Document B8xnEmLq4ZaO8KD7mkemNNGv8

EVALUATION OF MICRONUCLEI IN THE PERIPHERAL BLOOD OF CD-I MICE EXPOSED TO BENZENE Phase I Study 2000 ppm - 5 Day Evaluation Sponsored by The American Petroleum Institute Conducted by Raymond R. Tice, Ph.D. Medical Department Brookhaven National Laboratory Upton, NY 11973 Report Date: January 2, 1984 REDACTED* DBZ0029933 Project: Evaluation of Micronuclei in the Peripheral Blood of CD-I Mice Exposed to Benzene: Phase I Evaluation Purpose . To assess the utility of evaluating micronuclei (MN) in the peripheral blood of male and female CD-I mice exposed to benzene. Protocol At Hazleton Laboratories, CD-I mice were exposed to benzene (2000 ppm, 6 hr per day, 5 days), and a blood sample was obtained on the morning of day 6. A typical blood smear was made and stained with Giemsa. The slides were mailed to Brookhaven National Laboratory and received on November 14, 1983, along with peripheral blood smears made from rats also exposed to benzene. At this time, it was impossible to identify the species origin of individual slides. On December 6, 1983, the code for the slides was received, the mouse slides were removed, destained with methanol: glacial acetic acid (5:1) for approximately 10 minutes, and randomized before stain ing with acridine orange (AO) at a concentration of 0.125 mg/ml in phosphate buffer, pH 7.4 (staining protocol - stain for 5 min, rinse in distilled water, remove excess stain by a further rinse in buffer for 10 min, and place a coverslip on the slide using buffer). The AO stained slides were analyzed by one technician with no know ledge of the experimental design of the study. For each slide, the number of MN in 1000 polychromatic erythrocytes (PCE) and in 1000 normochromatic erythrocytes (NCE), and the %PCE in 1000 red blood cells were evaluated at 800x in a Zeiss Standard micro scope equipped with Epi-Fluorescence. The data were stored in a Vax 11/780 computer, the code broken, the animals grouped according to exposure conditions, and the data analyzed using nonparametric statistics with the BMDP Statistical Analysis Package. Results Table 1 lists the MN data on a per animal basis. Table 2 presents the group mean values'and standard error between animals for MN per 1000 PCE, MN per 1000 NCE and %PCE. When the data are statistically analyzed (see Table 3), the following con clusions can be made. 1. There are no technical difficulties associated with destaining the Giemsa stained slides and evaluating the presence of MN using the more sensitive AO staining technique. 2. There were no, significant differences in MN frequency in NCE or PCE, or in %PCE between concurrent controls and pretreatment controls. 3. Exposure to benzene (2000 ppm, 6 hr per day, 5 days) induced MN in the peripheral blood cells of CD-I mice. DBZ0029934 As expected, considering the short.duration time of the exposure, MN frequencies were more elevated in the PCE population than in the NCE population. Also, consistent with other data, male mice tended to ex hibit a greater genotoxic response than female mice. Exposure to benzene induced a significant depression in the %PCE in CD-I mice, the extent of the depression being equal in males and females. DBZ0029935 Table 1. Individual Animal Micronuclei Frequencies (continued) 3. Lab Acc. No. (8-29-83) 2586 2587 2588 2589 2590 2591 2592 2593 2594 2595 2596 2597 2598 2599 2600 2601 2602 2603 Lab Animal ' No. 619 625 626 629 631 686 692 693 695 703 652 657 658 663 737 740 744 746 * Sex M M M M M F F F F F M M M M F F F F % PCE* 2.4 7.5 2.9 2.4 2.8 4.2 4.0 5.0 2.8 1.6 1.0 0.7 0.4 4.2 2.5 0.3 2.1 1.6 . MN/1000 NCE** 2 3 0 1 1 1 1 0 1 0 '1 3 1 0 4 0 2 0 MN/1000 PCE*** 2 2 0 1 0 1 2* 0 1 8 3 3 9 3 1 0 6 2 * %PCE % polychromatic erythrocyctes in 1000 red blood cells. ** MN/1000 NCE = Number of micronuclei cells in 1000 normochromatic erythrocytes. . *** MN/1000 PCE - Number of micronuclei in 1000 polychromatic erythrocytes. DBZ0029936 Table 1. Individual Animal Micronuclei Frequencies (continued) 2. Lab Acc. No. (8-28-83) 2495 2496 2497 2499 2500 2501 2502 2503 2504 2505 2506 2507 2508 2509 2510 2511 2512 2513 2514 2515 2516 Lab Animal No. 601 605 608 616 670 673 678 679 681 633 640 642 643 647 648 716 717 721 723 724 727 Sex M M M M F F F F F M M M M M M F F F F F F % PCE* 2.8 1.1 2.9 2.1 1.5 0.8 2.0 3.3 3.2 5.2 0.5 1.3 0.5 0.3 5.4 1.0 1.5 0.6 1.1 2.3 2.9 MN/1000 NCE** 1 0 0 0 0 1 1 2 1 2 6 4 4 5 4 3 4 1 4 2 3 MN/1000 PCE*** 1 3 6 1 1 0 2 1 1 1 8 2 10 3 5 8 1 9 19 4 3 * %PCE = % polychromatic erythrocyctes in 1000 red blood cells. ** MN/1000 NCE => Number of micronuclei cells in 1000 normochromatic erythrocytes. *** MN/1000 PGE = Number of micronuclei in 1000 polychromatic erythrocytes. DBZ0029937 Table 1. Individual Animal Micronuclei Frequencies Lab Acc. No. (8-20-83) 2061 2062 2063 2064 2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 Lab Animal No. 04 05 11 14 08 34 36 37 44 56 62 08 09 12 14 21 26 28 30 40 43 Sex M M M M M M -M M M M M F F F F F F F F F F % PCE* 2.7 4.3 2.6 2.2 - 3.5 2.1 2.9 2.7 4.7 2.3 2.0 2.4 3.0 2.0 1.8 2.2 3.5 4.4 2.2 3.4 . MN/1000 NCE** 0 0 0 0 - 0 0 1 3 1 1 0 4 3 1 1 1 1 2 8 1 MN/1000 PCE*** 2 1 11 - 2 0 1 1 1 1 0 1 0 2 0 1 4 3 1 1 * %PCE = % polychromatic erythrocyctes in 1000 red blood cells. ** MN/1000 NCE = Number of micronuclei cells in 1000 normochromatic erythrocytes. *** MN/1000 PCE = Number of micronuclei in 1000 polychromatic erythrocytes. DBZ0029938 Table 2. Micronuclei Frequencies in the Peripheral Blood Cells of CD-I Mice Exposed to Benzene. Exposure Group Sex Number Animals MN/1000 NCE* MN/1000 PCE** . % PCE Pretreatment Controls Male Female M+F 10 10 20 0.60 0.31 2.20 0,74 1.40 0.43 1.10 0.18 1.00 + 0.30 1.05 0.17 Concurrent Controls Benzene Treated Male Female M+ F 10 ' 10 20 Male Female M+ F 10 10 20 0.80 0.33 1.00 0.21 0.90 + 0.19 1.70 0.56 1.70 0.73 1.70 + 0.45 3.20 + 0.61 2.30 0.50 2.75 0.40 4.80 0.96 5.30 + 1.80 5.05 1.00 *Micronuclei per 1000 normochromatic erythrocytes **Micronuclei per 1000 polychromatic erythrocytes *per cent polychromatic erythrocytes in 1000 red blood cells 3.0 + 0.3 2.7 0.3 2.8 0.2 2.8 0.6 3.2 + 0.5 3.0 0.4 1.5 0.6 1.7 + 0.3 1.6 + 0.3 DBZ0029939 Table 3. Statistical Analysis of MN Frequencies and %PCE in the Peripheral Blood Cells of CD-I Mice Exposed to Benzene A. Micronuclei Frequencies Pretreatment Controls M F M+F Concurrent Controls M F M+F Benzene Treated M F M+F Pretreatment Controls M F M+F PCE "P" VALUES 56 . .00* .54 .02* .42 00* Concurrent Controls M F M+F .61 .21 71 - .00* .06 .00* Benzene Treated M F M+F .00* .01* .53 .05* .01* .00* NCE "P" VALUES P values are calculated from the Kruskall-Wallis One-Way AN0VA using Chi-Square Distribution with one degree of freedom. * p value significantly different at less than or equal to 0.05. DBZ0029940 Table 3. Statistical Analysis of MN Frequencies and %PCE in the Peripheral Blood Cells of CD-I Mice Exposed to Benzene B. %PCE Determinations Pretreatment Controls M F M+F Concurrent Controls M F M+F Benzene Treated M F MfF Pretreatment Controls M F M+F Concurrent Controls Benzene Treated M F M+F .52 .45 88 M F M+F .02* .02* .00* .02* .01* .00* P values are calculated from the Kruskall-Wallis One-Way ANOVA using Chi-Square Distribution with one degree of freedom. * p value significantly different at less than or equal to 0.05. DBZ0029941