There are many different methods for testing the mildness of personal care products, and numerous factors that can affect the data collected. As a result, the definition of mildness will differ between laboratories. Therefore, one could argue that mildness is in the eye of the beholder. Recent work at the author’s company, shown here as a case study, has involved the development of mild body washes for sensitive skin, and examples of the tests used to describe the mildness of the products developed are given. Also, to understand skin’s responses to such tests, e.g., irritation and sensitization (see Figure 1), an overview of the mechanisms involved and skin conditions affected by cosmetic products are described, including skin barrier function and variations in skin sensitivity linked to skin disease, body site, ethnicity and age.
Skin Irritation and Irritant Contact Dermatitis
Mildness is most commonly associated with the absence of skin irritation. In contrast, an inflammatory response to a product is commonly associated with redness, heat, swelling and pain. Contact dermatitis is the medical term used to describe skin reactions resulting from exposure either to irritants, i.e., irritant contact dermatitis (ICD), or allergens, i.e., allergic contact dermatitis (ACD).
ICD is classified as a more immediate biological response to irritation. With ICD, the source of irritation may be a chemical, such as a cosmetic active, or abrasion to the skin. The immunological events in ICD start with the release of cytokines and other inflammatory mediators from activated or damaged skin cells. ICD involves the activation of the innate immune system and is distinct from ACD in that it does not involve the production of specific antibodies. Therefore, it does not involve the adaptive or acquired immune system. There are many different types of irritant contact dermatitis and degrees of severity,1 including, for example, chronic ICD, which is common on the hands and associated with work-related dermatitis. For a detailed review of the mechanisms of ICD and ACD see Rustemayer et al.2
In vivo irritation testing: While a range of human and animal models for testing skin irritation exists,3 the patch test remains the gold standard for the comparative evaluation of the irritation and sensitization potential of cosmetic products and ingredients. It usually involves the application of a product or solution to the skin, under occlusion, and the visual grading of the irritant response. Farage et al. provide a useful review of the development of patch test methods and the wide range of protocols and visual scoring methods used.4
Differences in patch test protocols still exist between laboratories. In order to address this, Mehling et al. have developed and cross-validated a protocol that allows the comparison of patch test results between laboratories.5 Their approach standardizes the number of panelists, the volume of the sample applied, the chambers used, the time points for skin evaluation, and the methods for grading skin reactions. The researchers also have introduced common positive controls, i.e., sodium laureth sulfate and sodium dodecyl sulfate, and negative controls, i.e, demineralized water. Using this approach, comparable data was generated across six test centers.
The primary method to evaluate skin irritation in patch tests is the use of visual grading scales; however, physical and biophysical skin testing methods are also available.4 These include spectroradiometers and chromameters to measure redness; laser Doppler flowmetry to measure blood flow; and evaporimeters to measure transepidermal water loss (TEWL). In general, it seems that all these techniques provide useful extra information but they do not improve the overall quality of the results obtained by visual grading.4
In situations where investigators look for inflammatory changes in skin that do not provide visible clinical irritation, more advanced techniques can be used. Perkins et al., for example, developed a tape stripping method for assessing skin irritation that involves the analysis of the stripped corneocytes for inflammatory markers.6 Also, Farage has investigated the use of a polarized light as an aid to visualizing low levels of irritation.7
Case study (part 1), soap chamber test on self-assessed sensitive skin: For the development of a mild body wash for sensitive skin, a soap chamber test was used to compare the potential irritancy of prototypes.8 In order to ensure mildness, blends of surfactants were used that would be expected to be mild to the skin while delivering sufficient cleansing and lathering. For the described work, 20-μL samples of 8% w/w product in water were applied to forearm skin, under occlusion, for five days; patches were applied for 23 hr on the first day, and six hours each for days 2–5. Skin erythema, scaling and fissures were assessed visually each day, one hour after the removal of the chambers. The total skin irritation was calculated by adding the three different attribute scores. Since the work focused on products for sensitive skin, tests involved subjects having self-perceived sensitive skin (n = 22). A questionnaire was used to recruit the panelists based on their responses to a series of statements regarding their skin’s response to cosmetic products.
Results showed that the test prototypes were significantly less irritating than the positive control, i.e., traditional bar soap (p < 0.001) (see Figure 2). All four products tested were significantly more irritating than the negative control (water) (p < 0.001). Also, the tests showed that the two test prototypes were less irritating than market benchmarks presented as mild/gentle body washes (p < 0.001). It was therefore concluded the given prototypes would be suitable for use on sensitive skin.
In vitro irritation testing: During the early stages of product development, it may not be practical for formulators to test the mildness of many prototypes with in vivo skin tests. Instead, the potential for skin irritation can be predicted using an in vitro assay. A number of assays have been developed to screen actives for their effects on proteins and to predict skin irritation. The collagen swelling test, for instance, measures the amount of extra water taken up by a collagen sheet/circle before and after treatment with a product.9–11 Products that can make collagen swell are believed to more likely be irritating.9, 10 The pH rise test looks at changes in the pH of a solution of bovine serum albumin after addition of product. The higher the pH rises, in theory, the more irritating the product.9 The zein test investigates the changes in water solubility of zein, a protein derived from corn, when a product is added.9 The more zein that is solubilized, in theory, the more irritating the product. Of course, the proteins of interest here are those in the stratum corneum (SC).
Squamometry is a technique that measures dye penetration into tape-stripped corneocytes.12 As with the test using collagen, it is assumed that the more the proteins in the SC swell from a treatment, the more potentially damaging and irritating the treatment is. Squamometry is claimed to identify the potential low or sub-clinical irritation produced by substances that have no immediate effect on TEWL.12
The red blood cell test is used for the rapid screening of the irritation potential of surfactants.13 The test uses the lysis of red blood cell membranes as well as cell protein denaturation to differentiate test compounds. Pape et al. claim a strong correlation between red blood cell tests data and in vivo eye irritation data.13
More recently, reconstructed epidermis systems have been developed that can screen actives and formulations for their irritation potential. Reconstructed epidermis consists of human epidermal cells cultured to produce human epidermis standardized in terms of thickness, terminal differentiation and reactivity to test compounds.14 While these models are complex and still being fully validated, they are already being used to predict skin irritation.11
A final and important point to note before leaving the area of skin irritation is that skin irritation from a cosmetic product can arise after a product has been removed. Loden et al. have shown, for example, that deposition of ingredients from mild soaps can form irritant reservoirs in the skin.15 Occlusion of the skin can therefore force the release of these ingredients and cause a delayed irritation response.15
Skin Sensitization and Allergic Contact Dermatitis
As noted, ACD is distinct from ICD in that it involves the adaptive immune system and the generation of antibodies to remove foreign substances from the body. The inflammation associated with ACD is an exaggerated interaction between an antigen and the immune system. Indeed, it is classified as a type IV hypersensitivity reaction.2
ACD is characterized by having an induction phase and an elicitation phase. In the induction phase, dendritic cells pick up allergens that have passed into the viable epidermis, transport them to the nearest lymph node, and present them to naïve T-cells. These reactions are mediated by inflammatory cytokines, chemokines and adhesion molecules. Once primed the T-cells produce antigen-specific effector T-cells that can quickly be recruited upon contact with the same allergen—elicitation. Upon re-exposure to the same allergen there is a strong immune system response. In most people, the reactions produced in ICD and ACD are clinically indistinguishable. Indeed, many of the principle inflammatory pathways essentially are similar.2 ACD differs in its immunological profile to atopic dermatitis (AD), commonly known as eczema. AD is characterized by elevated IgE levels and is classified as a Type I hypersensitivity reaction. However, it is argued that 80% of those with nonatopic ACD go on to develop higher IgE levels and so develop AD.16 So in some ways, ACD and AD are two ends of one spectrum.
In vivo testing for sensitization: Allergic reactions are routinely tested by dermatologists by patch testing, usually on the skin of the back.17 Individuals with a history of ACD are re-exposed to the suspected allergens under controlled conditions to verify the diagnosis. Thyssen et al. have reviewed the epidemiology of contact allergy across North America and Western Europe.18 Their review showed that the median prevalence of contact allergy to at least one allergen was 21.2%, with a range 12.5–40.6%. By far, the most important allergen was nickel, having a median prevalence of 8.6%; however, the fragrance mix set of allergens also was important.
Schnuch et al. have collected data on allergic reactions to commonly used preservatives.19 Although relatively low in incidence, preservatives are a significant trigger for skin reactions. Among those with suspected ACD, sensitization rates ranged from 1.5% for parabens to 5.3% for thiomersal. Chlormethylisothiazolinone/methylisothiazolinone, formaldehyde and methyldibromoglutaronitrile/phenoxyethanol had sensitization rates in the range of 2%.
Recently, the European Scientific Committee of Consumer Safety (SCCS) reviewed the epidemiology of contact allergy to fragrance ingredients.20 Its studies suggest that 1–3% of the population has contact allergies to fragrance ingredients. The SCCS further suggested that, on the basis of clinical evidence, the list of 26 fragrance substances in Annex III of the European Cosmetics Directive, i.e., the 7th Amendment, should be extended to include 82 fragrance substances.20 The study of patch test data helps formulators avoid known allergens. Indeed, there is evidence that the correct avoidance of allergens can reduce the incidence of ACD.21 However, historical data does not necessarily allow for the assessment of a new product’s potential for causing ACD. For this, it may be necessary to perform a human repeated insult patch test (HRIPT).22 HRIPTs usually involve an induction phase, where the test products are applied to the skin for 24–48 hr under occlusion, 5–15 times, 2–3 times during a week. There is usually then a break for 2–3 weeks before the products are applied again for 24–72 hr.23 The four most utilized HRIPTs include the Draize human sensitization test, the Shelanski-Shelanski test, the Voss-Griffith test and the modified Draize human sensitization test.23
Case study (part 2), Shelanski-Shelanski test on panelists with self-assesed sensitive skin: For the case study, the mild body wash developed for sensitive skin was tested via the Shelanski-Shelanski test.24 This involved 100 subjects having self-perceived sensitive skin, each of whom tested 10 products including prototypes and market benchmarks. The induction phase involved nine 24-hr exposures over three weeks. Products were applied as 0.5–1.0% solutions in water under occlusion. The protocol involved a one-week rest phase followed by a challenge phase, which aimed to induce elicitation, and involved exposure for 24 hr under the same conditions as the induction phase with assessment after 24 hr, 48 hr, 72 hr and 96 hr. After the challenge phase, no evidence of sensitization from any products was found. In other words, the degree of ICD-related irritation observed during the challenge phase was comparable to the induction phase. This suggested the prototypes had a low potential for sensitization.
Neurology of Pain, Itch and Stinging
Skin irritation is always, ultimately, related to simulation of the nervous system. It is therefore important to briefly consider the neurological origins of pain, itch and stinging. Free nerve endings in the dermis and epidermis, termed C-fibers and α-δ fibers, are involved in the sensation of heat, pain and itch. Usually, in ICD and ACD, it is the release of common inflammatory mediators such as histamine and substance P that simulate nerve endings in the skin and give the sensations of pain and itch; the release of histamine from mast cells during the inflammatory cascade typically initiate the pain response.
Topically applied compounds also can stimulate pain and itch by stimulating the release of neurotransmitters from nerve endings, which then stimulate inflammation through vasodilation and mast cell degranulation.25 Capsaicin, for example, from chili peppers, is believed to produce a stinging response via this mechanism, referred to as neurogenic inflammation. It also has become understood that a distinct subset of C-fibers are functionally important in itch,26 which are sensitive to histamine and other itch mediators. Yosipovitch and Barham point out there are interactions between the skin barrier and the nervous system;27 for example, restoration of the skin barrier function with moisturizers can potentially reduce dryness and itch.
Formulators should be aware that some cosmetic ingredients can cause stinging on the skin, and should be avoided for mild care products. This problem is well-known on facial skin, broken skin, diseased skin and “sensitive” skin; however, the biological mechanisms underlying the characteristic sting response are still not fully understood. Substances known to cause stinging include lactic acid, amyl-dimethyl-p-aminobenzoic acid, dimethyl phthalate, propylene glycol and phosphoric acid.28 The application of lactic acid to facial skin to elicit a stinging response has been widely used to diagnose “sensitive” skin,29 and facial sting tests can be used to test cosmetic products, in particular those for sensitive skin.
Factors for Mildness
Skin barrier disruption: As is known, the skin barrier is the first level of defense for the immune system. Disruption of the skin barrier by cosmetic actives can allow skin irritants or sensitizers to penetrate through the skin into the viable tissues. Therefore, a mild product should be one that does not affect skin barrier function. Potential skin irritants can reach viable tissue through three routes, via: hair follicles, with their sebaceous glands; eccrine sweat ducts; or across the SC. As appendages usually form only a small fraction, 0.1%, of the surface area of skin, they play only a minor role in overall skin penetration over long application times. However, these, so-called “shunt pathways” can be important on some skin sites, which will be described later, and allow penetration over short application times.
It is widely recognized that the barrier function of the skin resides mainly in the epidermis and is localized in the outermost layer, the SC.30 The SC is comprised of terminally differentiated, flattened cells termed corneocytes, which are rich in fibrous and amorphous proteins. Interspersed between the corneocytes are lipid bilayers that form a continuous, hydrophobic phase. Both the level of SC hydration and the integrity of the lipids are important to maintain a healthy barrier function.
Cosmetic products can affect the barrier function of the SC and, therefore, its resistance to irritant molecules. The physical effects of cosmetic ingredients on the skin barrier can be conveniently classified using the lipid-protein-partitioning (LPP) concept,31 which states that chemicals can act on the intercellular lipids and/or the proteins in the SC, and can also promote partitioning of chemicals into the SC. In addition to physical effects, cosmetic products can affect skin biology, the mechanisms of which are only now becoming understood. These changes to skin biology have been shown to affect the skin’s resistance to irritation.
Physical actions on intercellular lipids: Many cosmetic ingredients can affect the integrity of the intercellular lipids in the SC. Evidence of disruption to lipid structural organization by surfactants, for instance, has been demonstrated with a range of techniques, including differential scanning calorimetry and small angle x-ray diffraction;32 electron paramagnetic resonance (EPR);33 and transmission electron microscopy.34 Also, recent studies using Fourier transform infrared spectroscopy (FT-IR) have investigated the effects of surfactants on lipid packing arrangements.35 These showed that surfactants, such as sodium dodecyl sulfate, can reduce the degree of tight orthorhombic packing in skin lipids and thus, the authors propose, increase SC permeability.
The capability of surfactants to extract skin lipids can be tested using a lipophilic dye extraction method,11 which is based on the proposal that surfactants which extract more oil-soluble dye will be more damaging to the skin barrier. Other cosmetic ingredients that are surface-active or oil-soluble have the potential to disrupt intercellular lipids as well.36 Terpene essential oils, commonly found in fragrances, can disrupt SC lipids, acting as effective skin penetration enhancers.37 Also, fatty alcohols and fatty acids, used in a wide variety of skin products, can act on SC lipids and effectively enhance penetration.38
Even water is a good penetration enhancer. Warner et al. have shown by electron microscopy that water can disrupt SC lipids in a similar way to surfactants.34 Zhai and Maibach also reviewed the effects of occlusion and excess skin hydration on penetration, irritation and sensitization, and many examples are given of enhanced skin irritation as a result of occlusion.39
Physical effects on proteins in the SC: It is well-known that ingredients such as surfactants can have significant effects on the structure and function of skin proteins.40 Topically applied surfactant solutions can increase SC swelling, which results in greater water penetration into the SC and removal of natural moisturizing factors from the corneocytes.41
Biological effects: The pH of skin products is also known to have effects both on skin surface microflora and skin barrier function.42, 43 The pH gradient across the epidermis is now recognized as being important in regulating desquamation and in the generation of the lamellar lipid matrix.16 Kim et al. have shown that five weeks of treatment with skin care products formulated at pH 8 can increase TEWL and increase sodium lauryl sulfate-induced irritation, versus products formulated at a pH of 3 to 5.42 Coming at the subject from the opposite end, Hachem et al. have investigated the effects of hyperacidification of mouse skin with alpha hydroxyl acids. Their work suggests that reducing skin pH could potentially improve lipid processing and inhibit degradation of corneodesmosomes.44
Cosmetic products also can affect the enzymatic degradation of desmosomal proteins at the surface of the SC. For example, cleansing products containing sodium lauryl ether sulfate have been shown to inhibit enzymes in the SC that control desquamation, potentially leading to scaling, skin dryness and impaired barrier function.45
In some cases, the exact biological mechanisms through which cosmetic products affect barrier function are not fully understood. For example, the long-term use of skin moisturizers three times daily for four weeks has been associated with increased skin susceptibility to irritants such as sodium lauryl sulfate.46 Also, recently, it has been confirmed that aqueous cream BP, when used as an emollient twice daily for 28 days, can accelerate skin turnover and reduce skin barrier function;47 the data therefore suggests that its regular use as an emollient would increase the skin’s susceptibility to irritants.
As is generally known, there are major differences in the skin barrier structure and function between different body sites.48 Formulators should be aware of these when designing and testing formulations for mildness. For instance, Otberg et al. have shown that the density of hair follicles on the forehead is sixteenfold higher than on the forearm.49 This suggests that absorption through the skin appendages is probably much higher on the face. Water-soluble irritants that might struggle to penetrate the SC for example on the arm will find it easier to penetrate facial skin.
Also, skin barrier function is regulated both by the thickness of the SC and the size of the corneocytes.50 On some body sites such as the face, the SC is thinner and the corneocytes are smaller. As a result, the pathway for penetration between the corneocytes is shorter and skin barrier function is lower.50
Further, the application of surfactants such as sodium lauryl sulfate can increase TEWL as well as stimulate irritation, and recent investigations suggest that application of SLS to facial skin can increase TEWL much more than on forearm skin.51 This data suggests that facial skin is more susceptible to barrier disruption.
Combined, these studies confirm that facial skin is not only more permeable to potential irritants, but is also more susceptible to barrier disruption. If one also considers the higher density of nerve endings on the face, it is not surprising that 85% of consumers with self-perceived sensitive skin have experienced irritation on the face, versus 58% on the hands, or 23% on the torso.52
Skin Problems, Ethnicity and Age
In addition to site-to-site differences, there are various other factors that can affect consumers’ perception of product mildness. One of the most important factors is the condition of skin and the presence of any skin disease or malfunction. For example, many consumers suffer from AD or eczema. In the UK, 22% of women and 16% of men classify themselves as having or having had eczema, per internal company data. AD is known to impair skin barrier function and therefore, will make skin more prone to irritation.53 Add to this immune system hypersensitivity, which will cause more aggressive inflammatory reactions to allergens.
Another common skin complaint is “sensitive skin.” In the UK, 52% of women and 47% of men consider themselves as having sensitive skin, also per internal company data. Aside from the obvious causes of sensitive skin, i.e., contact dermatitis and AD, this condition in apparently healthy skin is not well-understood. However, reviews of current knowledge suggest it may be associated with poor skin barrier function and/or an over-sensitive neurosensory response.54
Case study (part 3), patch tests on panelists self-characterized as “atopic”: For the development of the body wash for sensitive skin, besides on self-perceived sensitive skin, patch tests were performed on panelists having AD (n = 31); these subjects have a history of eczema or asthma but no current skin problems. Patch tests on atopic panelists used 20-μL samples of 8% w/w product in water. Samples were applied to the outer upper arm under occlusion, for two consecutive periods of 23 hr. As before, skin irritation was assessed each day, 1 hr after removal of the chambers.
Figure 3 shows that all prototypes were, on average, significantly less irritating than the positive control, bar soap (p < 0.001), and similar in irritancy to the benchmark, presented as a mild and gentle soap (p > 0.05). It should be noted that a small number of atopic panelists showed extreme erythema responses to some treatments. These responses were not isolated to any particular treatment and perhaps relate to site-to-site differences in skin sensitivity. Overall, the patch test on atopic panelists showed that the prototypes were as mild as the benchmark and may be suitable for use by consumers with a history of atopy. Differences in structure and function and subsequent irritant response and susceptibility to cutaneous irritation can exist between different ethnic groups.55 In addition to this, aging can affect skin structure and function, and therefore irritant response.56, 57
This review has shown that skin irritation can be elicited through a number of mechanisms involving the immune system and the nervous system (Figure 1). Furthermore, it has demonstrated that the degree of irritation produced by a cosmetic ingredient can be strongly affected by barrier disruption, site-to-site skin differences, skin diseases and malfunctions, ethnic differences and age.
This review includes details on how the authors tested a mild body wash for sensitive skin. Mildness, in this case study, was confirmed using patch tests on sensitive and atopic skin, and through a HRIPT test on sensitive skin. In conclusion, mildness is definitely in the eye of the beholder; therefore, formulators wishing to formulate a mild cosmetic product should be careful to use the correct test methods and conditions to support the desired claims.
Send e-mail to CT_author@allured.com.
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