvent, water, an oxidizing agent, hydrogen peroxide, an emulsion stabilizer, cetearyl alcohol, and a surfactant, which in the case of Echoes developer is ceteareth-25 and in the case of Essensity is ceteareth-20. Regarding the differences found in the formulations, we can highlight the inclusion of beeswax in the Schwarzkopf formulation, which is a skin conditioner. The percentages of hydrogen peroxide used were, for each developer brand, 5.5% and 11.5%. In the case of Essensity, different volumes of hydrogen peroxide were used for the 5.5% and 11.5% formulations. For the formulation 5.5% was used a solution of hydrogen peroxide with 18 volumes, while in the 11.5% was used a solution with 38 volumes.
Table 4.1 - Subrina Professional Echoes Colour Developers Ingredients Subrina Professional Echoes Colour Developers Ingredients
Aqua PEG-40 Castor oil
Hydrogen peroxide Sodium cetearyl sulfate Cetearyl alcohol Disodium EDTA
Ceteareth-25 Sodium benzoate
Octyldodecanol Phosporic acid
Table 4.2 - Schwarzkopf Professional Colour Developer Essensity Oil Ingredients Schwarzkopf Professional Colour Developer Essensity Oil Ingredients
Aqua PEG-40 Castor oil
Hydrogen peroxide Sodium cetearyl sulfate Cetearyl alcohol Disodium pyrophosphate
Ceteareth-20 Potassium hydroxide
Cera alba (beeswax) 2,6 - Dicarboxypyridine Etidronic acid Isopropyl myristate
4.1.2. Reagents and Solvents
Purified water (prepared by reverse osmosis, at the Faculty of Pharmacy, Ljubljana)
Sodium chloride (INCI: sodium chloride, Lex Ltd., Koper) 4.1.3. Equipment
40
• Scale (Kern & Sohn GmbH, KB2400-2N, D-72336 Balingen, Germany)
• Water bath (IKA® RTC basic)
• Tewameter® TM 3000 (Courage & Khazaka GmbH, Köln, Germany)
o Probe Heater® PR 100 (Courage & Khazaka GmbH, Cologne, Germany)
o Condition of Ambient Sensor RHT 100 (Courage & Khazaka GmbH, Cologne, Germany)
4.2. Methods
4.2.1. Skin Preparation
In this work, the measurements were performed on pig ear skin, which had been properly stored in the freezer. The pig ear wasn’t totally used: the distal parts of the ear were divided into smaller parts.
On the day that the measurements were performed, the small pieces of the pig ear skin were taken of the freezer 10 minutes, before we started working with them, and left to thaw at room temperature. After this step, the skin was cleaned, if necessary, with gauze and distilled water to remove the dirt and with the help of a scissor the hair was cut off. The skin was separated from the cartilage with the help of a scalpel and split into 3 or 4 smaller pieces that can be adapted to Franz Cells.
4.2.2. Preparation of Receptor Medium
A physiological saline solution was used as a receptor medium, i.e. 0.9% sodium chloride. The 2,25g of sodium chloride were carefully weighed on the scale and added to a volumetric flask of 250mL. The purified water (prepared by reverse osmosis, Faculty of Pharmacy, Ljubljana) was added until it reached the ring graduation mark and mixed until there’s no powder to dissolve on the bottom of the flask.
4.2.3. Preparation of Franz Cells
Franz static diffusion cells represent the in vitro system, which are used for routine evaluation of dermal absorption and skin irritation. Each cell consists of a receptor moiety, where the skin sample is placed, and a donor part, where the test substance is applied to the skin. An appropriate membrane, in this case animal skin, is positioned so that the dermis is in contact with the receptor moiety and the epidermis is placed in the direction of the donor part.
The Franz cells were filled with 10mL of the previously prepared solution of the receptor medium using a micropipette (Transferpette® S, Brand GmbH & Co. KG,
41 a convex surface on the top, where the prepared skin is carefully placed. On top of the skin, the donor part is placed consistently with the receptor moiety. Finally, the excess of skin is cut off so that we can hinder further hydration with the water from the water bath.
The previously prepared Franz cells were placed in a water bath at 37ºC one hour prior to the samples application, with the water level on the receptor moiety. With the previous step we can simulate a human skin temperature, that is in fact about 32ºC on the surface of the skin.
Figure 4.3 - Complete Franz Cells 4.2.4. Sample Preparation
In this work, we evaluated ten different combinations of eight different coloured creams and four different developers. The combinations were the following:
• Combination 1: SP ECHOES 8/04 (colour 1) in SP 5.5% developer (developer 1), ratio: 1-1
• Combination 2: SP ECHOES 5/0 (colour 2) in SP 5.5% developer (developer 1), ratio: 1-1
• Combination 3: SP ECHOES 10/1 (colour 3) in SP 5.5% developer (developer 1), ratio 1:1
• Combination 4: SP ECHOES 10/1 (colour 3) in SP 11.5% developer (developer 2), ratio 1:1
• Combination 5: SP ECHOES 10/1 (colour 3) in SP 11.5% developer (developer 2), ratio 1:2
• Combination 6: SP ECHOES 6/77 (colour 4) in SP 5.5% developer (developer 1), ratio 1:1
• Combination 7: SP ECHOES 7/5 (colour 5) in SP 5.5% developer (developer 1), ratio 1:1
Donor part Skin sample Receptor medium
42 • Combination 8: Schwarzkopf Professional Essensity 10-19 (colour 6) in Schwarzkopf Professional Essensity Oil 11.5% developer (developer 4), ratio 1:2
• Combination 9: Schwarzkopf Professional Essensity 6-0 (colour 7) in Schwarzkopf Professional Essensity Oil 5.5% developer (developer 3), ratio 1:1
• Combination 10: SP Senseo 6/7 (colour 8) in SP 5.5% developer (developer 1), ratio 1:1
All combinations were prepared according the manufacturer’s instructions, regarding the ratio between the colour cream and the developer. These two products were weight on a scale and mixed together with the help of a mortar. The previously prepared combinations were directly applied in the skin sample through the donor part. Firstly, we applied a thin layer with the help of a swab, promoting the coating of the entire surface of the skin and preventing the formation of an intermediate layer of air between the skin and the hair dye sample, so that direct contact between the sample and the skin would be avoided. Finally, the rest of the hair dye sample was added using a plastic syringe until it reached an amount within the range of 0,4-0,6g.
4.2.5. Execution of the Measurements
The measurements of the transepidermal water loss (TEWL) were performed with a specific open chamber equipment which is the Tewameter® TM 3000 (Courage & Khazaka GmbH, Cologne, Germany). This device is connected to the computer unit Cutometer® MPA 580 (Courage & Khazaka GmbH, Cologne, Germany), which allows the use of several probes with the appropriate software (Software MPA). To fulfil this work, we used only one probe, the tewameter, which measures the current density of water vapor on the surface of the stratum corneum, that will travel through the probe into the atmosphere, which allows the continuous measurement of TEWL. Before performing the measurements, the tewameter probe is placed into the Probe Heater® PR 100 (Courage & Khazaka GmbH, Cologne, Germany), which has a pre-heating function, to achieve a temperature near to the skin temperature, around 35-37ºC. With the previous step, we can ensure the same temperature in the tewameter’s head probe and in the skin surface, to provide more stable measurements.
The Franz cells with the skin sample were placed in a water bath at 37ºC and after one hour the cells were removed from the bath and the measurement for the basal values of TEWL were performed. The donor part of each Franz cell was removed and the probe was horizontally placed in contact with the skin surface while the
43 measurements were carried out for 60 seconds. This procedure was performed for both types of tests, preliminary and hair dyes testing.
In the preliminary tests, after measuring the basal values, the water was applied on the skin sample. The three Franz cells used in this procedure were then placed on the water bath and after 35 minutes the water was removed from the skin with the help of a swab. The Franz cells were placed back in the water bath. After one hour, the measurements to obtain the final TEWL values were carried out using the same procedure described above to measure the basal TEWL values. This procedure was then repeated but, in this case, to carry out the preliminary tests with sodium dodecyl sulfate (SDS).
In the tests using different hair dye combinations, after measuring the basal values, the mixtures were applied on the skin sample and for each combination were used three Franz cells. These cells were placed, again, on the water bath and the hair dye combinations were carefully removed from the skin with the help of a swab after 35 minutes. According to manufacturer’s instructions, the combinations must be in contact with the hair from 30 to 45 minutes because only after this time it is possible to ensure colour reproduction. Furthermore, these products should not exceed this time of contact with the skin as they may be harmful to it. The Franz cells were placed back in the water bath for one more hour. Finally, we proceed to the final TEWL measurements, which were also carried out for 60 seconds.
