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

In a newly published risk assessment¹, scientists integrated mode of action and toxicokinetic data to develop an RfD protective of both cancer and noncancer effects from oral exposure to hexavalent chromium. This video abstract highlights the publication resulting from this research.

An updated risk assessment for oral chromium toxicity from Research Square on Vimeo.

¹Thompson CM, Kirman CR, Hays SM, Suh M, Harvey SE, Proctor DM, Rager JE, Haws LC, Harris MA. 2017. Integration of mechanistic and pharmacokinetic information to derive oral reference dose and margin-of-exposure values for hexavalent chromium. J Appl Toxicol [e-pub ahead of print] doi: 10.1002/jat.3545

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Ten factors for considering the mode of action of Cr(VI)-induced gastrointestinal tumors in rodents

Highlights:

  • Hexavalent chromium, Cr(VI), induces intestinal tumors in mice.
  • Eastmond enumerated 10 factors for determining a mutagenic mode of action.
  • These 10 factors are used to assess the mode of action for Cr(VI).
  • Analysis supports a non-mutagenic mode of action for Cr(VI).

Abstract:

The determination of whether a chemical induces a specific cancer through a mutagenic or non-mutagenic mode of action (MOA) plays an important role in choosing between linear and nonlinear low-dose extrapolation to derive toxicity criteria. There is no formal framework from the U.S. EPA for determining whether environmental chemicals act through a mutagenic or non-mutagenic MOA; consequently, most such determinations are made on an ad hoc basis. Eastmond [Mutat Res 751 (2012)] recently conducted a systematic investigation of MOA determinations by U.S. and international regulatory agencies and organizations, and identified ten major factors that influence them, including toxicokinetics, in vivo genotoxicity in target organs, data quality, and evidence for alternative MOAs. We have used these ten factors to evaluate mutagenic vs. non mutagenic MOA for gastrointestinal tumors induced by oral exposure to hexavalent chromium [Cr(VI)]. We also highlight similarities between Cr(VI) and other intestinal carcinogens previously determined to have non-genotoxic MOAs. Based on these analyses, we conclude that the MOA for Cr(VI) induced gastrointestinal tumors is non-mutagenic and that threshold risk assessment approaches are appropriate.

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Assessment of the mutagenic potential of hexavalent chromium in the duodenum of big blue® rats

Highlights:

  • An in vivo mutation assay informed by OECD Test Guideline 488 was conducted.
  • Big Blue® (TgF344) rats were exposed to 180 ppm Cr(VI) in drinking water for 28 days.
  • TgF344 rats were gavaged 20 mg/kg ENU on days 1, 2, 3, 12, 19 & 26.
  • On day 31, mutant frequency (MF) was assessed in duodenal tissue.
  • ENU increased MF 24-fold, whereas MF was unchanged by Cr(VI)

Abstract:

A cancer bioassay on hexavalent chromium Cr(VI) in drinking water reported increased incidences of duodenal tumors in B6C3F1 mice at exposures of 30–180 ppm, and oral cavity tumors in F344 rats at 180 ppm. A subsequent transgenic rodent (TGR) in vivo mutation assay in Big Blue® TgF344 rats found that exposure to 180 ppm Cr(VI) in drinking water for 28 days did not increase cII transgene mutant frequency (MF) in the oral cavity (Thompson et al., 2015). Herein, we extend our analysis to the duodenum of these same TgF344 rats. At study termination, duodenum chromium levels were below either the limit of detection or quantification in control rats, but were 24.6 ± 3.8 μg/g in Cr(VI)-treated rats. The MF in control (23.2 × 10− 6) and Cr(VI)-treated rats (22.7 × 10− 6) were nearly identical. In contrast, the MF in the duodenum of rats exposed to 1-ethyl-1-nitrosourea for six days (study days 1, 2, 3, 12, 19, 26) increased 24-fold to 557 × 10− 6. These findings indicate that mutagenicity is unlikely an early initiating event in Cr(VI)-induced intestinal carcinogenesis.

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High-Throughput Screening Data Interpretation in the Context of In Vivo Transcriptomic Responses to Oral Cr(VI) Exposure

A recently published study by ToxStrategies scientists in Toxicological Sciences aimed to contribute to the understanding of Cr(VI) MOA by evaluating high-throughput screening (HTS) data and other in vitro data relevant to Cr(VI), and comparing these findings to robust in vivo data, including transcriptomic profiles in target tissues. Evaluation of Tox21 HTS data for Cr(VI) identified 11 active assay endpoints relevant to the Ten Key Characteristics of Carcinogens (TKCCs) that have been proposed by other investigators. Four of these endpoints were related to TP53 (tumor protein 53) activation mapping to genotoxicity (KCC#2), and four were related to cell death/proliferation (KCC#10). HTS results were consistent with other in vitro data from the Comparative Toxicogenomics Database. In vitro responses were compared to in vivo transcriptomic responses in the most sensitive target tissue, the duodenum, of mice exposed to ≤180 ppm Cr(VI) for 7 and 90 days. Pathways that were altered both in vitro and in vivo included those relevant to cell death/proliferation. In contrast, pathways relevant to p53/DNA damage were identified in vitro but not in vivo. Benchmark dose modeling and phenotypic anchoring of in vivo transcriptomic responses strengthened the finding that Cr(VI) causes cell stress/injury followed by proliferation in the mouse duodenum at high doses.

This study demonstrates both the potential strengths and limitations of HTS data. Here, HTS data were retrospectively demonstrated to have potential utility towards identifying possible key events in chemical MOA that may be prioritized for further evaluation. Still, findings show that target tissue in vivo data remain a critical component for establishing the MOA for specific chemical-induced tumor outcomes. Future studies comparing in vitro versus in vivo responses induced by other chemicals with differing physicochemical properties and target tissues will further inform potential ranges of applicability for the utility of HTS in predictive toxicology.

 

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Reevaluation and Classification of Duodenal Lesions in B6C3F1 Mice and F344 Rats from 4 Studies of Hexavalent Chromium in Drinking Water

Abstract

Thirteen-week and 2-year drinking water studies conducted by the National Toxicology Program (NTP) reported that hexavalent chromium (Cr(VI)) induced diffuse epithelial hyperplasia in the duodenum of B6C3F1 mice but not F344 rats. In the 2-year study, Cr(VI) exposure was additionally associated with duodenal adenomas and carcinomas in mice only. Subsequent 13-week Cr(VI) studies conducted by another group demonstrated non-neoplastic duodenal lesions in B6C3F1 mice similar to those of the NTP study as well as mild duodenal hyperplasia in F344 rats. Because intestinal lesions in mice are the basis for proposed safety standards for Cr(VI), and the histopathology data are relevant to the mode of action, consistency (an important Hill criterion for causality) was assessed across the aforementioned studies. Two veterinary pathologists applied uniform diagnostic criteria to the duodenal lesions in rats and mice from the 4 repeated-dose studies. Comparable non-neoplastic intestinal lesions were evident in mice and rats from all 4 studies; however, the incidence and severity of intestinal lesions were greater in mice than rats. These findings demonstrate consistency across studies and species and highlight the importance of standardized nomenclature for intestinal pathology. The differences in the severity of non-neoplastic lesions also likely contribute to the differential tumor response.

 

http://tpx.sagepub.com/content/early/2015/11/13/0192623315611501.full.pdf+html

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Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy

Highlights:

  • Micronuclei were assessed in crypts of Swiss roll duodenal sections from B6C3F1 mice.
  • Oral gavage of 50 mg/kg cyclophosphamide significantly increased micronuclei.
  • Cr(VI) in drinking water (up to 180 ppm) for 7 days did not increase micronuclei.
  • Chromium was localized to duodenal villi by X-ray fluorescence microscopy.
  • γ-H2AX immunostaining did not differ between vehicle-and Cr(VI)-treated mice.

Abstract:

Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180 ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50 mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased γ-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

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