A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxicity and Relevance to Cancer Studies

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1 FUNDAMENTAL AND APPLIED TOXICOLOGY 32, (1996) ARTICLE NO A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxiity and Relevane to Caner Studies MICHAEL V. TEMPLIN, JEFFREY L. LARSON, 1 BYRON E. BUTTERWORTH, 2 KEITH C. JAMISON, JOEL R. LEININGER, 3 STEPHANE MERY, 4 KEVIN T. MORGAN, BRIAN A. WONG, AND DOUGLAS C. WOLF Chemial Industry Institute of Toxiology, P.O. Box , 6 Davis Drive, Researh Triangle Park, North Carolina Reeived Deember 15, 1995; aepted Marh 20, 1996 A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxiity and Relevane to Caner Studies. TEMPLIN, M. V., LARSON, J. L., BUTTERWORTH, B. E., JAMISON, K. C, LEININGER, J. R., MERY, S., MORGAN, K. T., WONG, B. A., CONOLLY, R. B., AND WOLF, D. C. (1996). Fundam. Appl. Toxiol. 32, Chloroform ats via a nongenotoxi-ytotoxi mode of ation to produe aner if given in doses and at dose rates suffiiently high to produe organ-speifi toxiity. In a reent study, hloroform failed to indue aner in male or female F-344 rats when administered by inhalation for 2 years at 90 ppm, 5. The present study was undertaken to define the onentration - response urves for hloroform-indued lesions and regenerative ell proliferation in the F-344 rat when exposed by inhalation and to orrelate those patterns of toxiity with the results from the inhalation aner bioassay. Male and female F-344 rats were exposed to airborne onentrations of 0, 2, 10, 30, 90, or ppm hloroform 6 hr/day, 7 for 4 days or 3, 6, or 13 weeks. Additional treatment groups were exposed 5 for 13 weeks or were exposed for 6 weeks and held until Week 13. Bromodeoxyuridine was administered via osmoti pumps implanted 3.5 days prior to neropsy and the labeling index (LI, perentage of nulei in S-phase) was evaluated immunohistohemially. A fullsreen neropsy identified the kidney, liver, and nasal passages as the only target organs. This study onfirmed that ppm is extremely toxi and would be inappropriate for longer-term aner studies. The primary target in the kidney was the epithelial ells of the proximal tubules of the ortex, with signifiantly elevated inreases in the LI at onentrations of 30 ppm and above. However, only a marginal inrease in the renal LI in the males was seen after exposures of 90 ppm, 5. Chloroform indued hepati lesions in the midzonal and entrilobular regions with inreases in the LI throughout the liver, but only at ppm exposures. An additional liver lesion seen only at the highly hepatotoxi onentration of ppm was numerous intestinal ryptlike duts surrounded by dense onnetive tissue. Enhaned bone growth and hyperellularity in the lamina propria of the ethmoid 1 Current address: Rhone-Poulen Rorer, Collegeville, PA To whom orrespondene should be addressed at CUT, P.O. Box 12137, 6 Davis Drive, Researh Triangle Park, NC Current address: Integrated Laboratory Systems. Researh Triangle Park, NC Current address: ZS Assoiates, Reading, England RG1 1Z5. turbinates of the nose ourred at the early time points at onentrations of 10 ppm and above. At 90 days there was a generalized atrophy of the ethmoid turbinates at onentrations of 2 ppm and above. Cytolethality and regenerative ell proliferation are neessary but not always suffiient to indue aner beause of tissue, sex, and speies differenes in suseptibility. A ombination of a lak of diret genotoxi ativity by hloroform, only a marginal indution of ell proliferation in the male rat kidney, and lower tissue-speifi suseptibility in the female rat is apparently responsible for the reported lak of hloroform-indued aner in a longterm inhalation bioassay with F-344 rats. 19% soiety of Toxiology Water intended for human onsumption is often disinfeted with sodium hypohlorite or hlorine dioxide. Chlorine and hlorine dioxide are also used as bleahing agents in paper prodution (Butler and Dal Pont, 1992). Chloroform (CHC1 3 ) is produed as a result of reations between hlorine and organi material present in water (Craun, 1993). Although formed in trae amounts, detetable onentrations of hloroform have been onsistently found in muniipal water supplies (Bunn et al, 1975; Symons et al., 1975). Chloroform has a low water solubility and relatively high volatility. Thus, hloroform initially present in waters an signifiantly ontribute to airborne onentrations (Andelman, 1985). Conentrations as high as ppm have been measured in ambient air, while onentrations in air in shower stalls and above swimming pools have been measured as high as 0.06 and 0.1 ppm, respetively (Singh et al, 1982; Aggazzotti et al, 1990; Jo et al, 1990). Chloroform has been subjeted to a substantial number of genotoxiity assays. In the most omplete and reent review of the literature it was onluded that neither hloroform nor its metabolites appear to diretly interat with DNA or possess genotoxi ativity (International Programme on Chemial Safety, 1994). Critial examination of both the genotoxiity data and patterns of toxiity leads to the onlusion that hloroform produes aner through a nongenotoxi-ytotoxi mode of ation (Eshenbrenner and Miller, 1945; Reitz etal, 1982, 1990; Rosenthal, 1987; Butterworth et al, 1992, 1995; International Programme on Chemial /96 $18 00 Copyright 1996 by the Soiety of Toxiology. All rights of reprodution in any form reserved. Downloaded from

2 110 TEMPLIN ET AL. Safety, 1994; Larson et al, 1993, 1994a,b,,d, 1995a,b, 1996; Templin et al, 1996a,b). The strength of the evidene that hloroform ats through a nongenotoxi-ytotoxi mode of ation (Butterworth et al, 1995) rests on the lak of hloroform DNA reativity and the observation that ytotoxiity and regenerative ell proliferation preede aner for every hloroform target site. As early as 1945, researhers noted the lose assoiation between the ytotoxiity and arinogeni ativity of hloroform (Eshenbrenner and Miller, 1945; Reitz et al, 1990). Chloroform aused a sustained ytolethal and regenerative ell proliferative response in the livers of female and male B6C3F, mie under gavage dosing onditions similar to those that lead to aner (National Caner Institute, 1976; Larson et al., 1994b,). Similar daily doses of hloroform administered in drinking water did not indue hepatoellular damage, proliferation, or liver tumors (Larson et al., 1994b; Jorgenson et al., 1985). This lak of arinogeni ativity was probably due to a slower dosing rate (the dose was frationated beause the animals drank several times during the day), resulting in a slower rate of toxi metabolite formation insuffiient to kill ells (Larson et al, 1994b). Complete loss of arinogeni ativity in a given target organ simply by hanging the vehile and dose rate would not be expeted of a mutageni arinogen. Mie in the drinking water study were hronially exposed to massive amounts of hloroform. If hloroform had been diretly mutageni, irreversible mutations would have aumulated even if they were formed at a slower rate. In a long-term study in whih male and female BDF, mie were exposed to hloroform atmospheres of 5, 30, or 90 ppm for 5, the only signifiant indution of tumors was observed at the highest onentration in the male mouse kidney (Yamamoto et al., 1994), whih is partiularly sensitive to the toxi effets of hloroform. In fat, the male BDF, mie in the aner study had to have exposure onentrations inreased stepwise starting at 5 ppm over a period of weeks to adapt to the 30 and 90 ppm onentrations. Exposure of male BDF, mie diretly to atmospheres of 30 or 90 ppm hloroform resulted in signifiant deaths from aute kidney failure (Templin et al., 1996a). Exposure under bioassay onditions produes nephrotoxiity and regenerative ell proliferation at this target site in male but not female BDF, mie, onsistent with the pattern of tumor formation (Templin et al., 1996a). Chloroform indues both liver and kidney toxiity in BDF, and B6C3F, mie. In ontrast to BDF, mie, B6C3F, mie arry the hepatoarinogen sensitivity gene (Hs) (Drinkwater, 1994), and hepatotoxi doses and dose rates of hloroform produe liver aner rather than kidney aner in this strain. This pattern of tumor formation illustrates the important priniple that the geneti bakground and target organ speifiity greatly influene tumor outome. Thus, indued toxiity and regenerative ell proliferation are neessary but not always suffiient onditions to indue aner in a given target organ. Chloroform given by gavage at the doses that produed kidney aner in male Osborne- Mendel rats also indues renal toxiity and regenerative ell proliferation in the kidney in this strain of rat (National Caner Institute, 1976; Templin et al., 1996b). A weak tumor response in the Osborne- Mendel male rat kidney was also seen when hloroform was given in the drinking water, but only at the highest onentration of 1800 ppm, whih also resulted in a 60% derease in water onsumption and a 25% loss in body weight (Jorgenson et al, 1985). Sine the highest onentration exeeded the maximum tolerated dose (MTD) and sine the nutritional status of this high-dose group was unertain, extrapolating the tumor response to lower doses is questionable. Effets seondary to water deprivation, altered nutrition, hloroform nephrotoxiity, or a ombination of these fators may also have been involved in tumor formation, whih ompliates interpretation of these results and the ability to ondut meaningful mehanisti studies. The kidney aner seen in the male Osborne-Mendel rats given hloroform by gavage suggested that the kidney might also be a target site in the F-344 rat. Cytotoxiity and regenerative ell proliferation our in the kidney of F-344 rats administered high doses of hloroform either by gavage or exposed via inhalation (Larson et al, 1994d, 1995a,b). A reently ompleted aner study reported no tumor indution in male or female F-344 rats exposed to hloroform atmospheres of 10, 30, or 90 ppm for 5 days/ week for 2 years (Yamamoto et al, 1994). The present study was undertaken to investigate the progression and onentration-response relationships for hloroform-indued lesions and regenerative ell proliferation in male and female F-344 rats during a 90-day inhalation exposure and to predit the profile of tumor indution that might be expeted with longer-term studies. These data define the toxiity profile for inhaled hloroform in F-344 rats and provide mehanisti insights that explain why no tumors were seen in the long-term inhalation aner study with this strain of rat (Yamamoto et al, 1994). MATERIALS AND METHODS Animals. This study was onduted under federal guidelines for the use and are of laboratory animals (National Institutes of Health, 1985) and was approved by the CUT Institutional Animal Care and Use Committee. Rats were housed in humidity- and temperature-ontrolled failities aredited by the Amerian Assoiation for Areditation of Laboratory Animal Care. Seven-week-old male or female F-344 rats [CDF(F-344)/CrBR] were obtained from the Charles River Breeding Laboratories, In. (Raleigh, NC). Rats were housed one per age in 8-m 3 stainless steel and glass inhalation hambers. Animals were randomized by weight and assigned to ontrol or treatment groups with separate hambers used for eah exposure onentration. Chambers were maintained on a 12-hr light-dark yle, with the light yle beginning at 3:30 AM and the dark yle at 3:30 PM Rats were alimated in the exposure hambers for 2 weeks and were 9 weeks of age Downloaded from

3 INHALED CHLOROFORM IN F-344 RATS 111 TABLE 1 Experimental Design for Male and Female F-344 Rats Exposed to Chloroform Vapors Number of rats at eah exposure duration Groups 4 days 3 weeks 7 6 weeks 7 13 weeks 7 13 weeks 5 13 weeks 6- week stop Male rats" Unlabeled BrdU-labeled Female rats Unlabeled BrdU-labeled 30* 30* 42" 42" 30* 42-30" 60' 30" 60' 15' 24' 15' 24' 16' W " Unlabeled rats were used for histopathologial evaluation. BrdU-labeled rats were used to evaluate LI and histologi hanges. Metabolism rats were used to evaluate hanges in the hloroform metaboli rate. * n = 5 rats per exposure level of 0, 2, 10, 30, 90, or ppm. ' n = 8 rats per exposure level of 30, 90, or ppm. " n = 5 rats per exposure level of 30, 90, or ppm. ' n = 8 rats per exposure level of 90 or ppm. 1 n = 9 rats per exposure level of 0, 10, or 90 ppm. * n 8 rats per exposure level of 0, 2, 10, or ppm; n = 5 for exposure levels of 90 or ppm. at the beginning of the exposures. NIH-07 rodent how (Ziegler Bros., Gardener, PA) and deionized filtered tap water were available ad libitum throughout the alimation and exposure periods. Food was hanged daily, immediately following the end of eah exposure period. Animals were weighed prior to the initiation of exposures and then biweekly for the duration of the study. Sentinel animals were housed in the animal faility as part of an ongoing surveillane program for parasiti, baterial, and viral infetions and were pathogen-free throughout the study. Generation and haraterization of atmospheres. Exposures were onduted in 8-m 3 hambers operated with a ontinuous flow of HEPA- and haroal-filtered air at 2000 liters/min and a stati pressure of 0.2 in. of water relative to the anteroom. The hamber environment was maintained at 22.2 ± 2 C and 50 ± 10% relative humidity. These parameters were monitored and ontrolled by a building management system (Andover Controls Corp., Andover, MA) (Wong and Moss, 1994). Temperature, relative humidity, airflow, and stati pressure were monitored ontinuously throughout the study and reorded every 15 min. Target exposure onentrations of hloroform were 0, 2, 10, 30, 90, and ppm. The exposure atmospheres were generated by a vaporization tehnique. Chloroform, >99.5% purity and stabilized with 0.006% amylenes (Aldrih Chemial Co., In., Milwaukee, WI), was stored in 10-gallon stainless steel pressure vessels. The 30, 90, and ppm atmospheres were generated using a nonpressurized system. Nitrogen, metered by a mass flow ontroller, was admitted into the vessel through a dip tube in whih the opening was loated below the surfae of the hloroform. The hloroformontaining nitrogen gas flowed through the vessel and into the supply air dut for the exposure hamber. In the 2 and 10 ppm hambers, nitrogen was used to pressurize the vessel to 5 psi and arry the hloroform into the hamber through a mass flow ontroller. Chamber onentrations of hloroform were monitored using a Miran 1A infrared gas analyzer. Certified gas standards of 2, 10, 30, 90, and ppm (Matheson Gas Co., Morrow, GA) were used to hek the alibration on a monthly basis throughout the exposures. Average analytial exposure onentrations were always within 4.5% of the target with standard deviations of no more than 2.7% from the mean (Larson et al., 1996). Experimental design. The number of animals and group designations within the experiment are outlined in Table 1. Rats were divided into groups exposed for periods of 4 days or 3, 6, or 13 weeks for male rats and 3 or 13 weeks for female rats. Daily exposures were onduted for 6 hr, from 4:00 to 10:00 AM, 7. To ompare the effets of a 7 exposure to the onventional 5 shedule, groups of rats were exposed to 30, 90, or ppm hloroform for 6 hr/day, 5 for 13 weeks. To investigate the reversibility of hloroform-indued alterations, additional groups of rats were exposed to 90 or ppm hloroform for 6 hr/day, 7 for the first 6 weeks, after whih rats were housed in the ontrol hamber for the remaining 7 weeks (6 weeks exposure, stop, 7 weeks holding). Designated subsets of rats were administered BrdU to label ells in S-phase (labeled groups) while others did not reeive BrdU (unlabeled groups). Tissues from rats that did not reeive BrdU were stained with hematoxylin and eosin (H & E). Both H & E- and BrdU-stained tissues were evaluated for histopathologial hanges. Rats were administered BrdU via osmoti pumps implanted approximately 3.5 days prior to neropsy (Alzet Model 2ML1, Alzet Corp., Palo Alto, CA). Osmoti pumps ontained a solution of 20 mg/ml filter-sterilized BrdU (Sigma Chemial Co., St. Louis, MO) in phosphate-buffered saline prepared on the day it was to be used. Osmoti pumps were implanted under asepti onditions as desribed in Eldridge et al. (1990). Tissue olletion and preparation. At neropsy, rats in the unlabeled groups were weighed and anesthetized with isofluorane administered by inhalation and then euthanized by exsanguination. Whole livers and both kidneys were immediately removed, weighed, and examined marosopially. Longitudinal setions of the left and median lobes of the liver and transetions of the left and right kidney were fixed in 10% neutral buffered formalin (NBF). Immediately following removal of the liver and kidneys, the heads of the rats were prepared as desribed previously (Morgan et al., 1991; Larson et al, 1994d) Nonnasal bones, sternum with rib, vertebrae, tibia, and femur were fixed by immersion in 10% NBF and dealified as desribed for the nose. In rats exposed for 3 or 13 weeks, a omplete tissue sreen was olleted and immersion-fixed in 10% NBF. Tissues inluded adrenals, brain, eum, ervix, olon, duodenum, ear anal, esophagus, eye with harderian gland, femoral-tibial joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mesenteri lymph nodes, ovaries, panreas, parathyroid gland, ribs, prostate, salivary gland, skin with mammary gland, siati nerve, seminal vesiles, spinal ord, sternum, stomah, spleen, testes, thigh musle, thymus, thyroid, trahea, urinary bladder, uterus, vagina, and vertebrae. Tissues were trans- Downloaded from

4 112 TEMPLIN ET AL. ferred to 70% ethanol after 48 hr, embedded in paraffin, setioned at 4-5 lim, and stained with H & E for mirosopi examination. Rats that reeived BrdU were anesthetized with 100 mg/kg sodium pentobarbital and the kidneys perfused in situ as desribed in Larson et al. (1993) using PBS as the buffer solution and 10% NBF as the fixative. The kidneys and liver were then removed, setioned, and fixed in 10% NBF. Nasal avities and bones were proessed as desribed by Larson et al. (1994). A omplete tissue sreen was olleted in rats exposed for 3 or 13 weeks. Histopathology. Chloroform-indued kidney histologi hanges were sored qualitatively for severity as follows: 0 = within normal limits, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe, where 1 through 4 indiate inreasing severity of the lesions ranging from enlarged proximal tubule ell nulei to vauolation in proximal onvoluted tubules and nerosis of individual proximal tubule epithelial ells. Chloroform-indued liver hepatoyte histologi hanges were sored qualitatively for severity as follows: 0 = within normal limits, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe, where 1 through 4 indiate inreasing severity of the lesions ranging from diffuse vauolation of hepatoyte ytoplasm to mitoti figures and individual and 2-3 adjaent hepatoyte nerosis. Chloroform-indued histologi hanges within the nasal passages were sored qualitatively for severity as follows: 0 = within normal limits, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe, where 1 through 4 indiate inreasing severity of the lesions ranging from edema in the lamina propria to loss of Bowman's glands, onnetive tissue proliferation in the lamina propria, mineralization of the basement membrane, and atrophy of the ethmoid turbinates and overlying olfatory epithelium. BrdU immunohistohemistry. BrdU-labeled tissues were mounted on ProbeOn Plus Slides (Fisher Sientifi, Pittsburgh, PA) to ensure adhesion during proessing. The immunohistohemial detetion of BrdU-labeled ells has been previously desribed (Eldridge et al, 1990). Briefly, setions were inubated for 1 hr at room temperature with an anti-brdu antibody (Beton-Dikinson, Mountain View, CA). After inubation with primary antibody, the slides were inubated for 30 min at room temperature with biotinylated horse anti-mouse IgG. Slides were then inubated with an avidin-biotin peroxidase omplex (Vetastain ABC peroxidase kit, Burlingame, CA) for 30 min at room temperature. The BrdU inorporation was visualized by a final inubation with the hromagen 3-amino-9-ethylarbazole (Zymed, San Franiso, CA) and ounterstained with hematoxylin. Soring of labeled nulei. A setion of duodenum, representing a tissue with a relatively high ell turnover rate, was inluded on eah slide to onfirm systemi delivery of BrdU to the tissues. Four regions of the kidney, ortex, outer stripe of the outer medulla, inner stripe of the outer medulla, and inner medulla were ounted as desribed in Larson et al. (1994d). In liver tissue setions, a minimum of 1000 hepatoellular nulei in the left lobe were ounted as desribed previously (Larson et al., 1994b). The labeling index (LI, perentage of ells in S-phase) was alulated by dividing the number of labeled nulei by the total number of nulei ounted with the result expressed as a perentage. The proximal portion of the dorsal sroll of the first endoturbinate was seleted as the nasal region to be quantified based on histologial hanges and qualitative assessment of sites of indued ell repliation as outlined in Mery et al. (1994). Labeled ells were ounted only in the lamina propria and adjaent periosteum. To assess possible alterations in bone growth at other osseous sites, the growth plate and periosteum were ounted in the tibia or femur, using whihever loation provided the most omplete setion on the slide, and the periosteum of one sternum was also ounted. The total number of labeled ells in the growth plate was determined as well as the growth plate width in millimeters aross the diaphyseal diameter. A 2-mm setion of periosteum was assessed for labeled ells, starting at the margin of the growth plate and progressing toward the diaphysis. The periosteum was evaluated in the region of the ompat bone of the sternum. Labeled ells, from growth plate to growth plate, were reorded and the distane in millimeters was determined. S-phase ounts were expressed as the number of BrdU-labeled ells/mm of bone following a modifiation of the unit length labeling index (ULLI) of Montiello et al. (1990). Statistis. The Williams test was used to determine signifiant differenes in organ weights, body weights, and the LI between treatment and ontrol groups (Williams, 1971, 1972). The Williams test is designed to establish the lowest dose that is signifiantly elevated over the ontrol in a dose-response study. Student's t test was used to determined statistial differenes in body and organ weights and LI between the exposure groups of 7 and 5 and between the exposure groups of 13-week ontinuous hloroform exposure and 6-week stop. Animal Health RESULTS Signs of mild dehydration were noted in some rats at the seond week of the study, primarily in the rats reeiving higher exposure onentrations. Slight hair loss, disharge from the eyes, and anogenital staining were noted at the later time points. These observations were primarily onfined to the higher onentration groups, and the observations were not always found in the same animals on onseutive weighing periods. Dose-dependent dereases in body weight gain were observed in hloroform-exposed rats at all time points (Fig. 1). The differene in body weight gain between ontrol and hloroform-treated rats appears to be attributable primarily to a transient weight loss in the hloroform-exposed groups. The initial weight loss was evident at all exposure onentrations at the Day 4 time point. By the third week, male rats exposed to 90 ppm and below had reovered from the initial weight loss and had gained somewhat relative to their starting weights. Male rats exposed to 90 and ppm exhibited a signifiant derement in body weight gain at all time points. The toxiity in the ppm group was partiularly severe and represented a 30% derement in body weight gain at the 13-week time point for exposures of both 7 and 5 days/ week (Fig. 1A). At 3 weeks, female rats exposed to ppm had a loss of approximately 10% of their initial body weight, and rats exposed to 90 ppm had an average weight only equal to that of the initial average weight at the start of the experiment (Fig. IB). By 13 weeks, all groups had inreased their body weight somewhat. However, female rats exposed to ppm for 7 or 5 exhibited a 23 or 16% derement in body weight gain, respetively. Rats exposed for the first 6 weeks and then held in the ontrol hambers until Week 13 were not different from ontrols. Organ Weights For the male rats the 3-, 6-, and 13-week average liver weights for the ontrols were 8.70 ± 0.20, 8.84 ± 0.58, and 9.88 ± 0.44 g, respetively. For the male rats the 3-, 6-, and 13-week average ombined kidney weights for the ontrols were 1.79 ±0.17, 1.78 ±0.15, and 2.09 ± 0.09 g, respe- Downloaded from

5 INHALED CHLOROFORM IN F-344 RATS days 3 wk (7 days/wk) 6 wk (7 days/wk) 13 wk (7 days/wk) 13 wk (5 days/wk) 13 wk (6-wk stop) Chloroform onentration (ppm) B 180»160-3 wk (7 days/wk) 13 wk (7 days/wk) 13 wk (5 days/wk) 13 wk (6-wk stop) Chloroform onentration (ppm) FIG. 1. Perentage inrease in body weight in (A) male or (B) female F-344 rats exposed to hloroform vapors for 4 days or 3, 6, or 13 weeks (males) or 3 or 13 weeks (females). Bars represent the mean inrease in body weight ± SD (n = 5-10 rats per group). Inreases in body weight are measured from the first day of exposure. For male rats the average starting weight for the ontrol group was ± 12.6 g. For the female rats the average starting weight for the ontrol group was ± 8.4 g. Rats were exposed 6 hr/day for 7 or 5. Additional rats were exposed for 6 hr/day, 7 for 6 weeks and then housed in the ontrol hambers for the remaining 7 weeks (6-week stop). Asterisks (*) denote groups that were statistially different from exposure- and duration-mathed ontrol groups (Williams test, p < 0.05). tively. No signifiant hanges in liver or kidney weight relative to body weight were noted at onentrations below 90 ppm (data not shown). Inreases of about 30% in relative liver weights were observed at 6 and 13 weeks with ppm exposures. Inreases of about 10% in relative kidney weight ourred with the 90 ppm and about 30% with the ppm exposures at 3, 6, and 13 weeks. For the female rats the 3- and 13-week average liver weights for the ontrols were 6.60 ± 0.9 and 5.27 ± 0.33 g, respetively. For the female rats the 3- and 13-week average ombined kidney weights for the ontrols were 1.59 ± 0.13 and 1.29 ± 0.11 g, respetively. No signifiant hanges in liver or kidney weight relative to body weight were noted at onentrations below 90 ppm (data not shown). Inreases of about 10% in relative liver weights were observed at 90 ppm at 3 and 13 weeks. Inreases of about 50% in relative liver weights were seen with ppm exposures at 3 and 13 weeks. These inreases were assoiated with a peridutular Downloaded from

6 114 TEMPLIN ET AL. TABLE 2 Kidney Lesion Sores and Inidene in Male or Female F-344 Rats Exposed to Chloroform Vapors Conentration (ppm) 4 days 3 weeks 7 6 weeks 7 13 weeks 7 days/ week 13 weeks 5 days/ week 13 weeks 6-week stop Male rats Female rats (0.5) 0.0 (0/5) 0.0 (0/5) 0.2 (1/5) 0.4 (2/5) 1.0(5/5) 0.3 (4/13) 0.4 (5/13) 0.5(6/13) 0.9 (12/13) 1.0(10/10) 1.9(10/10) 0.0 (0/8)" 0.5 (4/8) 1.0 (8/8) 1.4 (8/8) 1.4 (5/5) 1.2(5/5) 0.1 (1/12) 0.3 (4/13) 0.6(8/13) 1.0 (11/13) 0.5 (5/10) 2.0 (10/10) 0.6 (8/14) 0.8 (10/15) 0.5 (7/15) 0.6 (9/14) 1.2 (14/15) 1.4 (14/14) 0.4 (6/14) 0.7 (10/15) 0 7(10/15) 0.8 (12/15) 0.7 (10/15) 1.1 (14/14) 0.6 (8/14)* r 0.1 (2/15) 0.6 (6/13) 2.8 (13/13) 0.4 (6/14)'' < 1.8 (13/13) 0.4 (5/13) 1.4 (13/13) 0.6 (8/14)* r 1.1 (8/8) 1.4 (8/8) 0.4 (6/14)* f 0.9 (7/8) 0.8 (6/8) " Chloroform-indued kidney histopathologial hanges were sored qualitatively for severity as follows: 0 = within normal limits, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe, where 1 through 4 indiate inreasing severity of the lesions ranging from vauolation of proximal ell tubule (PCT) epithelium, enlarged PCT nulei, pyknoti PCT nulei, to individual tubule ell nerosis. Detailed desriptions of the lesions are given under Results. The first number in eah box is the mean lesion sore for the entire group of animals. The ratio in parentheses is that of the number of animals presenting with a lesion sore of I or greater, relative to the total number of animals evaluated in that group. 'Control animals are the same for all the 13-week studies. e Animals were not exposed at these time points. fibrosis (see below). Inreases of about 25% in relative kidney weight ourred with the 90 ppm and about 50% with the ppm exposures at 3 and 13 weeks. Male Rats Renal histopathology and ell proliferation. A summary of the renal histopathology sores is presented in Table 2. Areas of alterations were primarily onfined to the epithelial ells of the proximal onvoluted tubules (PCT) in the ortex. Male rats exposed to hloroform vapors for 4 onseutive days had minimal evidene of ellular alterations. Vauolation of PCT epithelium was found in 5/5 rats exposed to ppm, whereas rats exposed to 90 ppm and below were not onsidered to be signifiantly different from ontrols. After 3 weeks of exposure, sattered areas of vauolation within PCT epithelium were present in 12/13 rats exposed to 30 ppm, 10/10 rats exposed to 90 ppm, and 10/10 rats exposed to ppm. The PCT had a basophili appearane in rats exposed to ppm. A relative redution in hyaline droplets was noted in rats exposed to 30, 90, or ppm. Following 6 weeks of exposure, 11/13 rats exposed to 30 ppm, 5/10 rats exposed to 90 ppm, and 10/10 rats exposed to ppm had vauolation within the PCT epithelium. Fewer hyaline droplets were present in rats exposed to 30, 90, or ppm ompared to ontrols. After 7 hloroform exposure for 13 weeks, mirosopi kidney alterations were present in 14/15 rats exposed to 90 ppm and 14/14 rats exposed to ppm. In addition to sattered vauolation of the PCT, individual tubule ell nerosis and enlarged epithelial ell nulei were present in rats exposed to ppm. Rats exposed to 30 ppm and below were not signifiantly different from ontrol, with the exeption of fewer hyaline droplets in the 30, 90, and ppm groups. Rats exposed to hloroform vapor onentrations of 30 or 90 ppm 5 day/week for 13 weeks were not signifiantly different from ontrols. Sattered vauolation and nulear pyknosis of the PCT epithelial ells were found in 13/13 rats exposed to ppm. Fewer hyaline droplets were noted in rats exposed to 90 or ppm. In rats exposed to hloroform 7 day/week for 6 weeks and then held in the ontrol hambers until Week 13. sattered PCT epithelia with enlarged nulei in the kidneys were found in 8/8 rats exposed to 90 ppm and 8/8 rats exposed to ppm. Indued ell proliferation in the kidney ortex of rats at eah time point is presented graphially in Fig. 2A. Consistent with evidene of toxiity seen in the histopathologial evaluation, no inrease in the LI was found in rats exposed to hloroform vapors for 4 days. Following 7 exposure for 3 weeks the LI was elevated in the ortex of rats exposed to 30 ppm and above, reahing a value of Downloaded from

7 INHALED CHLOROFORM IN F-344 RATS i I 25 o I" I 15 Si. D days 3 wk (7 days/wk) 6 wk (7 days/wk) 13 wk (7 days/wk) 13 wk (5 days/wk) 13wk(6-wkstop) Chloroform onentration (ppm) B 8 a I 30 3 wk (7 days/wk) 13 wk (7 days/wk) 13 wk (5 days/wk) 13 wk (6-wk stop) Chloroform onentration (ppm) FIG. 2. Labeling index (LI) in the kidney ortex of (A) male or (B) female F-344 rats exposed to hloroform vapors for 4 days or 3, 6, or 13 weeks (males) or 3 or 13 weeks (females). Bars represent the mean LI ± SD (n = 5-10 rats per group). The LI is the perentage of nulei in S-phase identified in histologial setions stained immunohistohemially for BrdU. Rats were exposed 6 hr/day for 7 or 5. Additional rats were exposed for 6 hr/day, 7 for 6 weeks and then housed in the ontrol hambers for the remaining 7 weeks (6-week stop). Asterisks (*) denote groups that were statistially different from exposure- and duration-mathed ontrol groups (Williams test, p < 0.05). approximately 7-fold over ontrol in rats exposed to ppm. The LI in the ortex remained elevated in the rats exposed to 30, 90, and ppm 7 throughout the remainder of the study, indiating ontinual ytotoxiity and regenerative ell proliferation. At 6 and 13 weeks, the labeling index was approximately 2-, 4-, and 10-fold over ontrol in rats exposed to 30, 90, and ppm, respetively. The LI in rats exposed to 30 ppm 5 was not different from ontrol rats (Fig. 2A). At 90 ppm, the LI in 5 exposed rats was marginally inreased relative to ontrols, but the inrease was less than half that of rats exposed for 7. Rats exposed to ppm 5 days/ week had a LI approximately 7-fold over ontrol, a response that was similar to the 9-fold over ontrol value in rats exposed 7. Rats exposed to 90 or ppm hloroform vapors for 6 weeks and then held in the ontrol hambers for 7 weeks were not different from ontrols. An inrease in the LI was also found in the outer stripe of the outer medulla of rats exposed 7 to 90 or ppm for 3, 6, or 13 weeks and in rats exposed to Downloaded from

8 116 TEMPLIN ET AL. ppm, 5 for 13 weeks (Fig. 2A). The response was onsistently lower than that found in the ortex. Conentration-dependent inreases in the LI were not found in the inner stripe of the outer medulla or the inner medulla. Mean LI values ranged from 0.8 to 1.6 in the inner stripe and 0.1 to 0.4 in the inner medulla. The presene of foi of hroni progressive nephropathy (CPN) within the ortex was a onern in soring the LI in the male rats. An area of CPN was defined as having at least two adjaent tubules with a minimum BrdU LI of 30%. Both the inidene and LI within the foi were determined in the male rats. The area for determining the LI was restrited to the affeted tubules with a two-tubule surrounding border. Using these riteria, the average labeling index for foi of CPN was 20.0 ± 3.7%. In the 13-week ontrol rats there was evidene of at least one fous of CPN in 8 of 10 rats. A onentration-dependent derease in the inidene of foi of CPN in male rats exposed to hloroform vapors was present in this study with an inidene 6/10, 1/10, 0/10, and 0/ 4 for the 2, 10, 30, and 90 ppm groups, respetively. At ppm foi of CPN ould not be determined beause of the inreased regenerative ell proliferation throughout the ortex at this dose. When a lear distintion of the borders for a CPN foi ould be determined the area was exluded from the overall ortex LI. Although identifying a fous of CPN at the higher onentrations was sometimes diffiult where the LI was elevated throughout the ortex, we are onfident that the LI sores presented are aurate. A redistribution of the proportion of different ell types within the ortex was a possibility with hroni exposure to hloroform vapors beause of potential loss of a sensitive ell type. Proximal tubule ells, the primary target of hloroform-indued ytotoxiity, aounted for approximately 76% of the nulei in either ontrol or ppm hloroformtreated rats. Distal tubules, onneting tubules, and the nontubular portion were all present in approximately the same proportion in ontrol and ppm treated rats. Liver histopathology and ell proliferation. The inidene of hepati lesions and sores is presented in Table 3. Hepatoyte alterations were primarily onfined to the ppm exposed rats at all time points and in the 90 ppm exposed rats at the later time points. Mirosopi findings in the rats exposed 7 to ppm inluded sattered individual hepatoyte degeneration, mitoti figures, and midzonal vauolation. By 13 weeks, hepatoyte alterations were found in rats exposed 7 to 90 or ppm hloroform. In the rats exposed to 90 ppm, sattered vauolated hepatoytes were found in the livers of 6/15 rats, with the additional effet of single to multiple (2-3) hepatoyte nerosis in 9/15 rats. Mitoti figures and mild diffuse hepatoyte vauolation were noted in 15/15 rats exposed to ppm. Rats exposed to 30 ppm and below were not signifiantly different from ontrols. In rats exposed to hloroform vapors 5, sorable histologi lesions were found in the 90 and ppm groups. One rat exposed to 90 ppm had sattered vauolated hepatoytes and another had foi of single and multiple (2-3) hepatoyte nerosis. Diffuse hepatoyte vauolation, whih was most severe in the entrilobular and midzonal hepatoytes, was found in 13/13 rats exposed to ppm. In two of these rats, degeneration and nerosis of the first and seond layers of hepatoytes were present around the entral vein and affeted approximately 50% of the lobules. In addition, pale eosinophilia of the entrilobular to midzonal hepatoytes was found in some rats exposed to 30 and 90 ppm but was not inluded as a sorable lesion. Rats exposed to 90 ppm hloroform 7 for 6 weeks and then held in the ontrol hambers until Week 13 were not different from ontrols. In rats exposed to ppm for 6 weeks and then held until Week 13, sattered foi of single or multiple (2-3) hepatoyte nerosis with mild inflammatory ell infiltrate were infrequently found. Signifiant inreases in hepatoyte LI were found only in rats exposed to ppm hloroform (Fig. 3A). In the 7 exposures the hepatoyte LI was inreased by approximately 10-, 14-, 20-, and 25-fold over ontrols at 4 days and 3, 6, and 13 weeks, respetively. Rats exposed for 5 to ppm had a hepati LI similar to that of rats exposed for 7, with a 29-fold inrease over ontrols. Rats exposed to hloroform for 6 weeks and allowed to reover for 7 weeks did not have an elevated LI relative to ontrol animals. In male rats exposed to the highly hepatotoxi onentration of ppm hloroform, an additional nonhepatoyte lesion was found within the liver. The prevalene and severity of the lesions were greatest in the right lobe, although evidene of the lesion ould be found throughout all the lobes examined. These lesions were haraterized as glandular strutures lined by olumnar epithelium and goblet ells and surrounded by onnetive tissue. We have termed these lesions intestinal rypt-like duts with peridutular fibrosis to distinguish them from true holangiofibrosis. This lesion is similar to that seen following treatment with other hepatotoxiants suh as furan (Maronpot et al., 1991). This lesion appears to have a non-bile dut origin and a more detailed desription of this lesion and aompanying proliferative hanges has been presented in a separate manusript (Jamison et al., 1996). Nasal histopathology and ell proliferation. The severity and type of hloroform-indued nasal lesions were dependent on both onentration and duration of exposure (Table 4). The lesions were primarily onfined to the ethmoid portion of the nasal passages lined by olfatory epithelium. At the early time points, alterations involved the ventral and lateral regions of the ethmoid turbinates, while the entral aspets of the turbinates and nasal septum were unaffeted. Downloaded from

9 INHALED CHLOROFORM IN F-344 RATS 117 TABLE 3 Hepati Lesion Sores and Inidene in Male or Female F-344 Rats Exposed to Chloroform Vapors Conentration (ppm) 4 days 3 weeks 7 6 weeks 7 13 weeks 7 days/ week 13 weeks 5 days/ week 13 weeks 6-week stop Male rats Female rats (0/5) 0.0 (0/5) 0.4 (2/5) 0.4 (2/5) 0.3 (1/4) 0.0 (0/5) 0.0 (0/13) 0.0 (0/13) 0.1 (1/13) 0.0 (0/13) 0.2 (2/10) 1.8 (10/10) 0.0 (0/8) 0.0 (0/8) 0.0 (0/8) 0.4 (3/8) 0.8 (4/5) 2.0 (5/5) 0.2 (2/12) 0.1 (4/13) 0.2 (3/13) 0.0 (0/13) 0.3 (3/10) 2.0 (10/10) 0.1 (1/15) 0.2 (3/15) 0.0 (0/15) 0.1 (2/15) 1.0 (14/15) 3.9 (15/15) 0.1 (1/15) 0/1 (1/15) 0.0 (0/14) 0.0 (0/15) 0.8 (12/15) 3.0(15/15) 0.1 (1/15)* < 0.0(0/13) 0.3 (4/13) 2.4 (13/13) 0.1 (1/15) 0.0 (0/13) 0.3 (4/13) 2.0(13/13) 0.1 (1/15) 0.0 (0/8) 0.0 (0/8) 0.1 (1/15) 0.1 (1/8) 0.0 (0/8) " Chloroform-indued liver histopathologial hanges were sored qualitatively for severity as follows: 0 = within normal limits, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe, where 1 through 4 indiate inreasing severity of the lesions ranging from hepatoyte vauolation, degenerative hanges in hepatoytes, to hepatoyte nerosis. Detailed desriptions of the lesions are given under Results. The first number in eah box is the mean lesion sore for the entire group of animals. The ratio in parentheses is that of the number of animals presenting with a lesion sore of 1 or greater, relative to the total number of animals evaluated in that group. * Control animals are the same for all the 13-week studies. Animals were not exposed at these time points. With ontinued exposure, lesions were present throughout the entire ethmoid portion of the nose. Relatively few alterations were present in the anterior portions of the nasal avity or the posterior regions lined by respiratory epithelium. The type, severity, and distribution of the lesions were onsistent and usually present in all rats within a speifi onentrationand duration-exposed group. Rats examined following 4 days of exposure had lesions within the lamina propria haraterized by edema, loss of deep Bowman's glands, periosteal hyperellularity, and new bone growth in the proximal portions of the ethmoturbinates. The severity and relative distribution of the lesions were onentration-dependent, ranging from minimal involvement in rats exposed to 10 ppm to moderate to severe effets in rats exposed to ppm. Foal atrophy of the olfatory epithelium was noted in rats exposed to 90 or ppm. At 3 weeks, the most prevalent lesion in rats exposed to 10 ppm and higher was a onentration-dependent loss of the deep Bowman's glands and a mild to moderate edema in the lamina propria. Periosteal hyperellularity and new bone growth were no longer as apparent, and the reently formed turbinate bones appeared to be differentiating into mature bone. Mineralization of the basal lamina was noted in rats exposed to ppm. In the olfatory epithelium, foal edema and hanges from olfatory-to respiratory-type epithelium were observed. Signifiant lesions were not observed in the lamina propria or olfatory epithelium of rats exposed to 2 ppm for 4 days or 3 weeks. In addition to the lesions desribed in rats exposed for 4 days or 3 weeks, atrophy of the ethmoid turbinates was noted following 6 weeks of exposure. The appearane of redued turbinate size was minimally evident at 2 ppm and inreased in severity with higher onentrations. The spaes normally oupied by the nerves, glands, and other tissues that ompose the lamina propria were redued in size or ell numbers or were displaed by edema. Atrophy of the overlying epithelium had redued the thikness of the epithelial ell layer to approximately half that of the nonaffeted olfatory epithelium. At the 13-week time point, the overall effet of the lesions within the lamina propria and the olfatory epithelium gave the appearane that the turbinates oupied a redued volume in the nasal avity. Minimal atrophy was observed in rats exposed to 2 ppm. The response inreased in severity through the 10, 30, and 90 ppm exposed rats with moderate to severe effets in rats exposed to ppm. The type of response and the severity of the lesions were essentially the same in rats exposed to 30, 90, or ppm, 5 as that found in rats exposed 7. In rats exposed to 90 or ppm 7 for the first 6 weeks and then held in the ontrol hamber until Week 13, the lesions were not reversible, and lesion type and severity were similar to that found in rats exposed for either 6 or 13 weeks. The ULLI was determined in the proximal portion of the dorsal sroll of the first endoturbinate; this region was a primary target for the early hloroform-indued lesions in Downloaded from

10 118 TEMPLIN ET AL. 30 I o O ra D.15 [~] 4 days [3 3 wk (7 days/wk) H 6 wk (7 days/wk) 13 wk (7 days/wk) 13 wk (5 days/wk) 13 wk (6-wkstop) * Chloroform onentration (ppm) B wk(7 days/wk) B 13 wk (7 days/wk) [j 13 wk (5 days/wk) 13 wk (6-wkstop) 2 20 u O Chloroform onentration (ppm) FIG. 3. Hepatoyte labeling index (LI) in the livers of (A) male or (B) female F-344 rats exposed to hloroform vapors for 4 days or or 13 weeks (males) for 3 or 13 weeks (females). Bars represent the mean LI ± SD (n = 5-10 rats per group). The LI is the perentage of nulei in S-phase identified in histologial setions stained immunohistohemially for BrdU. Rats were exposed 6 hr/day for 7 or 5. Additional rats were exposed for 6 hr/day, 7 for 6 weeks and then housed in the ontrol hambers for the remaining 7 weeks (6-week stop). Asterisks (*) denote groups that were statistially different from exposure- and duration-mathed ontrol groups (Williams test. /; < 0.05). the lamina propria (Mery et al., 1994). A onentrationdependent ell regenerative response was present in rats exposed for 4 days to 10, 30, 90, or ppm hloroform (Fig. 4). The elevation in the ULLI ranged from an inrease of approximately 14-fold over the ontrols in rats exposed to 10 ppm to approximately 22-fold over ontrols in rats exposed to ppm. Rats exposed to 2 ppm were onsidered to be equal to ontrols. At the later time points of 3, 6, or 13 weeks, the ULLI was onsiderably lower ompared with the values at 4 days, and a onentration-dependent response was less evident. The ULLI was approximately 6-fold over ontrols in rats exposed to 10 ppm and higher for 3 weeks and 4-fold over ontrol in rats exposed to 10 ppm and higher for 6 or 13 weeks. Rats exposed to 2 ppm did not have a signifiantly elevated ULLI following 3, 6, or 13 weeks of exposure for 7. An inrease in the ULLI was found in rats exposed to 30, 90, or ppm 5 and rats exposed to 90 or ppm 7 for the first 6 weeks. For the 5 and 6-week stop groups a lear onentration Downloaded from

11 < INHALED CHLOROFORM IN F-344 RATS 119 TABLE 4 Severity of Nasal Lesions in Male F-344 Rats Exposed to Chloroform Vapors Conentration (ppm) 4 days 3 weeks 7 6 weeks 7 13 weeks 7 13 weeks 5 13 week 6- week stop (5/5) 1.0 (5/5) 1.4 (5/5) 2.0 (5/5) 3.0 (5/5) 3.8 (5/5) 1.3 (6/8) 1.4(5/8) 2.4 (8/8) 2.4 (8/8) 2.8 (5/5) 3.0 (5/5) 0.0 (0/7) 1.0 (7/8) 1 9 (8/8) 2 1 (8/8) 3.0 (5/5) 3.0 (5/5) 0.0 (0/10) 1.1 (10/10) 2.0(10/10) 2.0(10/10) 2.5 (10/10) 2.9 (10/10) 0.0 (0/10)* C 1.8 (8/8) 2.0 (8/8) 3.0 (8/8) 0.0(0/10)* 2.1 (5/8) 2.9 (8/8) Chloroform-indued histopathologial hanges in the ethmoid region of the nasal passage were sored qualitatively for severity as follows: 0 = within normal limits. 1 = minimal, 2 = mild, 3 = moderate, 4 = severe, where 1 through 4 indiate inreasing severity of the lesions. Nasal setions from rats exposed for 4 days or 3 weeks were assigned severity sores for lesions in the lamina propria ranging from edema and loss of Bowman's gland, penosteal hyperellulanty. to mineralization of the basal lamina In rats exposed for 6 or 13 weeks, severity sores were assigned for lesions ranging from edema and loss of Bowman's glands, olfatory metaplasia, basal lamina mineralization, to generalized atrophy of the ethmoid turbinates. Detailed desriptions of the lesions are given under Results. The first number in eah box is the mean lesion sore for the entire group of animals. The ratio in parentheses is that of the number of animals presenting with a lesion sore of 1 or greater, relative to the total number of animals evaluated in that group. * Control animals are the same for all the 13-week studies. Animals were not examined at these time points response was not evident, with the ULLI elevated only 2- fold over ontrols. Effets of hloroform on other tissues. No treatmentrelated lesions were observed in the sternebrae, vertebrae, tibia, femur, and artiular tissues of the knee. These sites represent bones that grow by intramembranous ossifiation, as do the bones of the ethmoid turbinates, and by endoondral ossifiation. A modest onentration-dependent derease rather than inrease in the ULLI in these bones was evident in the present study (data not shown). Histopathologial examination of H & E-stained tissues from these rats at these time points was onsistent with the signifiant derease in weight gain over this same period rather than a toxi response within the bone strutures. No treatment-related lesions were found in any of the other tissues that were taken in the full-sreen neropsy. Female Rats Renal histopathology and ell proliferation. Signifiant kidney lesions were onsistently found only in female rats exposed to hloroform vapor onentrations of ppm (Table 2). At 3 weeks 5/5 rats had sattered regenerating PCT epithelium affeting less than 10% of the ortex. Following 13 weeks of 7 ppm exposures, 14/14 rats had sattered PCT with anisokaryosis and megalokaryosis, whereas 13/13 rats exposed 5 had sattered PCT ells with enlarged nulei. The histologi hanges in the female rats at 30 ppm and above onsisted primarily of vauolation, in ontrast to the male rats where there was some evidene of nerosis. Female rats exposed to vapor onentrations of 90 ppm or lower for 7 or 5 as well as female rats exposed for 6 weeks to 90 or ppm then held until Week 13 all had low histologi sores and were not onsidered to be signifiantly different from ontrols. A lear onentration-dependent response in the LI in the kidney was found in the female rat (Fig. 2B). The LI in the ortex was inreased with onentrations of 30 ppm and greater at both the 3- and 13-week time points. At 3 weeks the LI was elevated 2-, 4-, or 10-fold over ontrols with 30, 90, or ppm exposures, respetively. The values were slightly higher at 13 weeks with the LI elevated 3-, 7-, or 12-fold over ontrols with 30, 90, or ppm exposures. The LI in the ortex of female rats exposed 5 was similar to that of a female rats exposed to the same onentration 7. The LI in 5 exposed animals was 2-, 7-, and 10-FOC with 30, 90, or ppm exposures. An inrease in the LI was also found in the outer stripe of the outer medulla of female rats exposed 7 to 90 or ppm for 3 or 13 weeks and in female rats exposed to ppm 5 for 13 weeks. However, the response was onsistently lower than that found in the ortex. Conentration-dependent inreases in the LI were not found in the inner stripe of the outer medulla or the inner medulla. Mean LI values ranged from 0.5 to 1.5 in the inner stripe and 0.1 to 0.7 in the inner medulla. Liver histopathology and ell proliferation. Following 7 exposures for 3 weeks, mild mirosopi alterations of midzonal vauolation were found in 4/5 female rats exposed to 90 ppm (Table 3). Centrilobular hepatoyte degeneration with single ell nerosis was found in 5/5 rats exposed to ppm. Rats exposed to 30 ppm and below Downloaded from

12 120 TEMPLIN ET AL. \~\ 4 days 3 3 wk (7 days/wk) 6 wk (7 days/wk) wk (7 days/wk) 13 wk (5 days/wk) 13 wk (6-wk stop) o o uj 400 \L Chloroform onentration (ppm) FIG. 4. Unit length labeling index (ULLI) in the proximal portion of the dorsal sroll of the first endoturbinate of male F-344 rats exposed to hloroform vapors for 4 days or 3, 6, or 13 weeks. Bars represent the mean ULLI ± SD (n = 5 10 rats per group). The ULLI is the number of nulei in S-phase in the lamina propria and adjaent periosteum The underlying turbinate bone was used for determination of length. Rats were exposed 6 hr/ day for 7 or 5. Additional rats were exposed for 6 hr/day, 7 for 6 weeks and then housed in the ontrol hambers for the remaining 7 weeks (6-week stop). Asterisks (*) denote groups that were statistially different from exposure- and duration-mathed ontrol groups (Williams test, p < 0.05). were not onsidered to be signifiantly different from ontrols. At 13 weeks, 12/15 rats exposed to 90 ppm hloroform 7 had areas of mild vauolation in midzonal hepatoytes with infrequent hepatoyte degeneration. Centrilobular to midzonal hepatoyte degeneration was found in 15/ 15 female rats exposed to ppm hloroform 7 for 13 weeks. Rats exposed to 30 or 90 ppm, 5 for 13 weeks were not signifiantly different from ontrols. In rats exposed to ppm, 13/13 had enlarged nulei in midzonal hepatoytes, entrilobular to midzonal hepatoyte vauolation, and entrilobular hepatoyte degeneration with single ell nerosis. Rats exposed to 90 or ppm 7 for 6 weeks and held until Week 13 were not onsidered to be signifiantly different from ontrols. The hepatoyte LI for female rats is given in Fig. 3B. An inreased LI was found only in the ppm exposures where the LI was inreased 10-fold over ontrols at 3 weeks and approximately 40-fold over ontrols with either a 7 or 5 exposure for 13 weeks. Female rats exposed to ppm for 6 weeks and evaluated at 13 weeks were not different from ontrols. The lesions haraterized by intestinal rypt-like duts with peridutular fibrosis as desribed above for the male rat liver were dramatially inreased in the livers of female rats exposed to ppm hloroform. These lesions were not seen at lower exposure onentrations of hloroform. Female Downloaded from rats exposed for 13 weeks 7 had a high density of variably sized 2- to 5-mm pale foi throughout all lobes. Mirosopially, the lesions were haraterized as glandular strutures lined by olumnar epithelium and goblet ells and surrounded by onnetive tissue. The prevalene and severity of the lesions was greatest in the right and audate lobes. The severity of alterations in livers of the female rats was greater than that of the males. A more detailed desription of this lesion and aompanying proliferative hanges has been presented in a separate artile (Jamison et al., 1996). Nasal histopathology and ell proliferation. The proliferative and atrophi alterations indued in the nasal passages of female rats exposed to hloroform vapor for 3 or 13 weeks were similar to those found in the male rat following 3 or 13 weeks of exposure (data not shown). Effets of hloroform on other tissues. The only treatment-related lesions observed in the full-sreen mirosopi tissue examination were in the kidney, liver, and nasal passages. Chloroform-assoiated lesions were not found in the nonnasal bones, sternebrae, vertebrae, tibia, femur, and artiular tissues of the knee, nor were these bone LI onsidered to be different from ontrol LI (data not shown). DISCUSSION Effets on Health and the Maximum Tolerated Dose In the study reported here rats were exposed to ppm beause a pilot study suggested that rats might be able to

13 INHALED CHLOROFORM IN F-344 RATS 121 tolerate this dose for longer periods (Larson et al, 1994d). Further, when we began we had no knowledge of the results from the Japan Bioassay Laboratory long-term inhalation study with F-344 rats (Yamamoto et al., 1994). Chloroform onentrations of 2 and 10 ppm had minimal effets on body weight gain, 30 and 90 ppm indued a minimal to mild derease in average body weight gain, and ppm had a signifiant effet on body weight gain (Figs. 1A and IB). The substantial initial weight loss and ontinued derement in weight gain observed at ppm demonstrate that this is an exessively toxi vapor onentration for both male and female rats and would exeed riteria for a MTD for longerterm studies. These observations onfirm 90 ppm as the appropriate highest onentration hosen for the hloroform inhalation aner bioassay with F-344 rats (Yamamoto et al., 1994). The results from the Japan Bioassay Laboratory inhalation study are valid and the use of higher exposure onentrations would not be justified. Chloroform-Indued Kidney Lesions A lear onentration response in the number of rats affeted, severity of histologial alterations, and inreased LT was present in the kidneys of both male and female rats exposed to hloroform vapors (Fig. 2, Table 2). The expression of ytohrome P450 2E1 in the rat kidney, the primary enzyme proposed to be responsible for the metabolism of hloroform to toxi metabolites, has been reported to be similar between male and female F-344 rats (Wilke et al., 1994). This may aount for the nearly equal sensitivity of the sexes to the renal ytotoxiity of hloroform. The LI in the ortex was signifiantly lower in male rats exposed for 5 than in those exposed to equal onentrations 7. However, the LI was similar in female rats exposed for either 7 or 5. These observations raise important issues as to appropriate designs for long-term studies. Inreased ell proliferation was not found in either sex of rats exposed for 6 weeks and then held until Week 13, indiating that the proliferative response is dependent on the presene of hloroform and represents regenerative growth as a result of repetitive ytolethality. A onentration of 10 ppm in the male and the female rat was determined to be the experimental no-observed-adverseeffet-level (NOAEL) within the proximal tubules of the ortex. No mirosopi alterations were found in either sex of rats exposed 7 to 10 ppm, nor was the LI within the proximal tubule epithelium elevated. CPN within the kidney ortex was not a signifiant fator in the olletion of the LI data in male rats. The foi in the ortex affeted by CPN were relatively small, involving one to five tubules, and ould be distinguished as distint areas of foal proliferation within the ortex. This pattern was in ontrast to hloroform-indued ell regeneration, whih was distributed throughout the ortex. Rats exposed to 30 or 90 ppm 5 as well as rats exposed to 90 or ppm for the first 6 weeks and then held in the ontrol hambers support the hypothesis that hloroform does not stimulate the CPN proess and atually may have an inhibitory effet. Chloroform-Indued Liver Lesions Signifiant mirosopi evidene of ytolethality and regenerative ell proliferation in the liver ourred only at the ppm exposure onentrations. This is in ontrast to the kidney, where histologial alterations and an inreased LI were found at onentrations as low as 30 ppm. Nonhepatoyte liver lesions haraterized as intestinal rypt-like duts with peridutular fibrosis were only found in rats exposed to the strongly hepatotoxi atmospheri onentration of ppm of hloroform. Interestingly, other liver ytotoxiants suh as furan produe the same lesion under severely hepatotoxi onditions (Maronpot et al., 1991). The furan-indued lesions also reportedly develop preferentially in the right and audate liver lobes. Maronpot et al. (1991) reported that 100% of the animals treated for 90 days with highly ytotoxi doses of furan and then maintained without further treatment developed holangioarinomas at 6, 12, and 18 months. Sine hloroform and furan have little strutural similarity, these lesions are apparently seondary to regenerative growth signals in the heavily damaged liver. Bile dut bromodeoxyuridine labeling indies as well as observations of the loations of the early lesions at the 3- and 6-week time points indiate that these lesions arose from a stem ell and not from bile duts (Jamison et al., 1996). These hloroform-indued lesions are different from the bile dut proliferative lesion produed by bile dut ligation followed by treatment with furan, whih more appropriately might be termed holangiofibrosis (Siria et al., 1994). If the F-344 rats had been exposed to ppm hloroform for 2 years, these lesions probably would have progressed to holangioarinomas. However, suh tumor indution would only be seondary to the extreme ytotoxiity produed at a dose in exess of the MTD with no relevane for extrapolating to lower doses. Chloroform-Indued Nasal Lesions The early response of periosteal hyperellularity and inreased ULLI in the lamina propria are onsistent with previously published 7-day inhalation studies (Larson et al., 1994d; Mery et al., 1994). Periosteal hyperellularity and inreased bone growth in this region have also been desribed following gavage dosing of hloroform to female F- 344 rats for 4 onseutive days (Larson et al, 1995b). However, hloroform does not appear to have a diret effet on bone growth in general sine there were no treatment-related effets on bone struture and growth at other sites. Late atrophy was present not only in the areas of the same Downloaded from

14 122 TEMPLIN ET AL. turbinates involved in the early proliferative response, but also in the entire ethmoid region of the nasal avity. A signifiant redution in the number of ells normally found in the lamina propria suggests that these ells are being eliminated. New bone growth is not a prominent feature, and the bone formed during the proliferative response has developed to the point that the original borders are no longer visible. The early lesions observed in the nasal passages of the rat are similar to those observed in the mouse (Larson et al, 1996). A ruial differene between the two speies is that the early proliferative lesions were transient and a late atrophi response was not apparent in the mouse. The observations of atrophy in the nasal turbinates of F-344 rats in the present study are onsistent with observations reported in a 2-year hloroform inhalation bioassay (Yamamoto et al, 1994). In an initial report of that study, the authors desribed nonanerous lesions suh as ossifiation of the nasal turbinates and metaplasia from olfatory to respiratory epithelium, alterations whih were present at the lower onentrations of 2 and 10 ppm. Further work will be needed to determine the speies speifiity of the response, inluding potential effets in humans. Researh questions inlude the issues of regional speifiity, regional dosimetry, effets on funtionality, and distinguishing between an effet and an adverse effet. Route-to-Route Extrapolations Route and duration of exposure have a temporal impat on the kidney response. Larson et al. (1993) reported approximately an 18-fold inrease in the LI in the ortex of male F-344 rats administered a single bolus dose of 180 mg/kg hloroform in orn oil. When multiple doses of hloroform were administered for either 4 onseutive days or 5 days/ week for 3 weeks, the LI was onsiderably lower (Larson et al., 1995a). This adaptation of the kidney to ontinued hloroform exposures was also seen in the B6C3F mouse kidney and ould represent a hange in the harater of the ells or loss of the ability to regenerate beause of severe toxiity (Larson et al., 1994). In ontrast, the pattern of LI indution in the F-344 rat kidney with inhalation exposures showed no inrease at the Day 4 time point, but the LI inreased and remained signifiantly elevated at 3, 6, and 13 weeks (Fig. 2). No inrease in the kidney LI was observed in male F-344 rats administered hloroform in drinking water for 4 days or 3 weeks (Larson et al., 1995a). Cell regeneration data have been effetively used by Larson et al. (1996) as an internal dosimeter to ompare the biologially effetive dose to the liver for different routes of exposure to hloroform in female B6C3F, mie. This omparison is more diffiult in the rat kidney, where the LI has a onsiderable time and route dependeny. Correlations between Cell Proliferation and Caner Events seondary to ytolethality and regenerative ell proliferation appear to be the driving fore for tumor formation for nongenotoxi-ytotoxi arinogens suh as hloroform (Butterworth et al, 1995). In every ase of hloroformindued aner examined thus far, there is a orresponding preeding toxi response with assoiated ativity suh as regenerative ell proliferation (Larson etai, 1994b; Templin etal, 1996a,b). There are, however, instanes where hloroform indues regenerative ell proliferation without subsequent aner in a given target organ. For example, hloroform produes liver and kidney toxiity in male B6C3F, and BDF, mie (Larson et al., 1994b; Templin et al., 1996a), but indues liver tumors in the former strain and kidney tumors in the latter (National Caner Institute, 1976; Yamamoto et al, 1994). These results illustrate the priniple that geneti bakground and target organ speifiity greatly influene tumor outome. Thus, indued toxiity and regenerative ell proliferation are neessary but not always suffiient onditions to indue aner in a given target organ. The ritial orrelation is between the shapes of the dose-response urves for regenerative ell proliferation and aner, and eah instane must be evaluated on a ase-by-ase basis. In evaluating the Yamamoto et al. (1994) study, the question to be addressed is why hloroform did not indue aner in the F-344 rats. Beause the same exposure onditions were used, the ell proliferation data from the urrent study provide a rare opportunity to ompare the results to those from a hloroform inhalation bioassay (Yamamoto et al, 1994). Lak of Genotoxi Ativity The first reason that hloroform did not indue aner in F-344 rats is that hloroform is not genotoxi. The weight of evidene indiates that neither hloroform nor its metabolites are DNA-reative (International Programme on Chemial Safety, 1994). A harateristi of a mutageni arinogen is that it produes aner in multiple tissues and speies (Ashby and Tennant, 1988). Chloroform was administered in very large amounts by inhalation to male and female F-344 rats for 2 years with no observed tumor indution (Yamamoto et al, 1994). This is not the mark of a genotoxi arinogen. In fat, a moderate amount of ell turnover ourred in the female rat kidney under bioassay onditions. If hloroform had even mild genotoxi ativity, the ombination of that ativity with the modest inrease in ell turnover would likely have produed at least some inrease in aner in that target site. Osborne-Mendel vs F-344 Rats Knowledge of the dose-response urves for indued regenerative ell proliferation provides some insight as to why hloroform administered at the MTD produed aner in Downloaded from

15 INHALED CHLOROFORM IN F-344 RATS 123 male Osborne-Mendel rats (National Caner Institute, 1976), but not in male F-344 rats (Yamamoto et al, 1994). Metaboli, pharmaokineti, and dosimetry studies suggest that hloroform is handled in a similar manner in Osborne- Mendel and F-344 strains (Staats et al, 1990). Data from both strains were used to develop a physiologially based pharmaokineti model for hloroform (Corley et al., 1990; Borghoff et al, 1994). In the bioassays with the Osborne- Mendel rat the tumors were lassified as tubular epithelial ell adenomas and adenoarinomas (National Caner Institute, 1976; Jorgenson et al., 1985). The results from the present study show that the proximal tubule epithelial ells are the primary targets in the F-344 rat kidney, indiating that the target organ and ell population at risk are the same between these two rat strains. Further, hloroform indues renal toxiity and regenerative ell proliferation to about the same extent in both male F-344 and Osborne-Mendel rats when given by gavage (Templin et al., 1996b). Chloroform indued kidney tumors in male Osborne- Mendel rats when administered by gavage (National Caner Institute, 1976). Thus, it was unexpeted when hloroform did not indue kidney aner in either sex of F-344 rats when given by inhalation (Yamamoto et al, 1994). Chloroform given by gavage at the doses that produed kidney aner in male Osborne-Mendel rats (National Caner Institute, 1976) also indues renal toxiity and regenerative ell proliferation in the kidney in this strain of rat (Templin et al., 1996b). In ontrast, hloroform given under the bioassay onditions of 90 ppm, 5 produed only a marginal proliferative response in the F-344 male rat kidney (Fig. 2). These data help explain the indution of kidney tumors in the Osborne-Mendel but not in the F-344 study and suggest that more than marginal inreases in ell proliferation oupled with persistent ytotoxiity are required to indue aner in this target organ. The onentration-response urves shown in Fig. 2 show substantial regenerative ell proliferation in the kidney at ppm hloroform. Were those onditions to be used in a aner study, they would in all likelihood yield kidney tumors. However, suh tumor indution would only be seondary to ytotoxiity seen in exess of the MTD with no relevane for extrapolating to lower doses. Male vs Female Kidney Responses The gavage study with Osborne-Mendel rats produed kidney tumors in male but not female animals (National Caner Institute, 1976). Similarly, hloroform administered by inhalation to BDF, mie indued tumors in male but not female rodents. In the ase of BDF, mie, males were very sensitive to the nephrotoxi effets of hloroform while female mie were resistant (Templin et al., 1996a). This nephrotoxi effet of hloroform is presumed to result from the prodution of toxi metabolites from hloroform in the kidney via the ytohrome P450 mixed-funtion oxidase system, and higher levels of P450 2E1 in male mie appear to be the basis for the inreased sensitivity seen in the males (Smith et al, 1984). Similarly, male but not female B6C3F, mie are sensitive to the nephrotoxi effets of hloroform (Larson et al, 1994b,, 1996). In ontrast, male and female F-344 rats exhibit approximately equivalent sensitivity to hloroform-indued kidney toxiity (Fig. 2, Table 2; Larson et al, 1995a,b). In fat, a modest amount of ell turnover was observed in the female rat kidney under the bioassay onditions of 90 ppm, 5 days/ week. Some male rat kidney arinogens appear to at by ausing aumulation of the male rat-speifi protein a 2a - globulin in the form of renal tubule protein droplets, resulting in inreased ell death and ell turnover (Borghoff et al, 1990). Beyond this, however, the kidneys of male rats are often the target organ for arinogenesis by hemials that do not indue a 2u -globulin and appear to be predisposed to tumor formation (Barrett and Huff, 1991). Thus, the lak of indution of kidney tumors in the female F-344 rats in the inhalation bioassay (Yamamoto et al, 1994) despite modest ell turnover at 90 ppm (Fig. 2) illustrates the importane of target organ suseptibility in hemial arinogenesis. Regenerative ell proliferation may also have been taking plae in the kidneys of the female Osborne-Mendel rats in the gavage bioassay (National Caner Institute, 1976). However, tumors were not produed beause indued regenerative ell proliferation is neessary but not always suffiient to indue aner beause of tissue-, sex-, and speiesspeifi suseptibilities. ACKNOWLEDGMENTS This work was supported in part by a researh grant from the Amerian Forest and Paper Assoiation, Washington, DC. The authors thank Catherine Sprankle, Beth Kegelmeyer, Rebekah Harden, Otis Lyght, Mary Morris, and Delorise Williams for quality tehnial assistane; Dr. Owen Moss, Arden James, Marianne Marshall, and Carl Parkinson for outstanding tehnial support in the generation of hloroform inhalation atmospheres; Paul Ross, Tim Shepard, Carol Bobbin, Rihard Masney, Elizabeth Humphrey, and Kathy Bragg for exellent animal are and treatment; and Dr. Barbara Kuyper for valued editorial assistane. REFERENCES Aggazzotti, G., Fantuzzi, G., Tartoni, P. L., and Predieri, G. (1990). Plasma hloroform onentrations in swimmers using indoor swimming pools. Arh. Environ. Health 45, Andelman, J. B. 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