Survival

Estimates of 27-week survival probabilities for male and female mice are shown in Table 3. Survival of all dosed groups of male and female mice was similar to that of the vehicle controls; all animals survived until the end of the study except one male administered 200 mg/kg.

Table 3

Survival of Haploinsufficient p16^Ink4a/p19^Arf Mice in the 27-Week Gavage Study of Benzene.

Body Weights, Clinical Findings, and Organ Weights

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 (Figure 4 and Tables 4 and 5). By week 13, mean body weights of males in the 25, 50, 100, and 200 mg/kg groups were 5%, 9%, 18%, and 19% less than that of the vehicle controls, respectively. 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. Male mice in the 100 and 200 mg/kg groups also had dark pigmentation of the nose.

Figure 4

Growth Curves for Male and Female Haploinsufficient p16^Ink4a/p19^Arf Mice Administered Benzene by Gavage for 27 Weeks.

Table 4

Mean Body Weights and Survival of Male Haploinsufficient p16^Ink4a/p19^Arf Mice in the 27-Week Gavage Study of Benzene.

Table 5

Mean Body Weights and Survival of Female Haploinsufficient p16^Ink4a/p19^Arf Mice in the 27-Week Gavage Study of Benzene.

Compared to the vehicle controls, the absolute liver and thymus weights of all dosed groups of males and the absolute right testis weights of 50 mg/kg or greater males were significantly decreased (Table C1). The relative thymus weights of 50 mg/kg or greater males were significantly decreased.

Hematology

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 (Tables 6 and B1). Male mice were more severely affected with a greater than 20% erythron decrease at 200 mg/kg compared to the 10% or less decrease in 200 mg/kg females. Also, at 13 and 27 weeks, the erythron decrease was accompanied by an increase in erythrocyte size, which was shown by the dose-related increase in mean cell volumes. The males were more affected than the females with an approximately 15% increase compared to approximately 4% at 200 mg/kg, respectively. Another consistent finding was a dose-related leukopenia, evidenced by decreases in the leukocyte counts. Males were affected at a lower dose and to a greater magnitude than females. For example, for 200 mg/kg males, leukocyte counts were approximately 20% of the control counts while those in 200 mg/kg females were approximately 68% of the control counts. The decreases in leukocyte counts were primarily related to decreases in the lymphocyte counts. Segmented neutrophil counts were also decreased in the 100 and 200 mg/kg groups at week 27. These findings were consistent with the predictable hematopoietic effects of benzene that can result in anemia, leukopenia, thrombocytopenia, or some combination of these.

Table 6

Selected Hematology Data for Haploinsufficient p16^Ink4a/p19^Arf Mice in the 27-Week Gavage Study of Benzene.

Pathology and Statistical Analyses

This section describes the statistically significant or biologically noteworthy changes in the incidences of malignant lymphoma and nonneoplastic lesions in the bone marrow, spleen, thymus, lymph nodes, skin, and mammary gland. Summaries of the incidences of neoplasms and nonneoplastic lesions are presented in Tables A1, A2, A3, and A4.

Malignant Lymphoma: The incidence of malignant lymphoma was significantly increased in 200 mg/kg males compared to the vehicle controls, and the incidence exceeded the historical control range in male mice from the current 27-week benzene and phenolphthalein studies and 40-week aspartame and glycidol studies (Tables 7, A1, and E1). Neoplastic cells infiltrated multiple organs including the spleen, thymus, lymph node, kidney, lung, and/or brain. No malignant lymphomas occurred in females.

Table 7

Incidences of Malignant Lymphoma in Male Haploinsufficient p16^Ink4a/p19^Arf Mice in the 27-Week Gavage Study of Benzene.

Bone Marrow: Significantly increased incidences of minimal to mild atrophy occurred in the 100 and 200 mg/kg males compared to the vehicle controls (Tables 8 and A2). Atrophy was characterized by histologically detectable decreased cellularity of femoral and/or sternal bone marrow with increased amounts of adipocytes and/or blood vessels. Dose-related increased incidences and severities of hemosiderin pigmentation occurred in males. There were no benzene-related effects on bone marrow in female mice.

Table 8

Incidences of Selected Nonneoplastic Lesions in Haploinsufficient p16^Ink4a/p19^Arf Mice in the 27-Week Gavage Study of Benzene.

Spleen: Dose-related incidences of lymphoid follicle atrophy were significantly increased in 100 and 200 mg/kg male mice (Tables 8 and A2). Lymphoid follicle atrophy occurred in a few females in the 25, 50, and 200 mg/kg groups (Tables 8 and A4). Lymphoid follicle atrophy was characterized by decreased lymphoid follicle size, decreased variety of cell types, decreased numbers of lymphocytes, and sometimes a decreased overall size of splenic profile. Affected spleens tended to have smaller amounts of lymphoid tissue than the vehicle control mice, as well as having primarily small lymphocytes with no germinal centers and fewer intermingled histiocytes.

The incidence of hematopoietic cell proliferation was significantly increased in 200 mg/kg males compared to vehicle controls, and hematopoietic cell proliferation occurred in all groups of females (Tables 8 and A2). Hematopoietic cell proliferation consisted of increased blood precursor cells in the red pulp that generally caused some enlargement of the spleen; however, in the 200 mg/kg males, the enlargement did not offset the lymphoid atrophy.

Thymus: The incidences of atrophy in the 100 and 200 mg/kg males were significantly greater than that in the vehicle controls (Tables 8 and A2). Atrophy was characterized by decreased size of the thymic profile, decreased numbers of lymphocytes, and loss of corticomedullary distinction.

Lymph Nodes (Mandibular, Mediastinal, and Mesenteric): Significantly increased incidences of atrophy (mandibular, mediastinal, and mesenteric) occurred in 100 and 200 mg/kg males (Tables 8 and A2). The incidences of atrophy of the mesenteric lymph node were significantly increased in 100 and 200 mg/kg females, and the incidence of atrophy of the mediastinal lymph node was significantly increased in 100 mg/kg females (Tables 8 and A4). Atrophy was characterized by decreased numbers of lymphocytes in the lymph nodes, decreased variety of cell-types in lymphatic nodules, increased sinusoidal spaces, and decreased medullary cords or germinal centers.

Skin: The incidences of skin pigmentation were significantly increased in all dosed groups of males and in 50 mg/kg or greater females, and the severity generally increased with increasing dose (Tables 8, A2, and A4). The males were more sensitive to this effect. The increased pigmentation (consistent with melanin) was detected in the epidermis of dosed mice from the paw but not the inguinal area. The pigmentation consisted of five or more separate foci per section of skin (minimal) to confluent regions over more than 1 mm (mild) to involvement of the entire epidermis (moderate).

Mammary Gland: Hyperplasia occurred in two female mice treated with 200 mg/kg benzene, but not in any other dosed groups (Tables 8 and A4).