Physical Obstruction of Nasal Cavities With Subsequent Asphyxia, Causes Lethality of Rats in an Acute Inhalation Study With Hydrophobic HMDZ Surface-Treated Synthetic Amorphous Silica (SAS).

The aim of the present study was to understand the mechanism of lethality associated with high dose inhalation of a low-density hydrophobic surface-treated SAS observed in some acute inhalation studies. It was demonstrated that physical obstruction of the upper respiratory tract (nasal cavities) caused the effects observed. Hydrophobic surface-treated SAS was inhaled (flow-past, nose-only) by six Wistar rats (three males and three females) in an acute toxicity study at a concentration of ~500 mg/m3 for an intended 4-hr exposure. Under the conditions of the test set-up, the concentration applied was found to be the highest that can be delivered to the test animal port without significant alteration of the aerosol size distribution over time. None of the test- material-exposed animals survived the planned observation time of 4 h; three animals died between 2 34 h after starting exposure and cessation of exposure at 3 14 h, two died after transfer to their cages and the remaining animal was sacrificed due to its poor condition and welfare considerations. Histology accomplished by energy dispersive X-ray (EDX) analysis demonstrated that test material particles agglomerated and formed a gel-like substrate that ultimately blocked the upper respiratory airways, which proved fatal for the rat as an obligatory nose breather. This observation is in line with the findings reported by Hofmann et al. showing a correlation between lethality and hydrophobicity determined by contact angle measurement. The aerosol characterizations associated with this study are provided in detail by Wessely et al.

Evaluation of developmental toxicity of Methyl Chloride (Chloromethane) in rats, mice, and rabbits.

Methyl Chloride (MeCl; Chloromethane) is a high production volume chemical (>1000 t/a) and is used as an industrial solvent. Based on cardiac lesions reported in developmental toxicity studies in mice, but not in rats, manufacturers decided to classify MeCl as a developmental toxicant, cat. 2. Recently, the European Chemical Agency required a developmental toxicity study in a non-rodent species. No developmental toxicity was observed in rabbits in the recently completed, GLP, OECD 414 guideline study. In view of the absence of cardiac effects in rats and rabbits, the purpose of this review is to consider whether the cardiac effects reported in mice should be considered real effects and, if so, their potential for relevance to humans. This paper provides substantive new evidence with data from a third species and shows that an evaluation of the integrated scientific evidence indicates the reported developmental cardiac effects in mice, if not an artifact, are unlikely to be relevant to humans. As such the classification of MeCl for developmental toxicity was reconsidered.

Hepatobiliary toxicity of furan: identification of furan metabolites in bile of male f344/n rats.

Furan, which occurs in a wide variety of heat-treated foods, is a potent hepatotoxicant and liver carcinogen in rodents. In a 2-year bioassay, furan caused hepatocellular adenomas and carcinomas in mice and rats but also high incidences of bile duct tumors in rats. Furan is bioactivated by cytochrome P450 enzymes to cis-2-butene-1,4-dial, an α,ß-unsaturated dialdehyde, which readily reacts with tissue nucleophiles. The objective of this study was to structurally characterize furan metabolites excreted with bile to better understand the potential role of reactive furan intermediates in the biliary toxicity of furan. Bile duct-cannulated F344/N rats (n = 3) were administered a single oral dose of 5 mg/kg b.wt. [(12)C(4)]furan or stable isotope-labeled [3,4-(13)C]furan, and bile samples collected at 30-min intervals for 4 h were analyzed by liquid chromatography-tandem mass spectrometry. A total of eight furan metabolites derived from reaction of cis-2-butene-1,4-dial with GSH and/or amino acids and subsequent enzymatic degradation were detected in bile. The main metabolite was a cyclic monoglutathione conjugate of cis-2-butene-1,4-dial, which was previously detected in urine of furan-treated rats. Furthermore, a N-acetylcysteine-N-acetyllysine conjugate, previously observed in rat urine, and a cysteinylglycine-glutathione conjugate were identified as major metabolites. These data suggest that degraded protein adducts are in vivo metabolites of furan, consistent with the hypothesis that cytotoxicity mediated through binding of cis-2-butene-1,4-dial to critical target proteins is likely to play a key role in furan toxicity and carcinogenicity.

Rapid and sensitive HILIC-ESI-MS/MS quantitation of polar metabolites of acrylamide in human urine using column switching with an online trap column.

The carcinogen acrylamide (AA) is formed during the processing of food. AA is metabolized to mercapturic acids, which are excreted with urine. A hydrophilic interaction liquid chromatography tandem mass spectrometry method (HILIC-MS/MS) using a zwitterionic stationary phase (Zic-HILIC) was developed and validated to quantitate the mercapturic acids of AA (AAMA) and glycidamide (GAMA), and AAMA-sulfoxide in human urine. In contrast to reversed phases, the application of Zic-HILIC resulted in efficient retention and separation of these highly polar compounds. Off-line sample workup was avoided by application of column switching with a Stability BS-C17 trap column prior to the analytical column, thus minimizing interferences with the urinary matrix. Limit of quantification values (LOQs) were 0.5 microg/L (AAMA), 2.0 microg/L (AAMA-sulfoxide), and 1.0 microg/L (GAMA) in human urine. Median concentrations in urine samples ( n = 54) of six nonsmoking human subjects were 24.0 microg/L (AAMA, 7.8-79.8 microg/L), 16.7 microg/L (AAMA-sulfoxide, 6.8-70.1 microg/L), and 3.82 microg/L (GAMA, 1.0-23.6 microg/L).

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk+/- mouse lymphoma cells.

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk+/- mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations>or=50 microM was reduced to <50%. In the dose range up to 25 microM, viability was >90%. Measures of comet-tail length and thymidine-kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 microM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at >or=100 microM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.

Biomarkers of furan exposure by metabolic profiling of rat urine with liquid chromatography-tandem mass spectrometry and principal component analysis.

Furan has been found in a number of heated food items and is carcinogenic in the liver of rats and mice. Estimates of human exposure on the basis of concentrations measured in food are not reliable because of the volatility of furan. A biomarker approach is therefore indicated. We searched for metabolites excreted in the urine of male Fischer 344 rats treated by oral gavage with 40 mg of furan per kg of body weight. A control group received the vehicle oil only. Urine collected over two 24-h periods both before and after treatment was analyzed by a column-switching LC-MS/MS method. Data were acquired by a full scan survey scan in combination with information dependent acquisition of fragmentation spectra by the use of a linear ion trap. Areas of 449 peaks were extracted from the chromatograms and used for principal component analysis (PCA). The first principal component fully separated the samples of treated rats from the controls in the first post-treatment sampling period. Thirteen potential biomarkers selected from the corresponding loadings plot were reanalyzed using specific transitions in the MRM mode. Seven peaks that increased significantly upon treatment were further investigated as biomarkers of exposure. MS/MS information indicated conjugation with glutathione on the basis of the characteristic neutral loss of 129 for mercapturates. Adducts with the side chain amino group of lysine were characterized by a neutral loss of 171 for N-acetyl- l-lysine. Analysis of products of in vitro incubations of the reactive furan metabolite cis-2-butene-1,4-dial with the respective amino acid derivatives supported five structures, including a new 3-methylthio-pyrrole metabolite probably formed by beta-lyase reaction on a glutathione conjugate, followed by methylation of the thiol group. Our results demonstrate the potential of comprehensive mass spectrometric analysis of urine combined with multivariate analyses for metabolic profiling in search of biomarkers of exposure.

Tools in metabonomics: an integrated validation approach for LC-MS metabolic profiling of mercapturic acids in human urine.

While for 1H NMR techniques there already exist common analytical and reporting standards, this does not apply to LC-MS metabolic profiling approaches. These standards are the more recommended when applying metabonomics to human biofluids, particularly urine samples, due to the high degree of biological variation compared to animals. A control study was performed, and urine samples of 30 healthy male and female human subjects were collected at intervals of 8 h twice a day for three consecutive days. Using selective multiple reaction monitoring in combination with a column-switching tool for the analysis of the mercapturate pattern, samples were screened for time and gender differences, the most common confounders. Data preprocessing parameters, alignment, scaling to internal standards, and normalization techniques were optimized by PCA, PLS-DA, and OPLS models. Great care was taken in the validation process of both analytical and chemometric protocols. Additionally, a problem of LC-MS, the combination of "different-batch" data to "one-batch" data could be solved by a batchwise scaling procedure. Based on these results, the use of metabolic profiling via mercapturates will be feasible for the detection of disease or toxicity markers in the future since mercapturates are important biomarkers of reactive metabolites known to be involved in many toxic processes.

Quantitation of mercapturic acids from acrylamide and glycidamide in human urine using a column switching tool with two trap columns and electrospray tandem mass spectrometry.

A sensitive and specific electrospray tandem mass spectrometry method using a column switching unit with two trap columns was established to quantify the mercapturates (MAs) of acrylamide (AA) and glycidamide (GA) in human urine. A specially endcapped material was applied for trapping the hydrophilic MAs and a pre-trap column was used to remove lipophilic compounds from the directly injected urine to protect the trap column. The limits of quantitation for AA-MA and GA-MA in urine were 0.5 microg/L and 1 microg/L, respectively. Urine was spiked with deuterated internal standards and injected directly into LC-MS/MS. Urine of smokers (n=13) revealed the highest concentrations of AA-MA and GA-MA in the range of 61-706 microg/L and 5-54 microg/L, respectively. Lower levels for AA-MA (14-102 microg/L) and GA-MA (1-11 microg/L) were detected in non-smokers (n=13).