The Potential Use of Non-Invasive Methods in the Safety Assessment of Cosmetic Products

The Report and Recommendations of ECVAM//EEMCO Workshop 361-3

Reprinted with minor amendments from ATLA 27, 515-537.

Vera Rogiers,4 Michael Balls,5 David Basketter,6 Enzo Berardesca,7 Christopher Edwards,8 Peter Elsner,9 Joachim Ennen,10 Jean Luc Lévêque,11 Marie Lóden,12 Philippe Masson,13 José Parra,14 Marc Paye,15 Gérald Piérard,16 Luis Rodrigues,17 Hans Schaefer,11 David Salter18 and Valerie Zuang5
4Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklacn 103, 1090 Brussels, Belgium; 5ECVAM, JRC Institute for Health & Consumer Protection, European Commission, 21020 Ispra (Va), Italy; 6Safety and Environmental Assurance Centre, Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1PR, UK; 7Department of Dermatology, University of Pavia, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy; 8Department of Dermatology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK; 9Department of Dermatology, FriedrichSchiller University, Erfurterstrasse 35, 07740 Jena, Germany; 10Department of Biophysics, Beiersdorf AG, Unnastrasse 48, 20245 Hamburg, Germany; 11L'Oréal Recherche, Centre Charles Zviak, 90 Rue du Géneral Roguet, 92583 Clichy Cedex, France; 12ACO, Hud AB, Box 542, 18215 Danderyd, Sweden; 73EVIC-CEBA, 48 Rue Jean Duvert, 33295 Blanquefort, France; 14Centro de Investigacion y Desarrollo, C/Jorge Girona 18-26, 08034 Barcelona, Spain; 15Colgate-Palmolive R&D, Avenue du Parc Industriel, 4041 Milmort, Belgium; 16Service de Dermatopathologie, Université de Liége, Centre Hospitalier Universitaire du Sart-Tilman, 4000 Liége, Belgium; 17Laboratory of Experimental Physiology, Faculdade de Farmacia da Universidade de Lisboa, Av. Fortas Armadas, 1600 Lisbon, Portugal; 18Cussons International Limited, Cussons House, Bird Hall Lane, Stockport SK3 OXN, UK

1ECVAM - The European Centre for the Validation of Alternative Methods. 2EEMCO (The European Group for Efficacy Measurements on Cosmetics and Other Topical Products) was created in 1994. It is a group of independent experts from private and public areas from different Member States of the European Union and with competence in the field of instrumental assessment of cosmetic products. EEMCO prepares overviews of existing evaluation methods and analyses the advantages and limitations of the existing techniques to provide general guidance to both the experimenter and the inspector.3This document represents the agreed report of the participants as individual scientists.

Address for correspondence: Professor V. Rogiers, Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium

Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy

Preface:

This is the report of the thirty-sixth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become better informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organization workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1).

The workshop on The Potential Use of Non-Invasive Methods in the Safety Assessment of Cosmetic Products was held in Brussels, Belgium, on 10-12 March 1998, under the co-chairmanship of Michael Balls (ECVAM), Gérald Piérard (the European Group for Efficacy Measurements on Cosmetics and Other Topical Products [EEMCO]) and Vera Rogiers (EEMCO). The participants included scientists working in both academia and industry.

The current status of clinical and instrumental assessment of the efficacy of cosmetics was reviewed, together with the potential of using non-invasive techniques in safety assessment with human volunteers.

Introduction

Cosmetic products are defined as "Substances or preparations intended to be placed in contact with the various external parts of the human body or with the teeth and the mucous membranes or the oral cavity, with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance 3 and/or correcting body odours and/or protecting them or keeping them in good condition. They must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use." (2). As used by the general population in daily life, cosmetic products usually pose no problems for human health. However, side-effects induced by cosmetics can sometimes occur. The frequency of side-effects is not exactly known, but the types of ingredients and finished products involved usually are (3).

Side-effects can be both local and systemic. Mainly local skin effects occur with cosmetic products, including irritation, contact allergy, urticaria and sunlight-induced reactions. Irritation is the most frequently observed side-effect of cosmetics (3, 4). It is clear that cosmetic companies and regulatory authorities should take all reasonable measures to minimise any harmful effects.

According to the Sixth Amendment to the European Union Cosmetics Directive, a dossier must be kept ready for inspection by the Competent Authorities, and must contain a toxicological file based on the safety assessment of the ingredients and the finished products (2). An efficacy file must also be retained, in which any claims made for the product must be substantiated. These claims can be supported in a variety of ways (5, 6). One of these is to conduct studies on human volunteers and measure the effects observed, either by clinical scoring or by using quantitative or semi-quantitative non-invasive methods.

The following considerations formed the background to the workshop.

  1. The developments made in bioengineering technology during recent years have been such that subtle changes in skin morphology and function can now be measured.
  2. The Sixth Amendment foresees a ban on the marketing of cosmetics containing ingredients or combinations of ingredients tested on animals after 30 June 2000 (as specified in Directive 97/18/EEC), provided that sufficient progress has been made in developing satisfactory methods to replace animal testing (7).
  3. A Seventh Amendment (in preparation) is likely to introduce an earlier ban on animal testing for finished cosmetic products. This would be based on the fact that the safety of a finished cosmetic product can be assessed from knowledge on the safety of its ingredients and by using methods that do not involve animal testing, except in very specific cases, for which an exception would be likely to be allowed.
  4. The value of animal or non-animal tests in predicting exposure in the human population might be limited. Therefore, confirmatory safety tests in humans might be scientifically and ethically necessary, provided that a safety assessment based on the ingredients had been carried out and had produced acceptable results.
  5. The Scientific Committee on Cosmetology and Non-Food Products recently approved ethical guidelines for the testing of cosmetic ingredients on human volunteers (8) and discussed a draft version on human testing of the irritative capacity of finished cosmetic products (9).

Members of ECVAM and EEMCO have stated that it is pertinent to evaluate and discuss the potential use of non-invasive methodologies in the safety assessment of finished cosmetic products on human volunteers and, in particular, in assessing the occurrence of skin irritation (10-14). Skin irritation, as already mentioned, is a local side-effect which is dependent on the concentration used, the exposure time, and some other specific-conditions. On the other hand, contact allergy is an immunologically based side-effect and is more difficult to predict, since it only affects predisposed individuals.

Since the terms "non-invasive" and "ethical" are open to different interpretations, some explanation is given of the sense in which they are used in this report.

Non-invasive

The term "non-invasive" carries a range of meanings in the literature, including, "without drawing blood", "without making contact", "without altering structure or function", "not penetrating the epidermis with material or with radiation", "not causing harm" and "maintaining the integrity of the organism, tissue or cell". All of these definitions can be insufficient in certain situations. Therefore, we have used "non-invasive" to mean "a procedure or instrument causing minimal and only temporary changes to structure or function, and in particular, not involving pain, incision or loss of blood".

Ethical basis

There are many ways of choosing and implementing an "ethical" basis for scientific and medical investigations on living organisms. There are also many ways of considering what should count as relevant similarities and differences between humans and other animals as subjects of such investigations, as is clear from reviews such as those by Midgley (15), Paton (16) and Gillon (17). In general, it is widely accepted that it is better, at least on scientific grounds, to conduct an investigation on humans, if the final product or procedure under investigation is intended to be used on, or applied to, human skin. This is because the structure and function of human skin and its appendages are sufficiently different to those found in other species for predictions extrapolated from tests conducted on other species to carry a significant risk of being qualitatively and/or quantitatively misleading. If such a risk of misinterpretation is unnecessary, given the available technology and other resources, then, even without other grounds, conducting an investigation on non-humans could be considered unethical. It is for this reason that the current potential of using non-invasive measurements on human volunteers in the safety assessment of cosmetics is reviewed in this report. Any such investigations should, of course, be conducted according to acceptable ethical standards, as described, for example, by Salter (18) and by Walker et al. (19).

The Clinical Assessment of Cosmetic Products

Normally, human volunteers with healthy, non-pathological skin are selected, and products are applied only when they are considered to be safe according to the toxicological profiles of their ingredients.

In principle, three levels of human volunteers study should be distinguished, according to the three levels of testing, i.e. safety, mildness and efficacy.

In safety testing, the ability of the test product to cause irritation is assessed in a small group of volunteers, and the data are compared with those for a similar product which has an extensive history of safe marketing. The test protocols typically involve some exaggeration of exposure, while using a regimen which involves a similar mode of application to that found during normal product use. In all circumstances, exposure to a product is stopped immediately if any signs of skin irritation appear. Evaluation of the test outcome includes a comparison of the time (number and frequency of exposures) required to elicit mild irritation, as well as an analysis of the skin irritation scores. While non-invasive bioengineering techniques could be applied to quantify the results, to make them more objective, and even to measure some subclinical symptoms, this has not been common practice to date. Visual assessments are usually applied, and although this type of assessment is subjective, good results can be obtained by trained experimenters (19, 20).

Skin compatibility and mildness testing in human volunteers can be carried out in a similar manner to that for safety testing, but must involve exposure (normal or slightly exaggerated) which closely mimics typical consumer use of the product. Since the purpose can simply be to demonstrate an absence of effects, it is not always necessary for the test to be comparative, i.e. to involve positive and negative controls. However, to enhance the sensitivity of the null response, the use of appropriate bioengineering techniques is rather more common in this situation and is to be encouraged.

Efficacy testing, which may be used for claim substantiation, can also be carried out on human volunteers These types of studies tend to be much more designed as normal in-use tests than those mentioned above. The need to formally substantiate the efficacy claims related to the product also requires objective measurement of the skin responses. Consequently, in addition to clinical measurements, including visual assessment and palpation, and an analysis of user questionnaires, appropriate skin bioengineering techniques are commonly employed in efficacy studies.

Signals to be Considered

When applying non-invasive methods, it is important to focus on the potential targets (stratum corneum, stratum Malpighii, dermis; Table I),to understand the theoretical risks on biological and clinical grounds. Therefore, a short description of the anatomy and physiology of the skin is given below.

Table I: Tentative Classification of the Most Commonly Used Non-Invasive Methods According to the Signals to be Detected

Non-invasive methods Main location of signals
Squamometry (quantitative) Stratum corneum
Corneosurfametry (quantitative) Stratum corneum
Transepidermal water loss (TEWL) Stratum corneum
Electrical methods for skin hydration Stratum corneum + stratum Malpighii + dermis
Microrelief Stratum corneum + stratum Malpighii + dermis
Laser-Doppler flowmetry (LDF) Dermis
Colorimetry Stratum corneum + stratum Malpighii + dermis
Narrow band spectroscopy Stratum corneum + stratum Malpighii + dermis
Ultrasound Dermis
Image analysis Stratum corneum + stratum Malpighii + dermis
Clinical (non-instrumental) assessment Stratum corneum + stratum Malpighii + dermis

Functional microanatomy of the skin

The skin is a multilayered organ, in which the stratum corneum represents the outermost layer, which covers and is produced from the living portion of the epidermis, the stratum Malpighii. Below the stratified keratinising epithelium, the dermis is formed by a richly vascularised connective tissue, where pilosebaceous follicles and sweat glands are located.

Within the epidermis, several cell types with different embryonic origins and functions are found. The keratinocyte corneocyte lineage, melanocytes and Langerhans cells will be considered in turn.

Keratinocytes and corneocytes
Keratinocytes form the bulk of the epidermis. They normally proliferate in the basal and epibasal layers and move progressively up through the stratum Malpighii toward the stratum corneum. During their ascent, they undergo differentiation, giving rise to the enucleated, flattened corneocytes which form the stratum corneum.

The stratum corneum is a compact, structurally heterogeneous, two-compartment system, in which layers of protein-enriched corneocytes are separated by a multilayered lipid-enriched extracellular matrix. The stratum corneum forms both a reservoir for, and a barrier to, the penetration of xenobiotics. Its thickness and molecular structure vary according to body region, and this affects its appearance and physical properties. In addition, genetic characteristics, ageing, skin diseases, and environmental hazards (for example, humidity variations, physical trauma, exposure to chemicals) alter the structure and function of the stratum corneum. Any topical product designed to be applied to the skin surface undoubtedly affects its quality and functional properties.

The most important adhesive force holding corneocytes together comes from the corneosomes, which are derived from the desmosomes that bind together the living keratinocytes. Corneosomes are normally subject to a programmed destruction after protease action. This permits the imperceptible casting off of single corneocytes from the skin surface and the continual renewal of the epidermis. Failure to degrade corneosomes correctly is the fundamental factor in most conditions where flaking is present. Such a feature results in a rough skin surface (xerosis), which is commonly called dry skin.

The maintenance of normal stratum corneum functions, including its turnover and permeability barrier homeostasis, is complex (21). It is regulated by many factors, including transepidermal water loss (TEWL) and various metabolic aspects which affect the keratinocytes. In fact, the epidermis generates a large number of biological response modifiers (BRM), which modulate the growth, maturation and apoptosis of keratinocytes. The leakage and diffusion of some BRM into the dermis can initiate vasomotor responses and inflammation.

The presentation of allergic and irritant contact dermatitis depends on intraepidermal damage. The form and extent of damage are dependent on the nature and concentration of the damaging agent, as well as on the conditions of exposure. An increasing number of chemical substances are now recognised as irritants and/or can become sensitisers.

Melanocytes
Melanocytes are regularly dispersed among the keratinocytes of the basal layer and contain melanosomes, the melanin-synthesising apparatus of the skin. Once loaded with melanin, melanosomes are transferred from the dendrites of the melanocytes to the cytoplasm of keratinocytes.

Melanosome size and degree of melanisation in epidermal melanocytes are genetically controlled, although non-genetic factors are undoubtedly important as well. Following the action of triggering factors or insults to the melanocytes, pigmentory changes can occur. Ultraviolet (UV) light, various BRM and hormones alter the skin colour related to melanin content and location. Epidermal melanosis results from increased melanin synthesis and transfer to keratinocytes. By contrast, melanoderma and certain types of ceruloderma result from melanocyte insults followed by melanin leakage into the dermis and melanosome phagocytosis by perivascular dendrocytes.

Both melanin distribution and production are altered during ageing. Chronological ageing is responsible for a progressive decrease in the density of active melanocytes. In contrast, photo-ageing induces a mottled pigmentation, with hyperactive foci adjacent to melanin-depleted areas. Such aspects are better appreciated when the skin is illuminated by long-wavelength UV light.

Langerhans cells
Langerhans cells are dendritic cells dispersed in the stratum Malpighii. They capture and process antigens which penetrate the skin, and then present them to T-lymphocytes in the initiation of a delayed-type immune response. Upon stimulation, they also release specific BRM. They represent the main resident cutaneous cell type involved in contact allergy.

Potential targets and location of signals to be detected

During the last two decades, an increasing number of non-invasive methods have been developed for objectively determining skin properties, so that subjective, visual or tactile evaluations of skin conditions can now be supplemented with quantitative measurements. This makes comparisons between results obtained in different parts of the world more feasible. It is also important to note that these new techniques permit, in certain cases, the quantification of skin properties and subclinical symptoms that are not perceptible to the human senses. However, standardization among instruments is at present imperfect, so measuring the same skin property with different instruments can give different results. The instruments from various companies, though based on the same principles, can use different scales. Hence, knowing that the transfer of scoring schemes between laboratories is difficult, the standardization and calibration of the instruments is a key issue in successfully applying these methods in efficacy testing, in skin compatibility and mildness assessments and, in particular, in safety testing.

In Table I, a tentative classification of the most commonly used non-invasive techniques has been made, according to the main locations of the signals to be detected. This classification is based on the events and signals produced when surfactants are applied to the skin. A cascade of biological events takes place, resulting in clinical signs after a certain length of time and according to a specific kinetic pattern. Many authors have demonstrated that these gross clinical signs actually mask a wide range of histological and functional changes (22). The first signals are caused by stratum corneum and membrane structural damage and can be detected by techniques such as corneosurfametry and squamometry. Stratum corneum functions then undergo measurable changes, and TEWL measurements and electrical methods for skin hydration become useful. At a later stage, many kinds of BRM are liberated and have further effects. They are the basis of erythema, skin roughness, changes in pigmentation, and the development of oedema and skin thickening.

Instrumental Assessment: Available Non-invasive Methodology

As already mentioned, the standardization of non-invasive methods is a key issue in their successful application in the safety testing of cosmetic products. This is necessary because of:

  1. environmental factors (for example, room temperature, relative humidity, light sources, air circulation);
  2. instrumental variables (for example, zero setting, calibration, probe properties, probe position);
  3. volunteer-linked factors (for example, age, sex, race, anatomical site, diurnal rhythm, skin type cleansing procedures, skin diseases, medication); and
  4. product-linked variables (for example, galenic form, dilution, amount per surface unit, frequency and mode of application, inclusion of blanks).

Only when these factors are taken into account in well-defined protocols can reproducible and relevant results be obtained.

Stratum corneum techniques: corneosurfametry and squamometry

Some minimally invasive methods have been designed to harvest the superficial part of the stratum corneum and to produce an objective and quantitative record of the tolerance of topically applied products (23).

The superficial corneocytes of the stratum disjunctum can be harvested by controlled, gentle rubbing of the skin surface. Another method consists of stripping by using adhesive tape. However, the commercially available adhesive tapes vary in their capacity to bind to the skin, so they are not usually suitable for accurate and reproducible corneocyte harvesting. A better method is to use appropriate clear self-adhesive coated discs applied to the skin under calibrated pressure for a defined period of time. Cyanoacrylate skin surface stripping is another method, which samples a thicker portion of the stratum corneum than the other procedures (24). All of these methods have their own advantages, limitations and pitfalls.

Corneocyte samples can be evaluated as they are taken or after staining with appropriate dyes. The nature of the assessments made varies according to the aim of the study. Ageing effects, xerosis, and the efficacy of cosmetic products can be assessed with a corneocyte sample by visual and microscopical observations, weight evaluation, optical measurements of light attenuation (25) and image analysis (11, 23, 26). Cyanoacrylate skin surface strippings are particularly suitable for the evaluation of the renewal dynamics of the stratum corneum (11). This type of sampling can also be used to study the contents of follicular openings. As a result, comedogenesis, comedolysis and bacterial load can be accurately quantified (27-29).

As already mentioned, the reliability and relevance of studying the stratum corneum are best demonstrated in the assessment of the interaction between surfactants and the skin. After single or repeated short-term applications of diluted surfactants, the stratum corneum is the first structure to exhibit changes related to the aggressiveness of the product. This can be conveniently quantified by using a variant of squamometry (30-33). Briefly, a clear self-adhesive disc is applied to the test site under controlled conditions. After careful removal, the sample is stained by anionic or cationic dyes according to the nature of the reaction to be studied.

Corneosurfametry and corneoxenometry were designed as screening tools devoid of potential hazards for humans (31, 34, 35), and are performed on cyanoacrylate skin surface strippings harvested from healthy volunteers. Samples are placed in contact with the test product (surfactant or another xenobiotic) under controlled conditions (duration, temperature, dilution). The subsequent staining and evaluation procedures are identical to those of the squamometry test.

False positive and false negative data might be obtained if the test product is not sufficiently removed from the sample during the rinsing procedure, as the product could alter the binding of the stain to the stratum corneum sample.

The main advantage of corneosurfametry and corneoxenometry is the avoidance of any hazard for the human volunteers, even when neat products are tested. A negative result assures safe use in humans as far as the integrity of the stratum corneum is concerned. A strong positive result predicts a clinical problem which would present as an inflammatory irritant reaction.

Transepidermal water loss measurements

TEWL, or better, "skin surface vapour loss" (28), represents the total water loss from the viable epidermis and dermis, diffusing through the stratum corneum to the skin surface and originating from the sweat glands below the thermal threshold for sweating. In practice, with careful choice of the measurement conditions, the contribution of sweat evaporation can be made very small (36).

Numerous publications have shown that TEWL measurement is a good indicator of the integrity of the barrier function. Damage is reflected by an increase in TEWL.TEWL measurements are usually based on the measurement of the water evaporation gradient developed from skin surface hygrosensors and thermistors present in an open probe at various distances from the skin surface.

There are a range of instruments, all of which provide results in g/m2/hour, although their calibrations might vary. It is therefore more accurate to talk of relative values than of absolute values. A number of important variables can affect TEWL measurements, including person-linked and product-linked factors, as well as environmental and instrumental variables (3741). Of particular importance is the probe temperature, since, if this is neglected, a 200-300% variation around the actual measured value can occur (39).

TEWL measurements have many applications in the cosmetics industry; for example, for the substantiation of claims for moisturising products (42-45) and the development of new ingredients for an effective barrier function (46-49). They are also used in clinical and pharmaceutical research to provide better understanding of the characteristics of normal skin (50), the development of skin disorders (51), and the ageing process (52, 53).

In fundamental research, TEWL measurements have been particularly useful in elucidating the functional role of the stratum corneum (54, 55) and the roles of the various lipids in the intercellular matrix of the stratum corneum (56-60).

With respect to the use of TEWL measurements in safety testing, most publications have dealt with various classes of surfactants. Predictions of the degree of the irritative response to cosmetic ingredients and finished products have been performed by using TEWL measurements. It has been clearly demonstrated that the TEWL is a good indicator of barrier damage due to irritation (59, 61, 62). Subclinical measurements are possible, and it is clear that TEWL assessment is more sensitive than visual assessment (63). Characterisation of the profile of irritancy (as a result of exposure to corrosive or non-corrosive irritants) by TEWL measurements has been proposed by Serup (64), and prediction of the percutaneous resorption of topically applied substances and products has been reported by Rougier (65).

Finally, TEWL measurements might also contribute to the development and validation of alternatives to the Draize skin irritation test, by providing data for use in in vitro/in vivo comparisons (66).

Electrical methods for estimating the moisturisation of the stratum corneum

Electrical methods for assessing the moisture content of the stratum corneum are based on measuring impedance, or conversely, conductance, as a function of one or more frequencies. Both impedance and conductance are frequency-dependent vector quantities, and the derivation of standard physical parameters such as resistance, reactance and capacitance is not simple (67-70).

Readings result in relative values, and care should be taken not to raise expectations for in uivo studies which cannot be justified (for example, inter-individual comparison of hydration values measured; 12).

Nevertheless, changes in stratum corneum hydration measured by electrical methods can be predictive of the irritation potential of topically applied compounds (41, 71-74). Indeed, subclinical irritant dermatitis can be detected by early changes (reduction) in stratum corneum hydration. In some experimental strategies, it has been shown that short-term surfactant application to the stratum corneum can also alter stratum corneum hydration and that this correlates well with the irritant potential of the compound tested (75). Stress tests (developed to assess stratum corneum hygroscopicity), such as the adsorption-desorption test, can be useful in evaluating subclinical changes in skin hygroscopicity and for predicting the onset of skin irritation (41, 76). Standardised procedures for hydration measurements have been published (12). The results obtained are strongly influenced by the properties of the skin surface, and the values measured decrease with increasing roughness.

Several different instruments are commercially available (12). The variables which affect the measurements are again related to the volunteers (77-79), to the environmental conditions (78, 80-82), and to the instrument used (82).

Microrelief measurements

The microrelief of skin is known to result from the three-dimensional organization of bundles of collagen present in the superficial dermis. The appearance of the skin surface and its geometric characteristics are also dependent on the presence of living epidermis and of the stratum corneum covering it. Thus, it seems logical to assume that all changes in the thickness, composition and structure of these layers would result in changes in microrelief.

The superficial dermis also contains capillaries that provide both the nutrition and the temperature regulation of the skin. All changes in blood flow, and in capillary porosity, are likely to result in changes (at least transient changes) in the state of hydration of the affected tissue, thus also modifying the microrelief seen at the skin surface.

Besides offering the possibility of clinical evaluation, microrelief can be measured by using various non-invasive techniques (14). Most of these methods use replicas (negative) or counter-replicas (positive) of the cutaneous surface. The relief profile is then scanned by either mechanical or optical profilometrical techniques. Usually, several lines need to be scanned. Today, two-dimensional models are often complemented by three-dimensional methods, which permit the quantification of skin anisotropy.

An alternative to the profilometric methods is surface image analysis of an appropriately lit replica, which permits a global data analysis of some relief parameters. Rotation of the samples is necessary to describe skin anisotropy.

The disadvantages of these techniques are often linked with the production of replicas and counter-replicas (83-85). The length of time allowed for the silicone polymer to harden, the presence of air bubbles at the surface of the replica, the thickness of the replica, and the colour of the replica (especially when optical measurements are involved), are all critical in the success of these techniques.

Mechanical profilometric techniques can be used to measure the amplitude of skin microrelief with a high degree of accuracy, but they are slow because of their low scanning speed. Optical profilometric measurements by the triangulation method with a linear sensor, measure the surface topography with a wide vertical range and involve faster scanning speeds.

Surface image analysis provides the mean density of the lines, the mean depth and the microrelief shape (86-88). This method can be automated, and the replicas can be automatically rotated. However, some data can be lost in the shadows, and the parameters obtained are not easily linked to classical standardized parameters (14, 23).

Microrelief measurements have been used to study the effects of topically applied surfactants. This is not surprising, since it is well known that the repeated application of surfactants to the skin modifies the barrier function, leaving the skin rough and dry (39). However, only a limited number of papers are available in the literature, which seems to suggest that other non-invasive methods (for example, measurement of colour, TEWL) are more advanced than quantitation of skin relief with respect to the objective determination of the effects of skin irritants.

Microscopical investigations of replicas of skin patches exposed to sodium lauryl sulphate can be used to evaluate the severity of the reactions, even with sodium lauryl sulphate concentrations as low as 0.02% (90 92).

In one study, the effects of three surfactants were distinguished by repeated patch tests and the subsequent use of cutaneous microrelief measurements (93). However, these results were not confirmed by others (94).

Some other applications of microrelief measurements after the application of irritants have been described by Agner & Serup (95). Modifications of skin relief following corticotherapy (96) and radiation (97, 98) have also been demonstrated.

It is possible that some of the methods listed above will be made redundant by new approaches made possible by the pattern projection analysers now being produced in Germany, Japan and the USA.

Skin colour measurements

Skin colour results from a combination of selective absorption and scattering of visible light wavelengths. Several major light-absorbing particles, referred to as chromophores, can be found in the skin. The colour of the epidermis is normally due to the presence of melanin (eumelanin and/or phaeomelanin) and, in rare instances, of carotenoids. Dermal chromophores can be found in the blood vessels of the skin (as oxyhaemoglobin, reduced haemoglobin and bilirubin).

In many human assays for evaluating skin response to topical products, redness is purported to be an indicator of inflammation. This concept is flawed when redness alone is used to assess all types of skin reactions. In fact, skin irritation can be non-erythematous in some instances.

Skin colour evaluations are often performed to determine the degree of physiopathological response of blood vessels to a variety of physical or chemical insults (99). Erythema is produced when exposure to irritants, allergens or short-wavelength W light causes blood vessels close to the skin surface to dilate. Oxyhaemoglobin in the erythrocytes in the blood vessels gives the skin a red colour. The visual characterization of erythema might be inaccurate when comparing cutaneous reactions in different subjects, because the colour of skin lesions generally depends on the colour of background normal skin. In fact, it is almost impossible to clinically detect the pink hue of discrete reactions in subjects with a dark complexion.

Traditionally, erythema has been assessed visually by trained observers according to predetermined arbitrary scales. Such assessments tend not to be consistent between studies or between observers. Additionally, the scale of increasing redness is not necessarily linear, which precludes the possibility of calculating an average value within a group of subjects.

Skin colour can be measured quantitatively by using reflectance techniques and spectrophotometry (13, 99,100). Reflectance colorimetry takes advantage of the CIE-L*a*b* standardised system. Spectrophotometry uses either the whole light spectrum or narrow bands to quantify the levels of haemoglobin (the erythema index) and melanin (the melanin index). Instrumental measurements of erythema with modern devices result in more-objective, more-reproducible and more-quantitative data than visual scoring.

Parameter a* and the erythema index are closely correlated with the degree of redness, and are consequently the most important measurements in the assessment of skin inflammation. In lightly pigmented subjects, the a* values and the erythema indices are linearly correlated. However, the erythema index becomes an overestimate when the melanin pigmentation increases. It is therefore advisable to measure the melanin index or the individual typology angle (101) at the same time, to better appreciate the significance of variations in recorded redness.

The major sources of variation for such colour measurements in groups of normal subjects include inter-individual differences and the test site used.

Comparative testing on the forearm should be randomised and obtained on the same longitudinal axis restricted to the mid-forearm (102). Furthermore, the position of the forearm, either horizontally or vertically oriented in an upward or downward direction, modifies the skin colour (103) and the sensitivity of the measurements. Differences in redness between an active inflammation site and a control site are likely to increase when the orthostatic blood pressure is decreased, i.e. when the forearm is held vertically upright (99).

It is important to consider the time-course of erythema when measurements are planned, as it can vary according to the nature of the test challenge. Depending on the nature and the severity of the inflammatory reaction, several distinct mediators, released by various cell types, can be involved in the erythemal response.

Some foods, medicines, neural and endocrine influences, the nycthemeral rhythm, environmental conditions and vascular diseases can considerably influence the data generated. The presence of chronic contact dermatitis, an acute allergic contact dermatitis on another part of the body, or a previous local skin challenge, can alter the skin's reaction to xenobiotics. These responses might be lessened or, conversely, boosted, according to local and systemic influences. Mast cell reactivity, identified by dermographism, and tachyphylaxia are other factors which influence the vascular response of the test site.

Laser-Doppler flowmetry

Laser-Doppler flowmetry (LDF) is a method which is able to provide continuous non-invasive measurements related to changes in microvascular perfusion, in terms of relative changes of blood volume and velocity. The method is based on the effects of the light on moving (mainly erythrocytes) and non-moving components of a limited volume of tissue.

When tissue is illuminated by a coherent, monochromatic low-powered light, such as that emitted by low-power lasers, only a small amount of light (about 3-7%) is reflected. The remaining 93-97% of the incident radiation is partly absorbed by various structures, and partly undergoes single or multiple scattering (104-106). A variable amount of this scattered light (more than 50% at 633-785 nm) is then re-emitted from the surface and is collected by a photodetector. The light recaptured by the photodetector produces the LDF raw signal.

Scattering results from the collision of light photons with either static or moving components of the tissue. The collision of one photon with a static structure causes a change in the direction of the photon without Doppler frequency shifting, whereas the collision of one photon with a moving structure (typically, an erythrocyte) causes a change in the direction of the photon with Doppler frequency shifting. As a result, the use of LDF produces an output signal which is proportional to the blood cell perfusion (or flux). This represents the movement of erythrocytes through the microvasculature.

Assuming a proportionality between erythrocyte number and blood volume, the LDF signal should be linearly related to the volume-velocity product of blood in the measured volume.

In relation to dermatology, the regional complexity of the microvasculature (107), its global variability in specific regions (such as fingers; 108), and the complex nature of light scattering in tissues, make LDF measurements suitable for characterizing only relative changes in blood flow. LDF data are also strictly site-specific. A probe with several integrated optical fibres is recommended for skin measurements, to minimise these variations and to provide more-relevant data (109).

LDF data have been shown to correlate well with visual scores in the assessment of patch test reactions, and to be particularly useful in detecting doubtful and non-visible responses. LDF is used in dermatology and related sciences to predict irritancy (110, 111), to evaluate patch test reactions, to assess topical products (anti-inflammatory and vasoactive drugs, sunscreens, detergent barrier creams), to monitor skin diseases (skin irritation and allergy, scleroderma, psoriasis, atopic dermatitis), and to quantify wound healing, burns and perfusion of skin flaps. Guidelines for the proper use of the instrumentation have been published (112).

Imaging techniques

Imaging techniques are highly diverse and can be classified according to their date of appearance in the scientific literature. These techniques include the B-scan Ultrasonic Method, Magnetic Resonance Imaging, Confocal Microscopy, and Optical Coherent Tomography. These methods are based on entirely different physical principles.

To date, only the ultrasound method has been the subject of technological development to the extent that it is used in numerous laboratories for product safety studies.

This method is based on the properties of ultrasonic waves (with a frequency higher than 20 KHz), which are partially reflected when they pass through the interface existing between two media with different mechanical properties. It is customary to say that an ultrasound image of the skin reproduces the echostructure of the various skin layers. In this type of image, a white point (or white line) represents the interface between a hard tissue and a soft tissue. A black zone represents homogeneous tissue, without any interface, whether it is hard or soft.

Skin images obtained by using this technique provide information related to skin morphology and the thickness of the various layers, as well as more-qualitative information with respect to tissue type. It is principally used in clinical research, in skin pharmacology and in studies on local toxicity (irritation/allergy). It can also be used to determine the depth of tumour invasion prior to excision. However, in this report, it is its application in measuring irritation which is of particular concern.

When examining an ultrasound image of the skin to which an irritant product has been applied, marked changes appear which affect both the thickness of different layers and the image contrast.

After the application of 1.5% sodium lauryl sulphate in a 24-hour patch test, the image obtained shows a very marked swelling of the dermis and epidermis. By using a high-performance system, it is even possible to obtain a clear image of epidermal acanthosis. Furthermore, dark zones distributed throughout the tissue are observed. These are very probably caused by the oedema which forms subsequent to inflammation (113-114).

Quantification of the grey levels of the image, which are indicative of the presence of water, is extremely difficult, as it is necessary to ensure that the ultrasound energy delivered to the skin has not changed between the "control" situation and the "exposed" situation. However, certain options are available, and various studies have been published with respect to the quantification of the grey levels before and after the application of weak irritants (115, 116). Nonetheless, it appears to be necessary to study a procedure for grey level quantification in detail, to ensure the reliability of this type of determination.

When determining the thickness of the various skin layers, the principal factor to be considered is epidermal hyperplasia. This is not yet possible, as the imaging systems currently available do not have sufficient resolution to enable a clear visualization of the epidermal layer, except in certain zones such as the hands. Further development is needed to permit a more rigorous approach for the determination of various parameters, and how they change following the application of very weak irritants.

Tensile properties assessment

The assessment of the mechanical properties of the skin is complex, yet is fundamental to understanding the physiology and pathophysiology of living skin, and ultimately to objectively assessing the effectiveness of topical products applied to the skin. The wide range of scientific disciplines involved in providing this specific knowledge illustrate the underlying difficulties which still exist with the state-of-the-art technology in this field.

The tensile functions of normal skin represent an important physiological characteristic, since they provide a degree of flexibility which is essential to movement and to resistance to rupture. The biomechanical anisotropy of human skin has been thoroughly studied (117, 118). Volunteer-linked variables in healthy (119 121) and diseased (76, 117, 122-124) skin have also been studied.

From a histological point of view, it is accepted that the mechanical characteristics of human skin result from the global contributions of connective tissue, dermis and hypodermic and, at least to some degree, from the epidermis. That is, the different levels of organization skin surface down to the deepest regions, including the hypodermic, determine the various mechanical characteristics of the skin (114, 125, 126).

However, knowledge of the pure mechanics of the skin has not significantly benefited the development of new techniques, despite the recent development of technological tools (some of which are now commercially available) specifically designed to assess the biomechanical characteristics of human skin (41).

These systems are based on the measurement of changes induced by the application of external forces to the skin surface and can provide quantitative indications of any changes. However, an objective appreciation on a purely mechanical basis is not yet possible, and the nature and significance of the information obtained is unclear, since there is no way of relating the quantitative data obtained to particular structures of the skin. Moreover, the available data are obtained via various techniques, applied under a range of experimental conditions, which precludes a comparison of the results.

Optimisation of the information provided by these systems should be considered essential for maximising the potential application and usefulness of this new technology. In addition, and independently of the technological advances expected in the future, the standardization of the measurement procedures currently used would represent a significant step forward.

Conclusions and Recommendations

  1. Efforts should be made to optimise the existing non-invasive methods and their use in human studies. Much variation exists in the protocols reported in the literature, and it is not always clear how they should be applied and how they could be combined with other techniques under optimal conditions.
  2. There is a great need for the optimization and standardization of the various protocols. This is a key issue, if non-invasive methods are to be increasingly used in the future in the safety and efficacy assessment of cosmetic products. It is therefore recommended that the most promising protocols for non-invasive quantification of skin properties and responses should be optimised and standardised at the European level.
  3. To minimise the risk to the consumer of adverse skin reactions caused by new cosmetic formulations, which are considered to be safe based on data on the safety of their ingredients, it is recommended that the safety of the formulation should be confirmed in human volunteer studies prior to product launch.
  4. Human volunteer testing should always be subject to strict ethical controls, as agreed by the Competent Authorities. It is recommended that a standard ethical protocol is defined, in which there is a clear definition of the conditions under which the safety, compatibility, mildness and efficacy testing of cosmetics on humans is permissible.
  5. Results obtained from the literature and the experiences of the workshop participants clearly indicated the possibility that some non-invasive methods for quantifying human skin irritant reactions (in particular, squamometry, corneosurfametry, TEWL measurement, electrical methods for stratum corneum hydration and LDF procedures) can be applied at a subclinical level. However, efforts are needed to better define what is meant by "a subclinical level", since human testing at this level is clearly more ethically justifiable than at the clinical level.
  6. The results generated by non-invasive methods of the type described in this report cannot stand alone. Thus, whenever instrumental assessment is being considered for testing the safety, compatibility, mildness or efficacy of cosmetic products, it is recommended that the results obtained are combined with those from clinical observations and/or from in vitro tests. It is also recommended that strategies are defined for optimising combinations of non-animal methods with human volunteer studies for complementary and confirmatory purposes.
  7. It is recommended that the general public and cosmetics companies are informed of the potential value of such strategies and methods. This could be achieved by producing educational material for general distribution and by setting up training courses for the companies concerned and especially for small and medium-sized enterprises.

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Appendix 1