Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

Two flavone 2'-glucosides from Scutellaria baicalensis.

Two new flavone glucosides, 5,2',6'-trihydroxy-6,7,8-trimethoxyflavone 2'-O-glucoside and 5,2',6'-trihydroxy-6,7-dimethoxyflavone 2'-O-glucoside were isolated from the aqueous methanol extract of the roots of Scutellaria baicalensis. From the extract, seven phenolics, 5,7,2',6'-terahydroxyflavone, 5,7,2',5'-tetrahydroxy-8,6'-dimethoxyflavone, skullcapflavone II, baicalin, baicalin methyl ester, wogonin 7-glucuronide and 3,5,7,2',6'-pentahydroxyflavanone were also isolated.

FD&C Red No. 105 (Rose Bengal B) neutralizes the thyroid tumor promoting effect of iodine-deficient diet in rats.

The effect of 3,4,5,6-tetrachloro-2',4',5',7'-tetraiodo-fluorescein sodium salt (Rose Bengal B or FD&C Red No. 105, molecular weight 1017.6) on the thyroid of rats treated with N-bis(2-hydroxypropyl)-nitrosamine (DHPN) and iodine-deficient (I-def) diet is studied. Six-week-old male F344 rats were divided into seven groups (20 each) and given a single subcutaneous injection of DHPN (2800 mg/kg body wt.). From week 2 to 20, I-def diet was given in combination with either FR105 (1.25, 5.0, or 20 mg/l) or potassium iodide (KI, 12.5, 50.0 or 200 micrograms/l) in drinking water. As a result, an amount of 1.25 mg/l of FR105 was slightly more effective than 200 micrograms/l of KI in terms of inhibition of the effect of I-def diet on thyroid weight, morphology, thyroid-related hormones and thyroid tumor development. It was calculated that 1 mumol/l of FR105 was slightly more potent than 1 mumol/l of iodide ion. As each FR105 molecule has four iodide residues, at least 25% of total iodide residues were calculated to be utilized by the rats given I-def diet.

Distribution and urinary excretion of aluminium injected with several organic acids into mice: relationship with chemical state in serum studied by the HPLC-ICP method.

The correlation between the in vivo tissue distribution or the urinary excretion of aluminium (Al) administered with four organic acids and the in vitro binding of Al to serum protein in the presence of the organic acids was studied: Aluminum was injected intraperitoneally into mice with an organic acid at a dose of 20 mg Al kg-1 (Al:ligand= 1:1 or 1:3). When the Al-citrate mixture was administered, hepatic and renal concentrations of Al at 3 h were low, while hepatic Al concentrations for the other three organic acids (malate, tartrate and isocitrate) were considerably higher. Compared to the sole administration of Al, urinary excretion of Al was at a high level in all organic acids. Aluminium added in vitro to control serum as an Al-citrate mixture was present only in a low-molecular-weight (LMW) fraction, while Al added with other organic acids was present in both the high-molecular-weight (HMW) and LMW fractions. Consequently, it was interpreted that Al in the LMW fractions was excreted into the urine and that a significant part of Al in the HMW fractions was transferred to the liver. The binding status of Al in serum observed in the in vitro study was also manifested in the in vivo samples obtained after the intravenous injection.

Spectroscopic characterization of SC-NTR: a subsidiary dye of allura red AC dye (FD&C red no. 40).

A major subsidiary dye in US certified Allura Red AC dye (FD&C Red No. 40) has been isolated by preparative high performance liquid chromatography. The paper chromatographic properties of the isolated dye indicate that it is the dye designated as SC-NTR in a previous paper (Marmion 1971). Spectroscopic analysis of the isolated dye is consistent with the disodium salt of 6-hydroxy-5-(2-methoxy-5-methyl-3-sulphophenylazo)-2-naphthalen esulphonic acid, which is an azo-coupling product between the meta-isomer of cresidine-p-sulphonic acid (CSA) and Schaeffer's salt (SS).

3-anilino-L-alanine, structural determination of UV-5, a contaminant in EMS-associated L-tryptophan samples.

An aniline derivative which corresponds to UV-5 in the preceding paper was isolated from the case L-tryptophan sample associated with eosinophilia-myalgia syndrome (EMS). By spectroscopic analyses, the structure was identified as 3-anilinoalanine. The compound was optically active, and the stereochemistry of alanine moiety was determined as L, by comparing the specific rotation with a synthesized 3-anilino-L-alanine.

The mutagenic constituents of Rubia tinctorum.

Twenty compounds were isolated from the roots of Rubia tinctorum which are used as a commercial source of madder color. Among these compounds, mollugin (1), 1-hydroxy-2-methylanthraquinone (2), 2-ethoxymethylanthraquinone(11), rubiadin (13), 1,3-dihydroxyanthraqunone (14), 7-hydroxy-2-methylanthraquinone (16), lucidin (17), 1-methoxymethylanthraquinone (18) and lucidin-3-O-primeveroside (19) showed mutagenicity with Salmonella typhimurium TA 100 and/or TA 98. Since the mutagenic compounds isolated are anthraquinone derivatives with the exception of compound 1, structure-mutagenicity relationships of the anthraquinones were also studied. The results suggested that the greatest activity is exhibited by 1,3-dihydroxyanthraquinones possessing methyl or hydroxylmethyl group on carbon 2.

In vitro effects of synthetic dyes on the syntheses of prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5-HETE).

The effects of thirty-one synthetic dyes (tar dyes in Japan), which are permitted in cosmetics, on three different enzymes were evaluated as an initial step in developing in vitro safety screens. The activities of the prostaglandin (PG)-synthetase from rabbit renal medulla and the lipoxygenases from potato tubers and guinea pig peritoneal polymorphonuclear leukocytes (PMN) were determined based on the biosyntheses of PGE2 and 5-hydroxyeicosatetraenoic acid (5-HETE), respectively. Xanthene dyes with halogen substituents exhibited marked inhibition on the PG-synthetase. Among the dyes which are permitted for any pharmaceutics and cosmetics, only the xanthene dyes exhibited inhibitory effects on the PG-synthetase at 2.5 mM. The xanthene dyes also showed inhibition and stimulation of potato lipoxygenase and PMN lipoxygenase, respectively.

Characterization of contaminants in EMS-associated L-tryptophan samples by high-performance liquid chromatography.

To identify chemical contaminant(s) associated with eosinophilia-myalgia syndrome (EMS), case and control lots of tryptophan were analyzed by HPLC with both UV and FL detection. Numerous contaminant peaks appeared on the chromatograms and some of them were identified as 5-hydroxytryptophan, indol aldehyde, indol, etc; from the retention time of authentic compounds. Among these, three peaks were significantly associated with case lots. One corresponds to di-tryptophan aminal of aldehyde (peak E). Others are unknown contaminants, UV-5 (FL-7) and UV-28 (FL-36). The structural elucidation and toxicological implication of UV-5 (FL-7) are currently in progress.

[Determination of thiabendazole (TBZ) in grapefruit].

The officially recognized gas chromatographic method (detector: nitrogen-phosphorus detector) for determination of thiabendazole (TBZ) in grapefruits, tends to give results that are higher than the actual values. This may be due to the fact that more of the TBZ in authentic TBZ solution is adsorbed on the column than TBZ in the sample solutions that contain a lot of impurities. Because of this difficulty, two liquid chromatographic methods were compared. If a fluorescence detector is available, the method of Nakazato et al. (ethyl acetate extraction and ion pair HPLC) is the best for preparation of test solution. When an ultraviolet detector is used, clean-up of the ethyl acetate extract is necessary. By the method of Kitada et al., the retention time of TBZ becomes shorter after injection of many samples. In view of these results, the method of Nakazato et al. is the methods-of-choice for TBZ in grapefruit.

Migration and material tests of some food-contact plastic wares made in Thailand.

Migration and material tests of food-contact plastic wares were carried out in compliance with the Thai Food Act. Sample materials studied were melamine resin, polyethylene, polypropylene, polystyrene, and polyvinyl chloride. Migration levels of phenol, formaldehyde, colors and heavy metals were determined. A test for vinyl chloride monomer in polyvinyl chloride was carried out. Migrant and residual levels in all samples were in compliance with the Act.

Release of formaldehyde and melamine from tableware made of melamine-formaldehyde resin.

The relationship between the concentrations of formaldehyde and melamine released into 4% acetic acid from dishes and bowls made of melamine-formaldehyde resin was determined. The average concentrations in the migration solution after the sample had been treated at 60, 80, and 95 degrees C for 30 min with 4% acetic acid were 0.0 +/- 0.1, 0.5 +/- 0.4 and 3.0 +/- 2.2 ppm, respectively for formaldehyde and 0.04 +/- 0.07, 0.21 +/- 0.20 and 1.19 +/- 1.18 ppm, respectively for melamine. The correlation between the concentrations of formaldehyde and melamine released at 95 degrees C was y=0.4858x-0.2728 (r=0.8860), where y is melamine concentration (ppm), x is formaldehyde concentration (ppm) and r is the correlation coefficient. The molar concentration ratios of formaldehyde to melamine (F/M ratio) were 15.4 +/- 11.6 at 80 degrees C and 14.9 +/- 10.1 at 95 degrees C. Hence the release of both migrants was affected by temperature but the F/M ratio was not affected. The release of both compounds was was increased on repetition of the migration test at 95 degrees C but their concentrations remained constant after the tenth and seventeenth repetitions of the treatment. During this period, the F/M ratio decreased according to the equation 1n y=-1.4344 1n x+3.7814 (r=-0.9984) for a sample before the tenth repetition of the treatment and remained between 1.7 and 1.9 after the twelfth repetition, where y is the F/M ratio and x is the number of repetitions of the treatment.

[Distinction between sodium chlorite and sodium hypochlorite by ion-chromatography].

A new method to distinguish between sodium chlorite and sodium hypochlorite by ion-chromatography is described. Under the operating condition (TSK-gel DEAE-5PW glass column 8.0 mm ID x 7.5 cm with an aqueous solution containing 20 mM sodium carbonate, 10 mM sodium hydroxide and 4 mM ethylenediamine) and electrochemical detector (Toso EC-8000, -200 mV, Ag-electrode), chlorite and hypochlorite anions were clearly separated and detected and the retention times (tR) were 6.0 and 4.6 min, respectively.

[Antimony and other heavy metals in metallic kitchen ware].

The antimony in metallic kitchen ware was determined. The content of this element in metals used for the production or repairing of utensils, containers and packaging which come in contact with foods is regulated and should be less than 5% in under the Japanese Food Sanitation Law. In eight metallic samples, antimony was detected in solder used for the production of a can for green tea and an eggbeater. The contents were 1.30% in the former and 1.90% in the latter. No antimony was detected in solder used for a cookie cutter. A sample of solder used for electric work, not for food utensils, contained 0.81% of antimony. In other metallic utensils which come in contact with food such as aluminum foil, a brass spoon, a stainless steel fork, a wire netting, and an iron rock for vegetable color stabilizing, antimony was not detected at a 0.05% detection limit. A qualitative test using rhodamine B also showed positive results in only three solder samples. Lead concentrations in solder used for the kitchen ware were from 39.3 to 51.3%. These concentrations were higher than the limit (20%) of lead content by the Law. No cadmium was detected in any samples.

[Modified method for the identification of sodium chlorite and sodium hypochlorite as food additives].

New methods for the identification of sodium chlorite and sodium hypochlorite were proposed. Especially, spectrophotometric method was found to be especially distinctive and effective for the identification of both food additives (Figs. 1-3).

Effects of food components and additives on the formation of nitrosamides.

The effects of food components and food additives on the formation of nitrosamides were studied. Citrate accelerated nitrosamide formation from nitrite and various precursors, such as ureas, urethanes, guanidines and amides. The accelerating effects of citrate on the formation of methylurea and ethylenethiourea were larger at higher pH values. The formation of most nitrosamides was not accelerated by thiocyanate, which is known to catalyze the formation of nitrosamines. Compounds having carboxyl groups tended to accelerate the nitrosation of methylurea. Nitrosation of methylurea was inhibited by thiols, polyphenols, alcohols, sorbic acid, dl-alpha-tocopherol etc., due to possibly competition for available nitrite.