Hexavalent chromium [Cr(VI)] compounds are classied as human carcinogens [Cohen et al. 1993; International Agency or Research on Cancer (IARC) 1990; National oxicology Program (NP) 1998], based on increased incidences o lung cancers in animals and humans ater inhalation exposures. Inhalation exposures occur primarily in occupational set- tings. Te highest exposure to Cr(VI) occurs occupationally among workers involved in chrome plating, chromate production, and stainless steel welding.

As a result o industrial contamination, concentrations o Cr(VI) in the drinking water and soil may be higher than concen- trations resulting rom natural sources alone, causing ingestion o higher concentrations by populations residing near these locations than by the general population. A Cr(VI) concen- tration o 580 µg/L was ound in a ground- water monitoring well in Hinkley, Caliornia (Pellerin and Booker 2000). Detectable lev- els o Cr(VI) have been reported in approxi- mately 30% o the drinking water sources monitored in Caliornia, which has a 1 µg/L detection limit or purposes o reporting [Caliornia Department o Public Health (CDPH) 2007a]; 86% o those sources had peak concentrations o≤ 10 µg/L. Te U.S. Environmental Protection Agency (EPA) has set a maximum contaminant level o 100 µg/L or total chromium in drinking water (U.S. EPA 2003). he limit in Caliornia and

Te toxicity o Cr(VI) in humans and ani- mals has been reviewed extensively (Agency or oxic Substances and Disease Registry 2000; Costa 1997; Costa and Klein 2006; U.S. EPA 1998). We identied only one lietime animal carcinogenicity study in the literature in which Cr(VI) was administered in the drinking water (Borne et al. 1968). Further analysis o the data (Sedman et al. 2006) revealed that in three generations o emale NMRI mice, the combined incidences o benign and malig- nant orestomach neoplasms were increased over controls; this study has several limita- tions, including high early mortality in the F0 generation as a result o ectromelia (mouse- pox) virus. A review o the ew epidemiologic studies that evaluated populations that were exposed to Cr(VI) in drinking water or in soil or slag ll concluded that these studies did not provide denitive evidence o an increase in cancer incidence or mortality rates (Proctor et al. 2002). However, a retrospective mortal- ity study in China ound higher incidences o lung and stomach neoplasms in people living near a chromium smelting plant compared with the general population (Zhang and Li 1987); statistical analysis o these data sup- ported this conclusion (Beaumont et al. 2008; Sedman et al. 2006).

health eects, including carcinogenicity, and the lack o adequate carcinogenicity studies by the oral route, the Caliornia Congressional Delegation, Caliornia Environmental Protection Agency, and Caliornia Department o Health Services nominated Cr(VI) to the NP or toxicity and carcinogenicity testing. We selected sodium dichromate dihydrate (SDD) or testing because it is the primary base material or the production o chromium com- pounds, is widely used in industrial applica- tions, and is the most water-soluble chromate.

Te NP previously conducted 3-month toxicity studies o SDD administered in drinking water (NP 2007). F344/N rats and B6C3F1 mice were exposed to 0, 62.5, 125, 250, 500, or 1,000 mg SDD/L. In these studies, both rats and mice had reduced body weight and water consumption and microcytic hypochromic anemia; the anemia was more severe in rats. Exposure-related histopathologic lesions included ulcers, epithelial hyper plasia, and squamous metaplasia in the glandular stomach o rats; epithelial hyperplasia o the duodenum o mice; and histiocytic cellular inltration in the liver, duodenum, and pan- creatic lymph node o rats and in the duode- num and mesenteric lymph node o mice.

We selected exposure concentrations or the 2-year studies o SDD ater review o the 3-month toxicity study data. Te highest exposure concentration or the 2-year stud- ies in rats and mice was limited by toxicity observed in the 3-month studies (NP 2007), and a wider spacing o exposure concentra- tions was selected to extend the dose–response curve. We added an additional low-exposure group [5 mg Cr(VI)/L] to the 2-year studies to provide a concentration closer to human exposure through contaminated drinking water. In this report we present the primary ndings o the NP chronic oral toxicity and

Address correspondence to M.J. Hooth, NIEHS, P.O. Box 12233, MD K2-13, Research riangle Park, NC 27709 USA. elephone: (919) 316-4643. Fax: (919) 541-4255. E-mail: hooth@niehs.nih.gov

Tis research was supported in part by the Intramural Research Program o the National Institutes o Health, National Institute o Environmental Health Sciences, under research project 1 Z01 ES045004-11 BB.

thelium that lines the oral cavity (oral mucosa and tongue) in male and emale rats, and o the epi- thelium lining the small intestine in male and emale mice. Cr(VI) exposure did not aect survival but resulted in reduced mean body weights and water consumption, due at least in part to poor pal- atability o the dosed water. Cr(VI) exposure resulted in transient microcytic hypochromic anemia in rats and microcytosis in mice. Nonneoplastic lesions included diuse epithelial hyperplasia in the duodenum and jejunum o mice and histiocytic cell infltration in the duodenum, liver, and mesen- teric and pancreatic lymph nodes o rats and mice.

carcinogenesis studies o Cr(VI) in male and emale rats and mice; a more detailed report on these studies is available as an NP techni- cal report (NP 2008a).

7789-12-0) was obtained rom Aldrich Chemical Company (Milwaukee, WI). he purity was determined using dierential scan- ning calorimetry, titration o the dichromate ion with sodium thiosulate and potassium errocyanide, speciation o the chromium ions using liquid chromatography/inductively cou- pled plasma-mass spectrometry, and poten- tiometric titrimetric analysis with sodium thiosulate. Based on these analyses, the overall purity was ≥ 99.7%. he dose ormulations were prepared approximately every 2 weeks by mixing SDD with tap water and stored at room temperature in sealed containers pro- tected rom light. Te dose ormulations were stable or 42 days under these conditions. Periodic analysis by ultraviolet spectroscopy with detection at 350–390 nm conrmed that all dose ormulations varied by < 10% o the target concentrations.

studies were conducted at Southern Research Institute (Birmingham, AL). Male and emale F344/N rats and B6C3F1 mice were obtained rom aconic Farms (Germantown, NY). Rats and mice were quarantined or 14 days, and were 6–7 weeks o age at the beginning o the studies. Animals were distributed randomly into groups o approximately equal initial mean body weights and identied by tail tattoo. Rats and mice were housed one (male mice), three (male rats), or ve (emale rats and mice) to a cage. Feed (irradiated NP-2000 waers; Zeigler Brothers, Inc., Gardners, PA) and tap water were available ad libitum. Animals were killed by carbon dioxide asphyxiation.

Animal use was in accordance with the U.S. Public Health Service policy on humane care and use o laboratory animals and the

Animals were treated humanely and with regard or alleviation o suering. hese studies were conducted in compliance with the Food and Drug Administration Good Laboratory Practice Regulations (Food and Drug Administration 1987).

50 emale rats and mice were exposed to SDD in drinking water at concentrations o 0, 14.3, 57.3, 172, or 516 mg/L (male and emale rats and emale mice) or 0, 14.3, 28.6, 85.7, or 257.4 mg/L (male mice) or 105–106 weeks. able 1 shows corresponding Cr(VI) concen- trations. Water consumption was recorded weekly or the rst 13 weeks and every 4 weeks thereater, with each water consumption

measurement covering a 7-day period. Animals were weighed initially, weekly or the rst 13 weeks, at 4-week intervals thereater, and at the end o the studies. Animals were observed twice daily and clinical ndings were recorded at 4-week intervals beginning at week 5.

Complete necropsies and microscopic examinations were perormed on all core study rats and mice. At necropsy, all organs and tis- sues were examined or grossly visible lesions, and all protocol-required tissues were xed and preserved in 10% neutral buered ormalin (eyes were initially ixed in Davidson’s solu- tion), trimmed and processed, embedded in paran, sectioned to a thickness o 4–6 µm, and stained with hematoxylin and eosin (H&E) or microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples rom each organ were examined. Te entire gastrointestinal tract was opened and potential lesions were collected or microscopic evaluation. Oral mucosa and tongue are not protocol-required tissues; however, because gross lesions in these tissues were diagnosed as neoplasms, the oral mucosa and tongue o all animals were evaluated histologically. Additional details regarding the pathology data generation, quality assurance review, and NP pathology working group are available in the NP technical report (NP 2008a). Details o these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses o the pathology data, the decision o whether to evaluate the diagnosed lesions or each tissue type separately or combined was based on the guidelines o McConnell et al. (1986).

rats and emale mice on days 4 (male rats only) and 22 and at 3, 6, and 12 months. At these time points, rats and mice were anes- thetized with CO2/O2, and blood was taken rom the retroorbital sinus.

probability o survival by the product-limit pro- cedure o Kaplan and Meier (1958). Animals ound dead o other than natural causes or missing were censored rom the survival analy- ses; animals dying rom natural causes were not censored. Statistical analyses or possible dose- related eects on survival used Cox’s (1972) method or testing two groups or equality and arone’s (1975) lie table test to identiy dose- related trends. All reportedp-values or the survival analyses are two sided.

We used the poly-k test (Bailer and Portier 1988; Piegorsch and Bailer 1997; Portier and Bailer 1989) to assess neoplasm and non- neoplastic lesion incidence. Tis test is a sur- vival-adjusted quantal-response procedure that modiies the Cochran-Armitage linear trend test to take survival dierences into account; we used a value ok = 3 in the analysis o site- specic lesions. ests o signicance included pairwise comparisons o each exposed group with controls and a test or an overall exposure- related trend. We used continuity-corrected poly-3 tests in the analysis o lesion incidence, and reportedp-values are one sided. Sub- and supralinear trends across doses or intestinal tumor rates were assessed with lack-o-t tests or the linear regression o intestinal tumor rate on dose (Neter et al. 1996).

We analyzed hematology and clinical chemistry data, which typically have skewed distributions, using the modiied (Dunn 1964; Williams 1986) nonparametric multiple

comparison methods o Shirley (1977). We used Jonckheere’s test (Jonckheere 1954) to assess the signicance o the dose-related trends and to determine whether a trend-sensitive test (Shirley 1977; Williams 1986) was more appropriate or pairwise comparisons than a test that does not assume a monotonic dose- related trend (Dunn 1964; Dunnett 1955). Beore statistical analysis, we examined extreme values identied by the outlier test o Dixon and Massey (1957) and eliminated implausible values rom the analysis.

emale rats showed signiicantly increased incidences o highly aggressive neoplasms o the squamous epithelium that lines the oral cavity (oral mucosa and tongue) at 516 mg/L (able 2). Specically, we observed increases or squamous cell carcinoma in the oral mucosa and or squamous cell papilloma or carcinoma (combined) o the oral mucosa or tongue o male and emale rats at 516 mg/L. here were squamous cell carcinomas in the oral mucosa o two 172 mg/L emale rats; this incidence exceeded the historical control ranges or drinking water studies and or all routes o administration (able 2). We did not observe non-neoplastic lesions in the oral mucosa.

Microscopically, the squamous cell carci- nomas were highly invasive, irregular masses. ypically, the carcinomas appeared to origi- nate in the oral mucosa o the palate adja- cent to the upper molar teeth (Figure 1A, B); in some animals they invaded the tongue, Harderian gland, and the sot tissues sur- rounding the nose, and in one case it pene- trated the maxilla and invaded the brain. Te squamous cell papillomas o the oral mucosa and tongue were exophytic masses that

projected rom the mucosa and consisted o irregular papillary prolierations o mature squamous epithelium supported by a core o brovascular stroma (Figure 1C).

a clear exposure concentration response or increased incidences o adenoma or carcinoma (combined) at all sites (combined) o the small intestine (duodenum, jejunum, or ileum; able 3). Tese increases were signicant at 85.7 and 257.4 mg/L in males and at 172 and 516 mg/L emales, the two highest exposure concentrations in each sex. In addition, the incidence in 57.3 mg/L emales exceeded the historical control ranges or drinking water studies and or all routes o administration (able 3). Tese increases were driven primar- ily by signicant increases in the incidences o adenoma o the duodenum in 257.4 mg/L males and in 172 and 516 mg/L emales; the number o mice with multiple adenomas was also signicantly increased at the high dose in both sexes (data not shown). In emales, the incidence o carcinoma in the duodenum was signiicantly increased at 516 mg/L. In the jejunum, the incidence o adenoma was increased in 516 mg/L emales. For both males and emales, we tested the combined incidence o adenoma and carcinoma or departures rom a linear dose response (data not shown). For males, the trend was linear, with no signicant departure rom linearity. For emales, the response was supralinear with a signicant departure rom linearity.

Adenomas were discrete, broad based, ocally extensive, plaquelike areas o proli- erating glandular epithelium that thickened the mucosa and protruded into the lumen (Figure 1D). Carcinomas were sessile, plaque- like neoplasms distinguished rom adenomas

by extensive invasion and eacement o the mucosa, underlying submucosa, and muscle layers (Figure 1E).

Low incidences o ocal epithelial hyper- plasia occurred in the duodenum o exposed male and emale mice (able 4). Although the increased incidences were not exposure concentration related or statistically signii- cant, we considered this lesion a preneoplastic lesion related to exposure to SDD because o its morphologic similarities to adenoma. Focal epithelial hyperplasias were ocal areas o pro- lierating glandular epithelium distinguished rom adenomas because they were smaller, less discrete, and conned to the supercial mucosal epithelium (Figure 1F).

Te incidences o diuse epithelial hyper- plasia were signicantly increased in the duo- denum o all exposed groups o male and emale mice (able 4). In the jejunum, the incidence o diuse epithelial hyper plasia was signicantly increased in 516 mg/L emales. In contrast to those o the controls (Figure 1G), the duodenums o exposed mice had short, broad, blunt villi and generalized mucosal hypercellularity that was particularly promi- nent in the villi (Figure 1H).

We ound signicant increases in histiocytic cell inltration in the duodenum and mesen- teric lymph node o rats and mice o both sexes, in the jejunum o emale mice, in the liver o male and emale rats and emale mice, and in the pancreatic lymph node o emale rats and male and emale mice; these data are presented in detail in the NP technical report (NP 2008a).

Te inltrating histiocytes were morpho- logically similar in tissues o rats and mice. Histiocytic iniltrates were characterized by the presence o individual, small clusters, and

7/49 (15.7)**
Females
Oral mucosa

torical control range or both drinking water studies and all routes but was not signifcantly increased compared with the concurrent control.eThe incidence exceeded the historical control range or drinking water studies but was not signifcantly increased compared with the concurrent control. *p ≤ 0.05, **p ≤ 0.01, and#p ≤ 0.001 compared with the control group by poly-3 test or a signifcant trend i assigned to a control group.