A toxicogenomic approach for the risk assessment of the food contaminant acetamide.

Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.

Scale-up and operation of a pilot-scale ammonia fiber expansion reactor.

Pretreatment and densification of agricultural residues at regional depots can simplify feedstock supply logistics for the production of biofuels in commercial biorefineries. We have previously reported the performance of a laboratory-scale (5 L) packed-bed ammonia fiber expansion (AFEX) reactor system, which showed significant promise for biomass pretreatment at distributed depots. In this paper, we describe the performance of a 90-fold larger pilot-scale packed-bed AFEX-reactor system, used to produce over 1,500 batches (~36 tons) of pretreated crop residues over a 5-year period. Virtually all unreacted ammonia was successfully removed from the biomass, and 76% of the ammonia was recycled and reused. Pretreatment performance at pilot scale was comparable to laboratory-scale, averaging 74% glucose and 75% xylose yield in a standard test compared with 71% and 73%, respectively. Other operating and maintenance aspects are also discussed.

Presence of Acetamide in Milk and Beef from Cattle Consuming AFEX-Treated Crop Residues.

AFEX treatment of crop residues can greatly increase their nutrient availability for ruminants. This study investigated the concentration of acetamide, an ammoniation byproduct, in AFEX-treated crop residues and in milk and meat from ruminants fed these residues. Acetamide concentrations in four AFEX-treated cereal crop residues were comparable and reproducible (4-7 mg/g dry matter). A transient acetamide peak in milk was detected following introduction of AFEX-treated residues to the diet, but an alternative regimen showed the peak can be effectively mitigated. Milk acetamide concentration following this transition was 6 and 10 ppm for cattle and buffalo, respectively, but also decreased over time for cattle while tending to decrease (p = 0.08) for buffalo. There was no difference in acetamide concentration in the meat of cattle consuming AFEX-treated residues for 160 days compared to controls. Further investigation is necessary to determine the metabolism of acetamide in ruminants and a maximum acceptable daily intake for humans.

The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue.

Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.

Effect of ammonia fiber expansion on the available energy content of wheat straw fed to lactating cattle and buffalo in India.

The seasonal lack of availability of lush green forages can force dairy farmers in developing nations to rely on crop residues such as wheat and rice straw as the major feed source. We tested whether ammonia fiber expansion (AFEX) treatment of wheat straw would increase the energy available to Murrah buffalo and Karan-Fries cattle consuming 70% of their diet as wheat straw in India. Forty lactating animals of each species were blocked by parity and days in milk and randomly assigned to 1 of 4 treatment diets (n = 10). Treatments were a nutrient-rich diet with 0 to 20% straw (positive control; PC) and 3 high-straw diets with various levels of AFEX-treatment: (1) 70% untreated straw (no AFEX), (2) 40 to 45% untreated straw with 25 to 30% AFEX-treated straw (low AFEX), and (3) 20% untreated straw with 50% AFEX-treated straw (high AFEX). The AFEX-treated straw was pelleted. Urea was added to the no and low AFEX diets so they were isonitrogenous with the high AFEX diet. Animals were individually fed the PC diet for 14 d followed by 7 d of adaptation to treatments, full treatments for 28 to 35 d, and finally PC diets for 21 d. Compared with buffalo fed the PC diet, those fed high-straw diets consumed 29% less feed dry matter, put out 16% less milk energy, and lost 0.8 kg/d more body weight; the AFEX treatment of straw did not alter intake or milk production but greatly ameliorated the body weight loss (-1.0 kg/d for no AFEX and -0.07 kg/d for high AFEX). In Karan-Fries cattle, high-straw diets decreased dry matter intake by 39% and milk energy by 24%, and the high AFEX diet increased intake by 42% and milk energy by 18%. The AFEX treatment increased digestibilities of organic matter, dry matter, neutral detergent fiber, acid detergent fiber, and crude protein by 6 to 13 percentage points in buffalo and 5 to 10 points in cattle. In conclusion, AFEX treatment increased the digestibility and energy availability of wheat straw for lactating buffalo and cattle and has commercial potential to improve milk production and feed efficiency when high-quality forages or grains are not available.

Exposure Assessment of Acetamide in Milk, Beef, and Coffee Using Xanthydrol Derivatization and Gas Chromatography/Mass Spectrometry.

Acetamide has been classified as a possible human carcinogen, but uncertainties exist about its levels in foods. This report presents evidence that thermal decomposition of N-acetylated sugars and amino acids in heated gas chromatograph injectors contributes to artifactual acetamide in milk and beef. An alternative gas chromatography/mass spectrometry protocol based on derivatization of acetamide with 9-xanthydrol was optimized and shown to be free of artifactual acetamide formation. The protocol was validated using a surrogate analyte approach based on d3-acetamide and applied to analyze 23 pasteurized whole milk, 44 raw sirloin beef, and raw milk samples from 14 different cows, and yielded levels about 10-fold lower than those obtained by direct injection without derivatization. The xanthydrol derivatization procedure detected acetamide in every food sample tested at 390 ± 60 ppb in milk, 400 ± 80 ppb in beef, and 39 000 ± 9000 ppb in roasted coffee beans.