Document mBmw0GRnXL78GMv8GZx3a11dJ

AA I 4^ K- 003dtf-0% DOV: CHEMICAL - > ~(r~) KloS* COM PA MY TEXAS DIVISION FREEPORT TEXAS Texas Medical Department Report No: TMD 64-3 Date: Oct. 15, 1965 PROGRESS REPORT: ^CONCLUSIONS: CHROMOSOME EVALUATION OF ANIMALS EXPOSED TO CHRONIC LEVELS OF ETHTLENIKINE AFTER 7 MONTHS EXPOSURE. D.J. Kilian, M.D. RESTRICTED R.L. Schwebel, D.V.M. .Marjorie S. Benge, B.S FOR USE WITHIN THE DOW CHEMICAL CO. Or. March 13-26, seven months post-exposure, the animals used in the chronic exposure study on ethylenimine, conducted at the Biochemical M Research Laboratory in Midland, were sacrificed. Bone marrow preparaV'tions following in vivo colchicine were taken on all species (except canines) as opposed to the conventional tissue culture methods used at the ore-exoosure and two month post-exposure phases. (See reports No. TMD 64-1 and TKD 64-2). 'Considerable time has been devoted to this project with 279 chromosome preparations made, 2790 slides stained and filed, 309 photomicrographs taken and six karyotypes prepared. All the slides have been so pre pared as to last indefinitely for future reference if the need anises'. Thorough investigation or each slide would take two people approxi mately five years to complete. Such time expenditure is impossible r*c unwx s 3 * not been possible because of the tremendous number of man hours in volved in such a project. s I 0 K. . l 1 ^ X, " s T-o. y / "V 4` t'vV-. <- k t' C-JUOS m <'/r. Distribution: !>t-\ f e AC-'4 r r c \~ Uky It' i\i "-V. ' K C~ r, ^ Texas Division:: Dr. E.3. Barnes, T.E. Brown, R. Buell, C.A. Christian, J.M. Leathers, H. McClure, M. Pruitt, K.M. Tolleson, Medical Department. Midland Division: B.B. Holder, M.D. Corporate Division: K.L. Gordon, M.D., R.H. Boundy, V.K. Rowe., D.D. Irish, R. Stewart, M.D., J. Johnson. Texas Bioproducts: V. Johnston, D.V.M. Midland Bioproducts: W.R. Dickson Pitr.an-Moore Division: 3. Slomka, M.D., J. Robertsen, D.V.M., T.L. Kerley, ?h.D. -- Tairas trvrston .'.rd*ar.a urvrsron n) mm& 00 14 O.ONr TDFNTTu; Zl~ 18 !SS5 CRESEARCH ndex 2 During our course of chromosome investigation the following experts outside the company have bean consulted at various times for their nel? and guidance on the technical aspects of chromosome preparation and*regarding the need and interpretation of the work. Mary L. Alexander, Ph.D., Genetics Foundation, Department of Zoology, University of Texas, Austin. Marvin Fuchs, M. D., F. A. C. ?., Internist and Allergist, Washington, D. C., and consultant to Chemirad Corporation, producer of ethylenimine for many years. A. Clark Griffin, Ph.D., American Cancer Society, Professor of Biochem istry, University of Texas, M. D. Anderson Hospital and Tumor Institute, Houston. Charles K. Hine, M.D., Professor of Occupational Medicine, University of California Medical Center. T. C. Hsu, Ph.D., Section of Cytology, Department of Biology, Universi ty of Texas, M. D. Anderson Hospitalsnd Tumor Institute, Houston. Don Micks, M. D., Professor, Department of Preventive Medicine and Public Health, University of Texas Medical Branch, Galveston. Harvey Rosenberg, M. D., Pathologist, Director of Laboratories, Texas Children's Hospital, Houston. Anil K. Sinha, Ph.D., Geneticist, Department of Pathology, Texas Children's Hospital, Houston. Wilson S. Stone, Ph.D., Professor, Head of Genetics Foundation, De partment of Zoology, University of Texas, Austin. - Doctor of Medicine Theiss, Medical Director, B. A. S. F., Ludwigshafen, Germany, producer of ethylenimine for the past 30 years. Wayne Truax, M. D., Assistant Professor, Hematologist and Cancer Chemotherapist, Department of Internal Medicine, University of Texas Medi cal Branch and former member of the Atomic Bomb Casualty Team at Hire shima, Japan. Shung-Jun Yang, Ph.D., Geneticist, formerly of the Department of Pathology, Texas Children's Hospital, now with Stanford University in California. DO 14?fVi7 CONFTDFNTTA 3 s: The attached charts are summaries of the results of the blood culture samples of the pre-exposure sad two month exposure studies (F.eport Zos. HZ) 64-1 and HZ) 64-2) and the blood culture and/or in vivo colchicine bone narrow preparations of the seven month ex posure group of each of the animals tested. These charts also in clude the progress to date on numbers of slide preparations made, cells photographed, and karyotypes done on each animal tested. Discussoon: At the terminal phase (seven month post-exposure) bone marrow preparations ware cone on rats, guinea pigs and rabbits by adninis- . tration of in vivo colchicine in dosages of 2.5 mgm/kg three hours prior to sacrificing-. In view of soma questionable findings in the blood chemistries, which were possibly due to the in vivo colchicine, conventional tissue cultures (as dona on the previous controls) and bone marrow preparations were done on the dogs. Fibrinogen increased in the tissue cultures to the extent that most of the preparations were not satisfactory for chromosome investigation. The bone marrow preparations were processed by placing marrow from the femur in Difco tissue culture media, adding three meg col chicine to each culture to arrest mitosis, incubating for two hours, than swelling the cells in hypotonic citrate for 30 minutes at 37.5C, and "fixing" in the usual manner with Camoy's. Control dog #905 (pre-exposure to air) and dog #908 (two-month exposure co~3.o ppm ethylenimine) have been taoroughly^investigatexL-- The remaining 560 slide preparations on canines have received only scattered attention. From the photographs made at this time no significant abnormalities have been seen. Three rabbits (#237, 252, 177) of the seven month exposure to 3.5 ppm and one rabbit h-179) of the unexposed controls have been thoroughly investigated. These four revealed an extremely high in cidence of_an.aup.loif.y. Because cf these findings a geneticist, Dr. Aral A* Sinna, iexas Children's hospital, was consulted. It was his opinion that these findings ware probably due to technique. Zona of the 830 slices prepared on the guinea pigs have been closely investigated. From the few photographs made at this time, no abnormalities have been seen. From the rats a total of 910 slides have been prepared for investigation. These are from the two month and seven month expo- -- S o ~ ^ps or._y. j.ne pre-exposure samples were unsatisfactory ^ __ ytootems. From the photographs made at this time no gross enromosome cnanges nave seen seen. no 1 V'eW -) .-.dnft.ifnt . 4 G w 3H lunv3 S being on recovery for three months bone marrow prepa- done onv nine~ ^ W rmatwsw which had1V*Apip AM beenW w -- -- e-- xp- osed to 5.0 ppm w -- r- n-- ---- Tan slides per animal ware prepared but have not a. o available on file are two bone marrow smears per each of 49" ,ts that had short term a::poava:e to ethylenimine. No c'nromo some nvestigafions have been made on tnese. \ O'-' \ >'-V- " ,T - * ' f^ ' .-oh in:'. C.ANIWI' r v.C,f V \\\. no. r*;:r- 'U "U rt* X F.r.f'o:.ur:r. v r o f1 V f." O uu>. cuuto:., ?! //'//<..i;. t* " rVC` *jAu; t'.wrni ostnci: 'VH. CWilM;. /'Hiosts nj wr v *f11- i^r>>- 3"o C* fo. t* <7i1 - I* i: <# 'em . Uo SMuj-lc; Hi M o u l-k> Semple j II AH'. COUiUOL':- EXI'u-UlUi r SC.* n cm Me. iiij Mbmj Vo K> ?o v. * -- -- ------------ ---------- -- M 91A r 90 6 /W '.'CC/nci >;V M 90S h 9 AH Few Mfciuj Hci S.-mpHs VO ?. VO ?. I SomeSen. ,'j ?.o 0 20 6 -- i-xrMiu i> Mbru) 20 16 Ho Simples 11 --.... V -TtFiKtU/.LMONTH t VTOUUE l-.A-.1l 1 i;__ "I) 'OO <-^ ; V 1' u oXa-i So /DV1 ^r O' CUt'lUilF. /-Hi //V PilrPAliM loUS S/-7/io:r/r n P- ^4 r.. j*rQ> vol J f.i 7h S'" ; j c* Severn- f'C'Cir due "io flbim. II ... . Csii:1'-,'j 11 Semm- Cmr'i ji.iiviM :' SOmr " pofiv due io fibrin. 11 11 ,1 V, :(l ` i 11 30 i i 30 t ?. ?.. ?. 2 W\\V\CEP Gc'vnpoor cim: it! fibrin II it n 50 i 30 i 2 2 o.as rm F 907 17.PD5UUU M 913 CMS PPM Exposure F M rornis <505 QIV n oa 2o *n "4 r-i a^ ~o \> z^ H .V) rs Menu) Msni) Maraj Momj 20 1 20 3 * t 20 2 5 20 1 iCO 30 / Soma Some 20 5 20 5 Some Some 20 A VO 6 #0 Semi"* poor <iu: Vo fibrin. Some. Soma -- poor due to fibrin u 11 30 i 2 11 30 i 2 30 i 2 11 SO i 2 360 /<> imFyiWh CLV.uuAS F'RV - ""VV C%* V /an; ;;x no. n.ROV'ZF. nu>. cufui;;. vl rv. [- /?h'osis M Z`>fs, 219, 7`y1 , 201, ?07 ' j f I/O, tm, ?f.i} zc.-i, 26'; AIR EXIWVi COWiKOtf M 250 Funcjui coni. 10 M 187 Mdrnj 10 M- 707,F-20C , F-272 %.'> fTil ekroairr M 229 M 232 M 2?8 F 177 F 257 F 252 Mam; M anij 10 10 0.45 [Til EXPOGUUi: M 173 Many M 100 Mamj M F- 236 ,F-290 10 io o>n viw typO'A'Rt M 299 Mani) 10 M 230 Mani) 10 M- 297, F-72A , F-747, F-262 70/M5 so il AO OF If. ;1 co* CD oO UHoV.POTCO CONTROL mr EXPO "0 COH't i ;0L5 GUINEA Pi&S PRf.- EX AKIMAL NO. EXPOSURE tiLD. CULTUfl. X- Ah1os is . ... ... ......... i 77 3.0 T7OJ 7IS< ~r> Q? jro: -o ,r 7tO*- 7A* 2 month EXPOSURE on*, annul;, T> un Ir/* Vp s r* O 2* CL i> P)ilosis ~o o O2 -*u :r CL O' Xo X y p NONE-- - Cental un&l .'8 59St-00 Manq 10 9 1 M 5907-01 Manq 10 2. M 59 67-01 HO SAMPLE HO SAMPLE M 5957-01, 5959-01, 5961 -01, 5967-00, 5964-00, b0( 6-00 i \ \F 0034-00. 6035-01, 603' ,00, 6037-01, 6039 01, 60' O-OC , 60' l-O), 6047-00, 604 5-01, 6044-00, 6045-01 I -TtRK IHAL7 MONTH E xposuue BLOOD CULTURE hhiosis B0HE M/.K..0VJ PRLPARATlOtlS 3" Z^?/ion's 7oSi C&t n < on O fC Xs -oi- 1% ' i -u7 ii J* ! i j l i X* *o c* \A \1 Mdnq Manq UO SAMPLE Maruj Plant] 10 | 10 l \ 60 i j i 110 1 i t Cf , ri \ w o 2 2 12 22 5.0 PPM exposure 1.7 PPM EXPOSURE M 5971 M 5968-00 Manq Manq 10 10 10 10 EXPIRED M 5972-01, 5973-01, 5971 -01, 5976 -00, 5977-01, 5978r00 F 6048-Qq 6049-01, 605(1 '-00, 6051 -01, 6053-01,6055-01, 605E 00, 6057-01 M 5980-00 Manq 10 6 ! M 5984-00 CIOTIED BLOO p-N< t qroilrih \ M 5901-01, 5987-01, 5983-01, 598 5 T 01, 5907-01, 59&E -00, 59 &< -ot, 5990-00 , 5991-01 F 0058-00, 6059-00, 606 9-00, oovO 6067-00, 606 POO, 6065 -01, 60 67-01,6068-00 Plant] Manq Mantj Many Manq Manq Manq 10 60 BO i 10 10 90 90 2 12 16 Z 1 18 18 1 0.7 PPM EXPOSURE M 5992-00, Manq j 10 l M 5975-01 Manq 10 l i i M 5993-01, 5994-00, 5996 -00, 5997 {oil 5999-01, 6000 -00, 600 -01, 6001-00 F 6070-00/ 6071-01, 6077-00,'. 607'i-01, 6074-00, 607 PCX) 607 -01, l 6078-00 , 607 91 -01.' -00, 6081-01 J Manq Manq Manq Manq to to 80 no 2 2 16 22 TOTALS 70 3? / 11 * 750 ISO GUINEA PIGS uiMF.yroc.'o COilTROl Air, EXPOSER cdhykols sex A til MAE NO. mexposure ILD. cuuur # Tlifosis 5936 [ M 2 MOUTH exposure OLD. CULTHP T PTfosts HOME ConlavmnaVcd M 5956-00 Many f-1 5963-01 Many M 5967-01 NO SAMPLE NO SAMPLE M 5957-01, 5959 01, 596101, 5962-00, 5964-00, 59K--00 F 6034-00 GOiS-ot, >035-00, 6037-01, 602-9-01, 604 0-0( 60- 1-01, 6042-00, 6043-01, 6044-00, 604 S- 01 !1 ' -TEFU41H AL~ 7 MONTH EXPOSURE BE 00 [7 culture TViorif DOME MARKOV/ PREPARATIONS TJ n eJjj- 5 Many Many HO SAMPLE Many Many 10 10 60 HO 5.0 PPM EXPOSURE M 5971 M 5968-00 Many Many EXP1RID M 5972-01, 6973-01, 5975-01, 5917-01,597*1-00 F 6048-00, 6049-01, 6050-00,*6051^01, 6053-01,605^-01, 6056-00, 6057-01 Many Mamj Many 10 60 60 .?. PPM exposure 14 5980-00 Many to M 5984-00 CLOTTED BLOO|D-N^ qrotblh M 5981-01, I 59 82-01, 5983-01, 59857i 01, 5967-01, 5986- F 6058-00, 6059-00, GOfcb-OO, 6061!00, 6062-00, 606(1 ,59&c 01 - ,6065 01 5990-00, 5991-01 6067-01,6066-00 Many Many Many Many 10 10 90 90 f;ONF T o.i pm EXPOSURE a M M M F TOTALS 5992-00, 5495-01 Many Many 599301, 5994 - 00 , 5990- 00, 5997j 01, 6070-00 6071-01, 607^-00,; 6073-01, 6078-00, 6079*01, j 608C -00, 5999-01, 6000 6074-00, 607 60&1-01 70 j 39 6001-01, 607 -01, 6002-00 } Many Many Many Many 10 10 80 HO 750 utiF^r'Otfb COilTTiULC Am EXT USER CO'.Ti'UOLS 1............ SR A 9) MAE NO. PREF.XP05URE Li LI). COUtUR, # Ahiosis -v Ss oos-'^* x;>r RATS 2 MOUTH exposure til VI VO COUII. cons ///;/?/,'p i7 IlA vr wn pn. r, D- po-* n = -to* * t*> J'hiosts -TEfUAmM.- 7 MONTH eXPOSHPst BLOOD CULTURE /'hiosis CORE MARKOV! PREPARATIONS 3?' /Viiosis on* j?jO-c<f o rU*- 4 uo^^ ': M 123-01, 124-11, 175-02 I? /-17 , 120-21, 130-01,| 132- =11, ljjj-02, 134-20 i Mang 100 n 157 Mang M 150 Mang (4 464 14 159,160, IGA, (05, (66, (67, 164, 70 F 459,462, 463, 4 GG, 4(>G 467, 4 69, ,4 60, 470 10 8 10 6 SA :rificed $/ R1F1CED |( HONE--lube broken; Mont) 80 Mang 90 3.5 F?n EXPOLAUit 14 191-00 Man*) 10 12 57 :rirced M 192-01 Mang 10 12 Si :fuficed IT 195-00, 196-01, 190-U, 199 02-, 20b-?.l, 202-22, 203-11J 204-02, 20542, 206-21 F 495-00, 496-01, 49`HO, 500 ?l, 001-00, 503-10, 5Q4-1|,50x12, 506-21 Mang Mang 100 90 O.AF PPM EXPOSURE M 279 Mang 10 6 57 R1FICFD H 230 Mang 10 10 $7 :r\f\ced M 233-10, 235 02, 257-00.23&-01, 2394Cl 240-H, 241-02, 242-18 F 543-12, 044-01, 545*10, 546T1, 0^7-02. 54&-20, 549 00, ^5l-l(J>, 55 S-02, 554-21 Mang Mang 80 100 0.15 PPM EXPOSURE M 265 14 266 Mang Mang 10 7 10 7 5A ;rirced SA R1FICED M 267-00, 268 01,270-11, 272-2|0, 273-01, 274-10,275-11, 2l6-q2, 277-12, 278,21 F 567-00, 568-01, 569-10 570-111,571-02 572-12, 573-00, 574-61, 578-20 TOTALS oo zo TJ 3 -P "n \> z S' HM >H > T BO 60 Mang Mang 100 90 830 SEX 5.0 PPM EXPOSURE ^ L>i M M M M F F 1F F ANIMAL no. 2OB-01 209*01 210-11 ?.ie>- \l IN VIVO coixmcme DONG MARROW T) -* %fSj k cnU __________________________________________ Poor * n k 10 to 10 to lhijlene.nTiine. - Recovery 3 Ho%. Post-Terminal Exposure REMARKS Bone marvoio preparations from both Temuri were mode in the same manner as described for the doqs in the "Discussion" section of fnis report, (rorofjraph 2) 612-10 513-00 614*01 516-11 517-02 Pom or * tf n to 1 Over an hour elapsed before -these femurs were to put info the media. Asa tesolV bone marrous 10 Smears were not attempted, Koweoer^ "Dr. Sadefc > to Pathologist, Midland Division, has some Smears to on the animals.