Reactions of sunscreen agents, octyl dimethyl-p-aminobenzoate (ODPABA) and octyl-p-methoxycinnamate (OMC), with hypochlorite in aqueous solution were investigated under the conditions that simulate swimming pool disinfection sites. Chlorination byproducts were determined by GC-MS. At a concentration of 9 µM, ODPABA reacted rapidly with free chlorine in the buffered solution at pH 7.0, OMC reacted with hypochlorite reasonably slowly under the same condition. ODPABA and OMC produced chlorine-substituted compounds as intermediates, which were decomposed to cleavaged products of ester-bond during the aqueous chlorination process. The chlorination intermediates of OMC exhibited weak mutagenic on Salmonella typhimurium TA100 strain without the S9 mix. The extent of the reactions depended on the chlorine dose, solution pH, and compound structures.

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tyl phthalate (DBP). 1)Some phthalate diesters induce testicular toxicity in the male reproductive tract. Jobling et al. showed that DBP binds to the estrogen receptor, displaces its natural ligand, and then acts as an estrogen antagonist.2) In addition, mono-n-butyl phthalate (MBuP) adversely affects development of the reproductive system in male rats.3) DEHP is a peroxisome proliferator that induces tumors in the rodent liver, and this effect is probably not exerted by DEHP itself but by one or more of its metabolites. 4)Phthalate diesters are metabolized to monoesters by hydrolases in many tissues, such as the pancreas, intestine, and blood.5-7) Generally orally ingested dialkyl phthalates are hydrolyzed by esterases in the wall of the small intestine and pancreatic lipases and not by gut flora. Absorption occurs almost entirely as the corresponding monoesters.5) Human saliva is a matrix of inorganic compounds, saliva proteins such as lingual lipase and α-amylase, oral flora, and cell debris. 8) However, to the best of our knowledge there are no reports concerning enzymatic hydrolysis of phthalate diesters in human saliva, except for our previous paper.9) The present work was therefore designed to characterize human salivary esterase in enzymatic hydrolysis of phthalate esters. These findings provide the information that will be required in considering the human health effects of phthalate esters. MATERIALS AND METHODSChemicals and Glassware ---Monoethyl phthalate (MEP), diethyl phthalate (DEP), and DEHP were purchased from Wako Pure Chemical Industries. (Osaka, Japan) and MBuP, mono-n-hexyl phthalate (MHxP), mono-2-ethylhexyl phthalate (MEHP), monobenzyl phthalate (MBeP), and di-n-hexyl phEnzymatic hydrolysis of phthalate esters in human saliva was investigated to characterize salivary esterase in the formation of monoesters from their diesters. The monoesters formed were analyzed by GC/ MS after incubation of phthalate diesters in the saliva. Hydrolytic activity in the supernatant obtained by centrifugation of the saliva at 1350 × g was equivalent to that in whole saliva, and the activity was inhibited by the addition of denaturing protein. The hydrolytic activity was dependent on the protein concentration in the supernatant. The optimum temperature and pH of the hydrolysis was 50°C and 8.2, respectively. In addition, the 80% acetone powder of the supernatant showed high substrate specificity for straight-chain alkyl group of phthalate diesters, especially the butyl group, whereas almost no specificity was seen for the 2-ethylhexyl and benzyl groups. These results indicate that not the oral flora but salivary esterase, such as lingual lipase, is involved in phthalate monoester formation from the diesters in human saliva, and do not act on the hydrolysis of monoester.

Human volunteers chewed polyvinyl chloride (PVC) products under controlled conditions for 60 min (four consecutive periods of 15 min), and then the amount of diisononyl phthalate (DINP) migration into the saliva was determined by HPLC. The PVC products consisted of a molded plate containing added DINP (500 mg/g) and five types of commercial toy containing 160-583 mg/g DINP (mean value 372 mg/g). The DINP migration rates ranged from 3.8 to 32.6 µg/cm 2 / hr (mean value 16.4 µg/cm 2 /hr). The DINP contents in the toy products did not correlate with the amount of in vivo migration. The DINP migration rates during the last 15-min period (45-60 min) decreased by 38-75% compared with the first session (0-15 min). In addition, in vitro DINP migration rates from the PVC products into saliva simulant during 15 min of rotary shaking were about 3.6-4.1-fold higher than the rates in vivo over 60 min, and remained essentially fixed for each sample.

The migration dialkyl phthalate was tested in volunteers who chewed polyvinyl chloride (PVC) toy products under controlled conditions. The PVC toy samples consisted of ball A containing 100 and 185 mg/ g di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (

-Reactions of nitrofuran antibiotics (nitrofurazone (NFZ) and frazolidone (FZD)) with hypochlorite in aqueous solution were investigated under the conditions that simulate wastewater disinfection. The chlorination byproducts were determined by high performance liquid chromatography. At -rite reaction was reasonably slow under the same pH. Nevertheless, the strong mutagenic parents disappeared completely after the hypochlorite reactions, and the chlorination byproducts were observed to exert a weak mutagenic effect on Salmonella typhimurium TA100 without S9-mix. The extent of the reactions depended on the chlorine dose, solution pH and compound structures.

We describe a method of mechanical agitation to determine rates of dialkyl phthalate migration from polyvinyl chloride (PVC) products into saliva simulant. The method consists of rotary shaking of a sample with 30 mL of saliva simulant (pH 7.0) at 35ῌ in a 50 mL glass tube at 300 rpm for 15 min, then measuring the amount of dialkyl phthalate in the saliva simulant by HPLC with a UV detector. The migration rates of diisononyl phthalate (DINP), di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) from PVC plates containing about 45῍ (w/w) plasticizer (molded in our laboratory) were identical. However, the migration rates from molded plates containing 13῍ (w/w) DBP were almost double those of DINP and DEHP at the same ratios. In addition, the amounts of DINP that migrated in vitro after rotary shaking for 15 min were equivalent to those in vivo determined in saliva from volunteers who chewed plates for 60 min. The migration rates of dialkyl phthalates from 11 commercially available toys ranged from 15.6 to 85.2 mg/cm 2 /h [relative standard deviation (RSD), 3 to 12῍].Key words: dialkyl phthalate; polyvinyl chloride (PVC) product; saliva simulant; rotation shaking; migration rate Introduction Dialkyl phthalates are widely used as plasticizers to impart softness and flexibility to normally rigid plastics such as polyvinyl chloride (PVC). Medical devices, toys and child-care articles are often made of PVC and contain diisononyl phthalate (DINP), di-2-ethylhexyl phthalate (DEHP) and/or di-n-butyl phthalate (DBP) as the predominant plasticizers 1)ῌ3) . Some dialkyl phthalates have testicular toxicity and other e#ects on the reproductive tracts of male and female animals 4) . The level of toxicity di#ers considerably depending on the nature of the alkyl chain 5) . Wine et al. have identified the testicular toxicity of DBP in rats by reproductive assessment using a continuous breeding protocol 6) . DEHP and DINP are peroxisome proliferators that induce tumors in the rodent liver 7) . Infants are uniquely exposed to dialkyl phthalates, especially DINP, since it is the major plasticizer in toys 3), 8) . The DINP and DEHP migration rates in children chewing or sucking PVC toys has been measured using two types of methods. Adult volunteers have chewed PVC products under controlled conditions (in vivo method) 9)ῌ12) and PVC toys have been shaken or impacted in a saliva simulant in vitro, but the migration rates varied considerably, with poor reproducibility. In addition, large amounts of data are di$cult to obtain in vivo for both practical and ethical reasons. Therefore, a simple and reproducible in vitro migration test is required.We found that the results of a 15-min in vitro migration test of DINP from PVC toys by mechanical agitation, especially rotary shaking, were well correlated with the results of in vivo tests over 60 min 14) . We also evaluated the e#ect of experimental conditions on the dialkyl phthalate migration rates into saliva simulant from PVC products using rotary shaking, and we have established ...

tyl phthalate (DBP), and/or diisononyl phthalate (DINP).1) These compounds have been identified as contaminants of environmental water, soil, and the atmosphere, and have been detected in human serum and plasma.2) Some phthalate diesters induce testicular toxicity and other effects on the male and the female reproductive tract.3) Zeiger et al. found that 16 phthalate diesters had no mutagenic activity in the Ames assay.4) Therefore the hepatocarcinogenic effect of DEHP must be exerted during the promotion phase, since this compound is not genotoxic. 5) Both DEHP and DINP are peroxisome proliferators that induce tumors in the rodent liver.6) This effect is probably not directly exerted by the diesters but by their monoesters and oxidative products.7) In addition, phthalate diesters are metabolized to monoesters by hydrolases in many tissues, such as the intestine, pancreas, and blood, 8) where they are absorbed almost entirely as the corresponding monoesters. 9)Although others have estimated the tumor initiation and/or promotion activity of DEHP, only a few have examined the in vitro promoting activity of other phthalate diesters and related monoesters at low doses. The present study screened the tumorpromoting activity of four phthalate diesters and three monoesters in vitro using BALB/c3T3 cocultured with transformed cells, as well as transformation assays using Bhas cells (v-Ha-ras-transfected BALB/3T3 cells). In addition, we evaluated the influence of monoesters formed by the hydrolysis of phthalate diesters on tumor-promoting activity in Bhas cells. MATERIALS AND METHODSReagents ---DBP and DINP were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). DiethylThe tumor-promoting activity of four phthalate diesters and three monoesters were tested in vitro in a screening assay using a transformed and non transformed BALB/c3T3 cell mixture and in a transformation assay using Bhas cells (v-Ha-ras-transfected BALB/3T3 cells). Di-2-ethylhexyl phthalate (DEHP), di-n-butyl phthalate (DBP), and their monoesters increased cell proliferation in the screening assay. Monon-butyl phthalate (MBuP) and mono-2-ethylhexyl phthalate (MEHP) promoted Bhas cell transformation, whereas the activities of DBP and DEHP were weak. The activity of MEHP was about 1/500 that of 12-Otetra-decanoyl-phorbol-13-acetate. MBuP and MEHP therefore showed tumor-promoting activity, whereas all the other phthalate esters tested, including di-isononyl phthalate, had little or no such activity. In addition, MBuP and MEHP were formed by hydrolysis in Bhas cells exposed to DBP and DEHP for 4 days. We postulate that MEHP plays a role in the slight ability of DEHP to promote cell proliferation.