TY - JOUR
AU - Roycroft, Joseph, H.
AB - Abstract Decalin (decahydronaphthalene) is a widely used industrial solvent known to cause male rat-specific α2u-globulin nephropathy. In this project, 13-week and two-year inhalation studies of decalin were conducted consecutively in both sexes of F344/N rats. The key objectives were to (1) characterize the 13-week toxicity of decalin in rats, with an emphasis on nephropathy in males; (2) compare the kidney concentrations of decalin, 2-decalone, and α2u-globulin in males over 2 to 13 weeks of decalin exposure; and (3) correlate male rat nephropathy observed in the 13-week study with renal carcinogenicity in the two-year study. F344 rats (M/F) were exposed via whole-body inhalation to 0, 25, 50, 100, 200, or 400 ppm decalin for 13 weeks. Urine was collected at weeks 2 and 6 for creatinine and decalol analyses and at week 12 for clinical urinalysis. Right kidneys were collected from male rats at weeks 2 and 6 and from both sexes at week 13, homogenates were prepared using the whole kidney, and these homogenates were analyzed for α2u-globulin, decalin, and 2-decalone. Left kidneys were evaluated for histopathology and cell proliferation utilizing a proliferating cell nuclear antigen technique and counting proximal renal tubular epithelial cells to determine cell labeling indices. Necropsies and histopathologic evaluations were performed at week 13. Decalin exposure caused increases in kidney weight, urinalysis parameters (protein, AST, LDH), kidney α2u-globulin concentration, and proximal convoluted renal tubular cell proliferation in males. These changes were accompanied by microscopic lesions (accumulation of hyaline droplets in cortical tubules, regeneration of proximal tubular epithelium, and granular casts in medullary tubules) clearly linked to α2u-globulin nephropathy. Both decalin and 2-decalone were related to increased α2u-globulin in male kidneys. Kidney concentrations of decalin, 2-decalone, and α2u-globulin in exposed females were negligible, while females excreted greater amounts of decalol metabolites in urine than males at weeks 2 and 6. There were no exposure-related microscopic lesions in females. For chronic exposure, F344 rats were exposed via whole-body inhalation to 0, 25, 50 (males only), 100, or 400 ppm decalin for two years. Chronic exposure induced a spectrum of nonneoplastic and neoplastic lesions in the renal cortex of males, ranging from regenerative lesions of chronic nephropathy to tubular carcinomas. Incidences of renal tubular adenoma, tubular carcinoma, combined tubular adenomas and carcinomas, cortical tubular hyperplasia, hyaline droplet accumulation, hyperplasia of pelvic epithelium, and mineralization in renal papilla were increased in exposed males compared to controls. There was a clear increase in the mean severity of chronic nephropathy in decalin-exposed males. It was concluded that the carcinogenic effect on the renal cortical epithelium of male rats exposed to decalin was related to increased turnover of this epithelium, resulting from the cytotoxic effects of α2u-globulin accumulation in the renal cortical tubular cell cytoplasm. decalin (decahydronaphthalene), 2-decalone, decalol, α2u-globulin, hyaline droplet, cell proliferation, hyperplasia, nephropathy, toxicity, carcinogenicity, sex, F344/N Decalin (decahydronaphthalene) is a widely used industrial solvent for fats, oils, resins, and waxes and is a replacement for turpentine in lacquers and oil paints (Longacre, 1987). A high potential for human inhalation or dermal exposure to decalin exists as a result of contact with crude oil or combustion products, during solvent manufacturing or use, or as an environmental contaminant. No serious industrial poisonings or data suggesting carcinogenicity in humans have been reported. Decalin is irritating to the skin, eyes, and mucous membranes, and causes central nervous system depression at high concentrations (Budavari, 1989). The 4-h LC50 for male Sprague-Dawley rats is 710 ppm (MacEwen and Vernot, 1977). Decalin is nonmutagenic in the Ames/Salmonella assay and in the mouse lymphoma assay and does not induce sister chromatid exchanges or micronuclei in canine peripheral lymphocytes (Benz and Beltz, 1980). The most recognized toxicity related to decalin exposure in laboratory animals is α2u-globulin-induced nephropathy in male rats. Decalin is one of a variety of chemicals that induce α2u-globulin accumulation (CIGA) in the male rat kidney (Bruner and Pitts, 1983; Mao et al., 1998; Stone et al., 1987). The synthesis of α2u-globulin is species-, sex-, and age-specific, in that the protein is synthesized in the liver under androgenic control in male rats. Low levels of α2u-globulin are detected in the male rat liver under the stimulus of testosterone at 35–40 days, and reach maximum levels by 60–80 days. Due to the development of hepatic insensitivity to androgen during aging, hepatic synthesis of α2u-globulin falls gradually after five months of age, and the levels are reduced by more than 90% by 22 months of age in male rats (Hard et al., 1993; Motwani et al., 1984; Roy et al., 1983). Neither α2u-globulin nor its corresponding mRNA is detectable in the livers of sexually intact female rats (Hard et al., 1993; MacInnes et al., 1986). α2u-Globulin is filtered by the glomerulus; approximately half the filtrate is normally reabsorbed into kidney proximal tubules, digested in cytoplasmic lysosomes, and reabsorbed back into the circulation. Lehman-McKeeman et al.(1990) demonstrated in vitro that α2u-globulin forms a complex with CIGA and becomes resistant to lysosomal hydrolysis, leading to its accumulation in tubular epithelium cytoplasm in the form of hyaline droplets. Considerable effort has been invested to establish the mechanistic basis for chemical-induced, male rat-specific nephropathy because of the implications of the potential end result, renal neoplasia, for human risk assessment (Borghoff et al., 1990, 1991; Swenberg, 1993). Although decalin exposure undoubtedly induces α2u-globulin accumulation, it is not clear which chemical moieties, either decalin alone or its metabolites or both, are responsible for in vivo binding to α2u-globulin. With fusion of two cyclohexane rings, both cis- and trans-decalin may be present in the commercial product (Olson et al., 1986). Decalin is extensively metabolized in vivo, potentially forming a variety of isomeric metabolites following exposure, such as 2-decalone and isomers of decalols (Longacre, 1987). When cis- or trans-decalin was administered to female rabbits, 67% (cis) or 53% (trans) of the dose, respectively, was eliminated in urine as decalol glucuronide conjugates (Elliott et al., 1966). In addition to decalols in urine, 2-decalone has been detected in the kidneys of male rats but not females (Elliott et al., 1966; Olson et al., 1986). α2u-Globulin nephropathy has been observed following short-term exposure to decalin using gavage and inhalation routes (Gaworski et al., 1985; Saito et al., 1992). However, there has been no study to determine if chronic exposure to decalin in male rats would result in significant increases in chronic toxicity and more importantly, renal carcinogenicity. Differentiating α2u-globulin-mediated renal tumors in male rats in a chronic study from other possible etiologies is critical for risk assessment because renal tumors induced by α2u-globulin nephropathy are not used in hazard characterization for the human population (Alison et al., 1994; Borghoff et al., 1996; Swenberg, 1993). Chronic exposure to other CIGA such as d-limonene and dichlorobenzene induced a low incidence of renal neoplasia in male rats (Borghoff et al., 1990; NTP, 1987, 1988). Several of these compounds also caused an increase in the severity and/or incidence of chronic progressive nephropathy not only in male rats but also in females, indicating that another mechanism of chronic renal toxicity might be operating in aging rat kidneys (Borghoff et al., 1996; NTP, 1986, 1987; U.S. EPA, 1991). Decalin was selected for inhalation toxicity and carcinogenicity studies by the National Institute of Environmental Health Sciences (NIEHS) based on the lack of long-term animal toxicity data, the high potential for consumer exposure, and its structural similarity to naphthalene or tetralin. This report presents findings related to renal toxicity and carcinogenicity following 13-week and two-year studies. The specific objectives were to (1) characterize the subchronic toxicity of decalin in male and female F344 rats, with an emphasis on nephropathy in males; (2) compare the concentrations of decalin, 2-decalone, and α2u-globulin in homogenates prepared from whole kidneys collected from males over 2 to 13 weeks of decalin exposure; and (3) correlate the nephropathy observed in the 13-week study with renal toxicity and carcinogenicity in the two-year study. MATERIALS AND METHODS Chemicals. Decalin (C10H18, decahydronaphthalene, CAS #91-17-8) was purchased from Sigma Aldrich Fluka (SAF) Bulk Chemicals (St. Louis, MO) for the 13-week study (cis:trans ratio of 35:65) and from Aldrich Chemical Company (Milwaukee, WI) for the two-year study (cis:trans ratio of 42:58). The relative purities of decalin for both studies were 100 to 101% by gas chromatography (GC), compared to independent high-purity (99%) standards obtained from Aldrich Chemical Co. Decalin was stored under a nitrogen headspace at room temperature; reanalyses performed at intervals throughout each study indicated no change in the relative purity of the test material. Unless cited otherwise, all other chemicals were purchased from either Sigma (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI), and were reagent-grade quality or better. Animals. Male and female F344 rats were obtained from Taconic (Germantown, NY) at ∼4 weeks of age and quarantined for 10 to 14 days before study. They were randomized, assigned to groups using body weight as a blocking variable, and housed in individual wire-mesh units in exposure chambers (Hazleton 2000, Lab Products, Inc., Harford System Division; Aberdeen, MD). Chamber environmental conditions (temperature, 75 ± 3°F; relative humidity, 55 ± 15%; airflow, 15 ± 2 cfm) were monitored at ∼4-h cycles for 24 h/day and maintained within acceptable ranges through the studies. Water and food (NTP-2000, Zeigler Bros., Inc., Gardners, PA) were available ad libitum, except food was withheld during exposures and urine collection. A 12-h light/dark cycle (light started at 0600 h) was maintained throughout the studies. Experimental Design 13-Week inhalation study. Rats (∼6 weeks old; total 20 rats/sex/group) were exposed via whole-body inhalation to 0, 25, 50, 100, 200, or 400 ppm decalin for 6 h plus T90/day (T90 ≅ 12 min), five days/week (exclusive of weekends and holidays) for up to 13 weeks. Animals were observed at least twice daily for moribundity and mortality, and their individual body weights and clinical signs were recorded weekly and at terminal sacrifice. At weeks 2 and 6 (five/sex/group) and week 12 (10/sex/group), rats were placed in metabolism cages (Lab Products Inc., Maywood, NJ) for 16-h urine collection immediately following exposure. Urine samples collected at weeks 2 and 6 were analyzed for volume, creatinine, and decalol isomers. Urine samples collected at week 12 were analyzed for the following analytes using Roche Cobas Fara methodologies: creatinine (Larson, 1972), protein (Silverman and Christenson, 1994), lactate dehydrogenase (LDH; Gay et al., 1968), aspartate aminotransferase (AST; Moss and Henderson, 1994), and N-acetyl-glucosaminidase (NAG; Yakata, 1983). Following urine collections at weeks 2 and 6, male rats were euthanized and kidneys were collected (five males/group). A complete necropsy was performed on both sexes at terminal sacrifice (week 13; 10/sex/group), selected organs (kidney, liver, thymus, testis, heart, and lungs) were collected and weighed. Homogenates prepared using the right kidney from male rats at weeks 2, 6, and 13 were subsequently analyzed for α2u-globulin, decalin, and 2-decalone. Homogenates prepared from the right kidney from female rats at week 13 were analyzed for decalin and 2-decalone. Left kidneys from both sexes were fixed in 10% neutral buffered formalin, processed, and stained with hematoxylin and eosin (H&E) for histopathologic evaluation. Left kidneys from male rats were also stained with Mallory-Heidenhain (MH) for protein evaluation and with anti-proliferating cell nuclear antigen (anti-PCNA) for determination of cell proliferation indices. Complete histopathologic evaluations were performed on all controls and 400-ppm rats. Target organs and gross lesions were examined in lower exposure groups until adverse effects were not observed. Kidneys from male rats were evaluated microscopically for all exposure groups. Two-year inhalation study. Rats (∼6 weeks old) were exposed via whole-body inhalation to 0, 25, 50, 100, or 400 ppm decalin for males (50/group, except 20 males at 400 ppm) and at 0, 25, 100, or 400 ppm for females (50/group) for 6 h plus T90/day (T90 ≅ 12 min), 5 days/week (exclusive of weekends and holidays) for two years. Note that in selecting these exposure concentrations for male rats, pathology findings from the 13-week inhalation study suggested that kidney lesions in male rats exposed to 400 ppm would be too severe, and that significant body weight loss and mortality would likely occur in male rats at this concentration during the two-year study. However, the feed for these studies was the relatively low-protein NTP 2000 diet (∼14% protein), and it was of interest to determine whether less severe male kidney lesions might be observed with this low-protein diet. Accordingly, the high exposure concentration for male rats in the chronic study was set at 100 ppm while the high exposure concentration remained at 400 ppm for female rats (50 rats/exposure/sex). An additional group of 20 male rats was exposed to 400 ppm to evaluate the effects of the low protein diet on the severity of kidney lesions. It was also necessary to include some males with the female rats exposed to 400 ppm to promote estrus cycling. Because male and female rats were housed in the same 400 ppm chamber as mice during a concurrently run two-year study, space limitations precluded using a greater number of male rats at this concentration. Animals were observed at least twice daily for moribundity and mortality and their individual body weights and clinical signs were recorded monthly through week 89, and every two weeks thereafter until terminal sacrifice. Complete necropsy and histopathological evaluations were done on all animals. Generation and monitoring of decalin exposure. Decalin was pumped through a preheater into the top of a heated glass column filled with glass beads. Heated nitrogen entered the column from below, assisting vaporization while transporting the decalin vapor to the chambers in heated lines to prevent condensation. Decalin exposure concentrations were monitored every ∼24 min during the exposure by on-line GC/FID (Hewlett-Packard [HP]-5890). The peaks for the cis- and trans-isomers were integrated separately, which showed no fractionation of decalin composition. The sum of the cis and trans peak areas was used to calculate the concentration of decalin for each determination. Calibration of the on-line monitor was achieved by quantitative determination of decalin in exposure chamber samples collected with adsorbent gas sampling tubes (ORBO 101; Supelco, Bellfonte, PA). The actual exposure concentrations for both 13-week and two-year studies were maintained within 5% of the target exposure concentrations. Renal cell proliferation. The antibody of proliferating cell nuclear antigen (PCNA; PC-10 clone) was obtained from DAKO (Carpenteria, CA). Sections of left kidneys and duodenum (as a positive control for cell proliferation), stained with anti-PCNA, were qualitatively (duodenum) and quantitatively (kidneys) assessed for proliferation labeling. Using a 20× objective and a 10 × 10 mm optical micrometer, ∼2000 proximal tubule nuclei in the cortex were counted from kidney sections and numbers of labeled and total (S-phase labeled and unlabeled) nuclei were recorded. Proximal tubules were counted from the lysosomal segment of the proximal convoluted tubule, referred as the P2 segment in the cortex of the kidney. The lysosomal segment of the straight segment of the proximal tubule was not counted. Identification of cells in the S-phase was based on cellular distribution and intensity of the reaction product. Proximal tubule nuclei with uniform deep red to black nuclear staining were recorded as positive for PCNA expression and counted as labeled. Cells in the G1 (those cells with minimal nuclear staining) and G2 (speckled nuclear and cytoplasmic staining) were not counted as labeled. This method is consistent with the method of Foley et al.(1991). Selection of the counting sites was done using the method of Larson et al.(1994): Starting at the second field in from the outer edge of the cortex at one pole of the kidney and moving from pole to pole across the cortex, all identifiable proximal tubular cell nuclei in every other field were counted until a total of ∼2000 proximal tubule nuclei were counted. If <2000 were obtained when the opposite pole of the kidney was reached, the grid was moved toward the convex surface of the kidney and counting was resumed using the same method as described above. The labeling index was calculated by dividing the number of labeled nuclei by the total nuclei, expressed as a percent. Diagnosis of proliferative lesions in kidney. In the kidney, hyperplasia, adenoma, and carcinoma are thought to represent a continuum in the progression of proliferative lesions of the renal tubule epithelium. Hyperplasias were generally focal lesions characterized by increased numbers of tubule epithelial cells forming multiple layers that partially or totally filled the tubule lumen and usually caused dilatation of the tubule. Adenomas were generally discrete expansile masses that were larger than hyperplasias (greater than the diameter of five tubules) and had a more complex structure. Carcinomas were less discrete and larger than adenomas with hemorrhage, necrosis, local invasion, and/or metastasis to distant sites (Hard et al., 1995). Analyses of α2u-Globulin, Decalin, and 2-Decalone in Kidney α2u-Globulin. Supernatants prepared from kidney homogenates were analyzed for α2u-globulin by competitive indirect ELISA (Borghoff et al., 1992), using ascites fluid containing anti-α2u-globulin monoclonal antibody, which was kindly provided by Dr. Susan J. Borghoff (CIIT Center for Health Research, Research Triangle Park, NC). Decalin. Kidney concentrations of cis- and trans-decalin were determined following cyclohexane extraction of kidney homogenates (∼100 mg) spiked with (cis + trans)-decalin-d18 (Aldrich Chemical Co.), using a mass selective detector interfaced to a gas chromatograph. Temperature programming (50 to 300°C) was used to conduct separations on a fused-silica capillary column (DB-5MS; 30-m × 0.25-mm ID; film thickness, 0.25-μm; J&W Scientific, Folsom, CA) with helium as the carrier gas. Selected ion mass chromatograms were obtained by monitoring characteristic ions for cis- and trans-decalin (m/z 138) and trans-decalin-d18 (m/z 156). Concentrations for total decalin isomers (cis and trans) were used for statistics and reporting. 2-Decalone. Kidney concentrations of cis- and trans-2-decalone (authentic standards for each isomer were purchased from Aldrich Chemical Co.) were determined following cyclohexane extraction of kidney homogenates spiked with 2-decalone-1,1,3,3-d4 (synthesized by Dr. John Cashman; Seattle Biomedical Research Institute, Seattle, WA) using GC/MS. Temperature programming (70 to 250°C) was used to conduct separations on a fused silica capillary column (DB-1701; 30-m × 0.25-mm ID; film thickness, 0.25 μm; J&W Scientific), with helium as the carrier gas. Selected ion mass chromatograms were obtained by monitoring characteristic ions for cis- and trans-2-decalone (m/z 152) and 2-decalone-d4 (m/z 156). Concentrations for total 2-decalone isomers (cis and trans) were used for statistics and reporting. Analysis of decalol isomers in urine. Urine samples (10 μl) were added to 0.2 M acetate buffer (100 μl; pH ∼5.4) containing an internal standard (1,2,3,4-tetrahydro-1-naphthol; Aldrich Chemical Co.) and incubated overnight at 37°C with ∼10 μl β-glucuronidase/arylsulfatase (Boehringer Mannheim, GmbH, Germany). Samples were extracted with cyclohexane and the eight possible decalol isomers were analyzed using GC/MS. Temperature programming (70 to 250°C) was used to conduct separations on a fused silica capillary column (DB-Wax; 30-m × 0.25-mm ID; film thickness 0.25 μm; J&W Scientific), with helium as the carrier gas. Selected ion mass chromatograms were obtained by monitoring characteristic ions for the eight decalol (m/z 136) isomers and 1,2,3,4-tetrahydro-1-naphthol (m/z 130). Six urinary decalol isomers were identified as trans,cis-1-decalol, trans,trans-1-decalol, trans,trans-2-decalol, trans,cis-2-decalol, cis,cis-2-decalol, and cis,cis-1-decalol (standards synthesized by Dr. John Cashman [Seattle Biomedical Research Institute], except cis,cis-1-decalol [Aldrich Chemical Co.]). No standards for cis,trans-1-decalol and cis,trans-2-decalol were available for isomer identification. Concentrations for total decalol isomers (all eight isomers) were used for statistics and reporting. Statistics. The Toxicology Data Management System (TDMS, supplied by NIEHS, Research Triangle Park, NC) was used to collect data and to perform statistical analyses for toxicology and pathology data. A modified Dunnett’s t-test using the Xybion Path/Tox System (Cedar Knolls, NJ) was used to compare exposed and control groups with respect to body and organ weights, and organ:body weight ratios. ANOVA using Statistical Analysis System software (SAS Institute, Inc., Cary, NC) was used to compare treated and control groups with respect to cell proliferation, α2u-globulin, decalin, 2-decalone, and total decalol concentrations, and clinical urinalysis measurements. Difference was considered statistically significant at p ≤ 0.05. RESULTS 13-Week Inhalation Study In-life observations. There were no exposure-related deaths, remarkable clinical signs, or differences in body weight gains for both sexes. The most significant finding at terminal sacrifice was an exposure-related increase in kidney and liver weights in males (Table 1). The organ weight increase was most substantial in male kidneys, as both the absolute and relative weights increased by ∼20%, while those of male liver weights increased by ∼11% above controls. The exposed females displayed no changes in kidney weights, but the relative liver weight increased at 400 ppm by ∼10% above controls (Table 1). Week 12 clinical urinalysis. On week 12, urinary concentrations of AST, LDH, and protein (but not NAG) of exposed males increased significantly relative to those of controls, particularly when normalized to creatinine concentration (Table 2). Compared to controls, AST activities increased by ∼5-fold, while LDH activities increased by ∼3-fold in decalin-exposed males. However, these increases were overall comparable in magnitude among exposure groups, showing no clear dose-response relationship. Increases in AST and LDH activities were also noted in females, but to a lesser degree than males and only at 100 ppm or higher (Table 2). Cell proliferation for male kidneys. The renal cell proliferation indices significantly (p ≤ 0.05) increased in all exposed male groups relative to the controls, except the 25 and 50 ppm groups at week 2 and 50 ppm group at week 13. However, the increases did not show any dose-response relationship and relative increases in exposed groups above the controls were remarkable (>∼2%) only for the 400-ppm group at week 6 (Fig. 1; Table 3). Renal cell proliferation indices at week 6 tended to be higher than those at weeks 2 and 13, while no substantial difference was observed between the latter two time points. Cell proliferation rates were not determined for the duodenum section. Histopathology, week 2, male kidneys only. Accumulation of hyaline droplets in renal proximal cortical tubules of exposed male rats was characterized by rounded eosinophilic droplets of variable size in the cytoplasm. Control males had minimal numbers of droplets visible. MH staining allowed clear separation of exposed groups from controls with blind evaluation. The majority of males in all exposed groups had clearly increased amounts of hyaline droplets compared to controls. Minimal regeneration of proximal tubular epithelium was observed in at least one animal in all decalin-exposed groups, with the exception of the 50-ppm group (Table 4). Foci of regeneration were characterized by basophilic tubules of slightly smaller diameter, containing an increased density of nuclei with an increase in mitotic figures. There was no clear increase in tubular regeneration with exposure concentration. Histopathology, week 6, male kidneys only. Control males necropsied at week 6 had more hyaline droplets than control males necropsied at week 2, when appreciated primarily with MH staining. As observed on MH and H&E staining, hyaline droplets from exposed males were larger than those of controls and those of exposed groups evaluated at week 2. Severity of proximal tubular regeneration was increased in groups exposed to 100 ppm or higher, compared with severity in groups at lower concentrations. Granular casts, consisting of granular cell debris and proteinaceous material, were present in the outer medulla at the junction of the inner and outer stripe of males exposed to 50 ppm or higher. Granular casts increased in severity and incidence with increasing exposure concentration. Histopathology, week 13, complete necropsy for both sexes. No exposure-related lesions were observed in female rats. Lesions related to decalin exposure were observed in the kidneys of the male rats. Number and size of hyaline droplets in male kidneys were related to exposure concentration; however it was difficult to differentiate the amounts among the 100-, 200-, and 400-ppm groups. The 50-ppm group had fewer droplets than rats exposed to 100 ppm and higher, and kidneys from the 25-ppm group were very difficult to differentiate from control kidneys on blinded evaluation based on the presence of hyaline droplets, even with the aid of the MH stain. Although not diagnosed separately, there was microscopic evidence of minimal proximal tubular degeneration accompanying hyaline droplet accumulation in some males exposed to higher concentrations. This lesion was characterized by swollen, granular, and disrupted cytoplasm, pyknosis, karyorrhexis, and sloughing of cellular contents into the tubular lumen. Foci of regenerating proximal tubules were present in only decalin-exposed rats. Males exposed to concentrations of 100 ppm or higher had regenerative lesions of similar severity, while those exposed to 50 or 25 ppm had fewer foci of regenerative tubules. Occasional granular casts filled dilated tubules in the outer zone of the renal medulla. Although minimal to mild, granular casts were easily detected and more clearly related to exposure concentration than the other lesions observed. Kidney decalin concentrations. In exposed females, kidney decalin concentrations increased ∼12 times between 25 and 400 ppm decalin at week 13 (Table 5). Kidney decalin concentrations from the 200- and 400-ppm groups were statistically higher than those of the lower exposure groups. Overall kidney decalin concentrations in females were substantially (∼40-fold) lower than those in males within the same exposure group. In exposed males, kidney decalin concentrations increased with increasing exposure concentration (Table 5, Fig. 2). At week 2, decalin concentrations at 100 ppm or less were not different from each other, but they were significantly (p ≤ 0.05) lower than those at higher exposure concentrations. At weeks 6 and 13, the concentration of decalin in the kidney increased with exposure concentration. Decalin concentrations increased substantially between weeks 2 and 6, but no increases were observed between weeks 6 and 13 at all exposure concentrations. Kidney 2-decalone concentrations. Kidney 2-decalone concentrations in exposed females (week 13 only) were below the limit of quantitation (LOQ: < 0.041 μg/g kidney). In exposed males, the 400-ppm group had slightly but significantly higher 2-decalone concentrations than the lower exposure groups at week 2 (Fig. 3). At weeks 6 and 13, 2-decalone concentrations in males tended to increase with increasing exposure concentration up to 100 ppm, but reached a plateau at higher concentrations. As with kidney decalin concentrations, 2-decalone concentrations at week 6 were significantly higher than those at week 2, with no further increases observed at week 13. Kidney α2u-globulin concentrations. Kidney α2u-globulin concentrations from control males slightly increased over time (0.15, 2.5, and 2.9 mg α2u-globulin/g kidney for weeks 2, 6, and 13, respectively). Those of exposed males appeared to increase to a greater extent with increasing exposure concentration (Fig. 4). However, due to considerable interanimal variability in the data, only the mean α2u-globulin concentrations from the 200- and 400-ppm groups were statistically greater than that from controls. Similar to the trends in kidney decalin and 2-decalone concentrations, α2u-globulin concentrations increased significantly between weeks 2 and 6, with no further increase at week 13. In order to determine whether both the parent chemical (decalin) and/or metabolite (decalone) were involved in α2u-globulin binding in kidneys, the mean kidney concentrations of decalin and 2-decalone from exposed male groups were converted to their molar concentrations, combined, and compared to those of α2u-globulin (Table 6). For this comparison, the α2u-globulin molar concentration was corrected, by subtracting the mean value from controls to discount the background levels of α2u-globulin in male kidneys at each sampling time. The ratio tended to increase at 400 ppm compared to the lower exposure groups at week 2, but in general no remarkable trend was noted in the ratio with exposure concentration. The grand mean ratio for all exposed groups was 0.99, or the unit value of 1, with individual ratios ranging from 0.34 to 2.0. These comparable molar quantities implied that the quantity of α2u-globulin in kidneys was closely linked with the combined quantity of decalin and 2-decalone. The relative amount of decalin versus 2-decalone varied with exposure concentration (data not shown). At exposure concentrations ≤100 ppm, there was a higher molar concentration of 2-decalone than decalin by up to two-fold, but this trend appeared to be reversed at higher exposure concentrations. At 400 ppm, there was a greater molar concentration of decalin than 2-decalone at all sampling times. Urinary excretion of decalol isomers. Urine volumes collected 16 h postexposure were not significantly different with regard to sex or exposure concentration (data not shown). Urinary creatinine concentrations were not affected by exposure concentration, but were significantly different as a function of sex and time, revealing that older animals excreted more creatinine, and that males had higher creatinine levels than females at later sampling times (Table 7). These trends in creatinine were expected, as body masses of animals increased over the period of ∼10 weeks and was more marked in males than females. Urinary concentrations for the combined decalol isomers increased in exposed groups with increasing exposure concentration (Table 7). The creatinine-normalized decalol was significantly higher at week 2 than 6 for both sexes because of relatively lower creatinine at week 2. However, the total amount of decalols did not change much between these time points in either sex. This was in contrast to the substantial increase in kidney decalin and 2-decalone concentrations between weeks 2 and 6 in exposed males. Females generally excreted 1.2- to 1.9-fold more total decalols than males at both times. Two-Year Inhalation Study In-life observations. All exposed male groups exhibited slightly lower two-year survival (40–47%) relative to controls (56%), except the 400-ppm group (70%). In females, only the 400-ppm group had slightly lower survival (56%) compared to controls (64%). Although differences in the mean body weights of surviving animals were generally minimal between the exposed and control groups in either sex, the 400-ppm male group had consistently lower body weights (∼3 to 7%) than controls, starting ∼ week 33 on study (data not shown). There were no significant clinical signs, in either sex, associated with decalin exposure throughout the study. Histopathology. Chronic inhalation exposure to decalin induced a spectrum of nonneoplastic and neoplastic lesions in the renal cortex of male rats, ranging from an increase in the severity of the regenerative lesions of chronic nephropathy to tubular carcinomas (Table 8). Incidences of renal tubule adenoma and adenoma or carcinoma (combined) were significantly increased in males exposed to 50 ppm or greater. Incidences of adenoma and adenoma or carcinoma (combined) in all exposed groups of males exceeded the historical ranges in controls (all routes of exposure) given the NTP-2000 diet (NIEHS, 2002). The incidences of carcinoma were also increased in exposed males and although these increases were not statistically significant, the incidences exceeded the historical control range. Two renal tubular cell carcinomas metastasized to the lung; one in a male rat exposed to 100 ppm and one in a male rat exposed to 25 ppm. There were no increased incidences of renal tumors in females that were related to exposure to decalin. There were no statistically significant increases in incidences of tumors of nonrenal tissues in either male or female rats that could be related to exposure to decalin. All exposed groups of males also had significantly increased incidences of hyperplasia (Table 8). The severity of nephropathy was greater in all exposed groups of males than in the controls (Table 8). Changes related to nephropathy consisted of a spectrum of lesions. These lesions included varying degrees of tubular dilation, proteinaceous tubular casts, atrophy, regeneration and hypertrophy of tubular epithelium, thickening of tubular and glomerular basement membranes, interstitial fibrosis, and varying numbers and aggregates of mononuclear inflammatory cells within the interstitium. The incidence of nephropathy was also significantly increased in 100-ppm females. Incidences of hyaline droplet accumulation were generally increased in exposed males, and the increases were significant in the 25- and 100-ppm groups. The hyaline droplets were similar to those seen in the 13-week study. Because the production of α2u-globulin in male rats declines later in life, an exposure-related increase in hyaline droplets was not expected at the end of a two-year study. The vast majority of the affected animals died or were sacrificed early, which might explain the increased incidences of the hyaline droplets. The incidence and severity of mineralization of the renal papilla were also increased in all exposed groups of males and the severity and incidence increased with exposure concentration. The mineral was present in tubules within the papilla and, in many instances, the profiles of the mineral were elongate (linear mineralization), which is characteristic of mineralization associated with α2u-globulin-induced nephropathy. The incidence of hyperplasia of transitional epithelium lining of the renal pelvis was significantly increased in all exposed groups of males. This change was generally mild and was characterized by increased thickness, often with papillary projections of the transitional epithelium lining the renal pelvis. DISCUSSION The kidney was the target organ in male F344 rats exposed to decalin for 13 weeks, at concentrations ranging from 25 to 400 ppm. This was evidenced by increases in kidney weights, urinary enzymes, and the severity and incidence of nephropathy in exposed male rats. The renal lesions observed in decalin-exposed males were clearly representative of α2u-globulin-induced nephropathy. There was a much greater accumulation of hyaline droplets in cortical tubules and regeneration of proximal tubular epithelium in all exposed male rats. Granular casts in medullary tubules were observed in one or more exposed males at 50 ppm or higher at week 6, and in all exposed groups at week 13 with incidence and severity increasing with exposure concentration. Granular casts, observed in the outer zone of the renal medulla, were a major characteristic of early α2u-globulin nephropathy. PCNA expression is greatly dependent on the cell cycle and that PCNA expression is increased and peaked at the time of S-phase where the PCNA production is greatest (Foley et al., 1991). Cell proliferation labeling indices were elevated for all decalin-exposed male rats, supporting evidence of an increase in renal cellular proliferation following exposure. As anticipated, the progression of histopathological lesions observed in the cortical region in male kidneys generally correlated well with changes in kidney α2u-globulin concentrations measured in the homogenates prepared from the kidney. Relatively milder lesions at week 2 were associated with α2u-globulin concentration substantially lower than those observed at week 13. The finding that females tended to eliminate more decalin, in the form of urinary decalols, than males was probably due to a substantially lower level of α2u-globulin (Dill et al., 2003). Low levels of decalin in female kidney homogenates likely reflect a passive tissue partitioning of decalin from blood. However, it is not known if other sex differences such as the rate or extent of decalin metabolism could also contribute to higher urinary excretion in females (Longacre, 1987). In addition to increases in kidney α2u-globulin concentration in male rat kidney homogenates between weeks 2 and 6, concentrations of decalin and 2-decalone also increased. Accumulations of both decalin and 2-decalone, in a similar molar ratio to α2u-globulin at all exposure concentrations, supported the hypothesis that both the parent and the metabolite bind to α2u-globulin, thereby leading to the accumulation of the protein in male kidneys. Within the total molar amount, there was a higher amount of 2-decalone than decalin at lower exposure concentrations (i.e., ≤100 ppm), but the relative amount of decalin increased with exposure concentration (i.e., ≥ 200 ppm). Several possible explanations might account for this observation. Metabolism of decalin to 2-decalone could be saturated at high exposure concentrations, leaving more decalin available for α2u-globulin binding. In a single inhalation study, the area under the blood decalin concentration curve increased supra-proportionally to the exposure concentration at ≥ 100 ppm (Dill et al., 2003). A lesser amount of decalin metabolized at saturating high exposure concentrations would result in relatively less 2-decalone being generated and thus competing for α2u-globulin binding. Although the actual binding affinities of decalin and 2-decalone to α2u-globulin were not measured, a higher amount of decalin would leave less free α2u-globulin for 2-decalone binding. Additionally, it is possible that formation of decalin-α2u-globulin complexes might delay the metabolism of decalin to 2-decalone. A combination of these different processes likely occurred with the net result that the relative quantity of the 2-decalone in the male kidneys decreased while that of decalin increased with increasing exposure concentration, but their combined molar amount was overall equivalent to that for α2u-globulin. Exposure to decalin at concentrations up to 400 ppm for two years did not result in significant in-life toxicity in males. A higher two-year survival of 400-ppm male rats compared to the other exposed or control males was likely associated with slightly but consistently lower mean body weights of the 400-ppm group. The association of low body weights with increased survival in laboratory animals has been recognized in the literature (Haseman et al., 1989). It appears that decalin exposure caused lower body weight gain, which contributed to the slightly higher survival for the 400-ppm group but the exposure-related pathological changes in kidneys did not increase moribundity or mortality during two years of exposure. However, it is noteworthy that the 400-ppm groups had a smaller number of animals used (20) compared to 50 males/exposure at other exposure concentrations. This might have increased the degree of uncertainty in the survival rate of the 400-ppm group. Chronic exposure to decalin induced a spectrum of neoplastic and nonneoplastic lesions in male kidneys, ranging from an increase in the severity of the regenerative lesions of chronic nephropathy to malignant renal tumors. The combined incidence of renal tubular carcinomas and adenomas in males was 1/50, 3/50, 7/49, 12/50, and 6/20 for the 0-, 25-, 50-, 100-, and 400-ppm groups, respectively. The combined incidence of these two tumor types in historical controls of male F344 rats fed NTP-2000 diet in NIEHS two-year studies is 3/906 (NIEHS, 2002). The increase in renal tumor incidence, coupled with the incidence of cortical tubular hyperplasia and the increased severity of chronic nephropathy in exposed males, clearly indicates a detrimental effect of chronic exposure to decalin on male kidneys. Linear mineralization of renal papillary epithelium and hyperplasia of transitional epithelium lining the renal pelvis were also clearly related to decalin exposure. This spectrum of lesions is characteristic of renal toxicity and carcinogenesis related to the chronic accumulation of α2u-globulin in cortical tubular epithelium (Hard et al., 1993). Hyaline droplets, although present in a relatively low incidence, were observed in cortical tubular epithelium of some males necropsied relatively early in this two-year study. Granular casts in the renal medulla were rarely observed in the chronic males. This lack of granular casts and hyaline droplets in male rats chronically exposed to CIGA was reported previously (Hard et al., 1993), and was considered to be related to the spontaneous decrease in α2u-globulin levels in aging rats. In summary, decalin inhalation exposure from 25 to 400 ppm for up to 13 weeks clearly induced male rat-specific α2u-globulin nephropathy. Both decalin and 2-decalone accumulated in the male kidneys but not in female rat kidneys along with α2u-globulin, suggesting that one or both of these compounds may bind to the protein. Following a two-year exposure, a spectrum of nonneoplastic and neoplastic urinary tract lesions involving the renal cortex was present in exposed male rats. It is concluded that the nonneoplastic lesions caused by chronic exposure to decalin were related to the carcinogenic effect on the renal cortical epithelium in male rats. This effect was considered to be related to excessive α2u-globulin bound to decalin or its metabolite in the cytoplasm of cortical tubular epithelial cells, resulting in cytotoxicity and increased renal cortical epithelium cell turnover. TABLE 1 Mean Body Weights and Kidney and Liver Weights from Male and Female Rats Exposed to Decalin for 13 Weeks . . Right kidney . Liver . Exposure concentration . Body weight . Organ weight . Ratio . Organ weight . Ratio . Note. Values are mean ± SD; n = 10. Exposure concentrations are given in ppm; all weights are in g. Ratio = organ weight/body weight × 1000.*p ≤ 0.05; **p ≤ 0.01. Males     0 302 ± 16 0.91 ± 0.06 3.0 ± 0.1 9.40 ± 0.42 31.1 ± 1.0     25 306 ± 29 0.97 ± 0.11 3.2 ± 0.1 9.92 ± 1.3 32.3 ± 2.0     50 295 ± 18 0.96 ± 0.07 3.3 ± 0.1** 9.88 ± 0.65 33.6 ± 2.0**     100 290 ± 14 0.94 ± 0.06 3.3 ± 0.1** 9.51 ± 0.67 32.8 ± 1.1     200 311 ± 20 1.05 ± 0.10** 3.4 ± 0.2** 10.5 ± 1.0** 33.8 ± 1.6**     400 303 ± 15 1.09 ± 0.08** 3.6 ± 0.2** 10.5 ± 0.64** 34.5 ± 1.1** Females     0 177 ± 8.7 0.61 ± 0.05 3.4 ± 0.2 5.38 ± 0.23 30.4 ± 2.1     25 174 ± 7.2 0.62 ± 0.03 3.5 ± 0.2 5.19 ± 0.18 29.9 ± 0.7     50 182 ± 7.7 0.64 ± 0.03 3.5 ± 0.1 5.81 ± 0.68 31.8 ± 3.0     100 181 ± 13 0.65 ± 0.04 3.6 ± 0.2 5.81 ± 0.84 32.0 ± 2.6     200 175 ± 9.5 0.63 ± 0.04 3.6 ± 0.2 5.61 ± 0.53 31.9 ± 1.7     400 173 ± 12 0.62 ± 0.04 3.6 ± 0.2 5.79 ± 0.46* 33.5 ± 1.0** . . Right kidney . Liver . Exposure concentration . Body weight . Organ weight . Ratio . Organ weight . Ratio . Note. Values are mean ± SD; n = 10. Exposure concentrations are given in ppm; all weights are in g. Ratio = organ weight/body weight × 1000.*p ≤ 0.05; **p ≤ 0.01. Males     0 302 ± 16 0.91 ± 0.06 3.0 ± 0.1 9.40 ± 0.42 31.1 ± 1.0     25 306 ± 29 0.97 ± 0.11 3.2 ± 0.1 9.92 ± 1.3 32.3 ± 2.0     50 295 ± 18 0.96 ± 0.07 3.3 ± 0.1** 9.88 ± 0.65 33.6 ± 2.0**     100 290 ± 14 0.94 ± 0.06 3.3 ± 0.1** 9.51 ± 0.67 32.8 ± 1.1     200 311 ± 20 1.05 ± 0.10** 3.4 ± 0.2** 10.5 ± 1.0** 33.8 ± 1.6**     400 303 ± 15 1.09 ± 0.08** 3.6 ± 0.2** 10.5 ± 0.64** 34.5 ± 1.1** Females     0 177 ± 8.7 0.61 ± 0.05 3.4 ± 0.2 5.38 ± 0.23 30.4 ± 2.1     25 174 ± 7.2 0.62 ± 0.03 3.5 ± 0.2 5.19 ± 0.18 29.9 ± 0.7     50 182 ± 7.7 0.64 ± 0.03 3.5 ± 0.1 5.81 ± 0.68 31.8 ± 3.0     100 181 ± 13 0.65 ± 0.04 3.6 ± 0.2 5.81 ± 0.84 32.0 ± 2.6     200 175 ± 9.5 0.63 ± 0.04 3.6 ± 0.2 5.61 ± 0.53 31.9 ± 1.7     400 173 ± 12 0.62 ± 0.04 3.6 ± 0.2 5.79 ± 0.46* 33.5 ± 1.0** Open in new tab TABLE 1 Mean Body Weights and Kidney and Liver Weights from Male and Female Rats Exposed to Decalin for 13 Weeks . . Right kidney . Liver . Exposure concentration . Body weight . Organ weight . Ratio . Organ weight . Ratio . Note. Values are mean ± SD; n = 10. Exposure concentrations are given in ppm; all weights are in g. Ratio = organ weight/body weight × 1000.*p ≤ 0.05; **p ≤ 0.01. Males     0 302 ± 16 0.91 ± 0.06 3.0 ± 0.1 9.40 ± 0.42 31.1 ± 1.0     25 306 ± 29 0.97 ± 0.11 3.2 ± 0.1 9.92 ± 1.3 32.3 ± 2.0     50 295 ± 18 0.96 ± 0.07 3.3 ± 0.1** 9.88 ± 0.65 33.6 ± 2.0**     100 290 ± 14 0.94 ± 0.06 3.3 ± 0.1** 9.51 ± 0.67 32.8 ± 1.1     200 311 ± 20 1.05 ± 0.10** 3.4 ± 0.2** 10.5 ± 1.0** 33.8 ± 1.6**     400 303 ± 15 1.09 ± 0.08** 3.6 ± 0.2** 10.5 ± 0.64** 34.5 ± 1.1** Females     0 177 ± 8.7 0.61 ± 0.05 3.4 ± 0.2 5.38 ± 0.23 30.4 ± 2.1     25 174 ± 7.2 0.62 ± 0.03 3.5 ± 0.2 5.19 ± 0.18 29.9 ± 0.7     50 182 ± 7.7 0.64 ± 0.03 3.5 ± 0.1 5.81 ± 0.68 31.8 ± 3.0     100 181 ± 13 0.65 ± 0.04 3.6 ± 0.2 5.81 ± 0.84 32.0 ± 2.6     200 175 ± 9.5 0.63 ± 0.04 3.6 ± 0.2 5.61 ± 0.53 31.9 ± 1.7     400 173 ± 12 0.62 ± 0.04 3.6 ± 0.2 5.79 ± 0.46* 33.5 ± 1.0** . . Right kidney . Liver . Exposure concentration . Body weight . Organ weight . Ratio . Organ weight . Ratio . Note. Values are mean ± SD; n = 10. Exposure concentrations are given in ppm; all weights are in g. Ratio = organ weight/body weight × 1000.*p ≤ 0.05; **p ≤ 0.01. Males     0 302 ± 16 0.91 ± 0.06 3.0 ± 0.1 9.40 ± 0.42 31.1 ± 1.0     25 306 ± 29 0.97 ± 0.11 3.2 ± 0.1 9.92 ± 1.3 32.3 ± 2.0     50 295 ± 18 0.96 ± 0.07 3.3 ± 0.1** 9.88 ± 0.65 33.6 ± 2.0**     100 290 ± 14 0.94 ± 0.06 3.3 ± 0.1** 9.51 ± 0.67 32.8 ± 1.1     200 311 ± 20 1.05 ± 0.10** 3.4 ± 0.2** 10.5 ± 1.0** 33.8 ± 1.6**     400 303 ± 15 1.09 ± 0.08** 3.6 ± 0.2** 10.5 ± 0.64** 34.5 ± 1.1** Females     0 177 ± 8.7 0.61 ± 0.05 3.4 ± 0.2 5.38 ± 0.23 30.4 ± 2.1     25 174 ± 7.2 0.62 ± 0.03 3.5 ± 0.2 5.19 ± 0.18 29.9 ± 0.7     50 182 ± 7.7 0.64 ± 0.03 3.5 ± 0.1 5.81 ± 0.68 31.8 ± 3.0     100 181 ± 13 0.65 ± 0.04 3.6 ± 0.2 5.81 ± 0.84 32.0 ± 2.6     200 175 ± 9.5 0.63 ± 0.04 3.6 ± 0.2 5.61 ± 0.53 31.9 ± 1.7     400 173 ± 12 0.62 ± 0.04 3.6 ± 0.2 5.79 ± 0.46* 33.5 ± 1.0** Open in new tab TABLE 2 Urinalysis in Male and Female Rats Exposed to Decalin for 12 Weeks . Exposure concentration (ppm) . Parameters . 0 . 25 . 50 . 100 . 200 . 400 . Note. Values are mean ± SD; n = 1. *p ≤ 0.05; **p ≤ 0.01. Males     Creatinine (mg/dl) 70.2 ± 38 65.9 ± 38 59.0 ± 36 58.0 ± 39 49.0 ± 22 62.9 ± 35     Protein:creatinine 1.23 ± 0.2 1.48 ± 0.27 1.71 ± 0.16** 1.79 ± 0.31** 1.65 ± 0.24** 1.63 ± 0.23**     AST (U/l) 10.6 ± 5.8 46.6 ± 26* 53.4 ± 37* 56.0 ± 38** 46.1 ± 28* 57.5 ± 37**     AST:creatinine 0.15 ± 0.02 0.73 ± 0.11** 0.90 ± 0.13** 1.0 ± 0.26** 0.89 ± 0.20** 0.88 ± 0.18**     LDH (U/l) 29.9 ± 18 77.9 ± 40 88.6 ± 59* 95.3 ± 59* 77.5 ± 41 102 ± 65**     LDH:creatinine 0.41 ± 0.07 1.25 ± 0.21** 1.48 ± 0.25** 1.73 ± 0.37** 1.52 ± 0.31** 1.57 ± 0.24**     NAG (U/l) 15.3 ± 10 16.5 ± 8.8 17.7 ± 9.1 17.5 ± 10 13.6 ± 6.7 17.9 ± 9.8     NAG:creatinine 0.34 ± 0.52 0.26 ± 0.04 0.33 ± 0.10 0.32 ± 0.05 0.27 ± 0.04 0.29 ± 0.03 Females     Creatinine (mg/dl) 40.1 ± 11 31.6 ± 14 35.1 ± 11 39.8 ± 15 38.5 ± 16 33.7 ± 12     Protein:creatinine 0.09 ± 0.02 0.09 ± 0.03 0.09 ± 0.03 0.10 ± 0.02 0.08 ± 0.02 0.09 ± 0.02     AST (U/l) 1.2 ± 1.1 1.4 ± 0.7 2.0 ± 0.9 2.0 ± 0.9 2.5 ± 1.0* 2.1 ± 1.1     AST:creatinine 0.03 ± 0.02 0.04 ± 0.01 0.06 ± 0.02 0.05 ± 0.02 0.07 ± 0.03** 0.07 ± 0.05**     LDH (U/l) 18.0 ± 6.6 21.5 ± 8.5 26.6 ± 11.5 30.9 ± 8.7* 35.9 ± 14.1** 33.3 ± 13.3*     LDH:creatinine 0.47 ± 0.21 0.70 ± 0.16 0.75 ± 0.16* 0.82 ± 0.19** 0.95 ± 0.10** 1.02 ± 0.33**     NAG 5.2 ± 1.8 4.2 ± 2.4 5.5 ± 2.0 5.7 ± 2.2 5.5 ± 2.5 4.6 ± 1.9     NAG:creatinine 0.13 ± 0.01 0.13 ± 0.02 0.16 ± 0.05* 0.14 ± 0.01 0.14 ± 0.02 0.13 ± 0.02 . Exposure concentration (ppm) . Parameters . 0 . 25 . 50 . 100 . 200 . 400 . Note. Values are mean ± SD; n = 1. *p ≤ 0.05; **p ≤ 0.01. Males     Creatinine (mg/dl) 70.2 ± 38 65.9 ± 38 59.0 ± 36 58.0 ± 39 49.0 ± 22 62.9 ± 35     Protein:creatinine 1.23 ± 0.2 1.48 ± 0.27 1.71 ± 0.16** 1.79 ± 0.31** 1.65 ± 0.24** 1.63 ± 0.23**     AST (U/l) 10.6 ± 5.8 46.6 ± 26* 53.4 ± 37* 56.0 ± 38** 46.1 ± 28* 57.5 ± 37**     AST:creatinine 0.15 ± 0.02 0.73 ± 0.11** 0.90 ± 0.13** 1.0 ± 0.26** 0.89 ± 0.20** 0.88 ± 0.18**     LDH (U/l) 29.9 ± 18 77.9 ± 40 88.6 ± 59* 95.3 ± 59* 77.5 ± 41 102 ± 65**     LDH:creatinine 0.41 ± 0.07 1.25 ± 0.21** 1.48 ± 0.25** 1.73 ± 0.37** 1.52 ± 0.31** 1.57 ± 0.24**     NAG (U/l) 15.3 ± 10 16.5 ± 8.8 17.7 ± 9.1 17.5 ± 10 13.6 ± 6.7 17.9 ± 9.8     NAG:creatinine 0.34 ± 0.52 0.26 ± 0.04 0.33 ± 0.10 0.32 ± 0.05 0.27 ± 0.04 0.29 ± 0.03 Females     Creatinine (mg/dl) 40.1 ± 11 31.6 ± 14 35.1 ± 11 39.8 ± 15 38.5 ± 16 33.7 ± 12     Protein:creatinine 0.09 ± 0.02 0.09 ± 0.03 0.09 ± 0.03 0.10 ± 0.02 0.08 ± 0.02 0.09 ± 0.02     AST (U/l) 1.2 ± 1.1 1.4 ± 0.7 2.0 ± 0.9 2.0 ± 0.9 2.5 ± 1.0* 2.1 ± 1.1     AST:creatinine 0.03 ± 0.02 0.04 ± 0.01 0.06 ± 0.02 0.05 ± 0.02 0.07 ± 0.03** 0.07 ± 0.05**     LDH (U/l) 18.0 ± 6.6 21.5 ± 8.5 26.6 ± 11.5 30.9 ± 8.7* 35.9 ± 14.1** 33.3 ± 13.3*     LDH:creatinine 0.47 ± 0.21 0.70 ± 0.16 0.75 ± 0.16* 0.82 ± 0.19** 0.95 ± 0.10** 1.02 ± 0.33**     NAG 5.2 ± 1.8 4.2 ± 2.4 5.5 ± 2.0 5.7 ± 2.2 5.5 ± 2.5 4.6 ± 1.9     NAG:creatinine 0.13 ± 0.01 0.13 ± 0.02 0.16 ± 0.05* 0.14 ± 0.01 0.14 ± 0.02 0.13 ± 0.02 Open in new tab TABLE 2 Urinalysis in Male and Female Rats Exposed to Decalin for 12 Weeks . Exposure concentration (ppm) . Parameters . 0 . 25 . 50 . 100 . 200 . 400 . Note. Values are mean ± SD; n = 1. *p ≤ 0.05; **p ≤ 0.01. Males     Creatinine (mg/dl) 70.2 ± 38 65.9 ± 38 59.0 ± 36 58.0 ± 39 49.0 ± 22 62.9 ± 35     Protein:creatinine 1.23 ± 0.2 1.48 ± 0.27 1.71 ± 0.16** 1.79 ± 0.31** 1.65 ± 0.24** 1.63 ± 0.23**     AST (U/l) 10.6 ± 5.8 46.6 ± 26* 53.4 ± 37* 56.0 ± 38** 46.1 ± 28* 57.5 ± 37**     AST:creatinine 0.15 ± 0.02 0.73 ± 0.11** 0.90 ± 0.13** 1.0 ± 0.26** 0.89 ± 0.20** 0.88 ± 0.18**     LDH (U/l) 29.9 ± 18 77.9 ± 40 88.6 ± 59* 95.3 ± 59* 77.5 ± 41 102 ± 65**     LDH:creatinine 0.41 ± 0.07 1.25 ± 0.21** 1.48 ± 0.25** 1.73 ± 0.37** 1.52 ± 0.31** 1.57 ± 0.24**     NAG (U/l) 15.3 ± 10 16.5 ± 8.8 17.7 ± 9.1 17.5 ± 10 13.6 ± 6.7 17.9 ± 9.8     NAG:creatinine 0.34 ± 0.52 0.26 ± 0.04 0.33 ± 0.10 0.32 ± 0.05 0.27 ± 0.04 0.29 ± 0.03 Females     Creatinine (mg/dl) 40.1 ± 11 31.6 ± 14 35.1 ± 11 39.8 ± 15 38.5 ± 16 33.7 ± 12     Protein:creatinine 0.09 ± 0.02 0.09 ± 0.03 0.09 ± 0.03 0.10 ± 0.02 0.08 ± 0.02 0.09 ± 0.02     AST (U/l) 1.2 ± 1.1 1.4 ± 0.7 2.0 ± 0.9 2.0 ± 0.9 2.5 ± 1.0* 2.1 ± 1.1     AST:creatinine 0.03 ± 0.02 0.04 ± 0.01 0.06 ± 0.02 0.05 ± 0.02 0.07 ± 0.03** 0.07 ± 0.05**     LDH (U/l) 18.0 ± 6.6 21.5 ± 8.5 26.6 ± 11.5 30.9 ± 8.7* 35.9 ± 14.1** 33.3 ± 13.3*     LDH:creatinine 0.47 ± 0.21 0.70 ± 0.16 0.75 ± 0.16* 0.82 ± 0.19** 0.95 ± 0.10** 1.02 ± 0.33**     NAG 5.2 ± 1.8 4.2 ± 2.4 5.5 ± 2.0 5.7 ± 2.2 5.5 ± 2.5 4.6 ± 1.9     NAG:creatinine 0.13 ± 0.01 0.13 ± 0.02 0.16 ± 0.05* 0.14 ± 0.01 0.14 ± 0.02 0.13 ± 0.02 . Exposure concentration (ppm) . Parameters . 0 . 25 . 50 . 100 . 200 . 400 . Note. Values are mean ± SD; n = 1. *p ≤ 0.05; **p ≤ 0.01. Males     Creatinine (mg/dl) 70.2 ± 38 65.9 ± 38 59.0 ± 36 58.0 ± 39 49.0 ± 22 62.9 ± 35     Protein:creatinine 1.23 ± 0.2 1.48 ± 0.27 1.71 ± 0.16** 1.79 ± 0.31** 1.65 ± 0.24** 1.63 ± 0.23**     AST (U/l) 10.6 ± 5.8 46.6 ± 26* 53.4 ± 37* 56.0 ± 38** 46.1 ± 28* 57.5 ± 37**     AST:creatinine 0.15 ± 0.02 0.73 ± 0.11** 0.90 ± 0.13** 1.0 ± 0.26** 0.89 ± 0.20** 0.88 ± 0.18**     LDH (U/l) 29.9 ± 18 77.9 ± 40 88.6 ± 59* 95.3 ± 59* 77.5 ± 41 102 ± 65**     LDH:creatinine 0.41 ± 0.07 1.25 ± 0.21** 1.48 ± 0.25** 1.73 ± 0.37** 1.52 ± 0.31** 1.57 ± 0.24**     NAG (U/l) 15.3 ± 10 16.5 ± 8.8 17.7 ± 9.1 17.5 ± 10 13.6 ± 6.7 17.9 ± 9.8     NAG:creatinine 0.34 ± 0.52 0.26 ± 0.04 0.33 ± 0.10 0.32 ± 0.05 0.27 ± 0.04 0.29 ± 0.03 Females     Creatinine (mg/dl) 40.1 ± 11 31.6 ± 14 35.1 ± 11 39.8 ± 15 38.5 ± 16 33.7 ± 12     Protein:creatinine 0.09 ± 0.02 0.09 ± 0.03 0.09 ± 0.03 0.10 ± 0.02 0.08 ± 0.02 0.09 ± 0.02     AST (U/l) 1.2 ± 1.1 1.4 ± 0.7 2.0 ± 0.9 2.0 ± 0.9 2.5 ± 1.0* 2.1 ± 1.1     AST:creatinine 0.03 ± 0.02 0.04 ± 0.01 0.06 ± 0.02 0.05 ± 0.02 0.07 ± 0.03** 0.07 ± 0.05**     LDH (U/l) 18.0 ± 6.6 21.5 ± 8.5 26.6 ± 11.5 30.9 ± 8.7* 35.9 ± 14.1** 33.3 ± 13.3*     LDH:creatinine 0.47 ± 0.21 0.70 ± 0.16 0.75 ± 0.16* 0.82 ± 0.19** 0.95 ± 0.10** 1.02 ± 0.33**     NAG 5.2 ± 1.8 4.2 ± 2.4 5.5 ± 2.0 5.7 ± 2.2 5.5 ± 2.5 4.6 ± 1.9     NAG:creatinine 0.13 ± 0.01 0.13 ± 0.02 0.16 ± 0.05* 0.14 ± 0.01 0.14 ± 0.02 0.13 ± 0.02 Open in new tab TABLE 3 Cell Proliferation Rates for Kidney in Male Rats Exposed to Decalin for Weeks 2, 6, and 13 . Labeling index × 100 . Exposure concentration (ppm) . 2 Weeks . 6 Weeks . 13 Weeks . Note. Mean ± SD; n = 5. 0 3.33 ± 0.47 3.16 ± 0.23 3.05 ± 0.42 25 4.17 ± 0.29 4.22 ± 0.30 4.52 ± 0.21 50 3.94 ± 0.64 4.82 ± 0.24 3.69 ± 0.62 100 4.27 ± 0.46 4.64 ± 0.57 4.08 ± 0.52 200 4.20 ± 0.65 5.03 ± 0.52 4.51 ± 0.41 400 4.89 ± 0.76 5.90 ± 0.62 4.47 ± 0.34 . Labeling index × 100 . Exposure concentration (ppm) . 2 Weeks . 6 Weeks . 13 Weeks . Note. Mean ± SD; n = 5. 0 3.33 ± 0.47 3.16 ± 0.23 3.05 ± 0.42 25 4.17 ± 0.29 4.22 ± 0.30 4.52 ± 0.21 50 3.94 ± 0.64 4.82 ± 0.24 3.69 ± 0.62 100 4.27 ± 0.46 4.64 ± 0.57 4.08 ± 0.52 200 4.20 ± 0.65 5.03 ± 0.52 4.51 ± 0.41 400 4.89 ± 0.76 5.90 ± 0.62 4.47 ± 0.34 Open in new tab TABLE 3 Cell Proliferation Rates for Kidney in Male Rats Exposed to Decalin for Weeks 2, 6, and 13 . Labeling index × 100 . Exposure concentration (ppm) . 2 Weeks . 6 Weeks . 13 Weeks . Note. Mean ± SD; n = 5. 0 3.33 ± 0.47 3.16 ± 0.23 3.05 ± 0.42 25 4.17 ± 0.29 4.22 ± 0.30 4.52 ± 0.21 50 3.94 ± 0.64 4.82 ± 0.24 3.69 ± 0.62 100 4.27 ± 0.46 4.64 ± 0.57 4.08 ± 0.52 200 4.20 ± 0.65 5.03 ± 0.52 4.51 ± 0.41 400 4.89 ± 0.76 5.90 ± 0.62 4.47 ± 0.34 . Labeling index × 100 . Exposure concentration (ppm) . 2 Weeks . 6 Weeks . 13 Weeks . Note. Mean ± SD; n = 5. 0 3.33 ± 0.47 3.16 ± 0.23 3.05 ± 0.42 25 4.17 ± 0.29 4.22 ± 0.30 4.52 ± 0.21 50 3.94 ± 0.64 4.82 ± 0.24 3.69 ± 0.62 100 4.27 ± 0.46 4.64 ± 0.57 4.08 ± 0.52 200 4.20 ± 0.65 5.03 ± 0.52 4.51 ± 0.41 400 4.89 ± 0.76 5.90 ± 0.62 4.47 ± 0.34 Open in new tab TABLE 4 Kidney Lesions in Male Rats Exposed to Decalin for Up to 13 Weeks . Exposure concentration (ppm) . Kidney organ, diagnosis . 0 . 25 . 50 . 100 . 200 . 400 . aIncidence. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Exposure week 2     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5a (1.0)b 5 (1.8) 5 (1.8) 5 (1.6) 5 (1.6) 5 (2.0)     Cortex, renal tubule, regeneration 0 1 (1.0) 0 2 (1.0) 1 (1.0) 3 (1.0) Exposure week 6     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5 (1.0) 5 (2.0) 5 (2.0) 5 (2.4) 5 (3.0) 5 (3.0)     Cortex, renal tubule, regeneration 1 (1.0) 4 (1.0) 5 (1.0) 5 (1.8) 5 (1.6) 5 (2.0)     Medulla, granular casts 0 0 1 (1.0) 4 (1.0) 5 (1.2) 5 (1.4) Exposure week 13     Number examined microscopically 10 10 10 10 10 10     Cortex, renal tubule, accumulation, hyaline droplet 10 (1.0) 10 (1.5) 10 (2.2) 10 (2.8) 10 (3.0) 10 (2.7)     Cortex, renal tubule, regeneration 1 (1.0) 10 (1.0) 10 (1.6) 10 (2.0) 10 (2.0) 10 (2.0)     Medulla, granular casts 0 2 (1.0) 7 (1.0) 8 (1.5) 10 (2.0) 10 (2.2) . Exposure concentration (ppm) . Kidney organ, diagnosis . 0 . 25 . 50 . 100 . 200 . 400 . aIncidence. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Exposure week 2     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5a (1.0)b 5 (1.8) 5 (1.8) 5 (1.6) 5 (1.6) 5 (2.0)     Cortex, renal tubule, regeneration 0 1 (1.0) 0 2 (1.0) 1 (1.0) 3 (1.0) Exposure week 6     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5 (1.0) 5 (2.0) 5 (2.0) 5 (2.4) 5 (3.0) 5 (3.0)     Cortex, renal tubule, regeneration 1 (1.0) 4 (1.0) 5 (1.0) 5 (1.8) 5 (1.6) 5 (2.0)     Medulla, granular casts 0 0 1 (1.0) 4 (1.0) 5 (1.2) 5 (1.4) Exposure week 13     Number examined microscopically 10 10 10 10 10 10     Cortex, renal tubule, accumulation, hyaline droplet 10 (1.0) 10 (1.5) 10 (2.2) 10 (2.8) 10 (3.0) 10 (2.7)     Cortex, renal tubule, regeneration 1 (1.0) 10 (1.0) 10 (1.6) 10 (2.0) 10 (2.0) 10 (2.0)     Medulla, granular casts 0 2 (1.0) 7 (1.0) 8 (1.5) 10 (2.0) 10 (2.2) Open in new tab TABLE 4 Kidney Lesions in Male Rats Exposed to Decalin for Up to 13 Weeks . Exposure concentration (ppm) . Kidney organ, diagnosis . 0 . 25 . 50 . 100 . 200 . 400 . aIncidence. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Exposure week 2     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5a (1.0)b 5 (1.8) 5 (1.8) 5 (1.6) 5 (1.6) 5 (2.0)     Cortex, renal tubule, regeneration 0 1 (1.0) 0 2 (1.0) 1 (1.0) 3 (1.0) Exposure week 6     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5 (1.0) 5 (2.0) 5 (2.0) 5 (2.4) 5 (3.0) 5 (3.0)     Cortex, renal tubule, regeneration 1 (1.0) 4 (1.0) 5 (1.0) 5 (1.8) 5 (1.6) 5 (2.0)     Medulla, granular casts 0 0 1 (1.0) 4 (1.0) 5 (1.2) 5 (1.4) Exposure week 13     Number examined microscopically 10 10 10 10 10 10     Cortex, renal tubule, accumulation, hyaline droplet 10 (1.0) 10 (1.5) 10 (2.2) 10 (2.8) 10 (3.0) 10 (2.7)     Cortex, renal tubule, regeneration 1 (1.0) 10 (1.0) 10 (1.6) 10 (2.0) 10 (2.0) 10 (2.0)     Medulla, granular casts 0 2 (1.0) 7 (1.0) 8 (1.5) 10 (2.0) 10 (2.2) . Exposure concentration (ppm) . Kidney organ, diagnosis . 0 . 25 . 50 . 100 . 200 . 400 . aIncidence. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. Exposure week 2     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5a (1.0)b 5 (1.8) 5 (1.8) 5 (1.6) 5 (1.6) 5 (2.0)     Cortex, renal tubule, regeneration 0 1 (1.0) 0 2 (1.0) 1 (1.0) 3 (1.0) Exposure week 6     Number examined microscopically 5 5 5 5 5 5     Cortex, renal tubule, accumulation, hyaline droplet 5 (1.0) 5 (2.0) 5 (2.0) 5 (2.4) 5 (3.0) 5 (3.0)     Cortex, renal tubule, regeneration 1 (1.0) 4 (1.0) 5 (1.0) 5 (1.8) 5 (1.6) 5 (2.0)     Medulla, granular casts 0 0 1 (1.0) 4 (1.0) 5 (1.2) 5 (1.4) Exposure week 13     Number examined microscopically 10 10 10 10 10 10     Cortex, renal tubule, accumulation, hyaline droplet 10 (1.0) 10 (1.5) 10 (2.2) 10 (2.8) 10 (3.0) 10 (2.7)     Cortex, renal tubule, regeneration 1 (1.0) 10 (1.0) 10 (1.6) 10 (2.0) 10 (2.0) 10 (2.0)     Medulla, granular casts 0 2 (1.0) 7 (1.0) 8 (1.5) 10 (2.0) 10 (2.2) Open in new tab TABLE 5 Group Mean (± SD) Concentration of (cis + trans)-Decalin Measured in Kidneys Collected from Rats Exposed to Decalin . . (cis + trans)-Decalin (μg/g kidney) . Target exposure concentration (ppm) . Exposure week . Males . Femalesb . Note. Rats were exposed for 2 (n = 5), 6 (n = 5), or 13 (n = 10) weeks. Females were only sampled following 13 weeks of exposure. At 13 weeks, kidney decalin concentrations in females were all significantly (p ≤ 0.05) different then in males in the corresponding dose groups. For males, kidney decalin concentrations at two weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same dose group. an = 4. 25 2 0.561 ± 0.25 — 6 7.33 ± 1.5 — 13 8.61 ± 2.2 0.126 ± 0.027 50 2 0.887 ± 0.49 — 6 13.3 ± 3.4 — 13 12.9 ± 2.1 0.301 ± 0.14 100 2 1.53 ± 0.87 — 6 19.4 ± 2.2 — 13 18.4 ± 3.8 0.501 ± 0.18 200 2 3.47 ± 1.3a — 6 31.0 ± 3.0 — 13 27.7 ± 3.3 0.828 ± 0.44 400 2 6.59 ± 1.5 — 6 39.0 ± 6.0 — 13 38.1 ± 6.6 1.46 ± 0.42 . . (cis + trans)-Decalin (μg/g kidney) . Target exposure concentration (ppm) . Exposure week . Males . Femalesb . Note. Rats were exposed for 2 (n = 5), 6 (n = 5), or 13 (n = 10) weeks. Females were only sampled following 13 weeks of exposure. At 13 weeks, kidney decalin concentrations in females were all significantly (p ≤ 0.05) different then in males in the corresponding dose groups. For males, kidney decalin concentrations at two weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same dose group. an = 4. 25 2 0.561 ± 0.25 — 6 7.33 ± 1.5 — 13 8.61 ± 2.2 0.126 ± 0.027 50 2 0.887 ± 0.49 — 6 13.3 ± 3.4 — 13 12.9 ± 2.1 0.301 ± 0.14 100 2 1.53 ± 0.87 — 6 19.4 ± 2.2 — 13 18.4 ± 3.8 0.501 ± 0.18 200 2 3.47 ± 1.3a — 6 31.0 ± 3.0 — 13 27.7 ± 3.3 0.828 ± 0.44 400 2 6.59 ± 1.5 — 6 39.0 ± 6.0 — 13 38.1 ± 6.6 1.46 ± 0.42 Open in new tab TABLE 5 Group Mean (± SD) Concentration of (cis + trans)-Decalin Measured in Kidneys Collected from Rats Exposed to Decalin . . (cis + trans)-Decalin (μg/g kidney) . Target exposure concentration (ppm) . Exposure week . Males . Femalesb . Note. Rats were exposed for 2 (n = 5), 6 (n = 5), or 13 (n = 10) weeks. Females were only sampled following 13 weeks of exposure. At 13 weeks, kidney decalin concentrations in females were all significantly (p ≤ 0.05) different then in males in the corresponding dose groups. For males, kidney decalin concentrations at two weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same dose group. an = 4. 25 2 0.561 ± 0.25 — 6 7.33 ± 1.5 — 13 8.61 ± 2.2 0.126 ± 0.027 50 2 0.887 ± 0.49 — 6 13.3 ± 3.4 — 13 12.9 ± 2.1 0.301 ± 0.14 100 2 1.53 ± 0.87 — 6 19.4 ± 2.2 — 13 18.4 ± 3.8 0.501 ± 0.18 200 2 3.47 ± 1.3a — 6 31.0 ± 3.0 — 13 27.7 ± 3.3 0.828 ± 0.44 400 2 6.59 ± 1.5 — 6 39.0 ± 6.0 — 13 38.1 ± 6.6 1.46 ± 0.42 . . (cis + trans)-Decalin (μg/g kidney) . Target exposure concentration (ppm) . Exposure week . Males . Femalesb . Note. Rats were exposed for 2 (n = 5), 6 (n = 5), or 13 (n = 10) weeks. Females were only sampled following 13 weeks of exposure. At 13 weeks, kidney decalin concentrations in females were all significantly (p ≤ 0.05) different then in males in the corresponding dose groups. For males, kidney decalin concentrations at two weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same dose group. an = 4. 25 2 0.561 ± 0.25 — 6 7.33 ± 1.5 — 13 8.61 ± 2.2 0.126 ± 0.027 50 2 0.887 ± 0.49 — 6 13.3 ± 3.4 — 13 12.9 ± 2.1 0.301 ± 0.14 100 2 1.53 ± 0.87 — 6 19.4 ± 2.2 — 13 18.4 ± 3.8 0.501 ± 0.18 200 2 3.47 ± 1.3a — 6 31.0 ± 3.0 — 13 27.7 ± 3.3 0.828 ± 0.44 400 2 6.59 ± 1.5 — 6 39.0 ± 6.0 — 13 38.1 ± 6.6 1.46 ± 0.42 Open in new tab TABLE 6 Mean Molar Ratio of Decalin and 2-Decalone Combined and α2u-Globulin In Kidneys from Male Rats Exposed to Decalin for Up to 13 Weeks . . Mean concentration (nmol/g kidney) . . Exposure concentration (ppm) . Exposure week . Decalin + 2-decalonea . Corrected α2u-globulinb . Mean ratioc . aTotal (cis + trans) isomers. bCorrected by subtracting the mean molar amount of α2u-globulin of the control group. cα2u-Globulin/(decalin + 2-decalone). 25 2 16.9 13.0 0.77 6 167 196 1.2 13 193 65.0 0.33 50 2 19.8 15.7 0.79 6 267 278 1.0 13 284 432 1.5 100 2 22.3 19.6 0.88 6 326 149 0.46 13 356 237 0.67 200 2 39.3 37.2 0.95 6 419 270 0.65 13 443 582 1.3 400 2 68.8 137 2.0 6 434 676 1.6 13 477 391 0.82 . . Mean concentration (nmol/g kidney) . . Exposure concentration (ppm) . Exposure week . Decalin + 2-decalonea . Corrected α2u-globulinb . Mean ratioc . aTotal (cis + trans) isomers. bCorrected by subtracting the mean molar amount of α2u-globulin of the control group. cα2u-Globulin/(decalin + 2-decalone). 25 2 16.9 13.0 0.77 6 167 196 1.2 13 193 65.0 0.33 50 2 19.8 15.7 0.79 6 267 278 1.0 13 284 432 1.5 100 2 22.3 19.6 0.88 6 326 149 0.46 13 356 237 0.67 200 2 39.3 37.2 0.95 6 419 270 0.65 13 443 582 1.3 400 2 68.8 137 2.0 6 434 676 1.6 13 477 391 0.82 Open in new tab TABLE 6 Mean Molar Ratio of Decalin and 2-Decalone Combined and α2u-Globulin In Kidneys from Male Rats Exposed to Decalin for Up to 13 Weeks . . Mean concentration (nmol/g kidney) . . Exposure concentration (ppm) . Exposure week . Decalin + 2-decalonea . Corrected α2u-globulinb . Mean ratioc . aTotal (cis + trans) isomers. bCorrected by subtracting the mean molar amount of α2u-globulin of the control group. cα2u-Globulin/(decalin + 2-decalone). 25 2 16.9 13.0 0.77 6 167 196 1.2 13 193 65.0 0.33 50 2 19.8 15.7 0.79 6 267 278 1.0 13 284 432 1.5 100 2 22.3 19.6 0.88 6 326 149 0.46 13 356 237 0.67 200 2 39.3 37.2 0.95 6 419 270 0.65 13 443 582 1.3 400 2 68.8 137 2.0 6 434 676 1.6 13 477 391 0.82 . . Mean concentration (nmol/g kidney) . . Exposure concentration (ppm) . Exposure week . Decalin + 2-decalonea . Corrected α2u-globulinb . Mean ratioc . aTotal (cis + trans) isomers. bCorrected by subtracting the mean molar amount of α2u-globulin of the control group. cα2u-Globulin/(decalin + 2-decalone). 25 2 16.9 13.0 0.77 6 167 196 1.2 13 193 65.0 0.33 50 2 19.8 15.7 0.79 6 267 278 1.0 13 284 432 1.5 100 2 22.3 19.6 0.88 6 326 149 0.46 13 356 237 0.67 200 2 39.3 37.2 0.95 6 419 270 0.65 13 443 582 1.3 400 2 68.8 137 2.0 6 434 676 1.6 13 477 391 0.82 Open in new tab TABLE 7 Creatinine and Decalol Isomers Excreted in Urine from Male and Female Rats Exposed to Decalin for Two or Six Weeks . . Males . Females . . . . Decalola . . Decalola . Exposure concentration . Week . Creatinine . ng/μg Creatinine . μg . Creatinine . ng/μg Creatinine . μg . Note. Urine collected for 16 h postexposure (mean ± SD; n = 5). Exposure concentrations are given in ppm. Creatinine is given in mg/dl. aCombined value for all eight isomers. 25 2 22.2 ± 4.8 86.0 ± 13 279 ± 34 12.8 ± 2.6 192 ± 32 462 ± 40 6 50.4 ± 37 62.7 ± 24 268 ± 99 29.6 ± 27 132 ± 24 474 ± 140 50 2 19.4 ± 10 207 ± 31 641 ± 140 17.0 ± 5.8 352 ± 92 854 ± 150 6 33.4 ± 19 125 ± 54 551 ± 190 35.4 ± 28 274 ± 38 980 ± 170 100 2 14.0 ± 2.6 527 ± 130 1470 ± 320 15.6 ± 4.2 735 ± 99 1730 ± 160 6 23.6 ± 7.7 237 ± 94 1240 ± 320 38.0 ± 31 579 ± 120 1830 ± 430 200 2 14.2 ± 3.0 893 ± 200 2550 ± 410 15.4 ± 8.6 1470 ± 220 3560 ± 440 6 22.0 ± 7.5 374 ± 200 1900 ± 640 23.6 ± 15 971 ± 210 3530 ± 670 400 2 19.4 ± 4.4 1710 ± 280 4960 ± 980 9.20 ± 1.6 2630 ± 280 5980 ± 430 6 32.2 ± 16 864 ± 280 4020 ± 1200 23.0 ± 9.3 2120 ± 140 6400 ± 770 . . Males . Females . . . . Decalola . . Decalola . Exposure concentration . Week . Creatinine . ng/μg Creatinine . μg . Creatinine . ng/μg Creatinine . μg . Note. Urine collected for 16 h postexposure (mean ± SD; n = 5). Exposure concentrations are given in ppm. Creatinine is given in mg/dl. aCombined value for all eight isomers. 25 2 22.2 ± 4.8 86.0 ± 13 279 ± 34 12.8 ± 2.6 192 ± 32 462 ± 40 6 50.4 ± 37 62.7 ± 24 268 ± 99 29.6 ± 27 132 ± 24 474 ± 140 50 2 19.4 ± 10 207 ± 31 641 ± 140 17.0 ± 5.8 352 ± 92 854 ± 150 6 33.4 ± 19 125 ± 54 551 ± 190 35.4 ± 28 274 ± 38 980 ± 170 100 2 14.0 ± 2.6 527 ± 130 1470 ± 320 15.6 ± 4.2 735 ± 99 1730 ± 160 6 23.6 ± 7.7 237 ± 94 1240 ± 320 38.0 ± 31 579 ± 120 1830 ± 430 200 2 14.2 ± 3.0 893 ± 200 2550 ± 410 15.4 ± 8.6 1470 ± 220 3560 ± 440 6 22.0 ± 7.5 374 ± 200 1900 ± 640 23.6 ± 15 971 ± 210 3530 ± 670 400 2 19.4 ± 4.4 1710 ± 280 4960 ± 980 9.20 ± 1.6 2630 ± 280 5980 ± 430 6 32.2 ± 16 864 ± 280 4020 ± 1200 23.0 ± 9.3 2120 ± 140 6400 ± 770 Open in new tab TABLE 7 Creatinine and Decalol Isomers Excreted in Urine from Male and Female Rats Exposed to Decalin for Two or Six Weeks . . Males . Females . . . . Decalola . . Decalola . Exposure concentration . Week . Creatinine . ng/μg Creatinine . μg . Creatinine . ng/μg Creatinine . μg . Note. Urine collected for 16 h postexposure (mean ± SD; n = 5). Exposure concentrations are given in ppm. Creatinine is given in mg/dl. aCombined value for all eight isomers. 25 2 22.2 ± 4.8 86.0 ± 13 279 ± 34 12.8 ± 2.6 192 ± 32 462 ± 40 6 50.4 ± 37 62.7 ± 24 268 ± 99 29.6 ± 27 132 ± 24 474 ± 140 50 2 19.4 ± 10 207 ± 31 641 ± 140 17.0 ± 5.8 352 ± 92 854 ± 150 6 33.4 ± 19 125 ± 54 551 ± 190 35.4 ± 28 274 ± 38 980 ± 170 100 2 14.0 ± 2.6 527 ± 130 1470 ± 320 15.6 ± 4.2 735 ± 99 1730 ± 160 6 23.6 ± 7.7 237 ± 94 1240 ± 320 38.0 ± 31 579 ± 120 1830 ± 430 200 2 14.2 ± 3.0 893 ± 200 2550 ± 410 15.4 ± 8.6 1470 ± 220 3560 ± 440 6 22.0 ± 7.5 374 ± 200 1900 ± 640 23.6 ± 15 971 ± 210 3530 ± 670 400 2 19.4 ± 4.4 1710 ± 280 4960 ± 980 9.20 ± 1.6 2630 ± 280 5980 ± 430 6 32.2 ± 16 864 ± 280 4020 ± 1200 23.0 ± 9.3 2120 ± 140 6400 ± 770 . . Males . Females . . . . Decalola . . Decalola . Exposure concentration . Week . Creatinine . ng/μg Creatinine . μg . Creatinine . ng/μg Creatinine . μg . Note. Urine collected for 16 h postexposure (mean ± SD; n = 5). Exposure concentrations are given in ppm. Creatinine is given in mg/dl. aCombined value for all eight isomers. 25 2 22.2 ± 4.8 86.0 ± 13 279 ± 34 12.8 ± 2.6 192 ± 32 462 ± 40 6 50.4 ± 37 62.7 ± 24 268 ± 99 29.6 ± 27 132 ± 24 474 ± 140 50 2 19.4 ± 10 207 ± 31 641 ± 140 17.0 ± 5.8 352 ± 92 854 ± 150 6 33.4 ± 19 125 ± 54 551 ± 190 35.4 ± 28 274 ± 38 980 ± 170 100 2 14.0 ± 2.6 527 ± 130 1470 ± 320 15.6 ± 4.2 735 ± 99 1730 ± 160 6 23.6 ± 7.7 237 ± 94 1240 ± 320 38.0 ± 31 579 ± 120 1830 ± 430 200 2 14.2 ± 3.0 893 ± 200 2550 ± 410 15.4 ± 8.6 1470 ± 220 3560 ± 440 6 22.0 ± 7.5 374 ± 200 1900 ± 640 23.6 ± 15 971 ± 210 3530 ± 670 400 2 19.4 ± 4.4 1710 ± 280 4960 ± 980 9.20 ± 1.6 2630 ± 280 5980 ± 430 6 32.2 ± 16 864 ± 280 4020 ± 1200 23.0 ± 9.3 2120 ± 140 6400 ± 770 Open in new tab TABLE 8 Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Rats in the Two-Year Inhalation Study of Decalin . Exposure concentration (ppm) . Parameter . 0 . 25 . 50 . 100 . 400 . Note. (T), terminal sacrifice. aNumber of animals with lesion. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. cHistorical incidence for two-year studies with controls given NTP-2000 diet: 3/906 (NIEHS, 2002). dNumber of animals with neoplasm per number of animals with kidney examined microscopically. ePoly-3 estimated neoplasm incidence after adjustment for intercurrent mortality. fObserved incidence at terminal kill. gBeneath the chamber control incidence is the p value associated with the trend test. Beneath the exposed group incidence are the p values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. hHistorical incidence: 0/906 (NIEHS, 2002). iHistorical incidence: 3/906 (NIEHS, 2002). *Significantly different (p ≤ 0.05) from the chamber control group by the Poly-3 test. **p ≤ 0.01. Males     Number examined microscopically 50 50 49 50 20     Cortex, renal tubule, hyperplasiaa 0 11** (2.3)b 11** (2.1) 15** (3.1) 5** (1.8)     Nephropathy, chronic 48 (1.4) 48 (2.3) 49 (2.6) 50 (2.3) 20 (3.0)     Cortex, renal tubule, accumulation, hyaline droplet 2 (1.5) 9* (2.9) 7 (2.0) 11** (2.6) 2 (2.5)     Papilla, mineralization 1 (1.0) 34** (2.4) 41** (2.9) 43** (3.1) 17** (3.3)     Pelvis, transitional epithelium, hyperplasia 1 (1.0) 8* (1.5) 8* (2.1) 10** (2.4) 5** (1.6)     Renal tubule, adenoma, multiple 0 0 0 0 1     Renal tubule, adenoma (includes multiple),c overall rated 1/50 (2%) 2/50 (4%) 6/49 (12%) 9/50 (18%) 5/20 (25%)         Adjusted rate (%)e 2.4 4.9 14.0 21.8 26.7         Terminal ratef 1/28 (4%) 0/23 (0%) 4/23 (17%) 5/20 (25%) 4/14 (29%)         First incidence (days) 733 (%) 644 680 617 716         Poly-3 testg p = 0.005 p = 0.492 p = 0.058 p = 0.007 p = 0.004     Renal tubule, carcinoma (includes bilateral)h 0 1 1 4 1     Renal tubule, adenoma or carcinoma,i overall rate 1/50 (2%) 3/50 (6%) 7/49 (14%) 12/50 (24%) 6/20 (30%)         Adjusted rate (%) 2.4 7.3 16.3 28.9 31.8         Terminal rate 1/28 (4%) 1/23 (4%) 4/23 (17%) 7/20 (35%) 4/14 (29%)         First incidence (days) 733 (T) 644 680 617 708         Poly-3 test p = 0.002 p = 0.296 p = 0.031 p < 0.001 p < 0.001 Females     Number examined microscopically 50 46 49 50     Nephropathy, chronic 44 (1.6) 45 (1.6) 48* (1.9) 48 (1.6) . Exposure concentration (ppm) . Parameter . 0 . 25 . 50 . 100 . 400 . Note. (T), terminal sacrifice. aNumber of animals with lesion. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. cHistorical incidence for two-year studies with controls given NTP-2000 diet: 3/906 (NIEHS, 2002). dNumber of animals with neoplasm per number of animals with kidney examined microscopically. ePoly-3 estimated neoplasm incidence after adjustment for intercurrent mortality. fObserved incidence at terminal kill. gBeneath the chamber control incidence is the p value associated with the trend test. Beneath the exposed group incidence are the p values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. hHistorical incidence: 0/906 (NIEHS, 2002). iHistorical incidence: 3/906 (NIEHS, 2002). *Significantly different (p ≤ 0.05) from the chamber control group by the Poly-3 test. **p ≤ 0.01. Males     Number examined microscopically 50 50 49 50 20     Cortex, renal tubule, hyperplasiaa 0 11** (2.3)b 11** (2.1) 15** (3.1) 5** (1.8)     Nephropathy, chronic 48 (1.4) 48 (2.3) 49 (2.6) 50 (2.3) 20 (3.0)     Cortex, renal tubule, accumulation, hyaline droplet 2 (1.5) 9* (2.9) 7 (2.0) 11** (2.6) 2 (2.5)     Papilla, mineralization 1 (1.0) 34** (2.4) 41** (2.9) 43** (3.1) 17** (3.3)     Pelvis, transitional epithelium, hyperplasia 1 (1.0) 8* (1.5) 8* (2.1) 10** (2.4) 5** (1.6)     Renal tubule, adenoma, multiple 0 0 0 0 1     Renal tubule, adenoma (includes multiple),c overall rated 1/50 (2%) 2/50 (4%) 6/49 (12%) 9/50 (18%) 5/20 (25%)         Adjusted rate (%)e 2.4 4.9 14.0 21.8 26.7         Terminal ratef 1/28 (4%) 0/23 (0%) 4/23 (17%) 5/20 (25%) 4/14 (29%)         First incidence (days) 733 (%) 644 680 617 716         Poly-3 testg p = 0.005 p = 0.492 p = 0.058 p = 0.007 p = 0.004     Renal tubule, carcinoma (includes bilateral)h 0 1 1 4 1     Renal tubule, adenoma or carcinoma,i overall rate 1/50 (2%) 3/50 (6%) 7/49 (14%) 12/50 (24%) 6/20 (30%)         Adjusted rate (%) 2.4 7.3 16.3 28.9 31.8         Terminal rate 1/28 (4%) 1/23 (4%) 4/23 (17%) 7/20 (35%) 4/14 (29%)         First incidence (days) 733 (T) 644 680 617 708         Poly-3 test p = 0.002 p = 0.296 p = 0.031 p < 0.001 p < 0.001 Females     Number examined microscopically 50 46 49 50     Nephropathy, chronic 44 (1.6) 45 (1.6) 48* (1.9) 48 (1.6) Open in new tab TABLE 8 Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Rats in the Two-Year Inhalation Study of Decalin . Exposure concentration (ppm) . Parameter . 0 . 25 . 50 . 100 . 400 . Note. (T), terminal sacrifice. aNumber of animals with lesion. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. cHistorical incidence for two-year studies with controls given NTP-2000 diet: 3/906 (NIEHS, 2002). dNumber of animals with neoplasm per number of animals with kidney examined microscopically. ePoly-3 estimated neoplasm incidence after adjustment for intercurrent mortality. fObserved incidence at terminal kill. gBeneath the chamber control incidence is the p value associated with the trend test. Beneath the exposed group incidence are the p values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. hHistorical incidence: 0/906 (NIEHS, 2002). iHistorical incidence: 3/906 (NIEHS, 2002). *Significantly different (p ≤ 0.05) from the chamber control group by the Poly-3 test. **p ≤ 0.01. Males     Number examined microscopically 50 50 49 50 20     Cortex, renal tubule, hyperplasiaa 0 11** (2.3)b 11** (2.1) 15** (3.1) 5** (1.8)     Nephropathy, chronic 48 (1.4) 48 (2.3) 49 (2.6) 50 (2.3) 20 (3.0)     Cortex, renal tubule, accumulation, hyaline droplet 2 (1.5) 9* (2.9) 7 (2.0) 11** (2.6) 2 (2.5)     Papilla, mineralization 1 (1.0) 34** (2.4) 41** (2.9) 43** (3.1) 17** (3.3)     Pelvis, transitional epithelium, hyperplasia 1 (1.0) 8* (1.5) 8* (2.1) 10** (2.4) 5** (1.6)     Renal tubule, adenoma, multiple 0 0 0 0 1     Renal tubule, adenoma (includes multiple),c overall rated 1/50 (2%) 2/50 (4%) 6/49 (12%) 9/50 (18%) 5/20 (25%)         Adjusted rate (%)e 2.4 4.9 14.0 21.8 26.7         Terminal ratef 1/28 (4%) 0/23 (0%) 4/23 (17%) 5/20 (25%) 4/14 (29%)         First incidence (days) 733 (%) 644 680 617 716         Poly-3 testg p = 0.005 p = 0.492 p = 0.058 p = 0.007 p = 0.004     Renal tubule, carcinoma (includes bilateral)h 0 1 1 4 1     Renal tubule, adenoma or carcinoma,i overall rate 1/50 (2%) 3/50 (6%) 7/49 (14%) 12/50 (24%) 6/20 (30%)         Adjusted rate (%) 2.4 7.3 16.3 28.9 31.8         Terminal rate 1/28 (4%) 1/23 (4%) 4/23 (17%) 7/20 (35%) 4/14 (29%)         First incidence (days) 733 (T) 644 680 617 708         Poly-3 test p = 0.002 p = 0.296 p = 0.031 p < 0.001 p < 0.001 Females     Number examined microscopically 50 46 49 50     Nephropathy, chronic 44 (1.6) 45 (1.6) 48* (1.9) 48 (1.6) . Exposure concentration (ppm) . Parameter . 0 . 25 . 50 . 100 . 400 . Note. (T), terminal sacrifice. aNumber of animals with lesion. bAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. cHistorical incidence for two-year studies with controls given NTP-2000 diet: 3/906 (NIEHS, 2002). dNumber of animals with neoplasm per number of animals with kidney examined microscopically. ePoly-3 estimated neoplasm incidence after adjustment for intercurrent mortality. fObserved incidence at terminal kill. gBeneath the chamber control incidence is the p value associated with the trend test. Beneath the exposed group incidence are the p values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. hHistorical incidence: 0/906 (NIEHS, 2002). iHistorical incidence: 3/906 (NIEHS, 2002). *Significantly different (p ≤ 0.05) from the chamber control group by the Poly-3 test. **p ≤ 0.01. Males     Number examined microscopically 50 50 49 50 20     Cortex, renal tubule, hyperplasiaa 0 11** (2.3)b 11** (2.1) 15** (3.1) 5** (1.8)     Nephropathy, chronic 48 (1.4) 48 (2.3) 49 (2.6) 50 (2.3) 20 (3.0)     Cortex, renal tubule, accumulation, hyaline droplet 2 (1.5) 9* (2.9) 7 (2.0) 11** (2.6) 2 (2.5)     Papilla, mineralization 1 (1.0) 34** (2.4) 41** (2.9) 43** (3.1) 17** (3.3)     Pelvis, transitional epithelium, hyperplasia 1 (1.0) 8* (1.5) 8* (2.1) 10** (2.4) 5** (1.6)     Renal tubule, adenoma, multiple 0 0 0 0 1     Renal tubule, adenoma (includes multiple),c overall rated 1/50 (2%) 2/50 (4%) 6/49 (12%) 9/50 (18%) 5/20 (25%)         Adjusted rate (%)e 2.4 4.9 14.0 21.8 26.7         Terminal ratef 1/28 (4%) 0/23 (0%) 4/23 (17%) 5/20 (25%) 4/14 (29%)         First incidence (days) 733 (%) 644 680 617 716         Poly-3 testg p = 0.005 p = 0.492 p = 0.058 p = 0.007 p = 0.004     Renal tubule, carcinoma (includes bilateral)h 0 1 1 4 1     Renal tubule, adenoma or carcinoma,i overall rate 1/50 (2%) 3/50 (6%) 7/49 (14%) 12/50 (24%) 6/20 (30%)         Adjusted rate (%) 2.4 7.3 16.3 28.9 31.8         Terminal rate 1/28 (4%) 1/23 (4%) 4/23 (17%) 7/20 (35%) 4/14 (29%)         First incidence (days) 733 (T) 644 680 617 708         Poly-3 test p = 0.002 p = 0.296 p = 0.031 p < 0.001 p < 0.001 Females     Number examined microscopically 50 46 49 50     Nephropathy, chronic 44 (1.6) 45 (1.6) 48* (1.9) 48 (1.6) Open in new tab FIG. 1. Open in new tabDownload slide Kidney cell proliferation index in male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5). FIG. 1. Open in new tabDownload slide Kidney cell proliferation index in male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5). FIG. 2. Open in new tabDownload slide Concentration of (cis + trans)-decalin in kidneys from male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5 [weeks 2 and 6], n = 10 [week 13]). Kidney decalin concentrations at 2 weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same exposure group. FIG. 2. Open in new tabDownload slide Concentration of (cis + trans)-decalin in kidneys from male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5 [weeks 2 and 6], n = 10 [week 13]). Kidney decalin concentrations at 2 weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same exposure group. FIG. 3. Open in new tabDownload slide Concentrations of (cis + trans)-2-decalone in kidneys from male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5 [weeks 2 and 6], n = 10 [week 13]). Kidney decalone concentrations at two weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same exposure group. FIG. 3. Open in new tabDownload slide Concentrations of (cis + trans)-2-decalone in kidneys from male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5 [weeks 2 and 6], n = 10 [week 13]). Kidney decalone concentrations at two weeks were all significantly (p ≤ 0.05) lower than at later sampling times within the same exposure group. FIG. 4. Open in new tabDownload slide Concentrations of α2u-globulin in kidneys from male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5). FIG. 4. Open in new tabDownload slide Concentrations of α2u-globulin in kidneys from male rats exposed to decalin for up to 13 weeks (mean ± SD; n = 5). This research has been funded by the National Institute of Environmental Health Sciences (NIEHS, Research Triangle Park, NC). It does not necessarily reflect the views of NIEHS, and no official endorsement should be inferred. 1 To whom correspondence should be addressed at Battelle, Toxicology Northwest, MS K4-16, P.O. Box 999, Richland, WA 99352. Fax: (509) 375-3649. E-mail: dillj@battelle.org. We thank Harvey Ragan for his input for urinalysis data and technical review of the manuscript and Kathy Debban for her technical assistance on α2u-globulin analysis. This work was supported by a contract from the National Institute of Environmental Health Sciences (N01-ES-55392), Research Triangle Park, NC. REFFERENCES Alison, R. H., Capen, C. C., and Prentice, D. E. ( 1994 ). Neoplastic lesions of questionable significance to humans. Toxicol. Pathol. 22, 179 –186. Benz, R. D., and Beltz, P. A. (1980). Cytogenetic toxicologic testing with dogs. 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TI - α2u-Globulin Nephropathy and Carcinogenicity following Exposure to Decalin (Decahydronaphthalene) in F344/N Rats
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfg028
DA - 2003-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/2u-globulin-nephropathy-and-carcinogenicity-following-exposure-to-03xljCDXDj
SP - 223
EP - 234
VL - 72
IS - 2
DP - DeepDyve
ER -