Development of a cumulative irritation model for incontinence-associated dermatitis.

Incontinence-associated dermatitis (IAD) is a painful yet preventable form of cumulative skin irritation prevalent amongst those with limited movement. Consequently, it has a significant impact on the quality of life for those affected as well as substantial cost implications. Prevention and intervention is typically through good skin hygiene regimes and regular use of barrier products. In this paper, we describe the development of an in vivo model of IAD in healthy volunteers by occluded application of alkaline synthetic urine to the volar aspect of volunteer's forearms for 6 h per day over a five-day period to reproduce the moist and irritant conditions causative of IAD. Irritation was assessed and quantified on a daily basis by a series of non-invasive biophysical measurements and compared to a contralateral saline-treated (control) site. Dermal irritation was assessed by subjective (visual) and objective measurements (laser Doppler and polarisation spectroscopic imaging, infrared thermography, skin reflectance spectroscopy, transepidermal water loss and skin surface pH). The provocation of reproducible, cumulative skin irritation was successfully demonstrated and quantified. This five-day model of irritation is considered appropriate for the initial clinical assessment of topical products to prevent or treat IAD.

Design and characterisation of a novel in vitro skin diffusion cell system for assessing mass casualty decontamination systems.

The efficient removal of contaminants from the outer surfaces of the body can provide an effective means of reducing adverse health effects associated with incidents involving the accidental or deliberate release of hazardous materials. Showering with water is frequently used by first responders as a rapid method of mass casualty decontamination (MCD). However, there is a paucity of data on the generic effectiveness and safety of aqueous decontamination systems. To address these issues, we have developed a new in vitro skin diffusion cell system to model the conditions of a common MCD procedure ("ladder pipe system"). The new diffusion cell design incorporates a showering nozzle, an air sampling port for measurement of vapour loss and/aerosolisation, adjustable (horizontal to vertical) skin orientation and a circulating manifold system (to maintain a specified flow rate, temperature and pressure of shower water). The dermal absorption characteristics of several simulants (Invisible Red S, curcumin and methyl salicylate) measured with the new in vitro model were in good agreement with previous in vitro and in vivo studies. Moreover, these initial studies have indicated that whilst flow rate and water temperature are important factors for MCD, the presence of clothing during showering may (under certain circumstances) cause transfer and spreading of contaminants to the skin surface.

The skin reservoir of sulphur mustard.

Studies of the percutaneous reservoir of sulphur mustard (HD) formed during absorption carried out during WWI and WWII are inconclusive. More recent studies have indicated that a significant amount of unreacted HD remains in human epidermal membranes during percutaneous penetration studies in vitro. The present study investigated the nature and persistence of the HD reservoir formed during in vitro penetration studies using dermatomed slices of human and pig skin (0.5mm thick). Amounts of (14)C-HD that (a) penetrated, (b) remained on the surface, (c) were extractable from and (d) remained in the skin after extraction were estimated by liquid scintillation counting (confirmed using GC-MS analysis). The results demonstrated that there is a reservoir of HD in human and pig skin for up to 24 h after contamination of the skin surface in vitro with liquid agent. At least some of this reservoir could be extracted with acetonitrile, and the amounts of extracted and unextracted HD exceed the amount required to produce injury in vivo by at least 20 fold. The study demonstrated the presence of a reservoir whether the skin was covered (occluded) or left open to the air (unoccluded). The study concluded that the extractable reservoir was significant in terms of the amount of HD required to induce a vesicant response in human skin. The extractable reservoir was at least 20 times the amount required per cm(2) estimated to cause a response in all of the human population, as defined by studies carried out in human volunteers during the 1940s.

In vivo skin absorption and distribution of the nerve agent VX (O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate) in the domestic white pig.

The purpose of this study was to characterize the skin absorption and distribution of VX (O-ethyl-S-[2 (diisopropylamino)ethyl] methylphosphonothioate) in the domestic pig in order to evaluate the animal as a potential model for assessing pretreatments against toxic anti-cholinesterase compounds. A liquid droplet (equivalent to a 2 x LD50 dose) of radiolabelled VX was applied to the inner ear-skin of each anaesthetized animal. Blood and tissue samples (liver, lung, kidney, heart and skin exposure sites) were obtained post-mortem. The amount of radioactivity in each sample was measured by liquid scintillation counting, from which the skin absorption rate and dose distribution of VX were calculated. A substantial proportion (22 +/- 3%) of the applied dose remained within the skin at the site of application. It is conceivable that strategies to minimize or remove this reservoir may be of benefit in the early treatment of VX-exposed casualties. Image analysis of autoradiographs of exposed skin sites indicated that each milligram of radioactive VX covered an area of 1.2 +/- 0.5 cm2. The average skin absorption rate of 14C-VX was 661 +/- 126 microg/cm2 per hour. Comparison of these data with previous studies suggests that human skin is less permeable to VX than pig skin, but VX spreads over a greater surface area when applied to human skin. Thus, paradoxically, while pig-ear skin is more permeable than human skin, the difference in skin surface spreading may lead to the absorption of an equivalent systemic dose.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Clinical manifestations of VX poisoning following percutaneous exposure in the domestic white pig.

Nerve agents are a class of organophosphorus chemicals that inhibit certain cholinesterase enzymes (ChE). If untreated, percutaneous exposure to nerve agents, such as VX (O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate) can cause paralysis, apnoea and death. Much of the information concerning the percutaneous absorption and subsequent toxicity of nerve agents has been obtained using various rodent models. However, the most relevant 'skin model' is arguably the pig. Therefore, the purpose of this study was to examine the clinical manifestations of VX intoxication in the domestic white pig following a 2 LD50 (120 microg/kg) percutaneous challenge. There was a consistent onset of signs (where present) in each animal: mastication was followed by miosis, salivation, fasciculations and apnoea. Whilst ChE activity did not correlate with the onset of signs, there was a qualitative relationship in that mastication preceded substantial ChE inhibition, miosis lagged behind the linear decrease in acetylcholinesterase (AChE) activity and fasciculations and apnoea occurred after maximum ChE inhibition had been attained (5-10% of normal). These observations may be of use for the triage of patients exposed to VX. In comparison with similar studies with GD, VX did not affect glucose utilization. However, VX was similar to GD in that it caused a mild hyperkalaemia and hyperphosphataemia, although the significance of this observation was not clear. There was substantial lateral diffusion of the initial droplet of VX over the application site, indicating that, when decontaminating exposed skin, attention should also be directed to areas peripheral to the original site of exposure.

In vitro skin absorption and decontamination of sulphur mustard: comparison of human and pig-ear skin.

The aim of this study was to evaluate the use of an in vitro skin diffusion cell system as a model for assessing decontaminants against the chemical warfare agent sulphur mustard (SM). The in vitro absorption rates of SM through heat-separated human (157 +/- 66 microg cm(-2) h(-1)) and pig-ear (411 +/- 175 microg cm(-2) h(-1)) epidermal membranes were in agreement with previous in vivo studies that quoted skin absorption rates of 150 and 366 microg cm(-2) h(-1), respectively. Decontaminants (fuller's earth, Ambergard and BDH spillage granules) were ranked in order of effectiveness by measuring the skin absorption rates and the percentage of applied dose of SM that penetrated human and pig-ear epidermal membranes. The effectiveness of fuller's earth measured in this in vitro study using human epidermal membranes was in agreement with a previous in vivo human volunteer study. Similarly, the effectiveness of fuller's earth and Ambergard measured in vitro with pig-ear epidermal membranes was in agreement with a previous in vivo study conducted on rats. However, there was complete disparity in the ranking of decontaminants between human and pig-ear epidermal membranes measured in vitro. Thus, although pig-ear skin may be a relatively good model for predicting the human skin absorption of SM, it is a poor model for testing decontamination systems. The results of this study further validate the use of Franz-type glass diffusion cells containing human epidermal membranes as a model for predicting in vivo human skin absorption.

Habituating pigs for in-pen, non-invasive biophysical skin analysis.

The purpose of this study was to develop a method for habituating pigs (Sus scrofa domestica, middle white strain) to enable non-invasive, biophysical measurements of dorsal skin to be obtained on a daily basis over a 7-week period, thus eliminating the need for anaesthesia or restraint. This was accomplished by associating measurements of transepidermal water loss (TEWL) and skin reflectance spectroscopy (SRS) with feeding times, and with positive reinforcement by allowing exercise outside the home pen. During the pig habituation period, a well-defined series of behavioural changes were observed that included dominant/ submissive leadership changes. Values of TEWL (6.29 +/- 1.25g.m(-2) x h(-1)) were in agreement with previous studies (7.56+/-2.90g.m 2 x h(-1)) obtained from unrestrained Yucatan hairless micro-pigs (Gabard et al. 1995). The coefficient of variance of TEWL and SRS measurements were comparable with those reported previously using anaesthetized pigs (Chilcott et al. 2000). These data imply that biophysical skin measurements obtained from unrestrained, conscious animals are comparable to those obtained from anaesthetized pigs and therefore, support the use of unrestrained pigs for non-invasive biophysical skin measurements. Habituating animals for in-pen, non-invasive, biophysical measurements has substantial implications for reducing and refining laboratory animal experiments in dermatological research without compromising animal welfare.

The electrostability and electrically assisted delivery of an organophosphate pretreatment (physostigmine) across human skin in vitro.

Physostigmine is a tertiary carbamate that is utilised as a pretreatment against organophosphate intoxication. Oral delivery of physostigmine is not practical due to high first pass metabolism and short elimination half life. Transdermal administration of physostigmine may circumvent such problems. The aim of this study was to assess the electrostability of physostigmine and the feasibility of electrically assisted transdermal drug delivery of physostigmine through isolated human skin in vitro. Buffered solutions of physostigmine (free base, salicylate and sulphate) were electrostable under conditions of iontophoresis and electroporation as measured by HPLC, although instability of the chloridised silver electrodes was observed. Physostigmine sulphate was chosen for further study as it appeared to prevent degradation of the electrodes. Under conditions of iontophoresis (0.8 mA cm(-2), applied for 5- or 2.5-min durations for a maximum period of 45 min over 8 h), the total quantity of physostigmine sulphate that penetrated was 6.5+/-2.3% and 3.9+/-1.7% (pH 5.0 and pH 5.5) of the total applied dose (2 mg). Physostigmine did not penetrate the skin when electroporated at a frequency of 0.1 Hz or 10 Hz (100 V, 1 ms pulse width, duration 1 s, repetition 5-10 s), but significant amounts were delivered at a frequency of 100 Hz, being 11.3+/-2.9% and 5.8+/-2.5% of the applied dose (pH 5.0 and pH 5.5, respectively). These data indicate that iontophoretic and electroporative drug delivery of physostigmine sulphate was buffer-dependent, an effect tentatively attributed to a combination of co-ion competition, mono/di-cation ratio and applied charge effects.

Non-invasive quantification of skin injury resulting from exposure to sulphur mustard and Lewisite vapours.

The severity and progression of skin lesions resulting from exposure to the chemical warfare agents Lewisite (L) and sulphur mustard (SM) have been investigated using the non-invasive biophysical methods of evaporimetry and reflectance spectroscopy in large white pigs in vivo. Erythema (redness) expressed immediately after exposure to L or SM vapours appeared to be related to the lesion severity as demonstrated by histopathological analysis. Skin brightness correlated well with scab formation whereas blueness (cyanosis) did not appreciably alter throughout the study. Rates of transepidermal water loss (TEWL) changed both with occlusion (during vapour exposure) and also mirrored the progression of macroscopic skin injury after 12 h. Whilst no single parameter could be used in isolation to ascertain the severity and subsequent progression of the skin lesions, measurement of erythema, skin brightness and TEWL could provide quantitative, non-invasive methods for determining the efficacy of antidotes or therapies to prevent the toxic effects of chemical warfare agents. However, neither colourimetry or TEWL provided a clinical evaluation of such lesions that were comparable with the prognostic capabilities of laser Doppler imaging.

Dermabrasion--a novel concept in the surgical management of sulphur mustard injuries.

Since its first use on the battlefields of Northern France during the First World War (1914-1918), sulphur mustard has remained a significant chemical threat to military forces around the world. Progress towards an effective treatment for these injuries has been slow due to the lack of suitable animal models upon which to study the toxicology and pathology. However, porcine and human skin are similar in structure and exposures to sulphur mustard vapour have been performed on porcine models to define the development and subsequent resolution of mustard-induced skin injuries. Yucatan miniature (n = 12) and large white (n = 6) pig models were used to assess the usefulness of mechanical dermabrasion in accelerating the naturally slow rate of healing of sulphur mustard vapour-induced injuries to the skin. Burn injuries underwent debridement at 4 days post-exposure and the resulting lesions were assessed at various time points up to 8 weeks post-abrasion. Rates of re-epithelialisation were accelerated in the dermabrasion (treated) vs the control (untreated) group by up to a factor of three (ANOVA: p = .0196, Yucatan; p = 0.165, large white pig). It was concluded that dermabrasion of sulphur mustard burns is a valuable procedure in the surgical management of these injuries.

Human skin absorption of Bis-2-(chloroethyl)sulphide (sulphur mustard) in vitro.

The purpose of this study was to measure the absorption and intra-epidermal fate of 35S-radiolabelled sulphur mustard (35SM) in human breast skin in vitro. Skin (full-thickness or heat-separated epidermis) was placed into static diffusion cells and was exposed to droplets of liquid 35SM or saturated 35SM vapour. Amounts of 35SM penetrating the skin were measured from which skin absorption rates were calculated. Unbound radiolabel was washed from the surface, extracted from the skin and analysed to determine the identity of the radiolabelled species in order to measure the extent of hydrolysis of sulphur mustard. Penetration rates of liquid 35SM measured in vitro (71-294 microg cm(-2) h(-1)) were in agreement with those measured previously in vivo using human volunteers (60-240 microg cm(-2) h(-1)). Rates of liquid 35SM skin absorption under occluded, infinite dose conditions were highest through heat-separated epidermal membranes (294+/-58 microg cm(-2) h(-1)) and lowest through full-thickness skin (71+/-14 microg cm(-2) h(-1)). Fluxes of saturated 35SM vapour (110+/-75 microg cm(-2) h(-1)) through heat-separated membranes were similar to those previously measured through human forearm skin in vivo (162 microg cm(-2) h(-1)). Although hydrolysis of 35SM did occur, both on the surface and within the skin, it accounted for only a small percentage of the total applied dose (<2.7+/-1.2%). The difference in total amount of liquid 35SM penetrated between occluded and unoccluded conditions in vitro (79+/-14%) was similar to that lost as vapour from unoccluded skin in vivo (80%). A substantial reservoir of 35SM (14-36% of the applied dose) was measured within heat-separated epidermal membranes for up to 24 h which may have significant implications for the management of personnel exposed to sulphur mustard.