Study Design

Groups of 15 male and 15 female mice were administered benzene in corn oil by gavage at doses of 0, 25, 50, 100, or 200 mg benzene/kg body weight 5 days per week for 27 weeks. The doses selected for use in the 27-week study were chosen to overlap those used for p53^+/− mice in the French et al. (2001) gavage study and for B6C3F₁ mice in the NTP (1986) 2-year gavage study.

Source and Specification of Animals

Male and female heterozygous B6.129-Cdkn2a^tm1Rdp N3 (i.e., haploinsufficient p16^Ink4a/p19^Arf) mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY) for use in the 27-week study. The N1 male mice homozygous null for the Cdkn2a deletion were backcrossed to inbred C57BL/6 females from Taconic Laboratory to produce male and female B6.129-Cdkn2^atm1Rdp haploinsufficient or p16^Ink4a/p19^Arf+/− mice (Serrano et al., 1996). The genetic background of these mice was: 80% C57BL/6, 19% 129/Sv, and 1% SJL. This line, designated 5003 by Taconic Laboratory, was embryo cryopreserved in 2003. Upon receipt, the mice were 3 to 4 weeks old. Animals were quarantined for 22 days and were 7 to 8 weeks old on the first day of the study. Before the study began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood samples were collected from up to five male and five female sentinel animals at 4 weeks and at study termination. The sera were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989a,b). All results were negative.

Mice were housed individually. Feed and water were available ad libitum. Cages and racks were rotated weekly. Further details of animal maintenance are given in Table 2.

Table 2

Experimental Design and Materials and Methods in the 27-Week Gavage Study of Benzene.

Clinical Examinations and Pathology

All mice were observed twice daily. Clinical findings were recorded weekly, to coincide with body weight collection, and at the end of the study. The mice were weighed initially, weekly, and at the end of the study.

Blood for hematology analysis was collected from the retroorbital sinus of mice under carbon dioxide anesthesia at 13 and 27 weeks. Samples were placed in micro-collection tubes (Sarstedt, Inc., Nümbrecht, Germany) coated with potassium EDTA. Hematocrit; erythrocyte, platelet, and leukocyte counts; mean cell hemoglobin; and mean cell hemoglobin concentration were determined with a Cell-Dyn^® hematology analyzer (Abbott Laboratories, Abbott Park, IL). Differential leukocyte counts were determined microscopically from smears stained with a Wright-Giemsa stain. A Miller Disc was used to determine reticulocyte counts from smears prepared with blood stained with new methylene blue.

Complete necropsies and microscopic examinations were performed on all mice. The heart, right kidney, liver, lung, right testis, and thymus were weighed. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. Table 2 lists the tissues and organs routinely examined.

Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy; the slide and tissue counts were verified, and the histotechnique was evaluated. For the 27-week study, a quality assessment pathologist evaluated slides from all tumors and all potential target organs, which included the bone marrow, lymph nodes, skin, spleen, and thymus.

The quality assessment report and the reviewed slides were submitted to the NTP Pathology Working Group (PWG) chairperson, who reviewed the selected tissues and addressed any inconsistencies in the diagnoses made by the laboratory and quality assessment pathologists. Representative histopathology slides containing examples of lesions related to chemical administration, examples of disagreements in diagnoses between the laboratory and quality assessment pathologists, or lesions of general interest were presented by the PWG chairperson to the NTP pathologist. Final diagnoses for reviewed lesions represent a consensus between the laboratory pathologist, reviewing pathologist(s), the PWG chairperson, and the NTP pathologist. Details of these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses of the pathology data, the decision of whether to evaluate the diagnosed lesions for each tissue type separately or combined was generally based on the guidelines of McConnell et al. (1986).

Survival Analyses

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes were censored; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Calculation of Incidence

The incidences of neoplasms or nonneoplastic lesions are presented in Tables A1, A2, A3, and A4 as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. The Fisher exact test (Gart et al., 1979) and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overall proportion of affected animals, were used to determine significance.

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973).

Mouse Peripheral Blood Micronucleus Test Protocol

A detailed discussion of this assay is presented by MacGregor et al. (1990). At 6.5, 13, 19.5, and 27 weeks, peripheral blood samples were obtained from male and female haploinsufficient p16^Ink4a/p19^Arf mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of up to 15 animals per dose group. In addition, the percentage of polychromatic erythrocytes among 1,000 total erythrocytes was determined for each animal as a measure of benzene-induced bone marrow toxicity.

The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the vehicle control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects. Because these studies were not repeated, the results of the micronucleus trials were accepted without replication.