Background:

Benzene is a widely used solvent and is used in the production of many chemicals and gasoline. Benzene is known to cause cancer in laboratory animals and leukemia in humans. We tested benzene in a genetically modified mouse strain that lacks two tumor suppressor genes as part of a study to determine if this mouse model could detect cancer-causing chemicals more rapidly than the standard 2-year rodent bioassay.

Methods:

We exposed groups of male or female haploinsufficient p16^Ink4a/p19^Arf mice by depositing solutions of benzene in corn oil directly into the animals’ stomachs through a tube five times per week for 27 weeks. The daily doses were 25, 50, 100, or 200 milligrams of benzene per kilogram of body weight; other animals receiving only corn oil served as the control groups. Tissues from 22 organs were examined for every animal.

Results:

Exposure to benzene caused malignant lymphomas in male haploinsufficient p16^Ink4a/p19^Arf mice receiving the greatest dose of benzene. Other effects seen in male mice included bone marrow atrophy, hematopoietic cell proliferation in the spleen, and atrophy of the thymus and lymph nodes. No increased incidences of cancer were seen in female haploinsufficient p16^Ink4a/p19^Arf mice, though atrophy of the mesenteric lymph nodes was observed.

Conclusions:

We conclude that benzene caused malignant lymphoma in male haploinsufficient p16^Ink4a/p19^Arf mice but not in females.

27-Week Study in Mice:

Groups of 15 male and 15 female haploinsufficient p16^Ink4a/p19^Arf mice were administered 0, 25, 50, 100, or 200 mg benzene/kg body weight in corn oil by gavage 5 days per week for 27 weeks. All animals survived until the end of the study except one male administered 200 mg/kg. Mean body weights of males administered 50 mg/kg or greater were generally less than those of the vehicle controls throughout the study, and those of 25 mg/kg males were less after week 13. Mean body weights of 200 mg/kg females were less than those of the vehicle controls after week 17. Treatment-related clinical findings in 25 mg/kg or greater males and 50 mg/kg or greater females included black, brown, or gray discoloration (pigmentation) of the feet. The thymus weights of all dosed groups of males were significantly decreased. At weeks 13 and 27, a dose-related decrease in the erythron occurred in males and females. The erythron decrease was shown by decreases in the hematocrit, hemoglobin, and erythrocyte count values in all dosed males and in the 100 mg/kg or greater females. Decreased leukocyte counts, primarily lymphocyte counts, resulted in a dose-related leukopenia in males and females. In males, segmented neutrophil counts were also decreased.

The incidence of malignant lymphoma was significantly increased in 200 mg/kg males compared to the vehicle controls.

In the bone marrow, significantly increased incidences of minimal to mild atrophy occurred in the 100 and 200 mg/kg males compared to the vehicle controls. In the spleen, there were significantly increased incidences of lymphoid follicle atrophy in 100 and 200 mg/kg male mice. The incidence of hematopoietic cell proliferation was significantly increased in 200 mg/kg males. The incidences of atrophy of the thymus in the 100 and 200 mg/kg males were significantly greater than those in the vehicle controls. In the lymph nodes, significantly increased incidences of atrophy (mandibular, mediastinal, and mesenteric) occurred in 100 and 200 mg/kg males, and the incidence of atrophy of the mediastinal lymph node was significantly increased in the 100 mg/kg females. The incidences of skin pigmentation were significantly increased in all dosed groups of males and in females dosed with 50 mg/kg or greater.

Genetic Toxicology:

The frequency of micronucleated erythrocytes was assessed at four timepoints during the 27-week study. Significant increases in micronucleated cells were observed at all timepoints, and the magnitude of the response correlated with duration of treatment.

Conclusions:

Under the conditions of this 27-week gavage study, there was clear evidence of carcinogenic activity^* of benzene in male haploinsufficient p16^Ink4a/p19^Arf mice based on the occurrence of malignant lymphoma. There was no evidence of carcinogenic activity of benzene in haploinsufficient p16^Ink4a/p19^Arf female mice administered 25, 50, 100, or 200 mg/kg.

Treatment of male and female haploinsufficient p16^Ink4a/p19^Arf mice with benzene was associated with toxicity to the hematopoietic system, lymphoid atrophy, and the accumulation of pigment in the extremities.