Strategies for the assessment of acute skin irritation potential.
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Animals↗
Biomedical subjects
M A Perkins
Publications and source records attributed to M A Perkins.
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.
Aspartate Aminotransferases↗
Application of a 4-h human patch test method for comparative and investigative assessment of skin irritation.
A human 4-h patch test has recently been developed for testing the irritation hazard potential of chemicals. The original method was developed for comparative irritation assessments relative to benchmark irritants using simple statistical tests. In this context, the method has been shown to be robust in intralaboratory testing over time. Recent interlaboratory testing has also established the consistency of the method in assessment of the relative irritation potential of selected chemicals. These data help to position the method as a suitable replacement for animal test methods in assessment of skin irritation hazard. In addition, the method has great utility for investigating different parameters of clinical skin irritation. Using kinetic response patterns and curve fitting analysis, we have compared the relative irritation potential of chemicals in greater detail, using as a basis the time required for test subjects to respond as well as the incidence of positive responses. Also, using the response to 20% sodium dodecyl sulfate (SDS) as a benchmark, we've been able to examine the intersubject variation in clinical skin irritation responses. In general, subjects most reactive to 20% SDS, in terms of the exposure time required to produce a positive response, were relatively more sensitive to a 2nd irritant chemical as well. However, this was not an absolute correlation in that some test subjects showed divergent patterns of response. The method was also used to compare directly the relative skin reactivity of different populations, based on race (Caucasian versus Asian) or on neurosensory skin sensitivity. Our results using this acute exposure test method indicate little difference in visually assessed skin irritation among these diverse human subpopulations.
Acetic Acid↗
Development of an in vitro method for skin corrosion testing.
National and international regulations require that chemicals must be properly classified, labeled, packaged, and transported based on their ability to damage or destroy tissue, e.g., skin. Traditionally, skin corrosion assessments were based on tests involving topical application of test substances to the skin of rabbits. In the present work, an in vitro skin corrosion test based on the use of reconstructed human skin cultures was developed as a potential replacement for in vivo rabbit skin tests for corrosion. In the in vitro method, test substances were applied topically to the stratum corneum surface of human skin cultures. Skin culture damage or cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. In time-course experiments, the time (in minutes) of test material exposure eliciting a 50% reduction of MTT metabolism (i.e., t50 value) was calculated. Using this method we evaluated 24 chemicals and found that the 9 corrosive chemicals were accurately distinguished from 15 strong, moderate, or mild skin irritants, using an in vitro cutoff of t50 < 3 min. Histologic examination of the cultures indicated gradations of epidermal necrosis quantitated using a specially designed grading scale, which correlated well with the corrosivity of treatment chemicals and cytotoxicity measurements. The predictivity of the method was confirmed and was consistent in skin culture models from two suppliers. Thus, the utility of human skin equivalent cultures as a screening tool for prediction of skin corrosivity appears to be independent of the commercial source of the cultures. We conclude that the in vitro assay using human skin equivalent cultures is a promising alternative to in vivo rabbit skin corrosion tests for assessment of the corrosivity of chemicals to human skin.
Animal Testing Alternatives↗
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.
Animal Testing Alternatives↗
Two miniature monitors for long-term ambulatory studies.
Two very low-power monitors are described. One typically limb-worn to record the level of physical activity of a person; the other, used with surface electrodes, to record three parameters of an EMG signal. The monitoring period can extend to days with a very accurate 'time of event' log. A novel method has been devised to double the resolution of the data gathered. A PC is used to store and process the collected data, which can be displayed or printed as one-, or four-channel, time-related histogram. The gathered data can also be analysed for trends and other statistical analysis. The hardware is relatively simple and made from low-cost components. Neither monitor uses a microprocessor for data gathering. The system is very easy to use requiring minimal operator training. Where 'no-frills' ambulatory monitoring is called for, the systems presented would compare very favourably with other commercially available monitors, either on cost, complexity of setting up, or on weight.
Circadian Rhythm↗
An approach for development of alternative test methods based on mechanisms of skin irritation.
Recent advances in techniques for culture of human skin cells have led to their potential for use as in vitro models for skin irritation testing to augment or replace existing rabbit skin patch tests. Our work is directed towards the development of cultured human skin cells, together with endpoints that can be linked to in vivo mechanisms of skin irritation, as in vitro models for prediction of human skin irritation, and for study of mechanisms of contact irritant dermatitis. Three types of commercial human skin cell cultures have been evaluated, epidermal keratinocytes and partially or fully cornified keratinocyte-dermal fibroblast co-cultures. Human epidermal keratinocyte cultures (Clonetics) were treated with product ingredients and formulations, and the extent of cell damage was assessed by incorporation of the vital dye neutral red. Cell damage correlated with human skin patch data for ingredient chemicals with the exception of acids and alkalis, but did not correlate with skin irritation to surfactant-containing product formulations. Cultures of human skin equivalents were evaluated as potential models for measurement of responses to test materials that could not be measured in the keratinocyte/neutral red assay. We developed a battery of in vitro endpoints to measure responses to prototype ingredients and formulations in human epidermal keratinocyte-dermal fibroblast co-cultures grown on a nylon mesh ('Skin2' from Advanced Tissue Sciences) or on a collagen gel ('Testskin' from Organogenesis). The endpoints measure cytotoxicity (neutral red and MTT vital dye staining, lactate dehydrogenase and N-acetyl glucosaminidase release, glucose utilization) and inflammatory mediator (prostaglandin E2) release. Initial experiments indicate a promising correlation between responses of the Skin2 model to prototype surfactants and in vivo human skin irritation. The responses of Testskin cultures to acids and alkalis help to prove the concept that a topical application model can measure responses to these materials. These results suggest that human skin cell models can provide useful systems for preclinical skin irritation assessments, as alternatives to rabbits, for at least certain classes of test substances.
Animal Testing Alternatives↗
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.
Acetylglucosaminidase↗
Evidence for ultrasonic finite-amplitude distortion in muscle using medical equipment.
Finite-amplitude distortion of ultrasonic waves from medical equipment has been observed to occur following transmission through calf muscle in human volunteers. Measurements were made using both dynamic pulse-echo imaging equipment and physiotherapy equipment. In both cases irradiation was carried out under operating conditions commonly used clinically. Pressure waveforms were measured at the skin surface using a broadband polyvinylidene difluoride membrane hydrophone. Using a pulsed, weakly focused 2.5-MHz beam with input peak pressure of 0.8 MPa and a pressure gain of 5.3 at the focus, the mean second harmonic peak magnitude (16 measurements) was 17 dB below the fundamental peak. A 1.1-MHz continuous wave therapy set with input peak pressure of 0.5 MPa showed mean second harmonic magnitude 23 dB below the fundamental.
Adult↗
The output of pulse-echo ultrasound equipment: a survey of powers, pressures and intensities.
A survey of the powers, pressures and intensities generated by ultrasonic pulse-echo equipment in clinical use has been carried out. Three conventional B-scanners, four linear-array scanners and four mechanically sectored scanners were included in the study. Measurements were made on a total of 22 transducers covering the nominal frequency range 2.25-7.5 MHz. On those instruments where an output power control was provided, two measurements were made: one at the maximum available power and a second at a lower power. On arrays with a variable transmit focus control, measurements were made at all available focus settings. In all, measurements were made on 38 separate focused pulsed ultrasonic fields. The measurements were carried out using a calibrated ultrasonic force balance, and a calibrated polyvinylidene difluoride (PVdF) membrane hydrophone. A very wide range of maximum powers, pressures and intensities were found. Powers from 0.5-80 mW were measured; spatial-average temporal-peak positive pressures at the transducer varied between 30 kPa and 1.15 MPa, and spatial-peak pulse-average intensities were in the range 3.6 X 10(3)-1.1 X 10(7) Wm-2.
Equipment Design↗
Portable aversion therapy stimulator.
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Aversive Therapy↗
Prevention of hearing under anaesthesia.
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Anesthesia, General↗
Hypothenar muscle action potential recorder.
Drugs which block the transmission of excitation from motor nerve to voluntary muscle fibre are used routinely in anaesthesia. The action of this group of drugs may be studied by stimulating a motor nerve and observing the evoked muscle response. Simple nerve stimulators are widely available and are designed to achieve supramaximal stimulation of a convenient peripheral motor nerve. Quantitative observation of the evoked muscle response is achieved by measuring either the action potential or the tension developed by the contracting muscle. While such techniques are commonly used for research and teaching purposes, they are not widely available to the practising anaesthetist. Electrocardiography, on the other hand, id frequently used in operating theatres and recovery rooms, so that medical personnel are accustomed to using ECG recorders and are trained to interpret the displayed information. We have developed an EMG pre-amplifier and signal processor to produce a signal which can be displayed by ECG equipment.
Action Potentials↗
Delayed response performance of mentally retarded patients.
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Adolescent↗