Biological evaluation and chemical characterization of photochemically crosslinked surgical gloves.
In recent work, we have developed a new pre-vulcanization process for NR latex based upon a UV initiated thiolene crosslinking reaction. The new technology allows the manufacture of dipped latex articles with the main focus on the production of surgical gloves without using any allergenic accelerator chemicals (e.g., thiurams, mercaptobenzothiazole or dithiocarbamates). Thiolene networks are known for their low oxygen inhibition and they are already used in other medical applications such as dental restoratives or bone cements (refs. 7 and 8).
The general reaction mechanism of the UV initiated prevulcanization of NR latex via the thiolene reaction is displayed in scheme 1. In the initiation step, a selected photoinitiator (PI) present in the latex matrix is excited by UV light which is followed by a bond cleavage to yield free radicals. In the presence of thiols, there is a sufficient hydrogen transfer from the thiol to the free photoinitiator radicals, resulting in the formation of thiyl radicals. Once formed, the thiyl radicals are capable of reacting with the C=C double bonds in the polyisoprene generating thioethers and carbon centered radicals. Further, thiyl radicals are formed due to a hydrogen abstraction from another thiol by the carbon centered radicals. The termination reactions involve radical coupling leading to disulfides, thioethers and covalent carbon-carbon bonds (ref. 9). Using multifunctional thiols, crosslinking is accomplished by this photochemical reaction.
When it comes to the UV illumination of liquid latex emulsions it has to be considered that NR latex comprises a very low light transmissivity. Thus, only thin latex films (<1 mm film thickness) can be irradiated homogenously. Taking this fact into account, the UV pre-vulcanization is carried out in a falling film photo reactor. The design of a falling film reactor makes a continuous and homogenous irradiation of the latex in thin films feasible. The technology is distinguished by low energy consumption because the crosslinking is carried out at room temperature and is accomplished within minutes. By using this falling film technology, UV pre-vulcanized latex is already produced in pilot plant quantities. However, the scale up from the pilot plant to a production line can be obtained easily because falling film reactors are already used on industrial scale processes such as water purification, water sterilization or preparative organic chemistry and photochemistry processes (refs. 10 and 11).
Surgical gloves are then made from the UV cured latex on a semi-industrial scale and exhibit excellent physical properties (e.g., tensile strength, ultimate elongation, force at break, aging stability). The present work is focused on the evaluation of the skin and biocompatibility of UV pre-vulcanized surgical gloves by acute dermal irritation/corrosion studies with rabbits, Nine Dose Buehler Test and cytotoxicity tests in compliance with ISO standards. In addition to these dermatological studies, the extractable and bio available amount of the crosslink chemical residues (photoinitiator and thiol) in UV precured latex gloves was investigated with analytical techniques such as high performance liquid chromatography coupled with mass spectroscopy (HPLC-MS) and elementary analyses.
Materials and chemicals
Natural concentrated rubber latex (high ammonia, 60 wt.-% dry rubber content) was purchased. The photoinitiator (PI) was supplied by BASF. Acetone, acetonitrile and dichloromethane were used in analytical grade and were supplied by Sigma Aldrich. All other reagents were from Sigma Aldrich and were used without further purification. For HPLC-MS analyses, water and acetonitrile of gradient grade from Acros Organics (Geel, Belgium) were used. All other reagents were from Sigma Aldrich and were used without further purification.
In the UV pre-vulcanization of NR latex, the photoinitiator and the thiol were emulsified in de-ionized water.
The emulsion of the crosslinking chemicals was added to 40 kg high ammonia natural rubber latex (40 wt.-% dry rubber content) comprising a concentration of 1.0 phr (parts per hundred of rubber) photoinitiator and TriThiol as crosslink agents, respectively. The latex formulation was then stirred by means of a magnetic agitator in a storage vessel at room temperature for two hours.
The UV pre-vulcanization was carried out in a tailor-made falling film reactor (figure 1). The pilot plant consisted of a reactor tube where a UV lamp was arranged centrically. An eccentric screw pump conveyed the NR latex formulation continuously from the storage vessel to the top of the falling film reactor and a continuous falling film was accomplished with a film thickness ranging from 0.2 to 0.5 mm. The thin latex film was irradiated and pre-vulcanized, respectively, in two illumination cycles with wavelengths between 240 and 460 nm.
[FIGURE 1 OMITTED]
Manufacture of UV-precured surgical gloves
0.5 phr of a phenolic antioxidant was added to the pre-vulcanized NR latex, and from this UV pre-vulcanized latex formulation, surgical gloves were manufactured on a semi-industrial scale using a tailor made dipping machine. In the first production step, the porcelain hand formers were cleaned with acid and alkaline solutions. After neutralizing and drying, the formers were dipped in a coagulant solution containing calcium chloride (coagulant), calcium carbonate (release agent) and cationic surfactants. The formers were dried and were then immersed in the UV pre-cured NR latex. The thickness of the latex gloves obtained from coagulant dipping ranged from 0.2 to 0.3 mm. In the next step, the gloves were dipped in hot water (leaching) to remove residual processing chemicals and to lower the protein levels in the final products. They were then immersed into powder slurry containing corn starch to avoid stickiness and automatically stripped from the formers. After packaging, the surgical gloves were gamma-sterilized with a [sup.60]Co--source (25 kGy nominal dose).
Biological testing--acute dermal irritation study with rabbits
The aim of this study was to examine the possible irritation and corrosion by the UV pre-cured glove following a single application to the intact skin of rabbits. The study was conducted in conformance with ISO 10993-10 (Biological evaluation of medical devices--Part 10: Tests for irritation and delayed-type hypersensitivity, 2002) at Austrian Institute of Technologies (AIT Seibersdorf).
Skin sensitization study
The Nine Induction Dose Buehler Test (the skin sensitization test according to E.V. Buehler) was performed in conformance with ISO 10993-10 (Biological evaluation of medical devices--Part 10: Tests for irritation and delayed-type hypersensitivity, 2002).
The in vitro biocompatibility of UV pre-vulcanized gloves was elevated on the basis of cell cytotoxicity. The cytotoxic tests were preformed in accordance with ISO 10993-5 (Biological evaluation of medical devices--Part 5: Test for in vitro cytotoxicity, 1999) and were carried out at Nelson Laboratories (Salt Lake City, UT).
Chemical testing--soxhlet extraction procedures
The UV cured surgical gloves were extracted to determine the residual TriThiol and photoinitiator levels, respectively. In these studies, surgical gloves pre-vulcanized with 1.0 phr photoinitiator and 1.0 phr TriThiol were characterized. In addition, a reference soxhlet extraction was carried out to estimate the total sulfur content and the thiol residues in non-crosslinked latex gloves without containing any crosslinking agents or other rubber additives. Each sample was weighed before being cut into approximately 1 x 1 cm pieces with a pair of scissors. Sample portions of about 15 g were placed in soxhlet cellulose cartridges and the extraction was carried out by using 200 mL acetone (HPLC grade). The solvent was refluxed for 24 hours under inert conditions. The solution was filtered through a microfilter with PTFE-membrane (0.45 [micro]m pore size) and the extracts were concentrated on a rotary evaporator (40[degrees]C, 450 mbar). The extract was vacuum dried at 40[degrees]C to constant mass, weighed and stored at 8[degrees]C for eventual analysis.
HPLC-MS determination of photoinitiator levels
Chromatography was carried out on the Thermo Electron LCQ Advantage MAX system equipped with vacuum degasser, quaternary pump, auto sampler (20 [micro]L sample loop) and UV-vis diode array detector (all from Thermo Electron, Waltham, MA). MS detection was performed on the Thermo Electron LCQ Advantage Max LC-MS/MS Ion Trap mass spectrometer equipped with an ESI (electrospray ionization) source. The separation column was a Hypersil Gold C 18 (150 x 2.1 mm ID, 3 [micro]m particle size) obtained from Thermo Electron.
A 10 [micro]L aliquot of each sample was injected into the HPLC-MS system. The mobile phase consisted of a gradient of water and acetonitrile. After 11 minutes of isocratic elution at 50% acetonitrile (elution of Lucirin TPO L), the gradient was run to 100% acetonitrile within one minute and kept at this level for 13 minutes to ensure complete elution ofresidues of the soxhlet extract. The column was maintained at 30[degrees]C and the flow rate was set to 200 [micro]L/min.
ESI detection was carried out in positive mode. No split was applied and the total flow of 200 [micro]L/minute was introduced into the MS interface. Nitrogen was used as nebulizer gas and as drying gas. The spray voltage was set to 6,500 V, the capillary voltage to 5 V, and the capillary temperature to 200[degrees]C. The tube lens offset was set to 40 V.
All analytes were dissolved in acetonitrile (gradient grade) giving 10 mg/100 mL stock solutions, kept in the dark at room temperature and filtered through a 0.2 [micro]m PTFE filter prior to injection.
Calibration of the UV-vis detector was performed with varying injected masses of photoinitiator in the range of 0.1 and 1.0 [micro]g. A strictly linear relationship was obtained between injected mass and the corresponding peak area with a coefficient of regression of [r.sup.2]=0.99975. Identification of photoinitiator in the soxhlet extract could be ensured by the aid of its MS signal of m/z = 317.
Elementary analysis" determination of TriThiol levels
The total sulfur content of the glove extracts was determined by C/H/N/S elementary analysis using an EA 1108 CHNS-O elemental analyzer by Carlo Erba Instruments. The determination limit for sulfur with sample amounts of 2-3 mg was about 0.05 wt.-% with an uncertainty below 0.02 wt.-%. The measurements were carried out at the Micro Analytical Laboratory, University of Vienna.
Results and discussion
During the skin irritation study, no symptoms of systemic toxicity in the animals were observed and no mortality occurred. The skin examination of the animals revealed that all areas treated with the test material and the negative control and all control areas were normal before the application and at each observation time. No erythema, eschar or oedema formation was observed. The calculated Primary Irritation Index was 0.0, classifying the UV crosslinked glove as non-irritant to skin.
The results of the Nine Induction Dose Buehler Test were decisive for the grading of the potential of sensitization. In this study, the test material treated areas of all animals of the negative control group and the test material group were normal. No test animal was regarded as irritated or sensitized. According to the results of the skin sensitization test, UV pre-vulcanized surgical gloves are considered non-irritant and non-sensitizing under the test conditions of this study.
Regarding the cytotoxicity of UV pre-cured latex, only mild cytotoxic reactions were observed after exposure to the undiluted extract of the UV prevulcanized surgical glove. The score of the control samples and the glove extract is provided in table 1. Herein the results of the positive and negative control demonstrate the sensitivity of the cells. With respect to the cytotoxicity of the glove extract, the visual examinations of the cells under the microscope showed that the cells were only occasionally lysed at a dilution of 1:2, indicating a slight toxicity. In dilutions of 1/4 and above, the cells had normal morphology, were able to re-attach on the tissue culture plate, and the growth could not be distinguished from non-treated cells of the negative control sample.
For the quantitative detection of the residual TriThiol levels, which are represented by the free and oxidized thiol-groups, elementary analysis was used. It has to be considered that NR latex contains proteins with thiol groups and other sulfur moieties itself. Hence, a reference extraction of non-crosslinked NR latex gloves containing no rubber additives was carried out. The total sulfur and TriThiol content of the reference glove extract obtained by elementary analysis was then subtracted from the analyte concentrations of the crosslinked glove extracts. The extractable chemical levels of non crosslinked gloves (containing no crosslinking chemicals) and UV pre-vulcanized gloves are summarized in table 2.
The results of the elementary analyses revealed that the TriThiol residues in UV crosslinked surgical gloves do not exceed 120 [micro]g/g glove. In our study, HPLC-MS was used for the quantitative determination of the photoinitiator levels in the glove extracts. It was found that 60 [micro]g/g glove photoinitiator can be extracted from UV pre-cured NR latex gloves. To get further information of the cleavage products of the photoinitiator, the work is still ongoing.
Due to the high solubility of the photoinitiator and the thiolcrosslinker in acetone, the chemical levels obtained with the soxhlet extraction procedure may be considerable higher compared to the bio available amount in skin contact. In that case, the extraction procedure does not fully reflect the bio available levels of chemical residues, but can give evidence of the free, not covalently bound crosslinking chemicals.
The photochemical pre-vulcanization of natural rubber latex is aiming at the manufacture of medical gloves, especially surgical gloves, without using any conventional sulfur processing agents such as activators or accelerators. Therefore, allergic contact dermatitis associated with accelerators such as dithiocarbamates, thiurams or mercaptobenzothiazole can be avoided.
In terms of extractable chemical residues, it could be shown that the total residual levels (180 [micro]g/g glove) of the crosslinking chemicals in UV cured gloves are far lower compared to the total accelerator contents found in conventional sulfur prevulcanized NR latex gloves (ref. 12).
To assess the biocompatibility, the UV cured surgical gloves were evaluated by irritation studies and skin sensitization tests in compliance with ISO standards. During the studies, no skin reactions involving erythema or oedema formation were observed in the test animals, classifying UV pre-vulcanized surgical gloves as non-irritant. The good skin compatibility is further proven due to the Nine Induction Dose Buehler Test because, according to the results of this study and to the Directive 2001/59/EC UV cured surgical gloves need not be labeled with "R43 may cause sensitization by skin contact." In addition, the cytotoxic tests revealed that UV pre-cured NR latex gloves exhibit a low cytotoxic potential. UV cured surgical gloves cannot be considered biocompatible based only on in vitro cells, but the low potential of cytotoxicity together with the good skin compatibility and low chemical residues provides evidence of considerable advantages against conventional sulfur cured medical devices.
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by Sandra Schlogl, (firstname.lastname@example.org) Polymer Competence Center Leoben; Armin Temel, Polymer Competence Center Leoben and Graz University of Technology; Raimund Schaller and Armin Holzner, Semperit Technische Produkte; and Wolfgang Kern, University of Leoben, Austria
Table 1--evaluation of the cytotoxicity of UV crosslinked surgical gloves (pre-cured with 1.0 phr photoinitiator and 1.0 phr TriThiol) Dilution in complete Score Score Sample cell medium sample 1 sample 2 Negative control Original extract 0 0 Media control Original extract 0 0 Positive control Original extract 4 4 Original extract 2 2 UV cured 1:02 1 1 surgical glove 1:04 0 0 1:08 0 0 1:16 0 0 Score Average Sample sample 3 Negative control 0 0 Media control 0 0 Positive control 4 4 2 2 UV cured 0 1 surgical glove 0 0 0 0 0 0 Table 2--quantification of extractable rubber chemicals by elementary analysis and HPLC-MS spectroscopy TriThiol Photoinitiator ([micro]g glove) ([micro]g glove) Glove type Elementary analysis HPLC-MS Non-crosslinked glove 0.06 -- containing no crosslinking agents UV pre-vulcanized glove 120 60 crosslinked with 1.0 phr crosslinking agents
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|Author:||Schlogl, Sandra; Temel, Armin; Schaller, Raimund; Holzner, Armin; Kern, Wolfgang|
|Date:||Nov 1, 2010|
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