Biomonitoring Equivalents for ethylene thiourea.

Ethylene thiourea, or ETU, is used in the rubber industry and is a degradation product and impurity in some fungicides. The general public may be exposed to low concentrations of residues of ETU in a variety of ways, including food treated with ethylene bis-dithiocarbamate (EBDC) fungicides or migration from rubber products. Biomonitoring of ETU in urine is useful for an assessment of integrated exposures to ETU across different sources and routes of exposure. In this evaluation, we review available health-based risk assessments and toxicological reference values (TRVs) for ETU and derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data. BEs were derived based on existing TRVs derived by Health Canada, yielding a BE of 27 µg of total ETU/L in urine associated with the Acceptable Daily Intake (ADI) and 6.7 µg/L associated with a 1e-6 cancer risk. These BEs are based on an analytical method that involves a digestion step to liberate conjugated ETU, thus producing 'total' ETU in urine. The BE values derived in this manuscript can serve as a guide to help public health officials and regulators interpret population based ETU biomonitoring data in a public health risk context.

Acrylonitrile's genotoxicity profile: mutagenicity in search of an underlying molecular mechanism.

Acrylonitrile (ACN) is a known rodent and possible human carcinogen. There have also been concerns as to it causing adverse reproductive health effects. Numerous genotoxicity studies at the somatic level in a variety of test systems have demonstrated ACN's mutagenicity; its potential to induce mutations in germ cells has also been evaluated. ACN is metabolized to reactive intermediates capable of forming adducts with macromolecules including DNA, a necessary first step in establishing a direct mutagenic mode of action (MOA) for its carcinogenicity. The mutagenicity of ACN has been well demonstrated, however, numerous studies have found no evidence for the capacity of ACN to induce direct DNA lesions that initiate the mutagenic process. Although ACN and its oxidative metabolite (2-cyanoethylene oxide or CNEO) have been shown to bind in vitro with isolated DNA and associated proteins, usually under non-physiological conditions, studies in mammalian cells or in vivo have provided little specification as to an ACN-DNA reaction. Only one early study in rats has shown an ACN/CNEO DNA adduct in liver, a non-target tissue for its carcinogenicity in the rat. By contrast, numerous studies have shown that ACN can act indirectly to induce at least one DNA adduct by forming reactive oxygen species (ROS) in vivo, but it has not been definitively shown that the resulting DNA damage is causative for the induction of mutations. Genotoxicity studies for ACN in somatic and germinal cells are summarized and critically reviewed. Significant data gaps have been identified for bringing together the massive data base that provides the basis of ACN's current genotoxicity profile.

Weight of evidence evaluation for chemical-induced immunotoxicity for PFOA and PFOS: findings from an independent panel of experts.

Immunotoxicity is the critical endpoint used by some regulatory agencies to establish toxicity values for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). However, the hypothesis that exposure to certain per- and polyfluoroalkyl substances (PFAS) causes immune dysregulation is subject to much debate. An independent, international expert panel was engaged utilizing methods to reduce bias and "groupthink". The panel concluded there is moderate evidence that PFOS and PFOA are immunotoxic, based primarily on evidence from animal data. However, species concordance and human relevance cannot be well established due to data limitations. The panel recommended additional testing that includes longer-term exposures, evaluates both genders, includes other species of animals, tests lower dose levels, assesses more complete measures of immune responses, and elucidates the mechanism of action. Panel members agreed that the Faroe Islands cohort data should not be used as the primary basis for deriving PFAS risk assessment values. The panel agreed that vaccine antibody titer is not useful as a stand-alone metric for risk assessment. Instead, PFOA and PFOS toxicity values should rely on multiple high-quality studies, which are currently not available for immune suppression. The panel concluded that the available PFAS immune epidemiology studies suffer from weaknesses in study design that preclude their use, whereas available animal toxicity studies provide comprehensive dataset to derive points of departure (PODs) for non-immune endpoints. The panel recommends accounting for potential PFAS immunotoxicity by applying a database uncertainty factor to POD values derived from animal studies for other more robustly supported critical effects.

Biomonitoring Equivalents for N,N-Diethyl-meta-toluamide (DEET).

N,N-Diethyl-meta-toluamide (DEET) is widely used as an effective mosquito and tick repellent. DEET is absorbed systemically after applications to skin. Once absorbed, DEET is rapidly metabolized with the predominant metabolite being m-dimethylaminocarbonyl benzoic acid (DBA). DEET and metabolites are predominantly excreted in urine after being absorbed systemically. Exposures to DEET are typically biomonitored via measures of DEET and DBA in urine. In this evaluation, we review available health-based risk assessments and toxicological reference values (TRVs) for DEET and derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data. BEs were derived based on existing TRVs derived by Health Canada, yielding 38 and 23 mg/L DBA in urine for adults and 57 and 34 mg/L DBA in urine in children for the acute oral and intermediate dermal TRVs, respectively. The BEs for unchanged DEET in urine are 21 and 12 mg/L in adults and 4.5 and 2.7 mg/L in children for the acute oral and intermediate dermal TRVs. The BE values derived in this manuscript can serve as a guide to help public health officials and regulators interpret population based DEET biomonitoring data in a public health risk context.

Biomonitoring Equivalents for glyphosate.

One of the most widely used herbicides worldwide, glyphosate is registered for use in many agricultural and non-agricultural settings. Accordingly, regulatory authorities develop toxicology reference values (TRVs) to conduct risk assessments for potential exposures. Exposures to glyphosate are typically biomonitored via measures of glyphosate in urine. However, measured concentrations of glyphosate in urine, with units mg/L urine, cannot be directly interpreted using the available TRVs as they are presented in terms of daily intake levels (e.g. mg/kg-bw per day). In this evaluation, we review available health-based risk assessments and TRVs for glyphosate and derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data. Biomonitoring Equivalents (BEs) are defined as the concentration or range of concentrations of a chemical or its metabolite in a biological medium (blood, urine, human milk, etc.) that is consistent with an existing health-based TRVs such as a reference dose (RfD) or tolerable daily intake (TDI). The BE values derived in this manuscript are screening values that can help public health officials and regulators interpret glyphosate biomonitoring data.

Best practices for exposure model peer review - A SciPinion advisory panel report.

The extent and rigor of peer review that a model undergoes during and after development influences the confidence of users and managers in model predictions. A process for determining the breadth and depth of peer review of exposure models was developed with input from a panel of exposure-modeling experts. This included consideration of the tiers and types of models (e.g., screening, deterministic, probabilistic, etc.). The experts recommended specific criteria be considered when evaluating the degree to which a model has been peer reviewed, including quality of documentation and the model peer review process (e.g., internal review with a regulatory agency by subject matter experts, expert review reports, formal Scientific Advisory Panels, and journal peer review). In addition, because the determination of the confidence level for an exposure model's predictions is related to the degree of evaluation the model has undergone, irrespective of peer review, the experts recommended the approach include judging the degree of model rigor using a set of specific criteria: (1) nature and quality of input data, (2) model verification, (3) model corroboration, and (4) model evaluation. Other key areas considered by the experts included recommendations for addressing model uncertainty and sensitivity, defining the model domain of applicability, and flags for when a model is used outside its domain of applicability. The findings of this expert engagement will help developers as well as users of exposure models have greater confidence in their application and yield greater transparency in the evaluation and peer review of exposure models.

A reproductive and developmental toxicity screening study of 1,3-butadiene in Sprague-Dawley rats.

1,3 Butadiene (BD) is an industrial intermediate used primarily in product manufacturing with the greatest exposure potential via inhalation. BD was evaluated for reproductive and developmental effects in a Good Laboratory Practice (GLP)-compliant, extended OECD 421 guideline study (completed 2003). Twelve-week old rats (12/sex/dose) were exposed via whole-body inhalation to BD vapor (0, 300, 1500, 6000 ppm) for 6 h/day, 7 days/week, starting 14 days prior to mating through the day prior to euthanasia (total exposures: 83-84 days for F0 males 60-70 days for F0 females). Select F1 offspring (1/sex/litter) were dosed 7 days (postnatal days 21-27 or 28-34), then necropsied. At 1500 and 6000 ppm, treatment-related facial soiling was seen in F0 males and females with decreased body weights/gains in F0 males. F1 males and females exhibited similar effects at 1500 and 6000 ppm. Importantly, the F0 generation had no evidence of altered sperm production, testicular effects, or ovarian atrophy, which were sensitive responses in mice. The no-observed-adverse-effect-level (NOAEL) is 300 ppm due to decreased body weight/gain and facial soiling at 1500 ppm, whereas 6000 ppm serves as a NOAEL for reproductive and developmental endpoints. This study contributes to the weight-of-evidence of differential BD reproductive toxicity in rats and mice.

Peer review of a cancer weight of evidence assessment based on updated toxicokinetics, genotoxicity, and carcinogenicity data for 1,3-dichloropropene using a blinded, virtual panel of experts.

A cancer weight of evidence (WOE) analysis based on updated toxicokinetics, genotoxicity, and carcinogenicity data for 1,3-dichloropropene was peer reviewed by a panel of experts. Historically, 1,3-dichloropropene has been classified in the U.S. as "likely to be carcinogenic to humans" via oral and inhalation exposure routes based upon the results of rodent cancer bioassays conducted in the 1980s. Contemporary studies led the authors of the WOE analysis to conclude that the currently manufactured form of 1,3-dichloropropene is not mutagenic and not carcinogenic below certain doses, pointing to a threshold-based approach for cancer risk assessment. SciPinion conducted a peer review of the WOE analysis using methods for assembling and managing blinded expert panels that maximize expertise while minimizing potential selection/participation bias. The process was implemented through a web-based application that poses a series of questions soliciting the experts' scientific opinions and observations about specific topics. The goal of the peer review was to have experts provide conclusions about the WOE for carcinogenicity classification of 1,3-dichloropropene, identify potential data gaps, and evaluate the validity of a threshold-based risk assessment for 1,3-dichloropropene. Based on a robust peer review of the current scientific information, a cancer WOE classification of "not likely to be carcinogenic to humans" is best supported for 1,3-dichloropropene. This conclusion is reached with a high degree of consensus (consensus score = 0.92) across expert panel members.

Biomonitoring Equivalents (BEs) for tetrabromobisphenol A.

Tetrabromobisphenol A (TBBPA) is a flame retardant used in a variety of products, including epoxy and polycarbonate resins. Relevant exposure to TBBPA has been assessed by measuring TBBPA in the blood of humans. Here, we derive Biomonitoring Equivalents (BEs) for TBBPA to interpret these, and future biomonitoring results for TBBPA in humans. The available toxicity risk values (TRVs) for TBBPA were all based on toxicology studies in rats. Several studies have been conducted in which TBBPA in blood of rats were measured following controlled oral doses of TBBPA. These data provide a robust relationship from which to derive BEs. BEs of 5.6 and 13.0 µg total TBBPA/L plasma were calculated for available cancer and non-cancer TRVs, respectively. Several studies have measured TBBPA in serum, with median concentrations less than 0.1 µg/L, indicating considerable margins of safety (MOS) for TBBPA based on the currently available biomonitoring studies.

Integration of mechanistic and pharmacokinetic information to derive oral reference dose and margin-of-exposure values for hexavalent chromium.

The current US Environmental Protection Agency (EPA) reference dose (RfD) for oral exposure to chromium, 0.003 mg kg-1  day-1 , is based on a no-observable-adverse-effect-level from a 1958 bioassay of rats exposed to ≤25 ppm hexavalent chromium [Cr(VI)] in drinking water. EPA characterizes the confidence in this RfD as "low." A more recent cancer bioassay indicates that Cr(VI) in drinking water is carcinogenic to mice at ≥30 ppm. To assess whether the existing RfD is health protective, neoplastic and non-neoplastic lesions from the 2 year cancer bioassay were modeled in a three-step process. First, a rodent physiological-based pharmacokinetic (PBPK) model was used to estimate internal dose metrics relevant to each lesion. Second, benchmark dose modeling was conducted on each lesion using the internal dose metrics. Third, a human PBPK model was used to estimate the daily mg kg-1 dose that would produce the same internal dose metric in both normal and susceptible humans. Mechanistic research into the mode of action for Cr(VI)-induced intestinal tumors in mice supports a threshold mechanism involving intestinal wounding and chronic regenerative hyperplasia. As such, an RfD was developed using incidence data for the precursor lesion diffuse epithelial hyperplasia. This RfD was compared to RfDs for other non-cancer endpoints; all RfD values ranged 0.003-0.02 mg kg-1  day-1 . The lowest of these values is identical to EPA's existing RfD value. Although the RfD value remains 0.003 mg kg-1  day-1 , the confidence is greatly improved due to the use of a 2-year bioassay, mechanistic data, PBPK models and benchmark dose modeling.

Comparison of Toxicity and Recovery in the Duodenum of B6C3F1 Mice Following Treatment with Intestinal Carcinogens Captan, Folpet, and Hexavalent Chromium.

High concentrations of hexavalent chromium (Cr(VI)), captan, and folpet induce duodenal tumors in mice. Using standardized tissue collection procedures and diagnostic criteria, we compared the duodenal histopathology in B6C3F1 mice following exposure to these 3 carcinogens to determine whether they share similar histopathological characteristics. B6C3F1 mice ( n = 20 per group) were exposed to 180 ppm Cr(VI) in drinking water, 12,000 ppm captan in feed, or 16,000 ppm folpet in feed for 28 days. After 28 days of exposure, villous enterocyte hypertrophy and mild crypt epithelial hyperplasia were observed in all exposed mice. In a subset of mice allowed to recover for 28 days, duodenal samples were generally indistinguishable from those of unexposed mice. Changes in the villi and lack of observable damage to the crypt compartment suggest that toxicity was mediated in the villi, which is consistent with earlier studies on each chemical. These findings indicate that structurally diverse agents can induce similar (and reversible) phenotypic changes in the duodenum. These intestinal carcinogens likely converge on common pathways involving irritation and wounding of the villi leading to crypt regenerative hyperplasia that, under protracted high-dose exposure scenarios, increases the risk of spontaneous mutation and tumorigenesis.

Risk assessment and Biomonitoring Equivalent for 2-ethylhexyl-2,3,4,5 tetrabromobenzoate (TBB) and tetrabromobenzoic acid (TBBA).

2-ethylhexyl-2,3,4,5 tetrabromobenzoate (TBB) is used as a flame retardant. Biomonitoring for TBB exposures include the metabolite, tetrabromobenzoic acid (TBBA), in urine. We derived a Reference Dose (RfD) for TBB and a Biomonitoring Equivalent (BE) for TBBA in urine. Three longer-term studies of oral gavage dosing of a commercial mixture BZ-54 (which includes 70% TBB) in rats were evaluated for deriving the RfD. The 95% lower confidence limits on the BMD associated with a 1 SD change from the mean (BDMLSD) values ranged from 77 to 134 mg/kg-day. The mean BMDLSD value of 91 mg/kg-day for maternal body weight changes was selected as the appropriate point of departure (POD), corresponding to a human equivalent dose (PODHEC) of 25 mg/kg-day. A total composite uncertainty factor (UF) of 300 yields an RfD of 0.08 mg/kg-day. A urinary mass excretion fraction (Fue) of 0.6 for TBBA following oral doses of TBB in rats was used to calculate BEs for TBBA in urine of 2.5 mg/L and 2.5 mg/g cr. Mean (5.3 × 10-6 mg/L) and maximum (340 × 10-6 mg/L) levels of TBBA measured in urine from human volunteers reported in the literature indicates margins of safety (MOS) are approximately 450,000 and 7,000, respectively.

Reduction of hexavalent chromium by fasted and fed human gastric fluid. II. Ex vivo gastric reduction modeling.

To extend previous models of hexavalent chromium [Cr(VI)] reduction by gastric fluid (GF), ex vivo experiments were conducted to address data gaps and limitations identified with respect to (1) GF dilution in the model; (2) reduction of Cr(VI) in fed human GF samples; (3) the number of Cr(VI) reduction pools present in human GF under fed, fasted, and proton pump inhibitor (PPI)-use conditions; and (4) an appropriate form for the pH-dependence of Cr(VI) reduction rate constants. Rates and capacities of Cr(VI) reduction were characterized in gastric contents from fed and fasted volunteers, and from fasted pre-operative patients treated with PPIs. Reduction capacities were first estimated over a 4-h reduction period. Once reduction capacity was established, a dual-spike approach was used in speciated isotope dilution mass spectrometry analyses to characterize the concentration-dependence of the 2nd order reduction rate constants. These data, when combined with previously collected data, were well described by a three-pool model (pool 1 = fast reaction with low capacity; pool 2 = slow reaction with higher capacity; pool 3 = very slow reaction with higher capacity) using pH-dependent rate constants characterized by a piecewise, log-linear relationship. These data indicate that human gastric samples, like those collected from rats and mice, contain multiple pools of reducing agents, and low concentrations of Cr(VI) (<0.7 mg/L) are reduced more rapidly than high concentrations. The data and revised modeling results herein provide improved characterization of Cr(VI) gastric reduction kinetics, critical for Cr(VI) pharmacokinetic modeling and human health risk assessment.

Human health screening level risk assessments of tertiary-butyl acetate (TBAC): calculated acute and chronic reference concentration (RfC) and Hazard Quotient (HQ) values based on toxicity and exposure scenario evaluations.

A screening level risk assessment has been performed for tertiary-butyl acetate (TBAC) examining its primary uses as a solvent in industrial and consumer products. Hazard quotients (HQ) were developed by merging TBAC animal toxicity and dose-response data with population-level, occupational and consumer exposure scenarios. TBAC has a low order of toxicity following subchronic inhalation exposure, and neurobehavioral changes (hyperactivity) in mice observed immediately after termination of exposure were used as conservative endpoints for derivation of acute and chronic reference concentration (RfC) values. TBAC is not genotoxic but has not been tested for carcinogenicity. However, TBAC is unlikely to be a human carcinogen in that its non-genotoxic metabolic surrogates tertiary-butanol (TBA) and methyl tertiary butyl ether (MTBE) produce only male rat α-2u-globulin-mediated kidney cancer and high-dose specific mouse thyroid tumors, both of which have little qualitative or quantitative relevance to humans. Benchmark dose (BMD)-modeling of the neurobehavioral responses yielded acute and chronic RfC values of 1.5 ppm and 0.3 ppm, respectively. After conservative modeling of general population and near-source occupational and consumer product exposure scenarios, almost all HQs were substantially less than 1. HQs exceeding 1 were limited to consumer use of automotive products and paints in a poorly ventilated garage-sized room (HQ = 313) and occupational exposures in small and large brake shops using no personal protective equipment or ventilation controls (HQs = 3.4-126.6). The screening level risk assessments confirm low human health concerns with most uses of TBAC and indicate that further data-informed refinements can address problematic health/exposure scenarios. The assessments also illustrate how tier-based risk assessments using read-across toxicity information to metabolic surrogates reduce the need for comprehensive animal testing.

Risk assessments for chronic exposure of children and prospective parents to ethylbenzene (CAS No. 100-41-4).

Potential chronic health risks for children and prospective parents exposed to ethylbenzene were evaluated in response to the Voluntary Children's Chemical Evaluation Program. Ethylbenzene exposure was found to be predominately via inhalation with recent data demonstrating continuing decreases in releases and both outdoor and indoor concentrations over the past several decades. The proportion of ethylbenzene in ambient air that is attributable to the ethylbenzene/styrene chain of commerce appears to be relatively very small, less than 0.1% based on recent relative emission estimates. Toxicity reference values were derived from the available data, with physiologically based pharmacokinetic models and benchmark dose methods used to assess dose-response relationships. An inhalation non-cancer reference concentration or RfC of 0.3 parts per million (ppm) was derived based on ototoxicity. Similarly, an oral non-cancer reference dose or RfD of 0.5 mg/kg body weight/day was derived based on liver effects. For the cancer assessment, emphasis was placed upon mode of action information. Three of four rodent tumor types were determined not to be relevant to human health. A cancer reference value of 0.48 ppm was derived based on mouse lung tumors. The risk characterization for ethylbenzene indicated that even the most highly exposed children and prospective parents are not at risk for non-cancer or cancer effects of ethylbenzene.

Safety assessment for ethanol-based topical antiseptic use by health care workers: Evaluation of developmental toxicity potential.

Ethanol-based topical antiseptic hand rubs, commonly referred to as alcohol-based hand sanitizers (ABHS), are routinely used as the standard of care to reduce the presence of viable bacteria on the skin and are an important element of infection control procedures in the healthcare industry. There are no reported indications of safety concerns associated with the use of these products in the workplace. However, the prevalence of such alcohol-based products in healthcare facilities and safety questions raised by the U.S. FDA led us to assess the potential for developmental toxicity under relevant product-use scenarios. Estimates from a physiologically based pharmacokinetic modeling approach suggest that occupational use of alcohol-based topical antiseptics in the healthcare industry can generate low, detectable concentrations of ethanol in blood. This unintended systemic dose probably reflects contributions from both dermal absorption and inhalation of volatilized product. The resulting internal dose is low, even under hypothetical, worst case intensive use assumptions. A significant margin of exposure (MOE) exists compared to demonstrated effect levels for developmental toxicity under worst case use scenarios, and the MOE is even more significant for typical anticipated occupational use patterns. The estimated internal doses of ethanol from topical application of alcohol-based hand sanitizers are also in the range of those associated with consumption of non-alcoholic beverages (i.e., non-alcoholic beer, flavored water, and orange juice), which are considered safe for consumers. Additionally, the estimated internal doses associated with expected exposure scenarios are below or in the range of the expected internal doses associated with the current occupational exposure limit for ethanol set by the Occupational Safety and Health Administration. These results support the conclusion that there is no significant risk of developmental or reproductive toxicity from repeated occupational exposures and high frequency use of ABHSs or surgical scrubs. Overall, the data support the conclusion that alcohol-based hand sanitizer products are safe for their intended use in hand hygiene as a critical infection prevention strategy in healthcare settings.

Interpreting estrogen screening assays in the context of potency and human exposure relative to natural exposures to phytoestrogens.

While the Environmental Protection Agency and the Organization for Economic Cooperation and Development have developed validated in vitro and in vivo screening assays to measure interaction of substances with estrogen, androgen and thyroid pathway components, to date, methods to contextualize such results in terms of potencies and actual human exposures are lacking. To place endocrine screening results in the context of potency and human exposure, we propose a method that entails (1) calculating a benchmark dose for a response measured in an endocrine screen; (2) estimating the human urinary concentration (biomonitoring equivalent, BE) expected to correspond to this dose (BEBMD ); (3) deriving the exposure:activity ratio (EAR) by comparing actual urinary values from human biomonitoring studies (e.g., National Health and Nutrition Examination Survey (NHANES)) to the BEBMD . Using OECD uterotrophic assay validation studies and NHANES results, we calculated EARs for genistein (EARGEN = 6.6 × 10(-4) ) and bisphenol A (EARBPA = 8.8 × 10(-7) ). The EARGEN is more than 700-fold greater than the EARBPA . Not only can these methods be applied to additional endocrine assays and compounds, they can contribute to weight of evidence decisions regarding the need for additional endocrine screening and testing-substances with low EARs may not warrant additional testing.

Evaluation of urinary speciated arsenic in NHANES: issues in interpretation in the context of potential inorganic arsenic exposure.

Urinary dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) are among the commonly used biomarkers for inorganic arsenic (iAs) exposure, but may also arise from seafood consumption and organoarsenical pesticide applications. We examined speciated urinary arsenic data from National Health and Nutrition Examination Survey (NHANES) 2009-2010 cycle to assess potential correlations among urinary DMA, MMA, and the organic arsenic species arsenobetaine. Urinary DMA and MMA were positively associated with urinary arsenobetaine, suggesting direct exposure to these species in seafood or metabolism of organic arsenicals to these species, although the biomonitoring data do not directly identify the sources of exposure. The magnitude of association was much larger for DMA than for MMA. The secondary methylation index (SMI, ratio of urinary DMA to MMA) observed in the NHANES program likewise is much higher in persons with detected arsenobetaine than in those without, again suggesting that direct DMA exposure is co-occurring with exposure to arsenobetaine. Urinary MMA was less correlated with organic arsenic exposures than DMA and, therefore, may be a more reliable biomarker for iAs exposure in the general US population. However, given the associations between both MMA and DMA and organic arsenic species in urine, interpretations of the urinary arsenic concentrations observed in the NHANES in the context of potential arsenic exposure should be made cautiously.

A chronic oral reference dose for hexavalent chromium-induced intestinal cancer.

High concentrations of hexavalent chromium [Cr(VI)] in drinking water induce villous cytotoxicity and compensatory crypt hyperplasia in the small intestines of mice (but not rats). Lifetime exposure to such cytotoxic concentrations increases intestinal neoplasms in mice, suggesting that the mode of action for Cr(VI)-induced intestinal tumors involves chronic wounding and compensatory cell proliferation of the intestine. Therefore, we developed a chronic oral reference dose (RfD) designed to be protective of intestinal damage and thus intestinal cancer. A physiologically based pharmacokinetic model for chromium in mice was used to estimate the amount of Cr(VI) entering each intestinal tissue section (duodenum, jejunum and ileum) from the lumen per day (normalized to intestinal tissue weight). These internal dose metrics, together with corresponding incidences for diffuse hyperplasia, were used to derive points of departure using benchmark dose modeling and constrained nonlinear regression. Both modeling techniques resulted in similar points of departure, which were subsequently converted to human equivalent doses using a human physiologically based pharmacokinetic model. Applying appropriate uncertainty factors, an RfD of 0.006 mg kg(-1) day(-1) was derived for diffuse hyperplasia-an effect that precedes tumor formation. This RfD is protective of both noncancer and cancer effects in the small intestine and corresponds to a safe drinking water equivalent level of 210 µg l(-1). This concentration is higher than the current federal maximum contaminant level for total Cr (100 µg l(-1)) and well above levels of Cr(VI) in US drinking water supplies (typically ≤ 5 µg l(-1)).