Natural mineral fibers: conducting inhalation toxicology studies-part B: development of a nose-only exposure system for repeat-exposure in vivo study of Libby amphibole aerosol.

BACKGROUND: Exposure to asbestos is associated with malignant and nonmalignant respiratory disease. To strengthen the scientific basis for risk assessment on fibers, the National Institute of Environmental Health Sciences (NIEHS) has initiated a series of studies to address fundamental questions on the toxicology of naturally occurring asbestos and related mineral fibers after inhalation exposure. A prototype nose-only exposure system was previously developed and validated. The prototype system was expanded to a large-scale exposure system in this study for conducting subsequent in vivo rodent inhalation studies of Libby amphibole (LA) 2007, selected as a model fiber. RESULTS: The exposure system consisting of six exposure carousels was able to independently deliver stable LA 2007 aerosol to individual carousels at target concentrations of 0 (control group), 0.1, 0.3, 1, 3, or 10 mg/m3. A single aerosol generator was used to provide aerosol to all carousels to ensure that exposure atmospheres were chemically and physically similar, with aerosol concentration as the only major variable among the carousels. Transmission electron microscopy (TEM) coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis of aerosol samples collected at the exposure ports indicated the fiber dimensions, chemical composition, and mineralogy were equivalent across exposure carousels and were comparable to the bulk LA 2007 material. CONCLUSION: The exposure system developed is ready for use in conducting nose-only inhalation toxicity studies of LA 2007 in rats. The exposure system is anticipated to have applicability for the inhalation toxicity evaluation of other natural mineral fibers of concern.

Natural mineral fibers: conducting inhalation toxicology studies - part A: Libby Amphibole aerosol generation and characterization method development.

BACKGROUND: Asbestos has been classified as a human carcinogen, and exposure may increase the risk of diseases associated with impaired respiratory function. As the range of health effects and airborne concentrations that result in health effects across asbestos-related natural mineral fiber types are not fully understood, the National Institute of Environmental Health Sciences has established a series of research studies to characterize hazards of natural mineral fibers after inhalation exposure. This paper presents the method development work of this research project. RESULTS: A prototype nose-only exposure system was fabricated to explore the feasibility of generating natural mineral fiber aerosol for in vivo inhalation toxicity studies. The prototype system consisted of a slide bar aerosol generator, a distribution/delivery system and an exposure carousel. Characterization tests conducted using Libby Amphibole 2007 (LA 2007) demonstrated the prototype system delivered stable and controllable aerosol concentration to the exposure carousel. Transmission electron microscopy (TEM) analysis of aerosol samples collected at the exposure port showed the average fiber length and width were comparable to the bulk LA 2007. TEM coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis further confirmed fibers from the aerosol samples were consistent with the bulk LA 2007 chemically and physically. CONCLUSIONS: Characterization of the prototype system demonstrated feasibility of generating LA 2007 fiber aerosols appropriate for in vivo inhalation toxicity studies. The methods developed in this study are suitable to apply to a multiple-carousel exposure system for a rat inhalation toxicity testing using LA 2007.

Internal dose of vanadium in rats following repeated exposure to vanadyl sulfate and sodium orthovanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant that exists in multiple oxidation states. Humans are exposed to vanadyl (V4+) and vanadate (V5+) from dietary supplements, food, and drinking water and hence there is a concern for adverse human health. The current investigation is aimed at identifying vanadium oxidation states in vitro and in vivo and internal concentrations following exposure of rats to vanadyl sulfate (V4+) or sodium metavanadate (V5+) via drinking water for 14 d. Investigations in simulated gastric and intestinal fluids showed that V4+ was stable in gastric fluid while V5+ was stable in intestinal fluid. Analysis of rodent plasma showed that the only vanadium present was V4+, regardless of the exposed compound suggesting conversion of V5+ to V4+ in vivo and/or instability of V5+ species in biological matrices. Plasma, blood, and liver concentrations of total vanadium, after normalizing for vanadium dose consumed, were higher in male and female rats following exposure to V5+ than to V4+. Following exposure to either V4+ or V5+, the total vanadium concentration in plasma was 2- to 3-fold higher than in blood suggesting plasma as a better matrix than blood for measuring vanadium in future work. Liver to blood ratios were 4-7 demonstrating significant tissue retention following exposure to both compounds. In conclusion, these data point to potential differences in absorption and disposition properties of V4+ and V5+ salts and may explain the higher sensitivity in rats following drinking water exposure to V5+ than V4+ and highlights the importance of internal dose determination in toxicology studies.

Inhalation exposure to multi-walled carbon nanotubes alters the pulmonary allergic response of mice to house dust mite allergen.

Background: Increasing evidence from rodent studies indicates that inhaled multi-walled carbon nanotubes (MWCNTs) have harmful effects on the lungs. In this study, we examined the effects of inhalation exposure to MWCNTs on allergen-induced airway inflammation and fibrosis. We hypothesized that inhalation pre-exposure to MWCNTs would render mice susceptible to developing allergic lung disease induced by house dust mite (HDM) allergen. Methods: Male B6C3F1/N mice were exposed by whole-body inhalation for 6 h a day, 5 d a week, for 30 d to air control or 0.06, 0.2, and 0.6 mg/m3 of MWCNTs. The exposure atmospheres were agglomerates (1.4-1.8 µm) composed of MWCNTs (average diameter 16 nm; average length 2.4 µm; 0.52% Ni). Mice then received 25 µg of HDM extract by intranasal instillation 6 times over 3 weeks. Necropsy was performed at 3 and 30 d after the final HDM dose to collect serum, bronchoalveolar lavage fluid (BALF), and lung tissue for histopathology. Results: MWCNT exposure at the highest dose inhibited HDM-induced serum IgE levels, IL-13 protein levels in BALF, and airway mucus production. However, perivascular and peribronchiolar inflammatory lesions were observed in the lungs of mice at 3 d with MWCNT and HDM, but not MWCNT or HDM alone. Moreover, combined HDM and MWCNT exposure increased airway fibrosis in the lungs of mice. Conclusions: Inhalation pre-exposure to MWCNTs inhibited HDM-induced TH2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

Effect of cell phone radiofrequency radiation on body temperature in rodents: Pilot studies of the National Toxicology Program's reverberation chamber exposure system.

Radiofrequency radiation (RFR) causes heating, which can lead to detrimental biological effects. To characterize the effects of RFR exposure on body temperature in relation to animal size and pregnancy, a series of short-term toxicity studies was conducted in a unique RFR exposure system. Young and old B6C3F1 mice and young, old, and pregnant Harlan Sprague-Dawley rats were exposed to Global System for Mobile Communication (GSM) or Code Division Multiple Access (CDMA) RFR (rats = 900 MHz, mice = 1,900 MHz) at specific absorption rates (SARs) up to 12 W/kg for approximately 9 h a day for 5 days. In general, fewer and less severe increases in body temperature were observed in young than in older rats. SAR-dependent increases in subcutaneous body temperatures were observed at exposures ≥6 W/kg in both modulations. Exposures of ≥10 W/kg GSM or CDMA RFR induced excessive increases in body temperature, leading to mortality. There was also a significant increase in the number of resorptions in pregnant rats at 12 W/kg GSM RFR. In mice, only sporadic increases in body temperature were observed regardless of sex or age when exposed to GSM or CDMA RFR up to 12 W/kg. These results identified SARs at which measurable RFR-mediated thermal effects occur, and were used in the selection of exposures for subsequent toxicology and carcinogenicity studies. Bioelectromagnetics. 39:190-199, 2018. © 2018 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Standardizing Extracted Data Using Automated Application of Controlled Vocabularies.

BACKGROUND: Extraction of toxicological end points from primary sources is a central component of systematic reviews and human health risk assessments. To ensure optimal use of these data, consistent language should be used for end point descriptions. However, primary source language describing treatment-related end points can vary greatly, resulting in large labor efforts to manually standardize extractions before data are fit for use. OBJECTIVES: To minimize these labor efforts, we applied an augmented intelligence approach and developed automated tools to support standardization of extracted information via application of preexisting controlled vocabularies. METHODS: We created and applied a harmonized controlled vocabulary crosswalk, consisting of Unified Medical Language System (UMLS) codes, German Federal Institute for Risk Assessment (BfR) DevTox harmonized terms, and The Organization for Economic Co-operation and Development (OECD) end point vocabularies, to roughly 34,000 extractions from prenatal developmental toxicology studies conducted by the National Toxicology Program (NTP) and 6,400 extractions from European Chemicals Agency (ECHA) prenatal developmental toxicology studies, all recorded based on the original study report language. RESULTS: We automatically applied standardized controlled vocabulary terms to 75% of the NTP extracted end points and 57% of the ECHA extracted end points. Of all the standardized extracted end points, about half (51%) required manual review for potential extraneous matches or inaccuracies. Extracted end points that were not mapped to standardized terms tended to be too general or required human logic to find a good match. We estimate that this augmented intelligence approach saved >350 hours of manual effort and yielded valuable resources including a controlled vocabulary crosswalk, organized related terms lists, code for implementing an automated mapping workflow, and a computationally accessible dataset. DISCUSSION: Augmenting manual efforts with automation tools increased the efficiency of producing a findable, accessible, interoperable, and reusable (FAIR) dataset of regulatory guideline studies. This open-source approach can be readily applied to other legacy developmental toxicology datasets, and the code design is customizable for other study types. https://doi.org/10.1289/EHP13215.

Mechanistic insights from the NTP studies of chromium.

Hexavalent chromium (Cr(VI)) is a contaminant of water and soil and is a human lung carcinogen. Trivalent chromium (Cr(III)), a proposed essential element, is ingested by humans in the diet and in dietary supplements such as chromium picolinate (CP). The National Toxicology Program (NTP) demonstrated that Cr(VI) is also carcinogenic in rodents when administered in drinking water as sodium dichromate dihydrate (SDD), inducing neoplasms of the oral cavity and small intestine in rats and mice, respectively. In contrast, there was no definitive evidence of toxicity or carcinogenicity following exposure to Cr(III) administered in feed as CP monohydrate (CPM). Cr(VI) readily enters cells via nonspecific anion channels, in contrast to Cr(III), which cannot easily pass through the cell membrane. Extracellular reduction of Cr(VI) to Cr(III), which occurs primarily in the stomach, is considered a mechanism of detoxification, while intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenicity. Tissue distribution studies in additional groups of male rats and female mice demonstrated higher Cr concentrations in tissues following exposure to Cr(VI) compared to controls and Cr(III) exposure at a similar external dose, indicating that some of the Cr(VI) escaped gastric reduction and was distributed systemically. The multiple potential pathways of Cr-induced genotoxicity will be discussed.

An ethanolic extract of black cohosh causes hematological changes but not estrogenic effects in female rodents.

Black cohosh rhizome (Actaea racemosa) is used as a remedy for pain and gynecological ailments; modern preparations are commonly sold as ethanolic extracts available as dietary supplements. Black cohosh was nominated to the National Toxicology Program (NTP) for toxicity testing due to its widespread use and lack of safety data. Several commercially available black cohosh extracts (BCE) were characterized by the NTP, and one with chemical composition closest to formulations available to consumers was used for all studies. Female B6C3F1/N mice and Wistar Han rats were given 0, 15 (rats only), 62.5 (mice only), 125, 250, 500, or 1000 mg/kg/day BCE by gavage for 90 days starting at weaning. BCE induced dose-dependent hematological changes consistent with a non-regenerative macrocytic anemia and increased frequencies of peripheral micronucleated red blood cells (RBC) in both species. Effects were more severe in mice, which had decreased RBC counts in all treatment groups and increased micronucleated RBC at doses above 125 mg/kg. Dose-dependent thymus and liver toxicity was observed in rats but not mice. No biologically significant effects were observed in other organs. Puberty was delayed 2.9 days at the highest treatment dose in rats; a similar magnitude delay in mice occurred in the 125 and 250 mg/kg groups but not at the higher doses. An additional uterotrophic assay conducted in mice exposed for 3 days to 0.001, 0.01, 0.1, 1, 10, 100 and 500 mg/kg found no estrogenic or anti-estrogenic activity. These are the first studies to observe adverse effects of BCE in rodents.

Systemic exposure and urinary excretion of vanadium following perinatal subchronic exposure to vanadyl sulfate and sodium metavanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant although there are limited data to assess potential adverse human health impact following oral exposure. In support of studies investigating the subchronic toxicity of vanadyl sulfate (V4+) and sodium metavanadate (V5+) following perinatal exposure via drinking water in male and female rats, we have determined the internal exposure and urinary excretion of total vanadium at the end of study. Water consumption decreased with increasing exposure concentration following exposure to both compounds. Plasma and urine vanadium concentration normalized to total vanadium consumed per day increased with the exposure concentration of vanadyl sulfate and sodium metavanadate suggesting absorption increased as the exposure concentration increased. Additionally, females had higher concentrations than males (in plasma only for vanadyl sulfate exposure). Animals exposed to sodium metavanadate had up to 3-fold higher vanadium concentration in plasma and urine compared to vanadyl sulfate exposed animals, when normalized to total vanadium consumed per day, demonstrating differential absorption, distribution, metabolism, and excretion properties between V5+ and V4+ compounds. These data will aid in the interpretation of animal toxicity data of V4+ and V5+ compounds and determine the relevance of animal toxicity findings to human exposures.

Evaluation of a semi-automated data extraction tool for public health literature-based reviews: Dextr.

INTRODUCTION: There has been limited development and uptake of machine-learning methods to automate data extraction for literature-based assessments. Although advanced extraction approaches have been applied to some clinical research reviews, existing methods are not well suited for addressing toxicology or environmental health questions due to unique data needs to support reviews in these fields. OBJECTIVES: To develop and evaluate a flexible, web-based tool for semi-automated data extraction that: 1) makes data extraction predictions with user verification, 2) integrates token-level annotations, and 3) connects extracted entities to support hierarchical data extraction. METHODS: Dextr was developed with Agile software methodology using a two-team approach. The development team outlined proposed features and coded the software. The advisory team guided developers and evaluated Dextr's performance on precision, recall, and extraction time by comparing a manual extraction workflow to a semi-automated extraction workflow using a dataset of 51 environmental health animal studies. RESULTS: The semi-automated workflow did not appear to affect precision rate (96.0% vs. 95.4% manual, p = 0.38), resulted in a small reduction in recall rate (91.8% vs. 97.0% manual, p < 0.01), and substantially reduced the median extraction time (436 s vs. 933 s per study manual, p < 0.01) compared to a manual workflow. DISCUSSION: Dextr provides similar performance to manual extraction in terms of recall and precision and greatly reduces data extraction time. Unlike other tools, Dextr provides the ability to extract complex concepts (e.g., multiple experiments with various exposures and doses within a single study), properly connect the extracted elements within a study, and effectively limit the work required by researchers to generate machine-readable, annotated exports. The Dextr tool addresses data-extraction challenges associated with environmental health sciences literature with a simple user interface, incorporates the key capabilities of user verification and entity connecting, provides a platform for further automation developments, and has the potential to improve data extraction for literature reviews in this and other fields.

Development and application of an LC-MS/MS method for the detection of the vinyl chloride-induced DNA adduct N(2),3-ethenoguanine in tissues of adult and weanling rats following exposure to [(13)C(2)]-VC.

In the 1970s, exposure to vinyl chloride (VC) was shown to cause liver angiosarcoma in VC workers. We have developed a new LC-MS/MS method for analyzing the promutagenic DNA adduct N(2),3-ethenoguanine (εG) and have applied this to DNA from tissues of both adult and weanling rats exposed to 1100 ppm [(13)C(2)]-VC for 5 days or 1100 ppm VC for 1 day. This assay utilizes neutral thermal hydrolysis and an HPLC cleanup prior to quantitation by LC-MS/MS. The number of endogenous and exogenous εG adducts in DNA from tissues of adult rats exposed to [(13)C(2)]-VC for 5 days was 4.1 ± 2.8 adducts/10(8) guanine of endogenous and 19.0 ± 4.9 adducts/10(8) guanine of exogenous εG in the liver, 8.4 ± 2.8 adducts/10(8) guanine of endogenous and 7.4 ± 0.5 adducts/10(8) guanine of exogenous εG in the lung, and 5.9 ± 3.3 adducts/10(8) guanine of endogenous and 5.7 ± 2.1 adducts/10(8) guanine of exogenous εG in the kidney (n = 4). Additionally, the data from weanling rats demonstrated higher numbers of exogenous εG, with ∼4-fold higher amounts in the liver DNA of weanlings (75.9 ± 17.9 adducts/10(8) guanine) in comparison to adult rats and ∼2-fold higher amounts in the lung (15.8 ± 3.6 adducts/10(8) guanine) and kidney (12.9 ± 0.4 adducts/10(8) guanine) (n = 8). The use of stable isotope labeled VC permitted accurate estimates of the half-life of εG for the first time by comparing [(13)C(2)]-εG in adult rats with identically exposed animals euthanized 2, 4, or 8 weeks later. The half-life of εG was found to be 150 days in the liver and lung and 75 days in the kidney, suggesting little or no active repair of this promutagenic adduct.

Validation and application of a method for the determination of total chromium in rat tissues by inductively coupled plasma mass spectrometry.

The validation of a method for the determination of chromium (Cr) in F-344/N rat tissues by inductively coupled plasma-mass spectrometry is described. Samples were analyzed after a rapid, open-vessel microwave digestion procedure. Performance of the method was evaluated using kidney tissue across a concentration range of 0.50-5.00 microg Cr/g tissue. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limits of detection and quantitation data. Data from a method cross-validation for B6C3F1 mouse kidney tissue are also presented. After validation, the method was applied to analyze samples collected in support of two chronic toxicity and carcinogenesis studies conducted by the National Toxicology Program.

Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure.

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276-290, 2020. © 2019 Wiley Periodicals, Inc.

Evaluation of 5-day In Vivo Rat Liver and Kidney With High-throughput Transcriptomics for Estimating Benchmark Doses of Apical Outcomes.

A 5-day in vivo rat model was evaluated as an approach to estimate chemical exposures that may pose minimal risk by comparing benchmark dose (BMD) values for transcriptional changes in the liver and kidney to BMD values for toxicological endpoints from traditional toxicity studies. Eighteen chemicals, most having been tested by the National Toxicology Program in 2-year bioassays, were evaluated. Some of these chemicals are potent hepatotoxicants (eg, DE71, PFOA, and furan) in rodents, some exhibit toxicity but have minimal hepatic effects (eg, acrylamide and α,ß-thujone), and some exhibit little overt toxicity (eg, ginseng and milk thistle extract) based on traditional toxicological evaluations. Male Sprague Dawley rats were exposed once daily for 5 consecutive days by oral gavage to 8-10 dose levels for each chemical. Liver and kidney were collected 24 h after the final exposure and total RNA was assayed using high-throughput transcriptomics (HTT) with the rat S1500+ platform. HTT data were analyzed using BMD Express 2 to determine transcriptional gene set BMD values. BMDS was used to determine BMD values for histopathological effects from chronic or subchronic toxicity studies. For many of the chemicals, the lowest transcriptional BMDs from the 5-day assays were within a factor of 5 of the lowest histopathological BMDs from the toxicity studies. These data suggest that using HTT in a 5-day in vivo rat model provides reasonable estimates of BMD values for traditional apical endpoints. This approach may be useful to prioritize chemicals for further testing while providing actionable data in a timely and cost-effective manner.

Toxicity and carcinogenicity of methyl isobutyl ketone in F344N rats and B6C3F1 mice following 2-year inhalation exposure.

Methyl isobutyl ketone (MIBK) is primarily used as a denaturant for rubbing alcohol, as a solvent and in the manufacture of methyl amyl alcohol. Inhalation of vapors is the most likely route of exposure in the work place. In order to evaluate the potential of MIBK to induce toxic and carcinogenic effects following chronic exposure, groups of 50 male and 50 female F344/N rats and B6C3F1 mice were exposed to MIBK at concentrations of 0, 450, 900, or 1800ppm by inhalation, 6h/day, 5 days per week for 2 years. Survival was decreased in male rats at 1800ppm. Body weight gains were decreased in male rats at 900 and 1800ppm and in female mice at 1800ppm. The primary targets of MIBK toxicity and carcinogenicity were the kidney in rats and the liver in mice. In male rats, there was increased mineralization of the renal papilla at all exposure concentrations. The incidence of chronic progressive nephropathy (CPN) was increased at 1800ppm and the severity was increased in all exposed groups. There were also increases in renal tubule hyperplasia at all exposure concentrations, and in adenoma and adenoma or carcinoma (combined) at 1800ppm; these lesions are thought to represent a continuum in the progression of proliferative lesions in renal tubule epithelium. These increases may have resulted from the increased severity of CPN, either through alpha2micro-globulin-dependent or -independent mechanisms. An increase in mononuclear cell leukemia at 1800ppm was an uncertain finding. Adrenal medulla hyperplasia was increased at 1800ppm, and there was a positive trend for increases in benign or malignant pheochromocytomas (combined). In female rats, there were increases in the incidence of CPN in all exposure concentrations and in the severity at 1800ppm, indicating that CPN was increased by mechanisms in addition to those related to alpha2micro-globulin. There were renal mesenchymal tumors, which have not been observed in historical control animals, in two female rats at 1800ppm. The relationship of these tumors to exposure to MIBK was uncertain. Hepatocellular adenomas, and adenoma or carcinoma (combined) were increased in male and female mice exposed to 1800ppm. There were also treatment-related increases in multiple adenomas in both sexes.

Influence of Helicobacter hepaticus infection on the chronic toxicity and carcinogenicity of triethanolamine in B6C3F1 mice.

Helicobacter hepaticus (H. hepaticus) infection causes hepatitis and increased hepatocellular neoplasms in male mice; although females are also infected, liver lesions are not typically expressed. In the 1990s, B6C3F1 mice from some chronic National Toxicology Program (NTP) studies were found to be infected with H. hepaticus. In these studies, there was hepatitis in many of the males, and there were more hepatocellular neoplasms in control males compared to studies with uninfected mice. In one of these studies, increased hepatocellular neoplasms at the high doses in male and female mice exposed topically to triethanolamine (TEA) provided the only evidence of carcinogenic activity. This study was repeated in mice free of H. hepaticus.However, the NTP mouse production colony and the diet differed between studies; these differences were the result of NTP programmatic decisions. In repeat study males, although control incidences were similar between studies, exposure did not result in increased hepatocellular neoplasms. In repeat study females, the control incidence of hepatocellular neoplasms was half that observed in the initial study, and these neoplasms were increased over controls at all doses. These data suggest that in the initial study, H. hepaticusinfluenced the induction of hepatocellular neoplasms in males, but not in females.

Toxicity, DNA binding, and cell proliferation in male F344 rats following short-term gavage exposures to trans-2-hexenal.

Hexenal is a genotoxic compound to which humans are exposed daily through the consumption of foods and beverages. The present studies were conducted to examine the relationships between the dose-responses of trans-2-hexenal-induced toxicity, DNA adduct formation, and cell proliferation. Male F344 rats were exposed by gavage to single doses of up to 500 mg/kg and killed 1, 2, or 4 days after dosing or were exposed to repeat doses of up to 100 mg/kg once daily for 5 days or 5 days per week for 4 weeks and killed 1 day after the end of the dosing period. Histologically, the primary observations were necroulcerative lesions, inflammation, and hyperplasia in the forestomach and inflammation in the glandular stomach. Hexenal-derived DNA adduct formation and cell proliferation were induced in the forestomach at doses of hexenal that also induced gastric toxicity; DNA adducts were not observed in the glandular stomach. These findings suggest that the toxicity of hexenal was limited to the site of contact (stomach) and that the observed DNA adduct formation and cell proliferation occurred in the setting of severe tissue damage.

Characterization of Zinc Carbonate Basic as a Source of Zinc in a Rodent Study Investigating the Effects of Dietary Deficiency or Excess.

Zinc deficiency and excess can result in adverse health outcomes. There is conflicting evidence regarding whether excess or deficient zinc in the diet can contribute to carcinogenicity. The objective of this study was to characterize zinc carbonate basic for use as a source of dietary zinc in a rodent toxicity and carcinogenicity study investigating the effects of zinc deficiency and excess. Because of the complex chemistries of zinc carbonate basic compounds, inconsistent nomenclature, and literature and reference spectra gaps, it was necessary to employ multiple analytical techniques, including Karl Fischer titration, combustion analysis, inductively coupled plasma-optical emission spectrometry, X-ray diffraction, infrared spectroscopy, X-ray fluorescence spectrometry, and thermogravimetric analysis to characterize the test article. Based on the collective evidence and through the process of elimination, the test article was found to be composed mainly of zinc carbonate basic with zinc oxide as a minor component. The zinc content was determined to be 56.6% (w/w) with heavy metals such as arsenic, cadmium, mercury and lead below the limit of quantitation of less than or equal to 0.01%. The test material was stable at ambient temperature. Based on the work described in this manuscript, the test article was suitable for use as a source of zinc in studies of deficiency and excess in the diet.