Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Assessment of the phototoxic potential of compounds and finished topical products using a human reconstructed epidermis.

The goal of this study was to design a model system for the assessment of phototoxic potential using a human reconstructed epidermis (HRE, SkinEthic Laboratories, Nice, France), by testing some representative phototoxic (P) and non-phototoxic (NP) compounds and finished topical products. The tissue response to 24-h application of 5-5000 microg/mL of the test agents in the presence and absence of UVA light was analyzed in terms of viability (Lactate Dehydrogenase release), pro-inflammatory activity (IL-8 release and mRNA expression) and morphology (histopathology). 8-Methoxypsoralen (P) and promethazin (P), but not sodium lauryl sulfate (NP) produced cytotoxicity concentration-response curves significantly different between irradiated and nonirradiated tissues. Only irradiated tissues showed morphological damage. Application of tetracyclin (P) in the culture medium, but not topically, induced similar signs of phototoxicity. 6-Methylcoumarine (weak P) was not cytotoxic, yet it increased IL-8 release and mRNA expression only following UVA irradiation. PUVA therapy creams containing 1% 8-Methoxy-psoralen (P) or coal tar (P) decreased viability and induced histologic damage in UVA-exposed tissues. In conclusion, the phototoxic potential of the tested agents was correctly predicted by using a tiered strategy that involves determining cytotoxicity, production of IL-8, and morphological damage following exposure of the HRE to the compounds and UVA light.

Cytokine profile of human bronchoalveolar macrophages and bronchial epithelial cells in response to inhalation particles of the cyclosporine derivative IMM 125.

Administration of antiasthmatic drugs in the form of inhalation particles may alter the cytokine network in the airways, independently of their pharmacological actions. Changes induced by drugs not well tolerated may potentially contribute to the immunopathology of the disease, a strongly undesirable effect. In this study, cell viability assays and characterization of the cellular profile of cytokines and chemokines were performed in order to investigate the response of human bronchoalveolar macrophages and bronchial epithelial cells in culture to inhalation particles of the cyclosporine derivative IMM 125. Interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-8 were assayed by enzyme-linked immunosorbent assay (ELISA) in the supernatants of bronchoalveolar macrophages, and RANTES, granulocyte--macrophage colony-stimulating factor (GM-CSF), and IL-8 in those of bronchial epithelial cells. Cells were studied both under basal and stimulated conditions (lipopolysaccharide and TNFalpha were used for activating macrophages and epithelial cells, respectively). The immunosuppressant FK 506 and the glucocorticoid Budesonide served as comparison. IMM 125 did not affect cell viability (except at high concentrations and long time periods). Moreover, IMM 125 did not induce an increase in the secretion of any of the cytokines and chemokines measured with respect to nontreated cells, except for a slight increase in IL-8, an effect that was also observed for FK 506, Budesonide, and inert latex particles, and was therefore regarded as nonspecific. Furthermore, IMM 125 significantly decreased the secretion of TNFalpha, IL-1beta by macrophages, and GM-CSF by epithelial cells, suggesting an antiinflammatory potential. In conclusion, the present in vitro results point to a good tolerance of human airways to IMM 125 inhalation particles.

Early changes in murine epidermal cell phenotype by contact sensitizers.

In order to develop an in vitro predictive assay for the detection of contact sensitizers, we investigated the possible modulation of the expression of cell-surface molecules in the early phases of treatment of murine epidermal cells (EC) with known contact sensitizers. After in vitro treatment of Balb/c EC with the strong contact sensitizer, TNBS, Langerhans cells (LCs) demonstrated a rapid up-regulation of CD45, CD40, CD32/16 (Fc gamma RII/III) and CD23 (Fc epsilon RII) molecules. CD45 and CD40 were also rapidly up-regulated on the dendritic epidermal T cells. Interestingly, after treatment with this severe sensitizer, a marked induction of CD40 expression was found on a CD45 negative population, most probably keratinocytes. In contrast to these cell-surface molecules, I-Ad/I-Ed and CD90.2 expression were unchanged. No change was observed on the expression of CD45 and CD40 after treatment with a mild or a weak contact sensitizer, citral and citronellal respectively. In contrast, like TNBS, they up-regulated the expression of CD32/16 and CD23 on LCs. The irritant sodium dodecyl sulfate had no effect on all these cell-surface molecules. Our results indicated that in vitro, chemicals with allergic potential induced early specific phenotype changes that may represent an early-activated state of the cells. This state may be responsible for initiating the afferent phase of contact sensitivity in vivo. Based on these findings, it might be possible to develop an in vitro assay to reduce the number of experimental animals for a fast screening of contact sensitizers and for discriminating between mild contact sensitizers and irritants.

Modulation of the activity of human monocyte-derived dendritic cells by chemical haptens, a metal allergen, and a staphylococcal superantigen.

For the development of mechanistic assays in immunotoxicology, the phenotype, cytokine production, and stimulatory function of dendritic cells (DCs) were assessed after incubation with the chemical haptens aminophenol, chlorpromazine hydrochloride, dinitrochlorobenzene (DNCB), and with the DNCB-corresponding tolerogen DCNB, the metal allergen nickel sulfate, the irritants sodium dodecyl sulfate and benzoic acid, as well as with staphylococcal enterotoxin B (SEB) and lipopolysaccharide (LPS). DCs were differentiated from human monocytes by in vitro exposure to GM-CSF and interleukin-4 (IL-4) for 7 days. Flow cytometric data revealed that only representative haptens increased the surface expression of HLA-DR, CD86, CD40, and of CD54 on DCs when compared to irritants or to the tolerogen. This event was associated with an increased ability of DCs to stimulate T cell proliferation. Moreover, after incubation with the haptens, but not with the irritants or the tolerogen, a higher production of TNF-alpha by DCs was observed. Under our experimental conditions, no release of IL-1beta, IL-10, or IL-12 was detected. Compared to the activation elicited by haptens, SEB strongly up-regulated HLA-DR and costimulatory molecule expression. In agreement with this effect, there was a marked release of TNF-alpha and a slight production of IL-12. IL-1beta and IL-10 were not detected in the culture medium. Finally, SEB-pulsed DCs showed a strong T-cell-stimulating activity. These data underline the activating potential of haptens versus irritants or a tolerogen on DC functions. The different levels of DC activation by haptens and SEB suggested that distinct cellular events were involved.

Comparison of the phagocytosis of two types of cyclosporin (SDZ OXL 400 and SDZ IMM 125) by alveolar macrophages from hamsters.

The aim of this study was to compare two types of cyclosporin (Cs) particles, SDZ OXL 400 and SDZ IMM 125, the latter being more hydrophilic, to understand their uptake by airway macrophages. Alveolar macrophages (AM), harvested by bronchoalveolar lavage (BAL) of hamster lungs, were cultured with two different doses (0.1 mg and 0.5 mg) for 1 h, 6 h, and 24 h. Control incubations without Cs particles or with latex particles were carried out simultaneously. Cell viability, cell activation (i.e., respiratory burst, interleukin-6 (IL-6) synthesis) and mean volume of particles phagocytosed per macrophage were measured. Both types of Cs particles did not modify the AM viability, and failed to induce IL-6 synthesis during phagocytosis but slightly decreased the cell oxidative respiratory burst. The comparison between SDZ OXL 400 and SDZ IMM 125 showed that for the lower dose the mean volume of both Cs types phagocytosed was similar at 1 h and 6 h. At 24 h an increase of the mean volume phagocytosed was seen for SDZ IMM 125 but not for SDZ OXL 400. For the higher dose the mean volume of SDZ IMM 125 phagocytosed was higher than SDZ OXL 400 at 1 h and 6 h and comparable for both types at 24 h. SDZ IMM 125 particles were phagocytosed more rapidly than SDZ OXL 400. The mean volume of phagocytosed latex particles increased with time and dose and was higher than for both Cs particle types. In conclusion, AM were seen to phagocytose particles of different physical properties (i.e., form, size, and shape), chemical properties (i.e., inert or peptidic) and degrees of hydrophilicity in a different manner.

Comparison of the rate of phagocytosis of orthorhombic cyclosporine A (CsA) and latex particles by alveolar macrophages from hamsters.

The aim of this study was to develop an in vitro model to estimate the clearance of pulmonary administered cyclosporine A (CsA). To do this we estimated the volume of CsA particles phagocytosed by alveolar macrophages (AM) lavaged from hamsters. AM were cultured with CsA particles at two doses of particles (0.1 mg or 0.5 mg) and at three incubation times (1 h, 6 h or 24 h). The AM were also incubated with or without latex particles. After incubation, AM were processed for light and electron microscopy and the mean volume of phagocytosed particles was estimated stereologically from micrographs of the cells. Here, however, the CsA particles were dissolved during the embedding process and only their negative images (vacuoles) could be detected. An indirect method was therefore developed. The volume of cytoplasmic vacuoles (called 'background' vacuoles) was estimated in control macrophages (without particles or with latex particles and subtracted from the total volume of vacuoles in macrophages incubated with CsA, which gave the volume of phagocytosed CsA. The volume of the 'background' vacuoles remained constant in all study conditions. At a dose of 0.1 mg CsA the volume phagocytosed per macrophage was 13.83 microns3 at 1 h, 8.43 microns3 at 6 h and 4.50 microns3 at 24 h. At a dose of 0.5 mg CsA, the volume phagocytosed varied from 26.59 microns3 at 1 h, to 4.13 microns3 at 6 h and 49.10 microns3 at 24 h. These results show no statistically significant dependence on time for either dose, and a statistically significant dose effect only at 24 h. With latex particles, the phagocytosed volume increased significantly with time and dose and was significantly higher than for CsA particles. This study showed that CsA particles are phagocytosed by AM from hamsters but to a lesser extent than latex particles. This difference could be correlated with physical properties, i.e. a difference between particle size and shape and/or chemical properties, latex particles being inert and CsA particles being peptidic. Moreover, different surface receptors on AM could be involved in the process of phagocytosis of CsA and latex particles.

Evaluation of the human cell line RPMI 2650 as an in vitro nasal model.

We have investigated a human nasal cell line, RPMI 2650 (ATCC), as an in vitro model to assess the absorption and tolerability of nasally administrated peptides. This cell line was cultured on different filters (i.e. Nunc, Costar, Falcon, Millipore, and Becton) which were uncoated or coated with collagen types I or IV, laminin, fibronectin and extracellular matrix. Cell line morphology, capability of forming a cell layer and phenotype were analyzed by light-, fluorescence- and electron microscopy. The morphological analysis showed that RPMI 2650 cells were forming cell clusters on one group of filters (i.e., Nunc, Costar, Falcon and Becton filters coated with collagen types I or IV, laminin and extracellular matrix). On the second group of filters (i.e., Becton and Millipore filters, coated with fibronectin) the cells had the tendency to spread in a cell layer. In both groups of filters, the cells never showed cell polarization, nor microvilli and tight junctions. Phenotyping of this cell line was performed by indirect immunofluorescence using monoclonal antibodies against human cytokeratins 10, 17 and 18 (markers of epithelial cells), desmin (markers of mesothelial cells) and vimentin (marker of mesothelial cells). Vimentin was strongly expressed, cytokeratins 10, 17 and 18 were weakly expressed, and desmin was not expressed. The human nasal cell line RPMI 2650 was not able to form a tight cell layer under these cell culture conditions. The limits of this cell line as an in vitro nasal model for drug absorption is discussed.

In vitro tolerability of human nasal mucosa: histopathological and scanning electron-microscopic evaluation of nasal forms containing Sandostatin.

An in vitro human nasal model was developed as a tool to study the local tolerability of nasal powder forms using excised nasal mucosa in a diffusion chamber. The suitability of this model was tested using Sandostatin (SMS) an octapeptide analog of somatostatin, as a reference drug enhanced by Avicel (microcrystalline cellulose) or lactose (100 mesh). The standard nasal spray vehicle was taken as a harmless control and 1% chenodeoxycholate (CDC) as a harmful control in terms of local tolerability. The extent of peptide permeation was determined by measuring SMS concentration in the receiving chamber. The labeling of SMS was detected by immunoperoxidase staining on cross sections. The local tolerability for all tested forms was assessed by histopathological examination and scanning electron microscopy. The apparent permeation coefficient allowed us to rank the absorption of the tested drug forms as Avicel > spray = lactose > 1%CDC. For all formulations, SMS was detected in the epithelium. No changes of the nasal mucosa could be observed with Avicel, lactose or nasal spray vehicle in the presence or absence of SMS. 1%CDC with or without drug showed an immediate destruction of the nasal epithelium. The validation of this in vitro model using human nasal mucosa will be further discussed as a tool for assessing the local tolerability of intranasally applied test substances.

Morphological features of human Reissner's membrane.

Light and electromicroscopic investigations of Reissner's membrane were undertaken on 10 cochleae from 6 patients with normal hearing for their age. The membrane consisted of two layers, an epithelium and a mesothelium separated by a basement membrane. The mesothelium was formed by a single thin layer which was intermittently discontinuous. The melanocytes were localized on the mesothelial side of the basement membrane. Their numbers was 2-4 times greater in the upper half of the basal turn and in the middle turn than elsewhere. The epithelium was much thicker and had more irregular features than the mesothelium. It was composed of two types of epithelial cells, flat and rounded. The flat cells were more regular in shape than the rounded cells and they were mainly distributed in the middle and apical turns. Judging from their structure they were in a resting state. The rounded cells covered a smaller area than the flat ones and had numerous microvilli. They assumed three different shapes, cuboidal, spindle-form and spherical and were arranged in four different patterns, namely bands, strands, whorls and clusters. The rounded cells were the most active according to the composition of the cytoplasm and dominated the cell population in the hook and the lower half of the basal turn where the age-related sensorineural degeneration is most apparent.

Human Reissner's membrane in patients with age-related normal hearing and with sensorineural hearing loss.

Morphological features of Reissner's membrane were investigated in 6 patients with age-related normal hearing (ARNH) and in 4 with sensorineural hearing loss (SNHL) stemming from various causes. The membrane consisted of an epithelium, a basement membrane and a mesothelium with melanocytes. There were two major forms of epithelial cells: flat and rounded. In all specimens, the rounded cells formed whorls, clusters, strands and bands. The bands were wider and the whorls larger in the basal turn and decreased gradually in size toward the apex. The number of melanocytes was 2-4 times higher in both the upper half of the basal turn and the lower half of the middle turn than in the rest of the turns. In specimens from patients with SNHL, whorls and clusters were both more numerous and larger, and the number of melanocytes was higher in the upper half of the middle turn and in the apical turn than that in the ARNH group. Ultrastructural examination of epithelial and mesothelial cells as well as of melanocytes showed more pronounced cellular degeneration in patients with SNHL than in those with ARNH. A possible correlation between structural alterations of Reissner's membrane and sensorineural degeneration is discussed.

Ultrastructural features of human Reissner's membrane.

Ultrastructural features of human Reissner's membrane were investigated in two groups of similarly aged patients. Five patients had age-related normal hearing (ARNH) and four patients had acquired sensorineural hearing loss (SNHL) from causes other than age. The membrane consisted of a mesothelium facing the perilymph and an epithelium facing the endolymph. The two cell layers were separated by a basement membrane. The mesothelium was formed by wide spread thin cells with a smooth surface. The epithelial cells assumed two different shapes, flat and rounded. Both epithelial cell types were covered with many short microvilli. In all specimens, the rounded cells were arranged in bands, strands, whorls and clusters. The size of bands and whorls was larger in the lower half of the basal turn and decreased gradually towards the apex. Bands and whorls were both larger in specimens from patients with SNHL than in those with ARNH and expanded up to the middle turn. In patients with SNHL, some flat cells had relatively few long microvilli. The epithelium showed more pronounced cellular changes in patients with SNHL than in those with ARNH and these alterations are discussed in relation to sensorineural degeneration.

Morphometric analysis of the cochlear nerve in man.

Fiber diameters were analyzed in the meatal segment of the cochlear nerve from 7 temporal bones obtained from 7 patients. Two patients had normal hearing for their age. Two had sustained noise exposure and one had presbyacusis of predominantly neural type. The cochleae displayed characteristic degeneration patterns. The other two manifested hearing loss of unspecified type. The fiber diameters ranged from 0.5 to 11 microns. The diameter distribution was unimodal in all seven nerves. The means of the diameters ranged from 4.2 to 5.5 microns. They were significantly different between patients with age-related normal hearing on the one hand and patients with noise induced hearing loss and neural presbyacusis on the other. The findings are discussed in relation to changes in nerve conduction speed and hearing loss; a possible correlation between the fiber diameter distribution and the tonotopical arrangement of the cochlea is suggested.

Ontogeny of Langerhans cells: phenotypic differentiation from the bone marrow to the skin.

We have performed double immunolabelings for cytofluorometric analysis and electron microscopy to investigate the coexpression of the CD1a (OKT6 and DMC1 monoclonal antibodies) antigen and the promonocyte/monocyte differentiation antigens CD14 (My4) or CD33 (My9) on putative bone marrow and umbilical cord blood precursors of the Langerhans cells (LC) and the epidermal LC. By cytofluorometric analysis, the percentage of CD1a+ cells which coexpressed the CD33 antigen was different from the bone marrow (5% of CD33+ cells are CD1a+), to the cord blood (3% of the CD33+ are CD1a+) and to the epidermis (the whole population of CD33+ LC are CD1a+). The ultrastructural morphology of the CD1a-expressing bone marrow, cord blood cells closely approximated that of a promonocyte/monocyte. Only LC epidermal were specifically recognized by the intracytoplasmic Birbeck granules. These CD1a+/CD33+ or CD14+ subpopulations found in three different locations (epidermis, bone marrow, cord blood) display a similar quantitative expression of the CD14 and CD33 antigens.

Langerhans cells of human mucosa.

The ontogeny of human LC and their presence in all Malpighian epithelia underline their important role in immunoregulation of the skin and mucous membranes. LC are also found in buccal and esophageal mucosa, in cornea and conjunctiva, in pulmonary, vesical, vaginal and cervical epithelia as well as in placenta villi. In all these Malpighian epithelia, the presence of DR + LC is necessary and essential for the surveillance function against allergo-antigens and the emergence of neo-antigens associated with malignant transformations.

A combined method for detection of cell surface marker expression and bromodeoxyuridine (BrdU) uptake by epidermal cells in suspension.

A rapid and convenient immunocytochemical method is presented for the simultaneous detection of surface antigen expression and S-phase cells using monoclonal antibodies and cells in suspension. The first reagent permits the nature of the cells to be determined while the second immunostaining stage reveals which of these cells are proliferating (BrdU-positive) cells. Staining and fixation conditions have been defined for the detection of cell surface markers by red fluorescence and BrdU labelling by green fluorescence. All the steps were carried out on cells in suspension and the membrane antigen distribution was not affected by the BrdU staining protocol. The method has been applied to two human epidermal cell subpopulations: Langerhans' cells and basal keratinocytes. This technique should have general applicability to the study of the cytokinetic characteristics of phenotypically defined cells in heterogeneous populations.

Langerhans cells in S-phase in normal skin detected by simultaneous analysis of cell surface antigen and BrdU incorporation.

We report on a double immunofluorescence staining for the detection of the Langerhans Cell (LC) population in S-phase. After trypsinization, the epidermal cell suspensions were enriched for LC and exposed to 10 microM BrdU for 2 h. Studies of BrdU labeled cells included the determination of their cell surface phenotype. Both membrane labeling and incorporated BrdU as revealed using anti-BrdU demonstrated the BrdU as revealed using anti-BrdU demonstrated the presence of LC in S-phase. We observed 6% of the epidermal LC in S-phase. This is a proof that LC are able to proliferate in the epidermal microenvironment. This technique, besides being rapid and free of radioactivity, allows the cytokinetic study of phenotypically defined LC in heterogeneous epidermal cell populations.

Culture of putative Langerhans cell bone marrow precursors: characterization of their phenotype.

We searched for the presence of human CD1-positive cells in bone marrow populations in order to characterize putative Langerhans cell precursors. Bone marrow progenitors were cultured in 0.8% methylcellulose supplemented with 10% granulocyte-macrophage (GM) colony-stimulating factor(s) GCT and HTB9. We compared the kinetics of these two factors and found that GCT was the more appropriate for our study. After 8 days of culture, colony-forming units of granulocyte-macrophages (CFU-GM) were tested for the presence of CD1-positive cells using the immunofluorescence technique. Positive cells were counted by cytofluorometric analysis: 9.4% CD1a (BL6), 13.4% CD1c (L161), 4.3% CD1b (NuT2), 4.6% CD2 (T11), and 25.5% CD33 (My9). Ultrastructural features and phenotype were then specified by the immunogold labeling technique using electron microscopy. A subpopulation of CD1-positive cells showed the ultrastructural morphology of bone marrow pro-monocyte/monocyte cells. By using well-characterized monoclonal antibodies, it was demonstrated that these cells expressed the following phenotype: CD14+, CD33+, CD4+, HLA-DR+, HLA-DP+, HLA-DQ-, OKT10-, CD2-. These data indicate that these bone marrow promonocyte/monocyte progenitors express a phenotype similar to that of epidermal Langerhans cells but the density of each antigen is much lower than that observed on mature skin dendritic cells.