Estrogenic activities of chemicals related to food contact plastics and rubbers tested by the yeast two-hybrid assay.

Food contact plastics and rubbers possibly contain many kinds of chemicals such as monomers, oligomers, additives, degradation products of polymers and additives, and impurities. Among them, bisphenol A, nonylphenol, benzylbutyl phthalate, styrene oligomers and hydroxylated benzophenones have been reported to possess estrogenic activities. In this study, other chemicals related to food contact plastics and rubbers, and their metabolites induced by the S9-mixture were tested for their estrogenic activities using the yeast two-hybrid assay. Among the 150 chemicals, 10 chemicals such as bis(4-hydroxyphenyl) methane, 4-cyclohexylphenol, 4-phenylphenol, 4,4'-isopropylidenediphenol alkylphosphite, two type of styrenated phenol (including mono type), tris(nonylphenyl) phosphite, 2,2'-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone and 2,4-diphenyl-4-methyl-1-pentene, their metabolites and the metabolites of 6 other chemicals, such as 2-(phenylmethyl) phenol, styrenated phenol (di and tri type), 1-(N-phenylamino)naphthalene, 4-tert-butylphenylsalicylate, nonylphenol ethoxylates and 2-methyl-6-tert-butylphenol, displayed estrogenic activities. All of them contained a phenol group in their chemical structures or formed one easily by hydrolysis or metabolism. However, most of the chemicals related to food contact plastics and rubbers, and their metabolites did not show any estrogenicity.

[Laboratory-performance study of the notified methods to detect genetically modified papaya (55-1)].

To investigate important factors affecting the reliability of the analytical results, proficiency tests were attempted for the histochemical method (GUS method) and the qualitative PCR method (PCR method) to detect genetically modified papaya (55-1) in the Japanease official method. The test samples were distributed to twenty-three laboratories that participated in the study and were examined according to the protocol. All the data collected from participating laboratories were statistically analyzed. In the PCR method, one negative sample was detected as positive using detection primers in one laboratory, though the sample was negative when checked using confirmation primers. Contamination might have occurred in the step of the preparation of the PCR sample solution using detection primers. In the GUS method, all the test samples were identified as expected. Thus, all the laboratories reported correct results overall.

A histochemical method using a substrate of beta-glucuronidase for detection of genetically modified papaya.

A histochemical assay for detecting genetically modified (GM) papaya (derived from Line 55-1) is described. GM papaya, currently undergoing a safety assessment in Japan, was developed using a construct that included a beta-glucuronidase (GUS) reporter gene linked to a virus coat protein (CP) gene. Histochemical assay was used to visualize the blue GUS reaction product from transgenic seed embryos. Twelve embryos per fruit were extracted from the papaya seeds using a surgical knife. The embryos were incubated with the substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc) in a 96-well microtiter plate for 10-15 hours at 37 degrees C. Seventy-five percent of GM papaya embryos should turn blue theoretically. The histochemical assay results were completely consistent with those from a qualitative polymerase chain reaction (PCR) method developed by this laboratory. Furthermore, the method was validated in a five-laboratory study. The method for detection of GM papaya is rapid and simple, and does not require use of specialized equipment.

[Material labeling of soft plastic toys and plasticizers in polyvinyl chloride products].

We investigated the labeling and content of plasticizers of soft plastic toys. First, we investigated material labeling in 96 products purchased in fiscal years 2000 and 2001. Among these products, 43% of those purchased in fiscal 2000 and 68% of those purchased in fiscal 2001 were labeled. We then investigated the kinds and amounts of plasticizers in 73 soft polyvinyl chloride (PVC) toys. Three kinds of phthalates and six other kinds of plasticizers were detected in the soft PVC toys. Diisononyl phthalate, di(2-ethylhexyl) phthalate, diisononyl adipate, and O-acetyl tributyl citrate were detected at high frequency, and in large amounts. The average total content of all plasticizers was 280 mg/g for the products purchased in fiscal 2000 and 227 mg/g for those in fiscal 2001. In fiscal 2001, antioxidants, 4-nonylphenol, and bisphenol A were investigated in addition to plasticizers.

[Identification of migrants from nitrile-butadiene rubber gloves].

Polyvinyl chloride gloves containing di(2-ethylhexyl) phthalate are restricted for food contact use. In their place, disposable gloves made from nitrile-butadiene rubber (NBR) are used in contact with foodstuffs. Some unknown substances were found to migrate into n-heptane from NBR gloves. By GC/MS, HR-MS and NMR, their chemical structures were confirmed to be 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (used as a plasticizer), 4,4'-butylidenedi(6-tert-butyl-m-cresol), a mixture of styrenated phenols consisting of 2-(alpha-methylbenzyl)phenol, 4-(alpha-methylbenzyl)phenol, 2,6-di(alpha-methylbenzyl)phenol, 2,4-di(alpha-methylbenzyl)phenol and 2,4,6-tri(alpha-methylbenzyl)phenol (used as antioxidants), and 2,4-di-tert-butylphenol, which seems to a degradation product of antioxidant. Migration levels of these compounds were 1.68 micrograms/cm2 of 2,4-di-tert-butylphenol, 2.80 micrograms/cm2 of 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, 46.08 micrograms/cm2 of styrenated phenols and 4.22 micrograms/cm2 of 4,4'-butylidenedi(6-tert-butyl-m-cresol) into n-heptane, respectively. The content of total styrenated phenols was 6,900 micrograms/g in NBR gloves.

[Inter-laboratory evaluation studies of notified ELISA methods for allergic substances (egg)].

Inter-laboratory evaluation studies were conducted for the notified ELISA methods for allergic substances (Egg). Standard extracts of egg spiked in extracts of sausage, sauce, cookie, bread and cereal at a level of 5-20 ng/mL as the sample solution were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of all three ELISA methods using an Egg Protein ovalbumin ELISA Kit (ovalbumin kit), an Egg Protein ovomucoid ELISA Kit (ovomucoid kit) and a FASTKIT Egg ELISA kit (Egg ELISA kit) were mostly less than 10%. Mean recoveries of the standard extract of egg were over 40% in the three ELISA methods. Repeatability relative standard deviations of egg standard solution in five food extracts were in the ranges of 18.7-25.5%, 18.6-41.8%, 21.3-43.3% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. Reproducibility relative standard deviations of egg standard solution in five food extracts were 16.8-35.1%, 19.6-35.8%, 24.7-51.1% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. The detection limits of all the ELISA methods were 4-5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of egg protein levels in extracts of sausage, sauce, cookie, bread and cereal.

[Laboratory-performance study of the notified methods to detect genetically modified maize (CBH351) and potato (NewLeaf Plus and NewLeaf Y)].

To investigate the key factors affecting the reliability of the analytical results, a laboratory-performance study was attempted for the notified methods to detect genetically modified (GM) maize (CBH351) and GM potato (NewLeaf Plus and NewLeaf Y). The test samples were designed as three pairs of blind duplicates, which included 0%, 0.1% and 1.0% GM maize (CBH351) or GM potato (NewLeaf Plus or NewLeaf Y). Fourteen laboratories participated in the study. The test samples were sent to the participating laboratories along with the protocol. The data were collected from all laboratories and statistically analyzed. For the 0% sample of the CBH351 maize, one laboratory reported a false-positive result. It was considered that contamination could have occurred via the common use of equipment or tools for the test. For the 0.1% samples of the NewLeaf Plus potato or NewLeaf Y potato, on the other hand, three laboratories reported false-negative results. It was presumed that these results were due to changes of the conditions of the electrophoresis and agarose-gel staining. The other laboratories reported appropriate results. It was considered that the method employed in this study was suitable for the assessment of laboratory performance.

[Identification of unknown substances in polyvinyl chloride gloves containing non-phthalate plasticizers].

The use of polyvinyl chloride gloves containing di(2-ethylhexyl) phthalate for food contact applications is restricted. In their place, polyvinyl chloride gloves containing non-phthalate plasticizers (PVC-NP) have been introduced. They contained unknown substances, so they were studied by GC/MS, HR-MS and NMR. The chemical structures of the unknown substances were confirmed to be diethylene glycol dibenzoate, triethylene glycol dibenzoate, dipropylene glycol dibenzoate, and alkylsulfonic acid phenyl ester, which are plasticizers. Including di-isononyl adipate, the contents of the plasticizers were 37.5-48.9% in the PVC-NP gloves, and their migration levels were 1,010-1,390 ppm into n-heptane. These are very high levels. These plasticizers are not widely used for food contact polyvinyl chloride throughout the world, and they have also not been registered as self-standards by Japanese manufacturers. Careful consideration will be necessary for the selection of a suitable plasticizer substitute.

[A detection method for recombinant DNA from genetically modified potato (NewLeaf Y potato)].

A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.