Document v6Yk7wD4zkB8aZ3MZndEjL4Yb

TECHNICAL TASK GROUP ON VINYL CHLORIDE RESEARCH Mr. Raymond j. Abramowitz Technical Director Ruco Division Hooker Chemicals Corporation P. O. Box 456 Burlington, N. J. 08016 (609) 499-2300 Ext. 70-221 Mr. Paul L. Armstrong Technical Department Manager Georgia-Pacific Corporation P. 0. Box 629 Plaquemine, Louisiana 70764 (504) 687-6321 Ext. 215 Dr. Zeb G. Bell, Jr. PPG Industries, Inc. One Gateway Center Pittsburgh, Pa. 15222 (412) 434-2585 Dr. Robert S. Brookman Firestone Plastics Co. Research Dev. & Tech. Ser. P. 0. Box 699 Pottstown, Pa. 19464 (215) 326-2000 Ext. 224 Mr. H. B. Carr Tenneco Chemicals P- O. Box 2 Piscataway, N. J. 08854 (201) 981-5176 B. W. Duck, M.D. British Petroleum Co. Ltd. Research Center-Chertsey Rd. Sunbury-cn-Thames Middlesex, England Tel. 85533 Ext. 451 Telex #266883 R. E. Eckardt, M.D. Exxon Corporation P. 0. Box 45 Linden, New Jersey 07036 (201) 474-3801 Mr. J. E. Ertel General Manager ROBINTECH Incorporated P. 0. Box 2342 Fort Worth, Texas 76101 (817) 332-7323 Ext. 23 Dr. A. W. Fuhrman Vice President Great American Chemical Corporation 650 Water Street Fitchburg, Mass. 01420 (617) 343-6973 Ext. 55 Dr. Mac Roy Gasque Olin Corporation 120 Long Ridge Road Stamford; Conn. 06904 (203) 356-2561 Dr. R. L. Gibson Gulf Oil Corporation P. 0. Box 1166 Pittsburgh, Pa. 15230 (412) 391-2400 * Dr. Walter D. Harris UNIROYAL, Inc. EMIC Building Naugatuck, Conn. 06770 (203) 573-2000 Ext. 2139 Dr. C. A. Johnson Goodyear Tire & Rubber Co. 1144 East Market St. Akron, Ohio 44316 (216) 794-7676 DTH 000011823 TTGVCR * M. N. Johnson, M.D. The B. F. Goodrich CompanyMedical Center 500 South Main Street Akron, Ohio 44318 (216) 379-3298 Dr. Flynt KennedyContinental Oil Company 1000 South Pine Drawer 1267 Ponca City, Okla. 74601 ('405) 762-3456 Ext. 4411 Dr. W. A. Knapp Allied Chemical Corporation P. O. Box 1057R Morristown, N. J. 07960 (201) 455-2814 Mr. William R. Krause Certain-Reed Products Corp. Pipe & Plastics Group P. O. Box 860 Valley Forge, Pa. 19482 (215) 687-5000 Ext. 7478 * Mr. Howard L. Kusnetz Shell Oil Company Box 2463 Houston, Texas 77001 (713) 220-2686 Mr. Robert W. Laundrie Mgr., Ecology, Safety & Health Engineering The General Tire & Rubber Company One General Street Akron, Ohio 44329 (216) 798-3227 Ext. 59 Mr. S. K. Law Process Engineer Kaysor-Century Corp. P. 0. Box 308 Saugus, Calif. 91350 (805) 259-2360 Mr. A. B. Lindquist Stauffer Chemical Co. Product Regulations Westport, Conn. 06880 (203) 226-1511 Mr. Avron B. Magram Director of Marketing Hatco Chemical Division W. R. Grace & Co. P. 0. Box 27 Fords, N. J. 08863 (201) 738-1000 Ext. 301 Dr. R. W. McBurney Diamond Shamrock Corporation T. R. Evans Research Center P. 0. Box 348 Painesville, Ohio 44077 (216) 352-9311 Ext. 401 * Dr. W. E. Rinehart Ethyl Corporation Medical Department 451 Florida Avenue Baton Rouge, La. 70801 (504) 387-0131 j Dr. George Roush, Jr. Monsanto Company Medical Department 800 N. Lindbergh Blvd. St. Louis, Mo. 63166 (314) 694-2188 Mr. Harry A. Russell Executive Vice President The Pantasote Company Greenwich Office Park II P. 0. Box 1800' Greenwich, Conn. 06830 (203) 661-0405 or 0406 Mr. H. L. Schmidt, Jr. Borden Chemical Div. of Borden, Inc. 180 East Broad Street Columbus, Ohio 43215 (614) 225-4882 DTH 000011824 TTGVCP * -3- * Dr. W. Mayo Smith Air Products and Chemicals, P. 0. Box 538 Allentown, Pa. 18104 (215) 395-7215 Inc. * Dr. T. R. Torkelson The Dow Chemical Company Corporate Medical Department 2030 Dow Center Midland, Mich. 48640 (517) 636-5197 * Mr. R. N. Wheeler Union Carbide Corporation Chemicals & Plastics Div. P. 0. Box 8004 S. Charleston, W. Va. 25303 (304) 747-2164 Research Coordinators T DTH 000011825 CYTOGENETIC STUDIES OF BONE MARROW CELLS FROM RATS EXPOSED TO VINYL CHLORIDE R. V. Johnston, D.V.M., D. J- Mensik, B.S., M. N. Pinkerton, B.S., E. B. Whorton, Jr., Ph.D.* Dow Chemical U.S.A. Texas Division - Freeport, Texas 77541 Industrial Health and Medicine Department Biomedical and Comparative Toxicology Research Laboratory Whorton - Associate Professor and Director, Medical Education, Computer Center, Preventive Medicine and Community Health Biostatistics, Univ. of Texas Medical Branch, Galveston, Texas 77550 DTH 000011826 CYTOGENETIC STUPES OF BONE MARROW CELLS FROM RATS EXPOSED TO VINYL CHLORIDE R- V. Johnston, D.V.M., D. J. Mensik, B.S., M. N. Pinkerton, B.S., E. B. Whorton, Jr., Ph.D. The Biomedical and Comparative Toxicology Research Laboratory of the Texas Division of Dow Chemical U.S.A. was asked by the Manufacturing Chemists Association to conduct cytogenetic studies on some of the rats which had been exposed to vinyl chloride at Industrial BIO-TEST Laboratories, Decatur, Illinois. The details of the exposure levels and pathological findings will be reported separately. Preliminary reports have appeared in Chemical Week (115:30, July 17, 1974) and in the Annals of the New York Academy of Sciences, Volume.246, page 219. MATERIALS AND METHODS Animals; The test animals were 20 male and 20 female Charles River CD outbred albino rats which were selected at random from larger groups of 100 each which are being used for carcinogenesis testing. At the time of harvest, the females weighed from 290 to 512 grams, and the males from 605 to 915 grams. The details are shown in Table 1. Exposure: At the time of harvest, the rats"had been exposed in chambers to vinyl chloride gas for 7 hours per day for 5 days per week for one year as follows: Group Exposure Level No. Males No. Females C None Ti 50 ppm t2 200 ppm T3 2500 ppm 5 5 5 5 5 5 5 5 DTH 000011827 togenetic Studies of Bone arrow Cells rrom Rats Exposed to Vinyl Cnloride page 2 Bone Marrow Collection Techniques; On September 5, 1975, approximately 12 hours following the last exposure and beginning approximately 3 hours prior to harvest, the rats were injected intraperitoneally with 1 mg/kg of col chicine (CalBiochem, Brookhollcw Station, Dallas, Texas 75247) using Hanks solution containing 1 mg/ml as shown in Table 1. Immediately prior to harvest, the rats were anesthetized in a car bon dioxide chamber and exsanguination was begun by severing the brachial artery. To obtain the bone marrow before it coagulated, the harvest was begun while the hemorrhage was in progress. The skin and muscles were removed from the right femur, and it was severed with a bone cutter. The bone marrow was aspirated into 6 ml of Hanks Media (Grand Island Biological Co., 3175 Staley Rd., Grand Island, New York 14072). The same technique was immediately followed on the left femur. Necropsy, gross pathology and collection of tissues for histopathology were performed by personnel of Industrial BIO-TEST Laboratories and will be reported separately. Cytogenetic Techniques: The bone marrow cells were processed at Industrial BIO-TEST Laboratories with hypotonic solution and fixative as outlined in Table 2. The fixed cell suspension was returned to the Texas Laboratories for slide preparation and evaluation. Ten slides were prepared from each animal (5 from each femur). The scoring of the slides for aberrations was done by three experienced scorers (#03 of our laboratory and #25 and #26 under DTH 000011828 /* cogenetic Studies of Bone 'arrow Cells _-.om Rats Exposed to Vinyl \,uloride page 3 contract at the Stanford Research Institute, Menlo Park, California) The techniques used are similar to those described in "Methods for the Analysis of Human Chromosome Aberrations" published by the World Health Organization, Geneva, 1973. The slides were coded so that the identity of the animal was unknown to the scorer. Ten metaphase spreads on each of 5 slides per animal were scored by scorer #03, whereas the other scorers each used a single slide to score 50 metaphase spreads. Only diploid or 2N-1 cells were scored. Statistical Findings: Since there were no multiple aberrations, miscellaneous aberrations, or chromosome aberrations, no analysis was necessary. Also since chromatid aberration rates were very nearly an image of the abnormal cell rates, the following analyses were done only for abnormal cells. The analysis variable considered was X=--1. a,b-PeJ-ls number of cells scored - on a per animal basis. Since the variable X is a proportion with an observed range of only .10, no transformation was used for analyses. Uniformly there were 50 cells scored for each animal by each of three scorers, or a total of 150 cells scored for each animal. There were 5 animals per Dose-Sex combination. The data were summarized as shown in Table 5, and the summary of the analysis of variance is shown in Table 6. Statistical Conclusions: At the 5% level of significance, there are indeed no signi ficant differences between sexes, among scorers, or among doses. There is some evidence (at the p = .09 significance level) that DTH 000011829 /togenetic Studies of Bom larrow Cells from Rats Exposed to Vinyl Chloride page 4 sexes may be different. The overall rate for males was .013, while the female rate was .018. At the p * .12 level of significance there was slight evidence that dose effects are sex dependent. It should be noted that both these implications could be due only to chance and "appear" largely because of the results for Dose (T3) females where the rate was .028. SUMMARY Studies by the Biomedical and Comparative Toxicology Research Laboratory of The Dow Chemical Company, Freeport, Texas, show that there was no significant increase in the chromosomal aberra tion rates of bone marrow cells of rats exposed to varying doses of vinyl chloride for one year at Industrial BIO-TEST Laboratories, Decatur, Illinois. /mhi 6-9-75 T DTH 000011830 SLE 1 IN VIVO COLCHICINE FOR RAT BONE MARROW CYTOGENETICS IN ONE-YEAR VINYL CHLORIDE INHALATION STUDY Decatur Research Laboratories Industrial BIO-TEST Laboratories, Inc. Decatur, Illinois Sponsored by Manufacturing Chemists Association Animal Wt.gms Sex 735 M 380 F 663 M 329 F 718 M 402 F 664 M 381 F 769 354 720 356 801 457 659 . 327 M F M F M F M F BIO-TEST Animal Treatment No. Group 54 C 178 C 299 T1 349 Tl 404 T2 594 T2 663 T3 742 T3 69 C 106 C 256 Tl 381 Tl 433 T2 524 T2 668 T3 735 T3 Biomed Culture No. vou^ia2 VC#2, 2A VC#3, 3A VC#4, 4A VC#5, 5A VC#6, 6A VC#7, 7A VC#8, 8A VC#9, 9A VC#10,10A VC#11,11A VC#12,12A VC#13,13A VC#14,14A VC#15,15A VD#16,16A Colchicine DoseJ ml/kg Time Harvest Time .74 6:15AM 9:15AM .38 f 9:25AM .66 II 9:40AM .33 II 9:45AM .72 n 10:05AM .40 M 10:08AM .66 ft 10:23AM .38 II 10:26AM .77 7:45AM 10:45AM .35 II 10:50AM .72 If 11:02AM .36 If 11:08AM .80 tl 11:23AM .46 M 11:25AM .66 (1 11:35AM .33 (1 11:39AM Code 42 47 52 58 66 61 69 54 49 71 73 60 76 44 80 62 915 M 354 F 618 M 366 F 736 M 341 F 624 M 507 F 84 149 205 397 459 550 694 732 C C Tl Tl T2 T2 T3 T3 VC#17,17A VC#18,18A VC#19,19A VC#20,20A VC#21,21A VC#22,22A VC#23,23A VC#24,24A .90 .35 .62 .37 .74 .34 .62 .51 10:00AM ft II It It It 11 It 1:05PM 1:08PM 1:10PM 1:15PM 1:30PM 1:35PM 1:45PM 1:50PM 79 53 74 56 78 64 45 75 605 '355 739 290 679 330 720 371 M F M F M F M F 55 200 264 303 469 569 609 728 C C Tl Tl T2 T2 T3 T3 VC#25,25A VC#26,26A VC#27,27A VC#28,28A VC#29,29A VC#30,30A VC#31,31A VC#32,32A 0.61 .36 .74 .29 .68 .33 .72 .37 12:00PM If t* II II II II If 3:10PM 3:14PM 3:23PM 3:25PM 3:35PM 3:40PM 3:50PM 3:53PM 68 65 70 55 50 41 46 63 649 M 398 F 696 M 512 F 694 M 387 F 757 M 453 F 39 160 279 369 489 599 639 717 C c Tl Tl T2 T2 T3 T3 VC#33,33A VC#34,34A VC#35,35A VC#36,36A VC#37,37A VC#38,38A VC #39,39A VC#40,40A .65 .40 .70 .50 .70 .39 .76 .45 2:00PM tl tf II If II II II 4:45PM 4:50 PM 4:55PM 5:03PM 5:07PM 5:10PM 5:20PM 5:25PM 72 57 67 77 59 51 48 43 1 #1 right femur 2 #1A left femur 3 1Dose - Stock 1 mg Colchicine/ml DTH 000011831 TABLE 2 IN VIVO RAT BONE MARROW CYTOGENETICS BIOMEDICAL AND COMPARATIVE TOXICOLOGY LABORATORY DOW CHEMICAL U.S.A. TEXAS DIVISION, FREEPORT, TEXAS I. MITOTIC ARREST A. Inject l.o mg/kg Colchicine 3 hours prior to sacrifice. B. Inject Colchicine intraperitoneally. C. Stock Colchicine: 1.0 mg/ml in Hanks Media. II. SACRIFICE AND BONE MARROW CELL RECOVERY A. Sacrifice animal with C02B. Remove all skin and most of the tissue from both femurs (try to avoid severing the femoral artery). C. Sever the femur at the distal end. D. Aspirate the bone marrow into a 10 cc syringe (with 20 gauge l3?" needle) containing 6 ml of Hanks Media. E. Place media and cells into a 15 ml centrifuge tube and mix cells vigorously making certain all cells are disassociated. III. HARVEST OF BONE MARROW CELLS A. Centrifuge cells for 10 minutes at 1500 rpm. B. Discard all supernatant. C. Add 6 ml of 0.075 M KCl and mix thoroughly with siliconized dispopette. D. Incubate cells in 37C water bath for 20 minutes. E. Centrifuge 5 minutes at 1500 rpm. F. Discard all supernatant. G. Add 5 ml of fresh fixative (3:1 methanol:acetic acid) and mix thoroughly. H. Refrigerate for minimum of 24 hours before making slides. IV. SLIDE PREPARATION A. Centrifuge fixed cells for 5 minutes and discard all super natant. B. Add 0.8-1.0 ml of fresh fixative and mix thoroughly. C. Drop cell suspension onto slides taken from 6% ethanol solution. D. Flame dry and then stain with 8% Giemsa at convenience. DTH 000011832 * o z aD5 Da u VC #1 9 17 25 33 VC #3 11 19 27 35 VC #5 13 21 29 37 VC #7 15 23 31 39 Eh 2PZ w 2Bi C C C C c t2 T2 t2 t2 T3 t3 t3 TABLE 3 MALE aTS SCORER 03 BIOMEDICAL RESEARCH LABORATORY CHROMOSOME AND CHROMATID SUMMARY SHEET a 1J0 g WU 8to CO uJ u o2 w <A CO twJ"---! MAJ VCuO. QCl) DEJ* HO2 OO2 2 E" CO CO u D\ O CO Cd 2-- 23fi OeM*t--N Ua 0,i o2 CO Cd M2 <A U m < CO Q VcJucoun~ Xo A < 2oA_ CtJOj'T-- CoO \O a zXo Co2O w2o XM u O CO 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 00 00 00 0 .02 0 .02 00 00 00 0 .04 0 .04 00 0 .04 0 .02 00 00 00 00 0 .04 00 0 .02 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 \ CAO Uiwn a g ou CO CJO u 0 0 0 .02 .02 0 0 0 .04 .04 0 .04 .02 0 0 0 0 .04 0 .02 (1) Multiple Aberrations -- a heavily damaged cell containing aberrations too numerous to count. (2) Miscellaneous Aberrations - a cell containing pulverizations, endoreduplications, etc. (3) Chromatid Aberrations -- includes breaks with fragment, breaks without frag ments, intrachanges, and interchanges. (4) Chromosome Aberrations -- includes breaks with fragments, breaks without fragments, dicentrics, rings, translocations, and inversions. (5) Abnormal Cells -- scored as one if a cell contained any of the abnormalities listed in notes (1), (2), (3) or (4). DTH 000011833 TABLE 3 MALI ATS SCORER 25 BIOMEDICAL RESEARCH LABORATORY CHROMOSOME AND CHROMATID SUMMARY SHEET |CULTURE NO. TREATMENT NO. CELLS SCORED MULTIPLE ABERRA TIONS/CELLS SCORED MISCELLANEOUS ABERRATIONS/CELLS SCORED (2) CHROMATID ABERRA TIONS/CELLS SCORED CHROMOSOME ABERRA TIONS/CELLS SCORED (4 ) NO. ABNORMAL CELLS/ CELLS SCORED (5 ) VC #1 9 17 25 33 VC #3 11 19 27 35 VC #5 13 21 29 37 VC #7 15 23 31 39 C 50 C 50 C 50 C 50 C 50 T1 50 T1 50 T1 50 T1 50 Ti 50 t2 50 T2 50 t2 50 t2 50 T2 47 T3 50 T3 50 *3 50 t3 50 T3 50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 .02 0 .02 0 0 00 0 .02 0 .02 0 .04 0 .04 0 .02 0 .02 0 0 00 0 .04 0 .04 0 .04 0 .04 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 0 .04 0 .04 0 0 00 0 0 00 0 0 00 0 0 00 Multiple Aberrations -- a heavily damaged cell containing aberrations too numerous to count. (2) Miscellaneous Aberrations -- a cell containing pulverizations, endoreduplications, etc. ^Chromatid Aberrations -- includes breaks with fragment, breaks without frag ments, intrachanges, and interchanges. (4) Chromosome Aberrations -- includes breaks with fragments, breaks without fragments, dicentrics, rings, translocations, and inversions. ^Abnormal Cells -- scored as one if a cell contained any of the abnormalities listed in notes (1), (2), (3), or (4). DTH 000011834 s33 SSL VC #1 9 17 25 33 VC #3 11 19 27 35 VC #5 13 21 29 37 VC #7 15 23 31 39 TABLE 3 MALL ATS SCORER 26 BIOMEDICAL RESEARCH LABORATORY CHROMOSOME AND CHROMATID SUMMARY SHEET Q 3 w8 Eh V) z MJ U 3 C 50 C 50 C 50 C 50 C 50 Ti 50 Ti 50 50 50 50 50 50 *2 50 To 50 50 50 T3 50 t3 50 50 50 M m to M" WCJJ --_ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 aa w to u D O \in z-- O <N 3 Eh u <2 U wWu Hma Ou2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 K< at w <ftg, in Q Jr'i M -- eh a <Q S o) W oz2 a8 -&2_ -Sfl. 0 .02 0 .02 .02 .02 0 .04 0 .04 .02 0 0 .04 .02 0 0 .04 .02 0 2 w m < in W S Om W'-' o O N. Q o z2 Su 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 toJ wm JQ <s a2 2o o z aw <CD w o 3wJ Z.LL. 0 .02 0 .02 .02 .02 0 .04 0 .04 .02 0 0 .02 .02 0 0 .04 .02 0 ^Multiple Aberrations -- a heavily damaged cell containing aberrations too numerous to count. ^Miscellaneous Aberrations -- a cell containing pulverizations, endoreduplications, etc. ^Chromatid Aberrations -- includes breaks with fragment, breaks without frag ments, intrachanges, and interchanges. (4) Chromosome Aberrations -- includes breaks with fragments, breaks without fragments, dicentrics, rings, translocations, and inversions. ^Abnormal Cells -- scored as one if a cell contained any of the abnormalities listed in notes (1), (2), (3) or (4). DTH 000011835 CULTURE NO. TREATMENT MULTIPLE ABERRA TIONS/CELLS SCORED (1 ) MISCELLANEOUS ABERRATIONS/CELLS SCORED CHROMATID ABERRA TIONS/CELLS SCORED CHROMOSOME ABERRA TIONS/CELLS SCORED (4 ) NO. ABNORMAL CELLS/ CELLS SCORED t5 ) TABLE 4 FEMAI 1ATS SCORER 26 BIOMEDICAL RESEARCH LABORATORY CHROMOSOME AND CHROMATID SUMMARY SHEET VC #2 10 18 26 34 VC #4 12 20 28 36 VC #6 14 22 30 38 VC #8 16 24 32 40 D 8 8 ui w w4 O oz C 50 C 50 C 50 C 50 C 50 T1 50 T1 50 T1 50 VTi 50 50 t2 50 T2 50 t2 50 T2 50 T2 50 t3 50 T3 50 T3 50 T3 50 T3 50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 00 0 .02 0 .02 0 .02 0 .02 0 .02 0 .02 0 .06 0 .06 0 0 00 0 0 00 0 .04 0 .04 0 .04 0 .02 0 0 00 0 .06 0 .06 0 .04 0 .04 0 0 00 0 .04 0 .04 0 .02 0 .02 0 0 00 0 .02 0 .02 0 .02 0 .02 0 .06 0 .06 ^Multiple Aberrations -- a heavily damaged cell containing aberrations too numerous to count. (2) Miscellaneous Aberrations -- a cell containing pulverizations, endoreduplica- tions, etc. ^Chromatid Aberrations -- includes breaks with fragment, breaks without frag ments, intrachanges, and interchanges. (4) Chromosome Aberrations -- includes breaks with fragments, breaks without fragments, dicentrics, rings, translocations, and inversions. ^Abnormal Cells -- scored as one if a cell contained any of the abnormalities listed in notes (1), (2), (3) or (4). DTH 000011836  oz w PEh <34 U VC #2 10 18 26 34 VC #4 12 20 28 36 VC #6 14 22 30 38 VC #8 16 24 32 40 TABLE 4 FEMAL' CORER 25 BIOMEDICAL RESEARCH LABORATORY CHROMOSOME AND CHROMATID SUMMARY SHEET a ws owu Eh to Z W3 sw Eh a < W2 o E* z C 50 C 50 C 50 c 50 c 50 T1 50 TTi1 50 50 T1 50 50 T2 50 T2 50 T2 50 T2 50 T2 50 T3 50 T3 50 t3 50 t3 50 t3 50 i i a2w) canJi--H &u3 \WUwQ HE IzD 2W 33 0M 0U Z Eh W 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ui 3 V) 3 \3wo o to zu z~ O CN 3< W'-* Eh 3w <2 Qw o2 to H aw oo X c oi 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CON w <m Ol 3-- Q 3<n M W-- Eh U <\Q S 01 W OZ2 KOO ZMO O Eh 01 0 0 .06 .02 .02 0 0 0 .02.02 .02 .02 0 .02 . .10 .02 0 0 .02 \ 3 05 W a 2 to U 3--> So Oc/> V3wu-.--erQ SO Uz i2d oo SMU OEM 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 s 01 3 3wmh* U-- 3a 22< ow2 ou Zffl oi C 01 3 OZ O3W 0 0 .06 .02 .02 0 0 0 .02 .02 .02 .02 0 .02 .02 .10 .02 0 0 .02 ^Multiple Aberrations -- a heavily damaged cell containing aberrations too numerous to count. (2) Miscellaneous Aberrations -- a cell containing pulverizations, endoreduplica- tions, etc. ^Chromatid Aberrations -- includes breaks with fragment, breaks without frag ments, intrachanges, and interchanges. (4) Chromosome Aberrations -- includes breaks with fragments, breaks without fragments, dicentrics, rings, translocations, and inversions. ^Abnormal Cells -- scored as one if a cell contained any of the abnormalities listed in notes (1), (2), (3) or (4). DTH 000011837 TABLE 4 FEMAL RATS SCORER 03 BIOMEDICAL RESEARCH LABORATORY CHROMOSOME AND CHROMATID SUMMARY SHEET 8 O tn z g EDhJ DO < gEh ihhWUnJ) #2 C 50 10 C 50 18 c 50 26 c 50 34 c 50 #4 *1 50 12 T1 50 20 T1 50 28 Tl 50 36 T1 50 #6 t2 50 14 t2 50 22 30 TTl2 50 50 38 TC 50 #8 T3 50 16 50 24 T3 50 32 t3 50 40 T3 50 i aa cn < J0w, \uuj->t- in Eh D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 cn w u D O \cn w z~ Z OtN ou tn QoOUucrn. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 < 2 cs H C<Q cn Q ahwJ<--n 0 .04 0 .06 0 0 0 0 0 0 .02 0 0 0 0 .02 .06 0 .08 0 2 OS u m < cn U hl~ 2 JT o ta iOzn `\uOT Qw Xu 0 0 0 0 0 0 .02 0 0 0 0 0 0 0 0 0 .02 0 0 0 a tn 0 .04 0 .06 0 0 .02 0 0 0 .02 0 0 0 0 .02 .06 0 .08 0 Multiple Aberrations -- a heavily damaged cell containing aberrations too numerous to count. (^Miscellaneous Aberrations ~ a cell containing pulverizations, endoreduplications, etc. (^Chromatid Aberrations -- includes breaks with fragment, breaks without fragments, intrachanges, and interchanges. (4) Chromosome Aberrations -- includes breaks with fragments, breaks without fragments, dicentrics, rings, translocations, and inversions. Abnormal Cells -- scored as one if a cell contained any of the abnormalities listed in notes (1), (2), (3) or (4). DTH 000011838 / Sex of Animal Scorer Number Males 03 25 26 Total Females 03 25 26 Total TOTAL VBLE 5 STATISTICAL SUMMARY C .009 .016 .012 .012 .02 .02 .012 .017 .015 Doses Ti T2 .016 .02 .02 .019 .012 .00 .012 .008 .004 .008 .024 .012 .004 .016 .028 .016 .016 .012 T3 .012 .008 .012 .011 .032 .028 .024 .028 .020 Total .012 .014 .014 .013 .015 .018 .022 .019 .016 Scorer 03 25 26 Total C .014 .018 .012 .015 Doses Ti T2 .010 .008 .014 .008 .022 .016 .020 .012 P = .50 T6, .022 .018 .018 .020 Total .014 .015 .018 .016 P 60 Sex of Animal Male Female Total C .012 .017 .015 Dose T1 T2 .019 .008 .012 .016 .015 .012 p = .50 T3 .011 _. 028 .019 T Total .013 .018 .016 09 DTH 000011839 f TABLE 6 ANALYSIS OF VARIANCE SUMMARY Source of Variation* df mean squares F ratio p-value Total 119 Sex Dose Sex by Dose Animals in Groups Scorers Sex by Scorer Dose by Scorer Sex by Dose by Scorer Scorer by Animal in Groups 1 3 3 32 2 2 6 6 64 .00108 .00028 .00073 .00034 .00022 .00007 .00026 .00026 .00045 3.176 .810 2.156 -- .490 .153 .568 .564 .09 ns .12 -- ns ns ns ns Hypotheses I* H0: *4n = HA: P = -09 H H Hq: ^03 = ^25 = ^26 P = .60 ha: At least one scorer is different III. Hq: Pc " Pi = ^2 = ^3 p = .50 Ha: At least one dose is different IV. Hq: Doses and Sexes are Independent ha: Dose effect is sex dependent * 1. Sex, Dose, Scorers considered Fixed Factors. 2. Animals considered random. Hq -- Null Hypothesis Ha -- Alternate Hypothesis ym -- Mean for males -- Mean for females 'T DTH 000011840