Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of food-flavouring substances uniquely used in Japan that belong to the class of aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups.

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of 'aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups' and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer's classification. The estimated daily intake of each of the four substances was determined to be 0.007-2.9 µg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250-600.0 µg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 µg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

The Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of acetal food flavouring substances uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 µg/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 µg/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800 µg/person/day. The daily intakes of the three class III substances exceeded the TTC (90 µg/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15 000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.

The JFFMA assessment of flavoring substances structurally related to menthol and uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on four flavoring substances structurally related to menthol (L-menthyl 2-methylbutyrate, DL-menthyl octanoate, DL-menthyl palmitate, and DL-menthyl stearate) uniquely used in Japan. While no genotoxicity study data were available in the literature, all four substances had no chemical structural alerts predictive of genotoxicity. Moreover, they all four are esters consisting of menthol and simple carboxylic acids that were assumed to be immediately hydrolyzed after ingestion and metabolized into innocuous substances for excretion. As menthol and carboxylic acids have no known genotoxicity, it was judged that the JECFA procedure could be applied to these four substances. According to Cramer's classification, these substances were categorized as class I based on their chemical structures. The estimated daily intakes for all four substances were within the range of 1.54-4.71 µg/person/day and 60-1250 µg/person/day, using the methods of Maximized Survey-Derived Intake and Single Portion Exposure Technique, respectively, based on the annual usage data of 2001, 2005, and 2010 in Japan. As the daily intakes of these substances were below the threshold of concern applied to class I substances viz., 1800 µg/person/day, it was concluded that all four substances raise no safety concerns when used for flavoring foods under the currently estimated intake levels.

[Residual pesticide concentrations after processing various types of tea and tea infusions].

The effects of processing to produce various types of tea or infusion on the levels of pesticide residues in tea were investigated for three insecticides (chlorfenapyr, pyrimiphos-methyl, and clothianidin). Tea plants were sprayed with one of the three pesticides and cultivated under cover. The levels of pesticide residues in tea decreased after processing according to the time and temperature of heating, as well as fermentation. Although significant differences were not observed among the three pesticides in the ratio of decreased of pesticide concentration after processing to green tea, clothianidin, which is a neonicotinoid insecticide and has a lower log Pow value, tended to be transferred more than the other two insecticides into infusions. However, no significant difference in the ratios of clothianidin transferred to infusions was observed among green tea with three different leaf sizes.

Analysis of trans-fat levels in total diet and one-serving samples using the verified GC-method and estimation of the intake in Japan.

In Japan, discussions on the regulation and labeling of trans-fat (TF) have under way for several years in the Food Safety Commission and the Consumer Affairs Agency. However, administrative measures for TF have not yet been taken, partly because of the insufficiency of scientific data in Japan. To provide data about the TF intake by Japanese, we determined the levels of TF contained in total diet samples and in food samples that were served as individual meals (one-serving samples). We analyzed 5 groups of total diet samples prepared in 11 regions throughout Japan, and 5 categories of one-serving samples using the GC-method after verifying its performance. The estimated daily intake of TF based on the analytical results of the total diet samples was around 500 mg and no significant difference was observed in the intake of the TF among the 11 surveyed regions. On the other hand, many one-serving samples classified into "hamburger", "pizza" and "Western food" categories contained more than 500 mg of TF per serving, the standard value in the labeling regulation in the United States. If these one-serving meals are taken to represent one meal out of 3 in a day, the intake of TF can easily be expected to exceed the daily intake estimated through the analysis of the total diet samples.

Determination method for total arsenic and partial-digestion method with nitric acid for inorganic arsenic speciation in several varieties of rice.

The determination method of total arsenic (As) and the speciation method of inorganic As for non-glutinous rice reported in our preceding paper were applied to several varieties of rice under optimized experimental conditions. In the determination of total As with ICP-MS, acetic acid was added to increase the sensitivity and an internal reference method with germanium was adopted to increase the precision. The extraction temperature in the partial-digestion method with nitric acid to speciate inorganic As was raised to 100 degrees C, because extraction efficiency over 90% was obtained from glutinous rice and colored rice at this temperature. In the investigation of polished and unpolished forms of non-glutinous, glutinous, and colored rice, the amounts of total and inorganic As were 0.04-0.54 mg/kg and 0.02-0.41 mg/kg, respectively. The color of rice was not related to total or inorganic As content. These results indicate that the inorganic As content in commercial rice should be carefully monitored.

[Surveillance of perchlorate level in leafy vegetables and bottled water].

Perchlorate (ClO(4)(-)) is both a naturally occurring and artificial compound, and it inhibits iodide uptake into the thyroid gland and disturbs thyroid function. It has been detected in many foods in the United States. In order to investigate perchlorate contamination in foods in Japan, perchlorate level in 82 leafy vegetable samples and 20 bottled mineral water samples was measured using a procedure based on the FDA's procedure, employing IC-MS/MS with (18)O(4)-labeled perchlorate as an internal standard. Among 82 leafy vegetable samples tested, perchlorate levels were under the LOQ (0.3 ng/g) in 3 samples and ranged from 0.3 ng/g to 29.7 ng/g in 79 samples. In 20 bottled water samples, perchlorate was under the LOQ (0.1 ng/mL) in 14 samples and ranged from 0.14 ng/mL to 0.35 ng/mL in 6 samples.

[Analytical method of dodine in agricultural products by LC/MS].

A method for the determination of dodine in agricultural products was developed by using liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). Dodine was extracted with acetonitrile and then acetonitrile-water (7 : 3) from a sample, and re-extracted with ethyl acetate. The extract was cleaned up on a PSA cartridge column (500 mg), and dodine was analyzed by LC/MS. In the case of oil seeds and nuts, hexane/acetonitrile-hydrochloric acid partition was performed to remove lipids before re-extraction with ethyl acetate. In the case of samples that contained a lot of chlorophyll, the eluate of the PSA cartridge column was further cleaned up on a graphitized carbon cartridge column (500 mg). The calibration curve was linear from 0.0001-0.02 microg/mL of dodine. The recoveries of dodine from sixteen kinds of agricultural products fortified at 0.1 mg/kg were 80.3-100.0%, and their relative standard deviations were 0.3-6.4%. The limits of detection (S/N=3) were 0.0006 mg/kg.

[Analysis of demeton-S-methyl, oxydemeton-methyl and demeton-S-methylsulfone in agricultural products by LC-MS].

We studied the simultaneous determination of demeton-S-methyl, oxydemeton-methyl, and their oxide demeton-S-methylsulfone, in agricultural products by liquid chromatography coupled with mass spectrometry (LC-MS). The sample homogenized with antioxidants L-ascorbic acid and butylhydroxytoluene was extracted with acetone. An aliquot of the crude extract was reextracted with ethyl acetate by using an EXtrelut column. After hexane/acetonitrile partitioning lipid-rich samples such as cereals, the extract was cleaned up on a PSA column or tandem graphitized carbon/PSA column, and determined by ESI-SIM mode LC-MS. Average recoveries (n=5) of compounds from ten kinds of samples fortified at the analyte concentration of 0.05 microg/g were from 73.8% to 102.5%, and the relative standard deviations were

Evaluation of modified PCR quantitation of genetically modified maize and soybean using reference molecules: interlaboratory study.

Real-time polymerase chain reaction (PCR)-based quantitative methods were previously developed and validated for genetically modified (GM) maize or soy. In this study, the quantification step of the validated methods was modified, and an interlaboratory study was conducted. The modification included the introduction of the PCR system SSIIb 3 instead of SSIIb 1 for the detection of the taxon-specific sequence of maize, as well as the adoption of colE1 as a carrier included in a reference plasmid solution as a replacement for salmon testis. The interlaboratory study was conducted with the ABI PRISM 7700 and consisted of 2 separate stages: (1) the measurement of conversion factor (Cf) value, which is the ratio of recombinant DNA (r-DNA) sequence to taxon-specific sequence in each genuine GM seed, and (2) the quantification of blind samples. Additionally, Cf values of other instruments, such as the ABI PRISM 7900 and the ABI PRISM 7000, were measured in a multilaboratory trial. After outlier laboratories were eliminated, the repeatability and reproducibility for 5.0% samples were <15.8 and 20.6%, respectively. The quantitation limits of these methods were 0.5% for Bt11, T25, and MON810, and 0.1% for GA21, Event176, and RR soy. The quantitation limits, trueness, and precision of the current modified methods were equivalent to those of the previous methods. Therefore, it was concluded that the modified methods would be a suitable replacement for the validated methods.

[Factors affecting the variance of copy numbers in real-time PCR as estimated from calibration curves].

The distribution interval of calibration curves in real-time PCR was successfully predicted from the variance of the Ct values, and the predicted distribution interval was consistent with the interval of the actual calibration curves. Although the width of the 95% confidence interval at the lower end was about 1%, the copy number estimated from individual calibration curves was distributed over a wider range, and the 95% confidence interval of the estimates of 20 copies was +/-40%. The variances of the Ct values at 20 copies were significantly larger than those at higher copy numbers, and this large variation increased the distribution interval of the calibration curves. The variation, however, can be attributed to the probabilistic distribution of copy numbers in the measurement system, and is impossible to reduce by improvement of the analytical methodology. The 95% confidence interval of the calibration curves obtained after omitting 20 copies was decreased and the measurement precision was improved.

[Estimation of the daily intake of nitrate based on analysis of total diet samples].

The concentrations of nitrates in total diet samples prepared at 11 places were determined according to the market basket method, and the daily intake of nitrates was estimated based on the analysis results. The mean estimated daily intake of nitrates was 200 mg/man/day (4.0 mg/kg/day) and exceeded the ADI (3.7 mg/kg/day) set by the JECFA by 8%. The principal sources of the nitrates were colored vegetables (7th food group) and other vegetables, seaweeds and mushrooms (8th food group), and the intake from these food groups accounted for more than 80% of the total intake. The reported concentrations of nitrates in individual foods and the estimated daily intake of these foods indicate that the intake of nitrates from spinach accounts for 38% of the ADI, that from Chinese cabbage for 10%, and that from Japanese radish for 20%.

[Development and evaluation of determination methods for boric acid in agar using ICP-AES and ICP-MS].

A new test method for boric acid in agar was developed. After digestion with nitric acid, the concentration of boron was measured by ICP-AES or ICP-MS with internal standards. Collaborative studies involving 5 laboratories were conducted to evaluate the new method by using a finely powdered agar sample and a standard sample (NIST SRM SRM1570a). The repeatability and reproducibility were very good. Moreover, HORRAT(R) values were less than 2. Therefore, it was considered that the new method is acceptable for the determination of boric acid in agar.

Orally administered apple procyanidins protect against experimental inflammatory bowel disease in mice.

Apple procyanidins (ACT) is a natural biologically active compound extracted from apple. Our recent studies have shown that ACT ameliorates the symptoms of atopic dermatitis and inhibits food-allergen-induced oral sensitization. The aim of this study was to investigate the potential protective effect and mechanism of action of ACT in a murine model of inflammatory bowel disease. We investigated the preventive effects of ACT in experimental models of colitis induced by dextran sulfate sodium (DSS) or oxazolone. Oral administration of ACT before DSS treatment attenuated the DSS-induced mortality rate and decreased body weight loss. ACT also prevented the body weight loss associated with oxazolone-induced colitis. Next we examined the effect of ACT on intraepithelial lymphocytes (IEL), which is a major T cell population in the intestine. Oral administration of ACT increased the proportions of TCRgammadelta and TCRalphabeta-CD8alphaalpha T cells in IEL and suppressed interferon gamma synthesis in stimulated IEL. In addition, ACT inhibited phorbol 12-myristate 13-acetate-induced secretion of interleukin 8 (IL-8) in intestinal epithelial cells. The combined anti-inflammatory and immunomodulatory effects of ACT on intestinal epithelial cells and IEL suggest that it may be an effective oral preventive agent for inflammatory bowel diseases.

[Effect of non-genetically modified (non-GM) soy varieties on the measured value of GM soy by a quantitative PCR method].

It is very important to examine the effect of non-genetically modified (non-GM) soy varieties, which constitute the matrix of the testing sample used to quantify GM soy (RRS), on the measured value of RRS by quantitative PCR methods. Therefore, we quantified the amount of RRS in powder-mixed samples containing 1 or 5% RRS prepared by using 10 different varieties of non-GM soy as the matrix. The results revealed that the measured values were not in agreement with the powder-mixing levels and that the extent of the difference depended on the variety of non-GM soy used as the matrix. The yields of DNA extracted differed among the soy varieties. On the other hand, analysis of DNA-mixed samples, that were prepared with the DNAs extracted from RRS and non-GM soy varieties, showed that the measured values of RRS were in agreement with the DNA-mixing levels. These results strongly suggest that the proportions of DNA derived from RRS and non-GM soy were not consistent with the powder-mixing ratio in the case of some non-GM soy varieties used as a matrix, resulting in the discrepancy between the measured values and the powder-mixing levels.

[Simultaneous determination of sedecamycin and terdecamycin in livestock products by LC/MS].

A simple and rapid LC/MS method for simultaneous determination of sedecamyin (SCM) and terdecamycin (TDM) in livestock products has been developed. SCM and TDM were extracted with acetonitrile. The extract was washed with n-hexane and then evaporated to dryness. The residue was dissolved in methanol, and injected into the LC/MS. The mass spectrometer was operated in the positive electrospray ionization (ESI) mode. LC separation was performed on a high-pH-resistant C18 column with 10 mmol/L carbonic acid-ammonia buffer (pH 10.0)-acetonitrile as a mobile pahse. The recoveries from swine muscle and liver fortified at the levels of 0.01 and 0.05 microg/g were 77-88%, and those from poultry muscle and liver fortified at the levels of 0.01 and 0.3 microg/g were 51-93%. The quantitation limits of SCM and TDM were 0.008 microg/g and 0.005 microg/g, respectively.

Biosensor immunoassay for the screening of dioxin-like polychlorinated biphenyls in retail fish.

Dioxin-like polychlorinated biphenyls (DL-PCBs) often make up the majority of the toxic equivalent (TEQ) contribution of dioxins found in fish samples. For the purpose of making risk assessments, it is therefore important to develop screening methods for determining TEQ concentrations of DL-PCBs in retail fish. We have developed a rapid biosensor immunoassay (BIA) for DL-PCBs that uses a surface plasmon resonance sensor (Biacore 3000). The BIA is highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118) that is generally the most abundant DL-PCB isomer found in fish. The fish extracts were first cleaned up on a multilayer silica gel column followed by an alumina column, then subjected to the assay. The quantitative limit of the assay was 1 ng PCB 118 per gram of tested sample. Dilution and recovery tests using purified fish extracts suggested that the matrix effect was minimized in the assay by diluting the analyzed samples. The assay results for retail fish samples (n=7) agreed well with those obtained by an enzyme-linked immunoassay (ELISA) using the same monoclonal antibody: ELISA has been already validated for determining DL-PCBs in fish samples, so BIA performs well in this analysis. Finally, BIA results for the TEQ concentrations of DL-PCBs in retail fish samples (n=10) correlated well with those obtained by high-resolution gas chromatography coupled to high-resolution mass spectrometry (r=0.89). Our method is therefore useful for screening retail fish to determine the TEQ concentrations of DL-PCBs.

DNA extraction method using a silica-base resin type kit for the detection of genetically modified papaya.

Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.