TWO-YEAR FEED STUDY OF CARCINOGENICITY feed study of carcinogenicity and chronic ... kasuke nagano, seigo yamamoto and taijiro matsushima ... (chba corn-ing 270,

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  • 247

    Vol. 31 No. 3

    The Journal of Toxicological Sciences,Vol.31, No.3, 247-264, 2006


    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


    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:

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    M. MATSUMOTO et al.

    Vol. 31 No. 3

    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.


    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 0.3C 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 -irradiation-sterilizedCRF-1 powdered diet (Oriental Yeast Co., Tokyo,Japan) in a spiral mixer for 20 min, and stored at 7Cuntil 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

  • Carcinogenicity and Toxicity of o-CNB.


    Vol. 31 No. 3

    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, N