Safety Assessment of Polyfluorinated Polymers as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 12 polyfluorinated polymers in cosmetic products; most of these ingredients have the reported function of film former in common. However, PTFE, the only ingredient that is reported as currently used in cosmetics, functions as a bulking agent and slip modifier, but not as a film former. The Panel reviewed data relevant to the safety of these ingredients under the intended conditions of use in cosmetic formulations, and concluded that PTFE and Hexafluoropropylene/Tetrafluoroethylene Copolymer are safe in cosmetics in the present practices of use and concentration described in the safety assessment; the data are insufficient to determine the safety of the 4 fluorinated-side-chain polymers and 6 fluorinated polyethers.

Genotoxicity of methyl acrylate and ethyl acrylate and its relationship with glutathione.

Methyl acrylate (MA) and ethyl acrylate (EA) had previously tested positive for mutagenicity in vitro, but in vivo studies were negative. One of the metabolism pathways of alkyl acrylates is conjugation with glutathione. The glutathione availability is restricted in standard in vitro test systems so that they do not reflect the in vivo metabolism in this respect. We investigated whether the addition of glutathione to the in vitro L5178Y/TK+/- mouse lymphoma mutagenicity test prevents alkyl acrylate's mutagenicity in vitro. We also investigated whether the quantitative relationships support the notion that the GSH supplemented in vitro systems reflect the true in vivo activity. Indeed, glutathione concentrations as low as 1 mM completely negate the mutagenicity of MA and EA in the L5178Y/TK+/- mouse lymphoma mutagenicity test up to the highest concentrations of the two acrylates tested, 35 µg/ml, a higher concentration than that previously found to be mutagenic in this test (14 µg MA/ml and 20 µg EA/ml). 1 mM Glutathione reduced the residual MA and EA at the end of the exposure period in the mutagenicity tests by 96-97%, but in vivo up to 100 mg/kg body weight MA and EA left the glutathione levels in the mouse liver and forestomach completely intact. It is concluded that the in-situ levels of glutathione, 7.55 ± 0.57 and 2.84 ± 0.22 µmol/g mouse liver and forestomach, respectively, can efficiently protect against MA and EA-induced mutagenicity up to the high concentration of 100 mg MA and EA/kg body weight and that the negative in vivo mutagenicity tests on MA and EA reflect the true in vivo situation.

A short-term inhalation study to assess the reversibility of sensory irritation in human volunteers.

Sensory irritation is an acute adverse effect caused by chemicals that stimulate chemoreceptors of the upper respiratory tract or the mucous membranes of the outer eye. The avoidance of this end point is of uttermost importance in regulatory toxicology. In this study, repeated exposures to ethyl acrylate were analyzed to investigate possible carryover effects from day to day for different markers of sensory irritation. Thirty healthy subjects were exposed for 4 h on five subsequent days to ethyl acrylate at concentrations permitted by the German occupational exposure limit at the time of study. Ratings of eye irritation as well as eye blinking frequencies indicate the elicitation of sensory irritation. These markers of sensory irritation showed a distinct time course on every single day. However, cumulative carryover effects could not be identified across the week for any marker. The rhinological and biochemical markers could not reveal hints for more pronounced sensory irritation. Neither increased markers of neurogenic inflammation nor markers of immune response could be identified. Furthermore, the performance on neurobehavioral tests was not affected by ethyl acrylate and despite the strong odor of ethyl acrylate the participants improved their performances from day to day. While the affected physiological marker, the increased eye blinking frequency stays roughly on the same level across the week, subjective markers like perception of eye irritation decrease slightly from day to day though the temporal pattern of, i.e., eye irritation perception stays the same on each day. A hypothetical model of eye irritation time course derived from PK/PD modeling of the rabbit eye could explain the within-day time course of eye irritation ratings repeatedly found in this study more precisely.

Ethyl acrylate: influence of sex or atopy on perceptual ratings and eye blink frequency.

Occupational exposure limits (OELs) are derived for protection from health hazards, assuming that exposed subjects are healthy adult workers. Whether differences in susceptibility to sensory irritation effects from airborne chemicals have to be taken into account is currently under discussion. Thus, we chose atopics as a healthy but possibly susceptible subpopulation that can be identified with a clinical test. To investigate the influence of sex or atopy on sensitivity to airborne chemicals, 22 subjects were exposed for 4 h to ethyl acrylate at three concentrations: 0.05 ppm (odor threshold; sham), 5 ppm (constant), and varying exposure between 0 and 10 ppm. Odor intensity decreased and eye irritation ratings increased in a dose-dependent manner, reflecting the time course of the exposure scenarios. The reports of moderate-to-strong eye irritation were verified by significant increases in eye blink frequency. Our results show that women reported subjective eye irritation to an increasing degree. However, these sex-related differences in ratings could not be verified by objective assessment of eye blink frequency. Atopic subjects reported higher odor intensity than non-atopic subjects, but only during the sham (odorous but not irritating) exposure condition. Differences in ratings on annoyance, and eye or nose irritation were not found. Furthermore, the study revealed that atopic subjects might belong to a group of subjects with frequent eye blink activity. Although the relative increase in blink rates was more pronounced in non-atopic subjects, atopic subjects had significant higher blink rates at the end of the exposure to varying ethyl acrylate concentrations. Our results do not support that atopy enhances chemosensory effects if only the increase of blink rates and not the absolute height are considered as adverse effect. Nevertheless, the results indicate that individuals with frequent eye blink activity should be distinguished from those with normal eye blink activity while investigating blink rates as objective parameter of eye irritation.

Methodological Implications and Repeatability of Nasal Nitric Oxide: Relevance for Challenge Studies.

There is an interest in assessing changes in nasal NO (nNO) levels as an effect marker of upper airways. In this study, we examined methodologic influences on short and long term repeatability of nNO levels assessed by a portable electrochemical analyzer. Nine atopic and eighteen healthy subjects were exposed for 4 h to ethyl acrylate concentration of 0.05 ppm (sham) and mean concentrations of 5 ppm (either constant 5 ppm or variable 0 to 10 ppm). Sampling of nNO was performed by using passive aspiration during both breath-holding (634 ppb) or calm tidal breathing (364 ppb, p < 0.0001). The intra-session (between-session) repeatability in terms of coefficient of variation was 16.4% (18.5%) using the tidal-breathing and 8.6% (13.0%) using the breath-holding method, respectively. Atopic subjects demonstrated a significant increase in nNO (breath-holding mean 16%, tidal-breathing mean 32%) after applying a constant ethyl acrylate concentration (5 ppm). Our findings suggest that the less elaborate tidal-breathing method might be sufficient to detect significant changes at a group level. Given a lower coefficient of variation of breath-holding we assume there is an advantage of that approach at an individual level. Further research is needed to validate the usefulness of nNO in the evaluation of irritative, non-allergic responses.

Assessment of the mode of action underlying development of forestomach tumors in rodents following oral exposure to ethyl acrylate and relevance to humans.

Chronic repeated gavage dosing of high concentrations of ethyl acrylate (EA) causes forestomach tumors in rats and mice. For two decades, there has been general consensus that these tumors are unique to rodents because of: i) lack of carcinogenicity in other organs, ii) specificity to the forestomach (an organ unique to rodents which humans do not possess), iii) lack of carcinogenicity by other routes of exposure, and iv) obvious site of contact toxicity at carcinogenic doses. In 1986, EA was classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC). However, by applying a MOA analyses and human relevance framework assessment, the weight-of-evidence supports a cytotoxic MOA with the following key events: i) bolus delivery of EA to forestomach lumen and subsequent absorption, ii) cytotoxicity likely due to saturation of enzymatic detoxification, iii) chronic regenerative hyperplasia, and iv) spontaneous mutation due to increased cell replication and cell population. Clonal expansion of initiated cells thus results in late onset tumorigenesis. The key events in this 'wound and healing' MOA provide high confidence in the MOA as assessed by evolved Bradford-Hill Criteria. The weight-of-evidence supported by the proposed MOA, combined with a unique tissue that does not exist in humans, indicates that EA is highly unlikely to pose a human cancer hazard.

An Adverse Outcome Pathway (AOP) for forestomach tumors induced by non-genotoxic initiating events.

The utility of rodent forestomach tumor data for hazard and risk assessment has been examined for decades because humans do not have a forestomach, and these tumors occur by varying modes of action (MOAs). We have used the MOA for ethyl acrylate (EA) to develop an Adverse Outcome Pathway (AOP) for forestomach tumors caused by non-genotoxic initiating events. These tumors occur secondary to site of contact induced epithelial cytotoxicity and regenerative repair-driven proliferation. For EA, the critical initiating event (IE) is epithelial cytotoxicity, and supporting key events (KEs) at the cellular and tissue level are increased cell proliferation (KE1) resulting in sustained hyperplasia (KE2), with the adverse outcome of forestomach papillomas and carcinomas. For EA, a pre-molecular initiating event (pre-MIE) of sustained glutathione depletion is probable. Supporting data from butylated hydroxyanisole (BHA) are also reviewed. Although there may be some variability in the pre-MIEs and IEs for BHA and EA, they share the same KEs, and evidence for BHA confers support for the AOP. Evolved Bradford Hill considerations of biological plausibility, essentiality, and empirical support were evaluated per OECD guidance. Although an MIE is not specifically described, overall confidence in the AOP is high due to well-developed and accepted evidence streams, and the AOP can be used for regulatory applications including hazard identification and risk assessment for chemicals that act by this AOP.

The role of ethyl acrylate induced GSH depletion in the rodent forestomach and its impact on MTD and in vivo genotoxicity in developing an adverse outcome pathway (AOP).

Adverse outcome pathways (AOP) and mode of action (MOA) frameworks help evaluate the toxicity findings of animal studies and their relevance to humans. To effectively use these tools to improve hazard identification and risk assessments for ethyl acrylate (EA), knowledge gaps in metabolism and genotoxicity were identified and addressed. For EA, hypothesized early key events relate to its irritation potential: concentration dependent irritation and cytotoxicity, progressing to regenerative proliferation and forestomach carcinogenicity after repeated oral bolus application in rodents. The current research quantitated glutathione (GSH) depletion to assess a kinetically-derived maximum tolerated dose (MTD) in the target tissue and used this information to conduct an in vivo genotoxicity study using current methods. In the mouse forestomach, gavage doses of EA caused GSH depletion to 47% of control at 20 mg/kg and 28% at 100 mg/kg. Cellular redox changes and histopathology support saturation of metabolism and an MTD of ∼50 mg/kg. No increases in point mutations or deletions occurred in the stomach or liver following a 28 day treatment of gpt delta transgenic mice at gavage doses up to 50 mg/kg/day. These results provide valuable information for evaluating AOP molecular initiating events or MOA key events for EA and other GSH depleting materials.

Tuning the Molar Composition of "Charge-Shifting" Cationic Copolymers Based on 2-(N,N-Dimethylamino)Ethyl Acrylate and 2-(tert-Boc-Amino)Ethyl Acrylate.

Copolymers of 2-(N,N-dimethylamino)ethyl acrylate (DMAEA) and 2-(tert-Boc-amino)ethyl acrylate (tBocAEA) are synthesized by reversible addition-fragmentation chain transfer polymerization in a controlled manner with defined molar masses and narrow molar masses distributions (Ð ≤ 1.17). Molar compositions of the P(DMAEA-co-tBocAEA) copolymers are assessed by means of 1 H NMR. A complete screening in molar composition is studied from 0% of DMAEA to 100% of DMAEA. Reactivity ratios of both comonomers are determined by the extended Kelen-Tüdos method (r DMAEA = 0.81 and rtBocAEA = 0.99).

Impact of Internal and External Factors on EBC-pH and FeNO Changes in Humans Following Challenge with Ethyl Acrylate.

Acute effects of ethyl acrylate exposure at 5 ppm for 4 h include changes of pH in exhaled breath condensate (EBC-pH) and exhaled nitric oxide (FeNO). So far, few data have been reported for atopic persons or the impact of the exposure conditions on biomarkers, e.g., constant versus variable application of irritants. Nine atopic and eighteen healthy volunteers without bronchial hyperresponsiveness were exposed for 4 h to ethyl acrylate concentrations of 0.05 ppm (sham), 5 ppm (constant concentration), and 0-10 ppm (variable, mean concentration of 5 ppm) in an exposure laboratory. A positive atopic status was defined according to specific IgE concentrations to common inhalant allergens (sx1 ≥ 0.35 kU/L). Biomarker levels were assessed before and after challenge and adjusted for levels after sham exposure (net response). Ethyl acrylate at constant, but not at variable concentrations induced a significant change in the net responses of EBC-pH and FeNO. Concerning FeNO, this could be observed only for atopic persons. The changes of biomarker levels were related to their baseline values. Biomarker responses to challenge with ethyl acrylate may be influenced by the patterns of application as well as baseline airway inflammation and atopic status of the volunteers.

Clinical Effects, Exhaled Breath Condensate pH and Exhaled Nitric Oxide in Humans After Ethyl Acrylate Exposure.

Ethyl acrylate is an irritant known to affect the upper airways and eyes. An increase of the eye blink frequency in humans was observed during exposure to 5 ppm. Studies on the lower airways are scant and our study objective was the evaluation of pH in exhaled breath condensate (EBC-pH) and nitric oxide in exhaled breath (FeNO) as markers of inflammation. Sixteen healthy volunteers were exposed for 4 h to ethyl acrylate at a concentration of 5 ppm and to sham (0.05 ppm) in an exposure laboratory. Clinical irritation symptoms, EBC-pH (at a pCO2 of 5.33 kPa) and FeNO were assessed before and after exposure. Differences after ethyl acrylate exposure were adjusted for those after sham exposure. 5 ppm ethyl acrylate induced clinical signs of local irritation in the nose and eyes, but not in lower airways. Exposure produced a subtle, but statistically significant, decrease in breathing frequency (1 breath/min; p = 0.017) and a lower EBC-pH (by 0.045 units; p = 0.037). Concerning FeNO, we did not observe significant changes compared to sham exposure. We conclude that local effects induced by 5 ppm ethyl acrylate consist of sensory irritation of eyes and nose. In addition, acute ethyl acrylate exposure to 5 ppm resulted in a net decrease of EBC-pH. Whether that can be interpreted in terms of additional lower airway irritation or already inflammatory alterations set in needs further investigations.

Characterisation and toxicological assessment of Neutral Methacrylate Copolymer for GRAS evaluation.

Neutral Methacrylate Copolymer is a fully polymerised copolymer used in the pharmaceutical industry to permit pH-independent delayed release of active ingredients from oral dosage forms. This function has potential use with food supplements and this article describes available information on the safety of the substance. Oral administration of radiolabelled copolymer to rats resulted in the detection of chemically unchanged copolymer in the faeces, with negligible absorption. Safety studies revealed no adverse toxicity following repeated administration at doses of up to 2000 mg/kg bw/d in a sub-chronic study in rats or 250 mg/kg bw/d in a sub-chronic study in dogs. No reproductive toxicity occurred at up to 2000 mg/kg bw/d in rats or rabbits. The substance shows no evidence of genotoxicity, has low acute toxicity and no irritation or sensitisation potential. An ADI value of 20 mg/kg bw was concluded from two alternative approaches. Daily exposure from use in dietary supplements is estimated as up to 10.0 mg/kg bw in adults and 13.3 mg/kg bw in children. There would therefore appear to be no safety concerns under the intended conditions of use. The information provided is intended to support an evaluation that the substance may be "generally recognized as safe" (GRAS).

Non-thermal plasma coupled with a wet scrubber for removing odorous VOC.

Odorous volatile organic compounds (VOCs) deteriorate the quality of life and affect human health. In this study, a process was developed to remove an odorous VOC using a combined non-thermal plasma (NTP) and wet scrubber (WS) system. The low removal efficiency of WSs and the large amount of ozone generated by NTP were resolved. Compared to the decomposition effects when using a WS and NTP separately, the NTP + WS system improved the removal efficiency of ethyl acrylate (EA) and significantly reduced ozone emissions. The maximum EA removal efficiency was 99.9%. Additionally, an EA removal efficiency of over 53.4% and a 100% ozone removal efficiency were achieved even at discharge voltages lower than 4.5 kV. Ozone catalysis was confirmed to occur in the NTP + WS system. Furthermore, we verified the removal of by-products such as residual ozone and formaldehyde, which is a representative organic intermediate of EA. This study demonstrates that the NTP + WS system is a green technology for removing odorous VOCs.

[Characterization of Humicola insolens cutinase-tachystatin A2 fusion protein and its application in treatment of recycled paper stickies].

With the decrease of forest timber resources, the recycling of waste paper has received increasing attention. However, the stickies produced in the process of waste paper recycling may negatively affect the production of recycled paper. The biological decomposition of stickies, which has the advantages of high efficiency, high specificity and pollution-free, is achieved mainly through the enzymatic cleavage of the ester bond in the stickies components to prevent flocculation. Cutinase is a serine esterase that can degrade some components of the stickies. Previous research indicated that the anchor peptide tachystatin A2 (TA2) is able to bind polyurethane. In this study, the cutinase HiC derived from Humicola insolens was used to construct a fusion protein HiC-TA2 by megaprimer PCR of the whole plasmid (MEGAWHOP). The enzymatic properties and the degradation efficiency of the fusion protein on poly(ethyl acrylate) (PEA), a model substrate of stickies component, were determined. The results showed that the degradation efficiency, the size decrease of PEA particle, and the amount of ethanol produced by HiC-TA2 were 1.5 times, 6.8 times, and 1.4 times of that by HiC, respectively. These results demonstrated that TA2 improved the degradation efficiency of HiC on PEA. This study provides a useful reference for biological decomposition of stickies produced in the process of recycled paper production.

Evaluation of the Effect of Ethyl Acrylate-Methyl Methacrylate Copolymer in Racecadotril Dispersible Tablet.

Objectives: Racecadotril is an anti-diarrheal drug that has the indication to reduce the secretion of water and electrolytes into the intestine. It has an unpleasant taste, when administered orally. The presenting study developed a pharmaceutical racecadotril dispersible tablet, which masked the unpleasent taste using wet granulation method. For this reason, the effect of the number of ethylacrylate-methylmethacrylate copolymers (Eudragit® NE 30D) in taste masking and in vitro dissolution of the finished product was investigated. Materials and Methods: Taste-masked racecadotril granules were prepared using Eudragit® NE 30D and the ratio between the amounts of racecadotril and Eudragit® NE 30D involved in the formulation was optimized. The products obtained in the dispersible tablet dosage form were evaluated in terms of taste and in vitro dissolution studies. In vitro dissolution profiles of the products obtained in this study were compared with reference product Tiorfan® granules for oral suspension manufactured by Bioprojet Pharma (Paris, France). A method of apparatus II (paddle), 900 mL, pH 4.5 acetate buffer + 1% sodium dodecyl sulfate (SDS) and 100 rpm at 37.0 ± 0.5°C was adopted. Results: Results of the studies have shown that the formulation should have Eudragit® NE 30D higher than 1% by weight of racecadotril to satisfy the taste-masking ability and the formulation should have Eudragit® NE 30D equal or lower than 10% by weight of racecadotril to have better release characteristic to be compatible with reference product. Conclusion: Our results demonstrated that a chemically long-term stable racecadotril dispersible tablet product, whose taste is efficiently masked using wet granulation method with an acceptable release profile was obtained with Eudragit® NE 30D ratio higher than 1% and equal or lower than 10% by weight of racecadotril. The developed formulation can increase patient compliance.

Processability evaluation of multiparticulate units prepared by selective laser sintering using the SeDeM Expert System approach.

3D printing in dosage forms fabrication is in the focus of researchers, however, the attempts in multiparticulate units (MPUs) preparation are scarce. The aim of this study was to fabricate different size MPUs by selective laser sintering (SLS), using different polymers, and investigate their processability based on the SeDeM Expert System approach. MPUs (1- or 2-mm size) were prepared with model drug (ibuprofen or caffeine), polymer (poly(ethylene)oxide (PEO), ethyl cellulose (EC) or methacrylic acid-ethyl acrylate copolymer (MA-EA)) and printing aid. Comprehensive sample characterization was performed and experimentally obtained parameters were mathematically transformed and evaluated using the SeDeM Expert System framework. The obtained samples exhibited irregular shape, despite the spherical printing object design. Polymer incorporated notably affected MPUs properties. The obtained samples exhibited low bulk density, good flowability-, as well as stability-related parameters, which indicated their suitability for filling into capsules or sachets. Low density values implied that compressibility enhancing excipients may be required for MPUs incorporation in tablets. Samples containing EC and MA-EA were found suitable for compression, due to high compacts tensile strength. The obtained results indicate that SeDeM Expert System may extended from powder compressibility evaluation tool to framework facilitating powders/multiparticulate units processing.

A study of Kollicoat® MAE100P film's structure and properties.

Generally, an organic-solvent-based film is denser and tougher than a corresponding aqueous-dispersion-based film. However, Kollicoat® MAE100P films prepared from aqueous dispersions had greater tensile strengths compared to the films cast from organic solutions. It was proposed that MAE100P polymer particles in aqueous media had a core-shell structure with a hydrophilic shell and a hydrophobic core. The hydrophilic shell was rich in ionized methacrylic acid (MAA) groups and the hydrophobic core primarily contained unionized MAA and ethyl acrylate (EA). As a result, ionized MAA formed a continuous phase which worked as a rigid frame and greatly improved the mechanical properties of aqueous-dispersion-based films. In order to prove this theory and investigate the effect of ionization level on this polymer system, the properties of pH, turbidity, zeta potential, and particle size of MAE100P dispersions were measured as a function of ionization level. The tensile strengths and thermal and mechanical properties of MAE100P films prepared from organic solution or aqueous dispersions of different ionization levels were investigated as well. FTIR was used to characterize the polymer films. Drug release in 0.1 N HCl from coated pellets was studied using the basket method. The experimental results showed that the original MAE100P polymer particles (if not specified, the ionization level is 6%) had a highly-charged surface. The properties of polymer aqueous dispersions were significantly changed by the ionization levels. Aqueous-dispersion-based MAE100P films or coats were stronger and comparable to or somewhat more effective in inhibiting drug diffusion than were organic-solvent-based coats. The tensile strength initially increased and then decreased with an increase of ionization level, while the water-uptake rate by the films continuously increased. Two endothermic peaks were observed in the DSC thermograms for cured MAE100P films. The high-Tg endothermic peak increased with an increase in ionization level, while the low-Tg peak didn't exhibit significant change except for the 18% ionization film. In the dynamic mechanical analysis, two relaxations in the storage modulus were observed in the aqueous-dispersion-based films. These data may suggest a two-phase structure in the form of a core-shell structure. The tensile-strength ratio for aqueous-dispersion-based films over organic-solvent-based films for MAE100P was close to that reported for films formed from polymer substances/particles with core-shell structures. In summary, the core-shell structure might result in a two-phase structure in the bulk MAE100P film prepared from aqueous dispersion. This special structure led to significantly-improved mechanical properties for aqueous-dispersion-based MAE100 films. The ionization levels had complicated effects on the polymer system by increasing the amount of ionic aggregates while also solubilizing the polymer and changing the mechanism of film formation.

Development, Characterization, Optimization, and In Vivo Evaluation of Methacrylic Acid-Ethyl Acrylate Copolymer Nanoparticles Loaded with Glibenclamide in Diabetic Rats for Oral Administration.

The methacrylic acid-ethyl acrylate copolymer nanoparticles were prepared using the solvent displacement method. The independent variables were the drug/polymer ratio, surfactant concentration, Polioxyl 40 hydrogenated castor oil, the added water volume, time, and stirring speed, while size, PDI, zeta potential, and encapsulation efficiency were the response variables analyzed. A design of screening experiments was carried out to subsequently perform the optimization of the nanoparticle preparation process. The optimal formulation was characterized through the dependent variables size, PDI, zeta potential, encapsulation efficiency and drug release profiles. In vivo tests were performed in Wistar rats previously induced with diabetes by administration of streptozotocin. Once hyperglycemia was determined in rats, a suspension of nanoparticles loaded with glibenclamide was administered to them while the other group was administered with tablets of glibenclamide. The optimal nanoparticle formulation obtained a size of 18.98 +/- 9.14 nm with a PDI of 0.37085 +/- 0.014 and a zeta potential of -13.7125 +/- 1.82 mV; the encapsulation efficiency was of 44.5%. The in vivo model demonstrated a significant effect (p < 0.05) between the group administered with nanoparticles loaded with glibenclamide and the group administered with tablets compared to the group of untreated individuals.

Proteomic analysis of haptenation by skin sensitisers: Diphencyprone and ethyl acrylate.

The potential risk of skin sensitisation, associated with the development of allergic contact dermatitis (ACD), is a consideration in the safety assessment of new ingredients for use in personal care products. Protein haptenation in skin by sensitising chemicals is the molecular initiating event causative of skin sensitisation. Current methods for monitoring skin sensitisation rely on limited reactivity assays, motivating interest in the development of proteomic approaches to characterise the skin haptenome. Increasing our mechanistic understanding of skin sensitisation and ACD using proteomics presents an opportunity to develop non-animal predictive methods and/or risk assessment approaches. Previously, we have used a novel stable isotope labelling approach combined with data independent mass spectrometry (HDMSE) to characterise the haptenome for a number of well-known sensitisers. We have now extended this work by characterising the haptenome of the sensitisers Diphenylcyclopropenone (DPCP) and Ethyl Acrylate (EA) with the model protein Human Serum Albumin (HSA) and the complex lysates of the skin keratinocyte, HaCaT cell line. We show that haptenation in complex nucleophilic models is not random, but a specific, low level and reproducible event. Proteomic analysis extends our understanding of sensitiser reactivity beyond simple reactivity assays and offers a route to monitoring haptenation in living cells.

Plasma polymerised nanoscale coatings of controlled thickness for efficient solid-phase presentation of growth factors.

The engineering of biomaterial surfaces and scaffolds for specific biomedical and clinical application is of growing interest. Certain functionalised surfaces can capture and deliver bioactive molecules, such as growth factors (GF), enhancing the clinical efficacy of such systems. With a custom-made plasma polymerisation reactor described here we have developed bioactive polymer coatings based on poly(ethyl acrylate) (PEA). This remarkable polymer unfolds fibronectin (FN) upon adsorption to allow the GF binding region of FN to sequester and present GFs with high efficiency. We systematically evaluate process conditions and their impact on plasma polymerised PEA coatings and characterise the effect of plasma power and deposition time on thickness, wettability and chemical composition of the coatings. We demonstrate that functional substrate roughness can be maintained after deposition of the polymer coatings. Importantly, we show that coatings deposited at different conditions all maintain a similar or better bioactivity than spin coated PEA references. We show that in PEA plasma polymerised coatings FN assembles into nanonetworks with high availability of integrin and GF binding regions that sequester bone morphogenetic protein-2 (BMP-2). We also report similar mesenchymal stem cell adhesion behaviour, as characterised by focal adhesions, and differentiation potential on BMP-2 coated surfaces, regardless of plasma deposition conditions. This is a potent and versatile technology that can help facilitate the use of GFs in clinical applications.