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.

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.

Recent European Food Safety Authority toxicological evaluations of major phthalates used in food contact materials.

During the 1980s and 1990s, and at the EU level, the Scientific Committee for Food evaluated a number of phthalates that were being used, or were requested for use, as additives in plastics. At this time, peroxisome proliferation was considered as the pivotal effect on which toxicological evaluation of these chemicals was based. At the end of 1990s, a general consensus has been agreed that rodents are highly sensitive to the phenomenon of peroxisome proliferation and that this particular effect should not be used for human risk assessment.

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.

Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

The threshold of toxicological concern (TTC) has been used for the safety assessment of packaging migrants and flavouring agents that occur in food. The approach compares the estimated oral intake with a TTC value derived from chronic oral toxicity data for structurally-related compounds. Application of the TTC approach to cosmetic ingredients and impurities requires consideration of whether route-dependent differences in first-pass metabolism could affect the applicability of TTC values derived from oral data to the topical route.

Disposition and metabolic profiling of bisphenol F in pregnant and nonpregnant rats.

The distribution of bisphenol F (4,4'-dihydroxydiphenyl-methane, BPF) was studied in female Sprague-Dawley rats. Pregnant and nonpregnant animals were gavaged with a single dose of 7 or 100 mg/kg [3H]BPF and were kept for 96 h in metabolic cages. The excretion of BPF residues occurred mainly in urine (43-54% of the administered dose), which was found to contain at least six different metabolites, and to a lesser extent in feces (15-20% of the administered dose).

Toxicological profile of cereulide, the Bacillus cereus emetic toxin, in functional assays with human, animal and bacterial cells.

Some strains of the endospore-forming bacterium Bacillus cereus produce a heat-stable ionophoric peptide, cereulide, of high human toxicity. We assessed cell toxicity of cereulide by measuring the toxicities of crude extracts of cereulide producing and non-producing strains of B. cereus, and of pure cereulide, using cells of human, animal and bacterial origins.

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.

Evaluation of anti-androgenic activity of di-(2-ethylhexyl)phthalate.

DEHP is a widely used platiciser in the manufacture of PVC-based materials. It is known to disrupt the reproductive tract development in male rats. We have performed the Hershberger assay with DEHP on an immature castrated rat model to check if DEHP antagonise the testosterone propionate androgenic effect on the accessory sex organs development.

Steroid activities comparison of natural and food wrap compounds in human breast cancer cell lines.

In this study, we tested and compared the endocrine disruption activities of compounds in materials used to package foods (bisphenol A, bisphenol F, and bisphenol A diglycidylether BADGE) with natural molecules (genistein, apigenin, kaempferol, and tangeretin) in the human breast cancer cell lines MCF-7 (ER(+)) and MDA-MB453 (AR(+); GR(+)). Octylphenol was also chosen as a xenoestrogen reference. Two compounds had no estrogenic activity: BADGE and tangeretin.

The autoradiographic test for unscheduled DNA synthesis: a sensitive assay for the detection of DNA repair in the HepG2 cell line.

We assessed the DNA-repair capacity of HepG2 cells, which were derived from a human hepatoma, by the unscheduled DNA synthesis assay, using the autoradiography protocol (UDS-AR). We evaluated DNA repair following exposure to direct mutagens (4-nitroquinoline-N-oxide (4-NQO), methyl methanesulfonate (MMS)), to mutagens requiring metabolic activation (benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), N-dimethylnitrosoamine (NDMA)) or to structurally related non-mutagens such as pyrene and 4-acetylaminofluorene (4-AAF). All positive compounds tested induced UDS in HepG2 cells.

Toxic interaction between hydroxyurea and 1-beta-D-arabino-furanosylcytosine on the DNA of a human hepatoma cell line (HEPG2).

Hydroxyurea (HU) and 1-beta-D-arabino-furanosylcytosine (AraC) are two compounds used to inhibit DNA repair in the comet assay and thereby increase its sensitivity. We used RNA synthesis and comet assays to assess the cytotoxic and genotoxic effects of HU and AraC in the HepG2 cells after 1, 5, or 21 h of exposure to concentrations used to inhibit DNA repair. HU was genotoxic between 2 and 10 mM after 1 h of exposure and cytotoxic after 21 h.

Influence of dietary soy isoflavones on the accessory sex organs of the Wistar rat.

We evaluated the effects of three rodent diets differing in soybean meal content on the response of the seminal vesicles, prostate and bulbocavernosus/levator ani (BC/LA) muscle to androgens and anti-androgenic compounds in the Hershberger assay. The diets tested were (1) L5, a semi-synthetic phytoestrogen-free diet, (2) DO4, 8.5% (w/w) vegetable protein and (3) DO3, 22.

Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays.

In the present study, DNA-damage and clastogenic or aneugenic effects of genotoxic compounds were examined in a metabolically competent human cell line (HepG2 cells) using the micronucleus and the comet assays. Compounds with various action mechanisms were tested: direct mutagens such as 4-nitroquinoline-N-oxide (4-NQO) and methyl methanesulfonate (MMS) and indirect mutagens requiring biotransformation to be active such as N-nitrosodimethylamine (NDMA), benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (2-AAF). The compounds were first tested for cytotoxicity by measuring their effects on RNA synthesis inhibition in HepG2 cells.

Effect of cis/trans isomerism of beta-carotene on the ratios of volatile compounds produced during oxidative degradation.

beta-Carotene is, when cleaved, an important source of flavor and aroma compounds in fruits and flowers. Among these aroma compounds, the main degradation products are beta-ionone, 5,6-epoxy-beta-ionone, and dihydroactinidiolide (DHA), which are associated by flavorists and perfumers with fruity, floral, and woody notes. These three species can be produced by degradation of beta-carotene through an attack by enzyme-generated free radicals and a cleavage at the C9-C10 bond.

Methods of in vitro toxicology.

In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment.

Uridine uptake inhibition assay: an automated micromethod for the screening of cytotoxicity.

The uridine uptake inhibition assay is a sensitive microassay for measuring cytotoxicity. This assay is normally performed with Hela S3 cells, which lack metabolic activity. In an earlier study, we adapted the test to HepG2 cells, a human hepatoma cell line that retains many hepatocyte characteristics, such as functional metabolic enzymes.

Estrogenic activity and metabolism of n-butyl benzyl phthalate in vitro: identification of the active molecule(s).

Some phthalates are suspected to disrupt the endocrine system, especially by mimicking estrogens. N-butyl benzyl phthalate (BBP) has estrogenic effects in vitro but not in vivo. The aim of this study was to identify the active molecule(s) (parent compound and/or metabolite(s)) involved in the estrogenic activities of BBP.

Uridine uptake inhibition as a cytotoxicity test for a human hepatoma cell line (HepG2 cells): comparison with the neutral red assay.

This study describes a sensitive microassay for measuring cytotoxicity based on the degree of inhibition of RNA synthesis in HepG2 cells. RNA synthesis is measured by the kinetic uptake of radiolabeled uridine. A large number of compounds were tested in a wide range of concentrations.

Metabolism of n-butyl benzyl phthalate in the female Wistar rat. Identification of new metabolites.

n-Butyl benzyl phthalate (BBP), a plasticizer used in polyvinylchloride (PVC) and other polymers, has been orally administered to female Wistar rats with four doses (150, 475, 780 and 1500 mg/kg body weight/day) for 3 consecutive days. Metabolites recovered in urines were analysed by gas chromatography-mass spectrometry (GC-MS) after 24, 48 and 72 hours. Six metabolites were identified.

Difference between guinea pig and rat in the liver peroxisomal response to equivalent plasmatic level of ciprofibrate.

Guinea pig was previously classified as a species nonresponsive to peroxisome proliferators. However, none of the previous reports was based on pharmacokinetic data. Here, after a comparative pharmacokinetic study between the guinea pig and rat, we evaluate the guinea pig liver peroxisomal response to ciprofibrate, a hypolipemic agent and a potent peroxisome proliferator in rat.

In vitro glucuronidation of peroxisomal proliferators: 2-ethylhexanoic acid enantiomers and their structural analogs.

In order to investigate the glucuronidation of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, by liver microsomes of several mammalian species including man, a gas chromatography method for the quantification of the corresponding glucuronides was developed. The variation coefficients for intra- and interassay repeatability were less than 3 and 7%, respectively. The rat liver UDP-glucuronosyl-transferase (UGT) presented similar Km and Vmax toward the two enantiomers.

Regio- and stereo-selectivity in the induction of peroxisome proliferation by substituted hexanoic acids.

Quantitative structure-activity relationship is an effective tool in order to predict drug potency. A similar approach is actually developed for peroxisome proliferation induced by substituted carboxylic acids issued from plasticizer metabolism in rats. The study is focused on acids found in rat urine after adipic diester dosings.

Toxicity of peroxisome proliferators.

Peroxisome proliferators are not a chemical class of compounds. They do not have a similar chemical structure but all induce characteristic effects in the liver of treated rats or mice. They produce within a few days a striking dose-dependent hepatomegaly accompanied by a characteristic proliferation of the peroxisomal and microsomal compartment as assessed morphologically and biochemically.

Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration.

The metabolic fate and pathway of the imidazole fungicide prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) were investigated in the rat after administration of oral single doses with radiolabelled molecules. At both dose levels (50 and 250 mg/kg body weight), virtually all of the ingested [14C-phenyl]prochloraz was excreted in the urine or faeces within 96 hr, the bulk of excretion occurring between 24 and 48 hr after dosing. Urinary elimination accounted for 61 and 68% of the respective initial doses.