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The Journal of Toxicological Sciences,Vol.31, No.3, 247-264, 2006
TWO-YEAR FEED STUDY OF CARCINOGENICITY AND CHRONIC TOXICITY OF ORTHO-CHLORONITROBENZENE IN RATS AND MICE
Michiharu MATSUMOTO, Yumi UMEDA, Hideki SENOH, Masaaki SUZUKI, Hirokazu KANO,Taku KATAGIRI, Shigetoshi AISO, Kazunori YAMAZAKI, Heihaciro ARITO, Kasuke NAGANO,
Seigo YAMAMOTO and Taijiro MATSUSHIMA
Japan Bioassay Research Center, Japan Industrial Safety and Health Association,
2445 Hirasawa, Hadano, Kanagawa 257-0015, Japan
(Received April 18, 2006; Accepted June 5, 2006)
ABSTRACT — Carcinogenicity and chronic toxicity of ortho-chloronitrobenzene (o-CNB) were exam-ined by feeding groups of 50 F344 rats and 50 BDF1 mice of both sexes o-CNB-containing diets for 2years. The dietary concentration of o-CNB was 0, 80, 400 or 2000 ppm (w/w) for rats and 0, 100, 500 or2500 ppm for mice. The 2-year administration of o-CNB produced a dose-dependent increase in inci-dences of hepatocellular adenomas and carcinomas in rats and mice of both sexes and hepatoblastomas inmice of both sexes. Incidences of altered cell foci in the liver were increased in the o-CNB-fed rats ofboth sexes. Metastasis from mouse malignant liver tumors occurred predominantly in the lung. The hepa-tocarcinogenic response to o-CNB was found to be more potent in mice than in rats. Marginally increasedincidences of renal cell adenomas in the 2000 ppm-fed female rats and renal cell carcinomas in the 2000ppm-fed male rats were noted, together with a significantly increased incidence of atypical tubule hyper-plasias. Spontaneous, age-related chronic progressive nephropathy was exacerbated in a dose-related man-ner, and caused the death of 47 male rats fed 2000 ppm before the end of the 2-year administration period.The highest dose levels of o-CNB except for the administration of 2000 ppm to male rats were thoughtto meet the criteria of the maximum tolerated dose set by both NCI and IARC guidelines. Causative fac-tors of o-CNB-induced carcinogenicity were discussed with reference to our previous rodent studies ofsubchronic toxicity of o-CNB and carcinogenicity and chronic toxicity of para-chloronitrobenzene.
KEY WORDS: ortho-Chloronitrobenzene, Tumor, Chronic toxicity, Mouse, Rat, Liver
INTRODUCTION
Ortho-chloronitrobenzene (o-CNB, CAS Regis-try No.: 88-73-3) has been used primarily as an inter-mediate in the production of drugs and dyes, and aslumber preservatives, fungicides and photographicchemicals (IARC, 1996; Chemical Daily, 2006). Theestimated annual production of o-CNB and para-chlo-ronitrobenzene (p-CNB) in 1985 was 60,000 tons inGermany, 40,000 tons in the U.S.A. and 30,000 tons inJapan (Booth, 2003). The annual production of o-CNBin Japan was 7,500 tons in 2004 (Chemical Daily,2006). Three thousand workers were occupationallyexposed to o-CNB in the U.S.A. between 1981 and1983 according to the National Occupational ExposureSurvey conducted by the National Institute for Occupa-
tional Safety and Health (NIOSH, 1983). The WorkingGroup of the International Agency for Research onCancer (IARC) reported that no data of human carcino-genicity for o-CNB were available (IARC, 1996). Abrief report is available on the induction of multipletumors in male rats fed o-CNB for 12 months and livertumors in male and female mice fed o-CNB for 10months (Weisburger et al., 1978). IARC has evaluatedo-CNB as being unclassifiable as to carcinogenicity inhumans (Group 3), because there is inadequate evi-dence in experimental animals for the carcinogenicityof o-CNB (IARC, 1996). o-CNB was reported to be apotent hematotoxicant similar to p-CNB (Nair et al.,1986; Travlos et al., 1996). It was found, however, inour previous study (Matsumoto et al., 2006a) that o-CNB exhibited less severe hemato- and spleno-toxicity
Correspondence: Michiharu MATSUMOTO (E-mail: [email protected])
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than did p-CNB, and that o-CNB was classified as apotent hepatotoxicant rather than as a hematotoxicant.We also reported that 2-year dietary administration ofp-CNB induced splenic and adreno-medullary tumorsin rats and vascular tumor in mice, along with chronichematotoxicity (Matsumoto et al., 2006b). o-CNB isthought to be genotoxic, since several in vitro studiesshowed positive bacterial mutagenicity of o-CNB withS9 activation (JETOC, 1997) and positive mammalianclastogenicity of o-CNB with cultured Chinese ham-ster lung cells (CHL/IU) without S9 activation(JETOC, 2005).
The present study was designed to provide dose-response data on rodent carcinogenicity and chronictoxicity of o-CNB for the health risk assessment of o-CNB-exposed workers. The carcinogenicity andchronic toxicity were examined by feeding F344 ratsand BDF1 mice of both sexes o-CNB-containing dietsat 3 different dose levels or a vehicle diet as a controlfor 2 years. The causative factors of o-CNB-inducedcarcinogenicity and chronic toxicity were discussedwith reference to the subchronic hepatotoxicity of o-CNB (Matsumoto et al., 2006a) and the carcinogenic-ity and chronic toxicity of p-CNB (Matsumoto et al.,2006b) reported in our previous rodent studies.
MATERIALS AND METHODS
The present study was conducted in accordancewith the Organisation for Economic Co-operation andDevelopment (OECD) Good Laboratory Practice(OECD, 1998) and with reference to the OECD Guide-line for Testing of Chemicals 451 “CarcinogenicityStudies” (OECD, 1981). The animals were cared for inaccordance with the Guide for the Care and Use ofLaboratory Animals (Institute of Laboratory AnimalResources, 1996). The present study was approved bythe ethics committee of the Japan Bioassay ResearchCenter (JBRC).
Test substanceo-CNB of guaranteed grade (>99% pure) was
obtained from Wako Pure Chemical Industries, Ltd(Osaka, Japan). The o-CNB was analyzed for purityand stability by both gas chromatography and infraredspectrometry before and after its use. These analysesindicated that neither impurities nor degradation prod-ucts were detected in the test substance.
Animals and husbandryFour-week-old F344/DuCrj rats (SPF) and
Crj:BDF1 mice (SPF) of both sexes were obtainedfrom Charles River Japan, Inc. (Kanagawa, Japan).After a 2-week period of quarantine and acclimation,the animals were allocated by a stratified randomiza-tion procedure into 4 body weight-matched groups,each comprising 50 rats and 50 mice of either sex. Theanimals were housed individually in stainless-steelwire hanging cages (170 mm [W] × 294 mm [D] × 176mm [H] for each rat and 112 mm [W] × 212 mm [D] ×120 mm [H] for each mouse) under controlled environ-mental conditions (a temperature of 22.8 ± 0.3°C and arelative humidity of 53 ± 2% with 15 to 17 air changes/hr) in barrier system animal rooms. Fluorescent light-ing was controlled automatically to give a 12-hr light/dark cycle. All animals had free access to filtered, UV-irradiation-sterilized drinking water supplied by anautomatic watering system.
Diet preparation and feedingA diet containing 80, 400 or 2000 ppm o-CNB
(w/w) for the rats and 100, 500 or 2500 ppm for themice was prepared once every 2 weeks by mixingfinely ground o-CNB with γ-irradiation-sterilizedCRF-1 powdered diet (Oriental Yeast Co., Tokyo,Japan) in a spiral mixer for 20 min, and stored at 7°Cuntil use. The highest dose levels of 2000 ppm for therats and 2500 ppm for the mice were chosen so as notto exceed the maximum tolerated dose (MTD) (Sontaget al., 1976; Bannasch et al., 1986), based on bothgrowth rate and toxicity in our previous study of 13-week toxicity (Matsumoto et al., 2006a). A feederfilled with the o-CNB-containing or vehicle diet in theindividual cages was changed once a week. The o-CNB concentrations in the powdered diet, as deter-mined by high performance liquid chromatography,were found to be 98.3 ± 2.4% of the target concentra-tion for the 80 ppm diet, 99.0 ± 2.6% for the 400 ppmdiet and 99.7 ± 4.4% for the 2000 ppm diet used for ratfeeding, and 99.8 ± 3.1% for the 100 ppm diet, 98.5 ±2.7% for the 500 ppm diet and 98.6 ± 3.8% for the2500 ppm diet used for mouse feeding at the time ofpreparation. As reported in our previous study(Matsumoto et al., 2006a), a temporal decrease in thedietary concentrations of o-CNB was 88.9% for the 50ppm diet and 80.4% for the 5000 ppm diet at roomtemperature on the 8th day after preparation, when theinitial concentrations at the time of preparation weretaken as 100%. The decreased concentrations wereattributed to the sublimation of o-CNB during the feed-ing. Groups of 50 rats and 50 mice of both sexes werefed the o-CNB-containing diets or a vehicle diet as a
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control throughout a 2-year administration period,starting at the age of 6 weeks.
Clinical observations, hematology, blood biochem-istry and pathological examinations
The animals were observed daily for clinicalsigns and mortality. Body weight and food consump-tion were measured once a week for the first 14 weeksof the 2-year administration period and every 4 weeksthereafter. Daily food consumption was calculated bysubtracting the weight of the remaining diet from thatof the initial diet, and dividing by the number of feed-ing days. The amount of o-CNB intake was calculatedfrom the daily amount of diet consumed, multiplied bythe time-averaged, observed concentration of o-CNBin the diet, and divided by the body weight. Animalsfound dead, moribund state or surviving to the end ofthe 2-year administration period received completenecropsy.
For hematology and blood biochemistry, the sur-viving animals were bled under ether anesthesia, afterthey were fasted overnight, for the terminal necropsy.Hematological parameters were measured with Auto-matic Blood Cell Analyzers (ADVIA120, BayerHealthCare, NY, USA and MICROX HEG-120N,Omron Co, Kyoto, Japan), and methemoglobin levelswere measured with a CO-Oxymeter (CHBA CORN-ING 270, Bayer HealthCare, NY, USA). Blood bio-chemical parameters were measured with an Auto-matic Analyzer (Hitachi 7080, Hitachi Ltd., Tokyo,Japan).
The organs were removed, weighed and exam-ined for macroscopic lesions at necropsy. The tissuesspecified in the OECD test guideline (OECD, 1981)were examined for histopathology. These tissues werefixed in 10% neutral buffered formalin, and embeddedin paraffin by routine procedures. Thin tissue sectionsof 5 μm thick were prepared and stained with hematox-ylin and eosin (H & E). The deposits of brown pigmentin the liver and renal proximal tubules of rats wereadditionally stained with Berlin blue.
Statistical analysisA positive trend in the dose-response relationship
for neoplastic incidence was analyzed by Peto’s test(Peto et al., 1980). The incidence of neoplastic andnon-neoplastic lesions were statistically analyzed byFisher’s exact test. However, the data from the 2000ppm-fed male rats were not included for either analysisby Peto’s test or Fisher’s exact test, since the adminis-tration of 2000 ppm to the male rats exceeded the
MTD. Body weight, food consumption, relative organweights, and hematological and blood biochemicalparameters were analyzed by Dunnett’s test (Yamazakiet al., 1981; Hamada et al., 1998). Survival curveswere plotted according to the Kaplan-Meier method,(Kaplan and Meier, 1958), and the log-rank test (Petoet al., 1977) was used to test a statistically significantdifference between any o-CNB-fed rat or mouse groupof either sex and the respective control. The death ratesdue to liver tumors along the time course of o-CNBadministration in the mice were also plotted.
When a rarely occurring tumor was observed,and when the incidence of the rare tumor in each of theo-CNB-fed groups was increased but was not statisti-cally significant compared with the concurrent,matched-control group by Fisher’s exact test, the bor-derline increase in the tumor incidence was tested for abiologically meaningful difference by comparing theobserved tumor incidence with the maximum tumorincidence of the JBRC historical control data. The useof historical control data for rarely occurring tumorswas similar to that described by Haseman et al. (1984and 1995). The historical control data were compiledfrom eight and seven 2-year bioassay feed studies of ratand mouse carcinogenicity conducted in JBRC,respectively, and were composed of 400 male and 400female F344/DuCrj (SPF) rats and 348 male and 349female Crj:BDF1 (SPF) mice. All the animals used, thediet and the experimental conditions were similarthroughout all of these studies. The nomenclature con-ventions, diagnostic criteria for neoplastic lesions,diagnostic processes by pathologists and finalization ofthe diagnosis were essentially identical.
RESULTS
Rat study1. Survival, body weight and food consumption
The survival rate of 2000 ppm-fed males was sig-nificantly lowered after the 76th week, and all of themdied before the end of the 2-year administration period(Fig. 1A). However, no significant difference in thesurvival rate was found between any of the other o-CNB-fed male groups and the control. The terminalsurvival rate was 80% for the male control, 80% for the80 ppm-fed males, 78% for the 400 ppm-fed males and0% for the 2000 ppm-fed males. The markedlydecreased survival rate in the 2000 ppm-fed malegroup was attributed to the increased number of non-neoplastic deaths, because 47 out of fifty 2000 ppm-fed males died of chronic progressive nephropathy
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Fig. 1. Survival curves of rats (A and B) and mice (C and D) and death curves due to liver tumors of mice (E and F)fed o-CNB-containing diets or a vehicle diet as a control for 2 years.
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(CPN) before the end of the 2-year administrationperiod. On the other hand, there was no significant dif-ference in the survival rate between any of the o-CNB-fed female groups and the female control (Fig. 1B).The terminal survival rate was 82% for the female con-trol, 84% for the 80 ppm-fed females, 90% for the 400ppm-fed females and 78% for the 2000 ppm-fedfemales.
The body weights of 2000 ppm-fed males weremarkedly suppressed after the 20th week (Fig. 2A).The markedly decreased growth rate was due to thedevelopment of severe CPN. The body weights of 2000ppm-fed females were decreased by 10% at the 78thweek and by 18% at the end of the 2-year administra-tion period, compared with the respective female con-trols (Fig 2A, Table 1). The 400 ppm-fed males exhib-
Fig. 2. Growth curves of rats (A) and mice (B) fed o-CNB-containing diets or a vehicle diet as a con-trol for 2 years.
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ited significantly decreased terminal body weight by10% compared with the control (Table 1), while the400 ppm-fed females did not. There was no differencein the terminal body weight between any of the 80ppm-fed male and female groups and the respectivecontrol. There was no significant difference in foodconsumption between any of the o-CNB-fed groups ofeither sex and the respective control except for the2000 ppm-fed male group (data not shown). The esti-mated amounts of o-CNB intake were found to
increase proportionally with an increase in the dietaryconcentration of o-CNB (Table 1).
2. Organ weights, hematology and blood biochemis-try
Relative liver weight was significantly increasedin the males fed 80 and 400 ppm, and in the femalesfed 400 and 2000 ppm. Relative spleen weight was sig-nificantly increased in the females fed 2000 ppm. Rel-ative kidney weight was significantly increased in the
Table 1. Chemical intake, terminal body weights and relative organ weights of rats and mice of both sexes fedo-CNB-containing or control diet for 2 years.
< Rats >2000 ppm400 ppm80 ppmControlGroup
Male991940Chemical intake (mg/kg per day)0394040Number of animals examined−355 ± 27**394 ± 26395 ± 47Terminal body weight (g)− 4.424 ± 0.471** 3.184 ± 0.408* 2.990 ± 0.672Liver (%)− 0.306 ± 0.085 0.393 ± 0.605 0.551 ± 0.684Spleen (%)− 0.961 ± 0.180** 0.769 ± 0.102 0.782 ± 0.273Kidneys (%)
Female1172240Chemical intake (mg/kg per day)39454241Number of animals examined
216 ± 21**256 ± 29266 ± 32265 ± 30 Terminal body weight (g) 7.314 ± 0.845** 3.571 ± 0.376** 2.972 ± 0.664 2.641 ± 0.416Liver (%) 0.522 ± 0.159** 0.255 ± 0.079 0.445 ± 0.925 0.324 ± 0.350Spleen (%) 1.257 ± 0.187** 0.785 ± 0.088** 0.717 ± 0.116 0.705 ± 0.071Kidneys (%)
< Mice >2500 ppm500 ppm100 ppmControlGroup
Male32954110Chemical intake (mg/kg per day)8173535Number of animals examined
30.6 ± 2.2** 40.0 ± 6.0** 49.2 ± 9.9 51.0 ± 8.4Terminal body weight (g) 28.286 ± 4.120** 12.890 ± 6.586** 5.101 ± 2.348 4.682 ± 3.366Liver (%) 0.393 ± 0.158* 0.737 ± 0.888** 0.308 ± 0.299 0.247 ± 0.205Spleen (%) 1.936 ± 0.160** 1.650 ± 0.180** 1.510 ± 0.841 1.281 ± 0.222Kidneys (%)
Female39669140Chemical intake (mg/kg per day)5263429Number of animals examined
26.5 ± 1.9** 33.0 ± 3.5** 37.9 ± 4.4 37.4 ± 5.8Terminal body weight (g) 33.269 ± 3.223** 12.174 ± 7.567** 4.614 ± 2.557 4.147 ± 1.235Liver (%) 0.550 ± 0.226 0.874 ± 0.901* 0.616 ± 0.883 0.477 ± 0.386Spleen (%) 1.721 ± 0.142** 1.569 ± 0.533** 1.221 ± 0.291 1.220 ± 0.279Kidneys (%)
Values are expressed as mean ± S.D.*and **: Significantly different from control at p<0.05 and p<0.01 by Dunnett’s test, respectively.− : The data could not be obtained because all of the animals died before the end of the 2-year administration period.
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males fed 400 ppm and in the females fed 400 and2000 ppm (Table 1).
A significant decrease in red blood cell count(RBC) along with a concurrent increase in reticulocyte
count occurred only in the 2000 ppm-fed females(Table 2). Hemoglobin concentration (Hb), hematocritvalue (Ht) and mean corpuscular hemoglobin (MCH)were significantly decreased in the males fed 400 ppm
Table 2. Hematological analysis of rats and mice of both sexes fed o-CNB-containing or control diet for 2 years.< Rats >
2000 ppm400 ppm80 ppmControlGroupMale
0394039Number of animals examined− 7.42 ± 1.18 8.30 ± 1.24* 7.44 ± 1.94RBC (106/μL)− 12.3 ± 1.8** 14.0 ± 2.1 12.9 ± 3.5Hb (g/dL)− 35.5 ± 4.8** 40.4 ± 4.8 37.1 ± 8.6Ht (%)− 48.1 ± 2.5** 49.2 ± 5.5* 51.8 ± 10.0MCV (fL)− 16.6 ± 1.0** 16.9 ± 1.4* 17.6 ± 2.8MCH (pg)− 949 ± 154** 833 ± 277 786 ± 274Platelet (103/μL)− 3.4 ± 1.6 3.6 ± 2.8 5.7 ± 6.8Reticulocyte (%)− 0.4 ± 0.2** 0.3 ± 0.1 0.3 ± 0.1Methemoglobin (%)
Female38454241Number of animals examined
6.71 ± 0.57** 7.80 ± 0.55 7.61 ± 1.48 7.82 ± 0.86RBC (106/μL) 12.2 ± 0.9** 14.1 ± 1.2** 14.2 ± 2.8 14.7 ± 1.6Hb (g/dL) 35.4 ± 2.4** 39.8 ± 2.9 39.7 ± 6.1 40.7 ± 3.6Ht (%) 52.9 ± 2.3 51.0 ± 1.7** 53.8 ± 9.4 52.3 ± 2.9MCV (fL) 18.2 ± 0.7** 18.1 ± 1.0** 18.8 ± 1.3 18.9 ± 1.0MCH (pg) 730 ± 115** 725 ± 123** 644 ± 154 612 ± 142Platelet (103/μL) 5.7 ± 1.4** 3.1 ± 1.4 4.6 ± 8.1 3.3 ± 2.9Reticulocyte (%) 1.3 ± 0.4** 0.4 ± 0.2** 0.3 ± 0.1 0.3 ± 0.1Methemoglobin (%)
< Mice >2500 ppm500 ppm100 ppmControlGroup
Male8143333Number of animals examined
8.92 ± 2.02 8.34 ± 2.75 9.56 ± 1.30 9.62 ± 1.13RBC (106/μL) 13.0 ± 2.7 11.6 ± 3.5 13.5 ± 1.7 13.8 ± 1.4Hb (g/dL) 41.7 ± 6.8 37.9 ± 9.8 43.1 ± 5.0 43.7 ± 4.5Ht (%) 47.6 ± 4.2 47.5 ± 7.4 45.3 ± 2.4 45.5 ± 2.2MCV (fL) 14.6 ± 0.6 14.2 ± 1.2 14.2 ± 0.7 14.4 ± 0.6MCH (pg)1658 ± 7111549 ± 5401723 ± 2951751 ± 430Platelet (103/μL) 8.0 ± 5.4** 9.4 ± 12.7** 2.8 ± 2.3 2.4 ± 1.6 Reticulocyte (%)
Female4253429Number of animals examined
10.00 ± 1.00 9.30 ± 1.57 9.32 ± 1.18 9.24 ± 1.29RBC (106/μL) 15.3 ± 1.2 13.4 ± 2.1 13.7 ± 1.7 13.6 ± 1.7Hb (g/dL) 47.8 ± 4.9 43.2 ± 6.5 43.2 ± 4.7 42.9 ± 4.3Ht (%) 47.9 ± 2.1 46.8 ± 2.7 46.5 ± 2.4 46.9 ± 3.8MCV (fL) 15.3 ± 0.5 14.4 ± 0.6* 14.7 ± 0.5 14.8 ± 0.6MCH (pg) 923 ± 5221168 ± 3541058 ± 3011097 ± 293Platelet (103/μL) 5.3 ± 1.0* 5.2 ± 3.9** 3.1 ± 2.7 4.7 ± 8.0Reticulocyte (%)
Values are expressed as mean ± S.D.* and **: Significantly different from control at p<0.05 and p<0.01 by Dunnett’s test, respectively.RBC: Red blood cell counts, Hb: Hemoglobin concentration, Ht: Hematcrit value,MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin.− : The data could not be obtained because all of the animals died before the end of the 2-year administration period.
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and in the females fed 400 and 2000 ppm. The methe-moglobin level was significantly increased in the malesfed 400 ppm and in the females fed 400 and 2000 ppm(Table 2). Total bilirubin and ALT were increased inthe 2000 ppm-fed females. γ-GTP was increased in themales fed 80 and 400 ppm and in the females fed 400and 2000 ppm. LDH was significantly decreased in themales fed 400 ppm and in the females fed 400 and2000 ppm. Creatinine was increased in the males fed400 ppm and in the females fed 2000 ppm. Urea nitro-gen was increased in the males fed 400 ppm and in thefemales fed 400 and 2000 ppm (Table 3).
3. HistopathologyThe incidences of hepatocellular adenomas and
carcinomas in both the males and females wereincreased in a dose-related manner, as indicated by asignificant positive trend by Peto’s test (Table 4). A sta-tistically increased incidence of hepatocellular ade-nomas was noted only in the females fed 2000 ppm.The incidence of hepatocellular adenomas in the 400ppm-fed males (14%) exceeded the maximum inci-dence of the JBRC historical control data for malehepatocellular adenomas (7 cases in 400 male rats witha maximum incidence of 6% in a single study). Theincidences of hepatocellular carcinomas in the 400ppm-fed males and 2000 ppm-fed females (6% and8%, respectively) exceeded the respective maximumincidence of the JBRC historical control data for hepa-tocellular carcinomas (null cases in 400 male andfemale rats). The hepatocellular adenomas were com-posed of a trabecular structure of hepatocytes, whichwere two or three layers thick. The hepatocellular car-cinomas were characterized by neoplastic hepatocytesforming several layers of irregular trabeculae havingpseudo-glandular or acinar structures (Photo 1). One ofthe four hepatocellular carcinomas in the 2000 ppm-fed female rats metastasized to the lung. The incidenceof acidophilic cell foci was significantly increased inthe males fed 400 ppm and in the females fed 400 and2000 ppm. The incidences of basophilic cell foci andspongiosis hepatis were significantly increased in the400 ppm-fed males, while the incidence of clear cellfoci was significantly increased in the females fed2000 ppm. Altered cell foci including acidophilic cellfoci, basophilic cell foci and clear cell foci were prolif-erative in nature, and classified as preneoplastic lesiondeveloping to liver tumor. Single cell necrosis and cen-trilobular hydropic degeneration were observed in boththe males and females fed 2000 ppm, whereas fatty
change occurred in the males fed 2000 ppm. Deposit ofbrown pigment occurred only in the liver of both malesand females fed 2000 ppm. The brown pigment wasobserved as dark brown droplets in the H&E stainingspecimen, and was stained negatively with Berlin blue.
The incidences of renal cell adenomas (4%) inthe 2000 ppm-fed females and renal cell carcinomas(8%) in the 2000 ppm-fed males exceeded the maxi-mum tumor incidence of the JBRC historical controldata for female renal cell adenomas (one case in 400female rats with a maximum incidence of 2% in a sin-gle study) and for male renal cell carcinomas (nullcases in 400 male rats), respectively (Table 5). Therenal cell adenomas were composed of nodular epithe-lial cells proliferating beyond the confines of the origi-nal tubule structure, and the sizes of the two benignrenal cell tumors were 0.7 and 1.5 mm at the widestdiameter. In contrast, the renal cell carcinomas werecomposed of pleomorphic epithelial cells, and the sizesof the 4 malignant tumors ranged from 3.0 to 7.0 mm.The increased incidences of atypical tubule hyperpla-sias at the proximal tubule as a proliferative and pre-neoplastic lesion were noted in both the males andfemales fed 2000 ppm.
CPN was the most prevalent non-neoplasticlesion found in the present study, which caused deathsin 47 males out of the 2000 ppm-fed group and 3 malesout of the 400 ppm-fed group before the end of the 2-year administration period, and severely affected the43 surviving males out of the 400 ppm-fed group. Theincidences and severities of CPN were higher in malesthan in females. The severity of CPN was increaseddose-dependently in both the o-CNB-fed males andfemales (Table 5), and the severe CPN was character-ized by glomerular sclerosis or atrophy without thenormal parenchymal tissue of the kidneys. In addition,cortical mineralization and urothelial hyperplasiaoccurred predominantly in the o-CNB-fed males. Thedeposit of brown pigment in the proximal tubules wasobserved in the proximal tubules of both males andfemales fed 400 and 2000 ppm (Table 5). The brownpigment was observed as dark brown droplets in theH&E staining specimen, and was stained negativelywith Berlin blue.
In the spleen of the o-CNB-fed rats of both sexes,capsule hyperplasia, angiectasis, engorgement oferythrocytes, increased extramedullary hematopoiesisand hemosiderin deposition were noted as signs ofhematotoxicity (Table 5).
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Mouse Study1. Survival, body weight and food consumption
The survival rates of 2500 ppm- and 500 ppm-fedmales were significantly lowered at the 73rd and 92nd
weeks, respectively, and thereafter (Fig. 1C). The sur-vival rate of 100 ppm-fed males was not significantlylowered compared with that of the male control group.The terminal survival rate was 70% for the male con-
Table 3. Blood biochemical analysis of rats and mice of both sexes fed o-CNB-containing or control diet for 2 years.<Rats>
2000 ppm400 ppm80 ppmControlGroupMale
0394039Number of animals examined− 0.18 ± 0.15 0.19 ± 0.09 0.23 ± 0.28Total-bilirubin (mg/dL)− 128 ± 105121 ± 114135 ± 147AST (IU/L)− 69 ± 42 52 ± 46 51 ± 37ALT (IU/L)− 166 ± 90*176 ± 96254 ± 296LDH (IU/L)− 35 ± 23** 13 ± 6 ** 6 ± 3γ-GTP (IU/L)− 0.9 ± 0.3** 0.6 ± 0.1 0.6 ± 0.1Creatinine (mg/dL)− 43.9 ± 31.7** 19.4 ± 3.2 18.0 ± 4.1Urea nitrogen (mg/dL)
Female38454241Number of animals examined
0.21 ± 0.03** 0.14 ± 0.02 0.29 ± 0.96 0.15 ± 0.05Total-bilirubin (mg/dL) 214 ± 261 126 ± 61162 ± 209169 ± 177AST (IU/L) 134 ± 117** 67 ± 28 73 ± 89 67 ± 70ALT (IU/L) 189 ± 73** 200 ± 70**285 ± 229268 ± 93LDH (IU/L) 77 ± 25** 6 ± 3** 3 ± 3 2 ± 2γ-GTP (IU/L) 0.6 ± 0.2* 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1Creatinine (mg/dL) 28.8 ± 12.7** 18.0 ± 4.2* 17.7 ± 4.8 16.1 ± 2.7Urea nitrogen (mg/dL)
<Mice>2500 ppm500 ppm100 ppmControlGroup
Male8143434Number of animals examined
0.38 ± 0.20** 0.29 ± 0.26* 0.14 ± 0.03 0.15 ± 0.07Total-bilirubin (mg/dL) 3136 ± 3412** 549 ± 590**156 ± 209306 ± 787AST (IU/L) 2400 ± 2502** 610 ± 759**120 ± 173234 ± 579ALT (IU/L)10515 ± 10479**7530 ± 10481**495 ± 693929 ± 2145LDH (IU/L) 74 ± 29** 3 ± 2* 1 ± 1 2 ± 1γ-GTP (IU/L) 21.0 ± 4.3 27.5 ± 22.7 21.3 ± 3.8 20.6 ± 3.0Urea nitrogen (mg/dL)
Female4263429Number of animals examined
0.58 ± 0.12** 0.24 ± 0.16** 0.16 ± 0.07 0.14 ± 0.03Total-bilirubin (mg/dL) 1432 ± 796** 449 ± 824**105 ± 122 94 ± 45AST (IU/L) 2115 ± 779** 480 ± 816** 51 ± 62 36 ± 27ALT (IU/L) 6228 ± 2802**2078 ± 4212**393 ± 528409 ± 395LDH (IU/L) 250 ± 30** 5 ± 8** 1 ± 1 1 ± 1γ-GTP (IU/L) 35.5 ± 15.1* 21.3 ± 9.8 15.2 ± 3.0 17.5 ± 5.2Urea nitrogen (mg/dL)
Values are expressed as mean ± S.D.* and **: Significantly different from control at p<0.05 and p<0.01 by Dunnett’s test, respectively.AST : Aspartate aminotransferase, ALT: Alanine aminotransferase, LDH: Lactate dehydrogenase,γ-GTP: γ-Glutamyl transpeptidase.− : The data could not be obtained because all of the animals died before the end of the 2-year administration period.
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M. MATSUMOTO et al.
Vol. 31 No. 3
Tabl
e 4.
N
umbe
r of
ani
mal
s be
arin
g he
patic
les
ions
in
rats
and
mic
e fe
d o-
CN
B-c
onta
inin
g or
con
trol
die
t fo
r 2
year
s. Fem
ale
Mal
e<
Rat
s >
Peto
tes
t20
00 p
pm40
0 pp
m80
ppm
Con
trol
Peto
tes
t20
00 p
pm a)
400
ppm
80 p
pmC
ontr
olG
roup
5050
5050
5050
5050
Num
ber
of a
nim
als
exam
ined
Neo
plas
tic l
esio
n↑↑
20**
2 0
0↑
1 7
3 2
Hep
atoc
ellu
lar
aden
oma
↑↑ 4
0 0
0↑↑
1 3
0 0
Hep
atoc
ellu
lar
carc
inom
aN
on-n
eopl
astic
les
ion
11**
2 1
2 4
6 7
9C
lear
cel
l fo
ci36
** 8
** 0
0 7
24**
3 2
Aci
doph
ilic
cell
foci
5**
9**
2229
120
** 7
6B
asop
hilic
cel
l fo
ci 1
0 0
0 0
35**
8 4
Spon
gios
is h
epat
is 6
* 1
0 0
18 0
0 0
Nec
rosi
s :
sing
le c
ell
0 0
0 0
16 0
1 0
Fatty
cha
nge
41**
0 0
048
0 0
0H
ydro
pic
dege
nera
tion
: ce
ntri
lobu
lar
44**
0 0
050
0 0
0D
epos
it of
bro
wn
pigm
ent
Fem
ale
Mal
e<
Mic
e >
Peto
tes
t25
00 p
pm50
0 pp
m10
0 pp
mC
ontr
olPe
to t
est
2500
ppm
500
ppm
100
ppm
Con
trol
Gro
up50
5050
5050
5050
50N
umbe
r of
ani
mal
s ex
amin
edN
eopl
astic
les
ion
↑↑38
**48
**22
** 8
↑↑34
**30
*29
*19
Hep
atoc
ellu
lar
aden
oma
↑↑48
**14
** 3
0↑↑
35**
2014
15H
epat
ocel
lula
r ca
rcin
oma
↑↑28
** 9
** 0
0↑↑
44**
35**
6 1
Hep
atob
last
oma
33 2
1 0
2011
1 7
Met
asta
sis
from
mal
igna
nt h
epat
ic t
umor
s b)
30 2
1 0
20 8
1 6
Lun
g 2
0 0
0 0
0 0
1B
one
mar
row
0 0
0 0
2 2
0 0
Peri
tone
um 2
0 0
0 0
0 0
0Pa
ncre
as 3
0 0
0 0
2 0
4O
ther
org
ans
Non
-neo
plas
tic l
esio
n37
**29
** 0
042
**42
**32
** 0
Hep
atoc
ellu
lar
hype
rtro
phy
: ce
ntri
lobu
lar
0 0
0 0
6*
18**
0 0
Nuc
lear
enl
arge
men
t :
cent
rilo
bula
r*
and
**:
Sign
ific
antly
dif
fere
nt f
rom
con
trol
at
p<0.
05 a
nd p
<0.
01 b
y Fi
sher
’s e
xact
tes
t, re
spec
tivel
y.
↑ an
d ↑↑
: Si
gnif
ican
tly d
iffe
rent
at
p<0.
05 a
nd p
<0.
01 b
y Pe
to’s
tes
t, re
spec
tivel
y.a)
: T
he d
ata
from
the
200
0 pp
m-f
ed m
ale
rats
wer
e no
t in
clud
ed f
or e
ither
ana
lysi
s by
Pet
o’s
test
or
Fish
er’s
exa
ct t
est,
sinc
e th
e ad
min
istr
atio
n of
200
0 pp
m t
o m
ale
rats
exc
eede
d th
e M
TD
.b)
: St
atis
tical
ana
lysi
s w
as n
ot a
pplie
d to
the
met
asta
sis.
Und
erlin
ed v
alue
s in
dica
te t
hat
the
tum
or i
ncid
ence
exc
eede
d th
e m
axim
um t
umor
inc
iden
ce o
f th
e JB
RC
his
tori
cal
cont
rol
data
.
Carcinogenicity and Toxicity of o-CNB.
257
Vol. 31 No. 3
Tabl
e 5.
N
umbe
r of
ani
mal
s be
arin
g re
nal
and
sple
nic
lesi
ons
in r
ats
and
mic
e fe
d o-
CN
B-c
onta
inin
g or
con
trol
die
t fo
r 2
year
s.Fe
mal
eM
ale
< R
ats
>20
00 p
pm40
0 pp
m80
ppm
Con
trol
2000
ppm
a)40
0 pp
m80
ppm
Con
trol
Gro
up50
5050
5050
5050
50N
umbe
r of
ani
mal
s ex
amin
edK
idne
yN
eopl
astic
les
ion
2 0
0 0
1 0
1 0
Ren
al c
ell
aden
oma
0 0
0 0
4 0
0 0
Ren
al c
ell
carc
inom
aN
on-n
eopl
astic
les
ion
5*
0 0
0 6
1 1
0A
typi
cal
tubu
le h
yper
plas
ia :
pro
xim
al t
ubul
e49
**45
**33
**20
5049
*48
43C
hron
ic p
rogr
essi
ve n
ephr
opat
hy(2
.7)
(1.4
)(1
.3)
(1.3
)(4
.0)
(3.2
)(2
.0)
(1.6
)(A
vera
ged
grad
eb) )
0 0
0 0
44 2
0 0
Min
eral
izat
ion
: co
rtex
7**
0 0
048
32**
1 0
Uro
thel
ial
hype
rpla
sia
: pe
lvis
49**
48**
0 0
4942
** 0
0D
epos
it of
bro
wn
pigm
ent
: pr
oxim
al t
ubul
eSp
leen
Non
-neo
plas
tic l
esio
n46
** 0
0 0
49 0
0 0
Cap
sule
hyp
erpl
asia
5*
0 0
016
0 0
0A
ngie
ctas
is27
** 5
* 1
0 3
11**
3 0
Eng
orge
men
t of
ery
thro
cyte
s28
**16
915
2 2
8*
2In
crea
sed
extr
amed
ulla
ry h
emat
opoi
esis
1223
**14
928
7*
2 1
Dep
osit
of h
emos
ider
in (
Mor
e th
an m
oder
ate)
Fem
ale
Mal
e<
Mic
e >
2500
ppm
500
ppm
100
ppm
Con
trol
2500
ppm
500
ppm
100
ppm
Con
trol
Gro
up50
5050
5050
5050
50N
umbe
r of
ani
mal
s ex
amin
edK
idne
yN
on-n
eopl
astic
les
ion
17**
4 0
032
**26
** 3
1D
epos
it of
hem
osid
erin
Sple
enN
on-n
eopl
astic
les
ion
43**
34*
1323
39**
37**
1418
Incr
ease
d ex
tram
edul
lary
hem
atop
oies
is45
**27
*23
1740
**21
**20
* 9
Dep
osit
of h
emos
ider
in
* an
d **
: Si
gnif
ican
tly d
iffe
rent
fro
m c
ontr
ol a
t p<
0.05
and
p<
0.01
by
Fish
er’s
exa
ct t
est,
resp
ectiv
ely.
a):
The
dat
a fr
om t
he 2
000
ppm
-fed
mal
e ra
ts w
ere
not
incl
uded
for
eith
er a
naly
sis
by P
eto’
s te
st o
r Fi
sher
’s e
xact
tes
t, si
nce
the
adm
inis
trat
ion
of 2
000
ppm
to
mal
e ra
ts e
xcee
ded
the
MT
D.
b):
The
val
ue i
n pa
rent
hesi
s in
dica
tes
the
aver
age
of s
ever
ity g
rade
ind
ex o
f th
e le
sion
in
affe
cted
ani
mal
s. T
he a
vera
ge o
f se
veri
ty g
rade
ind
ex w
as c
alcu
late
d w
ith a
fo
llow
ing
equa
tion.
Ave
rage
d gr
ade=
Σ (g
rade
× n
umbe
r of
ani
mal
s w
ith g
rade
)/nu
mbe
r of
ani
mal
s w
ith t
he l
esio
n.
Gra
de:
1: s
light
, 2:
mod
erat
e, 3
: m
arke
d, 4
: se
vere
.U
nder
lined
val
ues
indi
cate
tha
t th
e tu
mor
inc
iden
ce e
xcee
ded
the
max
imum
tum
or i
ncid
ence
of
the
JBR
C h
isto
rica
l co
ntro
l da
ta.
258
M. MATSUMOTO et al.
Vol. 31 No. 3
trol, 70% for the 100 ppm-fed males, 34% for the 500ppm-fed males and 16% for the 2500 ppm-fed males.The markedly lowered survival rates of 2500 ppm- and500 ppm-fed males were causally related to theincreased death rate due to malignant liver tumorsbefore the end of the 2-year administration period (Fig.1E). The 2500 ppm-fed females exhibited a signifi-cantly lowered survival rate after the 71st week (Fig.1D), which was attributed to the increased death ratedue to malignant liver tumors before the end of the 2-year administration period (Fig. 1F). There was no sig-nificant difference in the survival rate between any ofthe 500 ppm- and 100 ppm-fed females and the femalecontrol. The terminal survival rate was 58% for thefemale control, 68% for the 100 ppm-fed females, 52%for the 500 ppm-fed females and 10% for the 2500ppm-fed females.
The body weights of 500 ppm- and 2500 ppm-fedmales were significantly decreased by 22% and 40%compared with the male control, respectively. The 500ppm- and 2500 ppm-fed females exhibited signifi-cantly decreased terminal body weights by 12% and29%, compared with the female control, respectively(Fig. 2B, Table 1). There was no significant differencein growth rate between any of the 100 ppm-fed groupsof both sexes and the respective control (Fig. 2B).There was no difference in food consumption betweenany of the o-CNB-fed groups of either sex and therespective control (data not shown). The estimatedamounts of o-CNB intake were found to increase pro-
portionally with an increase in the dietary concentra-tion of o-CNB (Table 1).
2. Organ weights, hematology and blood biochemis-try
The relative weights of the liver, spleen and kid-neys were significantly increased in both the males andfemales fed 500 and 2500 ppm except for the spleenweight in the 2500 ppm-fed females (Table 1).
There was no significant change in any erythro-cyte parameter except for the significantly increasedreticulocyte count, which occurred in both the malesand females fed 500 and 2500 ppm (Table 2). AST,ALT, LDH, γ-GTP and total bilirubin were signifi-cantly increased in both the males and females fed 500and 2500 ppm (Table 3). Urea nitrogen was increasedonly in the females fed 2500 ppm.
3. HistopathologyThe incidences of hepatocellular adenomas and
carcinomas, and hepatoblastomas were increased dose-dependently in both sexes, as indicated by a significantpositive trend by Peto’s test (Table 4). The incidencesof hepatocellular adenomas was significantly increasedin all the o-CNB-fed male and female groups, whilethe incidences of hepatocellular carcinomas were sig-nificantly increased in the males fed 2500 ppm and inthe females fed 500 and 2500 ppm. The incidences ofhepatoblastomas were significantly increased in boththe males and females fed 500 and 2500 ppm (Photo
Photo 1. Hepatocellular carcinoma in the liver of a female rat fed 2000ppm o-CNB-containing diet for 2 years. The tumor is com-posed of irregular trabeculae having pseudo-glandular struc-ture. Bar indicates 200 μm. H & E stain.
Carcinogenicity and Toxicity of o-CNB.
259
Vol. 31 No. 3
2). In addition, the incidence of hepatoblastomas in the100 ppm-fed males (12%) exceeded the maximumincidence of the JBRC historical control data for hepa-toblastomas (3 cases in 348 male mice with a maxi-mum incidence of 2% in a single study). As shown inFigure 1E and F, the death rate due to liver tumorsoccurring before the end of the 2-year administrationperiod was increased in both dose- and time-dependentmanner. The o-CNB-induced hepatocarcinogenicitywas characterized by multiple tumor occurrences asevidenced by the simultaneous induction of hepato-blastomas and hepatocellular adenomas and/or hepato-cellular carcinomas in a single liver. There were 21males and 21 females simultaneously bearing the threetypes of liver tumor in the 2500 ppm-fed groups. All ofthese tumor-bearing animals except 5 males diedbefore the end of the 2-year administration, showingthe earliest death at the 68th week for the males and the63rd week for the females (Fig. 1 E and F). Forty-onepercent and 69% of the total malignant liver tumorsfound in the 2500 ppm-fed males and females, respec-tively, metastasized predominantly to the lung, fol-lowed by the bone marrow, peritoneum and pancreas.As non-neoplastic hepatic lesions, centrilobular hyper-trophy and nuclear enlargement of the hepatocyteswere observed in the o-CNB-fed mice of either sex.
The incidences of hemosiderin deposition in thekidney were significantly increased in the males fed500 and 2500 ppm and in the females fed 2500 ppm(Table 5). Significantly increased incidences of hemo-siderin deposition in the spleen were noted in all o-
CNB-fed male groups and in the females fed 500 and2500 ppm. The incidences of increased extramedullaryhematopoiesis in the spleen were significantlyincreased in both the males and females fed 500 and2500 ppm.
DISCUSSION
In 2-year bioassay studies of rodent carcinoge-nicity, it is of prime importance to select an appropriaterange of dose levels of the test substance, which per-mits a clear dose-carcinogenic response relationship inexposed animals. The highest dose level should be suf-ficiently high to statistically detect significant carcino-genic responses in a relatively small number of experi-mental animals, usually 50 rats or mice of either sexper group. In addition, the dose levels should be lowenough to allow the extrapolation of the observed car-cinogenic effects to effects that would occur at envi-ronmentally relevant levels below the experimentalrange of doses. According to the MTD criteria set byboth the National Cancer Institute (NCI) (Sontag et al.,1976) and the IARC guidelines (Bannasch et al.,1986), the highest dose should not induce toxic mani-festations that are predicted to reduce the lifespan ofthe animals except as the result of neoplastic develop-ment or a 10% or greater retardation of body weightgain, compared with the control. In the present study,the selection of the highest dose levels of 2000 ppm forrats and 2500 ppm for mice was predicted on the basisof both growth retardation and toxicity from our previ-
Photo 2. Hepatoblastoma in the liver of a male mouse fed 2500 ppmo-CNB-containing diet for 2 years. Bar indicates 100 μm.H & E stain.
260
M. MATSUMOTO et al.
Vol. 31 No. 3
ous study of 13-week toxicity (Matsumoto et al.,2006a). Since in the present study, the 2-year adminis-tration of 2000 ppm o-CNB to male rats caused 47non-neoplastic deaths due to CPN out of the 50 dosedmale rats, the highest dose level of 2000 ppm for malerats was thought to exceed the MTD according to thecriteria set by both NCI and IARC. The significantlylowered survival rates of the 2500 and 500 ppm-fedmale mice and the 2500 ppm-fed female mice wereattributed to the reduction in the lifespan of the mice asthe result of neoplastic development. Therefore, thedietary administration of o-CNB to all groups of ratsand mice except for the 2000 ppm-fed male rat groupwas thought to meet the MTD criteria with respect tothe survival rate. The highest dose levels of 2000 ppmfor female rats and 2500 ppm for both male and femalemice caused a greater than 10% decrement in the ter-minal body weights compared with the respective con-trols. The decreased body weight of 2000 ppm-fedfemale rat group was found to exceed the 10% decre-ment only after the final quarter of the 2-year adminis-tration period. The decreased terminal body weights by40% and 29% for the 2500 ppm-fed male and femalemouse groups, respectively, were thought to resultfrom the development of highly malignant and fatalliver tumors during the later administration period,since 47 out of the fifty 2500 ppm-fed male mice andall of the fifty 2500 ppm-fed female mice were foundto bear malignant liver tumors, and since 40% and 66%of the total male and female mice bearing the malig-nant tumors metastasized to other organs. Therefore, itcan be concluded that all of the highest dose levels ofo-CNB except for the administration of 2000 ppm tomale rats did not exceed the MTD. Haseman (1985)argued that the recommendation regarding the 10%decrement in body weight is empirical, however, and ifexceeded in practice does not necessarily invalidate the2-year carcinogenicity bioassay results. Even if the 2-year administration of 2000 ppm to male rats exceededthe MTD, a significantly positive dose-response rela-tionship for both hepatocellular adenomas and carcino-mas was found to hold for the groups of male rats fedo-CNB at dose levels below 2000 ppm, together withthe biologically meaningful increase in the incidencesof hepatocellular adenomas and carcinomas.
It was found evident in the present study that the2-year dietary administration of o-CNB produced adose-dependent increase in the incidences of hepato-cellular adenomas and carcinomas in rats and mice ofboth sexes and hepatoblastomas in mice of both sexes.The hepatocarcinogenic response to o-CNB was found
to be more potent in mice than in rats, since the inci-dences of malignant liver tumors were far higher inmice than in rats, and since more than half of the totalmalignant liver tumors metastasized to the lung, bonemarrow, peritoneum and pancreas in mice, while onlyone malignant liver tumor metastasized to lung in rats.The o-CNB-induced mouse hepatocarcinogenicity wascharacterized by the multiple occurrence of hepato-blastoma and hepatocellular adenoma and/or carci-noma in a single liver and the marked induction of ararely occurring hepatoblastoma, which was illustratedby 3 cases out of 348 male mice and null cases out of349 female mice in the JBRC historical control data.Mouse hepatoblastoma occurs in aged animals, asfound in the present study, unlike human hepatoblas-toma that primarily occurs in children under 3 years ofage (Turusov and Takayama, 1979; Frith et al., 1994).
It was found in this study that the incidences ofhepatoblastomas in the mouse groups fed all three doselevels of o-CNB and hepatocellular adenomas and car-cinomas in the rat groups fed the lower two dose levelswere higher in males than in females, indicating a cleargender difference in the o-CNB-induced hepatocarci-nogenicity. However, the high incidences of hepatocel-lular adenomas and carcinomas in the male and femalegroups of mice fed 2500 ppm hindered the gender dif-ference. Furthermore, the incidences of hepatocellularadenomas in the rats fed vehicle diet and all three spon-taneous liver tumors in the mice fed vehicle diet werealso found to be higher in males than in females in thisstudy, also indicating a gender difference in inducibil-ity of the spontaneous liver tumors. This result is con-sistent with both the NTP historical control data forspontaneous hepatic tumors in F344 rats (Haseman etal., 1990) and the JBRC historical control data forspontaneous hepatoblastomas in BDF1 mice. There-fore, it can be suggested that more sensitive hepatocar-cinogenic response to o-CNB in male rodents than infemale ones is causally related to higher inducibility ofthe spontaneously occurring hepatocellular tumors inmale rodents than in females.
No other bioassay study of rodent carcinogenic-ity for o-CNB has been reported to date except for abrief study by Weisburger et al. (1978), who reportedthat multiple tumors and liver tumors were induced bythe dietary administration of o-CNB to male rats for 12months and to male and female mice for 10 months,respectively. It is interesting to note on the basis of ourtwo previous studies (Matsumoto et al., 2006a and2006b) that the two isomers of chloronitrobenzenes, o-CNB and p-CNB, exhibited different types of rodent
Carcinogenicity and Toxicity of o-CNB.
261
Vol. 31 No. 3
carcinogenicity, presumably depending on the sub-chronic hepato- and hemato-toxicity of different carci-nogenic potency acting on the target organs. In our pre-vious study of 13-week toxicity (Matsumoto et al.,2006a), p-CNB was found to be a more potent hemato-toxicant than o-CNB, because significant increases inthe incidences of increased erythropoiesis in the bonemarrow and increased extramedullary hematopoiesisin the spleen in the rats and mice appeared at lowerdose levels of p-CNB than of o-CNB. The 2-yeardietary administration of p-CNB was found to producea dose-dependent increase in the incidences of splenicfibromas, fibrosarcomas, osteosarcomas, sarcomasNOS and hemangiosarcomas in male rats and fibrosar-comas and adreno-medullary pheochromocytomas infemale rats as well as the incidence of hepatic heman-giosarcomas in female mice (Matsumoto et al .,2006b). It can be suggested that p-chloroaniline, aprincipal metabolite of p-CNB, plays an important rolein the p-CNB-induced carcinogenicity, since p-CNBwas reported to be principally metabolized into p-chlo-roaniline (Bray et al., 1956; Rickert and Held, 1990),and since 2-year oral administration of p-chloroanilinehydrochloride by gavage was reported to inducesplenic tumors in male F344 rats (NTP, 1989a).
In our previous study of 13-week toxicity(Matsumoto et al., 2006a), o-CNB was found to be amore potent hepatotoxicant than p-CNB, because asignificant increase in serum ALT activity appeared atlower dose levels of o-CNB than of p-CNB, togetherwith the appearance of both necrosis and the hydropicdegeneration of hepatocytes only in the o-CNB-fed ratsand nuclear enlargement of hepatocytes with atypiaonly in the o-CNB-fed mice. Therefore, it can be sug-gested that the potent subchronic hepatotoxicity of o-CNB is causally related to hepatocarcinogenicity,while splenic tumors are the result of the persistence ofp-CNB-induced subchronic hematotoxicity. Sincechemically induced cell death followed by regenerativecell proliferation is recognized to be an importantmechanistic consideration for both genotoxic and non-genotoxic hepatocarcinogenicity (Butterworth et al.,1992), the persistence of potent hepatocellular toxicityresulting in cell death and subsequent regenerative cellproliferation induced by the long-term administrationof o-CNB to rats and mice might exert a cruciallyimportant role in the development of the liver tumors.
We have not yet obtained any further causativefactors to elucidate the o-CNB-induced hepatocarcino-genicity. However, a recent study of in vitro assay(JETOC, 1997) in which the bacterial mutagenicity of
o-CNB with TA98, TA100 and WP2uvrA was positiveonly in the presence of S9 activation seems to supportthe genotoxicity of metabolites produced by S9 activa-tion rather than o-CNB per se. It was reported that o-CNB was metabolized principally to 4-amino-3-chlo-rophenol and 3-amino-4-chlorophenol in the liver,while trace amounts of 2-amino-3-chlorophenol and 3-amino-2-chlorophenol were produced (Bray et al.,1956). These phenolic metabolites were reported to beconjugated with glutathione and glucuronide in theliver (Rickert and Held, 1990). It is inferred, therefore,that the mutagenic metabolites of o-CNB in the liverexert a crucially important role in the initial stage forthe formation of liver tumors.
An increase in the incidence of renal cell tumorsis thought to be related to treatment with o-CNB, sincethe incidences of renal cell adenomas in the 2000 ppm-fed female rats and renal cell carcinomas in the 2000ppm-fed male rats exceeded the respective maximumtumor incidence of the JBRC historical control data. Inaddition, the incidence of preneoplastic and prolifera-tive lesions, atypical tubule hyperplasias, was signifi-cantly increased at the same dose level. The renal celltumors found in the present study appear to be differentfrom the renal cell adenoma reported to be associatedwith CPN (NTP, 1989b; Kari et al., 1992; Hard et al.,1997; Hard, 1998; Seely et al., 2002; Hard and Seely,2005). First, while o-CNB induced not only benignrenal cell adenomas but also malignant carcinomas inthe present study, the CPN-associated tumor wasreported to be only a benign renal cell adenoma. Sec-ond, the sizes of the renal cell tumors (0.7 to 7.0 mm)found in the present study were larger than the CPN-associated tumors (0.4 to 1.0 mm) reported by Hardand Seely (2005). Third, the renal cell carcinomafound in the present study had a histopathologicallypleomorphic appearance. Fourth, since o-CNB wasreported to be a potent mutagen with S9 activation(JETOC, 1997), possible mutagenic metabolites of o-CNB, which were presumably biotransformed from acysteine conjugate of o-CNB metabolite, N-acetyl-S-(2-nitrophenyl)-L-cysteine (Bray et al., 1956), by β-lyase in the kidney, might act directly on the renalDNA, as suggested by Yamazaki et al. (2006) whodemonstrated positive nephrocarcinogenicity in maleF344 rats fed 1,4-dichloro-2-nitrobenzene, a structur-ally similar chemical of o-CNB, for 2 years. Therefore,it can be suggested that renal cell tumors are causallyrelated to the long-term administration of o-CNBrather than to CPN.
CPN was the most prevalent non-neoplastic
262
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lesion of chronic toxicity in the present study, as char-acterized by both a dose-dependent increase in theseverity of CPN and male predominance in its inci-dence and severity. CPN is a spontaneously occurringdisease progressing with increasing age and is specifi-cally observed in rats (Hard et al., 1997; Hard, 1998;Hard and Seely, 2005). Since a xenobiotic-inducedincrease in the severity of CPN was reported to beclosely associated with marginally increased inci-dences of atypical tubule hyperplasias and renal celladenomas (Hard et al., 1997; Hard, 1998; Seely et al.,2002; Hard and Seely, 2005), the possibility that theincreased severity of CPN found in the present studymight contribute to the marginally increased inci-dences of the renal cell tumors and atypical tubulehyperplasias cannot be entirely ruled out. However,this might not be likely to occur in o-CNB-inducednephrocarcinogenicity, as discussed in the precedingparagraph. The o-CNB-induced renal injuries werethought to be reflected by the increased serum levels ofcreatinine and urea nitrogen.
As another type of chronic toxicity, hematotoxic-ity of o-CNB appears to be evident in the o-CNB-fedrats, as indicated by a dose-related increase in methe-moglobin levels, a dose-related decrease in erythrocyteparameters and histopathological findings of capsulehyperplasia, extramedullary hematopoiesis and hemo-siderin deposition in the spleen. These chronic hemato-toxic responses to o-CNB were similar to the sub-chronic responses found in rats fed o-CNB for 13weeks (Matsumoto et al., 2006a), but were less severethan those observed in rats fed p-CNB for 2 years(Matsumoto et al., 2006b). However, the chronichematological signs and histopathological responsesthat appeared in the mice fed o-CNB for 2 years cannotbe interpreted as reflecting the hematotoxicity of o-CNB, because the mild hematotoxic effects thatoccurred in the mice fed o-CNB for 13 weeks(Matsumoto et al., 2006a) might be masked by theincreased incidence of highly malignant liver tumors.
In this study, the brown pigment was observed asdark brown droplets in both the liver and the proximalrenal tubules of o-CNB-fed rats of both sexes. Becausethe brown pigment was stained negatively with Berlinblue, which specifically stained ferric ions in tissues,the dark brown droplets observed in these two organswere thought to differ from the hemosiderin depositionthat occurred in both the spleens of rats and mice andthe kidneys of mice.
ACKNOWLEDGMENT
The present study was contracted and supportedby the Ministry of Health, Labour and Welfare, Japan.The authors are deeply grateful to Dr. KonosukeKumakura, Professor of Sophia University in TokyoJapan, for his discussion and encouragement through-out the present study.
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