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. Consequently in 2004, it was requested from the newly created European Food Safety Authority to perform a new evaluation of the mainly used phthalates on the basis of existing data. This paper summarizes evaluations of butylbenzylphthalate, dibutylphthalate, diethylhexylphthalate.

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. Hepatic cell lines and boar sperm, with cytotoxicity and sperm motility, respectively, as the end points, were inhibited by 1 nM of cereulide present as B. cereus extract. RNA synthesis and cell proliferation in HepG2 cells was inhibited by 2 nM of cereulide. These toxic effects were explainable by the action of cereulide as a high-affinity mobile K+ carrier. Exposure to cereulide containing extracts of B. cereus caused neither activation of CYP1A1 nor genotoxicity (comet assay, micronucleus test) at concentrations below those that were cytotoxic (0.6 nM cereulide). Salmonella typhimurium reverse mutation (Ames) test was negative. Exposure of Vibrio fischeri to extracts of B. cereus caused stimulated luminescence up to 600%, independent on the presence of cereulide, but purified cereulide inhibited the luminescence with an IC(50% (30 min)) of 170 nM. Thus the luminescence-stimulating B. cereus substance(s) masked the toxicity of cereulide in B. cereus extracts to V. fischeri.

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). Sulfatase treatment and subsequent high-performance liquid chromatography analyses suggest that the major urinary metabolite (more than 50% of the radioactivity present in urine) is a sulfate conjugate of BPF. At 96 h, BPF residues were detectable in all tissues examined with the largest amounts in the liver (0.5% of the dose). In pregnant rats dosed at day 17 of gestation, BPF residues were detected in the uterus, placenta, amniotic fluid, and fetuses (0.9-1.3% of the administered dose). Large amounts of radioactivity (8-10% of the dose) were still located in the digestive tract lumen at the end of the study. After administration of a single oral dose of [3H]BPF, 46% of the distributed radioactivity was excreted in bile over a 6 h period. In rats, BPF and/or its metabolites very likely undergo enterohepatic cycling, which could be responsible for the relatively high amounts of residues still excreted 4 days after BPF administration. This bisphenol is efficiently absorbed and distributed to the reproductive tract in female rats, and its residues pass the placental barrier at a late stage of gestation in rats.

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. DEHP significantly decreased the BC/LA muscles, the prostate, and the seminal vesicles relative weights from 100, 200, and 400 mg/kg bw/day, respectively. DEHP increased the liver relative weight from 100 mg/kg bw/day. A study was also performed on MDA-MB453 cell line stably transfected with pMMTVneo-Luc with DEHP and its major metabolites (MEHP and metabolites VI and IX) to identify anti-androgenic activity. Neither DEHP nor MEHP antagonised DHT activity in the MDA-MB453 transfected cells. In contrast, metabolites VI and IX were anti-androgenic in vitro. DEHP appeared not to be a 5alpha-reductase inhibitor and acted in an independent mechanism from the testicular production in the young rat.

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. With 4-NQO and MMS, a concentration-dependent increase in net nuclear grains per cell was observed, with 73 and 90% of cells, respectively, in repair at the highest concentration. B[a]P, 2-AAF and NDMA displayed similar dose-dependent UDS responses, but the percentage of cells in repair was lower (about 45%) than that for 4-NQO and MMS. We assessed the genotoxicity of the compounds tested by determining IC(5NNG): the concentration required to induce 5NNG. The compounds studied were ranked in order of IC(5NNG) as follows: 4-NQO = B[a]P > 2-AAF > MMS > NDMA. The UDS assay discriminated between mutagens and non-mutagens, as pyrene and 4-AAF failed to induce DNA repair. The present study demonstrates that UDS can be used as an endpoint for the detection of DNA damage 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. The presence of AraC (10, 50, or 100 microM) increased the DNA damage caused by HU (10 mM) suggesting a potentiation of the genotoxic effect. The interaction between the two inhibitors started after 5 h but was not dependent on the concentrations of AraC. Consequently, careful attention is required when employing a combination of HU and AraC, as their mechanisms of action could interfere with the interpretation of the data from genotoxicity assays.

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, 22.5% (w/w) vegetable protein. We determined the effects of dietary soy isoflavones after ten days of exposure and in animals fed L5 and DO3 diets throughout their lifetime (including the period of treatment with androgenic or anti-androgenic compounds). After ten days of exposure, we observed no effect of diet on the accessory sex organs of male Wistar rats. In contrast, diet affected the androgenic response to testosterone propionate in seminal vesicles and prostate. Seminal vesicles were the most sensitive organs. Vinclozolin caused a dose-dependent decrease in the relative weights of seminal vesicles, prostate and BC/LA regardless of diet. As vegetable proteins may contain high proportions of genistein and daidzein, two well-known oestrogenic endocrine disrupters that may alter the results of reproductive studies, we recommend the use of a standardised open-formula diet without soy isoflavones, such as L5, if the Hershberger assay is to be performed.

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. 4-NQO, B[a]P and 2-AAF were the most potent compounds; their IC(50) values were, respectively, 1.9 micro M (4h contact), 3.4 and 112 micro M after 20 h. MMS was mildly cytotoxic (IC(50)=0.9 mM) and NDMA had a weak effect (IC(50)=110 mM) after 4h contact. In the micronucleus and comet assays, concentrations required to obtain a significant genotoxic effect in HepG2 cells varied over a broad range, NDMA being active only at very high concentrations. To compare the sensitivity of the two assays, we measured the so-called FIC(2)-the concentration necessary to induce a 2-fold increase of the measured genotoxicity parameter. The data show that genotoxic effects were consistently observed at lower concentrations in the micronucleus test, except in the case of MMS. The measured FIC(2) values were 0.12 micro M (4-NQO), 0.17 micro M (2-AAF), 0.26 micro M (B[a]P) and 6.4mM (NDMA). MMS had such a weak effect in the HepG2 cells that we could not calculate its FIC(2) value. In the comet assay, FIC(2) values were observed, respectively, at 1.48 micro M (4-NQO), 3.67 micro M (B[a]P), 13.42 micro M (MMS) and 27 mM (NDMA). 2-AAF failed to induce DNA-damage in this assay. The present study shows that HepG2 cells could be a suitable tool for assessing the genotoxicity of direct and indirect mutagens and for establishing the lowest genotoxic concentration.

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. This study investigated the influence of cis/trans isomerism at the C9-C10 bond on the production of beta-carotene degradation compounds, first with a predictive approach and then experimentally with different isomer mixtures. beta-Carotene solutions containing high ratios of 9-cis-isomers produced more DHA, suggesting a different pathway than for the transformation of all-trans-beta-carotene to ionone and DHA. These results are important in the search for financially viable processes to produce natural carotene-derived aroma compounds.

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. This study describes a new automated protocol for the assay that makes it much more rapid. In the previous protocol, after the cells were treated with the test compounds and allowed to take up uridine for 30 min, samples were taken manually one by one and spotted onto 3MM Whatman paper. After drying, the paper sheet was then chromatographed in 5% (P/V) TCA for 2 h in order to precipitate and measure the total amount of RNA. In the new method, instead of paper chromatography, samples are transferred onto a 96-well microplate equipped with GF/C glass filters. Then, RNA precipitation by TCA is carried out with a manifold system, and the amount of radiolabeled uridine taken up by the cells is counted directly with a radioactivity microplate reader. This method makes it possible to screen many compounds simultaneously for cytotoxicity. To evaluate its sensitivity, we compared the IC(50) values obtained with new and original protocol for each eight toxic compounds. We found an excellent correlation between the two methods (r(2)=0.99). With the automated protocol, the uridine uptake inhibition assay is both sensitive and rapid enough for high-throughput daily screening.