Application of the threshold of toxicological concern (TTC) in the evaluation of drinking water contact chemicals.

The Threshold of Toxicological Concern (TTC) is an approach for assessing the safety of chemicals with low levels of exposure for which limited toxicology data are available. The original TTC criteria were derived for oral exposures from a distributional analysis of a dataset of 613 chemicals that identified 5th percentile no observed effect level (NOEL) values grouped within three tiers of compounds having specific structural functional groups and/or toxic potencies known as Cramer I, II and III classifications. Subsequent assessments of the TTC approach have established current thresholds to be scientifically robust. While the TTC has gained acknowledgment and acceptance by many regulatory agencies and organizations, use of the TTC approach in evaluating drinking water chemicals has been limited. To apply the TTC concept to drinking water chemicals, an exposure-based approach that incorporates the current weight of evidence for the target chemical is presented. Such an approach provides a comparative point of departure to the 5th percentile TTC NOEL using existing data, while conserving the allocation of toxicological resources for quantitative risk assessment to chemicals with greater exposure or toxicity. This approach will be considered for incorporation into NSF/ANSI/CAN 600, a health effects standard used in the safety evaluation of chemicals present in drinking water from drinking water contact additives and materials certified to NSF/ANSI/CAN 60 and 61, respectively.

Toxicological assessment of nattokinase derived from Bacillus subtilis var. natto.

Subtilisin NAT, commonly known as "nattokinase," is a fibrinolytic enzyme produced by the bacterial strain B. subtilis var. natto, which plays a central role in the fermentation of soybeans into the popular Japanese food natto. Recent studies have reported on the potential anticoagulatory and antihypertensive effects of nattokinase administration in humans, with no indication of adverse effects. To evaluate the safety of nattokinase in a more comprehensive manner, several GLP-compliant studies in rodents and human volunteers have been conducted with the enzyme product, NSK-SD (Japan Bio Science Laboratory Co., Ltd., Japan). Nattokinase was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 28-day and 90-day subchronic toxicity studies conducted in Sprague-Dawley rats at doses up to 167 mg/kg-day and 1000 mg/kg-day, respectively. Mice inoculated with 7.55 × 10(8) CFU of the enzyme-producing bacterial strain showed no signs of toxicity or residual tissue concentrations of viable bacteria. Additionally consumption of 10 mg/kg-day nattokinase for 4 weeks was well tolerated in healthy human volunteers. These findings suggest that the oral consumption of nattokinase is of low toxicological concern. The 90-day oral subchronic NOAEL for nattokinase in male and female Sprague-Dawley rats is 1000 mg/kg-day, the highest dose tested.

Derivation of an oral reference dose (RfD) for the plasticizer, di-(2-propylheptyl)phthalate (Palatinol® 10-P).

Di-(2-propylheptyl) phthalate (DPHP) is a high molecular weight polyvinyl chloride plasticizer. Since increasing production volume and broad utility may result in human exposure, an oral reference dose (RfD) was derived from laboratory animal data due to the lack of human data. In addition to liver and kidney, target organs were the thyroid, pituitary and adrenal glands in rats, recognizing that reproductive performance was not altered in two successive generations of DPHP-exposed rats. DPHP caused a reduction in pup and maternal body weights but not developmental or testicular effects typical of "phthalate syndrome." DPHP was not genotoxic. Due to the lack of carcinogenicity data, there is inadequate information to assess carcinogenic potential. The RfD of 0.1mg/kg-day was derived from the human equivalent BMDL10 of 10mg/kg-day for thyroid hypertrophy/hyperplasia in male F1 adults from the two-generation study. While in utero exposure did not alter sensitivity to thyroid lesions compared to subchronic exposures beginning at 6weeks of age, F1 adult males were the longest-term exposed population. The total uncertainty factor of 100x was comprised of intraspecies (10x), study duration (3x), and database (3x) factors but not an interspecies factor since rodents are more sensitive than humans to thyroid gland effects.

Derivation of an oral reference dose (RfD) for the nonphthalate alternative plasticizer 1,2-cyclohexane dicarboxylic acid, di-isononyl ester (DINCH).

1,2-Cyclohexanedicarboxylic acid, 1,2-diisononylester (DINCH), a polyvinyl chloride plasticizer, has food, beverage, and medical device applications that may result in general population exposure. Although no apparent toxicity information in humans was identified, there is a substantial data set in lab animals to serve as the basis of hazard identification for DINCH. Target tissues associated with repeated dietary DINCH exposure in lab animals included liver, kidney, and thyroid and mammary glands. In contrast to some phthalate ester plasticizers, DINCH did not show evidence of hepatic peroxisomal proliferation, testicular toxicity, or liver tumors in rats. Liver and thyroid effects associated with DINCH exposure were attributed to compensatory thyroid stimulation secondary to prolonged metabolic enzyme induction. The toxicological significance of mammary fibroadenomas in female rats is unclear, given that this common benign and spontaneously occurring tumor type is unique to rats. The weight of evidence suggests DINCH is not genotoxic and the proposed mode of action (MOA) for thyroid gland lesions was considered to have a threshold. No adverse reproductive effects were seen in a two-generation study. An oral reference dose (RfD) of 0.7 mg/kg-d was derived from a human equivalent BMDL10 of 21 mg/kg-d for thyroid hypertrophy/hyperplasia seen in adult F1 rats also exposed in utero. The total uncertainty factor of 30x was comprised of intraspecies (10×) and database (3×) factors. An interspecies extrapolation factor was not applied since rodents are more sensitive than humans with respect to the proposed indirect MOA for thyroid gland lesions.

Establishing a total allowable concentration of o-toluidine in drinking water incorporating early lifestage exposure and susceptibility.

o-Toluidine is a monocyclic aromatic amine present in the formulation of some materials that contact drinking water. NSF/ANSI 61 Annex A (2011) and US EPA (2005a) risk assessment guidelines were used to determine an acceptable drinking water level. Occupational exposure to o-toluidine is associated with an increased risk of bladder cancer but human disease rates could not be used to establish risk values due to inadequate exposure data and coexposures in the epidemiology cohorts. Chronic dietary exposure to o-toluidine hydrochloride was associated with benign and malignant tumors in both sexes of F344 rats and B6C3F1 mice. o-Toluidine is genotoxic in vitro and in vivo. A 10(-5) cancer risk level was extrapolated from the human equivalent BMDL(10) of 13mg/kg-day for the combined incidence of papillomas and carcinomas of the bladder transitional epithelium in female rats. Considering varying susceptibility to tumor development at different life stages, the unit risk was modified to incorporate potency adjustments for early-life exposures. A framework for lifestage adjustment is presented that makes assumptions evident. For this assessment, the lifetime unit risk derived was ∼2-fold greater than the unadjusted adult lifetime unit risk, and the resulting Total Allowable Concentration in drinking water is 20µg/L.

Hydroquinone: acute and subchronic toxicity studies with emphasis on neurobehavioral and nephrotoxic effects.

Hydroquinone (HQ) is a common water-soluble constituent of foods, an ingredient in skin lightening preparations, a photographic developer, and an antioxidant used in the preparation of industrial polymers. In this series of studies, aqueous solutions of HQ were given by gavage to male and female Sprague-Dawley rats to determine the acutely lethal dose, the clinical signs of behavioral toxicity associated with doses at or near a dose causing mortality, and the effects of the administration of dose levels resulting in acutely observable behavioral effects when administered 5 days/week for 13 weeks. The acute dermal toxicity of HQ in rabbits was also determined. For the acute oral toxicity study, groups of five male and five female rats were administered single oral doses of 375, 345, 315, or 285 mg/kg. At all dose levels, animals exhibited minor to moderate tremors and minor convulsions within the first hour after dosing. The acute oral LD50 value for both sexes combined was >375 mg/kg. Dermal application of 2000 mg/kg HQ to rabbits under an occlusive wrap for 24 h did not result in neurobehavioral effects or mortality. Subchronic exposure was accomplished by administration of doses of 200, 64, 20, or 0 mg/kg/day of HQ in water to groups of male and female rats study (10/sex/group). A functional observational battery (FOB) was used to detect neurobehavioral effects prior to HQ exposure and postexposure at 1, 6, and 24 h and 7, 14, 30, 60, and 91 days. Daily clinical observations were also recorded for each animal. Doses of 200 or 64 mg/kg HQ resulted in acutely observable behavioral effects including tremors and reduced activity. Tremors occurred within one hour of dosing and resolved by the 6-h examination. Brain weights were not altered by HQ administration, but mean terminal body weight was reduced approximately 7% for the 200 mg/kg males. Neuropathologic examination of the CNS and PNS, including special stains for myelin and axonal process, did not reveal any morphologic lesions associated with HQ administration or secondary to repetitive CNS stimulation by HQ. The nephrotoxic effects observed in Fischer 344 rats after HQ exposure was not observed in this study with Sprague-Dawley rats. Oral doses of >or=64/mg/kg HQ resulted in acute neurobehavioral effects indicative of CNS stimulation; however, subchronic exposure to dose levels that produced repetitive CNS stimulation by HQ did not result in an exacerbation of acute stimulatory effects over time or morphologic changes in the CNS or PNS or nephrotoxicity.

Conversion of tris(8-quinolinolato-N1, O8) aluminum to 8-hydroxyquinoline and activity in bacterial reverse mutation assays.

Tris(8-quinolinolato-N1, O8) aluminum (AlQ), an aluminum chelate of 8-hydroxyquinoline (8OHQ) is an important charge transfer molecule in semiconducting imaging devices. This study was conducted to evaluate AlQ and 8OHQ for the ability to induce reverse mutations, either in the presence or absence of mammalian microsomal enzymes, and to determine if AlQ decomposes or is metabolized to 8OHQ under assay conditions. The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (pKM101). The assays were conducted in the presence and absence of S9. AlQ doses were 1-1000 microg per plate while 8OHQ doses were 0.947-947 microg per plate to maintain molar equivalency. Stability studies were carried out for 4h at 37 degrees C under conditions designed to mimic mutation assays. Samples were analyzed by HPLC and LC/MS to tentatively identify potential metabolites of AlQ and 8OHQ. The results of the bacterial mutagenicity assay indicate that in the presence of S9, both AlQ and 8OHQ, caused increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA (pKM101). No increases were observed with any of the remaining tester strain/activation condition combinations. The stability study showed that AlQ degrades readily to 8OHQ under standard mutagenicity test conditions, and the positive test result with AlQ is due to the bioactivation of 8OHQ. In the presence of S9, 8OHQ is metabolized to one detectable product with molecular weight indicative of a one-oxygen insertion. 8OHQ N-oxide and 2,8-quinolinediol were ruled out as possible metabolites; 8OHQ epoxides and other quinolinediols were neither confirmed nor ruled out. Bacterial mutagenicity tests have not been shown to predict in vivo effects of 8OHQ; these assays are similarly expected to be poorly predictive of in vivo genotoxic and carcinogenic potential of AlQ.

Metabolic rate constants for hydroquinone in F344 rat and human liver isolated hepatocytes: application to a PBPK model.

Hydroquinone (HQ) is an important industrial chemical that also occurs naturally in foods and in the leaves and bark of a number of plant species. Exposure of laboratory animals to HQ may result in species-, sex-, and strain-specific nephrotoxicity. The sensitivity of male F344 versus female F344 and Sprague-Dawley rats or B6C3F1 mice appears to be related to differences in the rates of formation of key nephrotoxic metabolites. Metabolic rate constants for the conversion of HQ through several metabolic steps to the mono-glutathione conjugate and subsequent detoxification via mercapturic acid formation were measured in suspension cultures of hepatocytes isolated from male F-344 rats and humans. A mathematic kinetic model was used to analyze each metabolic step by simultaneously fitting the disappearance of each substrate and the appearance of subsequent metabolites. An iterative, nested approach was used whereby downstream metabolites were considered first, and the model was constrained by the requirement that rate constants determined during analysis of individual steps must also satisfy the complete, integrated metabolism scheme, including competitive pathways. The results from this study indicated that the overall capacity for metabolism of HQ and its mono-glutathione conjugate is greater in hepatocytes from humans than in those from rats, suggesting a greater capacity for detoxification of the glutathione conjugates in humans. Metabolic rate constants were applied to an existing physiologically based pharmacokinetic model, which was used to predict total glutathione metabolites produced in the liver. The results showed that body burdens of these metabolites will be much higher in rats than in humans.

The effect of oxygen tension on the cytotoxicity of hydroquinone and selected hydroquinone metabolites to isolated rat renal proximal tubular cells.

The cytotoxicity of hydroquinone (HQ) and several of its metabolites was studied using freshly isolated proximal tubular (PT) kidney cells from rats. Incubations were conducted for periods of up to 4 h at 37 degrees C, with cytotoxicity measured either as increased leakage of lactate dehydrogenase or as a decreased energy status, as determined by decreased ratios of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). Incubation atmospheres consisted of either 95% O(2)/5% CO(2), to promote cell viability in vitro, or 5% O(2)/5% CO(2)/90% N(2). Preliminary studies with bovine serum albumin (BSA) added to the incubation media indicated a lack of toxicity for HQ or its metabolites. For the tests discussed in this report, incubations were performed without the addition of BSA. Under 95% O(2) atmospheres, PT cells from male Fischer F344 rats were significantly more sensitive to HQ than those from male Sprague-Dawley (SD) rats, with decreases in ATP to ADP ratios seen as early as 0.5 h at a concentration of 0.5 mM. When incubations were performed under a 5% O(2) atmosphere, 2-(cysteine-S-yl)hydroquinone (Cys-HQ) and HQ toxicities were observed later (3-4 h) in the incubation period, occurred at higher concentrations, were similar in magnitude for the two strains, and were greater for Cys-HQ than for HQ. These results show that variations in oxygen tension can dramatically influence the toxicity of HQ and its metabolites. The specific compounds tested that were cytotoxic at a physiologically relevant oxygen tension (5%) were (in decreasing order of potency): Cys-HQ > 2-(glutathion-S-yl)hydroquinone > HQ. These results support an association of toxicity with metabolism through the glutathione pathway, with ultimate toxicity associated with the cysteinyl conjugate. Biochemical characteristics of PT cells from these two strains suggest a significantly greater capacity of cells from the SD rat to respond to oxidative stress.