Assessment of contact allergens by dissociation of irritant and sensitizing properties.

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose-response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay.     

Employment of cytology for in vitro skin irritation test using a reconstructed human epidermis model,Keraskin™

Skin irritation tests using reconstructed human epidermis (RhE) employ viability as an endpoint, but color interference or borderline results are often problematic. We examined whether the cytology of cells from treated RhE could determine skin irritancy. Six chemicals (three irritants; DnP, 1-B, PH, three non-irritants; DP, APA, HS) were evaluated in a RhE, Keraskin™. DP, HS, and PH were clearly classified with viability, but DnP, 1-B, and APA were often falsely determined, due to borderline values falling near the cutoff, 50%. In histology, the tissues treated with DnP, 1-B, and PH showed erosion of the stratum corneum, vacuolization, and necrosis in the basal layer. DP- and HS-treated tissues showed relatively normal morphology but APA induced necrosis similar to irritants. Cytology revealed that DnP, 1-B or PH depleted cells and induced irregular and abnormal cell shapes. In contrast, relatively regular and normal shapes and clear distinction between the nucleus and cytoplasm was observed for DP, APA and HS. To further confirm it, additional 10 substances, including false positives from OECD TG 439, were tested. Overall (16 substances in total), cytology: total area predicted the skin irritancy of test chemicals with the highest accuracy (87.5%) followed by cytology: cell count (81.3%), histology (75%) and viability (68.8%), confirming the utility of cytology as an alternative endpoint in the skin irritation test using RhE.     

Development of an in vitro corrosion/irritation prediction assay using the EpiDerm skin model.

A validation of the in vitro skin corrosion method using the EpiDerm skin model was performed using 12 recommended chemicals. All chemicals were correctly classified by OECD test guideline 431. In order to predict corrosion and/or irritation potential, additional compound exposure times and IL-1alpha measurements were included in a tiered testing approach. Four exposure times were performed followed by MTT (viability) and IL-1alpha measurement. This allowed classification of corrosive chemicals (OECD guideline 431) and those likely to be severe irritants. If the chemical was found to be corrosive or a severe irritant, no further experimental work was performed, otherwise a second experiment was performed using three further exposure times (same endpoints). The second experiment provided information on whether the chemical was likely to be a moderate/mild irritant. If the chemical was negative following both experiments, it was predicted as non-corrosive/non-irritating. A total of 12 chemicals were tested in the irritation or combined assay (five non-irritants, seven irritants). Specificity (% non-irritants concurring with EU classification) was 60% (MTT) and 100% (MTT+IL-1alpha). Sensitivity (% irritants concurring with EU classification) was 86% (MTT) and 86% (MTT+IL-1alpha). Accuracy (% chemicals correctly identified) was 75% (MTT) and 92% (MTT+IL-1alpha).     

Assessment of an ex vivo irritation test performed on human skin explants and comparison of its results with those of a 24-/48-h human patch test for the evaluation of cosmetics

When developing new cosmetics, it is extremely important to consider the safety of consumers. Absence of potential irritancy is generally assessed using an OECD TG439 compliant Reconstructed Human Epidermis (RHE) systems and MTT assays, resulting in an irritant/not irritant classification. To gain insight into the irritancy of molecules/finished cosmetic products and to predict the outcome of irritation tests performed on subjects whatever their nature, we developed a test that uses skin explants and histological analysis. Results showed that this irritation test is sensitive enough to accurately and repeatably detect known irritants. If the diverse origin of the skin explants used led to variability in the histological alterations scored, the overall grading of irritancy is highly reproducible. Finally, when testing 120 non-alcoholic cosmetics of various galenic forms, comparison of data between the ex vivo irritation tests and of a 24-/48-h human patch test revealed a single false negative, very close to the limit, and a 10% false positive rate. It was not possible to calculate the sensitivity of the ex vivo irritation test; however, its specificity was 89.9% and its accuracy was 89.1%. Similar results, with a slightly higher false positive rate, were found when testing 49 alcoholic cosmetics. These values exceed the minimum requirements of OECD TG439.     

In vitro skin irritation testing with human skin cell cultures

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have evaluated the effects of chemical irritants on human epidermal keratinocytes (NHEK) and on keratinocyte-dermal fibroblast (NHEK/DF) co-cultures. Cell viability in NHEK cultures, measured as incorporation of the vital dye neutral red (NR), was reduced in a dose-dependent manner in response to the chemical irritants tested. The half-maximal effective concentration (NR₅₀) values correlated with irritation scores in human patch tests with these materials. Certain materials were found to be incompatible with this test system. NHEK/DF cultures were treated with ten prototype surfactants, and were evaluated for cell viability (MTT incorporation), cytotoxicity (release of the enzymes lactate dehydrogenase and N-acetyl glucosaminidase), metabolism (glucose utilization), and inflammatory mediator (prostaglandin E₂) release. There was a close correlation of the dose-response characteristics for all the endpoints tested, and between the in vitro responses and human patch test scores for the surfactants tested. These results demonstrate the usefulness of human skin cell cultures and of cell viability, cytotoxicity, and inflammatory mediator release as endpoints, for the in vitro assessment of skin irritancy.     

Comparison of in vitro and in vivo human skin responses to consumer products and ingredients with a range of irritancy potential.

Human skin equivalent cultures were investigated as possible pre-clinical skin irritation screens to aid safety assessments for chemicals and product formulations, and to facilitate design of safe and efficient human studies. In vitro responses in human skin equivalent cultures were compared directly to in vivo human skin responses from historic or concurrent skin tests for representative chemicals and products, including surfactants, cosmetics, antiperspirants, and deodorants. The in vivo data consisted of visual scores (i.e., erythema and edema) from skin-patch tests and diary accounts of skin irritation from product-use studies. In the in vitro studies, cornified, air-interfaced human skin cultures (EpiDerm) were evaluated using methods designed to parallel human clinical protocols with topical dosing of neat or diluted test substances to the stratum corneum surface of the skin cultures. The in vitro endpoints have previously been shown to be relevant to human skin irritation in vivo, including the MTT metabolism assay of cell viability, enzyme release (lactate dehydrogenase and aspartate aminotransferase), and inflammatory cytokine expression (Interleukin-1alpha). For surfactants, dose-response curves of MTT cell-viability data clearly distinguished strongly-irritating from milder surfactants and rank-ordered irritancy potential in a manner similar to repeat-application (3x), patch-test results. For the antiperspirant and deodorant products, all the in vitro endpoints correlated well with consumer-reported irritation (r, 0.75-0.94), with Interleukin-1alpha (IL-1alpha) release, showing the greatest capacity to distinguish irritancy over a broad range. IL-1alpha release also showed the best prediction of human skin scores from 14-day cumulative irritancy tests of cosmetic products. These results confirm the potential value of cornified human skin cultures as in vitro pre-clinical screens for prediction of human skin irritation responses. A preliminary report of these results has been published.     

HSP27 as a biomarker for predicting skin irritation in human skin and reconstructed organotypic skin model

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessing parameters or endpoints are still not sufficient. To discover novel endpoints for skin irritation responses, 2DE-based proteomics was used to analyze the protein expression in human skin exposed to sodium lauryl sulfate (SLS) following the test protocol of the European Centre for the Validation of Alternative Methods (ECVAM) in the present study. HSP27 was up-regulated most significantly among the eight identified proteins, consistent with our previous reports. Acid and basic chemicals were applied on human skin for further validation and results showed that the up-regulated expression of HSP27 was induced in 24 h after the exposure. Skin-equivalent constructed with fibroblasts, basement membrane and keratinocytes was used to investigate the potential of HSP27 as a biomarker or additional endpoint for the hazard assessment of skin irritation. Our skin-equivalent (Reconstructed Organotypic Skin Model, ROSM) had excellent epidermal differentiation and was suitable for the skin irritation test. HSP27 also displayed an up-regulated expression in the ROSM in 24 h after the irritants exposure for 15 min. All these results suggest that HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.     

Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing

The potential of rat epidermal keratinocyte (REK) organotypic culture (ROC) with proper stratum corneum barrier as a model for screening skin irritants was evaluated. The test chemicals were selected from ECETOC database (1995) and the observed in vitro irritation potential was compared to ECETOC in vivo primary irritation index (PII), to EU risk phrases, and to the harmonized OECD criteria. Chemicals were applied onto the stratum corneum surface of ROC for 30 min and samples were taken from the underlying medium at 4 and 8 h after exposure. Cell membrane integrity (determined by LDH assay) and pro-inflammatory effect (determined by IL-1alpha release) were verified at both time points and correlated to PII values. The best correlation (R(2) = 0.831) was seen with LDH leakage test. Based on obtained data, chemicals were classified according to criteria defined by EU and OECD. From 12 chemicals, only two were incorrectly classified according to OECD criteria when using LDH leakage and IL-1alpha release as irritation markers. At the end of experiment, chemical-treated ROC cultures were fixed and histological changes were assessed. Typical signs for irritation were lightly stained cytoplasm, condensed nuclei, cellular vacuolization, eosinophilic cytoplasms, and blebbing. These irritation effects of chemicals were graded visually into four classes (A-D). The extent of morphological perturbations of the cultures mostly correlated with PII. The present results indicate the validity of the ROC model in predicting skin irritation potential of chemicals and show that the use of set of irritation markers with different mechanistic responses gives more information on irritation than if only one marker was used.     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.     

Prediction of ocular irritancy potential from dermal irritation test results

In a survey of the test files of Haskell Laboratory, it was found that of the materials that had been tested on rabbit skin for primary irritation or corrosivity, 60 were severe irritants or corrosive on the skin and had also been tested for ocular irritancy. Of these 60 materials only 39 were severe eye irritants, while 15 were mild irritants or non-irritants and the remainder were considered to be moderate irritants. Thus, to suggest that materials are eye irritants solely on the basis of their skin-irritation properties may be misleading, even when severe dermal irritants are considered.     

A COMBINED MURINE LOCAL LYMPH NODE AND IRRITANCY ASSAY TO PREDICT SENSITIZATION AND IRRITANCY POTENTIAL OF CHEMICALS

In an effort to establish a single, rapid screening procedure for the sensitization and irritancy potential of new chemicals, the parameters of a murine Local Lymph Node Assay and a mouse ear swelling irritancy assay were combined. To validate this assay, a range of chemical irritants and sensitizers were evaluated for their ability to elicit responses in B6C3F1 female mice. Chemicals were administered for four consecutive days to the dorsal and ventral surfaces of each ear. An increasein ear thickness served to predict irritancy, while [3H]thymidine uptake by cervical draining lymph nodes suggested sensitization. All chemicals known to be potent chemical sensitizers (oxazolone, 2,4-dinitrofluorobenzene, toluene diisocyanate) produced a marked lymph node cell proliferation in this assay. Animals exposed to irritating agents (sodium lauryl sulfate, croton oil, tetradecane, nonanoic acid, and benzalkonium chloride) experienced a significant increase in ear swelling. In addition, these irritating agents elicited lowlevel lymphocyteproliferation.In cases wherechemicals areconsidered tobeboth potent sensitizers and irritants (2,4-dinitrofluorobenzene, toluene diisocyanate, and benzalkonium chloride), robust increases in [3H]thymidine incorporation and ear swelling were demonstrated. Results were dose-responsive for all chemicals tested. The combined LLNA/ear swelling assay appears to be a reliable predictor of sensitization and irritancy potential, since it identified the activity of all eight chemicals tested. The advantages of this approach include a further reduction in the number of animals and time required to screen chemicals for both irritancy and/or sensitization potential. Although this assay does not have the capacity to discriminate between nonspecific lymph node proliferation and weak sensitizing ability of strong irritants, the information gained by the irritation component of the assay provides additional information when evaluating the significance of low-level lymphocyte proliferation in the LLNA. With further validation this assay could be useful as a common screening tool in the research and development of new chemical products.     

Eye irritation potential: palm-based methyl ester sulphonates

Aim: To evaluate eye irritation potential of palm-based methyl ester sulphonates (MES) of different chain lengths; C12, C14, C16, C16:18.

Methods: The Bovine Corneal Opacity and Permeability test method (BCOP), OECD Test Guideline 437, was used as an initial step to study the inducing effect of palm-based MES on irreversible eye damage. The second assessment involved the use of reconstructed human corneal-like epithelium test method, OECD Test Guideline 492 using SkinEthic? Human Corneal Epithelium to study the potential effect of palm-based MES on eye irritancy. The palm-based MES were prepared in 10% solution (w/v) in deionized water and tested as a liquid and surfactant test substances whereby both test conducted according to the liquid/surfactant treatment protocol.

Results: The preliminary BCOP results showed that palm-based MES; C12, C14, C16, C16:18 were not classified as severe eye irritants test substances with in vitro irritancy score between 3 and the threshold level of 55. The second evaluation using SkinEthic? HCE model showed that palm-based MES; C12, C14, C16, C16:18 and three commercial samples were potentially irritants to the eyes with mean tissue viability ≤ 60% and classified as Category 2 according to United Nations Globally Harmonized System of Classification and Labelling of Chemicals. However, there are some limitations of the proposed ocular irritation classification of palm-based MES due to insolubility of long chain MES in 10% solution (w/v) in deionized water.

Conclusion: Therefore, future studies to clarify the eye irritation potential of the palm-based MES will be needed, and could include; methods to improve the test substance solubility, use of test protocol for solids, and/or inclusion of a benchmark anionic surfactant, such as sodium dodecyl sulphate within the study design.     

Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.     

Alternative methods for eye and skin irritation tests: an overview

The evaluation of eye and skin irritation potential is essential to ensuring the safety of individuals in contact with a wide variety of substances designed for industrial, pharmaceutical or cosmetic use. The Draize rabbit eye and skin irritancy tests have been used for 60 years to attempt to predict the human ocular and dermal irritation of such products. The Draize test has been the standard for ocular and dermal safety assessments for decades. However, several aspects of the test have been criticised. These include: the subjectivity of the method; the overestimation of human responses; and the method's cruelty. The inadequacies of the Draize test have led to several laboratories over the last 20 years making efforts to develop in vitro assays to replace it. Protocols that use different types of cell cultures and other methods have been devised to study eye and skin irritation. Different commercial kits have also been developed to study eye and skin irritation, based on the action of chemicals on these tissues. This article presents a review of the main alternatives developed to replace the use of animals in the study of chemical irritation. Particular attention is paid to the reproducibility of each method.     

Relevance of in vitro tests to in vivo acute skin inflammation: potential in vitro applications of skin keratome slices, neutrophils, fibroblasts, mast cells and macrophages

In vitro tests on cells or keratome slices of skin may reproduce, or indirectly reflect, the first event in acute inflammation, the cytotoxic action of an irritant on epithelial/epidermal cells. Keratome slices of human or animal skin release enzymes, show histochemical changes and demonstrate increased or decreased utilization of isotope-labelled amino acids when exposed to chemicals, including surfactants, or bacterial toxins (Clostridium perfringens). The correlation with in vivo change is good for weak irritants and moderate to poor for strong irritants. Corrosive substances destroy the ability of the tissue to respond. Similar results have been obtained in tests on fibroblast cultures without or with an agar-keratin barrier. Neutrophils, or their separated granules, and mast cells have limited application in the prediction of chemical irritancy. The relevance and limitations of in vitro toxicological predictive tests are assessed in terms of the in vivo feature reflected by the in vitro test, the range of chemicals active in vitro compared to the in vivo responses, the ability to discriminate between intensities of reactions and the need for standards to compare results in different laboratories.     

Dual Characteristics of Skin Care Creams Evaluated by Two In-Vivo Human Experimental Models

The use of skin care creams is a well documented protection measure to reduce the risk of barrier damage and contact dermatitis from exogenous contact with skin irritants. Before choosing a skin care cream two aspects should be considered: a) Is the product able to reduce irritant reactions caused by the irritant, and b) is the product well tolerated, also on damaged skin. Both aspects can be evaluated by experimental models in human volunteers. We used two standard experimental designs to compare six commercially available skin care products: a) the chamber scarification test, designed to assess the irritancy potential, and b) the repeated short-time occlusive irritation test (ROIT), developed to evaluate the efficacy of skin care creams. The results showed that a high score in the chamber scarification test for skin irritation was not necessarily correlated to the products' ability to impede sodium lauryl sulfate (SLS)-induced irritant skin reactions. Three products were shown to both have a low irritancy potential and be capable of reducing skin barrier damage induced by SLS, and one product had both an irritant potential on scarified skin and also a modest capability to reduce skin irritation induced by SLS. The use of both test methods, chamber scarification and ROIT, gives valuable information on skin compatibility and efficacy of skin care creams. The clinical relevance of the test results can only be determined by comparing products with high and low scores in both tests in controlled clinical experiments with subjects at risk of developing irritant contact dermatitis.     

Can in-vitro assays predict chemically-induced skin damage?

Eleven chemicals with a known potential to damage skin as corrosive, irritants or non-irritants, have been tested for their ability to affect the function and viability of mammalian cells in three in vitro assays. The assays measured total protein content by Coomassie Blue R binding (CB), lysosomal integrity by Neutral Red uptake (NR), or mitochondrial function by MTT uptake/reduction. Two cell lines, Chinese Hamster Ovary cells (CHO) and Swiss Albino Mouse Fibroblasts (3T3), were compared. The results of all three assays agreed well with one another and with the observed number and morphology of the cells for each chemical tested. The CB tended to be the least sensitive and the NR the most variable of the three assays. Overall the MTT assay was most reliable, but may give anomalous results if used alone. CHO and 3T3 cells gave similar results, although 3T3 cells tended to be less sensitive than CHO cells. This may depend principally on the rate of division and proportion of dividing cells. Using the results from the MTT assay, corrosive chemicals were cytotoxic, since concentrations of less than 10 μg/ml reduced dye uptake by 50%. Chemicals which were skin irritants were moderately cytotoxic, since concentrations in the order of 50–500 μg/ml reduced dye uptake by 50%. Non-irritant chemicals were not cytotoxic up to 5 mg/ml. In decreasing order of cytotoxicity, the chemicals were ranked: tributyltin chloride, dibutyltin dichloride, silver nitrate, benzalkonium chloride, zinc monoglycerolate, sodium dodecyl sulphate, benoxaprofen, fenclofenac, n-hexane, butan-1-ol, 2-methoxyethanol. The categorization and ranking of the degree of skin irritation concurred with the degree of cell kill. These results indicate that such simple in-vitro tests could be used instead of animals to predict skin irritants at an early stage of hazard identification.     

Development of an oral mucosal irritation test using a three-dimensional human buccal oral mucosal model

The oral mucosa can become irritated by oral care products and lip cosmetics. Therefore, it is important to determine the irritation potential of their ingredients and products during safety evaluations. We developed a method for oral mucosal irritation test using EpiOral, which is a three-dimensional cultured model. Exposure of sodium lauryl sulphate (SLS) to EpiOral showed a dose-dependent decrease in cell viability. Under 120 min exposure conditions, SLS irritation was detected when 60% cell viability was set as a criterion. Evaluation of the irritancy of SLS and four other raw materials used in oral products at three laboratories under the above conditions confirmed good transferability of the test. Focused on the similarity of the oral and eye mucous, 32 chemicals categorised by the UN-GHS eye-irritation classification were evaluated to ensure the reliability of our criteria at these laboratories. The concordance rate between the UN-GHS classification and our test results was 100% for irritants and 60% for non-irritants. The good intra-laboratory reproducibility of our test was confirmed from the evaluation results of negative and positive controls, and the good inter-laboratory reproducibility was confirmed from the results of 32 chemicals. These findings showed that oral mucosal irritation can be evaluated using EpiOral.     

Interspecies comparisons of skin irritancy.

In a series of patch testing experiments, 24 familiar household materials or industrial chemicals were applied to intact and abraded skin of rabbits, guinea pigs, and humans for four hours. Skin responses were graded 4, 24, and 48 hr after application of the patches. A number of materials caused greatly different reactions from one species to another. Some caused tissue damage at abraded sites in the animals, but caused only mild reactions on either intact or abraded sites of humans. Hypochlorite bleach caused severe reactions on intact human skin, but produced considerably less reaction on both intact and abraded skin of rabbits and guinea pigs. Sodium tripolyphosphate and sodium carbonate caused vesiculation at abraded sites of one of six humans, but produced only slight reactions on animal skin. From these results it is concluded (a) that neither rabbit nor guinea pig skin should be relied upon exclusively to identify potentially hazardous irritants to human skin and (b) that the testing of abraded skin is unnecessary and can be misleading in the interpretation of the results, making the animal tests less, rather than more, reliable. Although neither species is entirely reliable, even if abraded site testing is omitted, the rabbit and the guinea pig appear to be similarly predictive as models for preliminary screening of household products and their chemical components for skin irritancy.     

In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants.

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have investigated the use of human skin fibroblasts for in vitro screening for skin toxicity. To assess the cytotoxic effects of surfactants, cell viability was measured by the NRU (neutral red uptake) assay and AB (Alamar blue) assay as in vitro methods. The skin irritation potential of surfactants by human skin patch test was assessed as in vivo methods. The close relationship was found between AB assay with human skin fibroblasts and human patch test (r=0.867). There was a relatively good agreement between the NRU and in vivo patch test (r=0.648). These results suggest that AB and NRU assay using cultured human fibroblast could be predictable methods for the irritancy of various surfactants in human.