Toxicology and Carcinogenesis Studies of o-Nitroanisole (CAS No.91-23-6) in F344Rats and B6C3F1 Mice (Feed Studies)
Chemical Formula: C7H7NO3
o-Nitroanisole is used as an intermediate for the preparation ofo-anisidineand in the manufacture of azo dyes. Toxicology and carcinogenesis studieswereconducted by administering o-nitroanisole (>99% pure) in the diet togroupsof male and female F344 rats and B6C3F1 mice for 14 days, 13 weeks, and 2years. Genetic toxicology studies were conducted in Salmonella typhimurium,Chinese hamster ovary cells, and mouse lymphoma cells.
Groups of five male and five female F344 rats receiveddietscontaining 0, 583, 1,166, 2,332, 4,665, or 9,330 ppm o-nitroanisole.Mean bodyweight gains and final mean body weights of males in the 4,665 and 9,330ppmgroups were lower than those of the controls. Absolute liver weights weresignificantly increased in males receiving 1,166 ppm or more and in femalesreceiving 583 ppm or more.
Groups of five male and five female B6C3F1 mice received dietscontaining 0,250, 500, 1,000, 2,000, or 4,000 ppm o-nitroanisole. Mean bodyweight gainsand final mean body weights of males that received 250 ppm and females thatreceived 4,000 ppm were significantly lower than those of the controls. Noother chemical-associated effects were observed.
Groups of 10 male and 10 female F344 rats receiveddietscontaining 0, 200, 600, 2,000, 6,000, or 18,000 ppm o-nitroanisole. Final meanbody weights and feed consumption by male and female rats receiving 6,000and18,000 ppm were lower than those of the controls. Hemoglobin andhematocritvalues were significantly lower and methemoglobin levels significantly higherin males in the 6,000 and 18,000 ppm groups than in controls. Absolute liverweights were significantly increased in females that received 200, 600, 2,000,and 6,000 ppm, absolute kidney weights were significantly increased in malesthat received 600, 2,000, and 6,000 ppm, and absolute spleen weights weresignificantly increased in males and females that received 6,000 and 18,000ppm.
Groups of 10 male and 10 female B6C3F1 mice received dietscontaining 0, 60,200, 600, 2,000, or 6,000 ppm o-nitroanisole. Final mean body weightgains,final mean body weights, and feed consumption by male and female micereceiving6,000 ppm were lower than those of the controls. Hemoglobin and hematocritvalues in males and females that received 2,000 or 6,000 ppm weresignificantlylower than those in the controls. The absolute and relative liver weights offemales in the 600 ppm group and relative liver weights of males and femalesinthe 2,000 and 6,000 ppm groups were significantly greater than those ofcontrols.
Lesions associated with exposure to o-nitroanisole were presentin theurinarybladder, spleen, kidney, liver, testis, and uterus of rats. Diffuse hyperplasiaof the transitional epithelium of the urinary bladder occurred in all male andfemale rats that received 6,000 and 18,000 ppm. A transitional cell papillomaoccurred in one male and transitional cell carcinomas occurred in two malesandthree females receiving 18,000 ppm. Congestion of the red pulp and capsularhyperplasia of the spleen and hepatocellular hypertrophy of the liver werepresent in males and females from the 18,000 ppm groups. Multifocaldegeneration and necrosis of the renal tubule epithelium with infiltration ofmononuclear inflammatory cells were present in male rats that received 600,2,000, and 6,000 ppm. At the 18,000 ppm level, degeneration of theseminiferous epithelium accompanied by loss of spermatogenic cells anddecreased numbers of spermatozoa were observed in the testes of male rats,while uterine atrophy was observed in female rats.
Hepatocyte hypertrophy of the centrilobular and midzonal regions of liverlobules was present in mice that received 200 ppm and increased in severityathigher exposure levels.
The doses selected for the 2-year study ofo-nitroanisole inrats were based on lower mean body weights, reduced feed consumption,andincreased severity of regenerative anemia in male and female rats receiving6,000 and 18,000 ppm during the 13-week study. Groups of 60 male and 60femaleF344 rats received diets containing 0, 222, 666, or 2,000 ppmo-nitroanisole.Groups of 60 male and 60 female B6C3F1 mice received diets containing 0,666,2,000, or 6,000 ppm o-nitroanisole. After 15 months, up to 10 animalsfromeach group were evaluated for chemical-related lesions.
Survival, Body Weights, Feed Consumption, and Clinical Findings
Survival ofmale rats receiving 2,000 ppm was significantly lower than that of the controlsdue to increased severity of nephropathy. Survival of 222 and 666 ppm malerats and all exposed female rats was similar to that of the controls. Survivalof groups of exposed male and female mice was similar to that of the controls.The final mean body weight of male rats receiving 2,000 ppm was lower thanthatof the controls. Final mean body weights of male and female mice thatreceived2,000 and 6,000 ppm were lower than those of the controls. Feedconsumption bymale and female rats was similar to that by the controls. The only clinicalfinding in male or female mice attributable to chemical administration wasdiscolored urine.
Neoplasms and Nonneoplastic Lesions
The incidence of mononuclearcellleukemia was significantly increased in male rats that received 666 and 2,000ppm and in female rats that received 2,000 ppm (males: 0 ppm, 26/50; 222ppm,25/50; 666 ppm, 42/50; 2,000 ppm, 34/50; females: 14/50, 11/50, 14/50,26/50).Nephropathy occurred in all male rats; the severity increased with exposurelevel. Focal hyperplasia of the renal tubule epithelium was present in threemales receiving 222 ppm and two males receiving 2,000 ppm. Renal tubuleadenomas occurred in one male from each of the 222, 666, and 2,000 ppmgroups,and renal tubule carcinomas occurred in two males from the 2,000 ppm group.Focal hyperplasia of the transitional epithelium of the urinary bladder waspresent in one female rat that received 222 ppm and two male rats and sixfemale rats that received 2,000 ppm. A transitional cell papilloma occurred inthe urinary bladder of one female rat from the 2,000 ppm group, and atransitional cell carcinoma occurred in another female from the 2,000 ppmgroup. The incidence of forestomach ulcers increased in male rats thatreceived 2,000 ppm, and the incidence of focal hyperplasia of theforestomachincreased with exposure level in male and female rats. In addition, squamouscell papillomas of the forestomach were present in one female receiving 222ppm, one male receiving 666 ppm, and one male and one female receiving2,000ppm, while squamous cell carcinomas were present in one male receiving 666ppmand one male and one female receiving 2,000 ppm. The incidences ofpituitarygland adenomas in male rats and mammary gland fibroadenomas in femaleratsdecreased with exposure level.
The incidence of cellular alteration in the liver was significantly increasedin exposed groups of male and female mice. The incidences of hepatocellularadenoma, hepatocellular adenoma or carcinoma (combined), andhepatocellularcarcinoma or hepatoblastoma (combined) were significantly increased inmalemice receiving 2,000 and 6,000 ppm. The incidences of hepatocellularadenomaor carcinoma were significantly increased in female mice that received 2,000ppm.
Groups of 60 male and 60 female F344 ratsreceived dietscontaining 0, 6,000, or 18,000 ppm o-nitroanisole for 27 weeks andwere thenmaintained on control feed without further chemical exposure for up to anadditional 77 weeks. Up to 10 rats from each group were evaluated for thepresence of chemical-related lesions at 3, 6, 9, and 15 months.
Survival and Body Weights
Survival of exposed male and female ratswassignificantly lower than that of the controls as a result of moribund deathsassociated with significantly increased incidences of urinary bladderneoplasms, primarily transitional cell carcinomas. All male rats that received18,000 ppm were dead by week 48 and all females that received 18,000 ppmweredead by week 61. Mean body weights of exposed male and female rats werelowerthan those of the controls throughout the study.
Neoplasms and Nonneoplastic Lesions
Hyperplasia of the transitionalepithelium of the urinary bladder was present in nearly all exposed male andfemale rats examined at the interim evaluations. A transitional cell carcinomawas first observed at the 3-month interim evaluation in a male rat thatreceived 18,000 ppm. At the 6- and 9-month interim evaluations, transitionalcell papillomas or carcinomas were observed in both exposed groups of malerats. Transitional cell carcinomas were observed at the 6-month interimevaluation in females receiving 18,000 ppm and at the 9-month interimevaluation in females receiving 6,000 and 18,000 ppm.
Adenomatous polyps of the large intestine were observed in a smallnumber ofexposed rats at the 6-, 9-, and 15-month interim evaluations. At the end ofthe study, the incidence of adenomatous polyps of the large intestine wassignificantly increased in all exposed groups and carcinomas of the largeintestine were present in four males and two females from the 18,000 ppmgroups. The incidence of hyperplasia of the transitional epithelium of thekidney pelvis was significantly increased in exposed male and female rats andtransitional cell papillomas were present in three males and one female thatreceived 18,000 ppm. Transitional cell carcinomas of the kidney were presentin one male receiving 6,000 ppm and six males and one female receiving18,000ppm. Transitional cell carcinomas of the urinary bladder were seen in nearlyall exposed male and female rats. Of the males and females receiving 6,000ppmwhich were without carcinomas, three males and one female had transitionalcellpapillomas.
Generalized centrilobular hypertrophy, focal hepatocellular necrosis,multifocal hepatocellular cytoplasmic vacuolation, and Kupffer cellpigmentation were observed in the livers of male and female rats at the 3- and6-month interim evaluations; however, only Kupffer cell pigmentation wasobserved at the end of the study. Congestion of the red pulp of the spleen wasobserved in nearly all exposed male and female rats at the 3-, 6-, and 9-monthinterim evaluations but the incidence was only slightly increased in the 18,000ppm groups at the end of the study. Degeneration and atrophy of theseminiferous tubule epithelium of the testes were observed at the 3- and6-month interim evaluations in all male rats receiving 18,000 ppm.
o-Nitroanisole was tested in two laboratoriesformutagenicity in Salmonella typhimurium strains TA97, TA98, TA100, TA1535,andTA1537 with and without exogenous metabolic activation (S9). Positiveresponses were observed at both laboratories in TA100 with and without S9activation. One laboratory found no increase in mutations, while the secondlaboratory detected a weakly positive response in TA1535 without S9. Nomutagenic activity was observed in the other tester strains.o-Nitroanisole waspositive in the mouse lymphoma assay for induction of trifluorothymidineresistance in L5178Y cells without S9 activation. In cytogenetic tests withChinese hamster ovary cells, o-nitroanisole induced a significantincrease inchromosomal aberrations at the highest dose tested in the presence of S9activation; sister chromatid exchanges were induced both with and withoutS9.
Under the conditions of these feed studies there was clearevidence of carcinogenic activity of o-nitroanisole in male and femaleF344rats that received diets containing 6,000 or 18,000 ppm for 6 months based onoverall increased incidences of benign and malignant neoplasms of theurinarybladder, transitional cell neoplasms of the kidney, and benign and malignantneoplasms of the large intestine. There was a chemical-related increasedincidence of mononuclear cell leukemia in male and female rats receivingdietscontaining 222, 666, or 2,000 ppm o-nitroanisole for 2 years. Marginallyincreased incidences of uncommon renal tubule neoplasms in male rats andforestomach neoplasms in male and female rats were considered uncertainfindings. There was clear evidence of carcinogenic activity ofo-nitroanisolein male B6C3F1 mice based on increased incidences of benign and malignanthepatocellular neoplasms. There was some evidence of carcinogenic activityofo-nitroanisole in female B6C3F1 mice based on increased incidencesofhepatocellular adenomas.
Increased severity of nephropathy in male rats, and increasedincidences offocal hyperplasia of the renal tubule epithelium and forestomach ulcers inmalerats, and of transitional cell hyperplasia of the urinary bladder, focalhyperplasia of the forestomach, and hyperplasia of transitional epithelium ofthe kidney pelvis in male and female rats were associated with exposure too-nitroanisole.
Synonyms: Methoxynitrobenzene, nitrophenyl methyl ether
Report Date: May 1993