Proposal of a flow scheme for the chemical-form-based quantitative analysis of chlorine compounds in pulp for sanitary products and verification of safety.

To determine the amounts and chemical forms of chlorine compounds in elemental chlorine-free (ECF) bleached pulp for sanitary products, a chemical-form-based quantitative analysis flow scheme was created. The scheme involves quantitative determination of compounds eluted in elution tests assuming dermal exposure. The results indicated that most of the chlorine compounds in pulp used for sanitary products were insoluble organically bound chlorine and water-soluble chlorides consisting of chloride ions. The total amount of chlorine obtained by analysis of the total chlorine in the pulp products was close to the sum of the amounts of water-soluble chlorides, residual chlorine, chloroacetic acids, and insoluble organically bound chlorine. Therefore, the balance of chlorine measured by the flow scheme was acceptable. Little residual chlorine was detected in the pulp products, suggesting that the chlorine dioxide used for bleaching was almost completely converted into inactive inorganic chlorides and organic chlorine compounds and that these substances do not affect the health risk posed by pulp products. A risk assessment of the detected chloroacetic acid and dichloroacetic acid revealed that there is no concern about the health risk posed by use of these products.

Investigation of the amount of transdermal exposure of newborn babies to phthalates in paper diapers and certification of the safety of paper diapers.

A risk assessment study of seven phthalates in paper diapers for newborn babies produced in Japan was performed. The diapers were purchased and the contents of the seven phthalates were determined and estimated amounts of exposure were calculated based on the eluted rate into artificial medium of urine or sweat, average weight of infants, and frequency of use. Di-2-ethylhexyl phthalate and di-n-butyl phthalate were detected in the topsheets and determined to be 0.6 µg/g and 0.2 µg/g, respectively. The daily estimated exposure volume was calculated to be in the range of 1.86 × 10(-10)-2.98 × 10(-6) mg/kg/day as follows: content of seven phthalates in the topsheet (0.1-1 µg/g) × eluted rate of phthalates into artificial sweat (0.0006-2.4%) × weight of the topsheet of a diaper (1.5 g) × the number of diapers used per day (12 sheets) × skin absorption rate (0.005-0.1)/average body weight (2.9 kg). For hazard assessment, we used 0.2-300 mg/kg/day for the seven phthalates based on the data available at international agencies. The margin of exposure to the seven phthalates was 6.71 × 10(4)-1.99 × 10(11), indicating that the risk of exposure to phthalates from the diapers produced in Japan was negligible.

Endocrine-mediated effects of two benzene related compounds, 1-chloro-4-(chloromethyl)benzene and 1,3-diethyl benzene, based on subacute oral toxicity studies using rats.

The purpose of this study was to investigate the endocrine-mediated effects of the benzene-related compounds with reference to Organization for Economic Co-operation and Development (OECD) Test Guideline No. 407. Rats were orally gavaged with 0, 10, 50, and 250 mg/kg/day of 1-chloro-4-(chloromethyl)benzene, and 0, 25, 150, and 1000 mg/kg/day of 1,3-diethyl benzene for at least 28 days, beginning at 8 weeks of age. Thyroid dysfunction was observed in rats given the 1,3-diethyl benzene. Serum T4 values increased in all groups of male rats and in the 1000 mg/kg group of female rats, and TSH values also increased in the 1000 mg/kg groups of both sexes after 28 days' administration. Decreased T3 values were observed in the 1000 mg/kg group of female rats after 28 days' administration, and hormone values increased in the 1000 mg/kg groups of both sexes after the 14-day recovery period. In addition, thyroid weight increased in the 1000 mg/kg groups and thyroid follicular cell hyperplasia was detected in one male rat from the 1000 mg/kg group after 28 days' administration. Endocrine-mediated effects, including thyroid dysfunction were not observed in any groups of rats treated with 1-chloro-4-(chloromethyl)benzene. Our results indicated that endocrine-mediated effects such as thyroid dysfunction were associated with some benzene-related compounds.

Enhanced OECD TG 407 in detection of endocrine-mediated effects of 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol.

The purpose of this study was to investigate whether the estrogenic effects were detected in the enhanced TG 407 if the estrogenic property was not so strong in the uterotrophic assay. The estrogenic property of 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol in the uterotrophic assay was slightly stronger than that of genistein or nonylphenol, but weaker than that of ethinyl estradiol. We performed a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) on 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol based on the OECD draft protocol. The test chemical, administered orally at doses of 0, 10, 50, and 250 mg/kg per day for at least 28 days, caused such estrogenic effects as abnormal estrous cycle, increased ovarian follicles, increased uterine epithelial height, and vaginal mucification in the 50 and/or 250 mg/kg groups. Moreover, follicular epithelial cell hyperplasia of the thyroid was detected in all male rats given the test chemical and in female rats in the 250 mg/kg group. It was concluded that the estrogenic effects were detected in growing rats given 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, and thyroid dysfunction was also observed as the endocrine-mediated effects.

Effects of in utero through lactational exposure to dicyclohexyl phthalate and p,p'-DDE in Sprague-Dawley rats.

Anti-androgenic chemicals alter sexual differentiation by a variety of mechanisms, and the mechanisms between phthalate esters and p,p'-DDE are considered to be different. We performed an in utero through lactational exposure assay using dicyclohexyl phthalate and p,p'-DDE to investigate the sexual differentiation of these chemicals. Pregnant CD (SD) IGS rats were given dicyclohexyl phthalate or p,p'-DDE orally from gestational day (GD) 6 to postnatal day (PND) 20, and the endocrine-mediated effects in dams and their offspring were examined. The reproductive performance of offspring was also examined. The doses of dicyclohexyl phthalate were 0, 20, 100, and 500 mg/kg/day, and those of p,p'-DDE were 5, 15, and 50mg/kg/day. Using the dicyclohexyl phthalate, a dam in the 500 mg/kg group showed dystocia and died. The viability index of offspring on PND 4 decreased in the 500 mg/kg group. Prolonged preputial separation, reduced ano-genital distance, increased areolas/nipple retention, hypospadia, decreased ventral prostate and levator ani/bulbocavernosus muscle weights and decreased testicular germ cells were observed in male offspring in the 500 mg/kg group. In the assay using p,p'-DDE, decreased viability index of offspring on PND 21, prolonged preputial separation in male offspring and early vaginal opening in female offspring were observed in the 50mg/kg group. The copulation and fertility indices decreased in the reproductive performance of offspring in the 50mg/kg group. The endocrine-mediated effects were detected in offspring of dams given 100mg/kg dicyclohexyl phthalate, and in offspring of dams given 20mg/kg p,p'-DDE. Our results suggest that the in utero through lactational exposure assay is a useful method to detect endocrine-mediated effects and that further comparative study between this assay and two-generation reproductive test are necessary when this assay becomes one of the definitive tests.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 3-amino-1,2,4-triazole based on the Organization for Economic Co-operation and Development draft protocols.

We performed a uterotrophic assay, the Hershberger assay, and the 28-day repeated-dose toxicity study (enhanced OECD test guideline no. 407) of 3-amino-1,2,4-triazole based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay, the test chemical was orally administered at doses of 0, 40, 200, and 1,000 mg/kg/day to castrated male Wistar rats for 10 consecutive days beginning on postnatal day 56, and no androgen agonistic and antagonistic changes were observed. Alternatively, when the test chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid follicular epithelial cell hypertrophy with increased thyroid weights was detected in the male and female rats in 25 and/or 125 mg/kg groups, and hypertrophy of the anterior pituitary cells with increased pituitary weights in male and female rats was also observed in the 125 mg/kg group. Furthermore, serum T3 and T4 values decreased and serum TSH values increased in male and female rats in the 125 mg/kg group. Therefore, 3-amino-1,2,4-triazole was concluded to have anti-thyroid acting as endocrine-mediated effects, but no estrogenic or androgenic effects. In addition, decreased body weight, and abnormal biochemical parameters attributed to thyroid, liver or kidney dysfunction were observed in male and female rats in the 25 and/or 125 mg/kg groups.

Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo.

The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), beta-naphthoflavone (beta-NF), indirubin, indigo, 3,3'-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and beta-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr (-/-)). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols.

We performed a uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study [enhanced Organization for Economic Co-operation and Development (OECD) test guideline No. 407] of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols. In the uterotrophic assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), the test chemical was orally administered to castrated male SD rats at doses of 0, 50, 200, and 1,000 mg/kg/day for ten consecutive days beginning on postnatal day 56, and no changes were observed. When this chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, an increase in thyroid weight was observed in the female rats in the 125 mg/kg group, an increase in serum thyroid-stimulating hormone (TSH) values in the male and female rats in the 125 mg/kg group, and a decrease in serum T3 and T4 values in the male rats in the 125 mg/kg group, and thyroid follicular epithelial cell hypertrophy was observed in some of the female rats in the 125 mg/kg group. These findings were concluded to be the result of endocrine-mediated effects of the chemical on thyroid function. In addition, increased liver weight, abnormal histological findings in the liver, and abnormal biochemical parameters related to liver function were observed in male and/or female rats in 5 mg/kg group and higher dose groups. The no-observed-effect level for 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) was concluded to be <5 mg/kg/day. In the uterotrophic assay of 3-(dibutylamino)phenol, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and no changes were observed. In the Hershberger assay of 3-(dibutylamino)phenol, the test chemical was orally administered at doses of 0, 50, 200, and 400 mg/kg/day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when this test chemical was orally administered at doses 0, 30, 100, and 300 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid weight increased in the male rats in the 300 mg/kg group, thyroid follicular epithelial cell hypertrophy was observed in a small number of male rats in the 300 mg/kg group, serum T3-values decreased in the female rats in the 300 mg/kg group, and a tendency for TSH-values to increase was observed in the male and female rats in the 300 mg/kg group. Therefore, 3-(dibutylamino)phenol was also concluded to have slight anti-thyroid acting effects as the endocrine-mediated effects. On the other hand, increased hemosiderin deposition in the spleen, increased spleen weight, hematological abnormalities, and squamous epithelial hyperplasia of the forestomach were detected in male and/or female rats in the 100 and/or 300 mg/kg groups, and thus the no-observed-effect level for 3-(dibutylamino)phenol was concluded to be 30 mg/kg/day.

Subacute oral toxicity study of bisphenol F based on the draft protocol for the "Enhanced OECD Test Guideline no. 407".

Since bisphenol F (4,4'-dihydroxydiphenylmethane) has been reported to exhibit estrogen agonistic properties in the uterotrophic assay, we performed a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) on bisphenol F based on the OECD draft protocols to determine whether it has endocrine-mediated properties. Bisphenol F was orally administered at doses 0, 20, 100 and 500 mg/kg per day for at least 28 days, but no clear endocrine-mediated changes were detected, and it was concluded to have no endocrine-mediated effects in young adult rats. On the other hand, the main effect of bisphenol F was concluded to be liver toxicity based on clinical biochemical parameters and liver weight, but without histopathological changes. The no-observed-effect level for bisphenol F is concluded to be under 20 mg/kg per day since decreased body weight accompanied by decreased serum total cholesterol, glucose, and albumin values were observed in the female rats given 20 mg/kg per day or higher doses of bisphenol F.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4 -[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols.

We performed an uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) of 4,4 -[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and the uterine weight of rats given the 1,000 mg/kg dose of the test chemical plus ethinyl estradiol decreased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 100, 300, and 1,000 mg/kg day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when the test chemical was orally administered at doses 0, 100, 300, and 1,000 mg/kg day for at least 28 days, a decrease in LH values in rats of both sexes and a decrease in FSH and estradiol values in female rats were detected in the 1,000 mg/kg group, and abnormal estrous cycles, uterine glandular atrophy, persistence of ovarian corpora lutea, vaginal epithelial mucification, and mammary glandular hyperplasia were also observed in one female rat in the 1,000 mg/kg group. Therefore, the uterotrophic assay used in this study showed that the chemical has the estrogen-antagonist properties, and some potentially endocrine-mediated effects were detected in growing rats based on the results of the enhanced OECD test guideline No. 407. However, the changes were observed in rats given a high dose of the chemical, 1,000 mg/kg day.

Comprehensive identification of cytochrome p450 isoforms from solubility-based fractionated rat liver microsomes.

Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms. The combination of proteomic analysis and the solubility-based fractionation would provide powerful tool for the expression analysis of the superfamily proteins having great similarities between the amino acids sequences.

Subacute oral toxicity study of diethylphthalate based on the draft protocol for "Enhanced OECD Test Guideline no. 407".

We performed a 28-day repeated-dose toxicity study of diethylphthalate based on the draft protocol of the "Enhanced OECD Test Guideline 407" to investigate whether it has endocrine-mediated properties according to this assay. Diethylphthalate was orally administered to SD rats at doses of 0, 40, 200, and 1,000 mg/kg/day for at least 28 days, but no endocrine-mediated effects were detected based on any of the parameters examined, suggesting that diethylphthalate does not possess endocrine properties according to this assay.

Subacute oral toxicity study of di(2-ethylhexyl)adipate based on the draft protocol for the "Enhanced OECD Test Guideline no. 407".

We performed a 28-day repeated-dose toxicity study of di(2-ethylhexyl)adipate (DEHA) based on the draft protocol of the "Enhanced OECD Test Guideline 407" to investigate whether it has endocrine-mediated properties according to this assay. DEHA was orally administered to SD rats at doses of 0, 40, 200 and 1,000 mg/kg/day for at least 28 days, and disturbance of the estrous cycle and increased ovarian follicle atresia were detected in the 1,000 mg/kg group.