Virunya S. Bhat

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.

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 C1–C8 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 ≥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 μg/L) was calculated for melamine in drinking water.

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.

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

Abstract 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.

Emergency do not consume/do not use concentrations for ferric chloride in drinking water.

The U.S. Congress [PL 107-188] amended the Safe Drinking Water Act and required each community water system serving more than 3,000 people to conduct vulnerability assessments. These assessments address potential circumstances that could compromise the safety and reliability of municipal water. Ferric chloride is used in coagulation and flocculation, and it is used to treat raw water with high viral loads, elevated dissolved solids or high bromide. Iron is an essential nutrient, but elevated concentrations of FeCl3 are corrosive as a result of hydrolysis to HCl. Based on a no-observed-adverse effect level (NOAEL) of 0.5% FeCl3 • 6H2O administered in drinking water to male and female F344 rats for up to 2 years, a do not consume concentration of 200 mg FeCl3 /L can be derived. Since instillation of 0.3 M (48.7 g/L) FeCl3 in saline to rodent vagina failed to elicit damage, a topical do not use concentration of 2000 mg FeCl3/L (600 mg Fe/L) can be assigned. The only FeCl3 data available to quantify ocular toxicity involved a pH 1 solution in rabbit eyes, but HCl instillation (pH 2.5) to rabbit eyes found permanent corneal ulceration after 10 min. The pH of FeCl3 in water at the do not use limit (2.4–2.6) is near the pH (2.0) considered corrosive by regulatory agencies. As direct eye contact with water at pH 4.5 or below increases complaints of ocular discomfort, emergency response plans that address FeCl3 in drinking water must account for Fe levels and the pH of the affected water.

Emergency Do Not Consume/do Not Use concentrations for potassium permanganate in drinking water.

Over the past decade, regulatory authorities and water purveyors have become increasingly concerned with accidental or intentional adulteration of municipal drinking water. Emergency response guidelines, such as the ‘Do Not Consume’ or use concentration limits derived herein, can be used to notify the public in such cases. Potassium permanganate (KMnO4) is used to control iron concentrations and to reduce the levels of nuisance materials that affect odor or taste of finished drinking water. Manganese (Mn) is recognized an essential nutrient, permanganate (MnO4 −) and manganous (Mn+2) ions are caustic, and the acute toxicity of KMnO4 is defined by its oxidant/irritant properties and by the toxicity of Mn. Ingestion of small amounts (4–20 mg/kg) of aqueous KMnO4 solutions that are above 200 mg/L causes gastrointestinal distress, while bolus ingestion has caused respiratory arrest following coagulative necrosis and hemorrhage in the esophagus, stomach, or liver. Dilute KMnO4 solutions (1–100 mg/L) are used as a topical antiseptics and astringents, but >1:5000 (200 mg/L) dilutions can irritate or discolor sensitive mucous membranes and direct skin or ocular contact with concentrated KMnO4 can perforate tissues. Based on clinical experience with 200 mg/L KMnO4, a Do Not Consume concentration of 7 mg/L KMnO4 (equivalent to 2 mg Mn/L) is recommended. Recognizing limited empirical data from which to calculate an ocular reference value, a skin contact ‘Do Not Use’ concentration of 30 mg Mn/L is recommended based on the skin irritation in some patients after a 10-min contact with 100 mg KMnO4/L.

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.

Derivation of an oral reference dose (RfD) for di 2-ethylhexyl cyclohexan-1,4-dicarboxylate (DEHCH), an alternative to phthalate plasticizers

Di 2-ethylhexyl cyclohexane-1,4-dicarboxylate (DEHCH, CAS 84731-70-4) is an ester of polycarboxylic acid assessed in a variety of mammalian toxicity assays as a substitute for phthalate ester-type plasticizers. An OECD 422 combined systemic toxicity study with a reproductive/developmental toxicity screening test in SD rats found minimal effects on the liver, spleen, and thyroid and no indication that DEHCH is a developmental or reproductive toxicant. In a 90-day feeding study in SD rats, no toxicologically relevant effects were noted. Low genotoxic potential of DEHCH is indicated by the lack of mutagenicity or clastogenicity in vitro. No studies assessing mode of action were identified. Where data gaps exist for DEHCH, a read-across approach was used to assess other toxicological endpoints of interest. Di-ethylhexyl terephthalate (DEHT, CAS 6422-86-2) and 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH, CAS 474919-59-0) have higher tiered studies to supplant the data lacking for health-based standard setting. DEHT and DINCH were chosen as the source substances due to similar physical/chemical properties and thus anticipated metabolism and toxicological characteristics. An oral reference dose (RfD) for DEHCH was calculated using the human equivalent NOAEL from the OECD 422 study. A total uncertainty factor of 100 was comprised of interspecies (3x), intraspecies (10x), subchronic to chronic (1x), LOAEL to NOAEL uncertainty (1x) and database uncertainty (3x) factors, resulting in an RfD of 0.3 mg/kg-day.