Comprehensive outlook of in vitro tests for assessing skin irritancy as alternatives to Draize tests

Journal of Dermatological Science 24 (2000) 77 – 91 www.elsevier.com/locate/jdermsci

Review article

Comprehensive outlook of in vitro tests for assessing skin irritancy as alternatives to Draize tests Hee Chul Eun *, Dae Hun Suh Department of Dermatology, Seoul National Uni6ersity Hospital, Seoul National Uni6ersity College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul, 110 -744, South Korea Received 20 December 1999; received in revised form 14 February 2000; accepted 16 February 2000

Abstract In vitro alternative methods have been verified for the possibility to assess cutaneous irritancy because humans cannot be direct initial experimental subjects and animal experimentation could be forbidden in the near future. Many kinds of cell cytotoxicity assays have been tried, revealing their own advantages and limitations. Cell function-based tests have been used less frequently than cytotoxicity assays. Three-dimensional culture systems are promising because they are closer to the actual in vivo skin, and some of them are commercialized nowadays. The ultimate objective of in vitro irritancy tests, which is the high degree of correlation with human in vivo test results, has been accomplished in many experimental settings. Before applying these in vitro methods we must consider several points, including cell sources, irritant characteristics, exposure time, endpoint of experiment, extrinsic factors affecting irritation, etc. In vitro skin irritancy tests have been developed continuously, and in the future they could assume a heavy responsibility of estimating the irritancy in human skin in vivo. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: In vitro; Irritancy; Skin; Test

1. Introduction Humans contact many kinds of chemicals in their lives, the majority of which have physiological effects. The number of chemicals used continues to increase as a result of the development of the chemical and biological sciences, and we have * Corresponding author. Tel.: +82-2-7602410; fax: +82-27427344. E-mail address: [email protected] (H.C. Eun).

become able to synthesize new chemicals for a wide variety of uses. Huge number of chemicals have been produced for use on a daily basis, which makes it essential that their intrinsic hazards are well defined. Worldwide, nations have developed their own regulations concerning the safe handling of chemicals. Naturally, people cannot be used as the initial experimental subjects, which has meant in practice that animals have been sacrificed to allow predictions to be made on the potential irritancy of chemicals to humans.

0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 0 9 3 - 1

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Environmental lobbyists and those who argue that animal experimentation should be forbidden, have found significant support in Europe and America, moreover, these movements are expanding on a global basis. The need for animal experimentation is being seriously questioned by powerful social, political, ethical and scientific groups. The chemical industry is under ever-growing pressure to replace animal experimentation with non-animal based methods for toxicological evaluation. The eye irritation method is one of the oldest methods of assessing chemical irritancy. Draize [1] who worked at the Food and Drug Administration (FDA) described in 1944 an irritancy grading system for use in evaluating the potential irritative effect of drugs and other materials intended for use in or around the eyes. Numerical scores were given for reactions of the cornea, conjunctiva and iris. The testing involved the use of Albino rabbits, and it was subsequently modified and refined by many investigators [2 – 5] with the scientific basis of predicting effects in humans [6]. In the case of the skin test, an area of the rabbit’s skin is shaved and covered with the chemical being tested. However, in addition to using animals, the Draize test has sometimes failed to identify problems which have occurred later in humans [7]. The need to develop alternatives for the Draize test has been apparent for some time. These alternative methods have used a diverse set of human and animal cells, tissues, organs, and even biochemical matrices, but unfortunately, they tend to model only a small part of the complex process examined by the Draize test. The aim of this article is to review the past references related to the alternative methods to the Draize test for the evaluation of skin irritancy.

2. Cell cytotoxicity assays These assays are amongst the most common of the in vitro bioassay methods used to predict the toxicity of substances to various tissues. They have been used to examine organ-specific damage and the tissue inflammatory potential of chemicals. There are many cell cytotoxicity assays.

2.1. Neutral red uptake assay (24 or 48 h exposure) [8] Neutral red, the marker used to determine cell viability, has been shown to be selectively retained by the lysosomes of living cells because of the pH differential between the inside of the lysosome and the surrounding cytoplasm. The amount of neutral red taken up by the population of keratinocytes is directly proportional to the number of viable cells in the culture. The test material-induced cytotoxicity (and cytostasis) is measured over a wide range of concentrations, and the concentration yielding a 50% reduction in neutral red uptake is used as a measure of the toxicity of the test materials. The neutral red uptake assay is generally good as a screening method and collaborative assay over the range of toxicities found in personal care or household products. It performs very well in terms of separating non-irritating materials from those at the higher end of the in vivo scale. Use of the assay should be limited to water-soluble materials. The toxicity of products with high or low pH may be markedly under-predicted. Available data supports the use of this assay for surfactant materials but attention should be paid to evaluating each product class. For example, fabric softeners may not perform well, yet shampoos do. The physical form should be considered during testing and it should be remembered that the toxicity of a solution will not necessarily predict the toxicity of the solid.

2.2. Neutral red release assay (5 – 30 min exposure) The uptake procedure was modified for shortterm exposures by Reader et al. [9]. Test materialinduced cytotoxicity is measured over a wide range of concentrations, and the concentration causing a 50% reduction in retained neutral red (within the population of cells) is used as the measure for comparative testing. The dose which causes a 50% reduction in dye retention is considerably higher than the dose of the same material which induces a 50% reduction in neutral red uptake. The neutral red release assay was devel-

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oped to assess the immediate toxic action of surfactants. The assay looks promising as a screening method for surfactant-based materials over the range of toxicities normally found in personal care products.

2.3. Cell protein assay [10] Surface-active agents would be expected to be cytolytic by disrupting cell membranes. Cell death in the population is measured by the total cell protein, relative to the control cultures. Depending on the incubation time with the test material, the cessation of cell replication may also contribute to the overall difference in protein content. The measurement of total protein in a population is only a crude measure of relative cell numbers. It relies on the relative growth of controls over the treated cultures to show differences in toxicity. However, it depends on the cell type used and the number of cell replications in the control. In addition, the dead cells may contribute significantly to the total protein because dead cells may either remain attached to the plastic or be lost to the medium. Test materials which kill and fix the cells will allow more cellular protein to remain in the dish than those which disrupt the membrane and release cytosolic proteins. Total protein is not a measure of current viability. Thus, dose – response curves tend to be very different for different types of cells and different classes of test articles.

2.4. Plasminogen acti6ator assay [11] Epithelial cells in culture secrete plasminogen activator into the medium. The presence of the enzyme is measured with a colorimetric substrate to determine the concentration of the enzyme. Decreases in the enzyme may come from an inhibition of protein synthesis or cell loss. The endpoint uses synthesis and release of the enzyme to monitor continued cell function (e.g. protein synthesis and transport) after a short exposure. The result integrates the functional activity of intact cells in the treated population during the 48-h period after treatment. Populations that do not die immediately may continue to produce the enzyme at a lower level.

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2.5. Dual dye cytotoxicity test [12] This assay is classified as a live–dead assay and evaluates the viability of cells on the basis of the presence of cytoplasmic esterases and the penetration of a damaged cell membrane by a substance, which would be excluded by a normal, intact cell membrane. Fluorescein diacetate is a dye used to evaluate the viability of cells in this manner. It readily diffuses into the cells, where the presence of esterase in the cytoplasm cleaves it, resulting in the creation of a compound which fluoresces green under UV light and which diffuses out of intact cells at a low rate. Ethidium bromide fluoresces red in UV light and does not readily penetrate the membrane of intact cells. This compound has a high affinity for nucleic acids and stains the nucleus of a dead cell red. This method is suggested to be useful in in vitro toxicity tests. Results obtained have proven to be reproducible and to have a good correlation with in vivo responses.

2.6. Trypan blue exclusion assay [13] Cells with intact membranes should exclude the dye Trypan blue and will remain unstained. Cells with damaged membranes will allow the dye to penetrate, staining the cytoplasmic components although some disagreement exists as to how much damage is required. For a given cell, the response is all or none. At present this assay does not appear to resolve differences among surfactants over a diverse range of toxicities. This method is regarded as a simple rapid screening technique for cell viability.

2.7. Red blood cell lysis assay [14] This method is based on short-term exposure to rabbit RBC with cytotoxicity (hemoglobin release) as the assay endpoint. Red blood cell lysis is an extreme example of a membrane damage endpoint. The release of hemoglobin is measured spectrophotometrically. Red blood cells are extremely fragile compared with nucleated cells, which have functioning cytoskeletons, so that the same exposure in one system is unlikely to give a

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similar response in the other. It may be useful to differentiate very mild surfactants from stronger ones.

2.8. MTT assay Tetrazolium dye MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide) and similar tetrazolium salts are converted from the oxidized to the reduced form by the NADH + dependent reaction catalyzed by succinate dehydrogenase [15,16]. In its oxidized form MTT is yellow and it turns blue-black on reduction. The concentration of the reduced form is then measured spectrophotometrically. Under stable conditions, the amount of MTT reduced per unit time is proportional to the cell number. It should be noted that this is also an indirect measure of cell viability and that test materials which increase mitochondrial activity also increase MTT reduction. MTT assay is prevalently used in various cell types and culture systems [17–20]. In our experience it is one of the excellent methods of cell viability measurement using cell culture [21].

2.9. Other cytotoxicity assays Release of cytoplasmic enzymes can be another measure of membrane integrity. For example, lactate dehydrogenase (LDH) is released into the culture medium and detected quantitatively with a chromogenic substrate [22]. The total available enzyme can be determined by lysing the population with 1% Triton X-100. Caution should be used with lactate dehydrogenase, as the enzyme may be readily inactivated. As membrane leakage is required, these assays often detect cells in the last stages of cell death. DNA synthesis measurements usually involve the use of radioisotopes such as tritiated thymidine. They are based on the assumption that the populations are randomly cycling so that the treated and control populations can be compared. The active, receptor-mediated transport of molecules into the cell can also be used to measure normally functioning cells in a population.

Uptake of uridine is a receptor-mediated process that requires an intact membrane, ATP and flux in the intercellular uridine pool (e.g. RNA synthesis) [23]. Uridine uptake is measured over a brief period so that the rate of uptake by the cells rather than a level of incorporation into RNA is taken as the endpoint. Under standard conditions, the uptake is expected to be proportional to the number of viable cells. As with the other endpoints discussed above, this assay endpoint would also be expected to measure morbidity as well as mortality. Alamar blue is a non-cytotoxic dye. Blue, nonfluorescent in its oxidized form, it is transported readily into living cells where it is reduced. In its reduced form Alamar blue is bright red, this form moves out of the cells into the surrounding media. The fluorescence is read on a plate reader with an excitation at 530 nm and an emission at 590 nm. It has the advantage of allowing the serial changes in the cell recovery rate to be checked because it does not affect cell viability [24,25]. The propidium iodide (PI) method is a quick, simple, and reproducible test for evaluating cell enumeration [26]. PI is a fluorochrome which binds to double-stranded DNA and RNA, primarily by intercalation. This technique is widely used in flow cytometry. Recently concerns about cytokines and other inflammatory mediators have been increasing. Acute skin inflammation is a complex process which involves resident epidermal cells, the cells of connective tissues, and blood vessels as well as invading leukocytes and lymphocytes interacting with each other under the control of cytokines and mediator networks. Keratinocytes, which consists of about 95% of the epidermal cell mass, act as both primary signal receivers and signal transducers, translating exogenous stimuli into endogenous signals [27–29], thereby initiating and controlling the body’s response both in the tissue and at the systemic level. The endogenous signals consist of various proinflammatory lipid- and peptide-mediators such as eicosanoids and cytokines. In addition, stimulated keratinocytes express intercellular adhesion molecules, which mediate

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the interaction of the cells with the inflammatory infiltrate [30]. Thus, the extraordinary signaling capacity of keratinocytes indicates a key role of this cell type in the induction and control of inflammatory responses. Signal release by keratinocytes is considered, therefore, to provide us with a suitable in vitro parameter related to irritancy. Although there are no specific cytokines, which represent the cutaneous irritant reaction, many researchers have tried to elucidate the release mechanisms of cytokines and other mediators [31–34].

3. Cell function-based assays Cell function-based tests measure responses of cells to sublytic concentrations of irritants. The fluorescein leakage assay is a measure of the barrier function of epithelial monolayers or multilayers. This has been used in several modified methods [35–37]. The rationale underlying the technique is based upon the assumption that irritancy is related, in part, to the ability of the epithelium to act as an effective barrier to potential irritants. Thus, the technical goal of the sodium fluorescein assay is to mimic, in part, the injury incurred by the stratum corneum by an irritant. The assay reflects several aspects of epithelial cell barrier function, for example, tight junctions/desmosomes and plasma membrane integrity. It is rapid, simple, and does not require specialized equipment. The disadvantages of the method are that an artefactual breach of the monolayer can yield false positive results, delayed effects may be underestimated, and there is no standardized procedure. The silicon microphysiometric method relies upon a light-addressable potentiometric sensor that allows indirect measurement of real-time changes in the cellular metabolism inside a flow through chamber. The instrument provides continuous monitoring of the rate at which living cells excrete acidic metabolites, which are a normal part of energy metabolism. The technique detects changes in cell metabolism that are likely to play a role in the local response to irritants [38]. A large number of cell types have been

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evaluated [39]. The endpoint is taken as the concentration of a test substance that causes a 50% reduction in the metabolic rate (MRD50). This is determined by measuring the metabolic rates of cells after exposure to increasing concentrations of a test substance and comparing the results to the pre-exposed metabolic rates.

4. Use of three-dimensional culture systems Conventional cell cultures, in which the cells are submerged in a culture medium, may be used to test the direct interaction between the test agent and the living cells. The extent of keratinocyte maturation in these cultures is lower than that of the epidermis in vivo, which allows the study of the effects of test agents only on the basal and suprabasal cells. In addition, this model is limited to water-soluble compounds. Therefore, a large number of preparations which may contact skin cannot be tested by this model. Furthermore, it is known that the time course and concentration dependence of a pharmacological response in vivo is strongly related to the barrier function of the stratum corneum. A good in vitro model, therefore, should adequately mimic the skin barrier function. Hence, topically applied agents should ideally be tested on a cultured skin substitute which morphologically, biochemically and functionally comes close to it’s in vivo counterpart. This condition can be met to a large extent when human keratinocytes are cultured at an air–liquid interface, attached to a biological matrix such as the dermis or a fibroblast-populated collagen matrix. Under these conditions a fully differentiated epidermis with a coherent horny layer is formed. The presence of a coherent layer of stratum corneum and the exposure of cultures to air permits a realistic application of skin irritants topically on the reconstituted epidermal surface. In this way, changes in tissue morphology, cell viability, keratin expression, lipid composition, barrier function and the modulation of cytokine production due to irritants may be evaluated. There are many publications, which detail the use of these three-dimensional culture systems [40– 48].

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5. Other assays

5.1. Skintex assay The Skintex dermal assay system is one example of the limited physico-chemical models, which have been used for the prediction of dermal toxicity [49,50]. Skintex uses the changes in relevant macromolecules on exposure to chemicals and formulations to predict in vivo irritancy and toxicity endpoints. The proposed mechanism of the Skintex assay involves alterations to the conformation and hydration of the ordered proteinbased macromolecular matrix, which suggests a relevance to the in vivo model, since there is a close relationship between the ability of a material to perturb proteins and its ability to produce skin irritation.

5.2. Microtox assay [51] This assay uses luminescent bacteria (e.g. Photobacterium phosphoreum, Vibrio fischeri ) as an indicator organism. The endpoint evaluated is the ATP-dependent light output from the bacteria. In principle, the bacteria reacts to the toxin by reducing the quantity of light emitted. The amount of light produced is roughly proportional to the number of bacteria present.

5.3. Tetrahymena motility assay [52] The assay is based on the supposition that specific cellular functions (in this case the motility of Tetrahymena) can be measured and that changes (decreases) in this function correlate with levels of irritation.

6. Correlation with skin irritation in vivo Given the complexity of the mechanisms involved in skin responses to irritants, it is especially challenging to develop an in vitro model that is predictive of skin irritation in vivo. Although current commercial skin culture kits are probably the most complex human organ culture available, they contain only the major skin struc-

tural cell types (epidermal keratinocytes and dermal fibroblasts) and do not contain the other cell types which are also important contributors to the skin inflammation mechanism in vivo (blood vessel endothelium, inflammatory cells, etc.). Nonetheless, there is an increasing appreciation of the complex and dynamic regulatory role played by skin keratinocytes and fibroblasts in terms of the inflammatory responses to irritants and sensitizers. Thus, it is reasonable and justifiable to use keratinocyte/fibroblast-based cultures as widely applicable in vitro models for skin irritation. In vivo tests for skin irritation, such as the Draize test, generally evaluate vascular endpoints of skin inflammation, namely erythema and edema. Since the human skin cell cultures currently available do not contain an intact vasculature, it is a research challenge to identify and validate in vitro endpoints that are of relevance to in vivo skin irritation. Generally the accuracy of an in vitro model is assessed by identifying a number of materials that have been previously tested for irritation in animals and then retesting the same materials in an in vitro assay. Data is conventionally analyzed by plotting the in vitro data on one axis and the animal data on the other. The mathematical relationship between the in vitro and in vivo scores, i.e., the algorithm that allows one to predict an in vivo score from an in vitro score, is called the prediction model [53–55]. Without such a predetermined relationship it is impossible not only to use an in vitro test correctly but also to perform a rigorous validation. One good example is the experiment in our laboratory using cultured human keratinocytes and its comparison with human patch test. We have conducted work on the comparison of in vitro with in vivo irritancy (Table 1) [56,57]. In weak and strong toxicity ranges, the ranking order of MTT and LDH relatively coincided. However, some discrepancies were found in shampoos of moderate toxicities. This may be due to the fact that in the in vivo situation irritants induce different mechanistic responses. Parish [58] obtained similar results using keratome slices of skin. The amounts of released acid phosphatase, lactate dehydrogenase and Nacetyl glucosaminidase were measured after expo-

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sure to chemicals, including surfactants, or bacterial toxins (Clostridium perfringens). In addition, histochemical changes and isotope utilization were determined. The correlation of these in vitro tests with in vivo change was good for weak irritants and moderate to poor for strong irritants. In the case of corrosive substances, they destroyed the ability of the tissue to respond appropriately. Wilhelm et al. [59] tested a spontaneously immortalized human keratinocyte line, HaCaT, as an in vitro model to predict the cutaneous irritation produced by anionic surfactants. For this purpose, a number of N-alkyl sulphate sodium salts with hydrocarbon chain lengths varying between C8 and C16 were studied for possible cytotoxic effects. The endpoints adopted to assess toxicity were the uptake of the vital dye neutral red and cell morphology criteria 24 h after dosing. A linear proportionality between keratinocyte number and neutral red uptake was established. All tested surfactants had cytotoxic effects as demonstrated by a decreased neutral red uptake, which showed a clear dose – response relationship. The in vitro cytotoxicity data proved to be highly reproducible when the test was repeated several weeks later. There were significant linear correlations between the IC-50 values and barrier damage (transepidermal water loss) and erythema (as evaluated by skin colour reflectance measure-

Table 1 Ranking order of irritancy by shampoos by in vitro and in vivo testsa Irritancy rankingb

1 2 3 4 5 a

Method MTT

LDH

Patch test

0.001%

0.001%

50%

100%

E B C A D

E B C A D

A E B C D

E A C B D

A, B, C, D, and E indicates five types of commonly used shampoos. b Irritancy ranking: 1\2\3\4\5 (more toxic\less toxic).

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ments). We think that the immortalized cell line is one of the ideal cells which compensate for the differences produced by different cell lines. Korting et al. [60] have already supported this idea. They evaluated the cytotoxic effects of surfactants on HaCaT cells, normal human keratinocytes and Swiss 3T3 embryonic mouse fibroblasts. The release of neutral red and the total protein content were affected in a dose-dependent manner in response to surfactant exposure. The three cell types showed a close correlation in the dose–response characteristics. In addition, the in vivo irritation potential of the surfactants was assessed in a soap chamber test using transepidermal water loss and skin redness as the endpoints. The neutral red release test showed a high correlation with in vivo responses while the total protein content did not correlate significantly. Bason et al. [50] tested several compounds using a dermal assay system. They compared in vitro data with human in vivo scores with respect to irritancy. For phenol, the Skintex system reproduced the in vivo dose–response quite accurately. However, it overestimated the irritant potential of trichloracetic acid, so that an analysis of the dose–response behaviour of this compound was not entirely relevant. This may represent a weakness in this assay system, but the anomaly is probably a result of the low pH of the test material used in the two-compartment model. At low pHs the membrane barrier is stripped from its support due to the corrosive effect of the acid, and the model system becomes invalid. Recently Demetrulias et al. [46] evaluated the ability of three-dimensional constructs to predict the skin irritation potential of surfactants. Four different laboratories were involved in the study. Fifteen substances, representing various surfactant categories and ranges of irritation potential, were tested. MTT assay was used to quantify viability in vitro. The time required to reduce the viability of each tissue to 50% of the distilled water controls was compared with mean erythema and edema scores from the clinical studies. No statistically significant interlaboratory variability was found. Only one false negative (lauramine

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Table 2 Causes affecting cell culture Culture system Cell type Media, factors Passage Donor (skin site, age, etc.) Irritation evaluation methods Irritant characteristics

oxide) was seen when non/mild and moderate/ severe irritant categories were assigned according to the in vitro scores. Many investigators have used animal skin to study the correlation of in vitro irritancy assessment with in vivo responses. Kemppainen et al. [61] studied the affect of liquid gun propellant on the barrier function of pig skin by measuring the penetration of [14C]benzoic acid following exposure to this chemical. Increase of permeability and the time course of return to normal barrier function were relatively similar between in vivo and in vitro experiments. Helman et al. [62] compared the in vivo irritancy of several irritants by evaluating histologic response 20 h later with microscopy. They performed a paralleled series of experiments by placing the discs of mouse skin on top of a stainless-steel ring support within the culture dish containing culture medium. Cellular enzyme leakage in the culture medium as well as histologic changes were determined. There were good correlations between in vitro and in vivo responses. The enzyme activity in the culture medium correlated well with the magnitude of histologic changes. Tachon et al. [63] compared in vitro and in vivo test methods for checking toxicity of hair dyes. They used Chinese hamster lung fibroblasts (V79) and determined the concentration required to reduce 50% of the growth (CI50). In vivo experiments were performed by intraperitoneal injection of the compounds into Swiss mice. The acute toxicity measured by LD50 showed a good correlation with CI50 in irritancy ranking orders of hair dyes. Other papers also deal with the relationship between in vitro and in vivo irritation test methods [64 – 66].

7. Considerations about in vitro irritancy tests In vitro toxicity testing systems have several advantages over in vivo systems [67]. They allow for control of environmental conditions, eliminate interactive systemic effects, and permit simultaneous or repeated sampling over time. They make it possible to perform complex interactive toxicity experiments and produce smaller quantities of toxic waste. Most of all, they reduce intact animal usage. However, they also have several disadvantages compared with in vivo toxicity testing systems [68]. Loss of in vivo organ morphology and functions as well as the absence of systemic influences are the foremost shortcomings. In addition, they are static systems resulting in progressive loss of function. In vitro toxicity tests are generally short-term studies, so that they have limitations in checking long-term toxicity. To use in vitro irritancy tests as good alternatives for in vivo irritancy, we must bear in mind the followings.

7.1. Factors affecting irritation In vivo and in vitro irritant reactions may be influenced by many conditions. Major extrinsic factors affecting irritation include membrane permeability factors (hydration, environment, regional variation, vehicle, membrane thickness), specific chemical irritant potential and the evaluation methods of irritant responses. Individual characteristics which affect the irritation experiments include age, sex, race and concomitant diseases [69].

7.2. Causes affecting cell culture responses Major causes of variation in the experiments using cell culture are listed in Table 2. As written before, a monolayer culture system may show different results from a three-dimensional culture system. According to investigators the culture system may be somewhat different and it must be written clearly in describing the experimental results. Cell type, of course, is one of the major determinants in experimental model. Fibroblasts show different irritant reactions from keratinocytes. Mucosa is clearly different from skin. We

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have experiences in making three-dimensional mucosa culture systems. We have reconstructed artificial buccal mucosa equivalents using keratinocytes and fibroblasts or de-epidermized dermis derived from non-cornifying buccal mucosa [48]. When treated with retinoic acid, the epidermis of buccal mucosa equivalents seemed to be less sensitive to retinoic acid than that of the skin. The effects of calcipotriol on the buccal mucosa equivalent and the skin epidermis were also different.

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Irritant reactions may vary according to the kinds of media, additional constituents, and calcium ion concentration in media (Fig. 1). Investigators must be alert to know whether the constituents in media or test materials could have an effect on the results. For example, tocopherol converts the color reaction in the MTT assay so that it may give false results. As the cultured cells also experience senescence according to the time lapse, passage in cell lines is also an important point that may be easily ignored. Irritant characteristics should be taken into considerations, because the solubilities and toxic ranges are different according to the chemical nature of irritants. In addition, the solvent itself can affect the irritation reaction. Experimental results also depend upon the investigators and the donors of skin sample so that there are variations among laboratories and cell lines (Fig. 2).

7.3. Considerations in cytotoxicity tests

Fig. 1. Different MTT results according to different batches of KGM. (A, B and C are different bottles of KGM). MTT assay was performed 72 h after 2× 104/cm2 cultured keratinocytes were allocated to each well of a 96 well plate. The same cell lines were used and the experiments were repeated 16 times. Data represent mean 9 S.D.

Fig. 2. Different MTT results according to cell lines (A, B, C, D and E are the different keratinocyte cell lines from different donors’ foreskins). MTT assay was performed 72 h after 2 ×104/cm2 cultured keratinocytes were allocated to each well of a 96 well plate. The experiments were repeated 16 times and they were tested by the same investigator. Data represent mean9 S.D.

Many assays use the same cell types with different exposure and endpoint protocols. Consequently they may provide subtly different information about the action of test materials on the cells. A number of key factors should be considered to meaningfully specify the method and, therefore, the quality of the data which it provides [70]. Firstly, how are the cells exposed to the test material? Secondly, what is the optimal time for exposure? Finally, what is the endpoint used to evaluate the effect of the test materials on the cell population? Most but not all test materials are diluted in aqueous medium but the time of exposure varies from 5 min to 48 h. Generally, those with long exposure times (i.e. ] 24 h) measure the effect at the end of the exposure period, e.g. the neutral red uptake assay. Neutral red release and Trypan blue exclusion assays measure toxicity immediately after short exposures, which means that toxicity must develop quickly if the endpoint is measured immediately after exposure. It should be remembered that the effective exposure time in vivo can be very short. Therefore, shorter exposure times (e.g. B 30 min) could be expected to be more closely approximate to experiences in vivo.

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three-dimensional culture, the doses giving 50% inhibition of DNA synthesis were a factor of ten lower than those required to release 50% of LDH. Thus, the endpoint chosen provides specific information about the health of the cell population, but the information provided by each endpoint is not necessarily the same. These considerations may be taken into a comparative study of the cytotoxicity of skin irritants on human oral and skin keratinocytes in monolayer culture model [71]. Cytotoxicity was evaluated by changes in mitochondrial metabolic activity (MTT assay) (Fig. 3) and plasma membrane integrity (LDH leakage) (Fig. 4) 24 h after exposure to irritants (sodium lauryl sulfate and benzalkonium chloride). Because these irritants

Fig. 3. Log dose – response curves determined by MTT assay for sodium lauryl sulfate (SLS) and benzalkonium chloride (BK) on skin and oral keratinocytes. Data are means and S.D.s of six experiments using cells from six different individuals with three replicates per dose group in each experiment. (a) SLS, (b) BK. *PB 0.05 (mean9 S.D.). — —, Skin keratinocyte; — — , oral cell.

The endpoint measure is intended to show the extent of damage caused by the test substance. Many endpoint measurements are possible. However, they may not provide comparable information. The range includes loss of membrane integrity, release of cytoplasmic enzymes, loss or decrease of metabolic activity, cessation/reduction of DNA synthesis or the inability to replicate. Cell division is one of the most sensitive endpoints, because it is one of the first processes to cease when a cell is exposed to a toxin. It is regarded that the relative sensitivity of these endpoints is generally cell replication\ metabolic \ membrane integrity. For example, in a comparative study using human fibroblasts in a

Fig. 4. Effect of sodium lauryl sulfate (SLS) and benzalkonium chloride (BK) on LDH leakage in cultures of skin and oral keratinocytes. Data are means and S.D.s of six experiments using cells from six individuals with three replicates per dose group in each experiment. (a) SLS, (b) BK; *PB0.05 (mean9 S.D.). — — , Skin keratinocyte; — — , oral cell.

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Fig. 5. (a) Effect of antimicrobial agents on cell proliferation of cultured human keratinocytes and fibroblasts measured by MTT. (b) Effect of antimicrobial agents on cell proliferation of cultured human keratinocytes measured by MTT and [3H]methyl thymidine incorporation. Cells were treated with the concentrations 0.01X. ‘X’ equals undiluted concentration (volume/volume%) of tested agent.

are highly water-soluble, they were mixed with culture media in varying concentrations. Dose – response curves were compared according to irritants and assay methods. We have concluded that the reaction patterns of the oral and skin keratinocytes to irritants were similar although benzalkonium chloride is more cytotoxic than sodium lauryl sulfate at the same concentrations. Our other application was a comparative study of cytotoxicity of topical antimicrobials to cul-

tured human keratinocytes and fibroblasts [72]. We used the MTT assay, LDH release and tritiated thymidine incorporation measuring DNA synthesis (Fig. 5). When the 50% inhibition concentration (IC50) of topical antimicrobials were compared, the order of cytotoxicity of the antimicrobials was obtained. In this study the tritiated thymidine incorporation appeared to be a more sensitive method in detecting cytotoxicity than MTT because the IC50 was lower in the former.

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7.4. Practical approach and selection of in 6itro irritancy tests First of all we must review data in existing databases from suppliers, trade organizations and the scientific literature. In addition, an analysis of the physico-chemical characteristics, such as extremes of pH or acid/alkaline reserve, may indicate a potential for skin irritancy. In certain cases additional skin irritancy data which were performed to assess the dermal safety of topically applied materials may be available. For example, skin irritancy data may be available from guinea pig or mouse skin sensitization studies or from rabbit percutaneous toxicity studies. However, the utilization of such data to assess skin irritation needs careful consideration. After reviewing historical data experimental models can be chosen. An in vitro test must satisfy the following things [73 – 76]. Firstly, it must appropriately model the exposure kinetics, i.e. it must accept the test material in the same physical form as the animal test, and it must remain in contact with the test material for the same amount of time. Secondly, the end points that are developed for the in vitro assay must be predictive of the underlying in vivo tissue responses and this relationship must be clearly understood. Other key factors to be considered include the physical characteristics of the products, the expected level of toxicity, the resolution required, the intended use of the resulting data, and the resources available. For example, if a material is not water soluble it would be fruitless to attempt to test it in monolayer cultures. A topical application assay in three-dimensional human keratinocyte cultures would be the logical choice in this situation because the test material will be applied directly to the surface of the target cells, ensuring exposure similar to that which would occur in vivo [77]. Studies using various in vitro irritancy tests have been published [71,78 – 84].

8. Conclusion The development of an in vitro irritancy testing model is a challenge to all of us because animal

models are likely to be prohibited in the future by regulations and law. The morphological and biochemical similarity of cultures and intact skin and the ability to reproduce relevant aspects of skin irritation mechanisms needs to be obtained. Considering all the points we mentioned above, it is clear that the monolayer culture system has limitations in the validation as an alternative method for in vivo irritancy. In that sense, three-dimensional culture seems to be more likely to be a potential candidate in the future, which is beyond this review.

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