[Survey of Total Mercury Content in Fishery Products].

The total mercury content in 112 fishery products, purchased from Osaka city during 2013-2018, was measured using a heating vaporization mercury analyzer. The average total mercury content in all tuna processed products was found out to be 0.115 µg/g (median=0.070 µg/g). Notably, albacore tuna exhibited the highest mercury content with an average concentration of 0.301 µg/g (median=0.296 µg/g). The total mercury concentration of the fishery products, except processed tuna and Kezuribushi (comprising shavings of dried and smoked mackerel, sardine, as well as horse mackerel), was generally low (<0.1 µg/g).The provisional tolerable weekly intake (PTWI) for mercury as set by the Joint FAO/WHO Expert Committee on Food Additives is 4.0 µg/kg body weight/week. On an average, for Japanese diet, the total weekly mercury intake from fishery products for a person weighing 50 kg was estimated to be 0.13 µg/kg body weight/week, which was 3.3% of the PTWI. These values indicate that it was not a problem to consume fishery products normally. However, albacore tuna cans have a relatively high total mercury concentration; thus, pregnant women consuming them daily may exceed the tolerable weekly intake of methylmercury (2 µg/kg body weight/week) as set by the Food Safety Commission of Japan.

[Reduction of Orange Allergen Cit s 2 Levels in Fresh Orange Juice with Pineapple Bromelain Enzymatic Treatment].

Oranges are consumed worldwide; however, they contain Cit s 2, a major profilin allergen. We aimed to reduce Cit s 2 levels by preparing mixed orange fresh juice with pineapple, as a convenient method for any kitchen. Cit s 2 levels in orange extracts digested with pineapple extract and its protease bromelain were evaluated with quantitative enzyme-linked immunosorbent assay. Cit s 2 levels decreased according to reaction temperature and time, which was inhibited by iodoacetic acid. Treatment with pineapple extract diluted 40-fold and 0.1 mg/mL of bromelain at 37℃ for 30 min contributed to reducing residual Cit s 2 levels below the cut-off of 15%, respectively. Since this condition can increase the proportion of orange juice and reduce the risk of ingesting the pineapple allergen bromelain, it is considered to be more practical. Broad utilization of proteases in hypoallergenic food products is expected following clinical studies for verification.

[Headspace GC/MS Analysis of Residual Solvents in Laminated Films Used for Food Packaging].

Multilayer laminated films may contain organic solvents derived from adhesives, printing inks and so on. A headspace-GC/MS analysis method for the simultaneous determination of 30 substances such as toluene, xylene, acetone, methyl ethyl ketone, ethyl acetate, butyl acetate, methanol, ethanol, was developed. A N,N-dimethyl formamide solution containing an internal standard substance was added to the sample. After leaving overnight at room temperature, the headspace gas was analyzed by GC/MS. This method was applicable to a laminate film made of various materials. The organic solvents such as toluene, ethyl acetate, and heptane were detected from 6 out of 42 food packaging bags made from laminated film on Japanese market using this method.

Effects of popping on nutrient contents of amaranth seed.

Amaranth seeds can be popped by heating. The traditional method of popping in a skillet is simple, but it is difficult to control the heating time and temperature. To overcome these disadvantages, we developed a fluidized bed continuous processing system based on hot air heating for producing popped amaranth seeds in bulk. Using this system, we evaluated the effects of heat treatment at 260 °C for 15 s on the contents of B-group vitamins and essential and trace elements in amaranth seeds. The results showed that the treatment did not affect the content of B-group vitamins, and the recovery for essential and trace elements was 97-196%. This popping system is useful for processing amaranth seeds in terms of the product quality and nutrition.

The development of dual-function solid-phase method as extraction and a decomposition reaction media for the determination of a formaldehyde releaser, imidazolidinyl urea, in cosmetics.

In this study, we present a simple method to determine imidazolidinyl urea (IU) in cosmetics using a solid phase as both a decomposition field and an extraction phase. IU is difficult to quantify because it is a mixture of allantoin-formaldehyde condensation products that are easily decomposed to release formaldehyde. In our method, IU is decomposed to allantoin and 1-[4-(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl]urea (4-HU) on an aminopropyl-bonded silica solid phase. Subsequent high-performance liquid chromatography enables quantification of the resulting allantoin and 4-HU. The quantified value was converted to the total allantoin amount on the basis of molecular weight, and the calculated value was compared with that of an IU reference standard to determine the contents. The calibration curves of the decomposed IU as allantoin and 4-HU were both linear over an IU solution concentration range from 0.05 to 0.65% (w/v). The recoveries from lotion, body soap, and conditioners, which contained 0.1%, 0.3%, and 0.6% (w/w) of IU, respectively, ranged from 88.2 to 107.5%. The relative standard deviation values for the recovery tests of six replicates ranged from 1.03 to 6.97%. The intra-laboratory precisions for the lotion and conditioner A containing 0.3% IU were 3.02 and 4.94%, respectively. This method was well validated and would be helpful in determining IU in cosmetic samples.

Determination of potential volatile compounds in polyethylene terephthalate (PET) bottles and their short- and long-term migration into food simulants and soft drink.

Head space (HS)-GC-MS was used to analyze possible migration of volatile compounds from polyethylene terephthalate (PET) bottles for soft drinks, and a total of six compounds were identified. Next, a rapid, simple, and accurate simultaneous method was established using purge-and-trap (PT)-GC-MS, to quantify their amounts in the liquid contents after short- and long-term storage in PET bottles. Starting with brand-new PET bottles, the maximum migration of 2-methyl-1,3-dioxolane into distilled water and 50 % aqueous ethanol after 2 years at 25 °C were 2.3 and 19 ng/mL, respectively. In commercially available bottled mineral water sold inside and outside Japan, we were able to detect 2-methyl-1,3-dioxolane in the same way. While nonanal was also detected in some products, 2-methyl-1,3-dioxolane was confirmed as the main volatile compound. Finally, the human exposure to 2-methyl-1,3-dioxolane was estimated based on the per capita intake of soft drinks in Japan and the migration amount in this study.

Comparison between mouse bioassay and HILIC-MS/MS for quantification of paralytic shellfish toxin in Japanese basket clams and mussels caught off coastal Osaka Bay in Japan.

The content and composition of paralytic shellfish toxins (PSTs) in Japanese basket clam (Corbicula japonica) and mussels (Mytilus galloprovincialis) from Osaka Bay, Japan, were investigated using a mouse bioassay (MBA) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), and the association between toxicity values of MBA and HILIC-MS/MS was verified based on research data. The overall toxicity in Japanese basket clam was lower than that in the mussel. The PSTs of Japanese basket clam and mussel consisted mainly of C1, C2, and gonyautoxins 1-4 (GTX1-4) taking toxins compositional differences as mol%. When multiplying the content of different toxins by the toxic equivalent factor (TEF), C2 and GTX1-4 accounted for more than 90% of total toxicity (MU TEF/g) based on the MU TEF score converted by TEF for the two species. The total content of C2 and GTX1-4 converted to toxicity was significantly correlated with the toxicity determined by MBA for the two species (r2 > 0.983). This study provides a suitable and ethical monitoring method to investigate toxicity in bivalves contaminated with A. tamarense by analysis of only predominant toxins, along with reducing use of MBA.

Skin sensitization potency and cross-reactivity of p-phenylenediamine and its derivatives evaluated by non-radioactive murine local lymph node assay and guinea-pig maximization test.

BACKGROUND: p-Phenylenediamine (PPD)-related chemicals have been used as antioxidants in rubber products, and many cases of contact dermatitis caused by these chemicals have been reported. OBJECTIVE: The aim of this study was to investigate relative sensitizing potency and cross-reactivity among PPD derivatives. METHODS: Five PPD derivatives, p-aminodiphenylamine (PADPA), N,N'-diphenyl-p-phenylenediamine (DPPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (DMBPPD), N-(1-methylheptyl)-N'-phenyl-p-phenylenediamine (MHPPD), and the core chemical PPD were evaluated for their sensitizing potency and cross-reactivity using the non-radioactive murine local lymph node assay (LLNA) and the guinea-pig maximization test (GPMT). RESULTS: PPD and all the derivatives were identified as primary sensitizers in both tests. The order of potency in the LLNA was as follows: IPPD and PADPA > PPD > DMBPPD and MHPPD > DPPD. In the GPMT, all six groups of animals sensitized with one of these chemicals cross-reacted to four other derivatives. Specifically, the five groups that have a common basic PADPA structure, that is PADPA, DPPD, IPPD, DMBPPD, and MHPPD, all reacted to each other at almost the same scores, while none of them reacted to PPD. CONCLUSIONS: The cross-reactivity profile found in the study was to some extent different from that in previous human data, where distinction between cross-reaction and concomitant primary sensitization is not always clear.

Concentrations of total mercury and methylmercury in red snow crabs (Chionoecetes japonicus) caught off the coast of Japan.

The total mercury (T-Hg) and methylmercury (MeHg) concentrations in red snow crabs (Chionoecetes japonicus) caught off the coast of Japan were analyzed. The T-Hg concentration ranged from 0.03 to 0.56 mg/kg (mean: 0.21 mg/kg) in the raw muscle, and 0.02 to 0.74 mg/kg (mean: 0.27 mg/kg) in the boiled muscle. The MeHg concentration ranged from 0.04 to 0.54 mg/kg (mean: 0.20 mg/kg) in the raw muscle. The mean ratio of MeHg to T-Hg was 0.88. The crab body weight was found to significantly correlate with the concentrations of T-Hg (r = 0.488) and MeHg (r = 0.490) (p ≤ 0.01). For the general population in Japan, the intake of MeHg from eating red snow crab was estimated to be lower than 0.013 mg/week, which was less than one-sixth of the tolerable MeHg intake (0.08 mg/week).

Estimation of dietary intake of biotin and its measurement uncertainty using total diet samples in Osaka, Japan.

Single-laboratory method performance parameters were evaluated for analysis of biotin in total diet samples by hydrolyzed extraction and microbiological assay with Lactobacillus plantarum. The method was shown to be accurate, repeatable, rugged, and applicable for the determination of biotin in a broad range of matrixes and concentrations of total diet samples. The measurement uncertainty was evaluated with all food sample groups by combining the relative uncertainty of precision, recovery from each food group, and interday variation factors calculated from the ruggedness test results. The estimated daily intake of biotin in Osaka city and its expanded measurement uncertainty were 70.1 +/- 11.2 microg/day. The value was 1.6 times higher than the current adequate intake of biotin in Japan.

Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement.

A new sildenafil analog has been identified in the softgel shell of a dietary supplement. The compound was investigated by UV spectroscopy and high-resolution MS analysis, leading to the proposed structure 1-methyl-5-{5-[2-(4-methylpiperazin-1-yl)acetyl]-2-propoxyphenyl}-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one. A synthetic reference compound with the proposed structure was prepared, and the two sets of analytical data were compared, confirming the structure of the new compound. The compound was named propoxyphenyl noracetildenafil from its structure and similarity with the known compound.

Immunomodulatory effects of mono-, di-, and trimethylphenols in mice.

The relationship of air pollutants with the increasing prevalence of allergic diseases is a matter of concern in developed countries. In this study, the immunomodulatory effects of mono-, di-, and trimethylphenols in mice were examined as regards two aspects. First, whether or not these chemicals act as sensitizers was evaluated by local lymph node assay. Of the 13 methylphenols tested, three dimethylphenol isomers (2,4-DMP, 2,5-DMP, and 3,4-DMP) were found to induce auricular lymphocyte proliferation after dermal application on both ears of mice. Cytokine production patterns in the supernatants of cultured auricular lymphocytes from mice showed these methylphenols to be contact sensitizers. Second, the effects of methylphenols on cytokine production profiles were examined using cultured splenocytes from immunologically naive mice. Under subtoxic conditions, eight methylphenols inhibited interferon-gamma (IFN-gamma) production significantly, while the effect on intreluekin-4 (IL-4) production was moderate, resulting in higher IL-4/IFN-gamma ratios in all of the tested chemicals, with the most prominent effect shown by 2,6-DMP. These results suggest that several methylphenols, especially dimethylphenol isomers, have potencies that affect the immune system, being immunogens themselves or modulators of the Th1/Th2 cytokine balance.

Development of monoclonal antibody-based ELISA for the quantification of orange allergen Cit s 2 in fresh and processed oranges.

The aim of this study was to develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) for the quantification of a major allergen (Cit s 2) in fresh and processed oranges. Purified recombinant Cit s 2 (rCit s 2)-small ubiquitin-like modifier (SUMO) was used for the production of mAbs. In the optimized ELISA, the recovery of rCit s 2 from Navel oranges or orange juice was 107-132%, and the intra- and inter-assay coefficients of variation were 3.1-8.8% and 4.4-11%, respectively. The Cit s 2 content in fresh oranges was determined to be 1,800±430ng/g, while this content was much lower in the processed foods. The developed ELISA demonstrated high reproducibility, sensitivity, and accuracy, and this assay may help individuals with orange allergy by determining Cit s 2 quantities in food products and controlling their Cit s 2 intake.

The Contents and Composition of Tetrodotoxin and Paralytic Shellfish Poisoning Toxins in Marine Pufferfish Canthigaster rivulata.

The contents and composition of tetrodotoxin (TTX) and paralytic shellfish toxins (PSTs) in skin, muscle, and internal organs of two samples of marine puffer fish Canthigaster rivulata from Wakayama prefecture, Japan, were analyzed. Liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography with post-column derivatization and fluorescence detection (LC-FLD) were used for the analysis of TTX and PSTs, respectively. For both samples, TTX and two analogues of PSTs, saxitoxin (STX) and decarbamoyl STX (dcSTX), were detected at levels over the limit of quantization (LOQ) only in the skin. These toxins in the muscle and internal organs were at trace levels, or not detected (ND). TTX contents were 11,000 and 13,000 ng/g (or 35 and 41 nmol/g), while PSTs contents were 168 and 460 ng/g (or 0.63 and 1.72 nmol/g) in the two skin specimens. The compositions of total toxin content were 98.2 and 96.0 mol% TTX and 1.8 and 4.0 mol% PSTs, respectively. Thus, the main contributor to toxin content in C. rivulata skin was TTX and the levels of PSTs toxicity in C. rivulata were very low. When the PSTs contents were converted into mouse unit score from the LC-FLD results, the resulting values of 1.0 and 2.8 MU/g of PSTs in C. rivulata skin were similar to those in Takifugu poecilonotus and Takifugu vermicularis in Japan, as determined in previous studies.

HPLC Analysis of Colorants Migrated from Children's Modeling Clays.

A method using high-performance liquid chromatograph (HPLC) was developed for the identification of colorants migrated from colored modeling clays, which are popular toys for children. Twelve permitted dyes and 25 non-permitted dyes were analyzed in 20 clays (10 wheat clays, 2 rice clays, 2 corn clays, 3 paper clays and 3 resin clays). As a result, 13 products which were labeled for children's use (under 6 years old) met the specifications of the Japanese Food Sanitation Law, while non-permitted colorants were eluted from 2 products. In additon, unknown colorants were eluted from 3 products for people over 6 years old, although these are not covered by the Japanese regulation. It was suggested that some type of clays contained pigments, which are generally used in printing ink and plastics.

Development and Validation of Simultaneous Analysis of Minerals and Toxic Elements in Foods by ICP-MS.

We have developed and validated an inductively coupled plasma-mass spectrometry (ICP-MS) method for the simultaneous analysis of minerals and toxic elements in foods. Food samples were digested by microwave irradiation to prepare solutions for measurement by ICP-MS. Optimal gas mode and internal standard for each element were selected as appropriate. The method was validated for eighteen elements in total using three certified standard reference materials, namely, Typical Japanese Diet, Rice Flour-Unpolished and Infant/Adult Nutritional Formula, according to the guidelines of the Ministry of Health, Labour and Welfare of Japan. The trueness and precision of the method were all within the acceptable limits, except for Na in Rice Flour-Unpolished.

Validation Study on a Rapid Method for Simultaneous Determination of Pesticide Residues in Vegetables and Fruits by LC-MS/MS.

A validation study was carried out on a rapid method for the simultaneous determination of pesticide residues in vegetables and fruits by LC-MS/MS. Preparation of the test solution was performed by a solid-phase extraction technique with QuEChERS (STQ method). Pesticide residues were extracted with acetonitrile using a homogenizer, followed by salting-out and dehydration at the same time. The acetonitrile layer was purified with C18 and PSA mini-columns. The method was assessed for 130 pesticide residues in 14 kinds of vegetables and fruits at the concentration level of 0.01 µg/g according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. As a result 75 to 120 pesticide residues were determined satisfactorily in the tested samples. Thus, this method could be useful for a rapid and simultaneous determination of multi-class pesticide residues in various vegetables and fruits.

Quantitative comparison of the results obtained by the multiple-dose guinea pig maximization test and the non-radioactive murine local lymph-node assay for various biocides.

We compared the results of the multiple-dose guinea pig maximization test (GPMT) and the non-radioactive murine local lymph-node assay (LLNA) for various biocides. Thirteen out of 17 positive biocides in the GPMT gave positive results in the LLNA. In the GPMT, the minimum first induction doses ranged over four orders (0.00005-0.5%), while elicitation-threshold doses, which were evaluated using an optimally sensitized group of animals in the multiple-dose studies, ranged over five orders (0.00006-2.8%). In the LLNA, minimum induction doses ranged over more than three orders (0.01-30%). With respect to 13 biocides that were positive in both the GPMT and the LLNA, results were quantitatively compared. When compared after conversion to corresponding area doses (microg/cm), the minimum doses required to elicit skin reaction in guinea pigs were always lower than that for induction in mice with all biocides. Correlation between minimum induction doses from the GPMT and the LLNA seemed poor (r=0.57), while that between minimum induction doses in the LLNA and elicitation-threshold doses in the GPMT was relatively good (r=0.73). The results suggest the possibility to estimate human elicitation-threshold doses, which are definitely lacking in the process of risk assessment for skin-sensitizers, from the data of the LLNA.

Allergenicity evaluation of p-chloro-m-cresol and p-chloro-m-xylenol by non-radioactive murine local lymph-node assay and multiple-dose guinea pig maximization test.

p-Chloro-m-cresol (PCMC) and p-chloro-m-xylenol (PCMX) are known to cause allergic contact dermatitis. For risk assessment of skin sensitizers, information on dose-response profiles in the induction and elicitation phases and cross-reactivity with analogous chemicals are important. In the non-radioactive local lymph-node assay (LLNA) using 5-bromo-2'-deoxyuridine instead of 3H-methyl thymidine, significant effect on lymph node cell proliferation was detected at 10% PCMC and 25% PCMX, while in the multiple-dose guinea pig maximization test (GPMT) at least one animal tested in the group was sensitized at a 5 ppm induction dose of either chemical. When mean skin reaction score in an animal group maximally sensitized with each allergen with the GPMT was plotted against log challenge concentration, linear regression lines with high correlations were obtained in both cases. The calculated elicitation threshold was lower for PCMC than PCMX. The area under the linear regression line between the threshold point and 1% of the elicitation concentration, another index of relative elicitation potency, was also greater for PCMC. Bidirectional cross-reactivity between PCMX and PCMC was detected in the GPMT. PCMC was thus identified in both LLNA and GPMT as a stronger sensitizer than PCMX in both the induction and elicitation phases. These results suggest that the non-radioactive LLNA is a simple and useful method for evaluating allergenicity in the induction phase, while the GPMT using a maximally sensitized animal group is more suitable for assessing the dose-response profile and cross-reactivity in the elicitation phase.

Discrimination of mushrooms causing food-poisoning incidents by using DNA sequence analysis.

In this study, the identification of mushrooms by using DNA analysis was investigated. Our analysis of internal transcribed spacer (ITS) regions revealed that a DNA-based method could be applicable for samples that are difficult to distinguish in terms of the morphological characteristics. PCR amplification using templates extracted from cooked samples gave sufficient fragments to analyze the sequence. However, treatment with simulated gastric fluid (SGF) for more than 30 min affected the analysis of the ITS region. Application to samples of vomit is also discussed.