EP3158555A1 - Synthetic skin - Google Patents

Abstract

The present invention relates to a synthetic skin, a method of production and use as a physical model, for the evaluation of processes on skin.

Description

SYNTHETIC SKIN

The present invention relates to a synthetic skin, a method of production and use as a physical model, for evaluating the effect or behaviour of substances on skin, and processes for removing the substances from skin, such as in the development of cleaning agents or decontaminants.

The application of a substance to an animal skin surface covers a wide range of scenarios. For example, intentional application of a chemical, product or material to a skin surface includes use, by a person, of cosmetics (e.g. make-up, deodorant), topical treatments (e.g. antibiotics, moisturisers) and other skin care lotions, gels or creams. There also exists the occurrence of unintentional or malicious contamination of a skin surface as a result of, for example, accidental spillages or exposure to undesirable substances.

The removal of substances from the skin surface can be achieved through a variety of processes, for example washing, using an absorbent material (e.g. wipes, cloths, tissues, powders), the application of a secondary cleaning substance (e.g. make-up remover), or a combination of such processes. Assessing the effectiveness of a skin decontaminant or a skin decontamination procedure can be achieved using a biological skin sample as a testing surface.

The term 'biological skin' includes, but not exclusively, mammalian skin, in particular human skin or porcine skin.

However, the use of biological skin is problematic due to the following reasons:

The inherent biological variance between different biological skin samples affects the relative absorption of a substance on the biological skin surface, reducing statistical differences between decontaminants and application processes. Biological variance may be as a result of differences in stratum corneum thickness (the most superficial layer of biological skin and the rate-limiting step to penetration of exogenous substances), differences in biological skin hydration levels, and differences in dermal appendages (e.g. number of follicles and skin pores).

Biological skin needs to be maintained at physiological conditions to ensure representative in vivo absorption rates. Furthermore, compositional changes of excised biological skin occur over time and as a result of storage. Ideal conditions to preserve the ultrastructure of biological skin in long-term storage would be to flash- freeze the biological skin, to prevent ice crystal damage and to promote vitreous ice formation during the freezing process, and then to store the biological skin in liquid , nitrogen. However, there are practical limitations in storing biological skin at lower temperatures, for example at -20°C. Under these conditions in the longer-term, the biological skin may be unsuitable for penetration studies due to dehydration and ice crystal damage, particularly if the biological skin is exposed to several freeze-thaw cycles.

In addition, biological skin, particularly human skin, is a limited source and not readily available.

Thus, there is a need to develop synthetic skins.

Synthetic skin is widely used in the pharmaceutical industry as membranes to simulate skin barrier function for transdermal drug permeation studies or as a support to measure the release rate of a drug from a delivery system. These synthetic membranes are sourced from the filter membrane industry and may be polymeric materials such as cellulose, silicone or polysulfone based materials. The filter membranes can differ in pore size, order of their pore structure, chemical composition and hydrophobicity. Although this provides a range of parameters that can be exploited to simulate the permeability of human skin for a given substance, there are a number of additional physiochemical and practical requirements for a skin surrogate, especially to enable it to be used as a platform for the evaluation of personal decontaminants. Ideally, the surface of a synthetic skin should preferably be topographically representative of biological skin. This feature would help ensure, for example, that spread of a substance on the synthetic skin surface correlates with its spread on a biological skin surface and, furthermore, that the amount of substance absorbed into the synthetic skin correlates with that into biological skin. This ensures that the initial drop fraction available to the decontamination process reflects that for biological skin.

The synthetic skin should preferably be of sufficient thickness to prevent substance breakthrough. This is important for quantitative studies of residual contamination.

The synthetic skin should preferably enable solvent extraction of all of the absorbed substance from its bulk without introducing any interferents into the extraction medium that would confound analytical quantification.

The synthetic skin should preferably be suitable for attaching to an undulating or geometrically complex surface, for example a head-form.

The mechanical integrity of the synthetic skin should be preferably be maintained upon exposure to a substance or a decontamination procedure.

The synthetic skin should preferably not induce breakdown of the substance applied.

The synthetic skin should preferably be prepared so that there are no significant inter-batch variations in a surrogate's chemical and physical properties.

Work towards addressing some of the above requirements has been reported. Charkoudian (Charkoudian JC. A model skin surface for testing adhesion to skin. J. Soc. Cosmet. Chem. 1988. 37:429-444; also disclosed in US5015431) reported a synthetic skin for testing adhesion of medical devices, which is produced by impregnating silicone with proteinaceous and fatty acid biomolecules, and a chemical fixative, to produce a chemically similar surface to human skin. The surface of this material was also modified to be topographically representative of human skin by casting on human skin. These attributes could impart the required surface wetting properties for a wide range of substances, whilst the silicone base provides suitable mechanical stability.

However, the application of a chemically complex synthetic skin to meet the aims of the present work is technically challenging. Solvent extraction of residual substances, on or absorbed into the synthetic skin, may also extract other molecules that could complicate analysis and lower experimental throughput (necessitating filtration or high resolution analytical techniques). Therefore, a chemically homogenous synthetic skin is desirable.

Thus, there is a need to develop improved synthetic skins.

The aim of the present invention is to develop improved synthetic skins, and especially skins more topographically representative of biological skin, and methods for production of such skins.

Accordingly, in the first aspect of the present invention there is provided a method for producing a synthetic skin, the method comprising the steps of: depositing a curable polymer on a non-biological material having a rough surface, so that the curable polymer contacts the rough surface; curing the polymer such that the rough surface imprints on to the polymer; and removing the non-biological material from the cured polymer to provide the synthetic skin.

The present invention relates to an improved synthetic skin, in particular wherein the surface of the synthetic skin simulant is topographically modified so that the spread of an agent on its surface is representative of its spread on biological skin, a method of production of the improved synthetic skin and its use as a physical model, for evaluating the effect or behaviour of substances on skin, and processes for removing the substances from skin, such as in the development of cleaning agents or decontaminants. In particular, the present invention is a suitable platform for assessing the efficacy of personal decontamination products and procedures, in the event of potential skin contamination by agents such as chemical agents, in particular chemical warfare agents or associated simulants.

The term 'agent' for the purpose of the present invention is known to the person skilled in the art and includes, but not exclusively, a chemical or biological agent, in particular a chemical agent, for example a chemical agent in a solid, liquid or gaseous phase, in particular a liquid chemical agent. The term 'agent' also includes chemical warfare agents or associated simulants.

Use of improved synthetic skin for purposes such as those outlined above may provide benefits that include: higher experimental throughput due to ease of use; greater availability, thus providing improved statistical power to resolve efficacy of decontamination products and/or procedures; enabling standardised methods, for example testing the efficacy of decontamination processes on skin, which is particularly advantageous for those countries that are restricted or limited in their use of biological skin; and correlating the spread of simulant on synthetic skin with the spread of live chemical agent (e.g. chemical warfare agent) the simulant represents on biological skin, thus providing a non-super toxic model to perform realistic decontamination protocols that do not require specialist facilities or resources.

The term 'depositing' includes, but not exclusively, using an applicator to uniformly draw a curable polymer over a non-biological rough surface, preferably at a slow and steady speed over the non-biological rough surface. The non-biological rough surface is preferably maintained in a substantially horizontal plane to ensure the curable polymer remains uniformly deposited on the non-biological rough surface. Alternatively, 'depositing' may include pjacing a non-biological rough surface down onto a curable polymer such that the non-biological rough surface is progressively rolled over the curable polymer and allowed to rest before polymer curing.

The term 'curable polymer' is known to a person skilled in the art and includes, but not exclusively, a synthetic and/or natural substance comprising repeating monomer subunits, that can be stored at suitable environmental conditions and duration to facilitate its hardening into a solid form. In particular, the term 'curable polymer includes silicone, which includes, but not exclusively, polymers comprising silicon, for example polymers containing silicon and additional elements such as carbon, hydrogen and/or oxygen. In particular, silicone includes polymerised siloxanes ('polysiloxanes'), comprising an inorganic-organic compound of chemical formula [R₂SiO]_n, structured as a silicon-oxygen backbone with additional organic groups bound to the silicon atoms.

The term 'rough surface' includes, but not exclusively, any non-biological material surface, for example an artificial or man-made surface, that displays a non-smooth, textured or undulating surface. Optionally, the rough surface includes strands of naturally-occurring or synthetic hair protruding from the surface, so that the hair strands become embedded in the curable polymer following depositing the curable polymer on a non-biological material having a rough surface.

The term 'curing' includes, but not exclusively, storing the curable polymer at suitable environmental conditions and duration to facilitate its hardening into a solid form. The term 'curing' in relation to silicone includes, but not exclusively, storing silicone in a liquid form at suitable environment conditions and duration to facilitate its hardening into a solid form. Suitable environmental conditions and duration for curing silicone are known to those skilled in the art, for example at room temperature e.g. approximately 23°C and a duration of up to or greater than 1 hour.

The Applicant has found that the method of the present invention offers a number of advantages. Firstly, this method is a simple and reproducible means of providing a synthetic skin with a topographically-modified surface that enables an agent to spread on its surface in a manner representative of said agent's spread oh biological skin. The Applicant has surprisingly found that using a non-biological material having a rough surface, as an embedding surface, facilitates the production of a synthetic skin with a topographically-modified surface that enables an agent to spread on its surface in a manner representative of said agent's spread on biological skin. For example, the Applicants have demonstrated that the drop spread of methyl salicylate (MS), a sulphur mustard (HD) simulant, on the roughened synthetic skin surface statistically matches MS drop spread on porcine skin. Furthermore, the present invention overcomes the need to modify the surface chemistry of the synthetic skin, or for the availability of biological skin as a casting surface for the synthetic skin.

Preferably, the curable polymer of the present invention comprises silicone. Further preferably, the curable polymer of the present invention is a commercially-available silicone, for example Dragon Skin FX Pro. This characteristic is advantageous as it aids the production of standardised methods for use by different laboratories to compare, for example, the efficacy of decontamination processes on skin. Silicone also has the advantageous properties of mechanical robustness, for example to decontamination procedures, and the ability to be produced in sufficient lateral sizes to cover body-forms. As shown by the Applicant, the synthetic skin of the present invention was demonstrated to be capable of being attached to a head-form and was mechanically stable during a decontamination drill wherein a General Service Respirator was donned and doffed during the decontamination procedure. Additionally, silicone enables high percentages of solvent extraction of the absorbed substance from its bulk. For example, absorbed MS can be solvent extracted from cured silicone with recoveries of 98% +/- 15% (95% CI).The applicant has found that a synthetic skin comprising silicone can be stored for approximately one month at ambient temperature (e.g. 21 °C) and air conditions.

Furthermore, the act of depositing the curable polymer on the non-biological material having a rough surface at a slow and steady speed overcomes the problem of air bubbles becoming trapped in the synthetic skin. In addition, the curing process also results in any remaining air bubbles rising out of the curable polymer, leaving a uniform polymer sheet at the end of the curing process. Air bubbles may damage the uniformity of the material, for example by producing regions on the synthetic skin that are void of roughened surface and/or thinner than the average thickness of the synthetic skin, the later characteristic conferring mechanical weakness which may resulting in rips and holes in the synthetic skin when being removed from the rough surface.

In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein the rough surface is an abrasive surface.

The term 'abrasive surface' includes, but not exclusively, a surface comprising grains or a similar hard substance, each of a size ranging from, for example, millimetre to submicrometre, adhered to a surface, particularly a flexible surface, such as paper, cloth, metal or plastic. Abrasive surfaces include those provided by, for example, coated abrasive surfaces such as sandpaper, emery cloths and other associated variants that provide a rough surface with abrasive qualities. The term 'abrasive surface' also includes surfaces that are moulded or prepared such that their surface is comparable in texture to coated abrasive surfaces.

In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein the abrasive surface comprises grains of average particle diameter 50 - 450 μιτι.

In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein the non-biological material comprises grit (i.e. grains) of standardised grit designation as defined, for example, by the European Federation of European Producers of Abrasives (FEPA), ISO 6344 (coated abrasives, size and tests) and/or the United States Coated Abrasive Manufacturers Institute (CAMI), said grit comprising coarse-grade grit, for example coarse-grade 40 (e.g. ISO/FEPA grit designation P40; CAM! grit designation 40; average particle diameter 425 μιη; coarse-grade particle range 336-425 μητι), medium-grade grit, for example medium-grade 70 (or similar e.g. ISO/FEPA grit designation P60, average particle diameter 269 μιη; CAMI grit designation 60; average particle diameter 265 μιτι; medium-grade particle range 190 - 265 μιτι), fine- grade grit, for example fine-grade 120 (ISO/FEPA grit designation P120, average particle diameter 125 μπι; CAMI grit designation 120; average particle diameter 1 15 μιτι; fine-grade particle range 1 15 - 162 im) or very fine-grade grit, for example fine- grade 150 (ISO/FEPA grit designation P150, average particle diameter 100 μιη; CAMI grit designation 150; average particle diameter 92 m; very fine-grade particle range 68-100 μηι). Alternatively, the grit is a combination of coarse-grade, medium- grade, fine-grade and very fine-grade grit.

In a further embodiment of the first aspect of the present invention, the non-biological material comprises grit of coarse-grade 40 or medium-grade 70.

In a further embodiment of the first aspect of the present invention, the non-biological material comprises grit of coarse-grade 40.

In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein the non-biological material is sandpaper.

These embodiments are advantageous as such abrasive surfaces are recognised as readily available, cost effective and easy-to-use, thus providing a simplistic and reliable means of permanently imprinting a roughened surface onto a curable polymer, to provide a synthetic skin with a topographically-modified surface that enables an agent to spread on its surface in a manner representative of said agent's spread on biological skin. Use of different abrasive surfaces, such as sandpaper comprising different grit grade (for example coarse-grade, medium-grade or very fine-grade), can be varied according to the agent to be tested. Thus, for example, the appropriate grit grade of the rough surface, for use as an embedding surface to produce a synthetic skin, can be determined by testing of a given agent's spread on the surface of the resultant synthetic skin, and its corresponding agent spread on biological skin. In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein the rough surface of the non- biological material is pre-coated with a non-stick substance.

The term 'non-stick substance' includes, but not exclusively, nano-particulate or liquid constitutions providing durability, smoothness and/or conferring the rough surface with an ability to repel foreign substances (i.e. 'super-phobic') such as oil, water, chemicals and soiling agents, prior to depositing the curable polymer on to the rough surface. Methods for pre-coating a rough surface with a substance or composition conferring non-stick or super-phobic properties are known to those skilled in the art and include vapour deposition or liquid coating.

In a further embodiment of the first aspect of the present invention, the non-stick substance comprises silicone dioxide (S1O2) liquid glass, one or more fluorine- containing chemical(s), manganese oxide polystyrene or zinc oxide polystyrene.

In a further embodiment of the first aspect of the present invention, the pre-coating substance is a silicon dioxide liquid glass super-phobic coating ('nanoglass'), for example Radaglass® liquid glass.

These embodiments are advantageous as the pre-coating substance eases the removal of the non-biological rough surface from the cured, roughened polymer surface, reducing the risk of mechanical damage to the synthetic skin and, furthermore, enabling the non-biological rough surface to be reused.

In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein non-bonded siloxane components are extracted from the cured polymer, the method comprising the steps of: immersing the cured polymer in an organic solvent; removing the cured polymer from the organic solvent; draining the cured polymer to remove non-bonded siloxane components; and air-drying the cured polymer. The term Organic solvent' is known to the person skilled in the art and includes carbon-containing compounds, in particular liquid compounds, used to extract soluble compounds from other substances. In particular, the term Organic solvent' includes compounds such as isopropanol, ethanol and ethyl acetate.

This embodiment is advantageous as this extracting step removes the presence of components, in particular non-bonded siloxane components, which may interfere with post-decontamination quantification of residual chemical or simulant, for example during gas chromatography analysis. Optionally, the embodiment is performed at least once, preferably three times, to ensure removal of non-bonded siloxane components. Further optionally, this embodiment includes a final quality control step comprising removing a section of the synthetic skin for subsequent analysis, for example gas chromatography analysis, to confirm the absence of interfering components. In a further embodiment, the organic solvent is isopropanol.

In a further embodiment of the first aspect of the present invention, there is provided a method for producing a synthetic skin, wherein hair is embedded into the synthetic skin. The embodiment could be achieved by initially incorporating the hair onto the rough surface of the non-biological material, said hair in turn becoming incorporated into the curable polymer upon deposition of the curable polymer on the non- biological material having a rough surface. For example, hair is firstly applied to the pre-coated, non-biological material having a rough surface by inserting each hair into a fabricated root appendage or hair bulb. Following application of the curable polymer, the rough surface is in contact with the curable polymer and the hair or, as required, root appendage, also embeds into the curable polymer. Detachment of the hair from the non-biological material, and subsequent removal of the non-biological material from the cured polymer, results in hair protruding from the cured, roughened polymer surface. Alternatively, for example, the hairs are embedded into the cured roughened polymer surface following removal of the rough surface. This embodiment beneficially provides a synthetic skin with hair, which more accurately represents skin regions on mammals, for example humans, and may enable the evaluation of the effect of hair on skin decontaminants, decontamination procedures on skin and the contact hazard presented to skin by object and material surfaces pre- and post- decontamination.

In a second aspect of the present invention there is provided a synthetic skin prepared according to the above-mentioned methods.

In a further embodiment of the second aspect of the present invention, there is provided a synthetic skin wherein the average thickness of the synthetic skin is greater than 0.35 mm. In a further embodiment, the average thickness of the synthetic skin is 1.11 mm ± 0.23 mm.

These embodiments are advantageous as the mechanical stability of the synthetic skin increased when the average thickness was greater than 0.35 mm. Unilateral ^" Nuclear Magnetic Resonance (NMR) proton density depth-profiling determined the depth at which the roughness was embedded into synthetic skin using the method of the present invention. A gradual increase in proton density from the surface of the silicone into its bulk over 0.25 mm was observed, indicative of the thickness of the surface roughness. Beyond this region, the silicone had a constant proton density. Furthermore, the mechanical stability of the roughened silicone significantly reduced when the silicone was less than approximately 0.35 mm i.e. a thickness that approached or impinged on the embedded roughness. Furthermore, an average thickness of synthetic skin of at least 0.35 mm was sufficient to mitigate simulant breakthrough upon contact pressures.

In a further embodiment of the second aspect of the present invention, there is provided a synthetic skin wherein hair is embedded into the synthetic skin.

In a third aspect of the present invention, there is provided use of a synthetic skin as a physical model, wherein the synthetic skin is prepared according to the above- mentioned methods. In a further embodiment of the third aspect of the present invention, the agent for testing using the synthetic skin as a physical model is a chemical agent. In a further embodiment, the agent for testing using the synthetic skin as a physical model is a liquid chemical agent.

The following chemicals, materials and methods were used in the examples that follow.

Chemicals and Materials

Methyl salicylate (MS) and tributyl phosphate (TBP) were purchased from Sigma- Aldrich (UK). Radaglass™ fibre protect S2 was purchased from Radal Technology (UK). PL red 515 (Petroleum Logistics, UK) was used to^' dye MS. Close-clipped porcine skin from the abdominal area of 3 healthy pigs (Sus scrofa, Yorkshire Landrace strain; weight range 15-20 kg) was dermatomed to a nominal 500 pm thickness using a Zimmer™ air dermatome (Zimmer LTD, Dover Ohio, USA). Skin specimens were stored flat and wrapped in tin foil on cardboard within a freezer (- 20°C) prior to use. The use of animals was conducted in accordance with the Animals (Scientific Procedures) Act 1986.

Nitrile and neoprene (polychloropene) polymer sheets were purchased from PAR Group Ltd. Translucent silicone and pigmented silicone (red) polymer sheets were purchased from SAMCO Ltd. Smooth-on Dragon skin FX Pro, Dragon Skin Medium 10, Dragon Skin Body Double brush-on silicones were purchased from Bentley Advanced Materials (UK).

The following Whatman membranes were purchased from VWR (UK): Cellulose nitrate (0.45 pm pore), Polytetraflurorethylene (PTFE) track-etched (WTP type ordered polypropylene support, 0.2 pm pore), Polytetraflurorethylene (PTFE) track- etched (TE type, randomly arranged polypropylene support 0.45 pm pore), Glass microfibre (1.6 pm pore), Nuclepore polycarbonate membrane (track etched; 0.4 pm pore), Anopore inorganic hydrophilic membranes (0.1 μητι pore). The following adhesives were procured to investigate the attachment of roughened silicone to a metal head-form: Wickes PVA Building adhesive (Wickes, UK), Wickes High Strength Contact adhesive (Wickes, UK), UHU Twist & Glue (general purpose) (Wickes, UK), 3M spray mount (3M, UK), Staples Liquid Glue Pen (general purpose) (Staples, UK), Monett Black Witch (quick drying neoprene adhesive) (donated by Dstl Physical Sciences Department).

Coarse-grade (40) and medium-grade (70) sandpaper was purchased from Wickes, UK. Grade P150 emery cloth was purchased from Radio-spares, UK.

The following absorbent wipes were procured to evaluate their effectiveness at removing methyl salicylate from roughened silicone: Clean room wipe (VWR, UK) and e-cloth (general purpose) (Enviroproducts Ltd®, UK).

Antistatic bar (Model 3024-F) was purchased from Fraser Anti-static techniques (Brampton, UK).

Preparation of Roughened Silicone

The commercial off-the-shelf silicone (Dragon Skin FX Pro (DSFXP)) was prepared in accordance to manufacturers instructions: liquid rubber Part B (polyorganosilioxanes, amorphous silica) was mixed thoroughly into Part A (polyorganosilioxanes, amorphous silica, platinum-siloxane complex) using a 1 :1 (ν/\ή ratio. The liquid silicone was deposited onto nano-glass (RadaglassTM fibre protect S2) pre-coated sandpaper (Wickes, UK), which was attached to a flat metal surface using tape along its periphery.

Sandpaper coarse-grade 40, medium-grade 70 or very fine-grade 150 was pre- coated by spraying nanoglass over its surface and drying at ambient conditions prior to roughened silicone preparation. The liquid silicone was slowly drawn over the sandpaper using a 30 mm width film applicator (Sheen Instruments, UK) set to 2.2 mm using adjustable micrometers. The slow motion ensured the mixture was not moved so fast that it 'pulled' creating excessive air bubbles. The silicone sheet was cured at 23°C for a minimum of 1 hr, during which the sandpaper acted as a mould to form a roughened topography, on to the surface of the silicone sheet. Any air bubbles in the silicone rose out of the silicone as it cured leaving a uniform sheet of silicone at the end of the process.

The roughened DSFXP silicone required further treatment so that it could be used for the post-decontamination quantification of residual simulant. This was due to non- bonded siloxane components that were extracted from the roughened silicone in I PA and which overlapped with the MS retention time in the gas chromatogram. This necessitated their extraction from the roughened silicone by immersion in isopropanol (1 ml/cm² isopropanol to silicone) and then air-drying under ambient conditions for 24 hrs prior to its use in experiments. This pre-conditioning typically required 3 consecutive extractions over 3 days in isopropanol, where the silicone sheet was drained as well as possible between extractions. To confirm absence of interfering peaks, a 25 cm² section of the silicone was removed and placed into 10 ml isopropanol for 12 hrs and then sampled for gas chromatography (GC) analysis (i.e. the final extraction was performed in the same or smaller volume of solvent that would be used to extract the silicone in the decontamination experiment).

The thickness of the roughened DSFXP silicone following the above processes was measured using a digital calliper as 1.11 mm ± 0.23 mm.

Synthetic Surrogate Screen

A high-throughput screen was developed to initially select the most suitable surrogates for downstream testing. The porcine skin or skin surrogate (25 cm² surface area) was placed onto an 11 \im pore Whatman Grade 1 filter paper laid flat against a glass plane. A 4 μΙ MS or TBP drop (dyed with 1 % PL Red 515 dye) was deposited onto the surface of the porcine skin/skin surrogate using a positive- displacement Eppendorf Multipipette. A glass block (25 cm², 87.7g ± 0.23g) was then placed onto the drop to provide an applied pressure of 0.35 kPa. The lateral spread area photographed through the glass weight was recorded at 15 minutes post- application of the glass weight. The area was measured using these photographs following scale-setting and freehand selection by Image J v.1 .40g software. The glass weight was then carefully removed to observe whether unabsorbed simulant remained on the porcine skin/skin surrogate surface. The porcine skin/skin surrogate was removed to observe whether simulant had broken-through onto the filter paper or was on the underside of the skin sample.

The above test was repeated 3 times for surrogate or skin sample. Note the porcine skin (500 μητι dermatome) was thawed and conditioned in 50 % ethanol in water (50:50 v/v) at 32°C for 1 h; it was then removed and excess fluid from the skin surfaces was blotted-off prior to performing the screen test. Rubber polymers were washed in isopropanol (3 x 3 s swirls) and then dried overnight prior to performing the test.

A subsequent test was performed under the same experimental conditions to test simulant breakthrough at higher applied pressure (10 kPa) for those surrogates where simulant had not broken through using the lower pressure.

Contact angles were obtained for all surrogates and porcine skin samples by depositing several 4 μΙ MS/TBP drops onto the sample surface. A camera (Cannon EOS 550D) equipped with a Wide-macro lens (28 mm F 1 .8 DG Macro EX Sigma) was aligned to capture cross-sections for each drop. Contact angles were obtained from photographs using Image J v.1 .40g software.

Contact Test

This test was designed to obtain quantitative information on the mass of liquid CW simulant absorbed into silicone or porcine skin following contact with an absorbent or non-absorbent contact medium.

The porcine skin/skin surrogate was placed onto a (lower) glass block and a 2 μΙ drop of MS was deposited using a positive-displacement Eppendorf Multipipette. For experiments using roughened silicone, a low-force Thermo Finnipippete was used to dispense a 2 μΙ drop carefully onto the surface. The MS mass was calculated from its density upon weighing the dispensed drop on the silicone surface. The initial contamination mass for drops deposited onto porcine skin was determined through an independent experiment where the operator mean mass dispensed was calculated from several drops deposited onto roughened silicone.

Following drop deposition, the contaminated silicone / porcine skin was contacted with either: an absorbent clean room wipe (CRW; VWR, UK) applied to the drop at 2.35 kPa (using one glass block weighing 87.7 g and a 500 g brass weight over a 25 cm² surface) for 3 seconds; or a non-absorbent glass block applied to the drop at 2.35 kPa (using one glass block weighing 87.7 g and a 500 g brass weight over a 25 cm² surface) for 3 seconds.

MS absorbed into porcine skin/skin surrogate were extracted using consecutive 24 h extractions, where the first extraction was in 10 ml propan-2-ol (IPA) and second or third extracts were in 11 .13 ml IPA until no MS was detected in the final extract by GC. The silicone was washed under 20 ml IPA flow (10 ml IPA on each side) to prevent MS carry-over between extraction vessels. The upper and lower glass blocks were placed into a jar and washed under a flow of propan-2-ol (10 ml). All extracts and washes were sampled and quantified by GC. For experiments using contact. medium an absorbent clean room wipe (CRW; WVR, UK) above, the MS in CRW was extracted in 20 ml IPA and washed with 10 ml IPA (each side) between extractions.

Extraction Fluid Carry-Over between Consecutive Extractions of Roughened Silicone

Following the contact test, the first extraction was performed in 10 ml IPA, which was dispensed from a calibrated dispenser (Dispensette, Brand) attached to the IPA Duran. Following first extraction, the silicone was removed and excess fluid drained before it was washed in IPA to minimise gross carry-over of MS between extractions. However, as silicone is an absorbent polymer, it carries over an amount of IPA into the next extraction vessel, which needs to be considered in calculations during MS quantification by GC. The amount of IPA carried over between extractions was determined from multiple measurements taken over three independent sets of experiments. One-way analysis-of-variance (ANOVA) found no significant differences in the mean amounts of IPA carried over between extractions in each of the experiment sets (a = 0.0.5; P = 0.42). Therefore, it was appropriate to take the mean of the entire dataset to determine the amount of IPA carry over, which was 1.13 ml; i.e. the volume of extraction solvent in second/third extracts was 11.13 ml.

Gas Chromatography

Quantification of MS in extracts and washes was performed using an Agilent Technologies 6890N Network GC System. Quantification of MS solutions was performed using an Agilent Technologies 6890 GC System. An autoinjector was used to introduce liquid samples into the capillary using on-column injection (2.5 μΙ volume, 50 °G). Samples were eluted from the capillary column (Agilent DB-WAX 30 m length x 0.530 mm megabore x 0.5 pm film thickness) using He carrier gas (10 ml min^"1 flow, 6.82 psi, 66 cm/s). A temperature program method was utilised: the oven was initially held at 50°C (2 min), followed by a 50°C/min ramp, and finally held at 150°C (8 min). Eluent was analysed using a flame ionisation detector (FID, 250°C).

Drop Spread on an Inclined Surface

This test was designed to observe the spreading behaviour of drops deposited onto the porcine skin or skin surrogate surface at an incline. A glass block was placed onto a weighted stand at a 51 ° incline. The porcine skin or skin surrogate (5 cm x 5 cm) was placed onto the inclined glass block and several 4 μΙ drops were deposited along the top (time^' = 0 min). A video camera was used to capture temporal drop spread over a ca. 1 min.

The porcine skin (500 pm dermatome) was thawed and conditioned in 50 % ethanol in water (50:50 v/V) at 32°C for 1 h; it was then removed and excess fluid from the skin surfaces was blotted-off prior to performing the screen test. Rubber polymers were washed in isopropanol (3 x 3 s swirls) and then dried overnight prior to performing the test. Roughened Silicon Application to Mannequin Head-Form

1. Application Method for Dyed Methyl Salicylate Spread Experiments

The roughened silicone was fixed to a metal head-form for immediate decontamination experiments. An undercoat of Dragon Skin FX Pro (DSFXP) silicone mix (Part A:B, 1 :1 ν/\ή was applied to the underside of the roughened silicone. The roughened silicone was then attached to the head-form using cable ties and goggles. This ensured the silicone maintained contact with the head surface and facial contours (particularly around the eye sockets, nose) as the "silicone-glue" undercoat dried overnight. The cable-ties and goggles were removed following the -drying process for Immediate Decontamination experiments.

2. Application Method for Quantitative Assessment of Post-Decontamination Residual Methyl Salicylate in Roughened Silicone

To optimise adhesion of the roughened silicone with the head-form, the roughened silicone was firstly overlaid on to the head-form and areas around the eyes, lower part of the nose and the left and right side of the mouth were cut away. The roughened silicone was then fixed to a metal head-form using a spray COTS adhesive that was pre-screened to ensure absence of interfering chromatographic peaks that would affect MS quantification by GC.

Spray mount (3M) was applied to the underside of the roughened silicone and swiftly transferred to the head-form, where it was fastened using the procedure outlined in the above section. This was necessitated by the adhesive quickly becoming brittle and foam-like due to latent heat during its application. Following drying of the adhesive and release of the silicone from the cable ties, tape was used at the edges of the silicone to further secure it to the head-form.

General Service Respirator (GSR) Filing to Mannequin Head-Form

The head-form was fitted with a GSR by Respirator Technicians at Dstl's Chamber Facility. The respirator size was 3 and the oro-nasal silicone rubber was size 3. The following strap adjustments were made: crown straps: position 8; side straps: position 15; and adjustable jaw straps: position 34. The GSR was applied to the head -form by inserting the mannequin chin into the yoke and then fitting the straps using the settings above.

Immediate Decontamination Procedures

The application of immediate decontamination procedures (blot-bang-rub) using DKP 1 -MK 1 was according to the manufacturer's instructions.

Sampling MS Vapour in GSR while Fitted to a Contaminated Head-Form

The GSR was applied to the head-form by inserting the mannequin chin into the yoke and then fitting the straps. A vacuum pump drew air from the inlet of the drinking straw, which was situated within the oro-nasal (inside) area of the GSR. This air was drawn through a series Of Tenax tubes that were attached, but-to-but, to the end of the drinking straw. This method de-risked the loss of analyte vapour to vacuum pump. The air flow was set to 15 L / min using a flow meter. The system was checked for leaks by closing the value to the straw, which was located outside the GSR, and ensuring the flow dropped to 0 L / min^*1. Sample tubes were analysed using different analytical approaches depending on the expected amounts of MS vapour evaporating from the roughened silicone surface, pre and post decontamination.

Unilateral NMR

Unilateral nuclear magnetic resonance (NMR) was used to obtain proton depth- profiles of pre-conditioned DSFXP silicone to measure impregnation of roughness. The instrumental parameters used to obtain the measurement are as follows. Depth profiles were obtained using a PM2 Profile NMR-MOUSE (Act Mobile NMR Solutions, Aachen, Germany). The NMR data was collected using an 8 mm x 8 mm (x-y dimensions) x 10 m (z direction) sensitive volume. The T1 (spin-lattice relaxation time) of the roughened silicone was used to inform the time required between repetitions of the basic sequence in the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence and to achieve the maximum sensitivity per unit time; a repetition time (Rj) of 1 .5 ^* T1 (630 ms) was used. The sensitive volume was positioned within the roughened silicone to measure the raw probe amplitude, which was used as the normalisation value (188 A.U), for each depth profile. The Fourier transform of 32 CPMG scans was averaged and used to obtain the signal amplitude at each depth, with a 10 μιη step size, and measurements repeated by resampling a minimum of 3 times. Thickness was determined to be the z-axis range within which a signal above that of noise was detected.

Statistics

The errors associated with repeat measurements were calculated with 95% confidence intervals (CI) of the mean, unless otherwise stated. A prior check for data normality was conducted using the Shapiro-Wilk test, before the following parametric tests were used: one-way analysis-of-variance (ANOVA) using the F-statistic to test for significance between the mean measurements of three or more groups, followed by a post-hoc Tukey test for significant differences between the means of subsets of groups; two-way ANOVA to determine whether there was a significant effect of two independent variables (group) on a dependent variable (the measurement) or interactions between two independent variables on the dependent variable.

Example 1

Synthetic Skin Surrogate Screen

A high-throughput screen was developed to initially select suitable surrogates for downstream testing. The test provided information on the following parameters that were used to compare with skin response:

1) Lateral spread of simulant on surrogate surface at 0.35 kPa applied pressure. Simulant spread on skin under applied pressure may be different than on a surrogate surface. This parameter is important for (a) understanding contamination spread during application of an immediate decontaminant, such as a wipe or DKP1 - MK 1 pad, where applied pressure is inherent to the process; (b) designing immediate decontamination experiments. For example, comparison of post-decontamination residual simulant in skin with the amount in a surrogate could be affected if the spread of the simulant extends beyond the surface area of the decontaminant in the surrogate and not on the skin. 2) Presence of unabsorbed simulant post-contact with a non-absorbent glass block. This provides rapid measurement of the relative absorbencies of candidate surrogates for different simulants that can be compared with skin.

3) Measurable contact angle on surrogate surface. Similar output as (2); provides a measure of relative absorbencies between candidate surrogates for different simulants when compared with skin, but in the absence of applied pressure.

4) Simulant breakthrough of surrogate under low (≤0.35 kPa) and higher applied pressures (10 kPa). It is undesirable for simulant to break through the surrogate under pressures that could be applied during a decontamination process. This would complicate experimental procedures for quantifying the post-decontamination residual hazard.

The data obtained from porcine skin and synthetic skin screen tests upon exposure to 4 μΙ drop of methyl salicylate (MS) or tributyl phosphate (TBP) is summarised as follows. MS drops deposited onto the porcine skin surface produced a contact angle. Therefore, these drops were available for smearing upon contact with a surface (such as a respirator, wipe, DKP 1 - MK 1 pad); this is an important attribute for a potential synthetic skin surrogate.

The hydrophobic and inert polytetraflurorethylene (PTFE) track-etched membrane (WTP type; laminated onto an ordered polypropylene support, 0.2 μιη pore) was the only tested membrane that elicited a contact angle upon MS or TBP deposition. The other membranes (PTFE TE type, glass microfibre, nuclepore, anopore) absorbed simulant rapidly upon surface deposition and were therefore not suitable as skin surrogates for use in immediate decontamination experiments. Another unfavourable factor was MS or TBP breakthrough at low applied pressure, which excluded glass microfibre, nuclepore and anopore membranes from further investigation.

Although PTFE (WTP type) membrane elicited a contact angle for the deposited drop, there was high variance associated with MS or TBP drop spread on this membrane; in some cases the spread was also limited by the lateral size of the membrane. This limitation in simulant spread can effect evaluation of post- decontaminant efficacy, particularly for wipes.

All of the rubber polymers elicited a contact angle for deposited MS or TBP drops on their surfaces unlike most of the filter membranes: Other factors which favoured all or some of the rubber polymers as a skin surrogate was the absence of simulant breakthrough and presence of unabsorbed simulant on their surface, which matched skin responses to the test conditions.

One-way ANOVA with a post-hoc paired comparisons test (Tukey) was used to determine significant differences between mean MS or TBP spread areas for all polymers and porcine skin tested. The lateral spread area of MS on the polymer rubbers was significantly greater than the spread area observed on porcine skin upon application of a non-absorbent glass block at 0.35 kPa for 15 minutes (P > 0.05). This was also the case for TBP spread on Dragon Skin Medium, Dragon Skin FX Pro (DSFXP) and pigmented silicone (P≥ 0.05). The greater simulant spread on these horizontal polymer surfaces compared with porcine skin was a significant issue on inclined surfaces for immediate decontamination experiments in further tests.

DSFXP silicone was further investigated as a potential synthetic skin surrogate due to: (a) absence of simulant breakthrough at high pressures (10 kPa) and (b) a droplet contact angle is formed upon its deposition onto the DSFXP surface. Additional benefits of DSFXP silicone were: translucent when cured, which broadens its application in experiments, particularly in the use of dyed simulants to determine contamination spread; can be procured as liquid silicone for easy application to body-forms; fast cure time (ca. 1 hr at 23°C); MS absorbed into DSFXP can be solvent extracted; and MS absorption into DSFXP is comparable with its absorption into porcine skin.

Example 2

Influence of Skin-Surrogate Topography on Drop Spread . Droplet Spread on Head-Form coated with Silicone

A head-form was brush-coated with DSFXP silicone mix (Part A:B, 1 :1 v/v) and cured at 23°C for 3 hrs. The silicone was within 1 mm thick across all areas of the head, which is suitable for ensuring quantification of post-decontamination residual MS by extraction. Ms droplet (4 μΙ) roll-off along facial contours, following its deposition on the silicone at the forehead location, was rapid and not representative of the behaviour of MS droplets on porcine skin. This was a critical issue to resolve to enable evaluation of decontamination procedures.

2. Drop Spread on an Inclined Surface

Tests were undertaken to observe whether there were differences between simulant drop spread on the inclined surface of (porcine) skin and DSFXP, which is the orientation of the majority of surfaces on a head-form. MS drops of 4 μΙ deposited onto the inclined porcine skin surface were static and did not noticeably spread during the timeframe of the experiment. In contrast, MS drops deposited onto the smooth DSFXP surface quickly 'ran' down the surrogate. TBP droplets on porcine skin had a tendency to spread throughout the furrows. However, on DSFXP, the drops rapidly spread towards its base at a relatively faster rate than to MS.

Example 3

Roughening of Silicone Surface to Modify Droplet Spread

The results from the surface inclined drop spread experiments demonstrated that the spread of simulants on DSFXP was not representative of drop spread on porcine skin. The surface topography of DSFXP was modified to address this issue. Liquid DSFXP was drawn down over a flat glass surface with a film applicator. Coarse- grade (ISO standard 6344, average particle diameter of 425 urn; Wickes '40') sandpaper was placed above the liquid DSFXP, followed by a glass panel and weight. This was removed after curing to leave a roughened surface to the silicone.

Further surface inclined drop spread experiments were performed using the roughened DSFXP silicone. Visualisation of MS droplet behaviour on this surface was more representative of its behaviour on skin. A quantitative comparison of drop spread on roughened DSFXP and porcine skin was conducted to support this observation. Samples of porcine skin or coarsened DSFXP were laid onto glass blocks. Note porcine skin was preconditioned in deionised water at 32°C for 1 hr. Each sample was challenged with a 2 μΙ_ drop of dyed MS before applying an upper glass block to provide a pressure of 0.35 kPa. The samples were photographed at 15 minutes to capture the spread of MS, which was measured using Image J software.

Table 1 presents data on area analyses between the substrate types and simulants. There was no significant difference between the mean MS spread areas on coarse ('40'-grade) and porcine skin (P = 0.65; a = 0.05); however there was a significant difference in TBP spread on silicone medium-grade (70) roughened DSFXP and porcine skin (P = 0.02; a = 0.05). The data demonstrates that the drop spread of MS is comparable with porcine skin without the requirement to modify the surface chemistry of the silicone.

Spread Area/cm²

Simulant Skin Type Exp t Exp 2 Exp 3 Mean ± Error

(95 % C! of the mean)

MS . Silicone (Coarse 40) 0.076 0.020 0.022 0.039 0.079

MS Porcine Skin 0.037 0.042 0.070 0.050 0.044

TBP Silicone (Coarse 40) 0.12 0.12 0.16 0.13 0.06

TBP Silicone (Medium 0.43 0.28 0.39 0.37 0.19

70)

TBP Silicone (Very Fine 0.83 0.47 _' 0.32 0.54 0.64

150)

TBP Porcine Skin 2.96 2.50 1.90 2.45 1.32

Table 1. Mean MS and TBP (2 μΙ drop) spread area (with ± 95 % CI) on porcine skin and roughened DSFXP (horizontal) surfaces 2 min after deposit. P values from independent samples T-test (a = 0.05) also displayed.

Example 4

Drop Splatter on Roughened Silicone

Although MS drop spread on roughened DSFXP mimicked that on porcine skin, there was significant droplet 'spatter' on its surface following simulant deposition with a positive displacement pipette (Eppendorf Multipipette). This spatter resulted in a random droplet spread pattern, as well as smaller volumes (microdroplets) separating from the initial drop and projecting away from the surface plane. It was postulated that a surface charge on roughened DSFXP could have elicited drop repulsion. This surface charge may have resulted from separation of the roughened DSFXP-sandpaper surfaces during its preparation, where charge exchange between the two surfaces could have occurred and/or attraction of charged ions from the air. The surface charge on the roughened silicone could be sustained due to its well-known properties as an electrical insulator. In addition, the electric field resulting from a surface charge can be enhanced by surface roughness. Repulsion between charged liquids deposited onto charged surfaces can cause liquid droplets to expel microdroplets upon contact. Thus, methods were pursued to minimise this surface charge.

Several pre-treatment methods were used to neutralise surface charge, which involved antistatic equipment or immersing the coarsened silicone into an ionic solution and then air drying at ambient conditions:-

1 ) Antistatic gun: The anti-static gun projects a stream of positive and negatively charged particles towards the substrate when a trigger is pressed. The antistatic gun was fired at the skin surrogate from a distance of approximately 15 cm to neutralise surface charge.

2) Anti-static bar: The anti-static bar generates an electrical field that causes air molecules in the vicinity of the bar to break down into positive and negative ions. The bar was operated at its optimum distance from the roughened silicone surface (40 - 100 mm). Charge neutralisation of the silicone took place under the following conditions:-

(a) The silicone was placed on a glass block at an incline during charge neutralisation.

(b) The silicone was neutralised by holding the material freely in air as the bar was applied for 30 s, before placing the silicone onto a glass block to conduct drop spatter experiments.

3) Tap water wash: Tap water contains many ions, which may neutralise surface charge. The roughened DSFXP sample was immersed in a tap water bath for 10 s before drying on filter paper.

4) Aqueous sodium hydroxide wash: The roughened silicone was immersed in a 1 M NaOH bath for 10 s before drying on filter paper. An additional pre-treatment was performed in the same manner as methods 3 and 4 using isopropanol (IPA) solvent. Although IPA is a non-ionic solution, it was used during the preconditioning of the roughened silicone to enable quantitative measurement of post-decon residual MS in this study.

Table 2 shows the mean (dyed) MS drop mass deposited (n = 6) (using a positive displacement pipette) onto roughened DSFXP silicone surfaces, which were conditioned using the above methods for charge neutralisation. Note a control experiment was performed to determine the mass of a 4 μΙ (dyed) MS drop, which was 3.49 mg ± 1 .23 mg (± 95% CI; n = 6), One-way ANOVA found no significant differences between mean MS mass dispensed on treated or non-treated roughened DSFXP (a = 0.05; P > 0.05).

Table 2. Mean MS drop mass deposited (n = 6) (using a positive displacement pipette) onto roughened DSFXP silicone surfaces, which were conditioned by different methods for charge neutralisation.

In the light of the above, an alternative approach was used to reduce drop spatter, which involved changing the drop dispensing method. MS drops were deposited onto a roughened DSFXP surface held at an incline on a glass block. Whatman filter paper was placed below the sample to capture and visualise dyed microdroplets repelled from the silicone surface. Replacing the positive displacement pipette with a low-force single application pipette (Thermo Finnipipette) resulted in MS drops dispensed onto the roughened DSFXP surface without spatter.

Example 5

Up-scaling and Optimising Preparation of Roughened Silicone

Reproducibility in the preparation of roughened DSFXP using the initial process described above was problematic due to trapped air bubbles in the silicone that damaged the uniformity of the material. These regions were thinner and void of roughened surface as well as being mechanically weaker, causing rips and holes as it was removed from the sandpaper. A modification was made to the DSFXP curing process to eliminate the problem of air bubbles becoming trapped in the surrogate. The method was successfully developed whereby liquid DSFXP was drawn over the surface of the sandpaper and cured. In addition, the sandpaper was pre-coated with nano-glass (RadaglassTM fibre protect) to ease the removal of cured DSFXP from the sandpaper, reduce the risk of mechanical damage to the silicone and to enable the sandpaper template to be reused. Nano-glass is a silicon dioxide (Si0₂) liquid glass super-phobic coating.

Unilateral nuclear magnetic resonance (NMR) proton density depth-profiling was used to determine the depth at which the roughness was impregnated into the DSFXP silicone bulk. A gradual increase in proton density from the surface of the silicone into its bulk over 250 pm was observed; this is indicative of the thickness of the surface roughness. Beyond this region the silicone had a constant proton density. The mechanical stability of the roughened silicone significantly reduced when the silicone was less than ca. 350 pm, i.e. thicknesses that approached or impinged on the impregnated roughness.

Example 6

Comparison of the Absorptivity of Roughened Silicone and Porcine Skin to MS The MS mass distribution following 3 second contact with a 2 μΙ MS drop on roughened silicone or porcine skin with CRW at 2.4 kPa was investigated, showing little difference in the mass distribution of MS measured with roughened silicone with that measured using porcine skin. This was supported by 2-way ANOVA of this dataset, confirming that the % mass of MS recovered was independent of the interaction between each skin type (porcine skin or roughened silicone) and each component sampled to determine the mass distribution (skin surface wash, bulk extract and CRW)) (a = 0.05; P = 0.93); i.e. the mass fraction recovered for each skin type was the same for each component sampled to determine mass distribution.

The contact test was repeated but without the absorbent CRW so that the contact medium was a non-absorbent glass block. The post-contact mass distribution using these contact parameters also showed little difference in the mass distribution of MS measured with roughened silicone with that measured using porcine skin. This was supported by 2-way ANOVA of this dataset. The % mass of MS recovered was independent of the interaction between each skin type (porcine skin or roughened silicone) and each component to determine the mass distribution sampled (skin surface wash, bulk extract and glass contact medium) (a = 0.05; P = 0.25); i.e. the mass fraction recovered for each skin type was the same for each component sampled to determine mass distribution.

The post-contact residual MS on the roughened silicone surface better represented the mass-distribution of MS on porcine skin compared with smooth silicone. Residual droplets of dyed MS were observed on the glass contact medium following contact with porcine skin or roughened silicone dosed with a MS drop (at 0.4 kPa for 15 min); in contrast, trace contamination was observed on the glass post-contact with a MS drop on smooth silicone under the same contact conditions.

The data above indicated comparable absorptive properties of roughened silicone with porcine skin through testing with one simulant (at one drop size) and two contact test conditions that used contact mediums of different absorptivity. The mass distribution of MS following a single 3 second contact with these mediums were the same when using either porcine skin or roughened silicone as the surface. The latter is particularly important in the case of multiple touches using the same contact medium, which is relevant during personal decontamination using a wipe / DKP 1 - MK 1 pad.

Example 7

Post Decontamination MS Spread on Synthetic Skin Coated Head-Form to Inform Method for Quantitative Assessment of Residual MS

Experiments were performed using dyed MS to measure its spread on roughened DSFXP coated head-form following (a) application of the blot-bang-rub procedure using a DKP 1-MK 1 pad or (b) donning / doffing GSR and then applying the blot- bang-rub procedure. This informed the design of downstream experiments for the targeted removal of contaminated silicone for post-decontamination quantification of residual hazard, where non-dyed simulant was used. Simulant spread data de-risked under-sampling of the residual hazard during silicone excision from the head-form and also minimised over-sampling. The latter can potentially improve the limit-of- detection for quantifying low residual hazards, as unnecessarily large pieces of excised silicone would require larger extraction solvent volumes and dilute residual MS.

The blot-bang-rub procedure was carried out as directed by the printed instructions on the DKP 1-MK1 packaging. The contamination spread area, on the FE (Fullers Earth) pad was measured following the above procedure using Image J software to provide an approximation of the spread on the silicone. The experiment was conducted on porcine skin (pre-conditioned in 50% ethanol in water at 32 °C for 1 hr before patted dry using filter paper), which was secured to the head form at the same location as for the silicone experiment (above left eyebrow, the experiment was also conducted with 4 μΙ TBP on porcine skin and roughened silicone but at a different location (right cheek).

Table 3 compares the dyed simulant spread areas on the DKP 1 -MK 1 pad following the above decontamination experiments. The spread of the 4 μΙ simulant drop (MS / TBP) was within 3.0 cm². Also, there was no significant difference in the spread area of MS on porcine skin compared with roughened DSFXP silicone (P =; 0.22; a = 0.05), but there was significantly greater spread of TBP on porcine skin compared with roughened silicone (P= 0.02; a = 0.05); these support earlier findings.

Table 3. Mean simulant spread area (with ± SEM) on DKP 1-MK 1 pad following blot- bang-rub of 4 μΙ dyed MS/TBP on roughened DSFXP silicone or porcine skin attached to a mannequin head.

Further spread tests were conducted on a coated head-form following an immediate decontamination drill using GSR. The processes used in this experiment were as follows. Roughened silicone was attached to the face of the head-form (ear-to-ear, chin-to-forehead). The roughened silicone was contaminated with 16 x 2 μΙ dyed MS drops. These drops were applied in a symmetrical manner using a low-force pipette (Thermo Finnipipette). The total time required to complete the contamination process was 2 minutes 30 seconds. The GSR was donned using the standard-operating- procedure and left for 10 minutes before it was removed. The blot-bang-rub procedure was then performed. Finally, the silicone was peeled-off the head-form and photographed for image processing and quantification of dyed MS spread.

The spread area for each MS drop could be measured directly on the silicone due to an improved visibility of the spread drop. Earlier experiments administered blot-bang- rub directly onto a single contamination site resulting in significant FE deposition and masking the spread drop. For this experiment, blot-bang-rub was administered over a larger area and multiple contamination sites, which ultimately deposited less FE per unit area. The greatest spread of dyed MS coincided with contamination sites that were in-line with the GSR seals; these drops had been under contact pressure.

The MS (dyed) spread data determined the contamination sites and guidelines for excising silicone sections from the head-form without under-sampling the post-decon residual contamination. Each excision was made around the contamination site within a 16 cm² area.

A number of additional observations regarding silicone were made during the above experiments: the roughened silicone did not move once attached to the head-form during the processes of donning/doffing GSR or applying the blot-bang-rub procedure using the DKP 1 - MK 1 pad; the roughened silicone was mechanically durable to the above processes; and there was no simulant breakthrough of the surrogate following the above procedures.

Example 8

Quantitative Assessment of Contamination Transfer to GSR during immediate Decontamination Action Drill

Following removal of the GSR from the MS contaminated silicone coated head-form, its internal cavities and rubber seals were swabbed using cotton buds pre-dipped in IPA. Separate swabs were used to swab 7 separate sections of the GSR, where contamination on the cotton buds was determined visually.

There was no contamination observed on four of the seven swabs. Trace contamination was observed on two swabs and positive contamination was observed on one swab. The findings indicated there was no or trace contamination around the peripheral face seals of the GSR. These seals are composed of butyl rubber and are less absorbent than the roughened silicone, which forced simulant into the synthetic skin under contact pressure. This was supported by the larger spread areas of dyed MS on the silicone at these peripheral face locations. There was MS drop partitioning between the roughened silicone surface and the oral-nasal seal (itself composed of silicone). Visual inspection of the synthetic skin following removal of the GSR observed proud drops remaining on the silicone surface. This indicated the absence of sufficient contact pressure by the oral nasal seal on this face location to result in MS absorption.

Follow-on experiments swabbed the GSR cavities with clean room wipe for subsequent solvent extraction and quantitative analysis of transferred simulant.

Example 9

Alternative Adhesives to enable Attachment of Synthetic Skin to Head-Form for Quantitative Assessment of Post-Decontamination Residual Contamination

The preconditioning process removed interferents from the silicone that leached into the extraction fluid and interfered with subsequent GC quantification of post-decon residual MS. Therefore, it was not possible to use the DSFXP silicone as an adhesive to attach the pre-conditioned roughened silicone to the head-form, since this would re-introduce the interferents. Alternative adhesives were investigated to attach the roughened silicone to the metal head-form (3M Spray Mount; Wickes PVA; Wickes High Strength; UHU Twist & Glue; Staples Liquid Glue Pen; Monett Black Witch). The range of Commercial-Off-The-Shelf adhesives had different bonding strengths, viscosities, application methods and drying times.

Each adhesive was tested to ensure absence of interfering peaks for GC quantification of MS (data not shown). The experiment was performed by depositing approximately 0.1 cm³ of the adhesive into a glass vial, which was left open to the air (at ambient conditions) to allow the adhesive to cure over 12 hours. Following this period, the cured adhesive was exposed to 10 ml IPA (the same volume used to extract MS from silicone sections) and then sampled for GC analysis at 12 hours post-exposure. PVA was the only adhesive that produced an interfering chromatographic peak and was not taken forward to further experiments. The strength of the adhesive to bond the roughened silicone to the head-form was assessed. In this experiment, the smooth (un-roughened) side of a 5 cm x 5 cm sample of preconditioned roughened silicone was coated with 0.32 g ± 0.04 g (S.D.) adhesive and then applied to the head-form. Note, each adhesive was assessed at the same head location (right or left side of forehead) to mitigate influence of topographical features on the measurements. The silicone was held in place with cable ties as the adhesive cured overnight (>12 hrs). Following this period, the cable ties were removed and the sample was subjected to blot-bang-rub using a CRW (instead of a DKP 1-MK .1 ), where its stability during this process was assessed and scored between 0 and 4 (in order of increasing stability).

The silicone sections were then carefully peeled away from the head-form. During this process, an assessment was made as to which surface the adhesive preferentially bonded with, by a qualitative assessment of the amount of glue on each surface of the interface (head-form vs. silicone). Spray mount showed 50:50 partitioning between silicone and head-form; high strength contact adhesive was mostly bound to silicone; neoprene adhesive was mostly bound to head-form.

The force required to peel the sections off the head-form was made by a single operator and scored between 0 and 4 (in order of increasing adhesive strength). Although the neoprene Black Witch adhesive provided the highest bonding strength and stability during the blot-bang-rub drill, it was supplied as a viscous liquid in a small volume. This makes it problematic to sufficiently cover roughened silicone of an area to fit the face of the head-form.

Although the stability and adhesive strength of 3M Sprayable Mount was lower than Black Witch, it was. sufficient to retain the silicone on the head-form during the blot- bang₇rub procedure. This adhesive was easily sprayed over the large surface area of the un-roughened side of the roughened silicone, which provided a practical solution towards attaching the silicone to the head-form; particularly since the geometrically complex features of the face required sufficient adhesive in the corresponding areas on the silicone. Based on the above, the 3M Sprayable Mount adhesive was selected for further investigation.

Example 10

MS Extraction from Adhesive-Backed Roughened Silicone

The adhesive on the underside of the roughened silicone may potentially affect the efficiency for extracting bulk MS by solvent extraction. The extraction efficiency of MS from adhesive-backed silicone was measured following a contact test (Table 4).

Samples of preconditioned roughened silicone (5 cm x 5 cm) were coated with an under layer of 3M Spray Mount adhesive. The samples were left to cure for 12 hours prior to performing the contact test. A 2 μΙ MS drop was deposited onto the adhesive- backed roughened silicone and then contacted with a glass block at 6 kPa applied pressure for 15 min. Following this period, the silicone was solvent extracted in 3 x IPA baths (first extract in 10 ml IPA, second or third extracts in 11. 3 ml IPA), where no simulant was detected in the final extract. Note, each side of the silicone was washed in 10 ml IPA to remove MS carry-over as it was transferred between extraction vessels.

The mean total recovery was 97.5% ± 14.7% (± 95 % CI) upon summing the amounts recovered from washing the non-absorbent contact medium (upper glass block) and the amount on/in silicone.

MS/pg

Experiment Initial Upper On/in Total % number contamination glass silicone recovery recovery mass block

1 2390 606.74 1668.20 2274.93 95.2

2 2290 352.37 1778.78 2131.15 93.1

3 2150 564.78 1676.24 2241.02 104.2

Mean - - - - 97.5

± 95 % CI of 14.7 mean

Table 4. MS recovery following extraction of adhesive-backed silicone samples post- contact.

Example 11

Method Validation: Simulation of Immediate Decontamination Drill with GSR using DKP 1-MK 1 and Determining Post-Decontamination Residual Hazard

This section reports the findings from an experiment designed to validate the method of measuring post-decontamination residual MS after performing an Immediate Decontamination drill on a head-form coated with roughened silicone. The head-form was contaminated with MS drops using a low-force pipette. After a short delay, the GSR was donned onto the head-form and remained in position for 5 minutes. This period was decided based on the time required to relocate to a safe position, decontaminate gloves, the outside surfaces of the respirator and areas of IPE clothing that may be touched to enable doffing of the respirator. During these 5 minutes, MS vapour from inside the GSR was sampled at 15 L / min (NATO AEP58 specified breathing rate). Following this period, the GSR was doffed and the head- form decontaminated using a controlled blot-bang-rub procedure with one DKP 1-MK 1 pad. This entailed blotting each contamination site once (knuckle-to knuckle method), banging each contamination site once to release FE (using opposite fresh side of the pad) and rubbing each contamination site once. Note the rubbing procedure involved rubbing the FE away from the head-form. The time taken to administer the blot-bang-rub procedure using the above parameters was 38 seconds.

The controlled method used to apply the blot-bang-rub process ensured that each contamination site was contacted with each step of the process using the minimum number of touches (x1). This provided a well-controlled baseline to determine the relative efficacies of any future modifications to the processes. The inside of the GSR was then decontaminated using the same DKP 1-MK 1 pad as was used for the head-form. The peripheral seal was banged to release the FE and this action was continued on all parts of the GSR working inwards. The visor area was wiped as well as the flaps of each seal. The GSR was then fitted onto the head-form and a second series of three Tenax tubes was immediately attached to the end of the drinking straw. The pump was started and the post-decontamination residual simulant vapour was sampled from inside the GSR for 5 minutes. The GSR was doffed after vapour sampling and the roughened silicone carefully excised.

This procedure confirmed that the roughened silicone can be attached to a head- form and is mechanically stable during an ID drill using in-service DKP 1 , where a GSR was donned/doffed during the decontamination process. The amount of MS on/in roughened silicone, post-decontamination, was quantified following excision from the head-form without under-sampling or over-diluting this residual hazard.

Claims

1. A method for producing a synthetic skin, the method comprising the steps of: depositing a curable polymer on a non-biological material having a rough surface, so that the curable polymer contacts the rough surface; curing the polymer .such that the rough surface imprints on to the polymer; and removing the non-biological material from the cured polymer to provide the synthetic skin.

2. A method according to Claim 1 wherein the rough surface is an abrasive surface.

3. A method according to Claims 1 -2 wherein the abrasive surface comprises grains of average particle diameter 50 - 450 μηι.

4. A method according to Claims 1-3 wherein the non-biological material comprises grit of coarse-grade, medium-grade, fine-grade or very fine-grade.

5. A method according to Claims 1-4 wherein the non-biological material comprises grit of coarse-grade 40 or medium-grade 70.

6. A method according to Claims 1 -5 wherein the non-biological material comprises grit of coarse-grade 40.

7. A method according to Claims 1-6 wherein the non-biological material is sandpaper.

8. A method according to Claims 1-7 wherein the rough surface of the non-biological material is pre-coated with a non-stick substance.

9. A method according to Claim 8 wherein the non-stick substance comprises silicon dioxide liquid glass, one or more fluorine-containing chemical(s), manganese oxide polystyrene or zine oxide polystyrene.

10. A method according to Claim 9 wherein the non-stick substance is silicone dioxide liquid glass.

11. A method according to Claim 1 , wherein the curable polymer comprises silicone.

12. A method according to Claim 1 1 further comprising the steps of: immersing the cured polymer in an organic solvent; removing the cured polymer from the organic solvent; draining the cured polymer to remove non-bonded siloxane components; and air-drying the cured polymer.

13. A method according to Claim 12 wherein the organic solvent is isopropanol.

14. A method according to any preceding claim wherein hair is embedded into the synthetic skin.

15. A synthetic skin prepared according to the method of Claims 1 -14.

16. A synthetic skin according to Claim 15, wherein the average thickness of the synthetic skin is greater than 0.35 mm.

17. A synthetic skin according to Claims 15-16, wherein the average thickness of the synthetic skin is 1 .1 1 mm ± 0.23 mm.

18. A synthetic skin according to Claims 15-17 wherein hair is embedded into the synthetic skin.

19. Use of a synthetic skin as a physical model, wherein the synthetic skin is prepared according to the method of Claims 1-14.

20. Use of a synthetic skin as a physical model according to Claim 19, wherein the agent for testing is a chemical agent.

21. Use of a synthetic skin as a physical model according to Claim 20, wherein the agent is a liquid chemical agent.