Development of updated RfD and RfC values for medium carbon range aromatic and aliphatic total petroleum hydrocarbon fractions.

Using total petroleum hydrocarbon (TPH) measurements as a tool for assessing potential human health risks associated with exposures to petroleum products in the environment poses unique challenges, as TPH represents highly variable and complex mixtures containing hundreds of individual chemicals with wide-ranging chemical and physical properties. Current risk assessment practice generally involves analysis of environmental samples for various TPH fractions and summation of risk across those fractions. The United States Environmental Protection Agency (USEPA) derived provisional toxicity criteria for low, medium, and high carbon range aromatic and aliphatic hydrocarbon fractions over a decade ago. These criteria have been used, in whole or in part, to derive risk-based cleanup levels for TPH contamination in soil and groundwater. Herein, we evaluate and update oral and inhalation toxicity criteria for two of these fractions - medium carbon range aromatics and aliphatics - using, where applicable, newer data, updated modeling techniques, and new/alternative analyses of certain endpoints, human relevance, and uncertainty. The results of the analyses support an ~10-fold increase in the USEPA provisional reference concentration (p-RfC) values from 0.1 mg/m3 to 1 mg/m3 for both medium carbon range aromatics (different uncertainty factor) and aliphatics (new study and different judgment of toxicity data from existing study). Compared to the USEPA provisional oral reference dose (p-RfD) values for the medium carbon range aromatics and aliphatics of 0.03 mg/kg-day and 0.01 mg/kg-day, respectively, the present analyses suggest the RfD for medium carbon range aromatics could be increased >6.6-fold to 0.2 mg/kg-day (updated modeling and different uncertainty factors), and the RfD for medium carbon range aliphatics could be increased ~20-fold to 0.2 mg/kg-day (new study). These updated toxicity criteria could be used by regulatory agencies to reevaluate risk-based screening levels or by risk managers to support cleanup levels for medium carbon range aromatics and aliphatics, while still ensuring adequate health protection.Implications: Petroleum products represent complex mixtures of hydrocarbons broadly comprised of aliphatic compounds (straight-chain, branched-chain, and cyclic alkanes and alkenes) and aromatic compounds such as benzene, alkylbenzenes, and polycyclic aromatic hydrocarbons. The complex nature of petroleum products presents challenges for assessing potential health risks associated with exposure to petroleum hydrocarbon contamination in the environment. It has been over ten years since the U.S. Environmental Protection Agency derived provisional toxicity criteria for low, medium, and high carbon range aromatic and aliphatic hydrocarbon fractions. In that time, risk assessment guidance and tools have evolved, and new studies have been published. Our analyses indicate that current provisional toxicity criteria for medium carbon range aromatics and aliphatics fractions are overly conservative by approximately an order of magnitude.

Assessing chemical carcinogenicity: hazard identification, classification, and risk assessment. Insight from a Toxicology Forum state-of-the-science workshop.

The Toxicology Forum convened an international state-of-the-science workshop Assessing Chemical Carcinogenicity: Hazard Identification, Classification, and Risk Assessment in December 2020. Challenges related to assessing chemical carcinogenicity were organized under the topics of (1) problem formulation; (2) modes-of-action; (3) dose-response assessment; and (4) the use of new approach methodologies (NAMs). Key topics included the mechanisms of genotoxic and non-genotoxic carcinogenicity and how these in conjunction with consideration of exposure conditions might inform dose-response assessments and an overall risk assessment; approaches to evaluate the human relevance of modes-of-action observed in rodent studies; and the characterization of uncertainties. While the scientific limitations of the traditional rodent chronic bioassay were widely acknowledged, knowledge gaps that need to be overcome to facilitate the further development and uptake of NAMs were also identified. Since one single NAM is unlikely to replace the bioassay, activities to combine NAMs into integrated approaches for testing and assessment, or preferably into defined approaches for testing and assessment that include data interpretation procedures, were identified as urgent research needs. In addition, adverse outcome pathway networks can provide a framework for organizing the available evidence/data for assessing chemical carcinogenicity. Since a formally accepted decision tree to guide use of the best and most current science to advance carcinogenicity risk assessment is currently unavailable, a Decision Matrix for carcinogenicity assessment could be useful. The workshop organizers developed and presented a decision matrix to be considered within a carcinogenicity hazard and risk assessment that is offered in tabular form.

An adverse outcome pathway for small intestinal tumors in mice involving chronic cytotoxicity and regenerative hyperplasia: a case study with hexavalent chromium, captan, and folpet.

Small intestinal (SI) tumors are relatively uncommon outcomes in rodent cancer bioassays, and limited information regarding chemical-induced SI tumorigenesis has been reported in the published literature. Herein, we propose a cytotoxicity-mediated adverse outcome pathway (AOP) for SI tumors by leveraging extensive target species- and site-specific molecular, cellular, and histological mode of action (MOA) research for three reference chemicals, the fungicides captan and folpet and the transition metal hexavalent chromium (Cr(VI)). The gut barrier functions through highly efficient homeostatic regulation of SI epithelial cell sloughing, regenerative proliferation, and repair, which involves the replacement of up to 1011 cells per day. This dynamic turnover in the SI provides a unique local environment for a cytotoxicity mediated AOP/MOA. Upon entering the duodenum, cytotoxicity to the villous epithelium is the molecular initiating event, as indicated by crypt elongation, villous atrophy/blunting, and other morphologic changes. Over time, the regenerative capacity of the gut epithelium to compensate declines as epithelial loss accelerates, especially at higher exposures. The first key event (KE), sustained regenerative crypt proliferation/hyperplasia, requires sufficient durations, likely exceeding 6 or 12 months, due to extensive repair capacity, to create more opportunities for the second KE, spontaneous mutation/transformation, ultimately leading to proximal SI tumors. Per OECD guidance, biological plausibility, essentiality, and empirical support were assessed using modified Bradford Hill considerations. The weight-of-evidence also included a lack of induced mutations in the duodenum after up to 90 days of Cr(VI) or captan exposure. The extensive evidence for this AOP, along with the knowledge that human exposures are orders of magnitude below those associated with KEs in this AOP, supports its use for regulatory applications, including hazard identification and risk assessment.

Early microRNA indicators of PPARα pathway activation in the liver.

MicroRNAs (miRNAs) are short non-coding RNA species that play key roles in post-transcriptional regulation of gene expression. MiRNAs also serve as a promising source of early biomarkers for different environmental exposures and health effects, although there is limited information linking miRNA changes to specific target pathways. In this study, we measured liver miRNAs in male B6C3F1 mice exposed to a known chemical activator of the peroxisome proliferator-activated receptor alpha (PPARα) pathway, di(2-ethylhexyl) phthalate (DEHP), for 7 and 28 days at concentrations of 0, 750, 1500, 3000, or 6000 ppm in feed. At the highest dose tested, DEHP altered 61 miRNAs after 7 days and 171 miRNAs after 28 days of exposure, with 48 overlapping miRNAs between timepoints. Analysis of these 48 common miRNAs indicated enrichment in PPARα-related targets and other pathways related to liver injury and cancer. Four of the 10 miRNAs exhibiting a clear dose trend were linked to the PPARα pathway: mmu-miRs-125a-5p, -182-5p, -20a-5p, and -378a-3p. mmu-miRs-182-5p and -378a-3p were subsequently measured using digital drop PCR across a dose range for DEHP and two related phthalates with weaker PPARα activity, di-n-octyl phthalate and n-butyl benzyl phthalate, following 7-day exposures. Analysis of mmu-miRs-182-5p and -378a-3p by transcriptional benchmark dose analysis correctly identified DEHP as having the greatest potency. However, benchmark dose estimates for DEHP based on these miRNAs (average 163; range 126-202 mg/kg-day) were higher on average than values for PPARα target genes (average 74; range 29-183 mg/kg-day). These findings identify putative miRNA biomarkers of PPARα pathway activity and suggest that early miRNA changes may be used to stratify chemical potency.

Chemical-specific maximum allowable levels for pesticide residues in dietary supplements.

Dietary supplements are regulated by the U.S. FDA as a subset of foods. Most botanical dietary ingredients do not have pesticide tolerances, resulting in the enforcement of zero tolerance or general maximum residue limits (GMRL), rather than utilizing science-informed tolerances. In the current study, chemical-specific maximum allowable levels (MALs) were derived for 185 pesticides by converting existing, authoritative-body human health effects criteria. MALs were derived for 96% of pesticides using criteria established by the U.S. EPA. If multiple authoritative-bodies had established human health effects criteria, the most scientifically-defensible criteria was selected, taking into consideration both carcinogenic and non-carcinogenic endpoints. Five pesticides (o-phenylphenol, pirimicarb, oxadixyl, tetradifon, o,p'-DDT), lacking criteria established by the U.S. EPA had criteria established by other authoritative-bodies that were utilized in the derivation of MALs. Two pesticides did not have any established human health effects criteria (o,p'-DDD and o,p'-DDE). In total, MALs were derived from existing criteria for over 98% of the pesticides in the present study. Consequently, it is demonstrated that human health effects criteria derived by authoritative-bodies can be effectively utilized to derive chemical-specific, science-informed MALs applicable to all food commodities, including botanical ingredients, thereby, minimizing reliance on precautionary zero tolerance and GMRLs.

Evolution of chemical-specific adjustment factors (CSAF) based on recent international experience; increasing utility and facilitating regulatory acceptance.

The application of chemical-specific toxicokinetic or toxicodynamic data to address interspecies differences and human variability in the quantification of hazard has potential to reduce uncertainty and better characterize variability compared with the use of traditional default or categorically-based uncertainty factors. The present review summarizes the state-of-the-science since the introduction of the World Health Organization/International Programme on Chemical Safety (WHO/IPCS) guidance on chemical-specific adjustment factors (CSAF) in 2005 and the availability of recent applicable guidance including the WHO/IPCS guidance on physiologically-based pharmacokinetic (PBPK) modeling in 2010 as well as the U.S. EPA guidance on data-derived extrapolation factors in 2014. A summary of lessons learned from an analysis of more than 100 case studies from global regulators or published literature illustrates the utility and evolution of CSAF in regulatory decisions. Challenges in CSAF development related to the adequacy of, or confidence in, the supporting data, including verification or validation of PBPK models. The analysis also identified issues related to adequacy of CSAF documentation, such as inconsistent terminology and often limited and/or inconsistent reporting, of both supporting data and/or risk assessment context. Based on this analysis, recommendations for standardized terminology, documentation and relevant interdisciplinary research and engagement are included to facilitate the continuing evolution of CSAF development and guidance.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.

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.

Comparative time course profiles of phthalate stereoisomers in mice.

More efficient models are needed to assess potential carcinogenicity hazard of environmental chemicals based on early events in tumorigenesis. Here, we investigated time course profiles for key events in an established cancer mode of action. Using a case study approach, we evaluated two reference phthalates, di(2-ethylhexyl) phthalate (DEHP) and its stereoisomer di-n-octyl phthalate (DNOP), across the span of a two-year carcinogenicity bioassay. Male B6C3F1 mice received diets with no phthalate added (control), DEHP at 0.12, 0.60, or 1.20%, or DNOP at 0.10, 0.50, or 1.00% (n = 80-83/group) for up to 104 weeks with six interim evaluations starting at week 4. Mean phthalate doses were 139, 845, and 3147 mg/kg/day for DEHP and 113, 755, and 1281 mg/kg/day for DNOP groups, respectively. Incidence and number of hepatocellular tumors (adenoma and/or carcinoma) were greater at ≥ 60 weeks for all DEHP groups with time and dose trends, whereas DNOP had no significant effects. Key events supported a peroxisome proliferator-activated receptor alpha (PPARα) mode of action for DEHP, with secondary cytotoxicity at the high dose, whereas DNOP induced modest increases in PPARα activity without proliferative or cytotoxic effects. Threshold estimates for later tumorigenic effects were identified at week 4 for relative liver weight (+24%) and PPARα activity (+79%) relative to the control group. Benchmark doses (BMDs) for these measures at week 4 clearly distinguished DEHP and DNOP and showed strong concordance with values at later time points and tumorigenic BMDs. Other target sites included testis and kidney, which showed degenerative changes at higher doses of DEHP but not DNOP. Our results highlight marked differences in the chronic toxicity profiles of structurally similar phthalates and demonstrate quantitative relationships between early bioindicators and later tumor outcomes.

Dose-response modeling of early molecular and cellular key events in the CAR-mediated hepatocarcinogenesis pathway.

Low-dose extrapolation and dose-related transitions are paramount in the ongoing debate regarding the quantification of cancer risks for nongenotoxic carcinogens. Phenobarbital (PB) is a prototypical nongenotoxic carcinogen that activates the constitutive androstane receptor (CAR) resulting in rodent liver tumors. In this study, male and female CD-1 mice administered dietary PB at 0, 0.15, 1.5, 15, 75, or 150 mg/kg-day for 2 or 7 days to characterize multiple apical and molecular endpoints below, at (∼75 mg/kg-day), and above the carcinogenic dose level of PB and examine these responses using benchmark dose modeling. Linear toxicokinetics were observed for all doses. Increased liver weight, hepatocellular hypertrophy, and mitotic figures were seen at 75 and 150 mg/kg-day. CAR activation, based on Cyp2b qPCR and pentoxyresorufin dealkylase activity, occurred at doses ≥ 1.5 mg/kg-day. The no-observable transcriptional effect level for global gene expression was 15 mg/kg-day. At 2 days, several xenobiotic metabolism and cell protective pathways were activated at lower doses and to a greater degree in females. However, hepatocellular proliferation, quantified by bromodeoxyuridine immunohistochemistry, was the most sensitive indicator of PB exposure with female mice more sensitive than males, contrary to sex-specific differences in sensitivity to hepatocarcinogenesis. Taken together, the identification of low-dose cellular and molecular transitions in the subtumorigenic dose range aids the understanding of early key events in CAR-mediated hepatocarcinogenesis.

Concordance of transcriptional and apical benchmark dose levels for conazole-induced liver effects in mice.

The ability to anchor chemical class-based gene expression changes to phenotypic lesions and to describe these changes as a function of dose and time informs mode-of-action determinations and improves quantitative risk assessments. Previous global expression profiling identified a 330-probe cluster differentially expressed and commonly responsive to 3 hepatotumorigenic conazoles (cyproconazole, epoxiconazole, and propiconazole) at 30 days. Extended to 2 more conazoles (triadimefon and myclobutanil), the present assessment encompasses 4 tumorigenic and 1 nontumorigenic conazole. Transcriptional benchmark dose levels (BMDL(T)) were estimated for a subset of the cluster with dose-responsive behavior and a ≥ 5-fold increase or decrease in signal intensity at the highest dose. These genes primarily encompassed CAR/RXR activation, P450 metabolism, liver hypertrophy- glutathione depletion, LPS/IL-1-mediated inhibition of RXR, and NRF2-mediated oxidative stress pathways. Median BMDL(T) estimates from the subset were concordant (within a factor of 2.4) with apical benchmark doses (BMDL(A)) for increased liver weight at 30 days for the 5 conazoles. The 30-day median BMDL(T) estimates were within one-half order of magnitude of the chronic BMDLA for hepatocellular tumors. Potency differences seen in the dose-responsive transcription of certain phase II metabolism, bile acid detoxification, and lipid oxidation genes mirrored each conazole's tumorigenic potency. The 30-day BMDL(T) corresponded to tumorigenic potency on a milligram per kilogram day basis with cyproconazole > epoxiconazole > propiconazole > triadimefon > myclobutanil (nontumorigenic). These results support the utility of measuring short-term gene expression changes to inform quantitative risk assessments from long-term exposures.

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.

Incidence of jaw lesions and activity and gene expression of hepatic P4501A enzymes in mink (Mustela vison) exposed to dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 2,3,4,7,8-pentachlorodibenzofuran.

This study assessed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8 tetrachlorodibenzofuran (TCDF) on the incidence of jaw lesions and on hepatic cytochrome P4501A (CYP1A) endpoints in mink (Mustela vison). Adult female mink were assigned randomly to one of 13 dietary treatments (control and four increasing doses of TCDD, PeCDF, or TCDF) and provided spiked feed for approximately 150 d (60 d prior to breeding through weaning of offspring at 42 d post-parturition). Offspring were maintained on their respective diets for an additional 150 d. Activity of hepatic CYP1A enzymes in adult and juvenile mink exposed to TCDD, PeCDF, or TCDD was generally greater compared with controls, but changes in other CYP1A endpoints were less consistent. Histopathology of the mandible and maxilla of juvenile mink suggested a dose-related increase in the incidence of jaw lesions. The dietary effective doses (ED) for jaw lesions in 50% of the population (ED50) were estimated to be 6.6, 14, and 149 ng/kg body weight (bw)/d for TCDD, PeCDF, and TCDF, respectively. The relative potencies of PeCDF and TCDF compared with TCDD based on ED10, ED20, and ED50 values ranged from 0.5 to 1.9 and 0.04 to 0.09, respectively. These values are within an order of magnitude of the World Health Organization toxic equivalency factor (TEF(WHO)) values of 0.3 and 0.1 for PeCDF and TCDF, respectively.

Toxicological review and oral risk assessment of terephthalic acid (TPA) and its esters: A category approach.

Polyethylene terephthalate, a copolymer of terephthalic acid (TPA) or dimethyl terephthalate (DMT) with ethylene glycol, has food, beverage, and drinking water contact applications. Di-2-ethylhexyl terephthalate (DEHT) is a plasticizer in food and drinking water contact materials. Oral reference doses (RfDs) and total allowable concentrations (TACs) in drinking water were derived for TPA, DMT, and DEHT. Category RfD and TAC levels were also established for nine C(1)-C(8) terephthalate esters. The mode of action of TPA, and of DMT, which is metabolized to TPA, involves urinary acidosis, altered electrolyte elimination and hypercalciuria, urinary supersaturation with calcium terephthalate or calcium hydrogen terephthalate, and crystallization into bladder calculi. Weanling rats were more sensitive to calculus formation than dams. Calculi-induced irritation led to bladder hyperplasia and tumors in rats fed 1000 mg/kg-day TPA. The lack of effects at 142 mg/kg-day supports a threshold for urine saturation with calcium terephthalate, a key event for calculus formation. Chronic dietary DMT exposure in rodents caused kidney inflammation, but not calculi. Chronic dietary DEHT exposure caused general toxicity unrelated to calculi, although urine pH was reduced suggesting the TPA metabolite was biologically-active, but of insufficient concentration to induce calculi. Respective oral reference doses of 0.5, 0.5, and 0.2 mg/kg-day and total allowable drinking water concentrations of 3, 3, and 1 mg/L were derived for TPA, DMT, and DEHT. An oral RfD of 0.2 mg/kg-day for the terephthalate category chemicals corresponded to a drinking water TAC of 1 mg/L.

Derivation of a melamine oral reference dose (RfD) and drinking-water total allowable concentration.

Due to its high nitrogen content, melamine has been used to adulterate food to increase apparent protein content. In 2008, thousands of Chinese infants consumed reconstituted formula derived from melamine-adulterated milk. Urinary-tract stones (comprised of melamine and uric acid) accumulated in some victims and lead to acute renal failure or death. Premature infants and children (<2 yr) have an increased susceptibility to ingested melamine. Due to incomplete reporting, the human data were inadequate to identify a no-observed-adverse-effect level (NOAEL) for melamine-induced pediatric urolithiasis. Urolithiasis, urinary bladder cystitis, and ulcerations were observed in F344 rats after subchronic or chronic ingestion of melamine at > or =72 mg/kg-d. Bladder epithelial damage was followed by epithelial hyperplasia that progressed to bladder papillomas and carcinomas in male but not female F344 rats or male or female B6C3F1 mice. Short-term assays suggest, at best, weak genotoxic activity, and kinetic data show that melamine is not metabolized. Since reliable exposure information was lacking from the clinical reports, an oral reference dose (RfD) based on urolithiasis in male rats after 13 wk of continuous melamine ingestion was calculated as a 10% benchmark dose (38 mg/kg-d). Incorporation of 10-fold interspecies and intraspecies (for the increased susceptibility of infants) uncertainty factors and a threefold database uncertainty factor (for the lack of immunological, neurological and reproduction toxicity data) yields an oral RfD of 0.13 mg/kg-d. Assuming the 70-kg adult consumes 2 L of drinking water daily, a total allowable concentration of 0.9 mg/L (900 microg/L) was calculated for melamine in drinking water.