OECD 414 supplementary prenatal developmental toxicity study of sodium molybdate dihydrate in the rat and benchmark dose evaluation.

In a continuing investigation of the potential for reproductive and developmental toxicity of molybdenum (Mo), consequent to the previous published OECD studies [1,2] and as directed by the European Chemicals Agency [3], a supplemental rat GLP-compliant Prenatal Developmental Toxicity (PNDT) study was conducted to investigate higher dose levels of sodium molybdate dihydrate (SMD) in an identical study design (OECD 414)[4] to Murray et al. 2014a [1], at dietary concentrations calculated to provide target Mo levels of 80 and 120 mg/kg bw/day (the maximum-tolerated dose). There was no effect on post-implantation loss, litter size, sex ratio or the incidence of external, visceral or skeletal fetal malformations or variations. Fetal weight was reduced proportionate to maternal dose. Minimal differences observed in the ossification status of some extremities of fetuses from females receiving 120 mg Mo/kg bw/day were confirmed as transient by skeletal examination of PND 21 pups from a further group of females receiving the same dose regime. There was no evidence of copper depletion in serum, placenta or liver. A benchmark dose evaluation using continuous and dichotomous approaches by combining the fetal body weight data from this study and the previous study determined that the BMD05 ranged from 47 to 57 mg Mo/kg bw/day, depending on the modelling approach and the BMDL05 estimates ranged from 37 to 47 mg Mo/kg bw/day. These levels are considered a more statistically robust point of departure for risk assessment for reproductive effects than the established NOAEL of 40 mg Mo/kg bw/day.

Refractory dissolved organic nitrogen accumulation in high-elevation lakes.

The role of dissolved organic matter (DOM) as either a sink for inorganic nutrients or an additional nutrient source is an often-neglected component of nutrient budgets in aquatic environments. Here, we examined the role of DOM in reactive nitrogen (N) storage in Sierra Nevada (California, USA) lakes where atmospheric deposition of N has shifted the lakes toward seasonal phosphorus (P)-limitation. Nuclear magnetic resonance (NMR) spectroscopy and isotope analyses performed on DOM isolated from Lake Tahoe reveal the accumulation of refractory proteinaceous material with a 100-200-year residence time. In contrast, smaller lakes in the same watershed contain DOM with typical terrestrial characteristics, indicating that proteins in Lake Tahoe are autochthonously produced. These data support the role of DOM as a possible sink for reactive N in these lake ecosystems and identify a potential role for DOM in affecting the inorganic nutrient stoichiometry of these environments.

A constrained maximum likelihood approach to evaluate the impact of dose metric on cancer risk assessment: application to ß-chloroprene.

ß-Chloroprene (2-chloro-1,3-butadiene, CD) is used in the manufacture of polychloroprene rubber. Chronic inhalation studies have demonstrated that CD is carcinogenic in B6C3F1 mice and Fischer 344 rats. However, epidemiological studies do not provide compelling evidence for an increased risk of mortality from total cancers of the lung. Differences between the responses observed in animals and humans may be related to differences in toxicokinetics, the metabolism and detoxification of potentially active metabolites, as well as species differences in sensitivity. The purpose of this study was to develop and apply a novel method that combines the results from available physiologically based kinetic (PBK) models for chloroprene with a statistical maximum likelihood approach to test commonality of low-dose risk across species. This method allows for the combined evaluation of human and animal cancer study results to evaluate the difference between predicted risks using both external and internal dose metrics. The method applied to mouse and human CD data supports the hypothesis that a PBK-based metric reconciles the differences in mouse and human low-dose risk estimates and further suggests that, after PBK metric exposure adjustment, humans are equally or less sensitive than mice to low levels of CD exposure.

Development and validation of a patient-reported disability measurement tool for patients with inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease can impact on a patient's ability to maintain normal physical and mental function, and fulfil their social, family and work roles. Aspects of disability in IBD have received little attention. AIM: To develop, validate and apply a questionnaire directed towards evaluating these disease aspects. METHODS: A literature review on disability in IBD was undertaken, and opinion about aspects of disability to measure was sought from six IBD-specialised gastroenterologists. A questionnaire was developed, and IBD patients completed the new disability questionnaire, the SF-36 and the short-IBD (SIBDQ - 10 point). A subgroup of patients completed the questionnaire again 4 weeks later. Healthy volunteers were studied as a control group. RESULTS: A total of 116 IBD out-patients were approached, of whom 81 (52 Crohn's disease and 28 ulcerative colitis) participated. Nineteen patients were re-evaluated at 4 weeks. Twenty-five controls were studied. All subscales demonstrated good Cronbach's alpha reliability and reproducibility. There was a significant inverse correlation between the disability score and the SIBDQ and between the disability score and the SF36 and a positive correlation with the Crohn's Disease Activity Index (CDAI) (all P < 0.001). Disability differed between ulcerative colitis and controls, but not between active and inactive disease. CONCLUSIONS: The new disability questionnaire is sensitive for detecting disability, is reliable and reproducible, and correlates with disease activity in Crohn's disease, but not ulcerative colitis. Further prospective testing is now needed in the longer term, larger patient populations and in different countries and ethnicities.

Comparison of the EU T25 single point estimate method with benchmark dose response modeling for estimating potency of carcinogens.

The T25 single-point estimate method of evaluating the carcinogenic potency of a chemical, which is currently used by the European Union (EU) and is denoted the EU approach, is based on the selection of a single dose in a chronic bioassay with an incidence rate that is significantly higher than the background rate. The T25 is determined from that single point by a linear extrapolation or interpolation to the chronic dose (in mg/kg/day), at which a 25% increase in the incidence of the specified tumor type is expected, corrected for the background rate. Another method used to obtain a carcinogenic potency value based on a 25% increase in incidence above the background rate is the estimation of a T25 derived from a benchmark dose (BMD) response model fit to the chronic bioassay data for the specified tumor type. A comparison was made between these two methods using 276 chronic bioassays conducted by the National Toxicology Program. In each of the 2-year bioassays, a tumor type was selected based on statistical and biological significance, and both EU T25 and BMD T25 estimates were determined for that end point. In addition, simulations were done using underlying cumulative probability distributions to examine the effect of dose spacing, the number of animals per dose group, the possibility of a dose threshold, and variation in the background incidence rates on the EU T25 and BMD estimates. The simulations showed that in the majority of cases the EU T25 method underestimated the true T25 dose and overestimated the carcinogenic potency. The BMD estimate is generally less biased and has less variation about the true T25 value than the EU estimate.

Comparison of cancer risk estimates for vinyl chloride using animal and human data with a PBPK model.

Vinyl chloride (VC) is a trans-species carcinogen, producing tumors in a variety of tissues, from both inhalation and oral exposures, across a number of species. In particular, exposure to VC has been associated with a rare tumor, liver angiosarcoma, in a large number of studies in mice, rats, and humans. The mode of action for the carcinogenicity of VC appears to be a relatively straightforward example of DNA adduct formation by a reactive metabolite, leading to mutation, mistranscription, and neoplasia. The objective of the present analysis was to investigate the comparative potency of a classic genotoxic carcinogen across species, by performing a quantitative comparison of the carcinogenic potency of VC using data from inhalation and oral rodent bioassays as well as from human epidemiological studies. A physiologically-based pharmacokinetic (PBPK) model for VC was developed to support the target tissue dosimetry for the cancer risk assessment. Unlike previous models, the initial metabolism of VC was described as occurring via two saturable pathways, one representing low capacity-high affinity oxidation by CYP2E1 and the other (in the rodent) representing higher capacity-lower affinity oxidation by other isozymes of P450, producing in both cases chloroethylene oxide (CEO) and chloroacetaldehyde (CAA) as intermediate reactive products. Depletion of glutathione by reaction with CEO and CAA was also described. Animal-based risk estimates for human inhalation exposure to VC using total metabolism estimates from the PBPK model were consistent with risk estimates based on human epidemiological data, and were lower than those currently used in environmental decision-making by a factor of 80.

Similarity calculations using two-dimensional molecular representations.

Molecular similarity calculations are important for rational drug design. Time constraints prevent these techniques being used on large data sets or on large molecules. By reducing the molecular representation to a two-dimensional form, the alignment of the molecules can be greatly speeded up. The accuracy of the resulting similarity values can be improved by using a neural network.

Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay.

The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number ( approximately 500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.

Unusual myocardial (myostromal) "repair" in cardiac transplant patient.

A 57-year-old man received a cardiac allograft for severe ischemic heart disease. His endomyocardial biopsy at eight weeks postoperatively showed a focus of unusual myocardial morphology characterized by small diameter myocytes associated with loose, myxoid appearing stroma and a myocytic mitotic figure. We feel this may represent a unique type of myocardial repair.

Non-cancer risk assessment for nickel compounds: issues associated with dose-response modeling of inhalation and oral exposures.

This report presents the results of noncancer dose-response modeling for inhalation and oral exposures to nickel compounds using the NOAEL/LOAEL and benchmark dose (BMD) approaches. Several key issues associated with the implementation of the BMD approach were examined. Primary among them are difficulties associated with use of data for which the dose-response shape is poorly defined: nonuniqueness of maximum likelihood estimates and lower bounds equal to zero. In addition, several generalizable properties of the "hybrid approach" for modeling continuous endpoints were identified. A hybrid modeling approach allows one to consider "biological significance" on an individual (rather than group) basis; differences between individual- and group-based biological significance in the definition of benchmark response (BMR) levels are elucidated. In particular, it is shown that BMDs defined using group-based BMRs may be more like LOAELs than NOAELs. Application of cross-chemical and cross-endpoint comparisons suggest that, for chronic inhalation exposure, nickel sulfate appears to be as toxic or more toxic than nickel subsulfide and nickel oxide, although the high response rates for the latter two compounds at the lowest chronically administered concentration make such conclusions problematic. A nickel reference concentration could be derived based on the most sensitive benchmark concentration for chronic inhalation exposure to nickel sulfate, 1.7 x 10(-3) mg Ni/m3 for lung fibrosis in male rats. Analyses of oral studies of nickel sulfate and nickel chloride suggest that an appropriate basis for the nickel oral reference dose would be a BMD of 4-5 mg Ni/kg/day, based on increased prenatal mortality. (Uncertainty factors were not determined and neither an RfD nor an RfC was derived in this paper.) The BMD approach provides appropriate quantitative support for toxicological judgment; this paper addresses specific issues associated with the role of the BMD approach in noncancer risk assessment. Resolution of these and other issues may require the accumulation of a number of case studies such as the one presented here.

Chest physiotherapy may be associated with brain damage in extremely premature infants.

OBJECTIVES: To determine whether a characteristic form of brain damage (encephaloclastic porencephaly) was associated with chest physiotherapy treatment in preterm babies. METHODS: A retrospective case-control study was undertaken among 454 infants of birth weight less than 1500 gm cared for during the 3-year period of 1992 to 1994. Thirteen babies of 24 to 27 weeks of gestation who weighed 680 to 1090 gm at birth had encephaloclastic porencephaly. Twenty-six control subjects were matched for birth weight and gestation. RESULTS: The patients received two to three times as many treatments with chest physiotherapy in the second, third, and fourth weeks of life as did control infants (median 79 vs 19 treatments in the first 4 weeks, p < 0.001). Patients also had more prolonged and severe hypotension in the first week than did control subjects (median duration of hypotension 4 vs 0.5 days, p < 0.01), and were less likely to have a cephalic presentation (31% vs 81%, p < 0.01). Since December 1994 no very low birth weight baby has received chest physiotherapy treatment in the first month of life in our nursery, and no further cases have occurred. CONCLUSIONS: Encephaloclastic porencephaly may be a previously unrecognized complication of chest physiotherapy in vulnerable extremely preterm infants.

Dose-response characteristics of uterine responses in rats exposed to estrogen agonists.

Assays for uterine response have played major roles in developing an understanding of estrogen-mediated processes and for identifying compounds with hormonal activity. Data from assays measuring increases in uterine wet weight in rats were evaluated in terms of their dose-response characteristics. Analysis using a Hill equation found inconsistent estimates for the ED50 (concentration giving half-maximal response) and n (steepness of response) among the assays. This variability reflects disparate assay protocols and limitations of the dose-response data collected in the experiments. Although uterine wet weight is easily measured, it arises from several physiological processes (e.g., water retention, cell proliferation). This contributes to the assay variability with different protocols. The potential use of the Hill equation for dose-response analysis to estimate a benchmark dose was also considered using these data sets as surrogates for receptor-mediated toxicological effects. Strengths and weaknesses were identified, but overall the Hill equation should likely become a favored option for determining a benchmark dose, particularly when a data set demonstrates a maximal response. For screening purposes, empirical analysis using the Hill equation provides adequate information for classifying and prioritizing compounds. To develop an understanding of how incremental exposures to compounds with estrogen agonist activities would affect intact adult females, quantitative analyses are required that account for the pharmacokinetics of estradiol and subsequent interactions of the receptor complexes in regulating the responses.

Pharmacodynamic model of the rat estrus cycle in relation to endocrine disruptors.

Several strains of laboratory rats have a high background incidence of mammary tumors and develop a persistent, anovulatory estrus condition at about 12 mo of age. The increased tumor incidence is believed to be associated with elevated estradiol (E2) and prolactin during the period of persistent estrus. A pharmacodynamic estrus cycle (PD-EC) model for the Sprague-Dawley rats has been developed in an attempt to analyze the physiological basis of early-onset persistent estrus and to examine the potential sites of interactions in the hypothalamic-pituitary-ovarian axis for endocrine-modulating xenobiotics that accelerate the onset of persistent estrus. This initial estrus cycle model focused solely on cyclical changes in E2 and luteinizing hormone (LH). An LH surge was scheduled when a hypothetical estrus cycle-related protein (EC-RP) under transcriptional control by the E2 receptor reached a critical concentration. In the model, aging-related cumulative hypothalamic E2 exposure impaired the LH surge by reducing the rate of production of the EC-RP. The progressively decreasing intercycle resynthesis rate leads first to longer, variable-length cycles and finally to persistent estrus at about 12 mo of age. This model construct is consistent with early-onset persistent estrus related to neonatal E2 exposures, with acyclicity associated with high-dose E2 exposure in the adult, and with persistent estrus conditions associated with exposures to xenobiotic endocrine modulators that are either weak E2 antagonists or weak E2 agonists. With further development these pharmacodynamic estrus cycle models should be useful in aiding risk assessments for compounds causing mammary-tissue tumors associated with persistent estrus states.

Benchmark dose analysis of developmental toxicity in rats exposed to boric acid.

Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference concentrations based on the use of no-observed-adverse-effect levels (NOAELs) divided by uncertainty factors. The benchmark dose (BMD) approach has been proposed as an alternative basis for reference value calculations. In this analysis of the developmental toxicity observed in rats exposed to boric acid in their diet, BMD analyses have been conducted using two existing studies. By considering various end points (rib XIII effects, variations of the first lumbar rib, and fetal weight changes) and various modeling approaches for those end points, the best approach for incorporating all of the information available from those studies could be determined. Particular emphasis has been placed on methods for combining data across studies and for combining potentially related effects (on rib XIII and on the first lumbar rib). The issues of study and end point selection are ones that will arise frequently in the process of estimating reference values. This example of boric acid suggests that the BMD approach provides a reasonable basis for appropriately comparing and combining study data, as opposed to ad hoc combinations of study results. Moreover, it is shown that the BMD approach can be used with combinations of end points considered to differ in severity. In this case, the preferred approach involved combining the data from the two studies, which were similarly designed and were conducted in the same laboratory, to calculate BMDs that were more accurate and more precise than those that could be derived from either study alone. It was determined that decreased fetal body weight provided the best basis for BMD calculations; BMDs calculated for fetal body weight changes were less than those for all other relevant end points. The appropriate BMD to use as the basis for boric acid reference dose calculation appears to be 59 mg/kg/day, which is very similar to the NOAEL observed in the second of the two studies (55 mg/kg/day). Although the first study failed to establish a NOAEL, the BMD approach could have been applied to that study, thereby avoiding the need for a repeat study. Similar BMD results were obtained in both studies.

Investigation of the impact of pharmacokinetic variability and uncertainty on risks predicted with a pharmacokinetic model for chloroform.

A sensitivity and uncertainty analysis was performed on the Reitz et al. (Toxicol. Appl. Pharmacol., 1990: 105, 443) physiologically based pharmacokinetic (PBPK) risk assessment model for chloroform. The analytical approach attempted to separately consider the impacts of interindividual variability and parameter uncertainty on the predicted values of the dose metrics in the model, as well as on liver cancer risk estimates obtained with the model. An important feature of the analytical approach was that an attempt was made to incorporate information on correlation between important parameters, for example, the observed correlation between total blood flow and alveolar ventilation rate. Using the published PBPK model for chloroform, the best estimate of the average population risk based on the preferred pharmacodynamic dose metric (PTDEAD), representing cell death, is 9.2 x 10(-7); this estimate is more than 500-fold lower than the risk estimate of 5.3 x 10(-4) based on an alternative pharmacokinetic dose metric (AVEMMB), which represents tissue adduct formation. However, when interindividual variability was considered the range of individual risks (from the 5th to the 95th percentile of the population) predicted with PTDEAD was extremely broad (from 3.0 x 10(-13) to 3.2 x 10(-4)), while individual risks predicted with AVEMMB only varied over a factor of four (from 1.9 x 10(-4) to 7.4 x 10(-4)). As a result, the upper 95th percentile of the distribution of individual risk estimates based on the preferred cell death metric were within a factor of three of the 95th percentile for the pharmacokinetic alternative. The crucial factor with respect to the much greater variability of chloroform risk estimates based on cell death is that the dose metric, PTDEAD, is exquisitely sensitive to variation of the parameters in the model defining the response of cells to the cytotoxicity of chloroform. Unfortunately, these key parameters are also highly uncertain, as well as strongly correlated. As a result it proved impossible to accurately quantify the additional impact of parameter uncertainty on the dose metrics and risk estimates for chloroform. In general, however, the approach used in this study should be useful for differentiating the impact of interindividual variability and parameter uncertainty on PBPK-based risk assessments of other chemicals where the sensitivity, uncertainty, and correlation of the key parameters are more limited.

Atypical aortic coarctation and innominate artery stenosis associated with clubfoot and lower leg ischemia in an infant.

We report findings in a male infant born with talipes equinovarus and ischemic necrosis of the left lower leg and foot associated with atypical coarctation of the ascending aorta and stenosis and hypoplasia of the proximal innominate artery. We hypothesize that a vasculopathy of unknown etiology occurred in utero that resulted in ascending aortic arch coarctation and innominate artery stenosis. The presence of a thrombus in the coarcted segment suggests that one or more emboli could have been responsible for vascular compromise and subsequent clubfoot deformity and lower leg ischemia. Alternatively, a vasculopathy similar to that hypothesized as occurring in the aorta might also have occurred in the left anterior tibial artery, causing structural deformity and soft tissue ischemia of the left lower leg and foot. We believe that this is the first report of this association of findings.

The application of benchmark dose methodology to data from prenatal developmental toxicity studies.

The benchmark dose (BMD) concept was applied to 246 prenatal-developmental toxicity (DT) datasets from government, industry and commercial laboratories. Five modeling approaches were used, 2 generic and 3 specific to DT models. BMDs for both quantal and continuous data were compared with statistically derived no observed adverse effect levels (NOAELs) to determine similarities. Quantal (Q) endpoints included litter responses (e.g., one or more dead or malformed implants), and QBMDs were calculated using a Q Weibull (QW) model. Two types of continuous (C) data were modeled, the proportion of implants affected per litter, and the change in fetal weight (both mean and distribution); continuous power (CP) and DT models were used to calculate CBMDs. QBMDs for a 5% change in response (QBMD05) were 6-fold lower, on average, than the corresponding NOAEL. CBMD05s on average were similar to the corresponding NOAELs, and CBMD05s from different models were similar to each other. Including litter size but not threshold improved the fit of the DT models. For fetal weight data, specific cutoff values were used to calculate BMDs that were similar on average to the corresponding NOAELs: (1) changes from the control mean (5% of the mean, 25th percentile of the control distribution, or a decrease of 0.5 standard deviation), and (2) a 5 or 10% decrease in the proportion of fetuses below the 5th or 10th percentile, respectively, of the control distribution. These results support the use of BMDs as providing a more consistent basis for risk assessment than do NOAELs.

Dose-response assessments for developmental toxicity. IV. Benchmark doses for fetal weight changes.

Recently, most attention on the application of benchmark dose (BMD) techniques to toxicology data has focused on quantal measures of response. Before the advantages of the BMD approach can be exploited in the risk assessment process, it is important that continuous measures of response also be modeled appropriately. In this study, we examined a variety of approaches to estimating BMDs for a change in fetal weight following chemical exposure from a total of 85 developmental toxicity experiments. We modeled the change in the mean fetal weight of a litter in response to treatment using a continuous power model, as well as reductions in the weight of individual fetuses within litters (defined as falling below a preset level) using a log-logistic model which incorporates litter size as a covariable and considers intralitter correlations. For the litter-based approach, several methods of defining a benchmark effect (BME) were considered, including a percentage change in mean litter weight, a change in mean litter weight relative to variability in the control group, and a reduction in the mean litter weight to some point on the control group distribution curve. For the fetus-based approach, we examined several BME options on the cumulative frequency distribution of the control fetuses for defining a low weight fetus and calculated several levels of additional risk. BMDs for four litter-based BMEs (a difference of 5% in mean fetal weight, a decrease to the 25th percentile mean weight of control litters, a decrease in the mean weight by 2 standard errors, and a decrease of 0.5 standard deviation units) and two fetus-based BMEs (a 5% added risk of weighing less than the 5th percentile of control weights and a 10% added risk of weighing less than the 10th percentile) showed strong similarities to each other and to statistically derived NOAELs. In addition to providing comparison with the NOAEL as a reference value, these analyses provided confirmation of the advantages of the BMD approach over the NOAEL in terms of the influence of dose spacing and dose selection. Combined with our previous analyses of quantal endpoints of fetal effects, this information provides a firm basis upon which to implement the benchmark dose concept in developmental toxicity risk assessments.

Considering pharmacokinetic and mechanistic information in cancer risk assessments for environmental contaminants: examples with vinyl chloride and trichloroethylene.

Risk assessments for vinyl chloride (VC) and trichloroethylene (TCE) are presented as examples of approaches for incorporating chemical-specific pharmacokinetic and mechanistic information into a more scientifically plausible cancer risk assessment. For VC, the evidence regarding mode of action includes direct reaction of a metabolite with DNA, resulting in DNA adducts and mistranscription, and cross-species target-tissue correspondence of a rare tumor type. Risk estimates for human exposure to VC predicted with a physiologically-based pharmacokinetic (PBPK) model and the linearized multistage (LMS) model were lower than those currently used in environmental decision-making by a factor of 30 to 50, and were more consistent with human epidemiological data. For TCE, there is evidence of increased cell proliferation due to receptor interaction or cytotoxicity in every instance in which tumors are observed, and the tumors typically represent an increase in the incidence of a commonly observed, species-specific lesion. Virtually safe exposure estimates for human exposure to TCE predicted with a PBPK model and a margin of exposure (MOE) approach were higher than those obtained by the conventional LMS approach by roughly a factor of 100. The MOE approach is recommended as an alternative to the LMS approach for chemicals with a carcinogenic mode of action which entails increased cell proliferation, leading to the expectation of a highly nonlinear cancer dose-response.