Humans are exposed to Cr(VI) through ingestion of contaminated water and soil; however, few data exist on the oral toxicity and carcinogenicity of Cr(VI). The NTP conducted 3-month (NTP 2007
) and 2-year (NTP 2008a
) studies of SDD administered in the drinking water to F344/N rats and B6C3F1 mice, to provide data on the potential for toxic and carcinogenic effects after ingestion of Cr(VI).
Chronic administration of SDD in drinking water did not affect survival or produce clinical signs of toxicity in rats or mice. We observed exposure-related reductions in body weight gain and water consumption for rats and mice in the highest exposure groups and attributed these changes partly to poor palatability of the dosed water. Several lines of evidence suggest that the animals were not dehydrated, including analysis of the water consumption data normalized to body weight and the complete lack of clinical observations or hematologic or clinical chemistry effects (NTP 2008a
) that typically indicate dehydration.
The NTP concluded that the exposure concentration-related significant increases in epithelial neoplasms of the upper alimentary tract (oral cavity) in male and female rats and of the lower alimentary tract (small intestine) in male and female mice provided clear evidence of carcinogenic activity of SDD in male and female rats and mice. We based this conclusion on the increased neoplasm incidences relative to concurrent controls and the rarity of these neoplasms ( and ) in historical controls. In both rats and mice, this conclusion was strengthened by similarities between the sexes. We observed no increases in nonneoplastic histopathologic lesions in either species suggestive of overt tissue damage due to the oxidant properties of Cr(VI).
We observed obvious species differences in the target tissues for the development of neoplasms between rats and mice. Of the 21 chemicals that have caused neoplasms of the oral cavity in NTP studies, none produced these neoplasms in male mice and only one, 1,2,3-trichloropropane (NTP 1993
), produced oral cavity neoplasms in female mice, demonstrating a greater sensitivity to the development of oral cavity neoplasms in rats relative to mice. Although slightly more common in rats, exposure-related increases of small intestine neoplasms in NTP studies are relatively rare in both species. The 2-year study of captan (National Cancer Institute 1977
) is the only other study performed by the NTP in B6C3F1 mice in which both benign and malignant intestinal neoplasms of epithelial origin have been definitely attributed to chemical exposure (Shackelford and Elwell 1999
Although the induction of neoplasms after exposure to SDD was limited to the alimentary tract, other data, including the toxicity to the erythron, provided evidence of systemic exposure and toxicity in male and female rats and mice exposed to Cr(VI) for 2 years. We also observed these lesions in the 3-month studies (NTP 2007
As part of the NTP 2-year studies on SDD (NTP 2008a
) and chromium picolinate monohydrate (CPM) (NTP 2008b
), which contains trivalent chromium [Cr(III)], total chromium content was determined in selected tissues and excreta of additional groups of male rats and female mice; these data will be presented in detail in an additional report. The goal of these studies was to examine the tissue uptake and distribution of Cr(VI) and Cr(III). Because Cr(VI) is reduced to Cr(III) both intracellularly and extracellularly and because analytical methods for the separate analysis of Cr(VI) or Cr(III) in biological samples are not available, the speciation of the tissue chromium after exposure to Cr(VI) was inferred by comparing total chromium concentrations in tissues of rats and mice exposed to similar doses of Cr(VI) or Cr(III). After oral exposure to Cr(VI), chromium accumulation was correlated with exposure concentration and duration in several tissues (NTP 2008a
). Similar doses of Cr(VI) and Cr(III) resulted in significantly higher tissue chromium concentrations with Cr(VI), indicating that chromium was absorbed and distributed to tissues of rats and mice as Cr(VI); these data are consistent with previous studies (Costa 1997
; Costa and Klein 2006
). The tissue concentration data were consistent with linear or supralinear (decreasing rate of response with increasing dose) dose responses. In the present studies, neither the oral cavity nor the small intestine was collected for total chromium analysis. However, other reports suggest that Cr(VI) is also likely to be absorbed in the small intestine to a greater extent than Cr(III) (Donaldson and Barreras 1966
; Febel et al. 2001
Reduction of Cr(VI) to the less permeable and bioavailable Cr(III) is thought to occur primarily in the stomach, as a mechanism of detoxification. Gastric reduction has been hypothesized to be efficient, such that oral exposure to Cr(VI) would not result in toxicity or carcinogenicity, except perhaps in the stomach (De Flora 2000
; De Flora et al. 1997
; Proctor et al. 2002
). Notably, in the 2-year study, no neoplasms or nonneoplastic lesions were observed in the forestomach or glandular stomach of rats or mice. However, the observed increases in neoplasms of the small intestine of mice and the toxicity to the erythron, histiocytic infiltration, and uptake of Cr(IV) into tissues of rats and mice suggest that, under the conditions of this study, at least a portion of the administered Cr(VI) was not reduced in the stomach. The significant disparity in the oral toxicity and carcinogenicity of Cr(VI) and Cr(III) in rodents, including the absence of increases in neoplasms or nonneoplastic lesions of the small intestine in rats or mice exposed to CPM (NTP 2008b
), provides additional evidence that Cr(VI) is not completely reduced in the stomach and is responsible for the observed effects.
Recently, De Flora et al. (2008)
have suggested that increases in neoplasms of the small intestine observed in mice in the present study are the result of saturation of the gastric reduction capacity. If such a threshold mechanism were to occur, the dose that saturated the reducing capacity would likely represent an inflection point on a sublinear dose response curve, with doses above the inflection point demonstrating an increasing rate of response per unit dose, because unreduced chromium would be transported into tissues. However, when we tested tissue concentration and mouse small intestine neoplasm data for linearity, data that were statistically nonlinear were supralinear (decreasing rate of response per unit dose).
A reduction capacity of about 84–88 mg Cr(VI)/day has been estimated for human gastric juice (De Flora et al. 1997
). This estimate was based on reported values of human secretion of gastric fluid per day during fasting and after consuming three meals per day in combination with experimental data on reduction of Cr(VI)/mL of gastric juice produced during these periods. Similar data are not available for Cr(VI) reduction by mouse gastric juice. However, assuming that Cr(VI) reduction is equally effective in mice and humans and that gastric secretion scales across species by body weight3/4
, then the Cr(VI) reduction capacity of gastric juice from a 50-g mouse would be approximately 0.4 mg/day (~ 8 mg/kg/day). This value is greater than all of the male mouse doses and is nearly equivalent to the average daily dose of Cr(VI) in the high-dose group of female mice in the 2-year drinking water study of SDD (). Collectively, the dose–response analysis and gastric reduction capacity calculations indicate that SDD induced neoplasms in the small intestine of mice at dose levels that did not exceed the estimated Cr(VI) reducing capacity for gastric juices in mice.
Cr(VI) is genotoxic in a number of in vitro
and in vivo
test systems (De Flora et al. 1990
; IARC 1990
); however, the mechanisms of genotoxicity and carcinogenicity are not fully understood. Because Cr(VI) as chromate structurally resembles sulfate and phosphate, it can be taken up by all cells and organs throughout the body through non-specific anion transporters (Costa 1997
). Once inside the cell, indirect DNA damage may occur through the generation of oxygen radicals during intracellular reduction of Cr(VI) through the more reactive pentavalent and tetravalent chromium to Cr(III) (O’Brien et al. 2003
); however, evidence of the role of reactive oxygen species in the genotoxicity of Cr(VI) is inconsistent (Chorvatovicova et al. 1991
; O’Brien et al. 2003
; Standeven and Wetterhahn 1991
; Zhitkovich 2005
). Cr(III), the final product of intracellular reduction of Cr(VI), has been shown to interact directly with DNA and other macromolecules to induce chromosomal alterations and mutational changes (O’Brien et al. 2003
; Quievryn et al. 2006
; Reynolds et al. 2007
; Zhitkovich 2005
). DNA adducts, DNA–protein cross-links, and DNA interstrand cross-links have all been identified as products of Cr(III)–DNA interactions. The relative contributions of the multiple, complex pathways of chromium-induced genotoxicity continue to be investigated.
In conclusion, the NTP 2-year study of SDD is the first and only lifetime study that clearly demonstrates the carcinogenicity of Cr(VI) in rats and mice after oral exposure. In addition, the hematology, histologic and tissue distribution data provide evidence of systemic exposure in rats and mice.