[Validation of Method for Nitrite Determination in Foods].

In general, nitrite in food is extracted under slightly alkaline conditions, deproteinized, and analyzed by a colorimetric method using color development by diazotization. However, depending on the sample, the sample solution may become cloudy and difficult to filter by the deproteinization treatment of the analytical method. Recently, an improved analytical method that solves these problems has been reported. Therefore, a validation study was performed on the improved analytical method was performed. The concentrations of sodium nitrite added to cod roe, fish sausage, and ham, which were not labeled with sodium nitrite, were set at the upper limits of the standards for use. We set the target values of 70-120% for trueness, less than 15% for intralaboratory reproducibility, and less than intralaboratory reproducibility for repeatability. As a result, the target values were met for the three samples verified: 88-92% for trueness, 2.0-3.0% for repeatability, and 3.2-4.3% for intralaboratory reproducibility. In addition, an interlaboratory study was conducted by eight institutes on the improved analytical method for nitrite. At each institution, sodium nitrite was added to the same three samples as in the validation study, at concentrations equivalent to twice the lower limit of quantification and the upper limit of the standards for use and analyzed in triplicate. The estimated trueness from the obtained analyses ranged from 82 to 95%, the repeatability ranged from 2.3 to 5.8%, and the inter-room reproducibility ranged from 3.5 to 11%. Thus, the improved analytical method could be useful for determining nitrite in foods.

[Validation Study on Migration Test for Plastic Food Utensils, Containers, and Packaging].

Migration test of food utensils, containers, and packaging is an important test method for confirming the safety and their compliance to the standards. However, there is little report on inter-laboratory study which was performed to evaluate the entire migration test, including migration operations and quantification. An interlaboratory study was performed participating 22 laboratories using 8 types of model synthetic resin samples containing 10 substances with a wide range of Log Pow values to evaluate the accuracy of the entire migration test. As a result, most of HorRat (r) values met the target criteria (0.3

[Interlaboratory Study on Evaporation Residue Test for Food Contact Products (Report 1)].

An interlaboratory study was performed to evaluate the equivalence between an official method and a modified method of evaporation residue test using three food-simulating solvents (water, 4% acetic acid and 20% ethanol), based on the Japanese Food Sanitation Law for food contact products. Twenty-three laboratories participated, and tested the evaporation residues of nine test solutions as blind duplicates. For evaporation, a water bath was used in the official method, and a hot plate in the modified method. In most laboratories, the test solutions were heated until just prior to evaporation to dryness, and then allowed to dry under residual heat. Statistical analysis revealed that there was no significant difference between the two methods, regardless of the heating equipment used. Accordingly, the modified method provides performance equal to the official method, and is available as an alternative method.

[Interlaboratory Study on Evaporation Residue Test for Food Contact Products (Report 2)].

An interlaboratory study was performed to evaluate the equivalence between an official method and a modified method of evaporation residue test using heptane as a food-simulating solvent for oily or fatty foods, based on the Japanese Food Sanitation Law for food contact products. Twenty-three laboratories participated, and tested the evaporation residues of nine test solutions as blind duplicates. In the official method, heating for evaporation was done with a water bath. In the modified method, a hot plate was used for evaporation, and/or a vacuum concentration procedure was skipped. In most laboratories, the test solutions were heated until just prior to dryness, and then allowed to dry under residual heat. Statistical analysis revealed that there was no significant difference between the two methods. Accordingly, the modified method provides performance equal to the official method, and is available as an alternative method. Furthermore, an interlaboratory study was performed to evaluate and compare two leaching solutions (95% ethanol and isooctane) used as food-simulating solvents for oily or fatty foods in the EU. The results demonstrated that there was no significant difference between heptane and these two leaching solutions.

Interlaboratory Study on Volatiles Test for Food-Contacting Polystyrene Products.

Using polystyrene, acrylonitrile-styrene resin and acrylonitrile-butadiene-styrene resin pellets as samples, an interlaboratory study was performed to evaluate the volatiles test method, based on the specifications described in the Japanese Food Sanitation Law for food-contacting polystyrene products. The study was conducted with the participation of twenty-one laboratories. Each laboratory quantified the contents of styrene, toluene, ethylbenzene, isopropylbenzene and propylbenzene in three test pellets using GC-FID, GC-MS or headspace-GC-FID. Statistical analysis revealed that the repeatability (RSDr) and reproducibility (RSDr) were 1.0-2.6 and 2.5-5.5% for the GC-FID method. The values of the performance parameters fulfilled the requirements (RSDr: 10%, RSDr: 25%), and the performance is sufficient for specifications testing. The RSDr and RSDr of results obtained using the GC-MS and HS-GC methods were 1.4-7.8 and 4.9-13%(GC-MS), and 2.0-2.6 and 3.3-6.9%(HS-GC-FID), respectively. The quantified levels were similar to those obtained with GC-FID. The study suggests that the GC-MS and HS-GC methods can be employed as alternative methods to the GC-FID method.

Interlaboratory Study on Caprolactam Test for Food-Contact Nylon Products.

The Japanese Food Sanitation Law sets a limit on the migration level of caprolactam for food-contacting nylon products. Here, we carried out an interlaboratory study in twenty laboratories to evaluate the performance of the official GC-FID test method and a GC-MS method as an alternative test method to the official method. Each laboratory quantified caprolactam in three test solutions in 20% ethanol as blind duplicates using GC-FID or GC-MS. The official method (GC-FID with absolute calibration) gave trueness, repeatability (RSDr) and reproducibility (RSDr) values of 96-97%, 3.3-5.4% and 4.0-6.7%, respectively. These values met the target criteria (trueness: 80-110%, RSDr: 10%, RSDr: 25%). The performance of the method was further improved by the introduction of heptalactam as an internal standard. As for GC-MS method, some values of the RSDr exceeded 10% when absolute calibration was used. However, when an internal standard was introduced, the trueness, RSDr and RSDr of GC-MS method were all acceptable at 94-96%, 2.0-4.4% and 7.0-9.4%, respectively. Therefore, GC-MS with an internal standard is available as an alternative test method to the official method.

[Interlaboratory Study on Zinc Test for Food-Contact Rubber Products].

Using six kinds of zinc solution in water and 4% acetic acid as samples, an interlaboratory study was performed to evaluate a zinc (Zn) test method for food-contact rubber products, based on the Japanese Food Sanitation Law. Eighteen laboratories participated, and quantified Zn in six test solutions as blind duplicates using flame atomic absorption spectrometry, induced coupled plasma-optical emission spectrometry or induced coupled plasma-mass spectrometry. Statistical analysis revealed that the trueness, repeatability (RSDr) and reproducibility (RSDr) were 97-103%, 0.7-4.9% and 1.7-8.9% by all measuring methods. The values of the performance parameter fulfilled the target value (trueness: 80-110%, RSDr: 10%, RSDr: 25%). The performance of these methods is sufficient for testing the adherence of samples to the specifications.

[Interlaboratory study on migration test of antimony and germanium for food-contact polyethylene terephthalate].

An interlaboratory study was performed to evaluate a migration test method of antimony (Sb) and germanium (Ge), based on the Japanese Food Sanitation Law for food- contact polyethylene terephthalate. Eighteen laboratories participated, and quantified Sb and Ge in three test solutions as blind duplicates using graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma-optical emission spectrometry (ICP-OES) or induced coupled plasma-mass spectrometry (ICP-MS). Statistical analysis revealed that the trueness, repeatability and reproducibility were 98-107%, 1.7-7.5% and 2.0-18.8% by using GF-AAS and ICP-OES. The performance of these methods is sufficient for testing the specifications. The performance parameters of ICP-MS were 99-106%, 0.7-2.2% and 2.2-10.5%, respectively. ICP-MS is available as an alternative measuring method. However, in some laboratories, the quantitative values of Sb were higher than the addition levels. We found that Sb in working solutions is absorbed on glass vessels. Careful control of concentration in working solutions is required for Sb analysis.

Heat treatment of egg white controls allergic symptoms and induces oral tolerance to ovalbumin in a murine model of food allergy.

SCOPE: Heated foods often present low allergenicity, and have recently been used in specific oral immunotherapy for food allergies. However, the influence of heating on tolerogenicity of food allergens is not well elucidated. Here, we investigated biochemical, allergenic, and tolerogenic properties of heated egg white (EW) using a murine model of food allergy. METHODS AND RESULTS: Raw EWs were treated at 80°C for 15 min (80EW, mild heating condition), 100°C for 5 min (100EW, cooking condition), or 121°C for 40 min (121EW, retort pouch condition), and freeze-dried. A transgenic OVA23-3 mice model expressing T-cell receptor specific for ovalbumin (OVA, a major EW allergen) induced Th2 cells and IgE production, and presented intestinal inflammation when fed untreated EW diet. 80EW-fed mice presented only moderate inflammation but high Th2 responses. 100EW-fed mice did not present inflammation but induced tolerance as seen in reduced T-cell responses and IgE levels. 100EW demonstrated higher digestive stability and slower absorption in intestine, compared with untreated EW and 80EW. 121EW was strongly aggregated, was not absorbed well, and developed Th1 responses without tolerance induction. CONCLUSION: OVA in EW treated only under a particular heat condition (e.g. 100°C for 5 min) lost allergenicity, but possessed tolerogenicity.

[Performance comparison of material tests for cadmium and lead in food contact plastics].

Based on the Japanese Food Sanitation Law, the performances of official and alternative material test methods for cadmium (Cd) and lead (Pb) in food contact plastics were compared. Nineteen laboratories participated to an interlaboratory study, and quantified Cd and Pb in three PVC pellets. in the official method, a sample is digested with H2SO4, taken up in HCl, and evaporated to dryness on a water bath, then measured by atomic absorption spectrometry (AAS) or inductively coupled plasma-optical emission spectrometry (ICP-OES). Statistical treatment revealed that the trueness, repeatability (RSDr) and reproducibility (RSDr) were 86-95%, 3.1-9.4% and 8.6-22.1%, respectively. The values of the performance parameters fulfilled the requirements , and the performances met the test specifications. The combination of evaporation to dryness on a hot plate and measurement by AAS or ICP-OES is applicable as an alternative method. However, the trueness and RSDr were inferior to those of the official method. The performance parameters obtained by using the microwave digestion method (MW method) to prepare test solution were better than those of the official method. Thus, the MW method is available as an alternative method. Induced coupled plasma-mass spectrometry (ICP-MS) is also available as an alternative method. However, it is necessary to ensure complete digestion of the sample.

[Denaturation of egg antigens by cooking].

Changes in egg protein contents by cooking were measured with an ELISA kit using Tris-HCl buffer in model foods including cake, meatballs, pasta and pudding made with whole egg, egg-white and egg-yolk. The egg protein contents were lowest in the deep-fried model foods of cakes and meatballs. Ovalbumin (OVA) was undetectable (<1 µg/g) and ovomucoid (OVM) was lowest in pouched meatballs, suggesting that processing temperature and uniform heat-treatment affect the detection of egg protein. Furthermore, egg protein contents were below 6 µg/g in the pouched meatballs and pasta made with egg-yolk, and OVA and OVM were not detected by Western blotting analysis with human IgE from patients' serum. On the other hand, processed egg proteins were detected with an ELISA kit using a surfactant and reductant in the extract buffer.

[Comparison between old and new methods for detection of allergenic substances (egg and milk)].

The old ELISA method for detection of allergenic substances (egg and milk) in Kanagawa prefecture from 2003 to 2007, employed before improvement of the food allergen labeling system, yielded detection rates of 20% for egg and 30% for milk. In 2005, after improvement of the labeling system, the detection rate using the new ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol increased by about 10% for egg, but decreased by half for milk. There were 4 positive samples (over 10 µg/g) for both egg and milk proteins, on account of contamination by ingredients at the manufacturing line and the lack of proper food labeling. In 2009, the contamination levels of egg and milk proteins in labeled commercial foods were low. In a comparison between the new and old methods with the same samples, both the new ELISA and Western-blot analyses showed an increase in the detection rate of egg protein. In relation to milk protein, the detection rates were decreased with the new ELISA, although the ELISA detection rate and consistency rates with Western-blot analysis were increased. On the other hand, in the case of a protein content below 5 µg/g, it was impossible to determine ovomucoid and casein by Western-blot analysis.