Mary A. Perkins

The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage

Analysis of bronchoalveolar lavage fluid (BALF) appears to be a sensitive approach to characterizing an acute inflammatory response within the lung. More work, however, is needed to determine if analyses of BALF endpoints can predict chronic responses (i.e., fibrosis). The objective of the present study was to compare the dose and temporal pulmonary response of a known fibrogenic agent, silica, and two known nonfibrogenic agents, aluminum oxide and titanium dioxide. Animals were instilled with silica (0, 0.2, 1.0, or 5.0 mg/100 g body wt), titanium dioxide (1.0 or 5 mg/100 g body wt), aluminium oxide (1.0 or 5.0 mg/100 g body wt) or saline. Animals (n = 5/group) were terminated 1, 7, 14, 28, and 63 days following instillation, and the BALF was characterized by biochemical and cellular assays. Histopathological changes were determined at 60 days after exposure. The biochemical results demonstrated BALF levels of lactate dehydrogenase (LDH), beta-glucuronidase (BG), N-acetylglucosaminidase (NAG), and total protein (TP) increased in a dose-related fashion at the earlier time points for all test materials, with the magnitude of change being greatest for silica. The temporal response for these parameters was significantly different for the two classes of materials. With time, the response for the fibrogenic dust steadily increased, while the levels for the nonfibrogenic dusts decreased toward normal values during the 2-month study period. Of the cellular changes, total cell numbers, neutrophils, and lymphocyte numbers were the most sensitive markers of the pulmonary response. As shown with the biochemical parameters, the cellular response to silica increased with time while that of the nuisance dusts did not. It was also found that, similar to inhalation studies, high doses of a nuisance dust may result in toxicity/inflammation. This toxicity at high dose levels emphasizes the importance of choosing relevant doses when comparing potentially fibrogenic and nonfibrogenic dusts. In conclusion, the persistent and progressive changes seen in the biochemical (LDH, TP, BG, NAG) and cellular parameters (total cells, neutrophils and lymphocytes) following silica administration correlated with the fibrotic response which occurred after exposure to this material. The less dramatic and transient changes seen with aluminum oxide and titanium dioxide correlated with the inert nature of these nuisance dusts. The results of this study indicate evaluation of BALF may provide a means to predict the chronic pulmonary response to a material.

A noninvasive method to assess skin irritation and compromised skin conditions using simple tape adsorption of molecular markers of inflammation.

Background/aims: We have developed a simple noninvasive method to assess inflammatory changes in human skin, even in the absence of visible clinical irritation. Our approach is based on a simple tape (Sebutape®) adsorption method to recover molecular mediators of skin inflammation (e.g., cytokines). This procedure has been used to investigate baseline cytokine levels on skin, to assess normal skin condition and to evaluate changes due to chemical insult, existing dermatitis, or sun exposure.

Pulmonary response to inhaled silica or titanium dioxide

The pulmonary response to mineral dust inhalation was investigated by characterizing markers of lung injury and inflammation, macrophage activation, dust clearance, and histopathology. Rats were exposed (6 hr/day x 5 days) to air or 50 mg/m3 crystalline silica (SiO2) or titanium dioxide (TiO2). At 7, 14, 28, and 63 days after exposure, bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, and N-acetylglucosaminidase, as well as cell number, type, and viability. Alveolar macrophages (AM) obtained in BALF were cultured with or without LPS and release of interleukin-1 (IL-1) and fibronectin was determined. Histopathology was conducted at 28 and 63 days. The exposure protocol resulted in 1.8-1.9 mg of mineral dust being deposited in the pulmonary region. Clearance of SiO2 was significantly less than TiO2. SiO2 increased BALF neutrophils (Days 14, 28, and 63), total protein (Days 28 and 63), and LDH and lymphocytes (Day 63). SiO2 increased AM-derived fibronectin release (Day 63) and LPS-induced IL-1 release (all time points), but not spontaneous release of IL-1. TiO2 did not change BALF biochemical or cellular parameters or AM secretory activity. Histopathology revealed minimal interstitial inflammation with SiO2 and no significant response in control or TiO2 rats. These results demonstrate the pulmonary response to inhaled SiO2 can be differentiated from the relatively innocuous TiO2 by changes in BALF markers of injury and inflammation further supporting the use of BALF analysis to make relative assessments of pulmonary toxicity. The stimulation of macrophage fibronectin release by the fibrogenic dust SiO2 and not TiO2 is consistent with a role for this glycoprotein in lung injury and repair. Last, the early and persistent effect of SiO2 on LPS-induced AM IL-1 release indicates this response may represent a sensitive early marker of dust-induced changes in the AM population.

Development and Intralaboratory Evaluation of an in Vitro Human Cell-Based Test to Aid Ocular Irritancy Assessments

A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornified mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation (r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.

A non-invasive tape absorption method for recovery of inflammatory mediators to differentiate normal from compromised scalp conditions.

Background/aims: A simple non‐invasive tape (SebutapeTM) adsorption method was used to recover inflammatory proteins from normal and compromised human scalp (i.e. dandruff and seborrheic dermatitis) in order to assess the inflammatory and immunologic changes relevant to these clinical conditions.

In Vitro and Human Testing Strategies for Skin Irritation

Abstract: Prior to the manufacture, transport, and marketing of chemicals or products, it is critical to assess their potential for skin toxicity (corrosion or irritation), thereby protecting the worker and consumer from adverse skin effects due to intended or accidental skin exposure. Traditionally, animal testing procedures have provided the data needed to assess the more severe forms of skin toxicity, and current regulations may require animal test data before permission can be obtained to manufacture, transport, or market chemicals or the products that contain them. In recent years, the use of animals to assess skin safety has been opposed by some as inhumane and unnecessary. The conflicting needs of the industrial toxicologist to (1) protect human safety, (2) comply with regulations, and (3) reduce animal testing have led to major efforts to develop alternative, yet predictive, test methods. A variety of in vitro skin corrosion test methods have been developed and several have successfully passed initial international validation. These have included skin or epidermal equivalent assays that have been shown to distinguish corrosive from noncorrosive chemicals. These skin/epidermal equivalent assays have also been modified and used to assess skin irritation potential relative to existing human exposure test data. The data show a good correlation between in vitro assay data and different types of human skin irritation data for both chemicals and consumer products. The effort to eliminate animal tests has also led to the development of a novel human patch test for assessment of acute skin irritation potential. A case study shows the benefits of in vitro and human skin irritation tests compared to the animal tests they seek to replace, and strategies now exist to adequately assess human skin irritation potential without the need to rely on animal test methods.

Evaluation of a quantitative clinical method for assessment of sensory skin irritation

Sensory skin irritation refers to the myriad of symptomatic complaints (e.g., sting and burn) frequently associated with inflammatory skin conditions or skin intolerance to various chemicals or finished products. Sensory irritation is an important factor in consumer acceptance of the products that they buy and use; however, from a safety testing and risk assessment standpoint, it has been difficult to evaluate. Recently, methods have been developed to more quantitatively assess sensory irritation using a semantically‐labeled scale of sensation intensity, the labeled magnitude (LM) scale. Using this device, studies were conducted to determine if test subjects’ perceptions of recalled or imagined sensory responses (from a series of survey questions) were related to their actual sensory reactivity to chemical challenge. Subjects were presented with 15 skin sensation scenarios of varying intensities and asked to record their self‐perceived recalled or imagined responses using the LM scale. Individual and mean responses to each of the 15 survey questions were compared within and across studies. Considerable variation was seen between subjects’ responses to the questions, particularly for questions pertaining to stronger stimuli (e.g., scalding water or skin lacerations). There was also little consistency seen in the pattern of individual responses across the questions. However, among 4 different study populations, the group mean scores for each of the 15 survey questions showed a high degree of consistency. Also, in spite of the variability in perceived responses to the recalled/imagined skin sensations, statistically significant dose‐response and time‐response patterns were observed in chemical (lactic acid and capsaicin) challenge studies. In one capsaicin study, a direct relationship was observed, among 83% of the study subjects, between the mean recall intensity scores and actual responses to subsequent capsaicin challenge. This pattern was not seen in a lactic acid challenge study. However, a similar relationship was seen in this study if only recall stimuli related to sting‐type responses were included in the analysis. Hence, use of recall/imagined skin sensation perception data for prediction of actual reactivity to chemical probes may have screening utility depending on the survey questions used. On the whole, the LM scale is of practical use for quantifying subjective sensory irritation responses. Combined with evolving noninvasive instrumental and bioassay procedures for identifying biophysical or inflammatory markers of sensory irritation, better methods are on the horizon for improving our sensory skin irritation testing and risk assessment capabilities.

Application of a Novel and Noninvasive Skin Sampling Technique for Analyzing Cytokine-Mediated Inflammation in Rosacea

A novel Sebutape™ skin sampling technique was recently developed and has been applied to the noninvasive study of skin inflammation. Using a simple procedure of Sebutape application and removal from the skin, we have previously detected changes in the profiles of cytokine adsorption from various forms of compromised skin and scalp, including, diaper dermatitis, UV exposed skin, surfactant-treated skin, dandruff, and seborrheic dermatitis. In the current study, we applied this method to the analysis of the chronic skin disorder, rosacea, a condition characterized by inflammation, but for which little is know about the inflammatory process, including the role of cytokines. Sebutape samples were collected from involved and noninvolved (control) facial skin sites of subjects with active rosacea of varying severity. These samples were compared with other samples obtained from the facial skin of normal subjects. The rosacea skin sites were characterized by significantly elevated recoveries of the cytokine, interleukin-1 receptor antagonist (IL-1RA) and also showed significantly elevated ratios of IL-1RA/interleukin-1α, when compared to either the noninvolved facial skin sites of the same subjects or to the skin of the normal subjects. These results are very consistent with those from our earlier studies and further support the utility of this method as the first truly noninvasive technique for diagnostic assessment of skin inflammation.