A systematic review and meta-analysis of patient data from the West Africa (2013-16) Ebola virus disease epidemic.

BACKGROUND: Over 28 000 individuals were infected with Ebola virus during the West Africa (2013-2016) epidemic, yet there has been criticism of the lack of robust clinical descriptions of Ebola virus disease (EVD) illness from that outbreak. OBJECTIVES: To perform a meta-analysis of published data from the epidemic to describe the clinical presentation, evolution of disease, and predictors of mortality in individuals with EVD. To assess the quality and utility of published data for clinical and public health decision-making. DATA SOURCES: Primary articles available in PubMed and published between January 2014 and May 2017. ELIGIBILITY: Studies that sequentially enrolled individuals hospitalized for EVD and that reported acute clinical outcomes. METHODS: We performed meta-analyses using random-effect models and assessed heterogeneity using the I2 method. We assessed data representativeness by comparing meta-analysis estimates with WHO aggregate data. We examined data utility by examining the availability and compatibility of data sets. RESULTS: In all, 3653 articles were screened and 34 articles were included, representing 16 independent cohorts of patients (18 overlapping cohorts) and at least 6168 individuals. The pooled estimate for case fatality rate was 51% (95% CI 46%-56%). However, pooling of estimates for clinical presentation, progression, and predictors of mortality in individuals with EVD were hampered by significant heterogeneity, and inadequate data on clinical progression. Our assessment of data quality found that heterogeneity was largely unexplained, and data availability and compatibility were poor. CONCLUSIONS: We have quantified a missed opportunity to generate reliable estimates of the clinical manifestations of EVD during the West Africa epidemic. Clinical data standards and data capture platforms are urgently needed.

Model studies of migration from paper and board into fruit and vegetables and into Tenax as a food simulant.

Four samples of paper and board (P/B) of a type used for packaging dry foods were subjected to migration tests using mushrooms, apples, potatoes and bananas, and using the polymeric powder Tenax as a food simulant. The P/B samples contained only low levels of diisopropylnaphthalene (DiPN) and diisobutyl phthalate (DiBP) and so the experiments were conducted after impregnating the P/B with added model substances. These were o-xylene, acetophenone, dodecane, benzophenone, DiPN and DiBP. Migration levels depended strongly on the nature of the substance and on the nature of the food and much less on the characteristics of the P/B, except insofar as they affected the contact area - flexible papers giving more extensive contact with the food than thick rigid board. Migration into Tenax was at least a factor of 10 higher than migration into the fresh fruit and vegetables. The food samples were placed in contact with the P/B and then overwrapped loosely with aluminium foil and so this correction factor will tend to be conservative compared with a more open storage of the packed foods. Washing, peeling or cooking the fruits and vegetables after contact with the P/B had a surprisingly small effect on contaminant levels in general, and no one processing step was effective in giving a significant reduction of all the types of chemicals studied. This was because either they had penetrated into the food (so resisting peeling), or were not freely water-soluble (so resisting washing) or were not particularly volatile (so resisting loss by evaporation during cooking).

Estimates of dietary exposure to bisphenol A (BPA) from light metal packaging using food consumption and packaging usage data: a refined deterministic approach and a fully probabilistic (FACET) approach.

The FACET tool is a probabilistic model to estimate exposure to chemicals in foodstuffs, originating from flavours, additives and food contact materials. This paper demonstrates the use of the FACET tool to estimate exposure to BPA (bisphenol A) from light metal packaging. For exposure to migrants from food packaging, FACET uses industry-supplied data on the occurrence of substances in the packaging, their concentrations and construction of the packaging, which were combined with data from a market research organisation and food consumption data supplied by national database managers. To illustrate the principles, UK packaging data were used together with consumption data from the UK National Diet and Nutrition Survey (NDNS) dietary survey for 19-64 year olds for a refined deterministic verification. The UK data were chosen mainly because the consumption surveys are detailed, data for UK packaging at a detailed level were available and, arguably, the UK population is composed of high consumers of packaged foodstuffs. Exposures were run for each food category that could give rise to BPA from light metal packaging. Consumer loyalty to a particular type of packaging, commonly referred to as packaging loyalty, was set. The BPA extraction levels used for the 15 types of coating chemistries that could release BPA were in the range of 0.00005-0.012 mg dm(-2). The estimates of exposure to BPA using FACET for the total diet were 0.0098 (mean) and 0.0466 (97.5th percentile) mg/person/day, corresponding to 0.00013 (mean) and 0.00059 (97.5th percentile) mg kg(-1) body weight day(-1) for consumers of foods packed in light metal packaging. This is well below the current EFSA (and other recognised bodies) TDI of 0.05 mg kg(-1) body weight day(-1). These probabilistic estimates were compared with estimates using a refined deterministic approach drawing on the same input data. The results from FACET for the mean, 95th and 97.5th percentile exposures to BPA lay between the lowest and the highest estimates from the refined deterministic calculations. Since this should be the case, for a fully probabilistic compared with a deterministic approach, it is concluded that the FACET tool has been verified in this example. A recent EFSA draft opinion on exposure to BPA from different sources showed that canned foods were a major contributor and compared results from various models, including those from FACET. The results from FACET were overall conservative.

Safety evaluation of mechanical recycling processes used to produce polyethylene terephthalate (PET) intended for food contact applications.

The development of a scheme for the safety evaluation of mechanical recycling processes for polyethylene terephthalate (PET) is described. The starting point is the adoption of a threshold of toxicological concern such that migration from the recycled PET should not give rise to a dietary exposure exceeding 0.0025 µg kg(-1) bw day(-1), the exposure threshold value for chemicals with structural alerts raising concern for potential genotoxicity, below which the risk to human health would be negligible. It is practically impossible to test every batch of incoming recovered PET and every production batch of recycled PET for all the different chemical contaminants that could theoretically arise. Consequently, the principle of the safety evaluation is to measure the cleaning efficiency of a recycling process by using a challenge test with surrogate contaminants. This cleaning efficiency is then applied to reduce a reference contamination level for post-consumer PET, conservatively set at 3 mg kg(-1) PET for a contaminant resulting from possible misuse by consumers. The resulting residual concentration of each contaminant in recycled PET is used in conservative migration models to calculate migration levels, which are then used along with food consumption data to give estimates of potential dietary exposure. The default scenario, when the recycled PET is intended for general use, is that of an infant weighing 5 kg and consuming every day powdered infant formula reconstituted with 0.75 L of water coming from water bottles manufactured with 100% recycled PET. According to this scenario, it can be derived that the highest concentration of a substance in water that would ensure that the dietary exposure of 0.0025 µg kg(-1) bw day(-1) is not exceeded, is 0.017 µg kg(-1) food. The maximum residual content that would comply with this migration limit depends on molecular weight and is in the range 0.09-0.32 mg kg(-1) PET for the typical surrogate contaminants.

Analytical screening studies on irradiated food packaging.

Foods may be irradiated in their final packaging and this process may affect the composition of the packaging and in turn affect the migration of substances into food. Headspace and liquid injection GC-MS and HPLC with time-of-flight MS have been used to identify and estimate levels of radiolytic products in irradiated finished plastic packaging materials. Fifteen retail packaging materials were studied. Investigations were carried out into the effect of different irradiation types (gamma and electron beam), irradiation doses (1, 3, 7 and 10 kGy) and dose rates (5 kGy s(-1) for electron beam and 0.4 and 1.85 kGy h(-1) for gamma) on the radiolytic products. Any differences seen in comparing the two ionising radiation types were attributed largely to the very different dose rates; for electron beam a 10 kGy dose was delivered in just 2 s whereas using gamma it took 5.4 h. Differences were also seen when comparing the same samples irradiated at different doses. Some substances were not affected by irradiation, others decreased in concentration and others were formed upon increasing doses of irradiation. These results confirm that irradiation-induced changes do occur in substances with the potential to migrate and that the safety of the finished packaging material following irradiation should be assessed.

Development of a new modelling tool (FACET) to assess exposure to chemical migrants from food packaging.

The approach used to obtain European Union-wide data on the usage and concentration of substances in different food packaging materials is described. Statistics were collected on pack sizes and market shares for the different materials used to package different food groups. The packaging materials covered were plastics (both flexible and rigid), metal containers, light metal packaging, paper and board, as well as the adhesives and inks used on them. An explanation as to how these data are linked in various ways in the FACET exposure modelling tool is given as well as an overview of the software along with examples of the intermediate tables of data. The example of bisphenol A (BPA), used in resins that may be incorporated into some coatings for canned foodstuffs, is used to illustrate how the data in FACET are combined to produce concentration distributions. Such concentration distributions are then linked probabilistically to the amounts of each food item consumed, as recorded in national food consumption survey diaries, in order to estimate exposure to packaging migrants. Estimates of exposure are at the level of the individual consumer and thus can be expressed for various percentiles of different populations and subpopulations covered by the national dietary surveys.

Methods of analysis for 2-dodecylcyclobutanone and studies to support its role as a unique marker of food irradiation.

Methods of analysis for 2-dodecylcyclobutanone (2-DCB) using gas chromatography with mass spectrometric detection (GC-MS), liquid chromatography with time-of-flight mass spectrometric detection (LC-TOF-MS) and LC with tandem MS (MS/MS) detection have been developed and optimised for maximum sensitivity to allow very low irradiation doses to be detected. The LC-MS/MS method, following derivatisation with 2,4-dinitrophenylhydrazine, was found to be the most sensitive technique and was used to determine the amount of 2-DCB formed from the model compounds palmitic acid, glyceryl tripalmitate and 1,3-dipalmitoyl-2-oleoylglycerol irradiated over a range of doses by two different irradiation sources (gamma and electron beam). The model compounds were also treated with a number of non-irradiation based processing techniques including heating in the presence and absence of oxygen, light, and redox active metal salts, in a conventional oven, microwave oven and pressure cooker. No 2-DCB was detected in any of the processed non-irradiated model compounds, reaffirming the hypothesis that 2-DCB is a unique radiolytic product that can be used as a marker of irradiation in foodstuffs.

Printing ink compounds in foods: UK survey results.

Three hundred and fifty foodstuffs packaged in printed paper/board were purchased from UK retail outlets. Solvent extracts of all foods and associated quality assurance samples were analysed by gas chromatography-mass spectrometry (GC-MS) to determine the presence and concentrations of 20 printing ink compounds: benzophenone, 4-methylbenzophenone, 2-methylbenzophenone, 3-methylbenzophenone, 4-hydroxybenzophenone, 2-hydroxybenzophenone, 4-phenylbenzophenone, methyl-2-benzoylbenzoate, 1-hydroxycyclohexyl phenyl ketone, 2-isopropylthioxanthone, 4-isopropylthioxanthone, 2,4-diethyl-9H-thioxanthen-9-one, 2,2-dimethoxy-2-phenylacetophenone, 2-methyl-4'-(methylthio)-2-morpholinopropiophenone, 4-(4-methylphenylthio)benzophenone, ethyl-4-dimethylaminobenzoate, 2-ethylhexyl-4-(dimethylamino)benzoate, N-ethyl-p-toluene-sulphonamide, triphenyl phosphate and di-(2-ethylhexyl) fumarate. The presence of one or more of the compounds benzophenone, 4-phenylbenzophenone, methyl-2-benzoylbenzoate, 1-hydroxycyclohexyl phenyl ketone, 2,2-dimethoxy-2-phenylacetophenone, 4-(4-methylphenylthio)benzophenone, ethyl-4-dimethylaminobenzoate, 2-ethylhexyl-4-dimethylaminobenzoate and triphenyl phosphate was confirmed in some food samples. Analysis of the associated packaging material was also carried out to confirm whether or not it was likely that the occurrence of these compounds in the foods was due to migration from the printed paper/board packaging. With the exception of triphenyl phosphate, detected in one foodstuff, all the packaging material contained the substance(s) found in the food.

Exploring the sensitivity of the zone of inhibition test for leachable biocides from paper and board food contact materials, and improvements thereof.

The zone of inhibition method to test the release of biocides from paper and board food contact materials was evaluated. The method tests the paper by placing a small specimen directly onto culture plates of Bacillus subtilis and Aspergillus niger. The principle is that any extractable biocide will diffuse from the paper into the surrounding nutrient medium and so inhibit growth of the microorganism in the vicinity. The test was found to have insufficient sensitivity for assuring food safety, where detection limits for migration at or below the mg l(-1) (parts per million) level are needed. Also, the test does not mimic the actual or foreseeable conditions of use since most paper/board materials are not intended for direct contact with an aqueous medium for up to 3 days at 30°C (B. subtilis) or 25°C (A. niger), which are the incubation conditions used. The sensitivity of the test was increased approximately 100-fold by preparing a concentrated extract of the paper to be tested and applying this extract to the assay via a blank paper carrier. This was done using methanol as a good solvent for most biocides, as a proof of principle. Other solvents or food simulants could be used to mimic the conditions of use intended for the particular paper/board samples under examination, e.g. contact with dry, fatty, aqueous or acidic foods, hot or cold. Twenty-four plain (unconverted) paper and board samples and 100 food packaging samples were evaluated using the modified procedure. The results revealed that the method has been developed to the stage where background cytotoxic action of normal paper constituents gives a weak response. Unlike the original method, therefore, the modified method with its improved sensitivity and the facility to link with the intended food contact conditions may be considered a suitable bioassay screening test to complement chemical analysis of paper/board for composition and migration.

Migration of melamine from can coatings cross-linked with melamine-based resins, into food simulants and foods.

Resins based on melamine-formaldehyde and related analogues such as methylolated melamine are used to cross-link coatings used inside food cans and on the metal closures of glass jars. Thirteen commercially coated cans and closures representing 80% of the European market were tested using simulants under realistic industrial heat-processing conditions for canned and jarred foods. The food simulants and the retort conditions used were 3% acetic acid for 1 h at 100 °C and 10% ethanol for 1 h at 130 °C. The highest migration level seen for melamine into simulant was 332 µg kg⁻¹. There was no detectable migration of the melamine analogues cyanuric acid (<1 µg kg⁻¹) or ammelide (<5 µg kg⁻¹) from any sample. Twelve of the thirteen samples released no detectable ammeline (<5 µg kg⁻¹) but the coating giving the highest release of melamine did also release ammeline at 8 µg kg⁻¹ with the higher of the two process temperatures used. Migration experiments into food simulant and foods themselves were then conducted using two experimental coatings made using amino-based cross-linking resins. Coated metal panels were exposed to the food simulant 10% (v/v) aqueous ethanol and to three foodstuffs under a range of time and temperature conditions both in the laboratory and in a commercial food canning facility using proprietary time and temperature conditions. The highest migration into a food was 152 µg kg⁻¹ from the first coating processed for a long time at a moderate sterilisation temperature. The highest migration into simulant was also from this coating at 220 µg kg⁻¹ when processed at 134 °C for 60 min, dropping to 190 µg k⁻¹ when processed at 123 °C for 70 min. Migration from the second coating was quite uniformly two to three times lower under all tests. These migration results were significantly higher than the levels of melamine extractable using 95% ethanol at room temperature. The experiments show that commercial canning and retorting can be mimicked in an acceptable way using laboratory tests with an autoclave or a simple pressure cooker. The results overall show there is hydrolytic degradation of the melamine cross-linked resins to release additional melamine. There is a strong influence of the temperature of heat treatment applied with foods or simulants but only a minor influence of time of heating and only a minor influence, if any, of food/simulant acidity.

Comparison of the migration of melamine from melamine-formaldehyde plastics ('melaware') into various food simulants and foods themselves.

A variety of melaware articles were tested for the migration of melamine into the food simulant 3% w/v acetic acid as a benchmark, and into other food simulants, beverages and foods for comparison. The results indicate that the acidity of the food simulant plays a role in promoting migration, but not by as much as might have been anticipated, since 3% acetic acid gave migration values about double those obtained using water under the same time and temperature test conditions. In contrast, migration into the fatty food simulant olive oil was not detectable and at least 20-fold lower than with the aqueous food simulants. This was expected given the solubility properties of melamine and the characteristics of the melaware plastic. Migration levels into hot acidic beverages (apple juice, tomato juice, red-fruit tea and black coffee) were rather similar to the acetic acid simulant when the same time and temperature test conditions are used, e.g. 2 h at 70°C. However, migration levels into foods that were placed hot into melaware articles and then allowed to cool on standing were much lower (6-14 times lower) than if pre-heated food was placed into the articles and then maintained (artificially) at that high temperature in the same way that a controlled time-temperature test using simulants would be conducted. This very strong influence of time and especially temperature was manifest in the effects seen of microwave heating of food or beverage in the melaware articles. Here, despite the short duration of hot contact, migration levels were similar to simulants used for longer periods, e.g. 70°C for 2 h. This is rationalized in terms of the peak temperature achieved on microwave heating, which may exceed 70°C, counterbalancing the shorter time period held hot. There was also evidence that when using melaware utensils in boiling liquids, as for stovetop use of spatulas, the boiling action of circulating food/simulant can have an additional effect in promoting surface erosion, increasing the plastic decomposition and so elevating the melamine release.

Determination of polyadipates migrating from lid gaskets of glass jars. Hydrolysis to adipic acid and measurement by LC-MS/MS.

Polyadipate plasticizers can be present in the polyvinylchloride (PVC) gaskets used to seal the lids of glass jars. As the gaskets can come into direct contact with the foodstuffs inside the jar, the potential exists for polyadipate migration into the food. The procedure and performance characteristics of a test method for the analysis of polyadipates in food simulants (3% aqueous acetic acid and 10% aqueous ethanol) and the volatile test media used in substitute fat tests (isooctane and 95% aqueous ethanol) are described. The PVC gaskets were exposed to the food simulants or their substitutes under standard test conditions. Studies were initially carried out using direct measurement of the polyadipate oligomers by liquid chromatography with time-of-flight mass spectrometric detection (LC-TOF-MS) but this was not practical due to the number of peaks detected. Instead, the migrating polyadipates were hydrolysed to adipic acid and measured by liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The amount of polyadipate that this measurement of adipic acid represents was then calculated. Method performance was assessed by analysis of gaskets from two types of jar lids by single-laboratory validation. Linearity, sensitivity, repeatability, intermediate reproducibility and recovery were determined to be suitable for checking compliance with the 30 mg/kg specific migration limits for polyesters of 1,2-propane diol and/or 1,3- and/or 1,4-butanediol and/or polypropylene-glycol with adipic acid, which may be end-capped with acetic acid or fatty acids C(12)-C(18) or n-octanol and/or n-decanol. The method was found to be much quicker than previous methods involving extraction, clean-up, hydrolysis, esterification, derivatisation and GC measurement, consequently saving time and money.

Migration of formaldehyde from melamine-ware: UK 2008 survey results.

Fifty melamine-ware articles were tested for the migration of formaldehyde - with hexamethylenetetramine (HMTA) expressed as formaldehyde - to see whether the total specific migration limit (SML(T)) was being observed. The SML(T), given in European Commission Directive 2002/72/EC as amended, is 15 mg kg(-1). Fourier transform-infrared (FT-IR) spectroscopy was carried out on the articles to confirm the plastic type. Articles were exposed to the food simulant 3% (w/v) aqueous acetic acid under conditions representing their worst foreseeable use. Formaldehyde and HMTA in food simulants were determined by a spectrophotometric derivatization procedure. Positive samples were confirmed by a second spectrophotometric procedure using an alternative derivatization agent. As all products purchased were intended for repeat use, three sequential exposures to the simulant were carried out. Formaldehyde was detected in the simulant exposed to 43 samples. Most of the levels found were well below the limits set in law such that 84% of the samples tested were compliant. However, eight samples had formaldehyde levels that were clearly above the legal maximum at six to 65 times the SML(T).

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.

A sensitive method for the determination of chlorine-36 in foods using accelerator mass spectrometry.

A method using accelerator mass spectrometry (AMS) has been developed to offer a more sensitive alternative to scintillation techniques for the determination of chlorine-36 ((36)Cl) in foods. The main problem in method development was the potential interference of the sulfur-36 ((36)S) isobar. This was overcome by reducing the sulfur level of acid digests of food by precipitation of chloride as silver chloride, then purification by washing, dissolution and reprecipitation to present silver chloride as the AMS target. The limit of detection was around 0.1 Bq kg(-1) and the limit of quantitation was around 0.2 Bq kg(-1). The AMS method was only semi-quantitative at the lowest levels of interest. To test the method a few samples of milk (five) and blackberries (three) collected from near two nuclear power stations as potential sources of contamination were analysed. Blackberries spiked at 0.2 Bq kg(-1) and milk spiked at 0.1 Bq kg(-1) could be distinguished from method blanks. There was no (36)Cl detectable in the unspiked samples.

New hierarchical classification of food items for the assessment of exposure to packaging migrants: use of hub codes for different food groups.

This paper describes development work undertaken to expand the capabilities of an existing two-dimensional probabilistic modelling approach for assessing dietary exposure to chemicals migrating out of food contact materials. A new three-level hub-coding system has been devised for coding different food groups with regards to their consumption by individuals. The hub codes can be used at three different levels representing a high, medium and low level of aggregation of individual food items. The hub codes were developed because they have a greater relevance to packaging migration than coding used (largely and historically) for nutritional purposes. Also, the hub codes will assist pan-europeanization of the exposure model in the future, when up to 27 or more different food coding systems from 27 European Union Member States will have to be assimilated into the modelling approach. The applicability of the model with the new coding system has been tested by incorporating newly released 2001 UK consumption data. The example used was exposure to a hypothetical migrant from coated metal packaging for foodstuffs. When working at the three hierarchical levels, it was found that the tiered approach gave conservative estimates at the cruder level of refinement and a more realistic assessment was obtained as the refinement progressed. The work overall revealed that changes in eating habits over time had a relatively small impact on estimates of exposure. More important impacts are changes over time in packaging usage, packaging composition and migration levels. For countries like the UK, which has sophisticated food consumption data, it is uncertainties in these other areas that need to be addressed by new data collection.

Use of overall migration methodology to test for food-contact substances with specific migration limits.

This work investigated if overall migration test procedures could also be used to test for the migration of specific substances from plastics. The overall migration test procedure used was the evaporative gravimetric method used with volatile food simulants. Thirty food-contact substances (additives and monomers) were tested for their chemical stability and volatile loss during the heated evaporation stage of the overall migration procedure. Eighteen of the 30 were determined in an acceptable yield. It is concluded that in the list of approximately 620 European Union substances that have specific migration limits of 5 mg kg(-1) or higher, and based on considerations of stability and volatility, more than half could be amenable to control using overall migration methodology. This is particularly the case for inert plastics with low intrinsic overall migration values of oligomers. This means that based on the overall migration test result found, testing laboratories could decide on a case-by-case basis if known additives and starting substances are covered by the overall migration result and no separate testing would be required for specific migration, with time and resource cost savings.

Development and single-laboratory validation of an HPLC method for the determination of cyclamate sweetener in foodstuffs.

A method for the determination of cyclamate has been developed and single-laboratory validated for a range of foodstuffs including carbonated and fruit-juice drinks, fruit preserves, spreads, and dairy desserts. The method uses the peroxide oxidation of cyclamate to cyclohexylamine followed by derivatization with trinitrobenzenesulfonic acid and analysis by a modified reversed-phase high-performance liquid chromatography-ultraviolet light (HPLC-UV). Cycloheptylamine is used as an internal standard. The limits of detection were in the range 1-20 mg kg(-1) and the analysis was linear up to 1300 mg kg(-1) cyclamic acid in foods and up to 67 mg l(-1) in beverages. Analytical recovery was between 82% and 123%, and results were recovery corrected. Precision was within experimentally predicted levels for all of the matrices tested and Horrat values for the combined standard uncertainty associated with the measurement of cyclamate between 0.4 (water-based drinks) and 1.7 (spreads). The method was used successfully to test three soft drink samples for homogeneity before analytical performance assessment. The method is recommended for use in monitoring compliance and for formal testing by collaborative trial.

Characterization of osteoblastic properties of 7F2 and UMR-106 cultures after acclimation to reduced levels of fetal bovine serum.

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.

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.