Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Safety assessment of food-contact paper and board using a battery of short-term toxicity tests: European union BIOSAFEPAPER project.

An European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing.

Characterization of the molar masses of hemicelluloses from wood and pulps employing size exclusion chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The molar mass parameters for arabino-4-O-methylglucuronoxylans, arabinohexenuronoxylans, 4-O-methylglucuronoxylans, hexenuronoxylans, and galactoglucomannans extracted from wood and pulps have been determined. To characterize different types of hemicelluloses, delignified wood (spruce, pine, larch, aspen, and birch) and chemical pulps (unbleached and totally chlorine-free bleached) were extracted with dimethyl sulfoxide (DMSO) or alkaline aqueous solutions. Size exclusion chromatography (SEC) with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) were employed to characterize the molar masses. The hemicellulose extracts were separated by SEC into fractions each containing components with a narrow range of molar masses and the average molar mass of each fraction subsequently determined by MALDI-MS. The molar mass parameters for the hemicelluloses were then calculated on the basis of the SEC distribution curves and MALDI-MS spectra. As expected, in most cases the hemicelluloses extracted from wood (holocellulose) exhibited higher molar masses than did the corresponding hemicelluloses from chemical pulps. The molar mass parameters for hemicelluloses isolated from pulps derived from cooking samples of the same batch of softwood chips decreased in the following order: ASAM pulp > MSSAQ pulp > kraft pulp. The lowest molar masses were demonstrated by the glucuronoxylans extracted from pulps obtained by cooking with acidic sulfite. The xylans from bleached kraft pulp were characterized by molar masses that were only slightly lower than those of the corresponding xylans from unbleached pulp. The xylans extracted into DMSO exhibited somewhat lower molar masses than did the corresponding xylans extracted into alkaline aqueous solutions. In all cases the range of molar masses demonstrated by the hemicelluloses investigated was found to be rather narrow, i.e., the polydispersity index Mw/Mn was found to be approximately 1.1-1.4.

Distribution of uronic acids in xylans from various species of soft- and hardwood as determined by MALDI mass spectrometry.

The distribution of 4-O-methylglucuronic acid residues along the polysaccharide chains of xylans isolated from birch, aspen, spruce, pine, and larch was studied here employing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the oligosaccharide mixtures obtained by partial acid hydrolysis. The hydrolyzates thus obtained were analyzed by MALDI-MS or by capillary electrophoresis as well as by size exclusion chromatography in combination with MALDI-MS. In the case of all softwood xylans examined, the major portion of the 4-O-methylglucuronic acid residues were distributed regularly on every seventh or eighth xylose residue, while a minor portion of these residues were attached to adjacent xylose residues in the beta-(1-->4)-D-xylopolysaccharide chains. In contrast, the 4-O-methylglucuronic acid residues in the hardwood xylans examined were found to be distributed irregularly within the xylan. Further support for these experimental findings with the xylans was obtained by simulation of the distribution patterns of the molecular masses of oligosaccharides arising from polysaccharides with the postulated distribution of uronic acid residues.

Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a nafion-coated probe.

Spectra of highly acidic oligosaccharides obtained by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be greatly enhanced in quality by coating the MALDI probe surface with a film consisting of a perfluorosulfonated ionomer (Nafion) prior to the addition of the sample-matrix mixture. For comparison, three mixtures containing highly acidic oligo- and polysaccharides derived from partial acidic hydrolysis of alginate, pectin, or carboxymethyl cellulose (CMC) were analyzed by employing probes with an uncoated gold surface or a surface coated with a Nafion or nitrocellulose film. The negative ion-mode MALDI spectra of the oligouronates (oligomers containing mannuronic/guluronic and galacturonic acid residues) obtained using uncoated or nitrocellulose-coated probes consisted of a series of broad, multiple peaks. These multiple peaks were assigned to the molecular ions of the nondissociated [M - H]- and partially sodiated [MnNa - H]-, where n = 1, 2, or 3, oligomers. In contrast, the corresponding spectra obtained with Nafion-coated probes contained only a single series of sharp peaks originating from the molecular ions ([M - H]-) of nondissociated oligomers exhibiting chain lengths of as many as approximately 15 uronic acid residues. The Nafion coating was apparently capable of removing the sodium counterions remaining in the deposit of the sample-matrix mixture on the probe, thereby greatly enhancing the signal-to-noise ratios of the peaks in the spectra. In a similar manner, higher quality spectra could also be obtained by using Nafion-coated probes for analysis of the oligouronates and CMC oligomers by positive ion-mode MALDI-MS.

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy.

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.

Analysis of carbohydrates in wood and pulps employing enzymatic hydrolysis and subsequent capillary zone electrophoresis.

An efficient method for determining the carbohydrate composition of extractive-free delignified wood and pulp is described here. The polysaccharides in the sample are first hydrolyzed using a mixture of commercially available preparations of cellulase and hemicellulase. The reducing saccharides in the hydrolysate thus obtained are subsequently derivatized with 4-aminobenzoic acid ethyl ester and thereafter quantitated by capillary zone electrophoresis (CZE) in an alkaline borate buffer with monitoring of the absorption at 306 nm. All reducing sugars (i.e., neutral monosaccharides and uronic acids) which occur as structural elements in the polysaccharides of wood and pulp can be quantitated in a single such analytical run, which can also determine the contents of 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (HexA) residues present in pulps obtained from alkaline processes. CZE analyses were performed using linear regression of standard curves over a concentration range spanning approximately three orders of magnitude. Carbohydrate constituents constituting approximately 0.1% of the dry mass of the sample could be quantitated. The overall precision of this analytical procedure--involving enzymatic hydrolysis, derivatization and CZE--was good (RSD=2.2-7.5%), especially considering the heterogeneity of the wood and pulp samples. The total yield of carbohydrates (93-97%) obtained employing the procedure developed here was consistently higher than that obtained upon applying the traditional procedure for carbohydrate analysis (85-93%) (involving acid hydrolysis and gas chromatographic analysis) to the same pulps. The trisaccharide HexA-xylobiose was the only HexA-containing saccharide detected using the conditions for enzymatic hydrolysis developed here (i.e., 30 h incubation at pH 4 and 40 degrees C); whereas mixtures of HexA-xylobiose and HexA-xylotriose were obtained when the incubation was performed at pH 5 or 6.

Oligosaccharides obtained by enzymatic hydrolysis of birch kraft pulp xylan: analysis by capillary zone electrophoresis and mass spectrometry.

Neutral and acidic oligosaccharides were obtained from an unbleached birch kraft pulp by treatment with a Trichoderma reesei endoxylanase pI 9 and subsequently characterized using capillary zone electrophoresis (CZE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The borate complexes of unsaturated acidic oligosaccharides having a 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (4 delta UA) residue linked to a beta-D-(1-->4)-xylooligosaccharide backbone were separated by CZE and detected by their UV absorption at 232 nm without prior derivatization. Pre-column derivatization with the chromophore 6-aminoquinoline (6-AQ) followed by CZE in alkaline borate buffer using detection based on absorption at 245 nm was used in the case of neutral xylosaccharides. Furthermore, MALDI-TOF-MS was employed to determine the molecular masses of both unsaturated and saturated acidic oligosaccharides. The acidic oligosaccharides released upon endoxylanase treatment of the birch kraft pulp were a (4 delta UA)-beta-D-xylotetraose, a (4 delta UA)-beta-D-xylopentaose, a (4-O-methyl-alpha-D-glucurono)-beta-D-xylotetraose, and a (4-O-methyl-alpha-D-glucurono)-beta-D-xylopentaose. Analysis after enzymatic hydrolysis with beta-xylosidase and alpha-glucuronidase from Trichoderma reesei strongly indicated that the uronic acid residue in these acidic oligosaccharides was linked to the D-xylose unit adjacent to the non-reducing D-xylose unit. The neutral xylosaccharides obtained after endoxylanase treatment of the pulp sample were D-xylose, beta-(1-4)-D-xylobiose and beta-(1-4)-D-xylotriose.

Efficient capillary zone electrophoretic separation of wood-derived neutral and acidic mono- and oligosaccharides.

Neutral and acidic monosaccharides, commonly present as structural units in wood-derived hemicelluloses, were derivatized by reductive amination using 6-aminoquinoline (6-AQ) and subsequently separated as their borate complexes by capillary zone electrophoresis. By using a quite concentrated (420 mmol 1(-1) alkaline borate buffer, a fused-silica capillary column with a small inner diameter (30 microns nominal I.D.) and a constant power of 1200 mW (corresponding to an applied voltage of approximately 21 kV), optimal separation was achieved. Under these conditions, the monosaccharides investigated were separated with a resolution, Rs, of 1.0-1.2 or greater. On-column UV detection at 245 nm was found to provide highly sensitive detection of the 6-AQ-derivatized monosaccharides. The minimum detectable concentrations were on the order of 10(-6) mol 1(-1) (corresponding to an estimated limit of detection of a few fmol). The linear calibration range of the method, including the 6-AQ derivatization step, was found to be about two orders of magnitude. Several neutral beta (1-4)-D-xylooligomers and acidic oligosaccharides containing 4-O-methyl-D-glucuronic acid units, which are common structural elements in hemicelluloses such as birch and spruce xylan, were also efficiently separated as 6-AQ derivatives, using the same buffer system. Finally, the usefulness of this analytical method has been demonstrated using a spruce wood xylan sample subjected to chemical and enzymatic hydrolysis.

Tobacco smoke chemistry. 2. Alkyl and alkenyl substituted guaiacols found in cigarette smoke condensate.

A series of alkyl and alkenyl substituted guaiacols, which comprise a group of biologically and organoleptically active compounds, have been synthesized. Mass spectra and GC retention times for these have been recorded and compared with those obtained for constituents of a weakly acidic fraction of smoke condensate derived from American blend type cigarettes. On the basis of these results, 25 guaiacols have been identified, 18 of which have not been detected in tobacco smoke condensate previously.

Synthesis of three 3-C-hydroxymethylpentoses with the D-ribo-, D-xylo- and L-lyxo-configurations. Identification of the latter with a monosaccharide isolated from phase I Coxiella burnetii lipopolysaccharide.

Three 3-C-hydroxymethylpentoses with the D-ribo-, D-xylo and L-lyxo-configurations, were synthesised via nitromethane addition for the first two and 1,3-dithiane addition for the last one, to appropriate 3-ulose derivatives. 3-C-Hydroxy-methyl-L-lyxose is identical with a monosaccharide component previously isolated from hydrolysates of the phase I Coxiella burnetii lipopolysaccharide.