Document jyqJ7Z5jeowvnn8MVnXq2KrVR

-57APPSNDEX 3 (C o n tin u e d ) CNDIVIOUAL AND SAN "OOO CONSUKPIQN O f ALS RATS NAlNTACJIED ON DIETS CONTAINING 2 , 4 , 5 - T "OR l TEARS Dose rt-jj 'icc/auy 30 -A n ic ia l Number 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 875 376 377 378 379 380 381 332 383 384 385 386 3S 7 388 389 390 391 392 393 394 395 396 397 398 399 900 901 902 903 904 905 906 907 908 909 MEANsSD 91294 26 25 23 26 23 26 21 25 25 23 U 5319 26 6 26 24 24 24 24 28 24 25 j 19322 26 27 27 26 25 27 30 27 27 23 350354 27 25 23 25 25 26 26 29 30 17 373382 28 23 27 21 n HD 24 NO 29 Days an T e s t an d C ram s.r?.ac./O av 4 0 6 - 4 3 4 - 4 6 9 - 4 9 7 - 532 - 6 0 4LQ 438 473 501 5 36 564 536392 26 27 20 26 77 J3 25 24 24 23 22 M 26 NO 26 27 L9 25 25 25 23 24 25 36 24 22 24 22 23 24 28 16 71* 26 24 25 24 21 21 36 24 24 23 17 26 25 29 2 5 Oead* 23 25 24 26 31 13 25 26 13 26 25 - 26 23 6 2 3 - '6 3 1 527' 655 23 19 2* 24 536590 ' )* NO 7L4713 Oead 20 20 28 28 33 ' 1 Otta& ' ' 28 17 . Dead* 1 20 30 13 23 . 25. 36 Dead --O ead ...... -- Oead" 1 ' 1 1 1,1-- 25 23 37* 22 19 . --Dead' - 1 Oe a d 24 -D ead- )ad* 49a 29 - 31 30 32 30 --O e a d - -O ead* 22 17 19 -13 Dead -------------- 27 26 23 ... 2 3 .. 23 21 20 20 --D ead 1,1 11 " 26 v 20 22 14 -Dead- 'W -Dead t m " ' 2 5 :1 27 : i ' 5 -3 2 6 :4 2 5 :2 2 6 :2 2 4 :6 4 t 4 2 5 .3 2 7 :6 Dead' Oead Dead d* Dead' 26:7 20:4 NO * Mo l a c a a v a i l a b l e . aV a lu e s e x c lu d ed from s t a t i s t i c a l a n a iy a is c a lc u la tio n s . 3S c a c is c tc a ly s i g n i f i c a n t d i f f e r e n c e :rom co a c ro L ^ a n u s in g D u n n e c t's c a s e , ?<0.Q5 SUS"l3 -258APPENDIX 3 (C o neiaued) INDIVIDUAL AND MEAN FOOD CONSUMPTION OF MALI RAIS MAINTAINED OF DIETS CONTAINING ' , , 5 - 1 ? 0 8 2 YEARS Ooae :i ; < z/d.iv A aixai Number 75- 0- a47 H Pays on Tasc a a c Q gaas/P.ac/D ay L I - 1 4 - 1 3 - 2 1 - 2 5 - 2 3 - 3 2 - 3 5 - 3 9 - 4 2 - 4 6 - 4 9 - 5 3 - 36-- 6 0 - 6 3 - 6 7 - 7 0 - 7714 13 21 25 23 32 35 39 42 46 49 53 56 60 63 67 ro 74 31 :o 310 311 24 23 26 26 27 26 26 26 26 26 26 23 23 2* 27 7 25 27 26 23 27 312 313 22 26 25 24 25 25 25 25 24 25 23 23 n 25 25 25 16 23 25 24 25 24 314 315 26 30 29 23 23 :s 27 23 26 24 24 25 23 27 27 25 26 25 25 24 26 26 316 317 24 26 22 26 27 25 25 27 26 26 20 25 27 26 25 : 25 24 ND 25 26 25 818 319 26 28 26 25 27 25 24 26 25 26 ND 25 27 26 26 ND 26 ND 26 24 26 26 ----- 3 2 0 24 29 27 # 23 . 29 23 27 23 26 27 27 26 s '2 9 27 23 27 26 ND 27 25 26 26 321 322 24 26 25 25 25 25 23 24 24 24 24 23 25 25 24 23 23 22 26 22 24 23 323 324 24 29 27 29 29 23' 23 30 27 27 27 23 30 30 23 27 23 26 23 28 29 23 325 326 827 24 26 26 26 25 25 24 26 25 24 25 26 27 25 25 25 25 ND 25 24 25 26' 328 329 24 25 25 25 26 25 24 26 24 24 25 25 27 25 25 24 24 24 25 24 24 25 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 353 359 MEANiSD z l 27*1 26*2 2 5 r2 1 7 :1 2 6 z l 2 5 s l 2 7 - 2 2 5 s l 25a2 25-1 27i: 26z2 26si 2 5 tl 25s3 24z2 2 6 ti 25z2 26z2 2 6 z l ND No d a t a a v a i l a b l e . -259APPENDIX 8 C a a c i n u e d ) INDIVIDUAL AND FOOD CONSUMPTION OF *AL XATS MAI2TAIN2D N 01 CTS CONTAINING 2 , i , 3 - T F 08 2 YARS 'Oose rag/`* g / d ay LO Anim ai Number 75- 310 311 312 813 3L4 315 316 317 318 319 320 321 322 323 324 3 5 326 327 328 329 330 331 332 333 334 335 336 33? 333 339 340 841 342 343 344 845 346 347 343 349 350 351 352 353 354 355 356 357 358 359 3L- 34- 33- H - 34 38 n 27 23 27 23 24 24 24 24 26 27 26 25 26 25 25 25 26 25 25 21 26 26 26 26 25 25 24 25 23 23 23 28 24 25 25 25 21 25 25 25 NEANsSO 2 6= 1 2 6 2 1 2 5 * 1 25=1 SD No d a c a a v a i l a b l e . 123* 27 21 27 25 25 26 27 23 24 23 26=2 D ay3 on T a s e acid O c a p a /a a c/O a y 123* 151- L30- 182- 203- 126 151 154 L32 136 207 207210 26 26 25 ND 25 26 29 24 23 24 ND 25 24 26 26 25 21 27 26 24 27 26 25 21 23 26 . 25 28 24 25 21 26 21 24 26 26 25 21 27 21 24 26 26 23 23 ND L7 23 27 23 27 27 29 23 26 29 21- 21 22 26 21 23 25 22 25 21 27 25 25 26 25s2 2 5 tl 21=1 27=1 21=J 2 4 r l 27=1 231235 29 25 26 27 27 26 28 19 24 21 25=2 235223 259253 SD 29 27 25 26 26 27 26 26 26 25 25 27 25 27 27 24 21 24 25 2 6 rl 26=1 263266 :ro 25, 25 25 27 27 24 ,27 23 26 25=1 23 7 291 25 25 25 27 25 24 21 27 24 25 25*1 oLlS't^' 260- APPENDIX 9 ( C o a c i n u e d ) ,* INDIVIDUAL AND MEAN *00 0 CONSUMPTION OF MALE U T S MAINTAINED ON OISTS CONTAINING 2 , 4 , 5 - T FOR 2 TEARS Oose n c /'A i/d a y 10 A niaal Number 75" 310 311 312 313 314 315 316 317 913 819 320 921 322 323 824 323 326 327 328 329 330 331 3 32 333 334 335 336 337 338 339 340 341 342 343 344 843 346 347 349 349 330 351 352 353 334 355 356 357 358 359 291294 . 29 25 37 27 27 24 25 29 25 25 MEANtSD 2 6 :2 315319 27 24 27 26 25 25 25 26 24 24 319322 28 25 28 27 2/ 26 26 29 27 27 350354 29 24 26 25 33 31 25 23 31 25 373332 39 24 27 25 28 25 24 27 23 NO :0 6 410 Days on T ese and O caam /R ac/D av 4 34 -- 49- *97- 532- 433 473 501 536 27 77 23 23 24 23 24 26 27 22 27 25 20 14 12 24 24 26 25 '* .............. . "* 1 22 13 22 22 ND 26 24 27 27 21 25 25 24 24 26 22 21 24 25 23 31 24 560564 538592 523527 631j5 5 29 U * 16 29 i t 24 19 23 24 26 29 20 ----------------------------Dead- "Oe a d .................. -- -- 25 -D ead-- IS 24 36 19 25 25 19 35 20 23 -D ead-- --Q e a d 25 -D ead-- 27 .................................................. -D e a d -- 5^ 22 20 15 2 7 :1 2 8 :3 2 7 :5 . 24:2 2 3 :4 16:3 24-1 2 9 :6 -D ead-- -- Dead- 2 3 :3 2 4 :1 -Dead-Dead- 636390 714713 -- Dead-- i D ead 24 22 26 26 - - 'D ead 23 20 Dead ND * No d a c a a v a i l a b l e . ^V alues ex clu d ed :ro a s c a c is e ic a i a n a ly s is c a lc u la tio n s . .1 .1 ,, .i J 1 9-4 51 (o A??ESfDIX i ( C o n t i n u a i ) INDIVtDUAL AMD AM'?OOD CONSUKPTON OF MAL* RATS MAIMTAIMED OF QtETS CONTAIMI'IG 2 , 4 , 5 - FOR 2 TSARS Dosa n s/< /d a v A a ta ai Mmnoar 75- 07 7li L itjj 13 13Vt 25 P ay a oo T gaa .and 3 c a n > a /3 a c /0 a y 25- 23- 3 2 - 35- 3 9 - 4 2 - 46-- 4 9- 53- 56- 6 0 - 6 3 - 5 7 - 70 - 7723 32 35 39 42 6 4 9 53 56 60 63 57 70 74 81 3 780 761 26 26 26 27 27 26 25 25 26 26 25 27 25 25 24 25 24 25 24 25 24 762 763 23 26 24 25 25 24 23 21 22 24 24 72 u 1 19 22 21 22 20 22 21 22 29 764 765 22 25 2* 24 26 23 24 23 24 23 23 24 25 25 24 24 . 24 23 24 22 23 23 766 767 25 27 26 29 30 29 27 26 26 28 26 26 33 28 23 26 25 23 26 26 34 23 768 769 25 31 30 23 30 29 23 30 27 27 23 23 31 30 29 MD 23 27 23 27 28 23 770 771 23 25 24 24 25 25 25 26 25 24 25 24 26 23 24 23 23 22 24 22 23 24 772 7 73 23 26 25 26 26 26 25 25 24 24 25 25 27 26 25 u a 25 24 25 24 25 ` 25 774 775 23 25 25 26 27 26 26 25 25 24 23 23 25 25 25 23 24 23 25 23 24 24 776 777 24 26 27 27 27 27 25 26 26 27 27 26 23 23 27 26 26 25 27 25 26 26 778 779 26 23 23 23 23 29 23 23 27 27 27 27 30 23 23 26 27 27 29 27 23 23 780 781 732 783 734 785 786 737 788 789 790 791 792 793 794 795 796 797 798 799 300 301 302 303 804 305 306 307 808 309 MEAMSO 24=1 Z6=2 2 6 2 2 b r i 2 7 2 26=2 26=2 25=2 25=2 25=2 25=2 25=2 2 3 r3 26=3 26=2 24=2 25=2 24*2 26=2 24=3 26=4 25=2 MD " Mo d a t a a v a l l a b l * . V a l u e s a x c lu d a d crota s e a e i s c i c a l a n a l y s i s c a l c u l a c i o c a . SU 5\1 APPENDIX 3 ( C o n c i n u a d ) INDIVIDUAL AMD MEAN "OQO CONSUMPTION OF MALE ?-ATS MAINTAINED ON DIETS CONTAINING 2 , 4 , i - T ?OR I 'CZk&S Dosa = a/k*/3av 3 A nim al Nuobar 75- 760 761 31- 34- 38- H 34 38 n 2 1 23 24 25 24 762 763 u a 22 22 21 764 765 23 24 22 23 766 767 29 27 27 26 788 27 23 27 30 769 770 771 22 24 23 24 772 25 25 24 25 773 774 775 23 25 24 24 776 777 25 27 26 27 778 779 27 23 27 27 780 781 732 733 784 785 786 737 788 739 790 791 792 793 794 795 796 797 793 7 99 300 301 802 303 304 905 306 307 308 309 MEANsSD 25= 2 2 6 * 2 25= 2 25= 2 U 9123 25 21 23 26 27 24 26 23 9J 26 24*2 Dava oa "33c and O caas/R ac/Q ay 123- 147- 151- 130- 132- 203126 151 154 132 136 207 207210 25 24 23 25 25 24 25 22 22 21 22 21 20 23 24 23 22 24 22 22 26 26 26 25 27 32 26 23 23 23 23 30 23 26 30 24 23 23 25 24 24 '2 5 26 25 23 26 24 26 27 23 24 22 25 24 29 15 27 25 25 SD 19 25 23 27 26 26 29 27 20 33 25=2 25=2 24*2 26=2 25*4 24=3 26*5 231235 26 22 22 27 29 25 24 23 27 23 25=2 235233 239263 25 25 22 21 24 23 25 26 23 29 25 23 27 25 25 24 27 27 27 28 26*2 25=2 253266 25 23 23 27 23 25 26 25 27 29 26=2 237291 25 23 23 23 24 27 24 26 27 25=2 V a lu e s ejcciudad r a s s c a c is e ic a l ta a ly s ia c a lc u la tio n * . -253- APPENDIX 3 ( C o n t i n u e d ) INDIVIDUAL AND SAM :OO0 CONSOMPTION OF MAI* 1ATS MAINTAINED ON DIETS CONTAINING 2 , 4 , j - r FOR 2 `iA3.S Dose a ic /k g /d a y J Suaoer 75- 760 761 762 763 764 765 ' 56 767 753 769 770 771 772 773 774 775 776 777 778 779 780 731 782 733 784 735 786 787 788 789 790 791 792 793 794 795 796 797 798 799 300 301 302 303 304 305 306 307 308 309 291294 27 24 27 30 25 25 24 29 29 315319 24 23 27 25 25 24 27 27 3L9322 25 350354 25 25 24 29 23 30 26 26 26 27 26 25 25 26 16 29 29 373332 25 24 31 39* 25 26 24 30 23 406*10 26 Paya oa Case and C ram s/R at/D av 69- 497- 532- 433 473 501 536 , .a 24 25 25 24 21 23 26 25 25 24 25 27 MU ' 26 25 20 22 25 25 24 24 24 22 22 3. a 26 29 30 20 MU 23 24 28 26 22 20 27 24 22 27 23 27 560554 2^ 588592 523627 23 551555 9J 536690 714713 -- --Dead-- 23 --D e a d -- 23 Ha 26 --D e a d -- 4L3 23 25 27 26 25 25 23 28 26 27 . 23 24 22 De a d - .......... 29 19 -- 25 22 -- 20 --D ia d - 25 L l1 21 Dead 34 71 12 Dead 23 Dead NO So d a t a a v a i l a b l e . ^ V a lu e s e x c lu d ed from s t a t i s t i c a l a n a l y s i s c a l c u l a t i o n s . n .APPSJDtX 9 I.V D m D l/A l AHO r o o O CONSWtPTION OF "EA. XATS >U.YTA*\VD OF DSTS CONTAIMNC 2 , , j - T ?QR 2 i EARS Dos aq /k g /Jay A nlsial M untr 75- 0- f- 14-- 1313 25 Paya on Tase and C caas/R ac/D ay 25- 23- 32- 35- 39- w-- 23 32 35 39 42 - 49 53 53- 56-- 6 0 - 6 3 - 6 7 - 7 0 - 56 60 53 67 70 il L73 910 911 23 22 19 20 25 25 21 17 17 20 19 13 17 0 16 17 17 L9 19 20 13 9 l: 913 L7 17 13 19 20 18 19 20 18 19 13 17 25 18 13 L9 17 17 17 16 L7 17 914 915 l; 17 L9 13 21 19 19 20 16 13 L3 13 13 13 13 19 13 19 17 L6 17 17 916 917 19 20 14 19 24 22 22 21 20 13 20 21 22 23 22 13 20 21 19 18 21 20 913 919 19 20 17 20 21 20 19 21 20 13 13 20 21 17 19 13 19- i a 19 13 19 19 920921 L7 13 13 17 19 -.18 16 13 16 17 17 L7 13 17 16 17 18 16 13 15 16 16 922... 18 923 . L7 21 21 24 25 22 24 20 22 13 17 19 18 20 19 17 16 18 16 13 17 924 923 17 19 19 17 23 22 21 20 13 13 20 19 24 17 18 17 16 13 13 16 19 13 926 927 17 18 13 18 19 13 17 19 16. 13 17 20 19 18 17 17 13 17 13 20 19- 17* 928 929 20 19 21 20 21 20 21 21 21 22 20 20 21 22 24 19 20 19 24 23 26 21 930 931 932 933 934 933 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 949 970 971 972 973 974 975 976 977 973 979 980 931 982 934 935 936 987 939 392 93 1 3 . 7 ; o ; 2 13*2 '.9 = 1 l l z l : i 3 -Q ;3 20=2 13=2 19 =2 1 9= 1 1 9 r l 2 1 :3 1 9 : 2 1 9 :2 1 3 :1 1 3= 1 13=1 19=2 1 3 : 2 19=3 13=L --1 1 J I I J ! SU 52-6 f-- t 26i' APPENDIX 9 ( C a r tc i n u e d ) INDIVIDUAL AND MSAfil FOOO CONSUMPTION OF FEMALE RATS MAINTAINED ON DIETS CONTAINING 2 , ; , 3 - T FOR : 't'SAAS A aiaal Dose Number rtg/kg/day 73 34 38 91 1 1 L23 0 910 911 19 21 17 17 19 912 19 16 L8 17 21 913 914 915 13 19 16 18 16 916 917 20 21 IS 19 19 918 919 13 19 19 20 19 920 921 15 17 16 17 18 922 13 13 17 17 18 923 924 925 L7 13 19 13 13 926 927 17 17 16 18 17 923 929 21 24 25 19 20 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 994 985 98 937 998 939 990 991 592 993 994 995 MEAN-SO 13=2 19=2 18=3 13 = 1 1 9 t l Days =n T ese and C ram a/R ac/D av 130- 132- :o 3 126 151 154 132 136 207 20 19 13 21 19 L7 20 17 17 20 17 L5 19 13 15 19 L3 17 20 23 19 20 19 25 21 20 21 19 19 19 16 16 19 17 L6 17 19 16 20 17 17 17 18 18 21 13 17 13 17 L6 19 17 13 21 19 19 20 20 19 20=2 19=2 17=2 20:1 13=1 17 207no 235 235233 20 21 19 19 20 19 36- 20 23 24 17 19 20 20 19 19 L9 13 23 - 19 21 16 13 21 20 17 19 13 17 20 Dead' 20=2 19=2 259263 19 15 17 13 22 13 18 13 17 20 253266 20 16 15 23 21 19 IS 20 17 20 237291 38; SD 19 22 21 16 18 31 16 13 .CD No d a c a a v a i l a b l e . ^ V alu es e x c lu d e d from a c a c ls c tc a i A n a ly s is c a l c u l a s i o a s . SUS^I '66-- AfPSNOtX 9 (C o n c in u e d ) INDIVIDUAL ANO MEAN ?OOD CONSUMPTION OF ;SMAL 8ATS MAINTAINED ON DIETS CONTAINING 2 , 4 , 3 - T "OR 2 EARS Dase av:; fcu/day 0 Numoer 75- 291294 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 923 926 927 923 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 953 959 960 961 962 963 964 963 966 967 968 969 970 971 97? 973 974 975 976 977 978 979 980 98 L 982 983 984 985 986 937 938 939 990 991 ND 16 20 24 21 13 13 17 18 20 993 995 MEAN SD L 5 :2 115319 19 17 16 23 21 16 13 17 17 20 13:2 319322 n 2i 19 24 21 19 20 22 18 22 21:2 350354 13 13 19 20 21 17 13 18 16 22 i? :3 373382 20 19 1L 26 17 U 71 7a 10 26 1 3 :6 406410 Oaya a a T ese and C caa s/S a c/D ay 634- 469- 497- 332- 4 38 473 301 336 19 19 U 21 17 19 16 13 19 16 16 19 17 23 20 22 22 23 22 19 L3 25 . 1 8 19 15 23 21 19 19 3* 19 16 18 12 20 16 17 16 17 18 22 22 22 23 560564 -.*0. a 23 23 47* 25 35 29 n 538592 23 19 20 22 23 22 17 26 93 523327 25 La 19 24 22 17 23 13 551355 24 L L3 oa 24 22 . 3L i 19 16 26 17 536690 713 Dead - ~ L9 15 14 Li 23 23 24 23 -- Deed-- 17 16 26 30 21 27 Dad 21 20 22 -O ead-- 19 --O ead-- 26 20 Oead* Oead -Oead- Oead" Oead Oead Oead---- Oead* -D ead- Dead Dead -D ead-O ead Oead* Dead ---- Dead-Oead -- .d- Dead Dead Dead Dead 19:3 13:2 Dead* ' 1 --- De a d " Z 0 :2 1 0 :3 24:5 21:3 :u 3 20 r 23:3 'fD * No d a c a a v a i l a b l e . V a lu e s excluded ;:o o s ia c is c ic a l a a a ly s ls c a lc u ia c io o a . "1 . I |. .. n -2 6 7 APPENDIX 9 (C o n tin u a ) INDIVIDUAI AND KEAN FOOD CONSUMPTION OF 72AJL2 3ATS MAINTAINED OF OISTS CONTAINING 2 , 4 , 3 - r 7 0 8 2 TSA8S A nim al Pays an Taso and G rana/ftac/O av Pose H uabar a- 11- 14- 13- 2 5 - 2 8 - 3 2 - 3 5 - 39 - 4 2 - 4- 49- 5 3 - 56- 60-- 5 3 - 6 7 - 70 - 7 7 - aa/'* i / d a v 75- l a i i 1 3 - 21 25 23. 32 35 39 42 4 49_ 53. 36 60 63 67 0 31 30 1096 1097 21 20 19 13 19 21 17 17 17 16 17 17 13 16 13 16 16 16 17 14 16 17 1098 1099 19 21 20 19 19 15 19 20 21 21 19 13 19 13 13 16 16 19 19 17 18 19 1100 21 20 20 19 19 20 13 13 21 13 18 21 MD 18 13 13 17 13 ia 16 19 17 1101 1102 1103 _ . 19 19 18 19 IS 13 17 13 23 13 L7 59 21 17 17 17 16 17 13 16 25 17 1103- 21 19 13 19 13 13 19 20 24 20 13 19 U 17 13 13 17 20 19 16 19 17 U 061107 - 17 13 14 17 17 17 15 17 17 16 16 15 16 21 15 14 14 . 17 15 14 16 17 1108 1109 18 17 19 13 19 24 13 19 23 20 13 19 22 L7 13 13 16 17 13 18 13 19 1110 LUI 19 24 13 19 19 13 16 13 17 17 13 13 19 16 16 17 17 17 16 16 16 MD 1112 20 22 21 19 19 19 13 19 20 19 19 13 19 16 19 17 - 17 19 - 18 -- - U -- -- 19 -- -XD---1113 1114 1115 19 20 13 18 2Q 31 17 20 19 20 13 19 20 ia IS 16 17 19 17 16- 19 18 U16 1117 1113 1119 1120 1121 1122 1123 1121 1123 1126 1127 1123 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 MEiUliSD L 9 : i 2 0 : 2 1 3 : 2 1 3 : 1 1 3 t l b 2 0 : 3 1 7 : l b 1 9 : l b 2 0 : 3 L 9 :2 L 3 : i 1 3 : 1 1 9 : 2 1 3 : 2 1 7 : 1 1 7 : 1 1 3 :1 1 3 : 1 1 3 : 1 1 5 :1 1 8 : 3 1 3 :1 SO So daca avallatila. *Valuas axeludad fro acaciadeal analysis- calculations. ^Statistically significant aviation fro control M a n using Qunnaet's Test, p<0 .0 5 . 9 -1 5 9 -3 -263 APPSNDIX 9 (C a n c in u e d ) INDIVIDUAI. AMD MEAM ? 0 0 0 CONSUHPTIO Oc FSMALS RAIS AINTAINED ON 0 I T 5 o o nta inin g : , 4 , i - r ?or 2 t a r s Dose a g /^ q /d a y A nim ai Suaoer 75- Dava on T ese and G casa/R ac/O av 3 1 - 3 4 - 3 8 - 9 1 - 1 1 9 - 1 2 3 - L 47- 151-- 1 3 0 - 32- 2 0 3 - 2 0 7 - 231- 2 3 5 - 2 5 9 - 2 6 3 - 237i 38 i i 95 123 126 13 L 134 132 136 207 10 235 238 263 266 291 30 1096 13 17 17 16 19 13 13 17 19 13 19 19 20 ia 17 16 O aad 1097 21 1098 1099 L 18 19 17 19 20 20 13 16 19 22 25 25 25 19 ? 20 1100 noi 1_7 16 17 17 2'' 17 17 L3 34" 19 24 20 19 18 19 13 19 1102 1103 18 L7 16 17 L3 la. L7 18 20 18 3 4 " SD 22 16 19 19 L7 1104 1103 19 19 18 17 19 17 19 U 19 19 17 20 L9 17 18 ia 22 1106 1107 16 17 15 14 17 16 16 16 14 17 19 ' 24 19 20 20 ' 19 17 ZIu o a 1109 17 19 20 19 22 23 20 22 20 20 32 29 27 25 26 20 ilio un 15 18 16 17 20 19 17 L 18 17 17 20 19 L9 i a ia 18 1112 1113 17 19 19 19 20 20 19 17 SD 17 24 23 20 20 18 14 13 1114 1115 13 19 18 17 13 LS 19 15 22 17 20 22 20 21 21 20 21 LU6 H I7 1113 U 19 L120 U21 1122 1123 1124 1123 1126 1127 L123 L129 1130 1131 1132 1133 1134 1135 1136 1137 1138 U 39 1140 1141 L142 1143 1144 1145 EANsSD 1 7 :1 1 8 :1 1 7 :2 1 7 :1 1 9 :2 1 9 :2 1 3 :1 1 7 :2 L 9:J 13:1* 2 0 :3 2 3 :4 2 1 :3 2 0 :3 19:2 19:3 1 9 :2 NO So d a c a a v a i l a b i . ^V alues axclu d td :oa s e a e ls c ic a l a o a iy s is c a lc u la d o a s . -269- APPENDIX 9 (C o n c ia u e d ) I NOIV USUAL AMD MEAM 7 0 0 0 C0NSL21PTICN O f :MAL 3ATS MAINTAINED ON DIETS CONTAINING 2 , 4 , 5 - T -OR 2 TEARS Dose B g/W day A nim al Number 75- 20 1.096 1097 1098 1099 1100 U01 1102 1103 1104 1103 1106 1107 uoa 1109 LUO LUI 1112 un 1U 4 1115 1116 1117 ins 1U 9 1120 U 21 1122 1123 U24 1125 1126 1127 U2S 1129 1U0 1U1 1132 1133 1134 1135 U 36 1137 1138 1139 1140 1141 1142 1143 1144 1145 KEAN2S0 291294 NO 20 20 19 19 17 23 19 19 17 19s2 313319 22 19 La L7 13 13 23 19 13 13 I9 t2 319322 22 24 20 L7 19 19 25 13 20 21 20*2 350334 373332 26 NO 20 22 17 13 20 17 19 19 23 23 22 25 17 16 20 17 21 23 2 1 i3 21*4 | o | o O' D ays ott ? e a c aa d C ram s/R ac/D a v 434- 469-- 497- 532- 433 473 501 536 L9 20 25 ~ 1 13 21 21 13 21 20 13 19 19 21 17 19 17 13 32 17 19 19 U L7 19 17 17 9` 15 17 20 20 25 21 22 19 17 16 17 13 19 19 9* 20 19 23 IS 23 20 -Dead- 19*2 19*1 19*5 21*5 L3*2 5O-- 564 538592 623527 22 22 ) 23 15 M 23 13 20 -Dead' 24 2C 23 33 19 571 20 20 21 21 21 24 17 20 29 19 22 20 17 15 "Dead-DeadOead* 551553 63690 714713 20 22 17 17 Dead-- " 17 30 22 20 13 13 503 28 23 403 22 17 -- 1 Dead* 16 NO 20 17 13 13 22 2* Di a i ^ ^ 23 23 13 21 13 19 Dead* -Dead- Dead Dead- -DeadOead* -- -Dead-Dead 24*4 19*2 :0*2 19*3 *3 ND * No d a c a a v a i l a b l e . v alu es excluded fron s c a d s c ic a l a n a ly s is c a lc u la c io a s. (pnnT3U 03) 6 nO K S iaV APPENDU 9 ( C o n t i n u e d ) a it tu H- G 'Sgu>c n P> hC o 3 le n I A lu oo ij) u<ii> ooo u3 * u J i'j|- ` o 0 ooooooo 00 CO Qo O 00 t t J 4 ) U N ( r o if >- u oOr JOoHCDOoOOso`>O4oWMo^SO|o<'.oW|> 4oI >t o'4^'JNoMro'^4 rOi-jrOJ-- O--fkOIQ--\Ot-- Oi-- OQi--\OQk-OrQ-\rOO--vOON"O* " O4O<N0iU>t'IUNrOll OU UOUOI UOI Ol UO iOU UOUOAOt - Ot~OP1Of" 0) S O' Ui ( U l-J h 1Cl D (O n O' Ir *- APPENDIX 9 (C o n tin u !) L...... I l I.... J L ... J L___S JJ *P 3 n h HU t* to Pn at- -- <4 At 1 Ca H- t* o at* * n s. * rat t* O 0 3M n 0 n 1 o js 1* (A 0 O O O O O O O O O O O O O O O CiO O O O O O O O O O O O O O F ( - ( ' ( ^ U U U U U U U U W U N A i t J N N f > ) P P N M H r ' r , r4 liit - u r o f - ` O ii)0 ),^ (iiU lf- W N (- '0 'i)to '^ a 'W t''U N H C D W 3 tO ^ o it N o It tVO 1 t** va It t43 It to aa u> 0 a Itto aa -*- j? ?aA 00 9 a 1iO*>1 o ta ,, t- A 1* VO ooCAA 1* 1-- r1--It U) It MM tIttoo a t*i VIKtj I--43 It It IO loi --4) 1006 1007 LOU 1015 too o DO t-- Ct tst H O to to o o Uu pi to too to l-v N to* to to* Is* to* OO 6001 BOOT |I 1 8Ut tst oo oo - o sO 4 43 sO 4> 0D V* 43 sO 4vO sOO' U Ais O- a < V IA |a b Pito VO 0 03 t .O to vO O o O 43 4 *' 1 G - PTb OB vO SO H 1 M o 3 1st to to o Ho tst to a p* o to to* o uo o to* oto tt Itos. IpS* Id 43 H oto to K>tst 00 lo to O r- tst a1st to o1st 43 Sto b1st 1st to to OO OB o 0B O to b '7to bto at P* to t0oB 00 VO o to 1 ( VO 03 VO o a to (A s | vO 0B VO a 00 It is >- to 00 b i i *-- to VO V0 to vO VO c "rIO Is* o 00 I bVO a P w to* o S ? 00 *4 c Oo VO b "Vau* a Ct so to CO OB otst VO p vO a* Oto 43 - VO 0B vO tr s r<n(at*uac1o2t - ce O IOs*tst to a to O'O'VO tst *4 00 a O'op<00 O' VO OB to It to VO p to SO as ISI 0B * V0 vo "VJIt s( p CO w <otst sO tt ^V VO O' H tsoi too >o O O' In ' to st as* (D "? lO a*O vO 3 "rvO 00 c 1st c; ot~ 0B 0B O' O' b G f* PitovO vO OD O' CO b at - s:;vO vO u> to i < a c | 1 8o oo 'A n >u cto ^ u O 'A P IO I- o i n* SS Iti o Zo*tpf!tok: 4> ari 0 a | pS g3>&6; to O3to -0 APPENDIX 9 ( C o n e i n u a d ) a .1530 *??S51DIX 9 (C o a e ia u e d ) )5>2 in* A p p a ro io Oose a z /'x z/d av A nim ai M uabar 0 75-594 75-595 75-597 75-598 75-599 75-O 7 5-O L 75-602 MEAN = 3.0. 30 7 5 -6 2 4 75-425 7S-.626 75-428 75-429 75-450 75-431 7 5 -4 3 2 MEAN rS .O . 10 75-414 75-417 75-413 75-419 75-420 75-421 75-422 75-423 MEAN iS.O . 3 75-404 75-407 75-408 75-409 75-410 7 5 -4 LI 7 5 - 6 L2 7 5 -4 1 1 MEAN sS .D . HEMATOLOGICAL VALUES OF MAIS JA IS OH DAY 3 2 0 F A 1 1 3 -1 1 9 0AY OCSTARY STUDY OF 2 , 4 , 5 - ? ?cv P ercent 55.0 53.5 53.5 55.0 53.5 52.0 5 5 .0 5 4 .0 54.2 1 .3 53.0 52.3 54.0 55.5 54.5 53.5 52.5 54.5 54.0 1.4 53.0 52.5 50.5 54. 5 53.5 30. J 52.0 53.0 53.2 1 .3 5 4 .0 5 0 .0 49.5 54. 5 52.0 5 2.0 53.5 32.3 52.5 2 .2 X '.O^/cna^ 3.31 9.13 9.32 9 . 45 3.32 3.39 9 .71 9.51 9 .21 0 .3 4 9 .21 9.34 3.39 9.33 3.93 9 .1 4 9. 44 9. 44 9.22 0 .20 3 .38 3 .52 3 .33 9.34 9 .48 9.77 9.30 9 .42 9.13 0 .5 2 3.62 3.74 3 .71 9.00 3.93 9 . 57 9 .2 2 3 .49 3 .91 0 .35 Hg zi 100 a l 1 4 .4 1 6 .0 1.6.3 1 7 .3 11.9 16.7 17.4 13.4 17.0 0. 7 16. d 1 6 .9 1 6 .3 17.3 1 7.1 17.2 17.3 1 7 .5 17.1 0.5 17.0 L 7 .0 1 6 .5 17.3 17.5 13.3 17.4 L 7 .5 17.3 0.5 17.1 16.1 17.1 17. J 17.1 19.1 1 7 .4 L 6 .9 17.3 0 .3 x :L0J / m J 12.3 U .6 1 2 .2 1 4 .5 12.9 1 5 .3 12.3 L 3 .3 1 3 .7 2 .2 12.1 1 2 .9 1 6.2 u.s 16.7 14.7 U .6 15.0 L 4.1 1 .3 15.4 16.3 16.3 1 2 .3 14*5 U .4 L 3 .6 15.3 1 5 .3 2 .0 14.1 1 5 .9 1 6 .3 9.9 U .9 17.5 1 3 .6 1 5.1 1 4 .5 2.3 MeuC 20 12 4 3 14 14 3 19 12 i? 10 19 15 15 3 ' 10 4 IO 3 16 3 6 5 11 20 10 11 17 6 7 13 Lo ,L 5 7 6 11 VBC D i f f e r e n c i a l Clo u n c (.) 1ras h 73 32 39 35 31 34 94 75 34 Mono 5 5 3 4 3 2 3 3 3 os in 0 0 3 i 2 0 0 3. 1 37 . 35 4 90 0 34 L 34 0, 93 2 38 2 92 2 38. 2 0 1 0 0 1 0 0 1 0 38 3 32 0 36 4 92 1 93 1 * 33 l 77 2 34 4 36 2 L 2 ? 0 1 5 12 j. 30 i L 90 39 2 1 12 35 1 33 0 34 1 93 0 91 0 1 0 0 0 2 8T 1 " 1 ilo s c a c i 3 c i c a l l y a i g a i . f i c a n e d i i f e c e n c a s fr o m c o n c r o i o o a n a o s i a g a n a l y s i s a c v a r i a n c e and. Oua n a c e 1a c a s e , ? < 0 ,.0 5 . 3aso 0 0 L 1 0 0 0 0 0 rh ro ta - X 10/aa^ 1 .2 3 5 L .3 3 0 1. 605 0.355 1 .5 2 0 1 .3 1 5 1 .5 2 5 1 .7 1 0 1 .3 9 3 0.265 R acicu lo cyces 0 .5 1 .3 L. 1 1.3 2 .0 1 .0 1 .0 i.O !.2 0 1.330 0 1.425 0 1.080 0 1.225 0 1.190 0 1.445 0 1.365 1 ' 1.275 0 1.298 0.128 1 .3 1.1 2.0 0 .7 1 .6 1 .3 0 .5 0 .4 1 .2 0 L.Q10 0 - 1.075 0 1.360 L 1.000 0 1.480 0 1.2S0 0 1.325 0 1.085 0 1.202 0.152 2 .4 2.'4 L.9 0 .7 0 .4 0 .3 1.2 0.2 1.2 0 1. 240 L 0.345 1 L. 080 0 1.245 L 1.200 0 L. 480 0 1.560 1 1.710 0 1.295 0.278 0 .6 0.3 0 .3 O . 0 .5 0 .4 0 .6 L .2 0 .6 -277- A f P S I D U 11 SMATOLOCICAL VALUES OP FEMALE S A tS ' ON OAY 33 OF A U 3 - L 1 9 DAY IETARY STUDY OF Oose A nim ai ne/fca/d av fuoer ?cv Percent 0 73-634 75-635 75-36 75-637 75-638 73-639 73*40 75-641 MEAN rS.O . 52.5 50.5 51.0 19.5 47.0 51.0 31.0 43.0 50.1 1.3 30 75-664 4 4 .5 75*5 51.5 75-66 4 9 .0 75-667 43.0 75-663 75-670 e 53-0 55.0 75-672 53.0 75-673 61.0 MEAN :S.O . 52.5 5.4 10 75-634 75-655 75-656 75-657 75-653 75-659 . 75-660 75-661 KEAN sS .3. 43.5 51.0 43.5 49.0" 49.5 5 0 .0 55.0 53.0 50.6 2. ] 3 75-644 75-645 75-646 75-643 75-649 75-651 75-652 75-653 MEAN S.D . 43.5 4 9 .0 4 7 .0 47.5 5 1 .0 50.5 56.0 51.3 50.1 2 .9 IfiC X LQ^/nm^ 3.44 7.33 9.10 7.79 3.05 3.12 3 .2 7 7.34 3.19 0.43 7.64 3.34 3 .3 8 3 .1 1 3.49 4 3.78 3.97 9.77 3.56 0.63 3.05 3.42 7.70 3.11 7.38 7.97 3.38 3.43 3.12 0.28 7.70 3.25 7.77 3.05 3.27 * 3.24 9 .1 1 7.79 3.15 0.45 SgD 2 /1 0 0 al L.i7 15..3 17,.3 15., 7 16., 4 15..3 16..0 15..5 16._i 0., 6 14..5 15..3 15..7 15..7 16..3 16..3 17,.7 13.,4 . 16.,4 1 ..2 15,.9 16..6 15,.J 15..3 15,. 6 15..3 16..1 16..4 15..9 0 ..4 15..0 15., 7 15,.4 15.. 4 16..0 16..7 17,.9 . 15.. 8 .... 16.. 0 0 ..9 'VBC x LO^-'oe ^ 12.7 U .O 12.5 12.0 14.5 13.0 9.4 14.0 12.4 1.6 3.6 13.5 15.6 9.9 10.5 12.7 9.5 15.7 U .O 2.3 11.5 13.4 15.2 12.3 7.3 11.9 9.5 10.5 L I.5 2.3 7.0 10.5 il. 6 16.5 13.7 1 1 .0 14.4 11.3 11.4 2 .2 .'eue 9 6 15 7 5 5 14 16 10 vac D if :i a r e n c i a l Counc CT L'/mon Mono S e s ia 39 1 91 2 35 3 90 L 90 2 94 0 31 ? 32 0 38 1 1 ]_ 0 j 2 1 2 2 T 10 87 1 10 87 2 13 30 0 U 33 0 2 97 0 3 38 2 17 31 2 9 39 0 11 37 1 2 1 2 1 1 2 0 2 1 9 90 8 0 13 36 0 7 39 2 3 39 2 3 95 1 3 91 0 7 39 1 3 97 0 7 91 1 1 1 2 1 1 0 3 0 1 12 10 7 4 10 1 14 9- a 33 0 37 0 92 94 0 39 0 98 0 85 1 91 . 0 91 0 0 3 0 1 1 0 0 a i tfo s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s c r o o c o n t r o l a s a o s u s i n g a n a l y s i s o f v a r i a n c e a n d D u n n e e t 's c a s e , ? < 0 .0 5 . laso 0 0 0 0 l 0 0 0 0 0 0 0 0 0 '0 0 0 0 0 0 0 0 l 0 0 0 0 0 0 1 0 1 0 0 0 Throabocyees s lo 1/ ^ 1.165 1.325 1.310 0.965 1.005 1.290 1.070 1.275 1.175 0.145 1.350 0.990 1.420 1.100 1.255 1.335 1.195 0.820 1.133 0.203 1.020 0.950 1.080 1.010 1.055 0.305 0.915 0.990 0.973 0.038 1.430 1.030 1.085 1.115 1.025 1.013 1.450 1 .0 0 0 1.144 0.187 P ad cu lo cytBS 0.7 1.5 0.9 2.1 1.7 1.7 1.2 2.7 0.9 0.7 1.4 1.5 1.7 1.4 1.2 1.6 1.3 2.2 ` 1.4 1.5 1.3 1.3 1.5 1 .0 i ,L 1.6 1.2 1.2 0.3 1.6 0.5 0.4 1.4 1. 1 1 (5 3 3 .7aAPPENDIX 12 HEMATOLOGICAL VALUES OF MALE PATS ON OAK 69 OF A TVO-iEAR DIETARY STUDY OF 2 ,4 5-? Dose .na/'xs/dav 0 vn ta a l Suaher PCV Percent 75-674 75-6 75 <6 -6 7 6 75-679 75-660 75-631 75-633 75-636 MEAN 3 .0 . 55.5 57.0 55.0 53.0 57.0 57.0 55.3 35.3 53.7 1 .4 use x LO^/ajm^ 9 .2 1 9 . 71 9.10 9.36 9.32 9.04 3.55 9 .0 0 9.22 .4 1 H? 3 a/100 *1 17.3 13.2 L7.2 16.9 18.5 17.6 16.1 17.1 17.4 .3 UBC 10x ^ / aan^ 9.5 Li. 3 LQ.S 14.6 3.9 15.0 17.0 10.3 12.3 2 .9 20 75-361 34.0 9 .2 6 17.5 12.3 73-363 30.3 9.4Q 17.9 13.0 75-364 55.0 9 .2 9 17.4 7.5 73-365 55.0 9 .6 3 17.9 12.5 73-366 54.5 9 .3 7 17.5 12.4 75-367 55.0 9 .2 6 17.2 14.3 75-363 54.0 3.56 17.2 14.3 75-370 54.0 9.51 17.6 15.0. MEAN -5 .0 . 5 4 .3 .3 9.29 ,, .3 2 17.5 .3 12.3 2 .4 L0 75-310 53.5 75-311 53.0 75-312 57.3 75-314 54.0 73-315 *9.5 75-317 59.5 75-613 57.0 73-620 54.0 MEAN 3 .0 . 55.o 3 .1 9.79 9.30 9 .5 3 9 .1 4 3.93 9 .5 1 9.67 3.93 9 .4 1 . 56 17.3 13.6 13.9 17.5 16.1 13.7 17.5 17.0 17. / 1 .0 12.3 12.3 12.9 12.9 11.0 15.3 L3.9 11.3 -2 .9 -1 .3 9 75-760 55.0 9.57 17.3 12.0 75-762 52.0 9.02 17.2 7.9 75-76 3 33.3 9.32 17.3 12.3 - 73 -7 6 4 - - 5 4 .0 -....... 9 .0 0 - 1 7 .0 ........... 9.9- 7S-766 53.0 9.55 13.2 17.9 75-767 59.5 9.36 L9.5 12.6 73-768 53.5 3.95 17.0 16.4 75-770 56.0 9 . 73 17.3 12.7 MEAN S .3. 55.4 -2. 4 9.44 . 39 17.3 . 3 12.7 3 .2 JSC D i f f e r s n t i a i Count ( S3 Neuc I'.g o n Mono l o s i n LI 66 2 2 15 -34 1 0 10 68 0 l 12 86 2 0 U 35 2 0 5 37 4 1 10 67 3 0 7 90 2 1 11 37 5 0 0 0 3 0 0 0 0 0 0 3 90 2 0 3 38 2 2 9 90 0 0 13 35 0 1 20 78 0 2 4 94 1 1 *9 88 2 0 12 35 0 2 a 37 1 1 0 0 1 1 0 0 1 0 0 13 61 0 l 16 63 L 0 12 86 2 0 10 S7 1 2 13 37 0 0 6 92 0 2 6 92 0 ' 15 31 1 L 1 2 36 1 1 11 89 0 0 16 31 1 2 9 90 0 0 9 38 1 L a 37 1 1 16 34 0 0 10 37 2 1 9 91 0 0 11 37 1 1 0 0 0 0 0 0 0 ? 0 0 0 1 1 0 0 0 0 0 Ho s t a t i s t i c a l l y s i j n l z i c a n c d i f f e r e n c e s :? a m c o n t r o l a e a n s u s i n g a n a l y s i s o '/ a r i a a c * a n d D u n n e c c 's t e s t . ? < 0 .0 5 . 3 0 C 7 tes 103/3m 3 1.080 1.100 1.040 0.990 1.275 1.295 1.250 1.590 1.204 . L94 1.335 1.200 1.130 1.000 1.250 1.235 1.435 1.475 1.276 . 169 1.660 1.445 1.535 1.070 0.320 1.525 1.040 1.440 1.292 .2 3 7 1.165 1.130 0.305 1.055 1.010 1.135 1.225 1.150 1.084 .1 3 1 .\e e lc u lo ' ,, 1.4 2.0 i.i 1.6 1.0 1.1 1.1 .9 1.0 1.5 1.3 1.2 1.4 .9 .9 .7 1.4 .7 1.4 2.3 1.6 1.5 L. 6 1.1 1.0 1.4 .9 1.6 1.3 L. 7 L. 7 1. 7 1 .1 J j 1 J 1 1 J i 279APPENDIJC 13 HEMATOUJCtCAL VALUES OF FEMALE RATS ON DAY 90 OF A TUO-YSAft DIETARY STUDY OF 2 , 4 , 5 - 7 Dose rg /W d a v 20 A nim al ?CV 75-910 75-912 75-913 75-9L6 75-917 75-919 75-920 75-921 MEAN 5 .0 . 48.0 47.5 50.5 50.5 49.0 49.0 50.0 50.0 4 9 .3 1 .1 73-1096 73-1097 75-1098 73-1100 73-1101 75-1102 73-1103 73-U 04 MEAN 8.0. 47.0 4 7 .0 50.0 5 0 .0 4 8 .0 53.0 51.5 44.0 4 8 .3 2 .9 10 75-1046 48.0 73-1047 51.5 75-1046 50.0 75-1049 50.0 75-1050 52.0 75-1051 51.0 75-1052 46.5 75-1054 53.5 MEAN 5 0 .3 8 .0 . 2.2 3 75-998 49.0 75-999 51.0 75-1000 49.0 75-1003 53.0 75-1004 4 8 .5 75-1005 53.5 75-1009 52.0 75-1010 49.5 MEAN S .D . 30. 7 2 .0 RBC X -,Q.3/.an3 7.55 3.23 3.52 7. 77 3.23 7.38 3 . 70 3.24 3.17 .3 8 9 .3 0 7.81 3.56 3.62 3.12 9.34 3.53 7,55 3.24 .3 9 7.51 3.45 3.08 3.27 9.35 3.53 7.60 8.39 3 .2 7 . `52 7 .6 0 8.20 7 .4 7 3.32 7 .3 0 3.30 a . 79 3.23 3.11 * . 46 t/io o 3i 15.0 15.3 16.1 15.3 15.3 15.5 16.7 , 15.9 15.3 .6 16.6 15.3 16.0 16.9 ISO 16.9 16.5 14.7 16.2 .7 14.9 16.3 15.9 15.7 16.8 16.3 15,6 17.7 16.2 .9 1 5 .5 16.4 15.6 16.9 1 5 .5 1 7.4 17.0 16.1 16.3 +. 7 TOC v 9.9 9.0 9 .4 10.3 7.3 7.7 7.3 13.0 9.4 1 .3 14.0 1 1 .3 10.6 14.0 16.0 9.9 10.9 .9 .4 ' 12.0 2 .4 3.1 15.2 1 6 .1 15.1 7. 7 10.1 9.0 10.3 11.5 3 .5 1 0 .5 1 4 .9 1 1 .2 1 0 .1 9 .1 1 2 .5 12.3 1 0 .1 11.3 * 1.3 vac Jlrzerenciai Count 1 N9U !.yiaoh MOflO 3a s o 12 37 0 7 92 3 95 U 37 10 90 0 15 35 0 3 92 0 9 90 0 9 90 1 1 0 9 91 0 3 35 3 91 0 3 7 90 1 13 30 3 3 3 91 0 69 91 0 0 92 1 1 9 39 1 1 0 o U 36 0 2 5 94 0 1 15 35 0 0 15 34 0 1 L6 78 1 5 9 91 12 30 9 37 00 26 20 12 36 1 1 0 0 0 0 0 0 0 2 0 9 90 1 0 10 37 1 2 3 93 2 2 6 89 1 4 6 91 1 l 6 92 1 1 to 39 0 1 13 36 1 0 3 90 1 1 Q 0 0 0 1 0 0 0 0 So s t a t i s t i c a l l y s l g o ii l c a a c d iita r e n c e s from c o n tr o l a e a n s u s in g a n a ly s is oi v a r ia n c e and O unnecc' s t e s t , p < 0 .0 5 . Throm b ocytes 10 1 .2 3 0 L. 130 1 .3 9 5 1 .0 4 5 0.965 1 .2 1 0 1 .3 7 0 1. 745 1.263 + .241 1 .2 9 0 1 .1 3 5 0.905 1 .0 1 0 0 .9 2 2 1 .0 1 0 i.U O 1 .1 5 0 1 .0 7 3 . 134 l.U O 1 .2 6 5 1 .3 1 5 L .5 0 5 1 .2 0 0 1 .2 2 0 1 .1 4 0 1 .2b0 1. 253 .1 2 1 1 .4 2 5 1.090 1 .1 2 0 1 .2 1 0 1 .1 4 5 0.995 1 .1 6 5 1 .2 1 5 1 .1 7 1 * .1 2 5 R eticulo c y te s 1 .4 1.0 1 .2 1. 7 2 .2 1 .5 L .4 1 .4 1 .4 1 .2 2 .1 1 .3 2 .1 1 .2 1.6 1 .2 2.5 1 .7 1 .2 .4 1. 7 2 .2 1.0 1.0 1 .9 1.9 1.1 1 .2 2 .0 1,7 1.3 1 .9 1 .6 1.3 1 .6 1.4 3-4 $ 3 ^ Vo j c a i s c i c a l i / 5 i ? n i i i c a n c d l i f a r e n c e s r o o c o n e r o i a e a a s u s i n $ a n a l o g i a oc v a r i a n c e a n d D u a n e c e 's c e n e . ? < 0 .0 5 . o OOOOQOOO 11- . i - e- O i - O O O r t .>NO) tUtOl --v O'C>U|--lOUOJCMJB0CU> Q> 09 U l U U l U A U A t C B (- f w < - r rt-j O t/ O O 'O *i> O *'1 1________ !.. i. : j l. . REMATOLOGICA!. VALUES 0 ? MALS HAIS ON DAY 3 6 4 OF A W O -EA & DLETAY STUDY OF 75-810 75-811 75-312 75-314 75-315 3 0 .5 35.5 47.0 53.0 50.0 3.79 3.39 7.46 3.39 3.65 16.7 17.1 1 5 .6 16.7 1 6 .5 tni s *!*-.- O M IA u iM U iu iu iu m u i O^I J0OIiiO0I1iOOi jO9I^iO9Ib*OOIi'OOI*l o o iM O > t> i^-u h U IU IU IU IU IU IU IU I jI. oI> o>oi> aI> oi- Io> OI(ti o )o> >( M N M |+ ;c - o> u h O ^ a o ti I * * ' U< Vi. Ul un (.' U l *> N i t - K H O O N O i O s N V b> U l O O O (ii U l- O ^ ui u |n> (.- O O O L - i O O O O (B n | t a n o cu w a w OI s m M m Ui u N t- i A f- u N r u Ui O I t Ui U U U u u a o Ui I- - t > U U ' | t (h O s ) o o 9 > t o u - t *' ^ O. *.- t ' O a> S | IVt* ut -\ u a s c bi s u O' u i- n i u u u u o a i u A p C C C C ^ P P U l i/> t ' S | U l u U l if i U f X Kit' uitisDO' ^ao|ut. #l--j fr- ii- iH- Bja O i--r-i B t J U t >0 U l - t-* O ' OI I O u I" N B i u O N O U M l' O O O O O O i-O VJ O* Vs O' K f" 14 B' le <->o4 us uQ1fa' Na t a' Ui u i nu uim |Iad io im r o a iv i o oooooooo |+ i - i-- t - t - r - o ls t-- CU O O O * O O t O O ' t i VJ OI M Ul C ' O ' O Sfit- U i u i u u p u i o |o O ti O' i- o-."'o833 B 3? 1 3.0 L I.2 U .3 L 3 .1 1 2 .3 20 77 L3 30 20 76 13 34 1.2 36 03 LL 13 21 02 0 0 0 0 0 L .5 1 3 1 .038 1 .0 4 0 0 .9 8 5 1 .0 5 5 M <U O |u NI - io 4 O O ' h O ' N O (T ! L.... - J :. I 281APPENDIX 15 HEMATOLOGICAL VALUES OF F CIALE RATS ON DAY 365 OF A TWO-YEAR DIETARY STUDY OF 2 , 4 , 5 -T Dose aa/k a/d av 0 A nim al Number PCV P e rc e n t 75-910 75-912 75-913 75-916 75-917 75-919 75-920 75-921 MEAN +S.D . 4 7 .0 4 8 .5 45.0 47.0 46.0 4 9 .5 4 7 .0 4 7 .0 47.1 + 1.4 RflC X 1, n0 6 f,am 3 7 .4 4 3 .2 0 7 .7 2 7 .1 0 7 .3 6 7 .9 0 7 .4 3 7 .5 1 7.58 + .3 4 KgB zJ 100 s i 1 4 .9 1 5 .9 1 5 .0 1 5 .2 14.3 1 5 .5 1 5 .0 1 5 .0 1 5 .2 + .4 VBC X l1r0t 3 /.o n 3 8 .3 7 .8 7 .5 1 5 .9 7. 7 1 0 .0 1 3 .4 1 5 .0 1 0 .7 + 3.3 WBC D i f f e r e n c i a l 'C o u n t (:) Neue Lvffioh Mono E o s i n B aso 20 78 2 0 16 80 2 1 17 78 1 4 21 75 2 2 13 84 1 2 20 75 1 4 9 86 2 3 9 39 0 1 16 81 1 2 0 1 0 0 0 0 0 0 0 T hrom b o c y te s 1 0 ^ /a n ^ 1 .0 2 8 0 .8 7 5 1 .0 8 0 0.825 0 .8 3 3 0 .9 5 3 1 .0 2 5 0 .3 8 3 0.938 + .098 R eticulo c y te s 1..5 1..2 1..0 1.,6 .5 .4 .9 1., 4 1..1 30 75-1097 48.0 7 .5 8 75-1098 75-1099 45.0 47.0 7 .1 8 7 .4 8 75-U O O 50.5 a . 62 75-1101 48.0 7 .9 3 75-1102 45.0 7 .2 4 75-1103 75-1104 . 51.0 4 6 .5 8 .1 9 7 .5 5 MEAN +S.D . 47.6 + 2.2 7 .7 2 + .49 15.6 14.6 14.8 17.0 1 6 .6 15.1 16.3 1 5 .5 1 5 .7 + .9 11.7 10.3 1 3 .3 10.1 1 7 .0 1 0 .3 7 .5 9 .3 11.3 + 2.8 12 82 3 3 10 87 0 3 28 72 0 0 9 89 1 1 8 89 1 2 14 82 2 1 7 87 2 3 15 32 2 1 13 84 1 2 0 0 0 0 Q 1 1 0 0 10 75-1046 44.0 7 .1 1 75-1047 5 0 .5 8.29 75-1048 4 6 .5 7.35 75-1049 46.0 7 . 70 75-1050 4 6 .5 8.41 75-1051 4 7 .5 7 .9 9 75-1053 4 6 .0 7 .9 3 75-1054 44.0 7 .5 1 MEAN +S.D . 4 6 .4 + 2.1 7 .7 9 + .45 3 75-998 44.5 75-999 48.5 75-1000 4 5 .0 75-1003 4 6 .5 75-1004 44.0 75-1005 52.0 75-1009 44.0 75-1010 46.0 7 .5 6 8 .4 6 7 .0 4 7 .6 8 7 .1 8 8 .2 4 8 .0 2 8.17 MEAN +S.D . 46.3 +2. 7 7 .7 9 + .51 14.3 1 6 .4 1 4 .9 1 5 .4 1 5 .8 1 5 .9 15.0 1 5 .0 1 5 .3 + .7 1 5 .0 1 6 .3 1 4 .4 1 5 .2 1 4 .1 1 7 .1 1 5 .0 1 5 .6 15.3 + 1 .0 8 .9 10. 7 7 .3 11.6 6.3 5 .6 1 0 .4 8.3 3 .6 + 2 .2 3 .3 1 3 .2 7 .3 9.1 8 .7 9 .3 9.1 1 1 .4 8 .9 + 2 .9 15 79 4 2 12 84 2 2 14 81 1 4 17 81 0 2 25 72 1 2 18 80 2 0 11 88 0 1 20 76 1 3 17 80 i 2 17 79 1 3 25 71 2 2 5 85 5 5 11 83 3 3 15 81 2 2 L3 86 1 0 25 68 2 5 12 87 1 0 15 80 2 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 No s t a t i s t i c a l l y s i g n i f i c a n e d i f f e r e n c e s fro m c o n c r o l s e a n s u s i n g a n a l y s i s o f v a r i a n c e a n d D u n n e t t 's t e s e , p < 0 .0 5 . 0 .8 1 8 0 .9 5 5 0 .9 6 8 0 .9 1 5 1 .1 2 3 0 .9 2 3 0 .7 1 0 0 .8 1 3 0 .9 0 3 .125 .5 2 .0 1 .0 1 .3 .7 .9 1 .7 1 .3 1 .2 1.175 1 .1 8 3 1 .1 3 5 1 .1 5 8 1 .1 1 8 0.738 0 .9 2 3 0 .9 1 5 1 .0 5 2 + .170 1 .0 5 8 0.968 0.310 1.050 0.963 0 .9 0 8 0 .9 9 5 0 .8 5 3 0 .9 5 1 .089 . 1.1 2 .1 1.0 .8 1 .4 1 .1 .9 .6 1 .4 .8 .; .7 .7 1 .2 1 .0 3ose ag /k g /d a v 30 10 3 -82* A??SNOtX 16 EMATOLOCXCAL VALUES OF MALE PATS ON 0A 726 OF A TWO-YEAR o e r r ARY STUDY OF 2 . 4 , 5 - r A oiaal Mumoer ?cv P ercent 75-6 78 7 3 -6 79 73-660 75-631 73-634 75-729 75-732 73-743 MEAN 3 . D. 4 5 .0 4 6 .0 4 3 .5 50.0 45. 3 3 .5 3 9 .0 4 6 .5 44.3 3 4 75-861 75-862 75-364 73-871 75-884 75-885 75-389 75-891 KEAN S .D . 43.0 5 0 .5 49.0 52.0 50.0 46.0 43.0 43.0 4 7.7 3 .4 75-814 75-319 75-344 73-350 75-3 15 KEAN S .O . 4 8 .5 51.0 46.5 43.0 47.3 3 .4 75-762 75-764 75-771 75-773 75-774 75 - 73 8 75-794 75-795 MEAN S .D . 4 8 .0 45.0 43.0 43.0 40.0 38.0 41.5 50.0 4 4 .2 4 .3 *5C :t lo V -a a ^ 6 .38 7 .7 1 7 .6 0 7 .4 2 7 .1 1 5.13 5.47 7.48 6.79 L .01 z/100 a i L 4 .6 L6. 4 1 5 .3 1 6 .3 1 5.1 11. 7 L 2 .7 1 5 .6 U .3 1. 7 JTBC x 10J /aa^ 1 3 .2 1 3 .2 1 0 .6 20.4 1 0 .9 5 .1 26.4 12.4 1 4 .3 6 .5 7.40 8.04 7.49 7 .9 2 7.28 7 .7 5 3.46 6.65 7 .5 2 .5 5 1 5 .5 16.3 1 5 .4 1 6 .2 15.1 1 4 .9 16.4 1 3 .5 15.4 1 .0 23-4 10.8 1 0 .4 10.4 9 .9 17.2 15.3 17.2 14.4 4 .8 8 .0 5 7 .6 0 7 .2 6 7.08 7 .5 0 .4 3 1 6 .3 18.0 1 6 .4 13.5 1 4 .9 24.3 14.6 14.2 Ho b l o o d c o u l d b e o b t a i n e d 1 5 .6 .9 17.6 5 .2 7 .1 6 7 .3 3 7 .7 3 7 .0 8 6.44 5.88 6 .8 5 7.91 7 .0 5 .6 6 1 5 .1 1 5 .0 16.1 14.3 L 3 .9 12.9 1 5 .2 16.3 1 4 .9 1 .2 17.7 2 1.3 U .O 1 0 .6 9 .3 9 .7 10.3 1 1 .5 a .9 4 .2 W3C O i f f e r e n c i a l o u r.c *T) M eut ,,/ranh MOCIO c a s in 3390 33 60 7 3 35 58 6 1 39 50 9 2 17 72 10 1 39 53 3 0 33 12 5 0 39 49 12 0 50 40 a 2 42 49 3 j. 0 0 0 0 0 0 0 0 0 50 41 3 1 31 62 4 3 26 64 9 1 11 86 3 0 21 76 3 0 53 35 11 24 67 9 0 47 45 6 2 33 59 7 1 0 0 0 0 0 0 0 0 0 43 45 10 2 43 49 7 43 45 3 4 37 S2 9 1 42 43 3 z 0 0 0 0 0 22 67 9 2 12 ?52 34 125 70 1 0 0 0 31 62 7 0 0 23 68 9 0 40 55 4 2 0 0 28 64 6 2 0 25 67 , 7 1 0 31 61 7 1 0 T hrom b o c y te s i.o' /a n '' 0.955 1 .3 5 0 0 .950 1 .1 1 5 1 .2 3 5 0.555 1 .3 5 0 1 .0 1 5 L .0 6 6 .2 6 2 1.000 1.005 0.385 0.755 0 .3 3 0 ' 0 .8 0 0 0.750 0.985 0 .3 7 8 .1 0 7 0.795 0.395 0.790 0.380 0.340 .055 1.340 0.940 0 .320 0.805 1 .0 2 5 0.375 0 .950 0 .700 0 .932 .1 9 3 R eticu lo cy tes T 2 .3 2 .2 2 .1 1 .4 1 .3 1 .3 1 .7 1 .3 1. 7 ' L.2 2 .8 L .6 1 .2 .3 2 .3 1 .4 S .4 2 .1 1 .7 1 .6 1 .8 2 .0 1.3 11..56 1.9 .3 1 .6 .2 1 .3 1.1 1 .1 N'o s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i r o n c o n t r o l l e a n s u s i n g a n a l y s i s o f v a r i a n c e a n d O u n n e c c 's t e s t , ?< 0 .Q 5 --> | --* I _J --) ! *] 9 -1 5 3 ^ 283APPENDIX 17 HEMATOLOGICAL VALUES OF FEMALE RATS ON DAY 727 OF A TWO-YEAR DIETARY STUDY OF 2 , 4 , 5 - T Dose ag /k g /d a y 0 30 A nim al Number PCV P ercent 75--9 1 2 75-914 75-918 75-926 75-927 75-929 75-935 75-936 MEAN S .D . 42.0 4 5 .0 5 0 .0 4 9 .5 50.0 53.0 44.0 45.0 47.3 3 .8 75-1098 75-1100 75-1102 75-1108 75-1110 75-1112 75-1116 75-1119 MEAN S .D . 47.0 4 3 .5 48.0 48.0 3 1 .5 46.0 43.0 48.0 44.4 5 .6 RBC x H T /an 6.38 7 .0 4 7 .1 8 7 .1 1 7 .4 6 7 .6 3 6.28 6.20 6.91 .5 5 6.03 5.65 6 .58 7 .3 3 3 .3 4 6.55 6 .0 9 6.72 6.04 L .20 Hgfl g/1 0 0 ml 1 3 .5 1 5 .0 1 6 .1 1 4 .9 1 6 .6 1 5 .0 13.8 14.3 1 4 .9 1 .1 15.2 14.2 1 5 .5 1 5 .4 9 .8 15.0 14.1 15.3 1 4 .3 1 .9 WBC x ,1,,03 /,mm 3 6 .9 9 .6 5 .1 7 .1 6 .8 1 0 .8 8.3 9.0 8 .0 1 .8 9 .1 9.2 7 .1 5 .8 9.2 8 .3 10.6 5 .6 3.1 1 .8 UBC D i f f e r e n c i a l C o u n t (7.) Neuc Mono Baso 24 71 4 1 41 47 11 1 32 59 72 31 58 74 14 79 4 3 14 76 3 2 29 63 8 0 13 78 8 1 25 66 7 2 0 0 0 0 0 0 0 0 0 46 49 4 1 0 37 55 71 0 18 75 5 2 0 15 77 6 2 0 21 78 1 0 0 19 69 11 1 0 35 59 5 1 0 -- -- -- -- -- 27 66 6 1 0 T hrom b o c y te s 1 .0 9 2 1 .0 0 0 1 .1 0 0 1 .0 2 5 1 .0 2 5 1 .1 1 5 0 .9 7 5 1 .4 6 5 1 .2 0 0 .156 1 .2 8 5 0.645 0.822 1 .3 6 5 1 .3 1 0 1 .4 0 8 1 .0 0 5 1 .2 3 5 1 .1 3 4 . 2 79 R eticu lo - 7. 2 .0 .3 .9 1 .0 1 .8 1 .7 1 .8 3 .6 1 .6 1 .6 1.4 1 .3 1 .2 1 8 .5 1.6 1 .8 2 .0 3 .7 10 75-1046 47.0 6.66 75-1056 45.0 6.69 75-1059 3 5 .0 3 .8 9 75-1070 49.0 7 .J 2 75-1072 4 5 .0 6 .6 9 75-1086 46.0 6 .8 0 75-1091 4 4 .0 6 .6 5 75-109 2 4 6 .5 6.68 MEAN S .D . 4 4 .7 4 .2 6.40 1 .0 3 14. 7 1 6 .0 1 0 .7 1 6 .3 15.1 1 5 .5 1 5 .4 1 5 .2 1 4 .9 1 .3 . 6 .8 4 .6 7 .2 6 .3 8 .5 6 .7 1 2 .2 1 3 .0 8 .2 2 .9 22 69 6 3 36 53 9 2 29 66 . 4 1 12 7S 9 4 28 61 11 0 19 71 5 5 26 66 6 2 40 57 3 0 26 65 72 0 0 0 0 0 0 0 0 0 3 75-99 7 so.o 6 . 74 1 6 .7 6 .4 12 77 7 4 0 75-999 4 9 .0 7 .2 6 1 6 .9 8 .3 24 73 3 0 0 75-1007 45.0 5.99 15.1 7 .4 17 74 3 L 0 75-1008 5 0 .5 6.88 1 6 .3 11.1 25 68 7 0 0 75-1010 47.0 6 .6 6 16.3 9 .2 53 40 6 1 0 75-1011 3 7 .0 5 .4 4 1 2 .1 12.3 32 59 9 0 0 75-1012 3 9 .0 5 .6 5 1 3 .1 8 .9 60 30 9 1 0 75-1017 44.0 6.03 ' 14.4 1 0 .3 35 58 7 0 0 MEAN S .D . 4 5 .2 5 .0 6.33 . 64 15.1 1 .3 9 .3 2 .0 32 60 7 1 0 No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s fro m c o n t r o l m ea n s u s i n g a n a l y s i s o f v a r i a n c e a n d D u n n e c c 's t e s t , ? < 0 .0 5 . 1 .1 9 2 0 .9 7 8 1 .0 8 2 0 .9 5 0 0 .8 7 8 0.962 1 .0 0 5 0.878 0.991 . 105 1 .1 5 2 1 .2 4 5 1 .1 4 8 1 .1 8 9 1 .6 3 5 1 .1 3 5 1 .3 9 0 0 .8 8 5 1 .2 2 2 .2 1 8 2.8 1 .9 5.4 2.0 1 .0 2 .1 1.0 2 .2 2 .3 1 .9 .9 1 .8 1 .2 1 .3 2 .5 0.5 1 .5 SU ' S 3 ^ -284- APPEND12 18 RESULTS OF URINALYSES OF MALE RATS ON DAY 32 OF A 118 -1 1 9 DAY DIETARY STUDY OF 2 ,4 ,5 - T D ose m q /k q /d av 0 30 10 3 A nim al N um ber S p e c ific G ra v ity a 73-594 75-595 75-597 75-598 75-599 75-600 75-601 75-602 MEANiS.D. 1.039 1.052 1.043 1.041 1.039 1.032 1.050 1.044 1 .0 3 0 c .006 75-624 75-625 75-626 75-623 75-629 75-630 75-631 75-632 MEANiS.D. 1.030 1.046 1.038 1.056 1.041 1.038 1.050 1.036 1 .0 2 7 ;.0 0 7 75-614 75-617 75-618 75-619 75-620 75-621 75-622 75-6Z3 MEAN+S.D. 1.063 1.052 1.029 1.028 1.044 1.043 1.052 1.038 1 .0 3 5 ;.0 0 5 75-604 75-607 75-608 75-609 75-610 75-611 75-612 75-613 MEANiS.D. 1.039 1.034 1.063 1.050 1.057 1.059 1.048 1.043 1 .0 3 8 t.010 pH 7.0 6.5 7.0 6.5 7.0 6 .5 7.0 6 .5 7 .0 6.5 7.0 7.0 6.5 6.5 6.5 6.5 6.0 6.0 7.0 7.0 6.5 6.0 6.0 6.0 6.5 7.0 6.5 7.0 6.0 6.5 7.0 6.0 Suqar aeg. aeg. aeg. neg. aeg. neg. aeg. neg. aeg. aeg. neg. aeg. neg. neg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. neg. aeg. neg. aeg. neg. aeg. ? ro ts in +++ 4-f +H+-H+T -H 1W ++ + +++ +-H+-H- ++ +-HtT -H+ +-H+++ -H-f + -f-H+ ++ -H-h -HH H1 -H-+ -H-r E atar.es neg. neg. aeg. neg. neg. neg. aeg. neg. neg. neg. neg. neg. neg. neg. aeg. neg. neg. neg. aeg. neg. neg. neg. neg. neg. neg. aeg. neg. neg. aeg. aeg. aeg. neg. O ccu lt 31oad aeg. aeg. m oderate aeg. aeg. aeg. aeg. aeg. Bilirubin aeg. aeg. neg. aeg. aeg. aeg. aeg. aeg. neg. neg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. sm all neg. aeg. m oderate neg. neg. neg. neg. neg. sm all neg. aeg. neg; neg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. aeg. neg. aeg. aeg. aeg. aeg. aeg. s t a t i s t i ca lly s i g n i f i c a n t d i ; i a r e n c a s from c o n c r o l nean u s in g a n a l y s i s o r v a r ia n c e and D u n n e tt1 c a s e p < 0 .0 5 . -285- APPENDIX 19 RESULTS OF URINALYSES OF FEMALE RATS ON DAY 83 OF A 118-119 DAY DIETARY STUDY OF 2,4,5-T D ose m q /kq /d ay 0 30 10 3 A n im a l N um ber S p e c ifi^ G r a v ity 7 5 -6 3 4 7 5 -6 3 5 75-636 7 5 -6 3 7 75-638 7 5 -6 3 9 75-640 75-641 M E A N iS .D . 1 .0 3 8 1 .0 2 1 1 .0 3 2 1 .0 2 8 1 .0 3 6 1 .0 3 3 1 .0 2 5 1 .0 2 4 1 ..0 4 3 + . 006 75-664 7 5 -6 6 5 7 5 -6 6 6 75-667 7 5 -6 6 8 75-670 75-672 75-673 M E A N iS .D . 1 .0 3 9 1 .0 2 1 1 .0 2 6 1 .0 2 6 1 .0 1 7 1 .0 2 5 1 .0 3 3 1 .0 3 0 1 .0 4 2 + .0 0 8 7 5 -6 5 4 75-655 7 5 -6 5 6 75-657 7 5 -6 5 8 7 5 -6 5 9 7 5 -6 6 0 75-661 M EA N + S.D . 1 .0 3 8 1 .0 3 2 1 .0 2 8 1 .0 3 4 1 .0 2 9 1 .0 3 9 1 .0 3 5 1 .0 4 4 1 .0 4 4 + .0 1 3 75-644 75-645 75-646 7 5 -6 4 8 75-649 75-651 75-652 7 5 -6 5 3 M E A N iS .D . 1 .0 5 6 1 .0 3 8 1 .0 2 7 1 .0 3 6 1 .0 3 8 1 .0 3 1 1 .0 4 7 1 .0 3 1 1 .0 5 0 i.0 1 0 EH _ 6 .5 6 .5 7 .0 6 .5 7 .0 7 .0 7 .0 6 .5 6 .0 6 .5 7 .0 7 .0 7 .5 6 .5 6 .5 6 .0 7 .5 6 .5 7 .5 7 .0 6 .5 6 .0 6 .5 6 .0 8 .0 7 .0 6 .5 6 .5 6 .5 6 .0 6 .0 6 .0 Suqar neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. P r o t e in tra c e tra ce + + 4-H++ tra ce + tra ce tra ce tra ce tra c e tra ce tra ce + -H- + + +-H++ + + + -H- +-H* -H* tra ce + -H-H-+ +++ K e to n e s neg. neg. neg. neg. neg. *n e g . neg. neg. O c c u lt B lo o d neg. neg. neg. neg. neg. neg. neg. neg. B ilir - neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. n e g .. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. a^ 3 t a t - s t l c a l l y s i g n i f i c a n t d if f e r e n c e s fro m c o n t r o l m ean u s in g a n a ly s is o f v a r ia n c e a n d D u n n e t t 's t e s t , p < 0 .0 5 . -286- A P F E N D I 2 2Q RESULTS OF URINALYSES OF MALE RATS OM DAT 89 OF A TWO-YEAR DIETARY STUDY OF 2,4,5-T D ose a g / '< a / d a v 0 30 A n im a l N um ber 75-674 75-675 7 5 -6 7 6 75-679 75-680 7 5 -6 8 1 7 5 -6 8 3 7 5 -6 3 6 M E A N + S .D . 7 5 -8 6 1 75-863 75-864 75-865 75-866 75-367 75-868 7 5 -8 7 0 M EA N + S.D . S p e c ific G ra v ity 1 .0 5 0 1 .0 4 4 1 .0 4 5 1 .0 5 4 1 .0 4 2 1 .0 4 7 1 .0 3 8 1 .0 3 6 1 .0 4 4 + .006 1 .0 5 4 1 .0 4 7 1 .0 5 0 1 .0 4 7 1 .0 3 0 1 .0 4 3 1 .0 3 9 1 .0 3 9 1 .0 4 4 + .0 0 8 a8 6 .5 6 .5 7 .0 6 .5 7 .5 6 .5 6.0 7 .0 6 .5 6 .5 7 .0 6 .5 7 .0 6 .5 6 .5 7 .0 rlu c o s e N eg. M eg. M eg. M eg. M eg. M eg. M eg. N eg. M eg. M eg. M eg. M eg. N eg. M eg. M eg. M eg. P ro te in H* Hr -H ++ -u T+ -Hr -HrH ++ Ti h -H -+ -H - Jm -H -+ -H* K eto n es M eg. M eg. M eg. M eg. M eg. M eg. M eg. N eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. O c c u lt 31ood M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. B ilir u b : M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. 10 7 5 -8 1 0 1 .0 3 8 7 5 -3 1 1 1 .0 2 8 7 5 -8 1 2 1 .0 4 9 75-314 1 .0 3 8 7 5 -8 1 5 1 .0 3 1 7 5 -8 1 7 1 .0 4 8 7 5 -8 1 8 1 .0 4 2 7 5 -8 2 0 1 .0 5 5 6 ,5 M eg. 6 .5 M eg. 6 .0 M eg. 6 .0 N eg. 6 .0 Meg. 6 .5 M eg. 6 .5 M eg. 6 .5 M eg. -w+ T t ! + ++ +-H-H+-H- M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. N eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M EA N + S .D . 1 .0 4 1 + .0 0 9 7 5 -7 6 0 7 5 -7 5 2 75-763 75-764 75-766 75-767 7 5 -7 6 8 7 5 -7 7 0 M EA M + S.D . 1 .0 4 5 1 .0 4 6 1 .0 3 2 1 .0 3 8 1 .0 4 7 1 .0 5 2 1 .0 3 4 1 .0 4 4 1 .0 4 2 + .0 0 7 7 .0 M eg. 7 .0 N eg . 6 .5 M eg. 6 .5 M eg. 6 .0 M eg. 6 .5 M eg. 7 .5 M eg. 6 .5 M eg. -H* + +-HH-+- -H- M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. N eg. N eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. M eg. :a n t d if f e r e n c e s fro m c o n t r o l m eans u s in g a n a ly s is o f v a r ia n c e and D u n n e tt 's t e s t , p < 0 .0 5 . ] -- j \ ..- 2 -1 -287APPENDIX 21 R E S U L T S O F U R I N A L Y S E S O F F E M A L E R A T S ON D A Y 9 0 TWO-YEAR DIETARY STUDY OF 2,4,5-T D ose m /kg /d av 0 30 A n im a l N um ber 75-910 75-912 75-913 75-916 7 5 -1 7 75-919 75-920 75-921 M EA N + S.D . 75-1096 75-1097 7 5 -1 0 9 8 75-1100 75-1101 75-1102 75-1103 75-1104 M EA N + S.D . S p e c ific G ra v ity 1 .0 3 5 1 .0 1 9 1 .0 4 4 1 .0 3 9 1 .0 2 3 1 .0 2 5 1 .0 2 8 1 .0 3 1 1 .0 3 0 + . 0 0 8 1 .0 2 1 1 .0 3 3 1 .0 2 9 1 .0 2 1 1 .0 2 9 1 .0 2 8 1 .0 2 4 1 .0 2 5 1 .0 2 6 + .0 0 4 pH 7 .0 7 .0 6 .0 6 .0 7 .0 6 .5 6 .5 6 .5 6 .5 6 .0 7 .0 6 .5 6 .0 6 .5 7 .0 6 .5 G lu co se N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. P ro te in T ra ce T ra ce T ra c e 4--H T ra c e T ra ce 4-HT ra c e T ra ce T ra ce 4-4T ra ce T ra ce T ra ce T ra ce T ra ce K eto n es N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. O c c u lt B lo o d N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. B ilir u b in N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. 10 75-1046 1 .0 3 6 75-1047 1 .0 2 1 75-1048 1 .0 3 0 7 5 -1 0 4 9 1 .0 1 7 75-1050 1 .0 2 3 75-1051 1 .0 3 0 75-1052 1 .0 1 7 75-1054 1 .0 2 4 6 .0 N eg. 6 .5 N eg. 7 .0 N eg. 7 .0 N eg . 6 .5 N eg. 7 .0 N eg . 7 .5 N eg. 6 .5 N eg. T ra c e + T ra c e T ra ce + 4-4* + 4-4-4 M EAN+S. D . 1 .0 2 5 + .0 0 7 3 75-998 1 .0 2 7 75-999 1 .0 4 0 75-1000 1 .0 3 0 75-1003 1 .0 3 1 75-1004 1 .0 3 4 75-1005 1 .0 2 6 7 5 -1 0 0 9 1 .0 2 6 75-1010 1 .0 2 1 6 .5 N eg. 6 .0 N eg. 6 .5 M eg. 6 .5 N eg. 7 .5 N eg. 6 .5 K e g . 6 .5 N eg. 7 .5 N eg . H* 4* T ra ce 4- T ra c e 4-H- T ra ce 4-4* M EAN+S. D . 1 .0 2 9 + .0 0 6 N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. S m a ll N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg V s t a ^ is c i c a l l y s i g n i f i c a n t d if f e r e n c e s fro m c o n t r o l m eans u s in g a n a ly s is o f v a r ia n c e and D u n n e t t 's t e s t , p < 0 .0 5 . SU5 ^ 3 -238 - -31 o aao o ao o 3 uu aa o o ao o ao o oooooooo o ao o o o o o R E S U L T S O F U R I N A L Y S E S O F MA M i R A T S ON D A Y 3 6 4 O F A T W O - Y E A R D I E T A R Y S T U D Y O F 2 , 4 S t a t i s t i c a l l y s i g i t i f lu ir n e il i f i t i L i i c e fro m c o n t r o l me un u s in g a n a l y s i s o f v a r i a n c e a n d D u n n e t t 's t e s t , p < 0 .0 5 . 3fl 20 24 C 20 20 24 24 33 33 33 33 Z ZZ ZZZZ Z 20 20 20 50 20 20 20 20 3 3333333 Z ZZZ Z ZZ Z i vn U 30 9O QO . ^ 20 00 20 20 30 --* 50 30 3 3 93 393 3 Z z Z Z Z SZ Z C/2 50 54 20 50 50 50 20 50 33 3339 3 3 Z ZZZZZZ Z 0 20 00 20 20 20 20 50 33339333 ZZZZZZZZ 20 3 ZZZZz z z z 20 20 20 fH 24 24 50 20 3 93 33 33 3 ZZ Z ZZZ Z 3 5024543420202024 93333333 ZZZZZZZZ sosoa)524co0 3333333 z zz zz zz 50 50 20 50 20 0 30 40 3 33 33 33 3 z z z zz z z z 20 20 20 20 20 20 20 30 3 33 333 33 zzzzzzzz 20 20 30 20 30 3 0 e o e o 33333333 zzzzzzzz mm a-i ! 4. + + t X + + + + + + +t T T ttt t t -r + f 3 20 2 O 2 0 X 20 32 *j 3 3 3 3 3 3 3 3 ~ zzzzzzzz CO CO 2 20 20 20 2 2 3 3 3 33 333 ZZZZZZZZ 2 0 2 0 2 f l 2 0 2 0 e 0 20 2Q 3 3333 333 ZZZZZZZZ 25 50 2 C 0 2 0 3 2 3333 3333 ZZZZZZZZ OOOOOOOO *1 ' 0 4 '0 ' 0 ' 0 < 0 0 ' 0 00000000 n>0 vO 'O 4 'O 'O ^ O ^w n^O O O O OOl^iO 'O vO 'O 'O OOOOOOOO 'O 'O 'O 'O v f iv O 'O 'O 00000000 29 c* oo oo C '^ O O O f l'^ O f--I "1 m i/N {N| **r >*7 + 1 OOOOOOOO 9H o --1 ^ ^ ^ O rtivinN 3(nc\i/i ON O o -r| OOOOOOOO -I .........................................................cn --4 W "H ^ ^ ^^ o f* on j-i m -e' -r nt -'-n 00000 00 - H --i - H -- --4 - ^ . -- o o - 33 r%N(S 3 o3 353-* u > T u n o o \ 0 ^ > y ' 0 = 0 sOOvO OOOOsO -- -- SI I I I i ! II i Z <0 z3!j Si '!4*>1* r,,--N v/1 .n-*>' /l 4r1- < M gN3O O O -O O O O > S =5 O O O 20 O !I!t !I1 j*> -.n \ n </n vn u*i z: < O --^4 -T u"i r-. =DO xi x i xi xi x iix :x x i z- ' j-1 'j-l -VN V"t v l '/N j-l --O < -- r-. r x ,**h ,-x, ,*. r r* : O NI4/1 jJ .-N. fx. r. O r* co en O X o i" i-- p x O Z' c >> 9 3 0X O -si O (-) <3 9X 5 APPENDIX 23 RESULTS OF IIKINALYSFS OF FEMALE HATS ON DAY 365 OF A TWO-YEAR DIETARY STUDY OF 2,4,5-T D o se ng/kg/day 0 30 A n im a l N um ber 75-910 75-911 75-915 75-916 75-917 75-919 75-920 7 5 -9 1 8 M EA N + S.D . 7 5 -1 1 0 5 75-1098 7 5 -1 0 9 9 75-1107 75-1108 7 5 -1 1 0 2 75-1103 75-1106 M liA N + S .D . S p e c ific G ra v ity 1 .0 3 6 1 .0 1 7 1 .0 1 5 1 .0 1 8 1 .0 2 3 1 .0 2 7 1 .0 3 9 1 .0 2 3 1 .0 2 5 + .0 0 9 1 .0 1 9 1 .0 2 7 1 .0 2 2 1 .0 2 3 1 .0 3 3 1 .0 1 5 1 .0 1 9 1 .0 1 5 1 .0 2 2 + .0 0 6 _ e!!_ 6 .5 6 .0 6 .5 6 .0 6 .0 6 .0 6 .0 5 .5 C lu c o s e N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. 6 .0 N eg. 6 .0 N eg. 7 .0 N eg. 6 .0 N eg. 5 .5 N eg. 6 .0 N eg. 6 .0 N eg. 5 .5 N eg. P ro te in 4-H++ ++ + i-v ++ + -H- + -H +++ -m - + -H-H+ + 10 75-1066 1 .0 3 1 6 .0 N eg. ++ 75-1056 1 .0 2 3 6 .0 N eg. -H -+ 75-1057 1 .0 2 2 6 .0 N eg. +++ 75-1058 1 .0 6 9 6 .0 N eg. + -t+ 75-1050 1 .0 2 2 6 .5 N eg. +++ 75-1051 1 .0 3 1 6 .0 N eg. ++ 75-1053 1 .0 2 6 6 .0 N eg. ++ 7 5 -1 0 5 6 1 .0 2 6 6 .0 N eg. +++ MEAN+S. D . 1 .0 2 9 + .009 3 75-998 1 .0 6 1 6 .0 N eg. + -H - 7 5 -9 9 9 1 .0 6 0 5 .5 N eg. H-+ 75-1000 1 .0 1 5 5 .5 N eg. + 7 5 -1 0 0 3 1 .0 2 3 6 .0 N eg. 75-1006 75-1005 7 5 -1 0 0 9 75-1010 1 .0 2 8 1 .0 6 6 1 .0 3 0 1 .0 3 0 6 .0 N eg. 6 .0 N eg. 6 .0 Neg. 6 .0 N eg. ++ +++ 4-H +++ M EA N + S.D . 1 .0 3 1 + .0 1 0 a No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s fro m c o n t r o l m e a n s u s in g a n a l y s i s o f K eto n es N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. Neg. ' O c c u lt B lo o d N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. B ilir u b in N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. S m a ll N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. 'a t e s t , p < 0 .0 5 . UrobJ11nogen Ehrlich Unlts/dl 0 .1 0 .0 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .0 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 289- APPENDIX 24 R E S U L T S O F U R I N A L Y S E S O F H A I.F R A T S ON D A Y 7 2 6 O F A TWO-- Y E A R D I E T A R Y S T U D Y O F 2 , 6 , , 5 - T D use "'liA n /d u y 0 10 A n im a L N u in li l' 7 5 -6 7 8 7 5 -6 7 9 7 5 -6 8 0 75-681 7 5 -6 8 6 7 5 -7 2 9 75-712 75-768 m i:AN 1 5 . 0 . 75-861 75-862 75-866 7 5 -8 7 1 75-886 75-885 75-889 75-891 Mt:AN l b . 1). S p e c ific G ra v ity a 1 .(1 2 5 1 .0 1 3 1 .0 1 9 1 .0 3 0 1 .0 1 6 1 .0 1 6 1 .0 2 9 1 .0 2 I F . 0 0 8 1 .0 1 5 1 .0 3 0 1 .0 2 9 1 .0 6 3 1 .0 3 3 1 .0 2 1 1 .0 1 7 1 .0 3 0 1 .0 2 7 3 .0 0 9 _ n lL 6 .0 6 .0 6 .0 6 .0 6 .0 7 .0 6 .0 6 .0 6 .0 6 .0 6 .0 6 .0 6 .0 6 .0 6 .5 G lucosu N eg. N eg. N eg. N eg. N eg. N eg. N eg. P ro te in No u r in e o b ta in e d + 3 -1 +H-H + -H -l i- H 1 H+++ K eto n es N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. 3+1 -t-H- . li- r -t-H +++ +++ t-H N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. O c c u lt B lo o d N eg. N eg. N eg. N eg. M od erate S m a ll N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. ; U lllru b li N eg. N eg. N eg. N eg. Neg. . N eg. N eg. N eg. N eg. N eg. + Neg. N eg. N eg. N eg. N eg. 10 75-816 1 .0 2 2 6 .0 N eg. 1-H - N eg. N eg. 75-817 1 .0 2 5 6 .0 N eg. H-+ N eg. N eg. 75-866 No u r in e o b t a in e d 75-850 75-815 1 .0 1 9 1 .0 2 0 6 .0 N eg. 6 .0 N eg. 1++ l t+ N eg. N eg. N eg. N eg. MIlAN ^ S . 1 ) . 1 .0 2 2 + .0 0 3 | 1 1 75-762 1 .0 2 6 6 .5 N eg. +++ N eg. N et*. 75-766 1 .0 1 6 6 .0 N eg. +1 N eg. N eg. 75-771 1 .0 1 8 6 .0 N eg. +++ N eg. N eg. 75- 771 1 .0 1 8 6 .0 N eg. N eg. N eg. 75-776 1 .0 2 1 6 .0 N eg. H I N eg. Neg . 75-788 1 .0 3 1 6 .0 N eg. +H N eg. N eg. 75-796 1 .0 3 3 6 .0 N eg. H-H N eg. N eg. 75-795 1 .0 3 2 6 .0 N eg . 3+1 N eg. N eg. M liA N lS .D . 1 .0 2 4 + .0 0 7 % , s L u L i s l I c a l i y s I g n i I' i c a n t d i f f e r e n c e s f r o i u c o n t r o l m e a n s u s i n g a n a l y s i s o f v a r i a n c e a n il D e n n e t t ' s t e s t , p < 0 . 0 5 . N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. Urobilinogen libiHell Units/ill 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 1 .0 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 1 .0 0 .1 0 .1 o .l 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 290- L....^ 1 . .. i I___ i 1 : L--J ..... J I----- i Ut 4X APPENDIX 25 R E S U L T S O F U R I N A L Y S E S O F F E M A L E R A T S ON D A Y 7 2 7 O F A T W O - Y E A R D I E T A R Y S T U D Y O F 2 . 4 . 5 - T P o se m g/kR/day 0 A n im a l N um ber 75-912 75-914 75-918 75-926 75-927 7 5 -9 2 9 75-935 75-936 M EA N ^ S.D . S p e c ific G ra v ity 1 .0 3 6 1 .0 3 2 1 .0 1 9 1 .0 1 5 1 .0 3 2 1 .0 4 2 1 .0 3 5 1 .0 2 4 1 .0 2 9 T .0 0 9 p ii G lu c o s e 6 .0 N eg. 6 .0 N eg. 6 .0 N eg. 6 .0 N eg. 6 .0 Neg. 6 .0 N eg. 6 .0 N eg. 7 .0 N eg. P ro te in +++ H+++ +++ +++ H -t K eto n es N eg. . Neg. N eg. N eg. N eg. N eg. N eg. N eg. O c c u lt B lo o d N eg. N eg. N eg. S m a ll N eg. N eg. T ra ce N eg. B ilir u b in N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. 30 75-1098 1 .0 3 0 6 .0 N eg. +++ 7 5 -1 1 0 0 1 .0 2 5 6 .0 N eg. +++ 75-1102 1 .0 2 5 6 .0 Neg. +++ 75-1108 1 .0 2 3 6 .5 N eg. -*+ + 7 5 -1 1 1 0 1 .0 1 5 6 .0 N eg. ++ 75-1112 1 .0 2 3 7 .0 N eg. -t-H - 75-1116 1 .0 3 9 6 .0 Neg. + 75-1119 1 .0 2 3 6 .0 N eg. -i-H - M K A N TS .D . 1 .0 2 5 T .007 N eg. N eg. N eg. N eg. N eg. N e g .. N eg. N eg. N eg. S m a ll N eg. N eg. La rg e N eg. T ra c e N eg. 10 75-1046 7 5 -1 0 5 6 1 .0 1 1 1 .0 5 4 6 .0 Neg. 6 .0 N eg. ++ N eg. N eg. N eg. N eg. 75-1059 75-1070 75-1072 75-1086 75-1091 1 .0 2 4 1 .0 3 1 1 .0 2 2 1 .0 3 9 1 .0 3 1 7 .0 6 .0 6 .0 6 .0 6 .0 N eg. N eg. N eg. N eg. N eg. +++ H-H- +++ +++ N eg. N eg. N eg. N eg. N eg. La rg e N eg. N eg. T ra ce N eg. r ln e o b ta in e d 7 5 -1 0 9 2 M EA N TS .D . 1 .0 3 0 T .0 1 4 3 75-99 7 75-999 1 .0 4 2 1 .0 3 0 6 .0 N eg. 6 .0 N eg. -H-+ +++ 7 5 -1 0 0 7 1 .0 4 4 6 .0 N eg. ++ 75-1008 1 .0 4 0 6 .0 N eg. -H + 75-1010 1 .0 3 6 6 .0 N eg. +++ 75-1011 1 .0 2 3 7 .0 N eg. -H + 75-1012 1 .0 2 0 6 .5 N eg. -t-H - 7 5 - 1 0 17 1 .0 2 1 6 .0 N eg. -H-+ MEANT S . D . 1 .0 3 2 T .0 1 0 N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. N eg. S m a ll N eg. Sm al 1 Neg Neg Neg Neg Neg Neg Neg Neg N eg. N eg. N eg. N eg. N eg. N eg. N eg. Neg Neg Neg Neg Neg Neg Neg Neg aNo statistical 1y significant differences from control means using analysi of variance and Dnnnett's test, p<0.05. Urobilinogen Ehrlich Unlts/di 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 0 .1 -291- 75-597 1 30 15.6 9 .1 0.58 5.8 0.37 100 O 55 i-i* O < J (U rt i lu H- ft P H0 0) O rt CD H- O PP 0 i-- P U c CA 0 H0 Q P0 n H* rt Hi - HCA O 0 HP P rt (A rt P* Hi tD Hi /\ CD O 1 0) O0 Ui O CD *h H O 0 O O P rt H O M b P 0 P CA H- 00 P 0 P "C CA HCA 0 en g n H P P H HO p. i =4; > U) IO M UU PPP 0PP CA (A P P MD "O O M3 P- iI I -* "O I-* O CJN OM o\ O O rh O Ni Ni P (A r t (A rt 0 rt 0 PC p. ^ 0. V 1+ K en P > o fe; -o en i Ln ND 00 en ro M en ro l+ U) oo 00 P> t> Ln MD CD ro en 4> 00 ro i+ ro Ln MD 4> -P' U) en en 00 H H 10 -o -n en io O ON P- M3 1+ H* M3 Ln 1+ OO H 1 O* O'* O ro M IO ro Ui H oO O OOO Ln 4> CT* CD *0 O n M ro MD 00 OO O H* ON O IO -P* 1+ H* 4> 00 1+ OO O H4 M3 Ln M 4> ON ON 00 OO O OO O H * IO * 0 Ln H ro Ln M ro OO OM ^1 MD 1+ M lO 0 " t o ON M H M O O en M -> O M t-* 1-* ON P- O 1+ H P' 0* H* io en -P' O' en en (JN oo O L ________ L. t L APPENDIX 26 Dose 2 ,4 ,5 -T ing/kg/day *-4 --4 -1-1 L/Il Ln Ln 11 Ln Ln 1 Ul Animal Number VO vO VO O n Ln -p- CO |0 H* Lo |0 |--* U N H Sample P e r io d CO CO PvO P- O CO CO CO 00 4> ro CO ro CO T o t a l Urine Vol. ro O' 48 h r . (ml) CO ro CO CO vO CO VD VO ro CO CO CO ro o o -o O ' 4> JO O ro vO to Creatinine |o O CO mg/48 h r . ro H* H* CO 00 VO ooo r--* M 00 CO H ooo H* M H* ONCo Coproporphyrin O O O yg/48 hr. Oo o CJNer Ln oo o o Oo Ln 4> CO Ln ro O'* o O O pg Coproporphyrin Ln SJ\ Ln o ON mg Creatinine to CO Ln ro CO M 4> CO ON ON CO O* Uroporphyrin 4> O Oi pg/48 h r . oo o o H* H* ON IO 00 ooO o I-* H ON Ln Ln O O o Ug Uroporphyrin O Ln Mro IO VO mg Creatinine - H* H H O n to IO O MM to CO VO Mo o M M d e l t a - ALA O O vO IO O O |Jg/48 hr. CO Ln Co P- ON CO p- CO ^0 IO o CO 4> -P- pg delta-ALA * Ln ro o mg Creatinine g2 O K ta K n n H H H O ON /-N o M hp > G iM M i/i h-* "K 00 H o 1 hi I--* o HG G vo w f d 1c* P > 5 K . ,h i O HH MO MM !H nP - " {a h n bo w HJ L n ;x ) H t-H O * 643 ^ < o -a t-i - t p' r t - -oI i / j H Oa poi Oh j 9e HO M L___ J J J J ... i I -293- A P P E N D I X 26 (Continued) URINARY EXCRETION VALUES OF CREATININE, COPROPORPHYRIN, UROPORPHYRIN AND DELTA-ALA (DELTA-AMINOLEVULINIC ACID) IN MALE RATS FOR UP TO DAY 106 OF A 118-119 DAY DIETARY STUDY OF 2,4,5-T COMBINED RESULTS OF THREE SAMPLE PERIODS2 E-< 1 m: *- >, * csi -o 00 QJ /--s C H *H Uw 3 H U 0 X u 0 CO H "*T 1 53 2 54 3 68 OJ c H U X H u CO 03 "T 0) ^ u 00 CJ s 28.1 21.6 30.6 75-625 1 44 28.2 2 56 29.7 3 59 34.8 75-626 1 50 32.0 2 54 30.2 3 48 29.8 75-627 1 43 25.8 2 51 27.0 3 55 27.0 75-628 1 42 28.1 2 23 26.4 3 32 33.0 MEAN S.D, 49b 28.8 11 3.2 u >* uu OX Cl o oo u <r CL ^ O 50 cj a. 46.0 39.0 52.0 u X. CL C u] ' o Cl 0 U fi. o CJ CC 50 a| s 1.64 1.80 1.70 u >% --M Cl X u o co Cl > 7 o^ U 00 8.4 9.4 1.9 u >i X CL u o c. o u 3 5 00 s 0.30 0.44 0.06 < <x xp i 3 0 0 u <r 3) 00 -o a 50 115 143 33.0 3 9 .a 57.0 0.72 7.4 0.26 1.31 13.0 0.44 1.64 3.9 0,11 60 73 94 56.0 1.75 12.0 0.38 110 44.0 1.46 16.0 0.53 84 54.0 1.81 2.7 0.09 130 34.0 1.32 7.1 0.28 60 31.0 1.15 10.0 0.37 62 41.0 1.52 2.2 0.08 99 45.0 1.60 6.9 0.25 60 30.0 1.14 14.0 0.53 156 53.0 1.61 1.2 0.04 113 4 3 .6b 1 . 48b 7.7b 0.28b 94 9.3 0.30 4.7 0.17 33 H g d e lta -A L A m g C r e a tin in e 1.8 5.3 4.7 2.1 2.5 2.7 3.4 2.8 4.4 2.3 2.3 3.7 2.1 5.9 3.4 3.3 1.3 'Sample Period ?/l = days 74- 76 of study n = days 99- 101 of study #2 = days 104-106 of study S t a t i s t i c a l l y s i g n i f i c a n t d iff e r e n c e from c o n tro l mean using a n a ly s is of v a ria n ce and D u n n ettTs T e st, p<0.05. m o c/ic r t-h r r P < r P H' H HP H* DO Op fl> h 4 H* {U n< {ri IX Ui H* o OQ cu rj H3 H fl) H* fl O rt P -P en rt p. ru Hin 1-* rt rtt - fl) 1 'O ru /\ p oo ru o Ln i-n < O 0 O o P rt H O h-1 0 P P P c/i P X) o fl) H HO P- 1 =5ti lo IO H* II II II PL IX X P Pp V4 0) P P h"4 nO '-J o vD -P- 4> i i 1M I-* O ON O h-* o> O o O H Hi Hi P P rt (0 rt Pi rr P P- p PL *<. PL "<J -C en H* Q fl) P p h-4 c en HU) 'O LH I On to l'O I-4 LO l'O M i+ H* LO -P- o ON LM Ut O H 4 ro LO Lo LO NO Lo -O 1+ N> no a\ H 4 LO N) LO nO NO ON O O' 4> LO LO LO -P'* O O 'O O O H- io VD P" CT\ ON or HOo ? IO 00 N) cr 4> to 4> O ON Ln OOO MMM O l'O N) O l'O P> l'O H* IO O n ro oO o OOo ON Ln -o vO Lo LO 1+ to Ui Mo M H* O ON -P- o 'O H* ON P> -~-J NO IO 1+ Oo H* CT NO Oo O O LO H 4 4> 4> 00 OOO O ro M Ln LO 4> H- H* Lo O on l'O MMM LO O O N) ON O MM to P'' 00 Ln o O 1+ H* LO vO "O U) LO l'O -P- ON 'O LO p' ro ON "O -o 75-617 Dose 2 ,4 ,5 -T o mg/kg/day -O -O -O Li1n cr* Ln 1 CTN L1n o\ Animal Number H* M H* (Tv Ln W |\) H U K) H W N) H Sample P e rio d P"* LO P * Ln P * N> r o H H* 0^ O P" Total Urine Vol. LO r o t o H* 0 0 r o 48 h r. (ml) olo LO La p- CO ro cr> O' ot o t--* H* H* Ln 00 NO o u> LO IO C r e a t i n i n e O 00 ing/48 h r . vO O o o .0 .0 ,0 ro ro ro CT* o L n o r o M H* L a IO O .O 0' r o r o IO C oproporphyrin 00 to O o oO pg/48 hr. ooO -o (Tv o\ Ln P" H* h~* o o H* (Tv Ov O P- O o O lig Coproporphyrin OO (Tv mg C r e a t i n i n e 00 LO vo t--* PLO Ln 00 M h-* vo LO O P* CTv Ln Uroporphyrin (Tv P- O pg/48 h r . Ooo o ro p- p * p- ooo oM VO IO o o o o |ig Uroporphyrin t--* ro mg C r e a t i n i n e p- P-* **o M 00 **4 CTv O O M VO O Ln P> O H* M delta-ALA O "4 VO VO Oo pg/48 hr. r o IO Ln Ln LO O P - vO O P - Ln o'* LO LO LO UR delta-ALA o P- LO La mg C r e a t i n i n e I OI W GH 5 oo O H H O O'* a o <3 t > O q I rd s M> W HK c n 00 H O 1 !Zi Mo H M r n c n us W M O > F H K MH t ! ahh una 0M w M H P* 1 o io h n K OO w hd en fa H MO Ci HJ 15 ni H ro CTv o o 0rt HP P D fX 1 ro VO P' 1 L -J I___ ! --1 : 1 i 1 UK delta-ALA mg C re atin in e -295- A P P E N D I X 26 (Continued) URINARY EXCRETION VALUES OF CREATININE, COPROPORPHYRIN, UROPORPHYRIN AND DELTA-ALA (DELTA-AMINOLEVULINIC ACID) IN MALE RATS FOR UP TO DAY 106 OF A 118-119 DAY DIETARY STUDY OF 2,4,5-T COMBINED RESULTS OF THREE SAMPLE PERIODSa Eh . 1 to * <r >> * ct3 CN t J 00 4J /-N CM H E => H J-i CO 4-1 0 CO H 56 46 47 44 24 31 56 43 38 42 36 45 29 28 34 40 10 > Cu M0 u j= Cu 0 00 u sr aC0U oo 2. 25.0 24.0 23.0 c >p*> *t-34. 0c) H Oc 3 01 4H a 0 CO JS M cu jc o oo 03 <T ^ * M 00 da 5.2 6.9 1.7 c M 0) < a 3 H C M O 4H-1 O j CO 0 M <uV ao 0 00 as 0.15 0.22 0.06 4.8 0.15 6.0 0.24 1.9 0.06 5.9 0.18 8.5 0.27 2.9 0.08 6.5 0.22 3.7 0.13 3.8 0.12 3.6 0.14 4.4 0.16 2.2 0.07 4.5 0.15 2.0 0.07 < t-3 U <X 1 CQ 00 U pH 0) 00 TJ a 110 99 122 no 100' 117 100 220 144 no 59 77 100 109 120 113 35 3.2 3.2 4.0 3.5 3.9 3.6 3.0 7.1 4.3 3.7 2.1 2.6 3.7 4.0 3.7 3.7 1.1 Sample Period #1 = days 74- 76 of s tudy #2 = days 99- 101 of study #3 = days 104-106 of study No s t a t i s t i c a l d if f e r e n c e from c o n tro l mean using a n a ly s is of v arian ce and D unnett's T e st, p<0.05. oLl b b l - 296- A P P E N D IX 27 U R IN AR Y EXC R ETIO N VALUES OF C R E A T IN IN E , CO PROPORPHYRIN, UROPORPHYRIN AND D E LTA -A LA (D E LTA -A M IN O LE V U LIN IC A C ID ) IN FEMALE RATS FOR UP TO D A Y 106 O F A 118-119 D A Y D IE T A R Y S T U D Y O F 2, 4, 5- T COMBINED RESULTS OF THREE SAMPLE PERIODSa I vn C^d * v 5 0) 4 cn ^ aO s0 Anima! Great: mg/48 u -9 5 2 75-634 ^3 O tH U a<* r--4 O. fsi cn 1 2 3 o > <u fi M U Z2 <U334 80-1 l-i SS "sTo 42 14 54 . <u c Li Hf i - f i 18.5 13.7 22.7 75-635' 1 34 16.3 2 62 19.8 3 56 20.2 75-636 1 39 20.7 2 24 20.2 3 40 23.6 75-637 1 28 17.6 2 39 23.0 3 50 25.0 75-638 1 34 16.3 2 42 20.2 3 42 22.3 MEAN 40 20.0 S.D `t l 2 3.1 U >> fi CL L* U OCoL .f i SO C^eoJ.-<"3s*o. 6 .2 3.4 4.1 Li a Lt O r> O Li CocJ. C1-J1 sc 50 0 . 34 0 . 25 0 . 18 10.0 11.0 9.6 0 . 61 0 . 56 0 . 48 7.1 0 . 34 8 . 1 0 . 40 8.5 0 . 36 7.5 0 . 43 8.3 0 . 36 7.7 0 . 31 7.6 0 . 47 7.8 0 . 39 9.3 0 . 42 7.7 0 . 39 2.0 0. 11 Lt >. f i Li CL f i LOaoUi. s-for 30 3 a. 2.2 0.9 1.4 >C a fi M -H 3u C1-J1 50 50 ' 3. 0 . 12 0 . 07 0 . 06 <: 1-3 H < JS I aj so J <T rH Cl 50 n3 40 36 92 -0o CuJ 5 50 s 2.2 2. 6 4.0 3.6 0 . 22 30 1.4 0 . 07 94 i . 5 0 . 07 .106 1 .8 .8 .2 2.2 0 . 11 40 1 .9 0.9 0 . 04 33 1 .6 1.1 0 . 05 88 3 .7 3.6 0 . 20 60 1.2 0 . 05 142 1.5 0 . 06 105 3.4 6.2 4 .2 2.8 0 . 17 40 1.3 0 . 06 102 1.4 0 . 06 105 2 .4 5 .0 4 .7 1.8 0 . 09 74 3 . 6 0.9 0. 06 36 1 .4 No s t a t i s t i c a l l y s i g n i f i c a n t d if f e r e n c e s from c o n tr o l means using a n a ly s is of variance and Dunnett's t e s t , p<0.05. Sample Period - #1 = days 74-76 of study ir2 = days 99--101 of study ir3 = days 104-106 of study j J S 1 --i j -- -- 1 - -l Dose 2 , 4 , 5-T mg/Ug/day Copropor] pg/48 hr -297- A P P E N D I X 27 (Continued) URINARY EXCRETION VALUES OF CREATININE, COPROPORPHYRIN, UROPORPHYRIN AND D E LTA-ALA (DELTA-AMINOLEVULINIC ACID) IN FEMALE RATS FOR UP TO DAY 106 OF A 118-119 DAY DIETARY STUDY OF 2,4,5-T COMBINED RESULTS OF THREE SAMPLE PERIODS3 lu u OJ 3 E 3 Z cfl E cH < o o c > 1-1 Q) /" N lu C H 0) PL U l uEu 3 <V I--I iH lu 5E * (0 uCaO S oo cn E-> - T OJ c iH J u *H u CO 0U) ^< r CO ue lu lu >1 3 PU alou olPuL l PL u5 1 u dO5-4J CJ 3o 1a Cg O iu 3>1 u u3 U P O 00 PL -T Olu 0 0 33 Pu cl u. 0ouoJ 4J C8 lSuJ 3U --OC< co E 3 Li <c1uo 3 <a or uDUa 00 3 U(Q e <H XJ <0 ua> -a CJ C c o S 30 75-664 1 15 14.3 5.4 0 .38 2.1 0 .15 10 C3.7 2 51 19.9 12.0 0 .60 0.8 0 .04 93 i+.3 3 43 18.9 8 . 6 0 .46 1.5 0 .08 - 75-665 1 59 16.5 8.7 0 .53 2.7 0 .16 40 Z . 4 2 56 16.8 13.0 0 .77 1 . 1 0 .06 122 7.3 3 50 21.5 10.0 0 .46 0.7 0 .03 80 3.7 75-666 1 46 14.3 8.5 0 .59 3.0 0 .21 50 3.5 2 43 14.6 8 . 8 0 .60 1.4 0 .10 8 8 3.0 3 37 16.3 4.1 0 .25 1.4 0 .08 220 1J.5 75-667 1 54 20.0 7.2 0 .36 2.9 0 .14 50 2.5 2 50 18.0 7.8 0 .43 1.2 0.07 107 5.9 3 67 - 11.6 - 1.5 - 130 75-668 1 18 10.3 4.6 0 .45 1 . 5 0.15 10 L . O 2 50 19.0 9.4 0 .49 1.9 0 .10 119 5.3 3 86 20.6 - - -- 120 5.8 MEAN S.D. 48 . 17.2 8 . 6 0.49 1.7 0 .10 8 8 4 .8 17 3.1 2.6 0 .13 0.7 0 .05 56 + _3.3 No s t a t i s t i c a l l y s i g n i f i c a n t d if f e r e n c e s from c o n tro l means using a n a ly s is of variance and D unnett's t e s t , p<0.05. aSample Period - #1 = days 74-76 of study if 2 = days 99-101 of study #3 - days 104-106 of study d s 5 '? 3 t 25 CO O O tHi Hi (A t M ID < tP rtt rt H- H- t nt>- Pt (A rt O H- HO P D tt Ot H H 1 O' VJ * r ..... LO fO M II II a tP tP pt CA (A (A M VD '- J O .O -P' J> 1 1 1 j ~0 h"* O CA OM CA O O Hl O H H i (A (A r t UJ r t t rt t P t p - ^ P - *< O IA ttt H ID H- rt Hi r t H- ** (A Ott rtt r t (A P r t H* Hi Hi *t /\ tD o ItD oO LA D (A Hi t O t O O t rt t O M t tItD (A t (A Ht- QQ IA H* CA i+ K en H o* hfe: * ^4 Ln Ln 11 ca CA 00La Ln -O CO tO J-* 1+ H LA ca O ON ca vO 4>* 1 ^ 4 ON CO *o Ln <o i+ IO to O *-4 LO ro ro o Ln -o O 1 IO IO 1" * O CA CA CO 4> 1+ l o CO IO H* I OO M 4> H Ui - o CA 1 0 0 CO oO LO IO 0 0 "O 1 H* M H* Lo CO O OOO Oo O LA CA CA LO r o 1--* 1+ H H* . ro co i+ oO o H* 00 O MM 1 LA LO OO OO -O LA 1 M M LA LA -O O OOO * O o co CA 0 0 o Hr o O - o ca "-4 CA ca r o i r--* M H Lo Ln CA O O 1+ H P 4> Lo Lo K) 1 e >4 La P' CA L O O ro o hS<SV Q L ........L APPENDIX 27 (Continued) --4 LI1/l Liin os as Ln Ln CTv Ln WN H WNH N> LO Ln 4> a s Ln Ln 4> vo ro Os LO H* N> H* 0 0 Ln o r o H* H* IO O MM O so H o Ln O so 00 00 t o Ln LO Oo O Ln 4> a s LO 0 0 Oo o P- -p0 0 VO O I'O N> H* M H* LO t o - a vO OOO o MO LO |SJ VO OOO o M ro CTs O L0 H4 O S O 00 0 H o H* VO Lo Ln -P" O O LO Ln Lo r o CJs Ln |N> LO vO i--1 Dose 2 ,4 ,5 -T mg/leg/day -*4 LIin as # Animal Number Ln W N H Sample Period P- 4> 4> T o t a l Urine Vol. Os vO 48 Hr. (ml) NJ h H *-0 t--* so Creatinine Ln a s mg/48 h r . Os Ln Coproporphyrin 00 V0 jig/48 h r. o O o )ig Coproporphyrin U ) Lo LO 4>* VO O mg Creatinine O M H* Uroporphyrin vO 4> s o |jg/48 h r . o O Q yg Uroporphyrin ooM 4> 00 O mg C re a tin in e 00 VO Os d e l t a - ALA 00 CD O yg/48 h r . Ln Lo delta-ALA ro a s h i mg C r e a t i n i n e t p> pd a iS p i Hi t-* H PI >U a in > pa Kj p i H o H* H o O /-~N O a s 2 pi o t-> PI h O >i eli t H* K p i H CD o r 1 o HI M H H4h n CD VO d N O CJ v> Hi> M H K ! H *2 O n HI M I M h Hn H '-- o o I CD M pd w Ht O X d Pi O 0* 0 *-< pi 0 pd o h9 o P I pd cn I'O a H !2 4> >* aH L11n C/l fVI ni O O pa o S H OH 1! I________j l______ J i .. .. J ] -299- A P P E N D I X 27 (Continued) URINARY EXCRETION VALUES OF CREATININE, COPROPORPHYRIN, UROPORPHYRIN AND DELTA-ALA (DELTA-AMINOLEVULINIC ACID) IN FEMALE RATS FOR UP TO DAY 106 OF A 118-119 DAY DIETARY STUDY OF 2,4,5-T COMBINED RESULTS OF THREE SAMPLE PERI0DSa I -m * >\ * T3 CN ^ O 0) j * O O Q M 0) J2 a z3 oj a c < 75-644 " o H (3 CU -C9COB* 1 2 3 0 > 01 C IH r-f 03 J Z HO -0a061 35 58 ( c H U . C .3 H U 00 oi < r a0u) eso 16.5 17.2 20.9 c H a o je o co w *<r CGaJUm3eo. 5.3 9.4 8.1 jo3 j c u o . *H a a) 00 eo e 0 32 0 . 55 0 39 m eu jz o CO eu <r 3o^ o3o 2.8 1.3 1.1 a o a| o 3. a0 0.17 0.08 0.05 <2 < -3 I CQ 0 0 U -<OU 380 40 61 87 330 3s0 2 .4 36 4 .2 75-645 1 35 16.5 8.0 0 48 3.1 0.19 20 1 .2 2 14 14.3 5.8 0 40 1 . 0 0.07 5 0 .4 3 62 21.9 7.0 0 32 1.5 0.07 112 5 .1 75-646 1 29 20.6 4.9 0 24 5.7 0.28 40 1 .9 2 30 17.7 8.5 0 48 1.7 0.10 42 2 .4 3 45 23-. 4 9.4 0 40 1.2 0.05 86 3 .7 75-647 1 2 3 14 25 38 12.0 9.0 0 75 16.5 11.0 0 67 17.9 8.4 0' 47 1.7 0.14 2.7 0.16 1.3 0.07 10 0.8 98 5 .9 72 4 .0 75-648 1 49 21.1 9.5 0 45 3.2 0.15 70 3 .3 2 45 19.4 11.0 0 57 1.4 0.07 75 3 .9 3 46 23.5 8.4 0 35 1.0 0.04 87 3 7 MEAN S.D . 39 18. 6 8. 3 0.46 2. 0 0.11 60 3 15 3. 3 1. 8 tO. 14 1. 3 0.07 33 1 No s t a t i s t i c a l l y s i g n i f i c a n t d iff e r e n c e s from c o n tro l means using a n a ly s is of variance and Dunnett's t e s t , p<0.05. Sample Period - #1 = days 74-76 of study #2 = days 99-101 of study #3 = days 104-106 of study O C/l h r r p) < PI rftl lu HP> rfil PO O P> (D If-l* pfl) ^<J PL f(/)l fOfflll 0ff0ll lu fl) h no r r P) - ri (/) rt H P*. (U 1 . M i r t M X J f(Ul A O f11l) O O (D Ml t O B o o r-i rr *< fOl rJ n> p) y P 0) HB OQ (1) fj fPl tfnl U> LO H LO OO 75-629 j3 O "O 'O "-4 'O Ln 1 L1in Liin U111 L11n on 0 n ch CTN CT I- 4 fl* fl* fl O Lo lo H O VO ^1 ^1 '-J '-O Litn Li1n Ln i Liin Liin On On ON On ON f lN> N> N ) ro f lLo N i O vo ^4 '-O L11n Liin Ln Liin O n On On ON f lLO LO LO LO LO N ) O 1+ f l `>0 A Lo <o IO O fl f l ro to H Ln Lo O Ln Ln Ln O il- fl* ON vo 00 fl* o K) *0 fl* A f-lf4l N> LO Ln O Ln O O 1+ fl* Ln H -fl LO fl* M fl fl* ON Ln *0 ON O Ln Ln o Ln 1f>l fl* O fl* fl* fl* fl O Lo IO io LO Ln Ln ON LO f l '-O LO 1+ fl t--1 fl* fl* fl* fl ho tJ o L/ Ln H LO N> U) Ln ^4 ro OO Lo 1+ fl Lo O fl lo *0 -*o ffll H* H LO O n fl fl LO ON vo 1r fl* ''O |0 fl* lo ro 4^ fl* I-* to ffll 00 ON o o o ro o ffll fl 00 IO fl* fl* fl* ro o Lo ON vo ON vo 00 o O O o o fl ro -o o ro A fl* H* VO LO fl LO Ln h - rfol OOOOo IIo fl* fl fl O O fl Lo f l 00 Ui fl Lo 0o O VO ON Ofol o Ln il OO ON OO O H* O O O CO CO Ui fl O fl Ln fl O ^4 fl o fl fl fl vo o 00 LO O Ln OfNl VO Ln 1+ OO M O O O M DIVO O O' vj 4> O 1+ O fl ro VO IO ro Pu f l fl ro ro IO Ln 00 o f l o -0 LO Ln IO ro 1+ O I-- 1 M M M M O Ln |0 H O v Ui IIoo of l o oo o o oOo o 00 ffll o Ln O 'O of l H oO oo Ln 'O oOOOO H Ln Of l OO Ln LO O 00 HoO o fl Ln lo O O Oo O O vO Hf*l fl 00 fl Lo o fl 955^ L l .... L-- APPENDIX 28 Dose 2 , 4 , 5-T mg/kg/day 1+ CO n K ^4 *VJ ^0 '-O C/l Ln O i cn c a O n O n c c ON Cn o O o o vO to CO K * o VO Animal Number 1+ H 4 H* MMM InJ CO Co o n O Co H Total Urine Vol. CO OO L o U i O o 24 h r . (ml) H H* H* CO M H* H* -* !-* Co CO oO Creatinine o n o CO U i 4^* H* VO mg 24 h r . i+ M u> Co M h4 M 'O c c CC vO vO Coproporphyrin as ooo Dg/24 hr. 00 i+ o -* CO M H* CO M M H4 O O CO CO H* VO s o SO 0 0 H a s O pg Coproporphyrin mg C re a tin in e Ih oo CO s o o o o o C-* SO 0 0 0 0 ON CO Uroporphyrin pg/24 hr. 1+ OO OO N ) '- i OO OO O O O O O H4 n J (J\ CT\ (J\ H pg Uroporphyrin mg C r e a tin in e <=i P I !2 id In HH CO O J2j O p? i-5 P i mm *-* O H PI 1o H4 DJ h0H0 O Mj H ft H* 00 o Io M hd H id V O co oO %s c/i M *-<3 %Ohrj 3 |V0 C2 4C o V* p i Ui o I H 3as H 2 1________J -301- A P P E N D I X 29 Dose 2,4,5-T mg/kg/day URINARY EXCRETION VALUES OF CREATININE, COPROPORPHYRIN AND UROPORPHYRIN FOR FEMALE RATS ON DAYS 118-119 OF A 118 -119 DAY STUDY OF 2 ,4 , c H cM o s >, --i U 01h c. c01 A o cB T-t 6 o.3 T-l oZ Oo. OtH o <r o o3 CM U CM CMB c EOl <CMT G.^< > 0) -> C a T-t M 3 JZ P. 3 C U C JZ trt U Mf 3 01 ^ u oo uS H u >, JZ IJ p JZ o oo U 3- JZ a> C. u H a H 4J 3 a . 0) O CJ 00 ao 3. B rt T -t u >T JZ u o . j= < ft, O^ M oo 35 3 . >, JZ -rt fti C U "H OU ft 3 01- l-i u au t5C 00 33 B 0 75-639 18.0 9.2 4.0 0 .43 0.5 0.05 75-640 22.0 7.5 4.0 0 .53 0.4 0.05 75-641 11.0 7.7 3.6 0 .47 0.2 0.02 75-642 19.0 8.6 6.1 0 .71 0.4 0.05 75-643 22.0 9.5 4.4 0 .46 0.6 0.06 MEAN 18.4 8.5 4.4 0 .52 0.4 0.05 S.D. 4.5 0.9 1.0 0 .11 0.1 0.01 30 75-669 25.5 8.9 7.9 0 .89 0.1 0.01 75-670 17.5 5.6 2.9 0.52, 0.2 0.04 75-671 34.0 8.2 4. 6 0 .56 0.3 0.04 75-672 10.0 7.5 9.9 1 .32 0.02 0.00 75-673 18.5 6.1 7.2 1 .18 0.1 0.02 MEAN 21.1 7.3 6.5 0 .89a o . i a 0.02 S.D. 9.1 1.4 2.8 0 .36 0.1 0.02 10 75-659 14.0 8.7 4.5 0 .52 0.3 0.03 75-660 21.0 7.6 5.3 0 .70 0.2 0.03 75-661 10.0 7.1 5.4 0 .76 0.1 0.01 75-662 13.0 8.6 5.7 0 .66 0.2 0.02 75-663 17.0 8.5 4.6 0 .54 0.3 0.04 MEAN 15.0 8.1 5.1 0 ..64 0.2 0.03 S.D. 4.2 0.7 0.5 0 .10 0.1 0.01 3 75-649 32.5 7.8 5.1 0 .65 0.5 0.06 75-650 13.5 9.0 4.2 0 .47 0.4 0.04 75-651 13.5 9.5 4.6 0 .48 0.5 0.05 75-652 14.0 6.2 3.5 0.56 0.3 0.05 75-653 15.0 8.6 4.3 0.50 0.2 0.02 MEAN 17.7 8.2 4.3 0 .53 0.4 0.04 S.D. 8.3 1.3 0.6 0 .07 0.1 0.02 S t a t i s t i c a l l y s i g n i f i c a n t d if f e r e n c e from c o n tro l mean using a n a ly s is of variance and D unnett's T est, p<0.05. -3G 2APPSHOIX 30 D osa g g /k g /d ay 0 URINAR* SXCRSTtON VA1USS o f C R E M I S I N E , COPROPORPSYRIN , U R O P O R ?K *R IS , ANO d a i c a - A L A ( d a i : a - a m i n o U v u l i o i c a c i d ) FOR MALE ANO FEMAL2 J A I S ON DA*S 9 5 - 9 7 OF A TVO-YEAR D tETAflX 3 T u D * OF 2 , 4 , 3 - t A nim ai yfum er Sax T ocal ( J r i n a '7oL. 43 hr.C rai) C re a c ia ir.e a g /4 3 h r. C ap ro p o rp h y rin u g /4 3 h r. ug C o p rap o rp n y ria/ ag C ra a c ia ia e U ro p o rp h y rin u g /4 3 h r. ug U ro o o rp h y ria/ mg C r a a c i a i a e d elca-A lA u g /4 3 h r. 7 5 --4 7 4 7 5 -4 7 5 7 5 -4 7 6 75-479 7 5 -4 3 0 MEAN S.D . M M M M N 22 12 20 23 44 20 no 2 3 .5 3 0 .1 2 2 .2 2 3 .3 3 3 .0 2 7 .5 4 .5 L 4 .0 2 2 .0 2 7 .0 1 7 .0 2 0 .0 2 0 .0 4 .9 . 50 .7 3 1 .2 2 .5 9 .6 1 .7 5 .2 7 2 .1 2 .7 4 .3 2 .1 1.7 2 .9 1 .1 .0 9 .0 9 . 22 .0 7 .0 8 .1 1 .0 6 130 130 130 130 140 152 26 ug d e lta -A lA / mg C r a a c i a i a a 5 .5 5 .0 5 .3 6 .2 4 .2 5 .5 0 .3 30 7 5 - 3 6 1 37 7 5 -3 6 1 31 7 5 -3 6 4 M 24 7 5 -8 6 5 M 33 7 5 -3 6 6 M 49 MEAN tS .D . 35 9 10 75 -3 L O 22 7S -3U M 30 7 5 -3 1 2 M 26 7 5 -3 1 4 M 53 7 5 -3 1 .5 M 35 MEAN S S .D . 34 14 3 7 5 -7 5 0 il 34 7 5 -7 4 2 M 31 7 5 -7 6 3 M 42 7 5 -7 6 4 M 37 7 5 -7 6 4 M 37 MEM sS .O . 36 4. 3 0 .7 2 7 .3 3 1 .0 2 3 .0 2 7 .9 2 9 .1 1 .6 2 3 .2 2 1 .0 2 3 .7 3 7 .7 2 4 .3 2 7 .1 6 .5 3 0 .6 2 7 .3 2 4 .3 2 3 .1 2 6 .3 2 7 .4 2 .2 3 6 .0 4 1 .0 3 4 .0 4 4 .0 4 3 .0 3 9 . 6a 4 .4 3 5 .G 2 2 .0 2 6 .0 4 4 .0 2 8 .0 3 1 .0a 3 .7 3 0 .0 2 7 .0 2 0 .0 1 3 .0 1 7 .0 2 2 .4 5 .3 1 .1 7 L .4 7 1 .1 0 1 .5 7 1 .5 4 1 .3 7 a .2 2 1 .2 4 1 .0 5 1 .1 0 1 .1 7 1 .1 3 1 .1 4 3 .0 7 .9 8 .9 9 .3 1 .6 4 . 65 .3 1 1 .1 7 2 .6 1 .9 2 .5 4 .4 3 .2 2 .9 0 .9 3 .4 2 .9 1 .6 2 .6 1 .3 2 .5 0 .3 2 .9 2-.0 2 .4 3 .0 1 .2 2 .5 0 .4 .0 3 .0 7 .0 8 .1 6 .1 1 .1 0 .0 4 .1 2 .1 4 .0 7 .0 7 " .0 7 .0 9 .03 .0 9 .0 7 .1 0 .1 1 .0 8 .0 9 .0 2 " 90 UO 160 30 140 U6 34 90 160 100 250 110 142 66 90 210 160 240 130 16 6 60 2 .9 3 .5 5 .2 2 .3 5 .0 3 .9 1 .1 3 .2 7 .6 4 .2 6 .6 4 .4 5 .2 1 .3 2 .9 7 .7 6 .4 3 .5 4 .9 6 .1 2 .2 0 7 5 -9 1 0 F 37 7 5 -9 1 2 F 63 7 5 -9 1 3 7 5 -9 1 6 t p 10 45 7 5 -9 1 7 ? 64 MEAN S .3 . 45 21 30 7 5 -1 0 9 6 r 46 7 5 -1 0 9 7 7 5 -1 0 9 8 75-L 1C O s* p 17 40 33 75-1101 F 17 MEAN S .D . 31 13 2 0 .0 1 7 .0 7 .5 2 2 .0 2 1 .3 1 7 .7 -fi.O 1 7 .5 1 4 .3 1 5 .6 1 3 .2 1 3 .6 1 4 .9 1 .7 6 .5 8 .1 2 .1 7 .6 8 .9 6 .6 2 .7 1 2 .0 3 .2 3 .2 3 .2 1 2 .0 9 .7 2 .1 ' .3 2 .4 3 .23 .3 4 .4 1 -3 7 .08 . 63 .5 5 .5 2 .62 .38 .6 5 * .1 4 1 .5 1 .6 1 .4 1 .3 L .6 1 .5 = 0.1 1 .2 L .3 0 .3 1 .2 1 .2 1 .1 0 .2 7 5 -1 0 4 6 7 5 -1 0 4 7 7 5 -1 0 4 8 75-L 049 7 5 -1 0 5 0 MEAN s.o. 7 e F p e 7 5 -9 9 3 7 5 -9 9 9 7 5 -lO C O 75-1003 7 5 -L 0 0 4 MEAN S .D . F ? 7 ? 51 14 23 60 50 41 19 44 36 57 50 4 49 11 2 1 .4 3 .0 1 7 .6 2 0 .4 2 0 .0 1 7 .5 5 .5 2 0 .7 2 0 .2 2 0 .1 2 1 .0 1 5 .0 1 9 .4 2 .5 8 .7 3 .0 1 0 .a 1 2 .0 7 .1 3 .3 3 .5 7 .3 9 .0 1 5 .0 9 .4 5 .2 9 .7 3.7 .41 .3 8 .6 1 .59 .3 6 .4 7 .1 2 .3 8 .4 4 .3 0 . 45 .4 1 .5 0 .i7 1 .5 0 .3 0 .3 2 .7 1 .1 1 .4 0 .3 2 .1 1 .0 3 .4 12 1? 1 .3 1 .0 caeiscically .can e frani cenerei, meaa u s m g an aj.y sis o: v a r i a n c a and D unnacc's case .0 3 .09 .1 9 .0 6 .07 .10 .0 5 .0 7 .09 .0 5 .09 .0 9 .0 8 .0 2 .0 7 .10 .0 4 .1 3 .06 .0 8 .0 4 .10 .0 5 . 17 .0 6 .0 3 .0 9 .0 5 210 1 0 .5 230 1 3 .5 20 2 .7 150 6 .3 170 7 .8 156 3 .3 32 4 .0 140 8 .0 170 1 1 .5 00 130 9 .3 140 1 0 .3 116 7 .9 66 4 .6 150 7 .0 UO 1 6 .2 U O 7 .4 200 9 .3 U O 7 .5 152 9 .6 29 3 .9 U O 7 .2 L00 3 .0 ISO 9 .0 120 5.7 150 1 0 .0 140 7 .4 31 2 .1 I 1 I V5 0 .5 5 -30 3 - APPENDIX 31 D oae m * /k /d a v 0 URINARY EXCRETION VALUES OF C R E A T IN IN E , COPROPORPHYRIN, UROPORPHYRIN, AND d e l c a - A L A ( d e l t a - a m i n o l e v u l i n i c a c i d ) FOR MALE AND FEMALE RATS ON DAYS 1 8 8 - 1 9 0 OF A TWO-YEAR DIETARY STUDY OF 2 , 4 , 5 - T T o tal A nim al U rin e V ol. Number Sex 48 h r.( m l) C re a tin in e n e/4 8 h r. C o p ro p o rp h y ria u /4 8 h r. Ug C o p ro p o rp h y r in / U ro p o rp h y rin m2 C r e a t i n i n e U i/4 8 h r. ug U ro p o rp h y rin / delca-A L A m2 C r e a t i n i n e 112/48 h r . 7 5 -6 7 4 7 5 -6 7 5 7 5 -6 7 6 75-679 75-680 MEAN + S .D . M M M M M 2 4 .0 4 4 .5 4 5 .0 2 7 .0 2 7 .0 33.5 +10. 3 2 6 .0 3 7 .0 3 1 .0 2 6 .0 3 1 .0 3 0 .2 + 4 .5 1 7 .0 2 9 .0 1 3 .0 1 5 .0 2 2 .0 1 9 .2 + 6 .4 .6 5 .7 8 .4 2 .5 8 .71 .6 3 + . 14 1 .6 3 .2 2 .5 1 .9 1 .8 2 .2 +0. 7 .0 6 2 .0 8 6 .0 8 1 .073 .0 5 8 .0 7 2 + .0 1 2 70 110 110 70 60 84 +24 Ug d e lc a -A L A / 2 .7 3 .0 3.5 2 .7 1.9 2 .8 + 0 .6 30 7 5 -3 6 1 M 3 0 .0 2 9 .0 7 5 -8 6 3 M 3 5 .0 3 0 .0 7 5 -8 6 4 M 3 7 .0 3 3 .0 7 5 -8 6 5 M 5 6 .0 3 0 .0 7 5 -8 6 6 M 4 0 .0 3 1 .0 MEAN + S .D . 3 9 .6 + 9 .9 3 0 .6 + 1 .5 3 9 .0 5 3 .0 4 6 .0 6 2 .0 37 ;0 4 7 .4a + 1 0 .3 1 .3 4 1 .7 7 1 .3 9 2 .0 7 1 .1 9 1 .5 5 a + .3 6 2 .0 2 .6 3 .0 3 .5 5 .9 3 .4 + 1 .5 .0 6 9 .087 .0 9 1 .1 1 7 * .1 9 0 .1 1 1 + .048 120 90 100 130 110 110 +16 4 .1 3 .0 3 .0 4 .3 3 .5 3 .6 + 0 .6 10 7 5 -8 1 0 M 31. 2 9 .0 7 5 -8 1 1 M 4 2 .0 2 5 .0 7 5 -8 1 2 M 5 2 .0 1 3 2 .0 7 5 -8 1 4 M 4 7 .0 3 6 .0 7 5 -8 1 5 M 3 8 .0 3 4 .0 MEAN + S .D . 4 2 .0 + 8 .1 31.2 + 4 .3 3 7 5 -7 6 0 M 4 5 .0 3 6 .0 7 5 -7 6 2 M 2 1 .0 2 4 .0 7 5 -7 6 3 M 4 6 .0 3 1 .0 7 5 -7 6 4 M 38.0 3 2 .0 7 5 -7 6 8 M 4 4 .0 2 3 .0 MEAN + S .D . 3 8 .8 + 1 0 .4 3 0 .2 + 4 .5 0 7 5 -9 1 0 F 2 0 .0 1 5 .0 7 5 -9 1 2 F 2 8 .0 2 0 .0 7 5 -9 1 3 F 4 6 .0 1 7 .0 7 5 -9 1 6 F 4 5 .0 2 3 .0 7 5 -9 1 7 F 6 6 .0 2 5 .0 MEAN + S . D 4 1 .0 + 1 7 .9 2 0 .0 + 4 .1 3 1 .0 2 6 .0 3 6 .0 3 2 .0 2 4 .0 2 9 .8 + 4 .8 2 9 .0 2 8 .0 2 5 .0 2 1 .0 2 5 .0 2 5 .6 + 3 .1 9 .3 1 1 .0 7 .4 1 2 .0 8 .2 9 .6 + 1 .9 1 .0 7 1 .0 4 1 .1 3 .8 9 .71 .9 7 + .17 .8 1 1 .1 7 .8 1 .6 5 .89 .8 7 + .19 .6 2 .5 5 .4 4 .52 .3 3 .4 9 + .11 1 .3 1 .6 2 .2 4 .0 3 .0 2 .4 + 1 .1 2 .7 1 .2 2 .8 2 .2 2 .5 2 .3 + 0 .6 1 .3 1 .5 1 .6 1 .7 1 .4 1 .5 + .2 .045 .0 6 4 .069 .1 1 1 .0 8 8 .0 7 5 + .025 .0 7 5 .0 5 0 .0 9 0 .0 6 9 .089 .0 7 5 + .0 1 6 .08 7 .0 7 5 .094 .0 7 4 .0 5 6 .077 + .015 160 80 130 140 90 120 +34 110 "0 120 110 140 110 +26 50 80 90 100 13 0 90 30 5.5 3 .2 4 .1 3 .9 2 .6 3 .9 + 1 .1 3 .0 2 .9 3.9 3.4 5 .0 3 .6 + 0 .8 3 .3 4 .0 5 .3 4 .3 5-2 44 3 .3 30 75-1096 F 5 4 .0 1 8 .0 75-109 7 F 5 5 .0 2 2 .0 7 5 -1 0 9 8 7 5 -1 1 0 0 F y 3 3 .0 4 0 .0 1 5 .0 1 8 .0 75-1101 7 4 4 .0 2 2 .0 MEAN +S. D. 4 5 .2 + 9 .4 1 9 .0 + 3 .0 1 7 .0 1 5 .0 1 0 .0 1 2 .0 2 8 .0 1 6 .4a + 7 .0 .9 4 .6 3 .67 .6 7 1 .2 7 .S 5a + .2 6 2 .0 1 .7 1 .5 1 .7 1 .4 1 .7 + .2 .111 .077 .1 0 0 .0 9 4 .0 6 4 .0 8 9 + .0 1 9 70 100 60 30 90 80 +20 3.9 4 .5 4 .0 4.4 4 .1 4 .2 0 .2 10 75-1046 F 3 8.0 2 0 .0 75-1047 F 4 8 .0 2 0 .0 75-1048 F 3 6 .0 1 5 .0 75-1049 F 5 1 .0 1 7 .0 75-1050 F 3 8 .0 1 7 .0 MEAN + S .D . 4 2 .2 + 6 .8 1 7 .8 + 2 .2 3 7 5 -9 9 8 F 4 4 .0 2 0 .0 75-999 F 2 4 .0 1 4 .0 75-1000 F 5 9 .0 1 8 .0 75-1003 F 7 6 .0 2 3 .0 75-1004 F 5 4 .0 1 8 .0 MEAN + S . D. 5 1 .4 + 1 9 .2 1 8 .6 + 3 .3 9 .1 9 .8 9 .2 1 0 .0 8 .0 9 -2 + .8 1 2 .0 7 .1 1 5 .0 1 1 .0 6 .6 1 0 .3 + 3 .5 .46 1 .6 .4 9 1 .1 .61 1 .6 .59 1 .2 .47 1 .1 .080 .0 5 5 .1 0 7 .0 7 1 .0 6 5 .52 1 .3 '.076 + .0 7 + .3 + .0 2 0 .6 0 1 .3 .51 1 .1 .8 3 1 .1 .43 1 .4 .3 7 1 .0 .0 6 5 .0 7 9 .061 .061 .056 .56 1 .2 .0 6 4 + .1 7 + .2 + .009 S t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e fr o m c o n c r o l ate a n u s i n g a n a l y s i s o f v a r i a n c e and D u n o e c t 's t e s t , p < 0 .0 5 . 90 90 90 70 80 30 10 80 60 30 1 "'O 100 90 +30 4 .5 4 .5 6 .0 4 .1 4 .7 -.3 3 .7 4 .0 T ` .J t '1 5 .6 4 .3 + 0 .3 gw iP L .1 su 5 2 BUST--' o U (A b fe I tili o o oo o U t *> t* 0- O >> O - O ' ,_1 *I--I--I I --I . H - o o o O < fiU }iO h o > 1% S I ` 5 b fe M UI ut ui IIiI J >P 4 i >U < I-* I- I-- I O' Li lb o M O ' O l o * LO O U i 00 O ' O CJ O O O 1t1-- o> Ul U ' O (U O ' 1- o o o o o o 1+ s o CS es n e n co Ui o OOOO H: O' o O cu U1 o s i s | U ooooo i 3/ . u O O' IUI- Us>i h-* ut la co o o c* o sii O o o o o o CD N o O O O o ooooo l i \- I- I-- - t-- C i - U ' O so <J> U. O O o o o O i i ' OD # s OS ooooo Ut O' H O SO ooooo 1+ 1-- I- I-- H* c-- r- u> o t CTs^ h-*o !-> O) O O s O O p o<5T\ H O ' ( I co <o f'j l Cu IO t- 1 tO t H JO ' .> <o M 'S si | + 1-- N N H 1 " h* - 1 Qo O ' U H M tu IIO CJ O O' p- C.I O C. O o OCu' O' s- O' 1- O. Cn en cu l oo Lo sO LO s j IO-- 11--- 1 -" UO OS| O O' O O' *.* 060 024 1+ ooooo s j s , U) f> fs> CO O 00 CJ 00 ri oo ooooo 00 SO - o O ' O ' P- io co -o O II o o o o oo o 11 o-* oCu 1c+o cd oo ooooo ' i ................ ., > 1- Sl> 1 i l u O U i 11 CJ C-i CJ L I U IJ u u u tu l u I-I U H 1I+-* U J *' Ii (PT=>F rTUTTn/iSTOCTOEE-rs-rsp) V T V -f= T 'P Otfv ` K IS iS iS W O T i ` KIiSUOaO'SiOD ` 2KIKI1V3D 20 S2.TTi;/i N0IX2K3K XVKIHO o y: X X X X h- *- -g a ooooo 'S 5 o t Ul Ul Ul U) lili i Oh 03 OUOi XXXXX )+ ^ - Ul O' o O O O O Ci i /Vi i /V> Q i QT\ O' O' O' O' O ' 1 - W h- XX XX X I I X O' O' O' O' 03 I1 Oj s ' i ' I N O O o ' o i> XXXXX o ooooo Q G 1+ Ul Ul O' o oooo o ooooo O' oa o o o o o is O Ui oooo 1+ f* IU ^ U o N N o 4> o a o a> M o tu o o o o o Ul o o o o o o S uu o o o o <--- o '*3 3` l>f-- |U O V- 1+ . O U> InTH? *" U1 U U i - M !- W H U l t- ' *u U 1+ N r> N N W H ca t-* *-- oo Ui O tu r-- iu *-- iu i-- V t- tu O Ci t ' cii O O O t-- O O 3 to O' Ul O O' Ui -o O O 'O O' n O O 0 0 * 1 -0 0 Ui OQ 03 00 Ui -u <> M Ul Ui U I-- CJ U oo tIUu Uol o oo o o UOi -O4'O-' *0-0*U00* o o O IU' 4f <0 O I--I LU O I t- r* i--*--*--j-- e ' S tU M H H N I-i |> U Ul 'O 'O tu CO ' N I Ci . 3lu ; Ul Ui i Cl O' O* t na.M 44 I J L. J -___ i :........J 305- APPENDIX 33 URINARY EXCRETION VALUES OF C R E A T IN IN E , COPROPORPHYRIN, AND UROPORPHYRIN FOR HALE AND FEMALE RATS ON DAYS 5 6 4 - 5 6 6 OF A TWO-YEAR DIETARY STUDY OF 2 , 4 , 5 - T Dose m g/kg/dav 0 Animal Number Sex 75-674 75-675 7 5 - 6 78 7 5 - 6 79 75-680 MEAN +S.D. M M M M H T o ta l Urine Vol. 48 h r . (ml) 47.0 53.0 46.0. 50.0 45.0 48.2 + 3 .3 C reatinine og/48 h r. 36.0 24.0 25.0 -- 35.0 30.0 +6.4 C o p ro p o rp h y rin ug/48 hr. 24.0 22.0 34.0 -- 34.0 28.5 + 6 .4 Ug C o p r o p o r p h y r i n / U r o p o r p h y r i n mg C r e a t i n i n e ug/46 h r. .67 .92 1.36 .97 .98 + .29 11.4 10.2 10.8 ___ - 11.4 11.0 + .6 ug U roporphyrin/ me C r e a t i n i n e .32 .43 .43 _____ .33 .38 t - 06 30 75-861 M 63.0 25.0 75-863 M 110.0 44.0 75-864 M 40.0 28.0 75-865 . M 127.0 25.0 75-866 M 129.0 17.0 MEAN + S .0 . 93.8* +40.1 27.8 +9.9 22.0 36.0 62.0 24.0 38.0 36.4 +16.0 .88 .82 2.21 .96 2.24 1.42 + . 73 8 .0 9.0 6.0 6 .2 9.6 7.8 +1.6 .32 .20 .21 .25 .56 .31 + .15 10 75-810 M 103.0 31.0 75-811 M ` 78.0 25.0 75-813 M 69.0 29.0 75-814 M 50.0 31.0 75-815 M 57.0 34.0 MEAN +S.D. 71.4 +20.7 30.0 + 3 .3 3 75-760 M 71.0 28.0 75-762 M 40.0 27.0 75-764 M 44.0 35.0 75-765 M 71.0 18.0 75-766 M 112.0 28.0 MEAN +S.D. 67.6 + 2 3 .8 ' 27.2 + 6 .1 0 75-911 F 94.0 12.0 75-912 F 61.0 23.0 75-913 F 27.0 . 14.0 75-914 F 17.0 16.0 75-917 F 69.0 ` 26.0 MEAN +S.D. 53.6 +31.5 18.2 +6.0 30 75-109 7 F 60.0 17.0 75-1098 75-1099 F y 58,0 96.0 22.0 22.0 75-1100 F 52.0 12.0 75-1102 F 129.0 13.0 MEAN +S.D. 79.0 +32.9 17.2 + 4 .8 10 75-1046 F 82.0 25,0 75-1047 F 40.0 20.0 75-1049 F 75.0 21.0 75-1050 F 68.0 24.0 75-1053 F 64.0 16.0 MEAN +S.D. 65.8 +16.0 21.2 +3.6 3 75-999 F 70.0 21.0 75-1000 y 95.0 2 4 . 75-1003 75-1007 F y 91.0 50.0 18.0 15.0 75-1008 F 56.0 28.0 MEAN +S.D. 72.4 +20.2 21.2 +5.1 30.0 24.0 22.0 58.0 30.0 32.8 +14.5 54.0 52.0 44.0 28.0 19.0 39.4 +15.3 7.6 14.8 6 .4 13.6 11.2 10. 7 Z3-7 20.0 28.0 18.2 12.0 18.4 1 9 .3d + 5 .7 19.8 12.2 12.6 17.2 20.0 16.4 + 3 .8 16.0 18.2 14.6 17.4 15.2 16.3 +1.5 .97 .96 .76 1.87 .88 1.09 + .45 1.93 1.93 1.26 1.56 .68 1.47 + .52 .63 .64 .46 85 .43 .60 + .17 L.18 1.27 .83 1.00 1.42 I.1 4 a + .23 .79 .61 .60 .72 1.25 .79 + .27 .76 .76 .81 1.16 .54 .81 + .22 14.6 10.4 9.8 16.8 12.6 12.8 +2.9 14.8 16.6 11.8 7.8 11.6 12.5 +3.4 4.6 6.6 4.0 4.0 7.4 5 .3 .6 7.4 11.2 12.4 4.2 9 .4 8.9 +3.2 7.0 6 .8 4.6 10.2 10.4 7.3 +2.5 5.4 6.6 7.6 1, U 7.0 6 .8 + .9 .47 .42 .34 .54 .37 .43 + .08 .53 .61 .34 .43 .41 .46 + .11 .38 .29 .29 .25 .28 .30 + .05 .44 .51 .56 .35 .72 .52* + .14 .23 .34 .22 .43 .65 .38 + .17 .26 .28 .42 .49 .25 .34 + .11 S t a t i s t i c a l l y s i g n if i c a n t in c re a s e fro c o n tro l aeaa by a n a ly s is o f v a ria n c e and D u n n etc's t e s t , p<0.05. 'S a m p l e n o t a n a l y z e d ; s a m p l e w a s b l o o d y d u e t o r a t h a v i n g s c r a t c h e d h i s s i d e a g a i n s t c a g e . 306- APPENDIX 34 Dose a s/k z /d a v 0 URINARY X CR ETIO N VALUES OF C R E A T IN IN E , COFROPORPHYRIN, UROPORPHYRIN, AMD d a i c a - A L A ( d e i c a - a m i n o l e v u U a i c a c i d ) FOR MALS AMD TzH ALS R A IS OM DAYS 6 9 8 - 7 0 0 OF A TWO-YEAR QtZTARY STUDY OF 2 , 4 , 5 - t lo c a l A nim al U ria Vol. Number Sex 48 h r . (ml) C raaclalne az/43 hr. C oorooorahyrin ^ /4 8 hr. ug C o p ro p o rp h y ria / Uronorphyrin az C raacialae uz/43 hr. dg U ro o o n jh y ria / deica-ALA az C rsacinine uz/43 hr. 7 5 - 4 73 75-479 75-430 75-434 75-732 MEAN S .D . M M M M M 71.0 33.0 35.0 74.0 138.0 90.2 27.4 13.0 22.0 32.0 28.0 24.0 24.3 5.4 17.0 L0. 24.0 12.0 9.9 14.6 6.0 .94 . 45 . 75 .43 .41 .50 .2 4 5.9 3.3 5.5 3.9 3.1 4, 7 +1.3 .38 .15 .20 .14 .13 .20 + .10 130 100 150 L00 130 120 +20 J* h a l c a - A L A / 7.2 4.5 4 .7 3.6 5 .4 5 .1 1 .3 30 75-361 M 129.0 12.0 75-363 M 154.0 7.0 75-364 M * 79.0 26.0 75-867 M 33.0 22.0 75-371 M 5 3 .0 31.0 MEAN S.D . 9 9 .6 40.9 19.6 9.9 15.0 7.8 32.0 19.0 40.0 22.3 13. Q 1.25 1.11 1.23 .36 1.29 1 .15a + .1 7 4 .0 4 .2 6 .4 2 .6 2.7 4 .0 1 .5 .33 .50 .25 .12 .09 .23 .20 130 170 UO 110 U 13 20 LO . 8 2 4 .3 4 .6 5 .0 3.5 9 .6 8 .7 1Q 75-314 M 23.0 24.0 75-315 M 76.0 27.0 75-318 M 127.0 19.0 75-319 M . 38.0 23.0 75-344 M 33.0 31.0 MEAN S .D . 64.4 39.4 24.3 4 .5 3 75-762 M 75.0 17.0 75-764 M 66.0 30.0 75-770 M 114.0 26.0 75-771 M 114.0 22.0 75-773 M 107.0 26.0 MEAN S.O . 95.2 22.9 2 4 .2 4 .9 7 0 75-912 53.0 15.0 75-913 ; 59.0 20.0 75-914 j 66.0 19.0 75-915 52.0 22.0 75-918 F 53.0 L9.0 MEAN +S.D. 57.6 +5.6 19.0 --+ 2 . 5 30 75-109 7 ? 23.0 Li.O 75-1098 F 61.0 15.0 75-1100 F 41.0 18.0 75-1104 F 35.0 14,0 75-1108 F 59.0 22.0 MEAN S.O . 54.3 2 1 .7 16.0 1 .2 13.0 12.0 2 .3 9 .4 12.0 9 .7 4 .4 1 5 .Q 10.0 16.0 20.0 L5.Q 15.2 3 .6 3.3 4.9 3.5 6.9 4.9 5.3 +1.9 1.2 9.0 7.4 14.0 3.5 3.0 9 .8 .54 .44 .12 .41 .39 .33 .1 6 .38 .33 .62 .91 .53 .66 .2 4 .25 .25 .45 .31 .26 .30 --+ . 0 9 .11 .60 .41 1.00 .39 .50 .3 3 4.7 2 .4 1 .4 2 .3 3.3 3.0 1 .3 2 .4 1 .6 3.7 3 .7 3 .4 3.0 .9 2 .2 1.1 2 .4 1.5 1 .1 1 .7 1.1 5.4 1.0 3 .1 1.7 2.5 1 .8 .20 .09 .07 .12 .12 .12 .05 .14 .05 .14 .17 .13 .13 .0 5 .15 .06 .13 .07 .06 .09 +.04 -- .10 .36 .06 .22 .08 .16 + .13 90 LIO LOO 70 120 100 20 130 110 250 210 220 130 50 130 140 120 130 140 130 `10 70 140 100 200 140 130 50 3.8 4 .1 5 .3 3.0 3.9 4 .0 3 .3 7. 3 .7 9 .6 9.5 3.5 7.3 2 .4 8. 7 7.0 6 .3 5 .9 7.3 7.0 +1.1 54 9 .3 5.6 L4.3 .4 3 .4 + 3 .6 -- ~! I *_J ... _. 10 75-1046 F 58.0 26.0 75-L047 T 43.0 20.0 75-1049 F 65.0 9.0 75-1056 7 77.0 19.0 75-1059 7 6 5 .0 a 16.0 MEAN +S.D. 50.8 +14.2 18.5 7 .0 13.0 7.6 3.6 10.0 11.0 9 .3 2 .3 .50 .38 .96 .53 .69 .59 + . 25 1 .6 2 .3 1.9 3.3 2 .3 2.5 +L. .06 .14 .21 .20 .13 .15 + .07 3 75-999 y 91.0 14.0 75-1007 ; 64.0 21.0 75-1008 7 72.0 21.0 75-1009 39.0 13.0 75-1011 ? 39.0 19.0 MEAN S .O . 71.0 21.2 18.6 + 2 .9 7.9 7.5 6.5 4.5 5.9 6.5 -2 .3 56 . 36 . 31 . 25 . 31 . 36 + .12 3.1 3.3 5 . 1. 2 .3 3; 4 1 .5 .22 .13 27 .39 15 13 z .7 Mo s c a c i s c i c a i i y s i g n i f i c a n e d i f f a r e n c e f r o a c o n t r o l a e a n s b y a n a l y s i s o f v a r i a n c e a n d O u n n e c c ' s - r e s e , ? < 0 . 0 5 . 1A n i a a i ?.oc i n c l u d e d La s c a c i s c i c s b e c a u s e o c b l o o d l a u r i n e j a m l e d u e : o a e u e z c e a a i i . 130 120 150 170 140 140 -20 190 190 130 liO 110 170 -30 5 .0 6 .0 16. 3.9 3 .3 9.1 +4.5 12.6 9.0 3.5 10.0 5.3 9 .4 -2 .3 S U $ (o ^ - 307- APPENDIX 35 * C L IN IC A L CH EM ISTRY VALU ES OF MALE RATS AT TERM IN ATIO N OF A 118 DAY D IETA R Y STUDY OF 2 ,4 , 5 - T D ose m g/ke/dav 0 30 10 3 A n im a l Number m g/100 m l BUN m U /m l SGPT 75-594 75-595 75-596 75-597 75-598 75-599 75-600 7 5 -6 0 1 75-602 75-603 MEAN iS .D . 22 16 16 32 21 21 16 18 23 19 21 5 53 50 62 68 52 64 42 54 42 48 54 9 75-624 75-625 75-626 75-627 75-628 75-629 75-630 75-631 75-632 75-633 MEAN S .D . 18 17 18 17 18 19 17 20 21 21 19 2 68 56 53 73 42 59 59 69 47 46 57 10 75-614 75-615 75-616 75-617 75-618 75-619 75-620 7 5 -6 2 1 75-622 75-623 MEAN S .D . 21 16 23 18 19 18 20 21 20 20 20 2 58 74 49 55 46 60 40 51 44 53 53 10 75-604 75-605 75-606 75-607 75-608 75-609 75-610 75-611 75-612 75-613 MEAN S .D . 22 21 17 21 18 17 16 25 23 18 20 3 45 47 49 53 57 55 46 56 42 49 50 5 m U /m l AP 115 92 155 102 84 122 101 149 85 103 111 25 144 109 94 199 90 137 72 114 105 105 117 36 90115 79 107 79 112 125 122 92 79 100 18 97 89 72 138 95' 115 133 139 125 138 114 24 m g/100 m l B iliru b in T o ta l m g/100 m l m g/100 m l B iliru b in B iliru b in D ire c t In d ire c t .2 .1 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 - .1 .1 0 .1 .1 0 .1 .1 0 .1 000 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 000 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 000 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 . .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 0 0 0 gm/100 m l gm /100 m l To tal P ro te in A lb u m in gm /100 m l G lo b u lin 6 .5 2 .1 4 .4 6 .2 2 .1 4 .1 6 .0 2 .1 3 .9 6 .6 2 .1 4 .5 6 .2 2 .1 4 .1 6 .6 2 .3 4 .3 6 .2 2 .3 3 .9 6 .5 2 .6 3 .9 6 .1 1 .6 4 .5 6 .7 2 .3 4 .4 6 .4 2 .2 4 .2 0 .2 0 .2 0 .2 6 .1 2 .0 4 .1 6 .6 1 .9 4 .7 6 .1 2 .1 4 .0 6 .2 2 .6 3 .6 6 .2 1 .8 4 .4 6 .3 2 .2 4 .1 6 .3 2 .2 4 .1 6 .4 2 .4 4 .0 5 .9 2 .2 3 .7 6 .2 2 .4 3 .8 6 .2 2 .2 4 .0 0 .2 0 .2 0 .3 6 .3 2 .3 4 .0 6 .3 2 .2 4 .1 6 .1 1 .7 4 .4 6 .5 2 .0 4 .5 6 .1 2 .1 4 .0 6 .7 2 .7 4 .0 5 .9 2 .2 3 .7 6 .6 2 .2 4 .4 5 .7 1 .9 3 .8 6 .3 2 .4 3 .9 6 .2 2 .2 4 .1 0 .3 0 .3 0 .3 5 .9 2 .1 3 .8 6 .2 1 .7 4 .5 6 .0 1 .8 4 .2 6 .5 2 .2 4 .3 5 .9 2 .4 3 .5 6 .6 2 .4 4 .2 6 .1 2 .2 3 .9 6 .6 2 .0 4 .6 6 .1 2 .3 3 .8 6 .5 2 .4 4 .1 6 .2 2 .2 4 .1 0 .3 0 .2 0 .3 No s t a t i s t i c a l l y s i g n i f i c a n t d if f e r e n c e s fro m c o n t r o l m eans u s in g a n a ly s is o f v a r ia n c e and D u n n e t t 's T e s t , p < 0 .0 5 . 308APPENDIX 36 CLINICAL CHEMISTRY VALUES OF FEMALE RATS AT TERMINATION OF A 119 DAY DIETARY STUDY OF 2,4,5-T D o se m g/Scg/day 0 30 10 3 A n im a l Number m g/100 m l 3 UN 75-434 75-635 75-636 75-437 75-638 75-639 75-640 75-641 75-642 75-643 MEAN tS.D. 18 17 19 22 20 18 16 22 13 22 19 2 75-664 75-665 75-666 75-667 75-668 75-669 75-670 75-671 75-672 75-673 MEAN 1S.D. 21 20 21 19' 21 19 17 28 19 21 21 3 75-654 75-655 75-656 75-657 75-658 75-659 75-660 75-661 75-662 75-663 MEAN rs.D. 22 27 23 23 21 23 17 26 23 18 22 3 75-644 75-645 75-646 75-647 75-648 75-649 75-650 75-651 75-652 75-653 21 19 27 16 19 ' 22 17 22 19 13 MEAN tS .D 20 3 aU/m l SGPT 47 45 50 40 43 42 42 46 43 43 44 3 46 42 51 56 42 46 38 42 51 48 46 5 53 66 49 61 37 54 44 30 55 58 56a 12 55 49 49 49 54 55 52 50 60 45 52 4 m U /m l A? 101 73 109 125 94 84 135 101 125 76 102 21 96 132 129 127 90 114 36 116 125 134 115 18 161 124 86 92 99 91 88 134 36 95 111 35 89 LL5 100 94 117 76 84 82 74 74 90 16 m g/100 m l B iliru b in To c a l m g/100 m l m g/100 m l 3 iliru b in B iliru b in D ira c s In d ira c s .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 0 .1 .1 0 .1 .1 .1 00 0 0 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 .1 0 .1 000 .1 .1 .1 0 .1 .1 .1 .1 .1 .1 .1 .1 .1 .1 .1 .1 .1 0 .1 0 .1 .1 o0 0 .1 0 0 0 0 0 0 .1 .1 0 0 .1 0 .1 .1 .1 0 .1 0 .1 .1 .1 0 .1 .1 0 .1 .1 0 i .1 0 .1 0 .1 .1 0 .1 .1 .1 0 .1 .1 00 0 0 ga/100 m l c t / 100 m l Tocai ? t o c aim A lb u m in gm/100 m l G lo b u lin 6 .6 2 .7 6 .5 2 .7 6 .2 2 .5 6 .7 2 .4 6 .8 2 .7 6 .5 2 .5 6 .4 2 .7 7 .1 3 .0 6 .2 2 .3 6 .8 2 .5 6 .6 2 .6 0 .3 0 .2 3 .9 3 .8 3 .7 4 .3 4^1 4 .0 3 .7 4 .1 3 .9 4 .3 4 .0 0 .2 6 .5 2 .6 3 .9 6 .8 2 .3 4 .0 7 .0 2 .6 4 .4 7 .6 2 .8 4 .8 6 .5 2 .7 3 .9 6 .8 2 .9 3 .9 6 .2 2 .6 3 .6 7 .0 2 .3 4 .2 6 .9 3 .0 3 .9 7 .0 2 .7 4 .3 - 6 .3 2 .3 4 .1 0 . 4 0 . 1 O'. 3 6 .3 7 .0 6 .3 7 .0 6 .6 . 7 .2 6 .6 7 .2 6 .9 7 .1 6 .3 0 .3 6 .4 7 .0 7 .1 6 .9 6 .5 6 .8 6 .9 7 .2 6 o6 7 .1 6 .8 0 .3 2 .5 2 .9 2 .4 2 .6 2 .2 2 .7 2 .5 3 .3 2 .9 2 .3 2.6 0 .3 2 .8 3 .0 2 .8 2 .7 2 .8 2 .9 2 .6 3 .1 2 .6 2 .7 2 .3 0 .2 3 .8 4 .1 3 .9 4 .4 4 .4 4 .5 4 .1 3 .9 4 .0 4 .8 4 .2 0 .3 3 .6 4 .0 4 .3 4 .2 3 .7 3 .9 4 .3 4 .1 4 .0 4 .4 4 .0 0 .3 a. S t a c is c ic a lly s ig n if ic a n t d iffe r e n c e fro m c o n tr o l mean u sin g a n a ly s is o f v a ria n c e and D unnect T e s t , ?< 0 .Q 5 . c U S ' l o 1/ -309APPENDIX 37 CLINICAL CHEMISTRY VALUES OF MALE RATS AT TERMINATION OF A TWO-YEAR DIETARY STUDY OF 2,4,5-T D ose m e/ke/dav 0 30 A n im a l N um ber m g/100 m l BUN 75-678 75-769 75-680 75-681 75-684 75-729 75-732 75-748 MEAN + S .D . 40 45 18 43 Dead Dead 171 16 56 58 mU / m l SGPT 35 55 37 32 -- -- 38 40 40 8 m U /m l AP 105 92 71 73 -- -- 114 83 90 17 75-861 75-862 75-863 75-864 75-867 75-871 75-882 75-884 75-885 75-889 MEAN + S .D . 42 20 171 18 36 15 21 12 36 26 40 47 48 263 49 93 33 133 51 67 69 L50 49 72 40 86 59 81 37 68 42 72 48 108 11 61 m g/100 m l B iliru b in T o ta l .3 .2 .2 .2 -- -- .2 .2 .2 0 m g/100 ml B iliru b in D ire c t .1 .1 .1 .1 -- -- .1 a ,i 0 m g/100 m l B iliru b in In d ire c t .2 .1 .1 .1 -- -- .1 .1 .1 0 .2 .1 .2 .1 .1 0 .2 .1 .2 .1 .2 .1 .2 .1 .2 . 1 .3 , i .2 .1 #2 . 1 00 .1 .1 .1 .1 .1 .1 .1 .1 .2 .1 .1 0 gm /100 m l T o ta l P ro te in gm /100 ml A lb u m in gm /100 m l G lo b u lin 5 .2 1 .4 5 .5 1 .6 5 .7 1 .9 5 .6 1 .6 ---- ---- 6 .5 ' 1 .6 5 .9 1 .6 5 .7 1 .6 0 .4 0 .2 3 .8 3 .9 3 .8 4 .0 -- 3 .9 4 .3 4 .0 0 .2 6 .1 1 .5 4 .6 5 .8 1 .9 3 .9 5 .4 1 .5 3 .9 6 .2 2 .1 4 .1 5 .9 1 .7 4 .2 5 .9 2 .4 3 .5 6 .1 2 .2 3 .9 6 .0 2 .4 3 .6 6 .4 1 .8 4 .6 6 .0 1 .6 4 .4 6 .0 1 .9 4 .1 0 .3 0. 3 0 .4 10 7 5 -8 1 4 12 42 103 75-819 53 37 95 7 5 -8 4 4 20 30 89 75-850 61 30 97 MEAN + S .D . 36 24 35 96 66 3 75-762 13 45 61 75-764 Dead -- -- 7 5 -7 7 1 18 35 50 7 5 -7 7 3 33 38 64 7 5 -7 7 4 77 38 106 75-784 75-788 16 38 117 Dead -- -- 75-794 19 40 74 75-795. 17 48 80 75-8QO 17 30 66 7 5 - 8 0 7 17 35 49 MEAN + S .D . 25 20 38 74 5 2A .2 .1 .2 . 1 .2 .1 .2 .1 .2 .1 00 .2 .1 ---- .2 .1 .2 .1 .2 .1 .1 0 ---- .1 .2 .1 .2 .1 .2 .1 .2 .1 00 .1 .1 .1 .1 .1 0 .1 -- .1 .1 .1 .1 -- .1 .1 .1 .1 .1 0 5 .7 5 .5 5 .7 5 .5 5 .6 0 .1 6 .0 -- 6 .2 5 .9 5 .8 6 .3 -- 6 .0 6 .0 5 .1 6 .3 6 .0 0 .4 1 .6 -.1 .5 1 .6 1 .5 1 .6 0 .0 1 .9 -- 1 .9 1 .7 2 .0 1 .9 -- 1 .4 1 .9 2 .3 2 .0 1 .9 0 .2 4 .1 4 .0 4 .1 4 .0 4 .0 0 .6 4 .1 -- 4 .3 4 .2 3 .8 4 .4 -- 4 .6 4 .1 2 .8 4 .3 4 .1 0 .5 No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s fro m c o n t r o l m ean s u s in g a n a l y s i s o f v a r i a n c e a n d D u n n e t t 's t e s t , p < 0 .0 5 . 310- APPENDIX 38 CLINICAL CHEMISTEY VALUES OF FEMALE FATS AT TEXMIMATION OF A TVO-TEAR DIETARY STUDY OF 2,4,i-T D ose 212/ '<2 / d a y 0 30 A n im al N um ber m g/100 m l BUN 75-912 73-913 75-914 75-915 75-913 ' 75-920 75-926 75-927 75-929 75-932 MEAN + S .D . 17 u 13 13 16 24 12 17 16 30 17 6 75-1097 75-1098 75-1100 75-1102 75-1104 75-1108 75-U 12 75-1115 75-1116 75-1117 75-1119 MEAN S .D . 16 30 27 Dead 22 15 13 22 18 17 3 19 6 mfj /m [ 3GPT 37 49 37 30 50 50 50 39 32 38 42 10 45 36 36 -- 34 45 43 30 27 38 37 37 6 aU /o i AP 40 78 42 36 91 36 93 38 47 184 68 47 48 76 68 -- 38 93 66 57 29 44 69 59 19 m g/100 m l B ilir u b in lo c a l .2 .2 .3 .2 .2 .3 .1 .3 .3 .2 .2 .1 .2 .2 .3 -- .2 .1 .2 .1 .1 .3 ,2 .2 .1 m g/100 m l B iliru b in D ire c t .1 .1 0 .1 .1 .1 .1 .1 0 .1 0 .1 .1 .1 -- .1 .1 .1 .1 0 0 0 .1 0 m g/100 m l B iliru b in In d ire c c .1 .2 .2 .1 ,2 0 .2 .2 .2 .2 .1 .1 .1 #2 -- .1 0 .1 0 .1 .3 .2 J. .1 ga/100 m l lo c a l ? ro c a in g a / 1 0 0 mL gm /100 m l A lb u m in G lo b u lin 7.4 2 .3 7.3 3 .5 6 .3 2 .9 7.0 2 .8 6 .5 2 .3 5.9 2 .1 6.1 2 .8 6.4 2 .1 5.3 2 .4 S .6 2 .3 6 .5 . 2 .6 0 .6 0 .4 4 .6 3.8 3.9 4.2 3.7 3.3 3.3 4.3 3.9 3.3 3 .9 e 0 .4 6 .0 6 .1 6 .2 -- 6 .4 6 .2 6 .4 6 .4 6.7 6.7 6.3 6.3 0.2 2 .6 3 .4 1 .8 4 .3 2 .9 3 .3 ---- 2 .9 3 .5 ' 2 .2 4 .0 2 .7 3 .7 2 .9 3 .5 3 .0 3 .7 1 .8 4 .9 3 .2 3 .1 2 .6 3 .7 0 .5 0 .5 10 7 5 -1 0 4 6 11 46 52 75-1047 17 30 75 75-1049 Dead -- -- 75-1056 18 41 57 7 5 -1 0 5 9 15 37 81 7 5-1 0 63 18 31 55 7 5 -1 0 70 16 40 34 75-1072 12 .5 6 72 7 5 -1 0 8 6 19 34 31 75-1091 14 30 44 MEAN 3 .0 . 16 3 40 56 9 18 3 75-99 7 17 34 43 75-999 11 36 58 75-1 0 07 19 26 31 75-1008 12 27 49 7 5-1 0 09 15 43 54 75-1010 62 45 76 7 5 -1 0 11 42 27 110 75-1012 340 94 114 75-1015 30 37 43 7 5 -1 0 1 7 15 51 43 MEAN +S. D. 56 101 42 63 20 28 .2 .1 .1 _ _. 3 .1 - . 2 ___ .4 .1 .3 .1 .2 .1 .2 .1 .2 .1 .2 .1 0 .1 .2 .1 .1 .1 .1 .2 .1 .1 .1 .1 .1 .3 .1 .2 .1 .2 .1 .4 .1 .2 .1 .3 .1 .2 0 .1 .2 0 .2 0 .2 .1 0 t2 .1 .1 .3 .1 .2 .2 .1 .2 .2 ,2 .1 6 .6 6 .3 5 .7 6 .7 5 .9 5 .3 6 .5 6 .3 6 .4 6 .4 0 .3 6 .5 6 .6 5 .9 5 .6 6 .5 6 .5 6 .4 5 .2 5 .7 6 .9 6 .2 0 .5 3 .0 2 .6 1 .3 3 .0 2 .3 2 .3 2 .9 2 .9 2 .9 2 .6 0 .5 2 .1 2 .5 2 .9 1 .8 2 .7 1 .4 1 .9 0 .9 2 .7 3 .3 2 .2 0 .7 3 .6 3 .7 5 .4 3 .7 3 .6 3 .0 3 .0 3 .4 3 .5 3. 7 0 .7 4 .4 4 .1 3 .0 3 .3 3 .8 5 .1 4 .5 4 .3 3 .0 3 ,6 4 .0 0 .7 No s t a t i s t i c a l l y s i g n i f i c a n t d i f f a r a a c a s f r o m c o a c r o i m e a n s u s i n g a n a l y s i s o f v a r i a n c e a n d D u n u s c c ' 3 c a s t , o < 0 . 0 5 . a (e -311- APPENDIX 39 TERMINAL ORGAN WEIGHTS AND ORGAN/BODY WEIGHT RATIOS OF MALE RATS AT TERMINATION OF A 118 DAY DIETARY STUDY OF 2 , 4 , 5 - T Dose I a a /lc a /d a v Number Fasted Body Weight -- s-- 0 75-594 415 75-595 449 75-596 484 75-597 452 75-598 468 75-599 484 75-600 517 75-601 478 75-802 458 75-603 460 MEAN rS.D. 466 27 Brain g/lOOg 1.84 1.60 1.86 1.91 1.86 1.86 1.95 1.88 1.82 1.87 1.85 0.09 0.44 0.36 0.39 0.42 0.40 0.38 0.38 0.39 0.40 0.41 0.40 0.02 Heart _ s _ ;i / l O Q g 1.15 1.33 1.38 1.42 1.44 1.48 1.50 1.33 1.51 1.41 1.40 0.11 0.22 0.30 0.29 0.31 0.31 0.31 0.29 0.28 0.33 0.31 0.30 0.02 O r g a n W e i g h c s (g and g / 1 0 0 g Body W e i g h t -) Liver Ridnevs Testes --a-- ft/ 1 00 _ s _ a/100a -- S-- it / lQ O a 10.51 12.44 11.30 11.62 11.77 12.29 14.26 12.52 17.10 11.74 12.55 1.87 2.53 2.77 2.34 2.57 2.51 2.54 2.76 2.62 3.73 2.55 2.69 0.39 2.52 3.50 3.25 3.74 2.93 2.96 3.47 3.19 4.09 3.38 3.28 0.44 0.61 0.74 0.67 0.83 0.63 0.61 0.67 0.67 0.89 0.73 0.70 0.09 4.02 3.53 4.42 3.88 3.75 3.96 4.55 4.36 3.97 4.04 4.05 0.31 0.97 0.79 0.91 0.86 0.80 0.32 0.88 0.91 0.87 0.88 0.87 0.06 Spleen a/100a 0.62 0.36 0.30 0.92 0.93 0.83 0.88 0.81 1.10 0.76 0.85 0.12 0.15 0.19 0.17 0.20 0.20 0.17 0.17 0.17 0.24 0.16 0.18 0.03 Thvnus a a/lOOa 0.36 0.29 0.39 0.28 0.30 0.39 0.27 0.53 0.30 0.38 0.35 0.08 0.09 0.07 0.08 0.06 0.06 0.08 0.05 0.11 0.07 0.08 0.08 0.02 30 75-624 464 1.84 0.40 1.35 0.2$ 12.78 2.76 3.85 0.83 4.19 0.90 o'. 75 0.16 0.33 0.07 75-625 442 1.82 0.41 1.38 0.31 11.79 2.67 3.68 0.83 3.59 0.81 0.91 0.21 0.31 0.07 75-626 510 1.85 0.36 1.48 0.29 14.44 2.83 3.97 0.73 3.64 0.71 1.14 0.22 0.42 0.08 75-627 470 1.82 0.29 1.23 0.26 12.50 2.66 3.30 0.70 4.22 0.90 0.71 0.15 0.29 0.06 75-628 524 1.93 0.37 1.79 0.34 17.90 3.41 4.31 0.82 4.21 0.80 1.04 0.20 0.42 0.08 75-629 447 1.91 0.43 i.49 0.33 12.71 2.85 3.39 0.76 4.63 1.04 0.86 0.19 0.31 0.07 75-630 536 1.98 0.37 1.34 0.25 15.03 2.80 4.31 0.80 4.21 0.78 0.85 0.16 0.41 0.08 75-631 444 1.70 0.38 1.33 0.30 11.29 2.54 3.03 0.68 3.82 0.86 0.72 0.16 0.38 0.09 - 75-632 440 1.79 0.41 1.35 0.31 13.71 3.12 3.94 0.90 3.74 0.35 0.99 0.23 0.48 O .U 75-633 461 1.92 0.42 1.41 0.31 12.35 2.68 3.66 0.79 " 3.87 0.34 0.93 0.20 0.31 0.07 MEAN iS.D . 474 36 1.86 0.08 0.39 0.02 1.41 0.15 0.30 0.03 13.45 1.94 2.83 0.26 3.74 0.42 0 . 79a 0.06 4.01 0.33 0.35 0.09 0.89 0.14 0.19 0.03 0.37 0.06 0.08 0.01 10 75-614 462 1.71 0.37 75-615 393 1.97 0.50 75-616 419 1.82 0.43 75-617 534 1.36 0.35 75-618 573 1.98 0.34 75-619 542 1.83 0.34 75-620 468 1.77 0.38 75-621 520 1.89 0.36 75-622 543 1.91 0.35 75-623 438 1.81 0.41 MEAN sS.D. 489 61 1.85 0.08 0.38 0.05 1.37 1.28 1.22 1.31 1.52 1.55 1.51 1.43 1.46 1.29 1.39 0.12 0.30 0.33 0.29 0.25 0.26 0.29 0.32 0.27 0.27 0.29 0.29 0.-02 13.01 9.77 10.38 14.87 15.64 15.15 11.93 14.82 15.89 11.02 13.25 2.32 2.81 2.48 2.46 2.79 2.73 2.80 2.55 2.35 2.93 2.52 2.69 0.17 3.28 3.10 2.90 4.32 3.39 4.03 3.14 3.69 3.36 3.45 3.57 0.46 0.71 0.79 0.69 0.81 0.68 0.74 0.67 0.71 0.71 0.79 0.73 0.05 4.42 3.85 3.52 3.70 4.06 4.35 4.43 3.82 3.96 3.80 4.00 0.32 0.90 0.98 0.84 0.69' 0.71 0.30 0.96 0.73 0.73 0.87 0.83 o .u 0.97 0.75 0.92 0.97 0.94 0.95 0.96 0.93 0.97 0 .89* 0.92 0.07 0.21 0.19 0.22 0.18 0.16 0.17 0.20 0.18 0.18 0.20 0.19 0.02 0.40 0.30 0.40 0.33 0.53 0.57 0.27 0.53 0.29 0.36 0.40 O.U* 0.09 0.08 0.10 0.06 0.09 0.11 0.06 0.10 O.OS 0.08 0.08 0.02 3 75-604 503 1.92 0.38 75-605 453 1.85 0.41 75-606 545 1.80 0.33 75-607 454 1.90 0.42 75-608 404 1.79 0.44 75-609 544 1.72 0.32 75-610 468 1.93 0.41 75-611 484 1.83 0.38 75-612 492 1.93 0.39 75-613 466 1.79 0.39 MEAN iS.D . 481 43 1.35 0.07 0.39 0.04 1.41 1.29 1.45 1.27 1.33 1.45 1.37 1.42 1.49 1.37 1.38 0.07 0.28 0.29 0.27 0.28 0.33 0.27 0.29 0.29 0.30 0.29 0.29 0.02 14.16 12.74 18.03 11.87 11.53 14.56 12.32 15.03 12.95 12.07 13.57 1.95 2.82 2.81 3.31 2.61 2.86 2.67 2.74 3.11 2.63 2.59 2.31 0.23 3.54 3.37 4.21 3.10 3.15 3.79 3.60 3.70 3.36 3.01 3.42 0.37 0.70 0.74 0.77 0.68 0.78 0.70 0.77 0.77 0.68 0.65 0.72 0.05 3.90 3.92 4.46 4.34 4.12 4.06 4.08 3.71 4.04 4.77 4.14 0.31 0.78 0.86 0.32 0.96 1.02 0.75 0,37 0.77 0.82 1.02 0.37 0.10 0.99 0.94 0.93 0.74 0.77 0.86 0.77 0.80 1.01 0.92 0.87 0.10 0.20 0.21 0.17 0.16 0.19 0.16 0.16 0.17 0.20 0.20 0.13 0.02 0.42 0.33 0.47 0.44 0.39 0.44 0.22 0.53 0.25 0.33 0.38 0.10 0.08 0.07 0.09 0.10 0.1C 0.08 0.05 0.11 0.05 0.07 0.08 0.02 S i g n i f i c a n t l y d i f f e r e n t from c o n tr o l means u sing a n a ly s is of v a ria n c e and Dunnecc's t e s t p<0.05. i o u mis in v in e ivc e n V i o noiiykikisi iY s i n n w G i i c s o i m ip jiar. ioos/ ktoho oky s ie j is urone tykiwesi l i ti l i ti *i o>. 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M H4UUAUOOW Ou cu - T- I O I J U 1> h - H O io*-.0^ rI* N IU H |U N 400000000 O O* O O O O O O O O O O OO O O O O O O O O O O o-. o - U O) N M 9 N ' OO O O O O O O O O O O ooooor -ooor -* OO o o o o o o OO O O O O O r O O O O O O O r- r ?e L 99 I OO OOOOOOOOOO i Is* Irt OO O O O O O O O O O O C N IJ I - U IJ ( j u f- (o - ig OO O O O O O O O O O O OO o o o o o o4 0 0 0 0 r- > N iu u Kj Il - Mi> lo OO OOOi o o o o o o OO O O O O O O O O O O t-- ( ' VI U l 'J N ' I ^1 ^ U O'. OOOOOOOOOO I OO O O O O O O O O O l OO O O O O O O O O O O 4000000000 OO O O O O O O O O O O OO O O O O O O O O O O OO O O O O O O O O O O O O U U ti 11U U Si NI Ui <9U 9UUUIOOMMMM4 OO O O O O O O O O O O OO O O O O O O O O O O O Cu K1lou |g |g (' Ki w 1' U -J f OtU-h' i 9t4(' U900 o o o c oooooooo OO O O O O O O O O O O H w I- ^ M lu4J U U 14(.'H OM BNOMBL) l'OHOMfi OO O O O O O O O O O O OO O O O O O O O O O O O tu UUU U MU NI Ot ' Li MNI ' t ' l ' *U| 4i 999 OO OOOOOOOOOO Uo*-P O-O"4- 1H` *O<1 HH Oo)o9 HcrhU' \ l____ L. I___ J ..... . . J -313- APPENDIX *1 TERMINAL ORGAN '-'EIGHTS AND ORGAN/BODY -EIGHT RATIOS OF MALE RATS AT TERMINATION OF A TWO-YEAR DIETARY ST'JDY OF 1 , 4 , 5 - T Dose Animal a g / k g / c a v Number Fasted Body Weignc Brain g/100c 75-673 ' 5-679 75-o3C 75-651 75-732 75-7-18 MEAN =S.D. 439 535 533 -99 458 529 508 2-3 1.95 1.94 2.06 2.08 1.98 1.98 2.00 :Q . 06 0.44 0.36 0.35 0.42 0.43 0.37 0.40 =0.04 75-361 75-562 75-863 75-864 75-367 75-371 75-882 75-884 75-885 75-389 75-391 75-894 75-904 75-906 75-907 75-909 MEAN rS.D. 436 536 331 650 418 652 638 551 -61 472 598 521 503 581 612 522 534 r92 2.02 2.13 1.97 2.10 1.92 2.08 2.06 2.06 1.75 1.89 2.10 2.03 1.96 2.13 L.98 1.97 2.01 20.10 0.46 0.36 0.60 0.32 0.46 0.22 0.32 0.37 0.38 0.40 0.35 0.39 0.39 0.37 0.32 0.37 0.39 :Q.07 ' 3-314 75-819 75-8475-350 MEAN :S.3. 599 445 580 503 522 =71 75-762 75-771 75-772 75-774 75-784 75-794 75-795 75-500 ' 5-307 MEAN rS.D. 474 556 503 453 573 512 532 791 593 35ilOO t.Ou 2.02 2.01 2.00 2.01 20.01 1.98 2.19 2.00 1.90 2.11 2.00 2.14 2.04 2.05 2.0sO.09 0.33 0.45 0.35 0.40 0.38 i 0 . 05 0.42 0.39 , 0.40 0.42 0.37 0.39 0.40 0.26 0.35 0.38 20.05 Heart : g/LOQg 2.14 2.52 2.12 1.93 2.06 1.66 2.07 r0.28 0.49 0.47 0.36 0.39 0.45 0.31 0.41 20.07 1.82 1.69 1.84 1.83 1.64 1.79 2.06 1.76 1.77 1.71 1.87 1.67 1.71 2.21 1.83 1.98 1.82* S O . 15 0.42 0.29 0.56 0.28 0.39 0.27 0.32 0.32 0.38 0.36 0.31 0.32 0.34 0 . 38 0.30 0.37 0.35 20.07 2.14 2.25 1.82 2.10 2.08 SO. 19 0.36 0.51 0.31 0.-2 0.40 20.03 1.71 1.81 1.75 1.79 2.07 1.94 2.00 1.90 1.88 1.87 20.12 0.36 0.33 0.35 0.40 0.36 0.38 0.38 0.24 0.32 0.35 sO.05 Organ '-'eights (g and g/lQQ g Body Weight) Liver . . Kidneys Testes g/lOOg -- g/100g g. ' l OOi ? 14.36 17.77 21.36 16.13 18.97 17.26 17.72 22.27 3.38 3.32 3.63 3.23 4.14 3.26 3.49 20.35 5.22 6.04 5.76 6.55 10.05a 5.73 5.36 20.49 1.19 1.13 ' 0.98 1.31 1.20a 1.08 1.14 2 0 .il 2.64 4.38 4.01 4.23 4.09 4.22 3.93 20.65 0.60 0.82 0.68 0.85 0.39 0.80 0.77 20.11 16.49 14.58 15.32 18.90 11.87 14.58 15.83 12.63 2 5 .10a 15.48 17.19 14.47 14.85 16.95 16.73 17.02 15.52 21.81 3.78 2.49 4.63 2.91 2.84 2.24 2.48 2.29 5.44a 3.28 2.87 2.78 2.95 2.92 2.73 3.20 2.96 20.61 5.71 4.95 4.32 5.34 3.20 4.20 4.30 4.04 5.94 5.46 6.11 4.94 5.-24 4.37 4.63 7.71 5.03 21.05 1.31 0.34 1.31 0.82 0.77 0.64 0.67 0.73 1.29 1.16 1.02 0.95 1.04 0.75 0.76 1.45 0.97 rO .2 6 3.24 3.98 1.07 4.27 2.46 4.90 2.88 4.76 3.62 3.85 4 . 74 3.76 4.50 4.13 4.12 4.24 3.78 20.99 0.74 0.63 0.32 0.66 0.59 0.75 0.45 0.86 0.79 0.32 0.79 0.72 0.39 0.71 0.67 0.30 0.70 20.15 18.50 18.30 17.83 17.74 18.09 20.36 3.09 4.11 3.08 3.53 3.45 20.49 5.39 5.73 5.11 6.66 5.72 20.68 0.90 1.29 0.88 L.32 1.10 20.24 4.17 4.24 4.12 4.22 4.18 20.05 0.70 0.95 0.71 0.84 O.SO rO.12 14.20 18.62 17.78 1 3 ." la 16.43 18.23 15. c7 23.33 16.55 17.59 2.99 3.35 3.53 3.03a 2.37 3.56 2.93 2.95 2.79 3.12 20.31 3.33 5. 4 7.21 7.25 4.46 6.07 4.64 4.38 5.27 0.81 1.02 1.43 1.60 0.78 1.19 0.37 0.55 0.39 5.42 21.23 1.02 20.33 3.41 4.31 4.74 1.33 3.60 3.97 -.33 -.09 2.78 3.62 il.0 3 0.72 0.73 0.94 0.29 0.63 0.78 0.81 0.52 0.47 0.66 20.20 Spleen -- */100e 0.96 1.43 1.51 1.34 1.25 1.28 1.29 20.19 0.22 0.27 0.26 0.27 0.27 0.24 0.25 :0.02 1.34 1.54 0.56 1.28 0.62 1.13 1.18 1.26 1.09 1.40 1.30 1.27 1.32 1.20 1.25 0.96 1.20 20.30 0.31 0.26 0.17 0.20 0.15 0.17 0.18 0.23 0.24 0.30 0.30 0.24 0.26 0.21 0.20 0.13 0.23 20.05 1.38 1.63 1.23 i . 23 1.38 20.21 0.23 0.38 0.21 0.24 3.27 :0.08 1.00 1 1 1.37 1.25 1.14 1.10 1.34 1. ^ j 1.05 : . ?i 20.13 0*21 0.25 0.27 0.28 0.20 0.21 0.25 0.19 0.18 0.23 20.04 Thymus g/iOOg 0 ." 0.09 0.03 0.15 0.06 0.09 0.10 20.03 0.03 0.02 0.01 0.03 0.01 0.02 0.02 zO.Ol 0.09 0.15 0.11 0.09 0.09 0.15 0.10 0.15 0.15 0.09 0.11 0.13 0.11 0.12 0.11 O.OS 0.11 20.03 0.02 0.02 0.03 0.01 0.02 0.02 0.01 0.03 0.03 0.02 0.02 0.03 0.02 0.02 0.02 0.01 0.02 20.01 0.12 0.10 0.16 O.OS h "' 20.04 0.02 0.02 0.03 0.02 n'i r . 01 0.09 C .ll 0.80 0.09 0.16 0.14 0.19 0.21 0.12 0.13 20.05 0.02 0.02 0.01 0.02 0.03 0.03 0.04 r> 0.02 0.02 20.01 -e ig h ts excluded from c a lc u l a t i o n s due to tumor o r mass. s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e from c o n t r o l mean u s in g a n a l y s i s of v a r ia n c e and D u n n e ct's t e s t , o<0.05. c U . < 0 l -JU - A??EHDIX 42 7SSMIMAL O&CA# -EIGHTS AtfD 0RCAN/300T -EIGHT RATIOS OF F2ALS. RATS At TSRWI^AIIQ.N OF A TJQ-'dAA OIETAA sTVOT O F 'I U . j - T /<23 o s s A fliaa i u s a ay Suaoec ja a e e d 3iy -a i? a t -- i-- 3 75-912 379 75-9L3 75-914 368 399 7 5 - 9 LS 3 6 6 75-9L3 338 75-420 402 75-425 233 75-427 369 75-429 332 73-432 523 75-935 467 ... 7 5 - 4 3 6 400 75-939 426 73-942 369 73-943 531 75-446 447 73-947 304 75-946 34L 75-931 75-952 75-954 177 434. 572 75-96L 546 75-965 323 75-466 532 75-471 407 73-472 75-973 412 274 75-480 75-481 532 324.. 75-985 75-987 505 420 75-489 492 75-491 -63 75-992 276 ' 5-993 275 75-495 497 SIAS =6.0. 419 =93 3 ra in i/LOQg -- S-- L.34 1. 79 1.32 1.91 1.75 1.31 1.73 1.58 1.79 1.33 1.33 1.74 1 .3 1 1.36 1.32. 1.79 1.35 1.33 1.34 1.30 i . 79 L.31 1.91 1.79 1.35 1.37 1.30 1.71 1.70 1.73 2.10 1.38 1.32 L.36 1.97 1.76 0.48 9 .4 9 0.46 9 .5 2 9 .5 2 0.45 0.33 0.51 9.47 0.J5 440 . 3 9 a. 0 .4 2 0 .5 0 0.34 0.40 0 .6 1 0 .3 4 0.49 0 .4 1 0 .3 1 0.2S 0 .5 9 0 .3 2 0 .4 5 0.45 0.36 0.32 0 .5 2 0 .3 4 0.50 0.38 0.39 0.67 0.71 0.35 1.32 =0.07 0. -6 =0.11 oa 75-1097 501 1.33 0.37 75-L098 7 5 - 00 71-U 04 334 364 377 1.72. 1.32 1.31 0.52 0.50 0.48 75-1108 402 1.76 0.44. 75-1112 423 1.30 0.42 7 5 -L U 5 434 1.39 0.44 75-1116 407 1.76 0.43 75-1117 377 1.37 0.50 7 5 - 1 1 1 9 .\* \6 1.37 0.55 75-1121 450 1.31 0.40 75-1123 531 1.72 0.27 75-1124 291 1.90 0 35 75-1130 457 1.34 0.40 75-1132 343 1.32 0.53 75-1133 4-3 1.91 0.43 75-1134 313 1.38 0.59 75-1133 440 L.37 0.43 75-1143 75-1144 75-1145 426 527 334 1.31 L.34 1.97 0.43 0.35 0.59 MEAN 41i 1.33 0 46 =3.3. =30 =0.06 =0.09 10 7 5 --1 0 4 6 4 4 5 1.73 0.40 75-104 7 391 1.32 0.47 75-1056 313 1.77 0.57 75-1059 319 1^32 0.57 75-1063 395 1.39 0.48 75-1070 427 1.33 0.44- 75-1072 323 1.37 0.58 75-1086 414 1.35 0.45 75-1091 351 1.37 0.53 7 5 -1 0 9 2 493 1.32 0.37 73-1095 410 1.35 0.45 MEAN 389 1 .3 4 3.43 = 5 .0 . =53 = 0 .0 4 =0.07 3 7 5-997 434 1.39 0.44 '3-499 421 1.33 9 .-5 75 -1 0 0 7 6L8 1.93 9,31 73-1008 44} 1.92 0 .- J 75-1009 311 1.93 9.62 75-1010 -1/ 1.35 9 44 75-1011 405 ...` 7 9 .-4 7 5 - L O i : 27 L l . . `i 0.35 7 3 - 1 0 1 5 =13 1.35 0.30 75-1 0 1 7 356 1.33 3.51 *3-1013 75-1019 390 333 1.90 1.33 3.-9 3 .-9 3-1020 Oi 1.34 5 5L * ; - 1 0 U 523 1. =4 0.23 36 L 1.31 2 0 7 3 - 1 0 2 5 ` 03 1.15 2.35 75-1035 -09 : . n 0.44 " - 1 0 3 7 337 1.34 0.55 3 - 1 0 4 5 - - 3 1 36 3 .-2 :S .3. 414 1.36 0 .-? =71 =0 05 :0.09 daart --i - g / i O Q a > L .U 1.19 1.15 1.10 1.20 1.35 1.03 1.58 1.42 1 .3 1 1.17 1.31 l.U 1.63 1.21 1.36 1.36 1.78 L .21 l . 31 1 .5 2 1.19 1.16 1.36 1.40 1.09 1.41 L. 14 0 .9 6 l.U 1.31 1.19 L. 23 1.05 L .U 1.22 1.23 =0.20 0.30 0 .3 2 3.29 0.30 0.35 0.34 0.36 0.43 9.37 0.35 0.25 0.33 0.27 0.44 0.23 0.30 0.45 3.33 0.32 0.30 0 .2 7 0.18 0 .3 6 0 .2 3 3.36 0.27 0.51 0.21 0.30 0.22 0.31 0U4 0.28 0.38 0.40 0.25 0.32 =0.07 3 r * a a - e i g n c a ;'g ci d = / l O O z 3 o d y . ' a ; ; h ; ) 11 v ,, ' l i d -.BVS J=arus/O var -- i U 0 i - i -- i i 4-0g -U _ 1! IQOg 10.33 ?.C1 12.22 9.32 3.23 13.63 7. -6 11.00 12.50 14.21 11.37 13.90 LS.19 12.27 13.69 L i . 19 U .47 20.08 3.77 16.07 15.66 15.33 10.57 13.60 13.48 10.00 U .47 U .33 10.33 16.95 11.22 19. :o 24.22 3.43 5.39 12.90 13.07 =3.92 2.36 2. - 3.36 2.55 2.45 3.40 2.54 2.98 3.27 2.72 2.54 3.43 3.36 3.32 3.52 3.40 4.11 3.71 2.33 3.71 2.74 2.38 3.27 3.37 3.31 2.43 4.19 2.51 3.19 3.35 2.57 3.3a 5.23 3.06 2.14 2.50 3.12 =0.54 2.35 2.54 3.35 2.49 2 .5 1 3.49 2.53 3.04 2.92 3.31 2.70 3 -il 2.55 3.34 2.33 2.97 3.49 3.46 2.57 4.16 3-27 3.33 3.73 2 .4 2 3.01 3.08 3.63 2.40 2.97 2.31 2.96 3.13 3 .5 1 2.14 2.13 2.42 3.00 =0.4 7 0.75 3.69 3.34 2. si 0.77 0.37 0.90 0.32 0.78 0.53 0.53 , 0.33 0 .6 Z 0.90 0.54 O . 1.15 0.64 0.63 0.96 0.57 0 .5 2 1.16 0 .4 4 0.74 0.75 1.33 0.45 0.92 0.56 0 . 70 0.54 0.76 0.78 0.79 0.19 0.75 =0.20 2.26 1.05 0 .3 3 0 .7 1 :.3 2 a 1.73 1.09 2.43 0.52 0.5 7 1.34 1.23 0 .5 1 L. 72 0 .7 1 0 .9 9 l.U 0.35 0 .7 2 L.OO 1.15 0.56 0 .9 7 0.53 0.75 3 .4 5 a 5 .52a 2 .9 2 a 3 .7 4 0.77 1.15 0.33 0.3 3.43 0 .2 2 l.U 0 .9 9 = 0 .-3 0-50 0.23 0.22 0.19 3. 94 0.43 0.39 0.57 0.15 3.U 0.22 0.31 0.14 0.47 o .u 0.22 0.37 0.16 0.19 0.23 0.20 0.09 0.30 O .U 0.19 0 .3 4 a i.sz* 0 .5 5 a 0,23 0.15 0.27 0.L7 0.14 O .iS O .U 0.23 0.25 =0.14 1.25 1.34 1.29 1.08 1.38 1.33 1.25 1.12 1.40 1.26 1.33 1.20 1.30 1.15 1.23 1.29 1-15 1.34 1-19 1.10 1.38 1.26 =0.10 0.25 0.40 0.35 0.29 0.34 0 ,3 1 GU9 0.27 0.37 0.37 0.30 0.19 0.45 9.25 0.36 0.29 0.36 0.30 0.23 0.21 0.41 0.32 =0.07 16.39 15.31 10.06 12.59 9.12 13.17 16.31 14.31 13.37 9.54 12.08 12.75 3.75 13.06 11.-5 12.71 3-54 12.39 14.96 14.51 10 .o O 12.55 =2 . 4 7 3.37 4.59 2.76 3.33 2.27 3.07 3.37 3.52 3.68 2.34 2.58 2.02 3.01 2.36 3.34 2.34 2.72 2.32 3.51 2.75 3.17 3.10 =0.57 3.05 3.55 2.54 3.02 2.72 3 .2 2 2.50 1.72 3 .SI j . 36 2.33 3.70 3.11 2.98 3.17 2.79 2.55 3.27 3. 06 2.59 3.59 3.00 =0.35 0.51 1.09 0.70 0.80 0 .5 3 0.75 0.60 0 .6 7 3.93 1.00 0.63 0.43 L.37 0.55 0.92 0.62 0.30 0.74 0.72 0.49 1.07 0.76 =0.19 0 .5 7 l.U 1.21 L. 22 L. 30 1.55 0.33 2.45 1 .1 3 0 .3 0 0 .3 a 0 .7 1 1.08 ' 0.57 0.70 0 .3 5 1.30 0 .6 6 0 .3 1 0 .3 0 2.25 1.11 =0.51 O .U 0.33 0.33 0.32 0.32 0.36 0.19 0.60 0.30 0.24 0.20 O .U 0.37 0.12 0.20 0.19 0.57 0.15 0.19 0.15 0.57 0.29 =0.16 1.36 2.46 1.20 1.10 1.27 1.19 L .O l 1.33 1.28 1.66 1.15 1.27 0.31 0.37 0.38 0.35 0.32 0.23 0.31 0.32 0.36 0.34 0.28 0.33 =9.04 10.42 11.53 11.73. 3 .3 1 12.41 11.73 3.57 10.23 .9 .4 3 14.20 12.52 11.07 S i . 73 2.34 2.96 3.75 2.76 3.14 2.76 2.56 2.43 2.70 2.38 3.05 2.36 =0.33 3.13 2.71 3.73 2.76 2.34 2.70 2.52 3.02 2.31 3.50 2.74 2.96 =0.39 0.72 0.59 1.19 0.37 0.72 0.53 0.78 0 .7 2 0.30 0 .7 3 0 .6 / 0 .7 8 =0 15 1.23 0 .3 0 1.12 1.52 1.16 0 .3 3 1.38 0.71 1.18 0 32 1.35 1.09 =0.31 0.23 0.20 0.36 0 .5 1 0.29 0.19 0.43 0.17 0.33 O .U 0.33 0.29 =0.11 c. u 1.56 1-37 1.17 1.33 1.13 L .*) L.*0 1.09 L. 1 1 1.52 . . 21 1.23 1.27 1.-3 1.23 1.33 1. i r 1 9. - 1.30 =0.15 0.36 0.32 0.19 0.30 9.36 9.34 0 . 34 0.40 0.20 9.31 3.54 5.52 . -4 0 .5 5.-0 9.33 3.30 3.55 3.37 3.32 =9.06 12.05 12.05 23 . 5 19.76 4.06 15.29 13.33 3.2'* 13.44 9.77 i . 42 . J 10. / 3 21.91 15.-3 -2 .=3 15-01 12.13 1 2 . 10 10.32 2.73 2.36 2.33' 4,41 1. 32 4.05 3.30 3.04 2.39 2 . 74 - >j 3 1 . -4 :. ?3 4.15 4.27 3.35 3.-2 3-=0 :.7 : 3.31 =0.32 2.96 3 .25 2.54 3.32 2.50 4. 22 4.07 2.55 2.53 2.55 2.50 - . 51 2 53. J5 2.90 -.39 2.*3 3 - 55 3 -17 ) . 12 : 0 .55 0 .5 8 3.77 0.-2 0 .7 4 0.30 1i ..0o0i 0 .9 8 0 .-3 0.30 50 . 5 7 3- 6 /. ? 2 3.53 0.30 . 30-63 1.05 3.71 0 . 79 =0 . 12 0 .J7 1 1.92 0.61 0.59 1.07 0.59 ' :f 2 .55 0.54 1 .1 . -.23 2.72 : . 58 .37 0 . =9 2.55 1.33 '5 0.52 3.45 =0.s2 ' O .I3a 0.46 0.10 0.15 j . 35 3.17 3.29 3.25 0.09 3.32 0.36 0 ..3 7.27 3.15 3.19 ) . 33 3. - j 3.29 3 . 12 *1 :O .U :;o s c a c i s c i c a l l y s xcluaed 1i g r a i i = a a c = : : C 0 . C4 : :o a ; ilc u ld c ic 03 i'J* :r= a =on=rol :a =aeor =r naan jsio? - n a i y s i 5 3S VSC i r . c e 4 M '3D u r i n a c : ;a s = , ?<3.05. io L a e c i a- 0.33 0.52 0.97 0.50 3.98 0.98 0.73 3.36 0.33 1.49 0 .5 0 66 0.75 0.97 3.75 0 .9 8 0 .7 4 1.67 0 .3 6 3.33 0 .3 2 0.50 0 .7 3 0.53 0.96 LU5 0 .9 8 0 .5 1 0.53 0.69 0 .7 1 0.79 0.37 3.50 0 .5 3 0.37 - 7a O .i; 3.24 0.14 3.29 3 .2 4 3.23 0.23 0 .2 2 '3.-3 3.14 0.17 3.13 0.26 110 . 1 4 n 0.25 0 .3 1 0.23 0.19 0.14 0 .0 9 0.24 3.12 O .U 0.30 0.36 0.10 0.18 0.14 0.17 0.16 0.19 0.22 0 .2 1 0.17 0.36 =0.36 .i ` =0.08 1.07 1.03 0 .7 4 0.76 0 .3 5 0 .9 0 1.06 0 .6 6 0.77 L. 15 0.95 0 .5 6 0 .7 2 0.54 0 .3 6 0 .7 5 0.95 ' A 3.59 0 .3 4 3.33 0 .3 5 =0.18 0 .2 1 0.31 0.20 0.20 0.21 0U1 0.24 0.16 QUO 0.340.21 0.10 0.25 0.12 0.25 0.17 QUO 0.25 0.14 0.16 3.25 0.21 =0.06 3.97 1.14 0 . 79 0.71 0.96 0 .3 0 0.73 3.32 0 .3 2 0 .3 1 0 .7 2 0.35 =0.13 0 .2 2 0.29 0.25 3 .2 2 0 .2 4 0.19 O .U 0.20 0.23 0.13 0.18 0 .2 2 =0.03 1.01 11 . C 2 ., 3.79 3 66 3.59 1.51 3.33 3.71 3 56 3-63 3.71 0 5L 3.7 5 2.23 1 ) . 79 3.79 1.35 3. 53 =3.4 2 0.23 0.24 0.23 aj -! =" 0 . L4 0.37 0 . :1 3.19 3.1; 0.13 3.14 .1 j 0. =4 10 . 2 2 J j.U 3.24 0.12 ='3-12 . sziqp-s >.2Q 3.10 0.1Q 0.17 3.10 0.05 0.10 0.07 0.09 0.02 0.09 O .U 0.06 0.06 0.07 0.08 0.19 O .U . o .u 0,04. 0 .0 7 0.09 0.06 0.060.07 0.07 O3..3U4, 0.06 0.08 O .U 0.38 0.08 0.07 0.03 0.09 0.09 =0.04 0.05 0.03 0.03 3.05 0.03 0.04 0.02 0.02 0.00 0.02 0.03 0.01 0 .0 2 (3.Q1 0.02 0.02 0.03 000...000111 0 .0 2 0 .0 1 0 .0 2 0 .0 1 0 .0 2 0 .3 2 0.02 0 .0 2 0.02 3.03 . 3.112 0I 01. =0.01 QUO 0 .0 7 0 .U 0 .0 9 0.04. 0.15 0 .0 9 0 .0 8 O .U 0.05 0.20 0 .0 6 0.09 0.05 0.06 0,10 o .u 3.10 0.07 0.03 0.09 3.09 = 0 .0 4 0.02 0.02 0.03 Q .02 0.01 0.04. 0.02 0.02 00..0041 0.04 0.01 0.03 0.01 0.02 0.02 0.04 0.02 0.02 0.01 0.03 3.02 =0.01 0.10 3.38 0.30 0.10 0.08 o .u 0.17 O .U O .U 0.07 0.11 =0.03 0.02 0.02 0.03 0.03 0.02 0.03 0 .0 5 0.04 0.03 0.03 0.02 0.03 sC .Q l .0 .1 0 0.15 0.06 3.29 0.12 1 *7 3.11 0 .3 4 3.03 3.09 0.17 . 09 '4 ;.:a . 37 0.07 O .U 0.09 : 0 .34 0.32 3 .Cl o.j : 3.04 0.32 0.03 0.02 ',1i .'1 'i 1 'L .1 3.01 1 2.22 -! r . 3.02 .a - p =0.01 su $ 1 0 I'rmlcd11* ." wfrf. Ti'/*/- VoJ. 57. pf*. ^15 tu ' - I C Pw fgjm oo Pre** Utvl in C*^j C H riu in 00l>42M ;7 j.U(Q1.(i:ni\Af r 5s v RESULTS OF A TWO-YEAR CHRONIC TOXICITY AND ONCOGENIC STUDY OF RATS INGESTING DIETS CON TAINING 2,4,5-TRICHLOROPHENOXYACETlC ACID (2,4,5-T) R . J. K o c ib a , D . G . K eyes, R . W . L isow e, R . P. K a l n in s , D . D . D ittenber, . C E . W a d e , S. J. G orzim sxi, N . H . M a h l s * a n d B. A . Sc h w etz T o x ic o lo g y R e s e a r c h L a b o r a to r y (a n d M id la n d A n a ly tic a l L a b o r a to r y ') , H e a lth a n d E nvironm ental R esearch, D ow C hem ical U S A , B uilding 1803, M idland, M l 48640, U SA - (R e c e ive d 27 D ecem ber / 973} A b s t r a c t-- G r o u p s o f S p r a g u e - D a w l e y ra ts (50 m a le s a n d 50 fem ales) w/ere m a in ta in e d o n diets s u p p ly in g 3, 10 o r 30 m g 2 . 4 ,5 - T /k g b o d y w e ig h t/d a y fo r u p to 2 y r , w ith a n i n t e r i m a u t o p s y ( o n a n a d d i t i o n a l ten m ales a n d tan fem ales per g ro u p ) after 113-119 cays. T h e highest d o se level w as associated w ith s o m e degree o f toxicity, including a decrease in b o d y -w eig h t g ain a n d increases in relative kidney w e ig h t, in th e v o l u m e o f u r in e e x c r e te d a n d ir. th e u r i n a r y e x c r e t io n o f c o p r o p o r p h y r i n a n d u r o p o r phyrin, plus slight m o rp h o lo g ica l changes in the kidney, liver a n d iur.gs. T h e kid n ey ch an g es involved, p rim arily , the p resence o f m ineralized deposits in the renal pelvis. 'P a ra m e te rs n o t adversely affected b y this d o s e level in clu d ed d e ath rate, food co n su m p tio n , th e o c cu rre n ce o f p alp ab ie masses, h a em ato - iogical indices (red-celi c o u n t, haem o g lo b in , packed ceil volum e, to ta l a n d differential while-celt counts, throm bocytes and reticulocytes), the results of routine urine analyses, u rin a ry excretion of creatinine and 5-am inolaevulinic acid, serum -chem U try values (urea nitrogen, giutam lc-pyruvic-transam lnase and alkaline-phosphatase activities, bilirubin, total protein, album in and globulin), weights of organs other th a n the kidneys, tu m o u r incidence a n d gross an d m icro sco p ic m o rp h o lo g y o f ail the o rg an system s e x am in ed , w ith the ex cep tio n o f those m en tio n ed above. A t th e in te rm e d ia te dose level (10 m g/kg/day) only m inim al effects w ere noted, prim arily an increased incidence of m ineralized deposits in the renal pelvis an d , in th e m ales a n d onfy d u rin g the early p h ase o f the stu d y , a n increase in urin ary excretion _ o f c o p ro p o rp h y rin . A : the low er do se level (3 m g/k g /d ay ) th ere-w ere n o ch an g es th at were considered to be related to treatm ent thro u g h o u t the 2-yr period. Thus, this study revealed no oncogenic response in rats, even w hen the d u ratio n o f 2.4,5-T adm inistration extended over m ost of their lifespan at a dosage high enough to induce-toxicity. . Etf.E-l IN TR O D U CTIO N - 2 ,4 ,5 -T ric h lo ro p h e n o x y a c e tic ' a cid (2,4,5-T) has been u sed a b o u t 25 yr as a selective herbicide in the m anagem ent of certain crops, pasture, rangeland and fo rests. In th e p ro d u c tio n o f 2,4,5-T, the s ta rtin g m aterial for the aro m atic p o rtio n o f the c o m p o u n d is t e i r a c h l o r o b e n z e n e , w h ic h is s u b s e q u e n t l y h y d r o lysed to 2 ,4,5-trichlorophenol. In the p ro d u c tio n of the trichlorophenol, several im purities m ay be form ed u n d e r u n fav o u rab le co n d itio n s. O n e of these im p u ri ties is 2 ,3 ,7 ,S -te !ra c h lo ro d ib e n z o -;7 -d toxin ( T C D D ) w hich m a y be fo u n d a t low levels in the trich lo ro - plienol. O v er the past decade, a co n certed effort has b een m a d e to m in im iz e Ihe level o f T C D D in 2,4,5-T, an d c o m m ercial 2,4,5-T cu rre n tly co n tain s no m ore than 0 1 ppm T C D D . V arious toxicological studies h ave been conducted w ith 2,4.5-T o v er the past 25 yr. T h e single oral d o se LD, ,4.5-T en re p o rte d to b e 339 m g /k g in m ice, 381 m g/leg in g u in ea-p ig s, 500 m g /k g in rats a n d 310 m g /k g in c h ick s. ( R o w e o H y m a s, 1954) . T h e L D j 0 i n d o g s o f ICO m g , /k, r e p o e d b y D r i l l . <S:: H i r - \ a t z k a (1 9 5 3 ), is l o w e r k h a n in 0 t h e : sp ec ie s, m o s t \ p ro b a b ly b e c a u s e o f the ir s lo w e r r; itc o f u rin a ry elim - \ Lena s u c h a s 2^ O - T ( P i p e r , R o s e , G e a r in g , 1973). - T h e r c o m p o u n d 2 ,4 ,5 -T is re a d ily a b s o r b e d a n d rap id ly ex creted by m a m m a ls, including man.,. T h e half-life values for the clearan ce o f `'C activity from the p la sm a o f ra ts given single oral doses of iabeiled 2 , 4 ,5 - T a t d o s e s o f 5, 30, ICO a n d -200 m g / k g w e r e 4-7, 4-2, 19-4 a n d 25-2 hr, re s p e c iiv e iy ( P ip e r er ai. 1973). T h e half-lives for' elim ination from th e b o d y o f t h e s e r a t s w e r e 1 3 - 6 , 1 2 T , 19-3 2n d 2 2 - 9 h r , r e s p e c t iv e ly . Ir. d o g s g i v e n '"` C - l a b e i l e d 2 ,4 ,5 - T a t 5 m g / k g . haif-life values for clearan ce from the p la sm a a n d c le aran c e fro m th e b o d y w ere 77-0 a n d 56-6 hr, re s p e c tiv e ly ( P ip e r er al. 1973). T h is lo w er ra te o f c l e a r an ce p ro b a b ly explains th e greater toxicity o f 2,4,5-T in dogs th a n in rats. W h e n five, h u m a n v o lu n te e rs in g e sted 2,4,5-T, c o n ta in in g less th a n 0-05 p p m T C D D , in a d o se o f 5 m g /k g (G e a rin g . K r a m e r , S c h w e tz, R o se Sc R o w e, 1973), e ss e n tia lly all th e 2,4,5-T w a s a b s o r b e d a n d e x creted u n c h a n g e d in th e u rin e, w ith a half-life o f 23-1 hr. T h is ra te o f e x c r e tio n re sem b les m o re closely th at o b se rv e d in tn e ra t th a n th a t in the dog. In an u n p u b lish e d su b ac u te toxicity stu d y c o n ducted previously by this laboratory, no ad v erse effects -were c o s e m e d 2 , 4 , 5 - T a t d o s e Ilse v e l s o f 3 o r 10 m g /k g /d n y fo r 90 days. A t h ig h er d o s e levels (30 o r 100 m g /k g /d ay ), th e re w ere in creases in ' liver a n d k id n ey w eights, elevations in seru m e n z y m e s 205 NIT i n . i'. i i *. ill : S f- * ' i- iF;N - i I -s > Ji T e - N -. * * i > - . . it riv ". r Ji,--.1:11 - v v ll;!'! j v V r ilklif. '.- :i 'L/'C l *!i; U ; ! < . y ; 'i { \ - - : CV IjiS T T L r!Si N U N n ;rx --/rt: ;}) f f V i . ' N , TiUF . tLr lUU. A. v r-.; !? l i i - I 7 ' 3 " ; li;i!i S 'i - U T IT T lid : ; L -Atm) j \ (i - sy ii. x! m i!fj i 8 i ;} !/ if 2 0 6 R. 1 . Kociba, D. G. Keyes. R. W. Lisowe, R. P. Kalnins, D. D. DirrENneR, C E. Wads e t a l. a n d o th e r in dications of slight toxicity. Drill Si H ir- EXPERIMENTAL a tz k a (1953)' fo u n d n o a d v e rs e efTects in d o g s given 2-5 o r 10 m g 2 ,4 ,5 -T /k g for 5 d a y s / w k for 3 m o n t h s ; G ro u p s o f 100 rats (50 m ales a n d 50 females) w ere f o u r d o g s c o u l d n o t t o l e r a t e a h i g h e r d o s e level o f m a i n t a i n e d fo r u p to 2 y r o n d ie ts s u p p l y i n g 3, 10 2 0 m g /k g an d died d uring the e x p e rim e n t o r 30 m g Z 4 ,5 -T /k g b o d y w eight/'day. T h e co n tro l H ig h m a n , G a in e s Si S c h u m a c h e r (1976) re p o rte d g ro u p for this p a rt of th e stu d y c o n sisted o f 86 m ales re su lts o f s h o rt-te rm toxicity stu d ie s in m ice using a n d 86 fem ales. A n a d d itio n a l ten ra ts o f each sex sam p les o f 2,4,5-T w ith varying co n te n ts o f T C D D . w ere included for each treatm en t a n d co n tro l g ro u p T h e se studies, w h ic h w ere c o n d u c te d in c o n ju n c tio n for the in te rim kill o n d a y s U S a n d 119. w ith teratology studies, d e m o n stra ted m ultiple toxic T e st m aterial. T h e p u rifie d s a m p le o f 2,4,5-T w as effects fro m tre a tm e n t w ith d o ses o f 120 o r 60 m g su p p lie d by T h e D o w C h e m ic a l C o m p a n y , M id lan d , 2.4.5-T /k g ; lo w er d o se levels w ere n o t stu d ied. M I. T h e identity o f the 2,4,5-T w as verified, p rio r to Sm ith, Schw etz, M u rray , C raw ford, Jo h n , K o cib a the initiation of the study, by elem ental analysis, gas Si H u m isto n (1973) recently rep o rted a three-gene- ch ro m ato g rap h y , so lid -p ro b e m ass spectrom etry, gas ra tio n re p ro d u c tio n stu d y in ra ts ingesting 2,4,5-T. c h ro m a to g ra p h y -m a s s s p e c tro m e try , n u c le ar m a g N o a d v erse effects w ere seen at a d o se level o f 3 m g/ netic re so n a n c e sp e c tro m e try a n d infra-red sp ectro "kg/day. H ig h e r d o s e levels o f 2,4 ,5 -T w h ic h w ere suffi m e try . W ith in the lim its o f precisio n o f e ac h o f these cien t to cau se signs o f toxicity h a d no effect o n the * techniques, the m aterial w as essentially 2,4,5-trichloro- reproductive capacity of th e rats, except for a tend phenoxyacetic acid. A ssay by stan d a rd procedures in ency to w ard a red u ctio n in n eo n atal survival at dose d icated ap p ro x im ately 99% 2,4,5-trichlorophenoxy- levels o f 10 a n d 30 m g /k g /d a y . acetic acid. O th e r p h en o x y acid im purities am ounted S everal stu d ies h a v e b e en c o n d u c te d in m ice ov er to 1-3% (w /w ) b y gas c h ro m a to g ra p h y . U sing gas th e p a s t 10 y r to assess th e p o te n tia l o n c o g e n ic ity of c h r o m a to g r a p h y - m a s s s p e c tro m e tr y , T C D D , hex3- 2.4.5- T . Innes, U U an d , V alerio, P etru ceili, F ishbein, chlorodfbcnzo-pi-dioxin, h ep tach lo ro d ib en z o -p -d io x in H a r t , P a lo tta , 3 a ie s , F a lk , C a r t , K le in , M itc h e ll Sc a n d o c t a c h lo r o d ib e n z o - p - d io x in w e re n o t d e te cte d , P e te rs (1969) re p o rte d n o increase in th e in cid en ce of th e lim its of d e te ctio n b e in g 0-33, 0 1 2 , 0 4 0 a n d t u m o u r s in tw o s t r a in s o f m ic e g iv e n t h e m a x i m u m 0 4 0 p p b (jig /kg), re s p e c tiv e ly . T h e re c ry s ta lliz a tio n to le ra te d dose (21-5 m g /k g /d a y ) o f 2,4,5-T (containing approxim ately 30 p p m T C D D ) by stom ach tube from so lv en t te lra c h lo ro e th y le n e , w as p re se n t a t a level of 022%. .day 7 to d ay 28 o f age. A fter the m ice w ere w eaned D iet p rep a ra tio n . T h e te st d iets w e re p re p a re d b y a t 28 d a y s o f age, 2 ,4 ,5 -T w a s m ix e d d ire c tly in- th e d is s o lv in g a n a p p r o p r i a t e a m o u n t o f 2 ,4 ,5 -T in diet a t a co ncentration o f 60 p p m for approxim ately reagent-grade acetone a n d m ixing the solution 18 m o n t h s . I n a s t u d y c o n d u c t e d fo r t h e N a t i o n a l th o r o u g h l y w ith g r o u n d l a b o r a t o r y c h o w to g iv e a C an c e r Institute b y the B ionetics R esearch L a b o ra 1% prem ix. T his prem ix, after d ry in g ,'w as m ixed w ith to ries (1S6S) b u t n e v e r p u b lis h e d , w e a n lin g m ic e (tw o r a t c h o w to p re p a re d ie ts s u p p ly in g 3, 10 a n d 30 m g strains) w ere given a single d o se o f 215 m g 2,4,5-T /kg 2,4,5-T /kg b o d y w eight/day. T h e b o d y w eights an d b y sc injection an d w ere o bserved for the follow ing fo o d c o n su m p tio n of 20 rats of each sex per gro u p 18 m o n t h s . T h e a u t h o r s c o n c l u d e d t h a t t h e r e w a s n o w e r e u s e d as th e b a s is fo r p r e p a r i n g th e s e d iets. C o n in crease due. to 2,4,5-T in the in cid en ce o f tu m o u rs. trol rats w ere su p p lied w ith g ro u n d la b o ra to ry c h o w In a n initial p u b licatio n , M u ra n y i-K o v a c s , R u d ali treated "with a n e q u iv a len t a m o u n t o f acetone. P o r . Sc I m b e r t (1976) r e p o r te d re s u lts o f th e ir s tu d y o f tw o tio n s o f e a c h p r e p a r e d d ie t f r o m e a c h d o s e level w e re in b r e d s tra in s o f m ice ( C 3 H f a n d X V I I G ) g iv e n c o lle cte d d u rin g m o n th s 5, 7, 12, 19 a n d 23. a n d 2.4.5-T (100 m g/litre) in th e d rin k in g -w a te r fo r 2 an aly sed for 2,4,5-T. m o n th s from 6 w k o f age. T h e m ice w ere subsequently E xp e rim e n ta l d esig n . M a l e a n d fem ale S p ra g u e -- f e d d i e t s c o n t a i n i n g SO p p m 2 , 4 , 5 - T ( a p p r o x i m a t e l y D a w l e y r a t s , 7 - 8 w k o l d ( f r o m S p a r t a n R e s e a r c h A n i . 12 m g /k g /d ay ) for the rest o f the lifetim e study. O n m als, H aslett, M I) w ere ra n d o m ly p laced (using a the basis of their m eth o d o f c o m b in in g tu m o u r types, table o f ra n d o m n u m b ers) In su sp en d ed w ire-bot . these a u th o rs re p o rte d a. statistically significant, in to m e d cages (tw o rats/cag e) for a 1 1 8 /1 19-day an d cre ase in tu m o u r in cid en ce in C 3 H f m ic e fo llow ing a 2 -y r c h ro n ic toxicity stu d y . In d iv id u a l rats w ere 2 ,4 ,5 -T tr e a t m e n t , b u t n o s ig n if ic a n t d iff e r e n c e s in id e n tifie d b y m e ta l e a r ta g s a n d d o s e levels w e r e a lso . t u m o u r i n c i d e n c e i n t h e m i c e o f t h e X V I I G s t r a i n . i d e n t i f i e d b y t o e ^ l i p p i n g . F o o d ( P u r i n a L a b o r a t o r y T n a su b se q u e n t c o m m u n ic a tio n , M u ra n y i-K o v a c s, C h o w , R a lso n -P u rin a C o m p a n y , St. L o u is, M O ) a n d / R u d a li S i I m b e r t (1977) r e p o rte d th a t n o in c re a se in w a te r w e r e a v a ila b le ad lib . - tu m o u rs was n oted in o th e r studies in w hich they C lin ica l o b serva tio n s. R a ts w e re o b s e r v e d for their .. g a v e t h e s a m e t w o s t r a i n s o f m i c e a s e r i e s o f n e o n a t a l g e n e r a l s t a t e o f h e a l t h a n d f o r p o s s i b l e t o x i c o l o g i c a l sc in jectio n s o f 2,4,5-T. M u ra n y i-K o v a c s el cl. (1976) resp o n ses d u rin g th e s tu d y , o b s e rv a tio n s being ; stated, how ever, that the m o u se m a y not b e th e best recorded w h e n .th e rats w ere w eighed. F r o m m o n th / e x p e r im e n ta l m o d e l fo r te s tin g th e c a r c i n o g e n ic ity o f 6, o b s e r v a ti o n s w e re a ls o r e c o r d e d a t e a c h m o n th ly 2.4.5- T a n d re c o m m e n d e d fu r th e r te stin g in g re a te r e x a m in a tio n for th e d e te c tio n o f p a lp a b le m asses. c u m b e r s o f a n i m a l s a n d in o t h e r s p e c i e s , s u c h as- th e B o d y w e i g h t s w e r e r e c o r d e d a t a p p r o x i m a t e l y w e e k ly rat. in terv als for all th e ra ts s c h e d u le d fo r th e in te rim kill I n o r d e r to e v a lu a te th e lo n g - t e r m to x ic ity a n d at 11S /l 19 d a y s a n d a lso for' 20 r a ts o f e a c h sex in o n c o g en ic potential of 2,4,5-T in rats, th e study the co n tro l and test g ro u p s d u rin g th e first 3 m o n th s rep o rted here w as c o n d u cted . It involved the dietary of the 2-yr part of the study. T hereafter, b o d y weights a d m in is tr a tio n - o f 3, (0 o r 30 m g 2 ,4 ,5 -T /k g /d a y to o f th e la tte r ra ts w e re r e c o r d e d a t a p p ro x im a te ly rats for u p to 2 yr. m o n th ly intervals. F o o d c o n su m p tio n w as recorded P -c C h ro n ic toxicity of 2,4,5-T in rats 207 a p p ro x im a te ly tw ice w eek ly for all the rats sch ed u led s to m a c h , sm all a n d large intestines, m esen teric ly m p h for th e in te rim kill a n d d u rin g the first 4 m o n th s o f node(s), skeletal (thigh) m uscle, salivary gtaud, testes, the 2-yr stu d y for 20 rats o f each sex from each group. epididym is, accessory m ale sex glands, u rin ary b la d F o r th e rest of the 2-yr study food co n su m p tio n was der, uterus, ovary, trachea, oesophagus, aorta, th o ra re c o rd e d fo r 'u p to 20 rats o f e ac h sex p er g ro u p for 1 v.'k/m onth. cic ly m p h node(s), thym us, lungs, bronchi, in te g u m e n t, th y ro id gland, p a ra th y ro id glands, a d ip o se tis B lo o d sam p les for haem ato lo g ica! d e te rm in a tio n s sue, eyes, ad ren al gland(s). an d any grossly o b serv ed w ere c o lle cte d fro m the tail veins o f eight rats of each lesion, w ere preserved in form alin fixative. T h e sex a n d g ro u p , o n d a y s 32/S3 for the rats sch ed u led , w eig h ts o f the liver, kidneys, brain, heart, th y m u s, for th e in te rim kill a n d o n days S9/90, 364/365 and sp leen a n d testes o r ovaries/uterus (as a unit) w ere 7 2 6 /7 2 7 for those, in th e 2-yr stu d y {w hen .g ro u p sizes re c o rd e d for all the rats. perm itted ). T h e total ery th ro c y te co u n t, total a n d dif All rats dying o r killed during the co u rse o f the ferential leucocyte co u n ts, th ro m b o cy te an d reticulo 2-yr stu d y w ere also subjected to a gross p ath o l g ical cyte c o u n ts, p a c k e d ceil volum es a n d h aem o g lo b in e x am in atio n . R epresentative parts o f the skull (includ c o n ce n tratio n s w ere d eterm in ed using a u to m a ted ing n asal tu rb in ates an d ear canal) along w ith any te c h n iq u es (C o u lte r C o u n te r M o d el Z B l, from g ro ss lesions suggestive o f a significant p ath o lo g ical C o u lte r E lectronics, H ialeah , FL ) o r m an u al p ro process of tu m o u r form ation were collected from each cedures. ra t a n d p re se rv e d in fo rm alin fixative. U rin e sam ples w ere collected from the sam e A te rm in a l a u to p sy w as c o n d u c te d o n all ra ts su r n u m b e rs o f an im als a n d at the sam e tim es as the viving the 2-yr period of treatm ent. T h e o rg an s a n d b lo o d sam ples. T h e specific gravity a n d p H o f the tissues w eighed an d preserved an d the p ro ced u res u rin e a n d th e p resence o r absence o f glucose, protein, used for these an im als an d for those au to p sied d u rin g "k eto n es, b iliru b in a n d occult b lo o d w ere d ete rm in e d the 2-yr p e rio d w ere as described for th e interim (A m es B ililabstix o r Multisti-x, from A m e s C o m p a n y , au to p sies. A t the term inal autopsy, sm ears o f peri E lk h a rt, I N ; T S M ete r, from A O O p tical, Buffalo, p h e ra l b lo o d a n d fem oral b o n e m a rro w w ere p re N Y ) a t e a c h o f th e s e tim e s , a n d u r in a r y u r o b il in o g e n p a r e d fo r m o s t rats, a n d filed fo r fu tu r e re f e r e n c e if w a s d e te r m in e d o n d a y s 364 /3 6 5 a n d 7 2 6 /7 2 7 o f tre a t n e e d e d . P o r tio n s o f fat, liver a n d k id n e y for p o ssib le m ent. ' 2 ,4 ,5 - T a n a ly s is w e r e s a v e d fr o m a m a x i m u m o f five U rin a ry levels o f creatinine, c o p ro p o rp h y rin an d ra ts o f each sex a n d group, at b o th the in terim an d u ro p o rp h y rin w ith o r w ith o u t -am inolaevulinic acid term inal autopsies. (6-A L A ) w ere d e te rm in e d by B io-Science L a b o ra H isto lo g ic a l e x a m in a tio n o f tissues. P a ra ff in -e m b e d tories, V a n N uys, C A . R ats w ere h o u sed individually ded sections of tissues were stained w ith h aem ato x y lin in m e ta b o lis m cages for co llectio n o f 24- o r 4 8 -h r a n d eosin. S ectio n s o f the preserved tissues fro m the u rin e sam p les, w h ic h w ere tra n s p o rte d in glass ja rs c o n tro l a n d hig h -d o se g ro u p s killed o n d a y s 118/119 w r a p p e d in a l u m i n i u m foil and. p a c k e d in d r y ice. a n d f r o m all tr e a te d a n d c o n tr o l r a ts d y in g o r k ille d U rin e s a m p le s w ere c o llected fro m rive rats o f e a c h d u rin g th e 2-yr stu d y o r killed a t .term in a tio n w ere - sex a n d g ro u p o n th re e o ccasio n s before d a y 106 o f su b jec te d to histological ex am in atio n , b u t th o s e tak en tre a tm e n t a n d o n c e ju s t p rio r to th e in te rim kill in fro m th e lo w er-d o se g ro u p s a t the interim kill w ere . th e s u b sid ia ry s tu d y , a n d fro m fo u r, o r five ra ts o f n o t e x am in ed , b e ca u se o f a lack o f any d efin ite tre a t e a c h s e x a n d g r o u p a f t e r 9 5 - 9 7 , 1SS--1 9 0 , 3 6 1 - 3 6 3 , m e n t - r e l a t e d e f f e c t s in t h e h i g h - d o s e g r o u p . A d 5 6 4 -5 6 6 a n d 6 9 8 -7 0 0 days of the 2-yr tre a tm e n t. d itio n a l sections of liver a n d kidney from m o st c o n U rin e v o lu m e s w ere re c o rd e d for each individual rat.. trol a n d to p -d o se rats killed after 118/119 d ay s w ere A t b o th the in terim an d term inal autopsies, seru m stain ed for lipid c o n ten t using Oil R ed O . A d d itio n al sam ples w ere collected from a m axim um o f ten rats o f each sex an d g ro u p for the d eterm in atio n o f urea n itro g en (B U N ), activities of g lu tam ic-p y ru v ic tran s s e c tio n s o f k id n e y s ta k e n fro m five to s e v e n fe m a le rats from th e c o n tro l a n d high-dcse g ro u p s at the term in al kill w ere stained w ith Oil R ed O , by M a l a m in a se (S G P T ) a n d alk alin e p h o s p h a ta s e (SAP),, total, d irect and indirect bilirubin, an d total protein, lo ry 's re a c tio n for iron, w ith Z iehl N e eise n stain for acid fastness, by D ah l's m e th o d for calciu m a n d by a lb u m in a n d g lo b u lin . A u to m a te d p ro c e d u re s w ere th e p e rio d ic -a c id -S c h iff reactio n (L u n a, 1968). used for these determ inations (T echnicon A uto- S ta tis tic a l eva lu a tio n o f d ata. H a e m a to lo g ic a l, u ri A nalyzer, T ec h n ic o n C o rp o ra tio n Rye, NY). Pose m o rtem exa m in a tio n . R ats sch ed u led for the n a ry a n d clinical chem istry param eters, b o d y weights, o rg a n w eights an d o rg a n /b o d y w eight ratios w ere interim a u to p sy w ere deprived of food overn ig h t after an aly sed statistically by a one-w ay analysis o f vari t r e a t m e n t f o r 1 1 S o r 1.19 d a y s a n d t h e n k i l l e d b y a n c e f o l l o w e d b y D u n n e t t 's T e s t , w i t h P < 0 0 5 ( S t e e l d e c a p ita tio n . T h e eyes o f all ra ts w e re e x a m in e d , by 8c T o r rie , 1960). D a ta o n m o rtality , p a lp a b le m asses, m ean s o f a glass slide pressed gently against the c o r g ro ss p ath o lo g y , h isto p ath o lo g y an d tu m o u r inci nea u n d e r b rig h t flu o rescen t illu m in atio n . T h e eyes d e n c e in the rats of the 2-yr stu d y w ere a n a ly se d using . f r o m a m a x i m u m o f f iv e r a t s o f e a c h s e x a n d g r o u p F i s h e r 's E x a c t P r o b a b i l i t y , P < 0 -0 5 , o n e - s t d e d te s t w ere p re se rv e d in Z e n k e r's fixative a n d th o se from (Siegel, 1956). F o r gross p athological o b serv atio n s, the re m a in in g rats w ere fixed in 10% buffered fo rm a statistical e v alu atio n o f the cu m u lativ e fin d in g s for lin. A c o m p le te g ro ss p a th o lo g ic a l e x a m in a tio n w as p e rfo rm e d o n all th e ra ts by a v e te rin a ry p a th o lo g ist. the entire 2-yr study involved com p ariso n of the d ata of each o f the treatm en t groups against th o se o f the R e p re se n ta tiv e sec tio n s o f all m a jo r o rg a n s a n d tis sues, in c lu d in g liver, kidneys. heart, p a n crea s, spleen, b rain (cereb ru m , cerebellum and b rain stem ), spinal cord, p e rip h e ra l (sciatic) nerve, pituitary gland. co n tro l g ro u p of that sex. T he d ata w ere inspected an d those suggestive of a difference from the control were a n a l y s e d s t a t i s t i c a l l y . F o r h i s i o p a t h o l o g i c a l o b serv atio n s and tu m o u r incidences, the cu m u lativ e v rii-.- f C f.; . a. x.'...- ;l! h": b irl ! v Y iifr: s it: M f .\ i i .J ii, t.. V N p.r iSr:Ui piiV' i-.. " H srV . r` "i IP k h y -i.' >-S } \ t-- tr*>. V i- !U r_3 * * :=O - !<--l 'i 8V? I U -i y. P fx.rii It' 'U: r i_Z" .] 7 7 T - . 1 i; R . J . K o c t n .v , D . G . K g y s s , R . W . L is o w k , R . P . K a l n i n s , D . D . D t n r.N H E K . C . E . W a u e e t u l. h VJ .- i 'i ; '.' . I !*'. : ' /. T a b l e 1. S u m m a r y o f b o d y -w e h jh ls , c lin ic a l o b s e r v a tio n s a n ti o r g a n w e ig h ts In r a ts f e d d ie ts c o n ta in in g 2 ,4 ,5 - T f o r u p to 2 y r f \ D ose of 2 ,4 ,5 -T (m g /k g /d a y ) .* ' | ' * ; ` N o. of ra ts/ g ro u p } 26. - V V '/ B o d y w e ig h t (g) o a d a y K 53- 95 i 314 ii- .; ' U rin a ry e x c re tio n T e rm in a tio n o f 1 1 8 /1 1 9 -d ay stu d y T e rm in a tio n o f 2-yr stu d y T o ta l 4 8 -h r u r in e v o l. (m l) ; C o p ro . p o rp h y rin (p g /1 8 hr) U ro p o rp h y rin (/<g / 4 8 h r ) C o p ro p o rp h y rin < 1-8/48 h r ) U ro p o rp h y rin (l-g /4 8 h r) 0J 10 30 : .... ' B 6 + 1 0 ' i ;,i. 3 8 5 2 1 4 4 4 3 0 ; 4 9 8 36 i '6 2 7 53 50 + 10 385 25 4 4 7 4: 3 4 5 0 } 41 ; 6 3 | 59 5 0 + 10 386 25 419 36 508 43 | 6 3 5 59 50 + 10 . 3 2 5 21 4 3 7 3 0 . 4 9 2 38 ! . 6 1 6 50 M u le s 38 8 4 0 10 . 36 i 14 ' 4 9 11* 17-5 4 9 2 3 1 4-2 24 6 9 6* 4 3 6 9 3* 4 1 1-8 4-5 2 0 5 0 3-1 7-7 4-7* 14-6 6 0 ' 15-2 3 6 9 7 4-4 2 2 S 13 0 F e m a le s 0 86 + i0 -. 2 J 3 1 4 ' 2 7 8 -jt 1 6 3 01 17 361 28 . 9-6 i-9 3 / . 5 0 + 1 0 , ; 2 5 4 1 2 2 7 9 1 5 3 0 6 1 7 3 6 3 i - 2 6 NE . 10 3 3-5 ' NE . 1 0 1 . 5 0 + 1 0 2 5 3 1 5 '< 2 7 3 1 5 2 9 9 17 '!' 3 6 5 2 8 'f NE . 9 2 08 I -. N E 30 50 + 10 2 4 4 12* 266 1 5 * - 288 17- v 347 27* : NE 16 4 7-0* NE 5-8 1-9 6-5 2 3 9-8 2-3 8 0 9-8 4 7 1-8 30 0 9 3 0 1-3 4 0 t 1-5 NE NE NE t N E * N o tr c a tm c n i- r c la le d effect t S e le c te d to d e p ic t th e effects c o n s id e r e d to b e r e la te d to tr e a tm e n t.1 } In 2 -y t + 1 1 8 /1 1 9 -d ay stu d ie s. It V a l u e s a r c m e a n s S D a n d t h o s e m a r k e d w i t h a n a s t e r i s k d if f e r s i g n i f i c a n t l y ( b y a n a l y s i s o f v a r i a n c e a n d D u n n e l t 's T e s t ) f r o m c o n t r o l v a l u e s : * P < 0 0 5 . K id n e y w e ig h ts: 1 1 8 /1 1 9 d a y stu d y (g /IO O g b o d y w eig h t) 0 70 0 09 0 72 0 05 0 73 0 05 0-79 0 06 * NE NE ' NE :':v- !'; V i> i;' ^\ ' v W ' * "-`f A \iy- ^Vi'-Ir,r v V V . I 17 " * 1 1 C h ro n ic toxicity of 2,4,5-T in rats 209 d a ta of each dose g ro u p w ere c o m p a re d w ith those of the c o n tro l g ro u p of that sex. T h e exact nu m b er o f tissues e x a m in e d w as u sed as th e to ta l g ro u p size in each analysis p e rfo rm ed . T h e d a ta sverc inspected an d those suggestive of a statistical difference from the co ntrol w ere analysed. RESULTS D ie ta r y c o n te n t o f 2,4 J - T T h e resu lts o f a n a ly se s o f feed s a m p le s o n five o c c a s io n s in d ic a te d t h a t th e d o s a g e lev els o f 3, 10 a n d 30 m g 2 ,4 ,5 -T /k g /d a y e q u a t e d w ith a p p r o x im a te ly 35, 230 a n d 677 p p m o f 2,4,5-T in th e diet. R e p e a te d a n a lyses o f the feed sam p les indicated generally g o o d agreem ent betw een the in tended an d the analytically m e a su re d c o n c e n tra tio n s o f 2,4,5-T. O n o n e occasion, analysis o f the diet for the g ro u p o f rats given 3 m g/ kg/day indicated a so m ew h at higher th an expected 2,4,5-T co n ten t, b u t this w as c o n sid e re d to h ave a negligible im pact on the o u tc o m e o f the study. P alpable m asses and a nim al observations T h e first p a lp a b le m a ss w a s n o te d in a m ale rat o f the co n tro l g ro u p . T h e re w ere no statistically sig nificant differences betw een control a n d treated g r o u p s o f m a l e r a t s e x c e p t d u r i n g m o n t h s 13 a n d 14, w hen, th e g r o u p given 10 m g 2 ,4 ,5 -T /k g /d a y h a d an increased incidence of palpable masses. T his w as con s id e r e d o f n o to x ic o lo g ic a l sig n ific a n ce , b e c a u s e o f its isolated occurrence and lack of a dose- or tem poralr e s p o n s e r e la tio n s h ip . T h e r e w e re n o d iffe re n c e s in th e in cidence ra te s of p a lp a b le m asses in g ro u p s of tre a te d fem ales ex cep t for statistically significant in c re ase s d u r in g m o n th s 9 a n d 10 in th e g r o u p receiving 3 0 m g / k g / d a y a n d d u r in g m o n th 14 in th a t g ro u p receiv in g 10 m g /k g /d a y . T h e s e o b s e rv a tio n s w ere c o n sid ered o f n o toxico lo g ical significance, since they w ere isolated occurrences an d did not occu r during the latter p h a se s o f the study. E x a m in a tio n o f the rats d u rin g th e co u rse o f th e 2-yr stu d y rev ealed no o v e rt clinical evid en ce o f toxicity a ttrib u ta b le to these th re e levels o f tre a tm e n t w ith 2,4,5-T. B o d y w eig h ts H a e m a to lo g y and urine a n a lyses T h e re -were no statistically significant differences R e p e titiv e e v a lu a tio n o f all h a e m a to lo g ic a l p a r from c o n tro l m e a n s in the m e a n b o d y w eights o f any am eters revealed no statistical differences from con o f the treated m ale a n d fem ale rats in the 113/119 trol values at a n y d o se level a t a n y tim e d u rin g the d ay study. M e a n b o d y w eights of fem ales given 30 m g co u rse o f the su b ch ro n ic o r ch ro n ic p arts o f the study. 2.4.5- T /k g /d a y s h o w e d a statistically sig n ificant d e R o u tin e urine analyses also revealed no changes con cre ase fo u r tim es d u rin g th e c o u rse o f th e 2 -yr stu d y sid ered to b e related to ingestion o f 2,4,5-T. O n on e ( T a b l e 1), a n d t h o s e o f t h e m a l e s o n t h i s d o s e s h o w e d o c c a s i o n t h e r e w a s a s t a t i s t i c a l d e c r e a s e in t h e s p e c ific a tren d to w a rd s slightly low er values d u rin g m o st of g ra v ity o f th e u rin e o f m a le ra ts g iv en 10 m g 2,4,5-T/ th e 2 -y r p e r io d . M a le s a n d fe m a le s g iv e n 3 o r 10 m g k g /d a y , b u t b e c a u s e o f its is o la te d o c c u r r e n c e an d 2.4.5- T /k g /d a y d id n o t differ statistically from th e . lack o f a d o se re sp o n se this w as c o n sid e re d to be c o n tro l b o d y w eights, except at o n e p erio d w hen the o f no toxicological significance. m e a n b o d y w eig h ts o f m a ie s g iv e n 10 m g '2,4,5-T /kg/ R epetitive evaluation o f urinary porp h y rin s d id - d ay w ere statistically increased. T h is isolated observa reveal o b serv atio n s th at w ere co n sid ered to be related t i o n w a s c o n s i d e r e d o f n o t o x i c o l o g i c a l s i g n i f i c a n c e s . t o t h e h i g h e r d o s e l e v e l s o f t r e a t m e n t ( T a b l e 1). I n F o o d consum ption, d a ta - . m a l e s r e c e i v i n g 3 0 m g 2, 4, 5- T / k g / a a y , t h e r e w e r e s t a t istically significan t in creases in to ta l u rin e v o lu m e and T h e r e w e r e n o c o n s i s t e n t d e v i a t i o n s i n t h e r a t e o f i d t h e e x c r e t i o n o f c o p r o p o r p h y r i n a n d u r o p o r p h y r i n . th e f o o r h e o n s u m p e io n o f m a le s o r fem ales g iv en a n y ' In m ales receiv in g 10 m g /k g /d a y , th ere w a s a signifi- of the th ree dose levels d u rin g the c o u rse o f either .c an r increase in c o p ro p o rp h v rin excretion, b u t only th e 1 1 8 /1 19-day o r th e 2-yr stu d y . T h e few s p o ra d ic in. th e early p h a se of th e stu d y . In fem ales receiving cases in w h ic h .th e re w as a statistical in crease o r d e 30 m g 2,4,5-T/kg/day, excretion of c o p ro p o rp h y ria crease in fo o d c o n s u m p tio n b e tw e e n th e c o n tro l a n d w as increased at m o st o f the sam pling tim es; excre various treatm en t g ro u p s follow ed no consistent tio n o f u ro p o rp h y rin sh ew e d a statistically significant trend, a n d w ere co n sid ered o f no toxicological signifi decrease after 119 d a y s b u t a n in crease after 565 days. cance. t _ .V ' ' - . 5 - 7 " N o s i g n i f i c a n t d i f f e r e n c e s i n t o t a l u r i n a r y v o l u m e o r M o rta lity ... :'v - ' ' f . : : - :.T he in c id e n c e o f d e a t h s in t h e g r o u p o f m a le - r a ts given 30 m g 2,4,5-T /kg/day sh o w e d a statistically sig nificant decrease d u rin g the latter p art o f the 2-yr . i n t h e e x c r e t i o n o f c r e a t i n i n e , c o p r o p o r p h y r i a , u r o p o rp h y rin o r 5-A L A w ere recorded, for m ale rats on 3 m g /k g /d a y o r for fem ales o n 3 o r 10 m g /k g /d a y at . any tim e during the study. . study, a finding c o n sid e re d to b e sec o n d a ry to the less sev ere c h ro n ic ren al d isease in th ese ra ts. T h e . m o rta lity in (he fem ales g iv e n th is h ig h d o se level' sh o w ed no difference from the c o n tro l p a tte rn . F o u r C lin ica l ch em istry . . . . . . . .' 'V . '. . "T erm in al" analyses o f seru m sam ples fo r T3UN, S G P T , SA P, bilirubin (total, direct an d indirect), total o r five tim e s d u r in g th e s tu d y th e re w e re statistically significant in creases in d e ath s in m ales a n d fem ales given 10 m g Z 4 ,5 -T /k g /d a y . w hile m ales given 3 m g/ k g /d a y sh o w e d statistically significant d ecreases at four intervals. F e m a le s o n this low d o se level sh o w ed no differences from c o n tro l values. T h e se differences in m o rta lity p a tte rn s in ra ts given 3 o r 10 m g 2.4,5 -T / k g /d a y sh o w e d no d o se-re lated effects a n d w ere c o n p ro tein , a lb u m in a n d globulin after 113/119 days or 2 yr sh o w ed no alteratio n s co n sid ered to be related to 2,4,5-T tre a tm e n t a t a n y d o se level S G F T show ed a statistically significant in crease in the g ro u p of fem ales given 10 m g /k z /d a v lor 119 d ay s, o u t this w as c o n s id e r e d to b e o f n o to x ic o lo g ic a l s ig n ific a n c e , in view o f its isolated n ature, the lack ot d o se response andi the a b se n c e ot a n y sim ila r fin d in g a t tn e end sidered to be o f no toxicoioeical significance. of the 2-yr study. 2 1 0 R. 3. Kociba, D. G. Keyes, R. W. Lisowe. R. P. Kalnins, D. D. Dittenrer, C. E. Wade ei al. T a b ic 2. S u m m a ry o f m a jo r gro ss a n d h isto p u th o lo g ico i o b serva tio n s in rats m aintained on d iets co n ta in in g 2,4 J - T fo r 2 y rf N o. of rats aflccted/no. exam ined M ales Fem ales Lesion D ose (mg/kg. d a y ). . . 0 3 10 30 0 3 10 30 K idney M ineralized deposits in pelvis Localized reaction adjacent to mineralized ........ d e p o s i t s M oderate to severe chronic renal disease Increased p ig m e n t in renal tu b u la r cytoplasm M ineralization secondary to renal disease seen in: pulm onary alveoli ' ' m yocardium . .. m y o c a r d i a l b l o o d v e s s e l s gastric m ucosa and muscularis 56/86 13/86 5/86 12/86 25/85 Liver '. . Enlargement ' . . Focal biliary hyperplasia Periportal inflam m ation " F o cal aggregates o f reticulo-endotheliai cells adjacent to degenerate o r necrotic ' hpatocytes M ultiple foci o f h e o ato ce llu la r alteratio n (swollen hpatocytes) Focal hepatocellular cytoplasm ic vacuolization 11/86 9/86 29/86' 34/86 NE NE 28/50 NE 8/50 3/50 6/50 13/49 3/50 6/50 7/50* NE NE 20/50 NE NE 1/86 1/50 5/50* 8/50- NE 30/50 NE NE 22/50* NE 25/86 6/86 0/S5 19/50 6/50 0/50 22/50 2/50 0/50 37/50* 1/50 11/50* 4/50 2/50 5/50 9/49 . 0/50* 0/50 0/50* 1/50* 7/50 8/50 9/50 1/50* 14/50* 28/50* NE NE NE NE - NE ' NE NE NE NE NE NE . NE " -' NE NE NE . NE NE NE . NE . NE NE N E N E ' 36/86 17/50 21/50 34/50* NE NE 12/50* 8/50* 15/86 4/50 6/50 NE NE 3/50NE Lungs Focal interstitial inflam m ation Focal accum ulations of alveolar m acrophages' - in alveoli - Focal cholesterol clefts " '' Focal accum ulations of secretory material " in alveoli .. 21/86 23/366/36 5/50* 5/50* 27/50- 16/50 13/50 29/50* 4/50 . 5/50 - 9/50* N E ..N E NE 0/36 NE N E- NE - - NE NE NE NE - NE 'N E .'. . 2/50 '2 /5 0 4/50* C ardiovascular system Dilated/flaccid ventricle Periarteritis 'L e ft atrial throm bosis ;' . . . . .-.___ a. . vO> 22/86 15/50 7/50 9/50. 0/86 1/50 2/50 .: - 2 0 / 8 6 1 2 / 5 0 8 / 5 0 S/50 NE NE NE - 12/86' 6/50 4/50 3/50 NE NE NE N E = N otreatm ent-related enect tS elected from the results o f ex am in atio n o f all o rg arr sy ste m s, to depict the effects considered to be related to treatm en t. T h e tu m o u r in cidence in ail the o rg a n system s was co n sid ered to be unaffected by an y level o f treatm en t. Values m arked with an asterisk differ significantly (by Fisher's Exact Probability Test) from the corresponding control incidence: * p < 0-05. O rgan v/etghts .v ; A statistically significant increase in the relative w eight of the k id n ey s o f m a le .ra ts killed after being: g i v e n 3 0 m 'g 2 , 4 , 5 - T / k g / d a y f o r 1 1 S d a y s w a s c o n s i d e r e d to b e r e i a t e d t o t r e a t m e n t [ T a b l e 1). N o o t h e r organ-w eight changes w ere n o ted at the interim autopsies. A t the term inai a u to p sy after the 2-yr treat m ent, the only statistically significant difference was a decrease in th e a b so lu te h e a rt w eight of m ales given 30 m g/V g/dayi this w as co n sid ered to be o f highly questionable toxicological significance because of an absence of any discernible gross o r histooathological c h an g es in these h earts. N o a lte ra tio n s c o n sid ered to be related to tre a tm e n t w ere fo u n d in the w eights o f b rain , liver, .testes, spleen, th y m u s o r u teru s/o v aries at a n y tim e o r at a n y d o s e level. Cross and kisicpatkoiogical observations T h e m a jo r p a th o lo g ic a l o b se rv a tio n s c o n sid ered to b e re la te d to 2,4 ,5 -T tre a tm e n t a re s u m m a riz e d in T a b le 2. F u ll d e ta ils o f th e g ro ss a n d h is to p a th o lo g ical findings for b o th the 2-yr an d the sh o rter stu d y a r e o n file w ith th e a u th o rs . T h e k idneys o f fem ale rats receiving 30 m g 2,4,5-T/ kg/day show ed gross an d m icroscopic evidence o f an in c re a se d in c id e n c e o f m in e ra liz e d d e p o sits in th e r e n a l p a p illa e o r p e lv is (F ig . 1); th e se m in e r a liz e d deposits w ere som etim es accom panied by a localized reaction o f the ad jacen t renal-pelvis epithelium . O th e r ren al effects a ttrib u te d to this dose o f 2,4,5-T in clu d ed a n in c re a se d c o n te n t o f p ig m e n t (F ig. 2) w ith in th e cytoplasm o f the proxim al convoluted tubular e p ith e lial ceils (in fem ales), a n d a d e crea se ;n th e sev e rity o f th e c h r o n i c n e p h r o p a t h y (F ig . 3) t h a t n o r m a l l y o c c u r s s p o n t a n e o u s l y , esp e c ia lly n th e m a le s, ;n th is stra in of rat. T h e special stains a p p lied to sections of kidneys o f the control and high-dose g roups indi cated sim ilar staining reactions for both groups. F i g . J. M i n e r a l i z e d d e p o s i t in t h e r e n a J p a p i l l a a d j a c e n t t o ah e r e n a l p e l v i s o f t h e k i d n e y o f a r a t g i v e n 3 0 m g 2 , 4 , 5 - T / k g / d a y f o r Z y n H a e m a c o x y l i n a n d e o s i n x ICO. I j j i ' 21! ifl 212 213 n- *"*'**r ` arr*i i 'T f - ` S b c M w i r ! > y p ^ d tfo!:Vfr .f f ra| * "" ^ - W d a y , ompri;,, s a sIight d e e r y >p rp , s a a n d p e n p o n a f n r la m m a o o n . H a e m a lo x y lin a n d s o sin x 100. i f s. h' r :1 214 i 1, i- i `i TV.' .V T C h ro n ic ioxiciiy of 2,4,5-T in rats / 215 e x c e p t th a t th e in c re a se d p ig m e n t n o te d in th e ren al in th e in c id e n ce o f b o th p eriarteritis a n d left a tria l tu b u la r cells o f the hig h -d o se g ro u p gave a n increased th ro m b o sis, b u t these o b serv atio n s m ay n o t have p o s itiv e re a c tio n w ith M a llo ry 's stain foe iro n (see b e e n th e result o f 2,4,5-T tre a tm e n t. F ig . 2). A t th e in te rm e d ia te d o se level (10 m g /k g /d a y ), N o o th er gross or histooathological changes c o n th e fem ales s h o w e d a n in c re a se d incid en ce o f m in e ra s id ered to be a ttrib u ta b le to 2,4,5 -T in g estio n w ere lized d e p o sits in th e renal pelvis o f fem ale rats a n d d e te cte d in an y o rg an s. statistically significant increases in the incidence o f m inim al degenerative and inflam m atory changes, in T u m o u r incid en ce cluding focal renal tu b u lar a tro p h y and renal ag g re T h e in cid en ce o f tu m o u rs in m ale a n d fem ale rats g a t i o n s o f l y m p h o i d cells. T h e la tte r in c re a s e s , h o w is d e ta ile d in T a b l e s 3 a n d 4 a n d s u m m a r i z e d in ever, w ere c o n sid e re d to be a reflection o f a n overall T a b le 5. T h e s a m e spectrum ' of tu m o u rs o b serv ed d e c re a s e in th e sev erity o f th e g e ria tric c h ro n ic ren al h isto ric a lly in ra ts o f this strain w ere n o te d in the d isea se n o te d in th is g ro u p . A statistically significant liver, n a sa l tu rb in a te s /h a rd palate, lungs, p a n crea s, d e c re a s e in th e in c id e n c e o f m in e ra liz e d d e p o sits in k id n e y , u rin a ry b la d d e r, testes, o v ary , u teru s, m u s - th e re n a l p elvis o f m a le ra ts given 10 m g 2 ,4 ,5 -T /k g / c u lo -sk eleta l tissue, o ra l cavity, to n g u e , saliv ary d ay m a y o r m ay n o t have been the result o f treat glands, sto m ach , sm all intestine, large intestine, su b m en t. In th e m ales a n d fem ales o n 3 m g /k g /d ay th ere c u ta n eo u s tissues, integum ent, m a m m a ry g lan d , ear w ere n o renal lesions of an y type th at w ere considered canal, b rain , peripheral nerves, pituitary giand, cranial to be related to treatm e n t. cavity, ad ren al glands, eye, lym ph nodes, th y m u s, T h e decrease in the severity o f the sp o n tan e o u s spleen, m esentery, thyroid an d p a rath y ro id glands. c h ro n ic n e p h ro p a th y n o ted in m a le rats receiving S tatistical analyses o f these d a ta sh o w ed th at th e inci 3 0 m g 2 ,4 ,5 -T /k g /d a y (Fig. 3) w as a c c o m p a n ie d b y a dence, o f e a c h type o f tu m o u r in a n y o f th e 2 ,4,5-T - s e c o n d a ry d e crea se in the in c id e n ce of m in e ra liz a tio n tre a te d g ro u p s w as c o m p a ra b le to th at in th e c o n tro l o f the p u lm o n a ry alveoli, m y o card iu m , m y o c a rd ia l g ro u p , w ith the exception o f the incidence o f inter- b lo o d vessels, a n d gastric m u c o sa a n d m uscularis. follicular C-ceil a d en o m a o f the thyroid, w h ich w as A lth o u g h th e k id n ey w as th e p rim ary targe: organ, in creased significantly in females given 3 m g /k g /d ay . th e liver w as also affected slightly by lifetim e tre a t T h is o b serv atio n w as not considered to be related m e n t w ith 30 m g 2,4,5-T /kg/day, the m aies sh o w in g to treatm en t, since there was no dose re sp o n se and a d e crea sed incidence o f liver e n larg em en t a n d a n th e fem ale co n tro l g ro u p had an unusually low inci increased incidence o f focal biliary h y p erp lasia a n d d en ce o f thyroid a d en o m as co m p ared to the h istorical p e r i p o r t a l i n f l a m m a t i o n (F ig . 4),. a n d . th e fe m a le s a n c o n t r o l d a t a (u p to 17%). T h u s , n one, o f th e t u m o u r s in creased in cid en ce o f focal agg reg atio n o f reticu lo listed in T ab les 3 a n d 4 w ere considered to be related en d o th e lial cells ad ja ce n t to d e g en e ra te o r n ecro tic to tre a tm e n t w ith an y o f th e dose levels o f 2,4,5-T. h e p a t o c y t e s a n d a. d e c r e a s e d i n c i d e n c e o f m u l t i p l e fo c i N e i t h e r w e r e t h e t o t a l n u m b e r s o f t u m o u r s p e r g r o u p , o f h e p a to c e llu la r c h an g e (sw ollen hepatocytes). T rie the: av erag e n u m b e rs p e r ra t or the tim es o f o b s e rv a in cid en ce o f focal hep ato cellu lar' c y to p la sm ic v a cu o tio n o f tu m o u rs affected by any o f the levels o f tre a t liza tio n w as d e crea sed in th e livers o f m ales given m e n t, these- d a ta in each case being c o m p a ra b le to 10 o r 3 0 m g / k g / d a y . A l t h o u g h th e i n c i d e n c e o f g r o s s l y t h o s e o f th e c o n t r o l g r o u p s ( T a b l e 5). 'visible liver n o d u le s w as h ig h er in th e c o n tro l g r o u p ' o f fem ales th a n in the tre a tm e n t groups, this c o n tro l incidence w as also so m ew h at higher than th at gener D ISC U SSIO N a l l y e n c o u n t e r e d i n o u r c o n t r o l s . L i v e r s o f r a t s g '. v e n D u rin g the course o f this 2-yr study, th e 2,4,5-T 3 m g 2 ,4 ,5 -T /k g /d a y sh o w e d no c h an g e s a ttrib u ta b le tre a tm e n t h a d n o ad v erse effects o n the o n se t o f m o r to treatm en t. tality, food co n su m p tio n , the d ev elo p m en t of o alp ab le T h e lungs sh o w ed so m e changes that, directly or m asses, h aem atology, routine urine analyses, u rin a ry indirectly, m a y have been the result, o f tre a tm e n t w ith ex cretio n of creatinine o r -A L A , seru m clinical 30 m g 2 ,4 ,5 -T /k g /d ay ; the m ales h a d an increased in ch em istry , o rg an w eights (except the relative w eight cid en ce o f focal p u lm o n a ry interstitial in fla m m a tio n , o f th e k id n e y a t the in te rim kill in rats given 30 m g / focal accu m u latio n s of alveolar m acro p h ag es an d kg/day), tu m o u r incidence and gross an d m icro sco p ic c h o le ste ro l clefts, w hile a few fem ales s h o w e d focai m o r p h o lo g y o f all o rg a n sy stem s o f th e b o d y e x ce p t a c c u m u la tio n s o f secreted m aterial in the alveoli. A t th e k idney, liver an d possibly lung. N ev erth eless, the the in te rm e d ia te a n d lo w er d o se levels th e re w e re no h ig h e st d o se level (30 m g 2 ,4 ,5-T /kg/day) p re d ic ta b ly p u lm o n a r y c h a n g e s a ttrib u ta b le to tre a tm e n t, with, c a u s e d s o m e degree o f toxicity. T h is in c lu d e d a d e the p o ssib le e x ce p tio n o f an in creased in cid en ce o f cre ase in b o d y -w eig h t gain, increases in to ta l u rin e focal interstitial fibrosis in th e lungs o f fem ales on v o lu m e, u rin a ry c o p ro p o rp h y rin a n d u ro p o rp h y rin , the in te rm e d ia te d o se level. As this c h an g e w as n o t a n in crease in relative k id n ey w eights a n d m o r p h o seen at the h ig h e r d o se level, it w as of highly q u e s logical a lte ra tio n s in the k idney, liver a n d po ssib ly tio n ab le toxicological significance. lung. E x a m in a tio n o f the tissues of the c ard io v a sc u la r In the kidney, these changes consisted m ainly of system rev ealed n o lesions th a t c o u ld definitely be m in e ra liz e d deposits in the renal pap illae o r pelvis, a ttrib u te d to the ingestion o f 2,4,5-T. In the g ro u p so m etim es acc o m p a n ie d by a resultant localized re a c given 30 tn g /k g /d .ty , the fem ales sh o w e d a statistically tion of the adjacent ep ithelium of the renal pelvis. sig n ific a n t in c re a se in th e in c id e n ce o f h e a rts w ith a T h e c h r o n ic n e p h ro p a th y n o rm a lly e n c o u n t e r e d in d i l a t e d , f l a c c i d v e n t r i c l e , b u t t h e o p p o s i t e t r e n d a g e d r a t s w a s d e c r e a s e d in. r a t s g i v e n 3 0 m g 2 , 4 , 5 - T / o c c u rre d in the inales. M aie s given eith e r 0 o r 30 m g / k g /d a y . M o rp h o lo g ic a l alte ra tio n s in the iiver a ttr i kg/day sh o w ed an ap p aren t trend to w a r d a decrease b u te d to treatm ent w ith 30 m g /k g /d ay in clu d ed an ' ' ' " T a b i c 3. T u m o u r In c id e n c e In m a le r a n m a in ta in e d o n d ie ts c o n ta in in g 2 ,4 ,5 - T f o r up to 2 yr to O' # N o. of rats found to be affected * in: ,` ' : v. ' M onths 13-18 M o n th 19-tcrmiiiftl kill W hole period of study (lotal)t Tum ours or " >' D o s e ( m g / k g / d a y ) . 0 3 . 10 30 0 3 .1 0 30 0 3 10 30 tum our-like lesions Ho. of rats exam ined. 14 12 22 9 :. 69 37 27 41 86 50 50 50 Hepatocellular carcinom a 1 0 0 0 0 .. 3 l 0 1 3. 1 0 1 K c r a l o a c a n l h o m a o f n a s a l l u r b i n a t c s / h a r d p a l a t e . ': 1 ; .. 0 - 0 ; 0 : - 0 ... 0 0 0 1 0 0 0 Squam ous tell carcinom a of hard palate ,. . 0 0 0. 0 1. 1 I. 0 1J 10 M ixed adenom a of hard palate .' o 0 . : o . 0. l r. .. 0 - 0 . ' 0 , L 0 0 0 Pulm onary adenom a .. v. o t o : o . O' -rO 0 .. : 0 0 10 0 Pancreatic acinar udenomn -j ' 2 ;2 ' 2 ; ' 3 . . 2 3 . 15 ; 7 . " 17 i 26 17 9 20 P a n c r e a t i c a c i n a r a d e n o c a r c i n o m a -1, >> 0 , o . p o 0 ; 2 . . 0 0 : 0 2 .0 0 0 Pancreatic idct-edl adenom a v; o s i ' 2 1 0 ' ' 12 : ". 8 : 6p 7: 13 I ' a n t i e a i i c is le t - c e l l a d e n o c a r c i n o m a 1 ' :... 1 ' r : . 0 ; t o \ ' 0 0- o '1 0 1 U e n a l pelv is U u n d i i o n a l - e d l a d e n o m a p . \ ` [ .d, .v o .. 0 0 ,. 0 :0 1 0 ' 1 . 0 0 Renul tubular adenom a f 'a p i l l a r y a d e n o m a o f r e n a l p e lv is . V: ,>i ''V; !J-- o> . :o 0 o o :. : 0 , 0 \ 0 /, 1 -0 0 r0 1 0 0 11 Renal carcinom a i Transitional-cell carcinom a of urinary bladder I 'v . ; . o , " .` l ' 0 .o . 6 .! 1 i. 0 " 0 ...: 0 1 10 0 0 0 0 0 7 l 1 0 0 0 7 0 0 1 0 0 with metastasis . , 0 0, 0 ; . 0 1 0 0 0 10 0 0 Interstitial-cell a d en o m a of testis .. ; . ,-i ' ! 0 .: 0 , 0, 1: 4 ' t, > . " 2 r2 4 1. 2 3 M esotheliom a o f testis . ; o ' / ' * / ; f-i, 0 , . o Or Or 1 ii 0. o :0 000 Sertoli cell tu m o u r o f testis .v ;' v - u ':.. '1 : 0 ' a 0 .V . 0 0 '0 o. 0- 000 C h o n d rosarcom a of rib o .-. o y ' . o ' . 0 l : . o o 0 000 Oral papillom a ' - 1 ' n- . ' .r O '- o ; . O' 0 ' 1, . 0 O' 0. 1 0 0 0 S q u a m o u s - c e l l c a r c i n o m a o f t o n g u e ' 0. 0 0, 1 1.. l 0 3 l 10 4 Salivary-gland adenom a ' 0. 0 ; ' 0 II o: 1 0 0 0 .0 0 0 Squam ous papilloma of stom ach m ucosa ' 0 0 , 0 3 1 0 0 3 1 0 0 M u c o e y s t a d c n o c a r c i n o m a o r a d e r r o c a r c i n o m a o f small intestine 0. o: 0 0 2 "2 0 0 2 2 0. 0 fib ro sarco m a of small intestine .,' 0 r0 0 0 0 ; o o .1 0 00 l Lym phosarcom a of small intestine *. , 0 0 l 0 1 0 0 0 10 l 0 Undilierenliaicd sarcom a of small intestine ; :0 0 0 1. . 0 0 0 0 0 0 0 I Lym phosarcom a of small or large intestine with ) mciasUbis *. * ' - 0 Papillary adenom a or m ycocysladenom a of small intestine ., ` 0 Subcutaneous fibrom a : v - : ` 1 Subcutaneous adenocarcinom a *. ` . 0 M alignant fibrous histiocytom a with m etastasis i o: M alignant fibrous histiocytom a w ithout m etastasis -o Subcutaneous liposarcoma ' i0 Subcutaneous m yxom a 0 0' 01 0 1 0 .0 :0 0 0 . . ' 0 0 0 0 0. 0 0 ;o 1 o .0 i 0. 1; 1 '0 . 0 : 3 , 7/5 'l . 1. ,,- 0 :o 0i' 0 0 .. 1 0 0. 1 . 5 0 . .o o 0 0 o .: :o 0 10 d 0 3 0 0 0 1 0 1, 1 4 0 0 1 0 0 0 1 8/6 0 1 0 0 1 0 0 2 0 0 0 0 0 l 0 3 1 0 0 1 0 Vf 'l 1r 79 u 8 IS / o p IK m 3 p 50 g i ja y K r 'Z p o o 03 ? p m i 5 -- i ' *!' " ;i : 1 ' 'd" ' '"u cr `Vfr'"-'7 ; y F .'7 \- -.TV.-.-: f.*..:.c* r >>-"r v.V1 -n.` v*-, ji: <; . ^ ', r , ' i . :.V<- * r-' r 4;' :ir ''' _\ s'; 'l ; s'.'. V; , it I C h ro n ic toxicity of 2,4,5-T in rats I'M ,v . . 1< ! {' l, 1l' Ii Subcutaneous carcinom a . i 1 . 0 0 : o '< 0 ! o 0 0 l000 1 M am m ary-gland fibroadcnom a/adcnofibrom a 0 0- 1 0. '' 1 2 10 1 12 l 1 M am m ary-gland adenocarcinom a without metastasis ; 0 0 . ; 0 :o 10 0 0 10 00 M am m ary-gland adenocarcinom a with metastasis : 0 i . 0 0 .! 0 :i ' 1 0 0 0 10 0 0 M amm ary-glaml carcinosarcom a Squam ous papilloma of integument 0 0, 0 0 w jo 0 t 10 "11 0 . 0 0 .1 0 1 10 l0 00 11 C u ta n c u u s adnexal c a rc in o m a w ith m e tastasis . v -o 0 * 1 ; ! o 0' 0 :. 0 0 0 10 f ibrosarcom a adjacent to ear lag " i0 0 !, 1 i0 00 0 0 0 0 10 Cutaneous adnexal cystadenom a : 0 0 t> 0 10 0 0 10 0 0 Zym bal-gland carcinom a of ear canal S ubauricular umlifTcicntiaicd sarco m a w ith l 0 ;i: 0 0 r.3 0 l 14 0 11 metastasis 0 0 o 0 :: 1 :0 0 0 10 00 A strocytom a o r m ixed glial n eoplasm o f b rain G ra n u la r cell m y o b la s to m a o f brain M ediastinal m alignant s c h w a n n o m a o r m alig n an t 0l 0 0 : o 1. 0 0 ?0 I l 0 ' h;i o 1 1 l 0 111 1 1 .1 schw annom a of cranial nerve 0 0 ` 0 0. ; 1 1 0 0 1 1 0 0 Schw annom a of cranial nerve .rC 0 0 0 0 0 0 1 0 0 0 1 0 Pituitary adenom a formation(s) -,i 2 . 1 3 0 ' 2 0 9 10 10 . 22 10 13 10 P itu itary a d en o c a rc in o m a w ith o u t In va sio n o f b ra in - - o 0 jv, 1 . 0 4 2. 1 3 4 2 2 3 P ituitary a d e n o c a rc in o m a will) in v a sio n o f b ra in ., ; 0 0. 0 l: ri 0 0 0 10 0 1 N curo lib io iu a in cranium . 0 ,r 0 '! 0 . 0 i 0 o 0 10 0 0 A denom a of adtdial cortex . .. 0 0 l 0 .i 3 13 1323 A denocarcinom a of adrenal co rtex . 0 0 A drenal p b aeo c b ro rn o cy to m a -- u n ila te ra l o r b ila te ra l 3 ? 0 4 0 , * 1 ! 33 10 0 1 10 14 19 36 10 0 12 IB 20 A drenal p h a e o c h ro m o c y lo in a -- m alig n an t 0 0 ! 0 0 '. 7 4 1 1 8 4 1 1 Adicnal ganglioneurom a ' . . 0. 0 0 0 0 0 0 10 0 0 1 Retro-ocular squam ous-cell curcinonra 0 0. 0 0 0 0 0 10 0 0 1 Im raorbital undilfercnlialed sarcom a , 0000 0 0 10 0 0 10 Mesenteric lipoma 00 00 1 0. 0. 0 10 0 0 Generalised lym phosarcom a . ; ;. 0 1 0 0 10 01 0. 2 10 0 Thym ic lym phosarcom a '0 0 0 0 .0 1 .0 0 0 10 0 Splenic lym phosarcom a T hyroid inter follicular C-ccIl adenom a *. 0 0 .0 0 1 .0 :0 . l 01 0 0 0 0 0 :0 1 6 .3 2 6 ' 6 327 Thyroid imcrfollicular C-ccll adenocarcinom a w ithout m etastasis 0 0 0. 0 1 1 .0 0 1 100 ''there w o e no statistical differences (by F ish er's E xact P ro b ab ility Test, P < 0 0 5 ) b etw een c o n tro l va lu es and th o se for an y treated g ro u p s. W h ere m u ltip le lu m o u r s ilic sam e classification o c c u r re d in in d iv id u a l rals, lire d a u a re listed as lire to ta l no. o f t u m o u r s o f th a t ly p e /n o . o f ra ts b e a rin g (hem . 1 D uring m onths 1-6 there was only one tu m o u r noted (a generalized ly m phosarcom a) in one control rat. T u m o u rs occurring during m onths 7-12 included one pancreatic a c i n a r a d e n o m a ( c o n t r o l ) , o n e p a n c r e a t i c i s l e t - c e l l a d e n o m a ( c o n t r o l ) , o n e s u b c u t a n e o u s f i b r o m a (10 m g / k g / d a y ) a n d o n e a d r e n a l p h a c o c h r o m o c y t o m a , m a l i g n a n t ( c o n t r o l ) . All of these tu m o u rs are included in the total tabulatio n . \ 217 ri j. s <> 'T LJV .r'A ir r ' :1v' I ' M4-) /. T a b ic 4. 7mou/- hic/i/e/icc in fe m a le ra ts m a in ta in ed on d iets co n ta in in g 2 ,4 ,5 -T fo r up to 2 y r I K> DO N o. of ruis found to be affcctcdt in: 't . M onlhs 13-18 M o n th 1 9 -lcrm in al kill W hole period of study (lotal)f 1 Tum our or " , Dose (m g/kg/day)... 0 .3 10 3 0 0 3 . 10 3 0 0 3 10 30 tum our-like lesions N o . o f r a t s e x a m i n e d . . . 17 11 ' 11 9 67 37 36 3 9 86 50 50 50 R . J. K o c i h a , D . G . K s y e s , R . W . L i s o w h , R . P . K a l n i n s , D . D . D i t t h n r h r , C . H. W a d s e t a i Il H epatocellular hyperplastic nodulc(s) . 1 '0 1 , 0 3 2 2 14 2 j Ilcpaiocclluluar carcinoma(s) . 1 0 .' 0 ' 0 1 0 0 02 0 a P ancreatic islet-cell a d e n o m a " ' ' 1 0 0 0 7 1 3 28 1 P a n c r e a t i c is le t- c el l a d e n o c a r c i n o m a ; , . .. . 0 0 0 .0 0 00 10 0 Pancreatic acinar adenom a 0 . o 0 0 :0 0 1 00 0 1 Renal haem angiom a 0 0 \ .0 .0 0 o 0, 1 0 0 !1 . G r a n u l o s a l - c c l l n e o p l a s m o f o v a r y 0 0 .0 0 4 1 . ' 1 0 4 1 1 Strom al polyp of uterus ' ' '1:" * ' . , 1 - o ' 2/1 1 13 4 8 / 7 11/10 14 4 H M alignant schw annom a-- uterus (metastasis), or vagina 0 ' 0 . ;0 :0 ' 1 0 0. 1 1 0 |.j A d c i i o c a i c i n o m u o f u l e r u s 0 00 0 : 4' 0 1 04 0 ;s M y x o s a r c o m a o f u l e r u s 0 0 c, ./ 1 0 0 :0 : 0t 0 0 0 U < Schw annom a of ulerus . ' |V 0 1 0 ;0 0 0: 1 00 1 ;) Leiom yosarcom a of uterus with metastasis 0. 0 0 00 0 1 00 0 (| Iibcom a of uicrus/vuginu , - 10 0 ,. 0 0 l, t' 0: 0 1 1 fi P a ra v a icbral rh a b d o m y o sarco m a w ith m etastasis 0 .0 0 .1 0' 0 -0 .0 0 0 ? l ' i b r o m a o f g i n g i v a , r, 0 00 0 :o 0 0 10 0 *1 Squamous-cell carcinom a of tongue " .0 .0 0 0 0 .. 0 0 10 0 C . A d e n o m a o f t o n g u e *'si o 0 It- o - i o i r -. 0 ' 0 ; - 0 1 0 vj *, , Squam ous polyp or papillom a of stom ach o 0 ..V 0 ,0 - 0 : 3 .'i l 0 .0 3 1 : . M tieoeystadcnoearcinonia or adenocarcinom a of small j . : : , . if i n t e s t i n e Lym phosarcom a of small mestine 0 ` 0 -" 0 0 0 0 0 0 o 0a 1 .0 r' 1 0 0, i 0 1 0 i l eiom yom a or leiom yosarcom a o f small Intestine 0 ' O' 0 0i 0 1- 0 1 0 Subcutaneous lipoma 0 00 0 .0 1 0 00 1 Subcutaneous fibroma o . 0 0 . 1 ' o : 0 0 -: 0 0 0 l Subcutaneous fibrosarcoma -i . .. ' .0 00 o 0- 0 0 10 0 i S u b c u ta n e o u s c arcin o sarco m a . !- ., . 1: - . ... * S u b c u t a n e o u s m y x o s a r c o m a - . ' : M arnm ary-gland fibroadenorna/adcnofibrom a 0 0 25/13 0 0 9/7 0 0 12/9 0 0 . 11/7 0. 0 113/52 1 1 3S/24 0 Q" 49/28 0. 0 43/25 0 0 138/65 1. 1 47/31 M am m ary-gland adenom a 3/2 1 0 M am m ary-gland adenocarcinom a w ithout metastasis ' 3/2. - ,0 -0 M am m ary-gland adenocarcinom a with metastasis 0 10 11 11 11 12/8 -4 1 6/5 4 1 4/3 1 0 8/7 15/10 7 /6 5 7/6 4 21 2 ; M am m ary-gland cystadenom a M am m ary-gland fibroma ,'-v , 0 1 1 mO 0 s0 !0 !o . 2 . 1 3 1. 6 /4 3 32 3/2 4 2 1 . M am m ary-gland cysiribroadcnoina/cysladcnofibrom a 0 0 0 10 13/11 1 0 /7 9 /7 5 1 3 /1 1 1 0 /7 M am m ary-gland unclassified m alignant neoplasm , . j . 1 t (carcinoma) t0 0 !o 0 0 0 01 0 1 3 0 3 0 1 0 1 11/9 0 l 1 1 1 0 0 0 0 0 0 1 0 2 1 0 1 0 12/11 1 0 0 0 0 0 1 1 1 0 0 0 0 1 0 0 0 0 0 62/38 4/3 1 0 6 /4 3 9 /7 0 1. 0 0 1 I 0 0 55/33 9/8 6 4 3 3 /2 5 . 00 j ^ J;: .'.t/W* * :V . U--'i 'C * ^ 1(- ' rH.'VWr .*!. I ... ,i ... *. -i.'ji v**< ' I M am m ary-gland cystadcnocarcinom a 0 S ipiam ous p apillom a or k c ra io a c a n lh o m a of- : inic^uiaciit ? ? ,> 0 Zym bal-gland carcinoma of car canal .` i ' 0 A denom a of Zym bal gland f- 0 Pituitary adenom a formation(s) s Pituitary adenocarcinom a w ithout extension or invasion of brain 2 Pituitary adenocarcinom a with extension of brain . - 1 Pituitary adenom a arising from pars interm edia 0 N eurofibrosarcom a in cran iu m . Vj- 0 A denom a of adrenal cortex i; 2 Adrenal phneoehrom ocytom a, unilateral 1 Adrenal phacochrom ocytom a, m alignant ` 0 .Mesenteric lipom a ' ; 0 Abdoniinal haeinaitgiosarcom a v ' 0 Granulocytic leukaemia * ;0 Generalized m ouocylic leukaem ia .: '0 Generalized lym phosarcom a .. ** Iniia-abdoiniiul lym phosarcom a or carcinosarcom a'' "1 with m nastasis 0 Lym phangiom a of mesenteric lym ph node : 0 Thym ic lym phosarcom a 0 Splenic myeloid leukaem ia .0 Thyroid inicrfolliculur C-ccll adenom a '"/ v , < 0 T hyroid inter follicular C-ccll ad en ocarcinom a b e without metastasis Vo T hyroid intcrfollicular C-ccll ndcnocarcinom a with metabUi'iis 0 Parathyroid adenom a *' ; ; o I 0 .0 -0 0 0 3 30 i0 i0 v 1 Id 0 I, o .0 ,0 :0 0 0 ' 0 < 1 o 0 ;o 0 :0 0' i o 01 0 ' 10 - 0 : 0 . 'O ' ' 1 V, ` : ; . 'j |ij 0 I .0 .0 :o 00 0 o .0 1 0 ; :) ! 00 0 ; 0 j. * 00 . O' . i :- 2 0. 1 0 0 3; $ 34 t Si O' ! ? < 0 :a 4 0 !!!' 0 2 ' .1' 4 0 ! 5 0 i1' 0 11 '0 i: 0 0 0 ' . 0. 3! 0 :i in 1 \ 0 v0 0 0 1 '! 1 i 1 0 i0 .1 3 . ,0 0 0 ;3 1. ;1 .1 i 0 0/ l 0 0 00 0 0 0 0 1 0 . 0 20 . .. O 16 635 222 0 0, 0 0 1- 0 2 4: 1 32 2 000 000 000 0 t0 1 0 ; 0 0 0l 0 0 0 0` ; 00 10 7 i 2 0 0 1 0 5 2 1 0. 42 0 0 1 23 6 5 1 .0 6. 6 1 l 0 0 o 2 V- 6 2 0 0 2 3 0 0 0 0 1 .1 10 10 00 01 3 10` 2 0 .1 3 0 101 0 0 1 1 ____i.___:___ __ /2 0 0 0 0 0 0 20 3 2 0 1 4 2 0 0 1 1 0 l 0 0 0 0 2 0 1 0' l 0 0 0 19 5 2 0 0 3 2 0 0 0 0 0 1 0 0 1 0 6 1 0 1 t W here m ultiple turnouts of llic sam e classification occurred in individual rats, the data arc listed as the total no. o f tu m o u rs o f that lype/no. o f rats bearing them. lO u rin g m onths 1-6, there was only one tu m o u r n o ted (a generalized lypiphosarcom a) in one rat given 3 m g/kg/day. T u m o u rs occurring during m onths 7 -12 included one s tio m n l p o ly p o f the u te ru s (10 m g /k g /d a y ). o n e m a m m a r y - g la n d lib r o a d c n o m a /a d c n o f ib r o m a in b o th the 10- a n d 3 0 -tn g /k g /d ay g ro u p s , o n e m a m m a r y - g la n d a d e n o c a rc in o m a with m etastasis (3 0 m g /k g /d a y ) a n d o n e m a m m a ry -g la n d unclassified m a lig n a n t n e o p la sm (carcin o m a) (control). All o f these tu m o u r s are in clu d ed in the total tabulation. T h e v a l u e m a r k e d w i t h a n a s t e r i s k d if f e r s s i g n i f i c a n t l y ( b y F i s h e r 's H x a c t P r o b a b i l i t y T e s t , < 0 0 5 ) from the control value. C h ro n ic toxicity of 2.4.5-T in rats ,1 } \' i\ -V' t) r'M w \T \ ,. ' V. t v\ l` ; ii- ' I '" r f r ' i . i j.ft . *) r* T iT j- v i ' ------ -- ^ .z r f f c y vT,ciT* - w- w . w.-.x . . . ------- ............................. -r~rr~ -- -- -- --------- -- _- -- 1 I ! " L -1 -i (i . : d ti i * ;vj Hi :i-y! . \ i: 'i5 'i VL- 'i -2 ra 3 -:V1 V ; t. :.-.i I ; -/ Ct"r *2 " y'fk 220 R . J. i C o a o A , D.. G . K e yes , R. W . L iso w f , R . ? . K a l n i n s , D . D. D i t t c N b e r , C . E. W a D al. E F- ' f c T a b i c 5. S u m m a r y o f dc;;a o n c ~ 'r ' Dose of 24,5-T (m g/kg/day) N o. of rats/group n o . 0; tum ours/ group 0 86 172 3 50 89 10 50 71 30 50 97 0 86 " 308 3 50 140 10 50 . . 147 30 50 151 ' t r; 4. n n in tc iin e d a n d ia ls c o n ta in in g 2 N . 5 - T f o r u p to 2 y r :o. o f tu m o u r-b e arin g rats/rats a u lo p sied u '- ring m onths r a t r - V ; 7 - t 2 13--l S 19-24 M aiIts 2-0 1/1 1/2 3 /1 4 60/63 1-8 0/0 0/1 9/12 26/23 1-4 0/0 l /l 15/22 20/23 1-9 0/0 0/0 6/9 24/25 Fem ales 3-6 0/0 1/2 16/17 30/31 2-8 l/l o/l 10/11 18/18 2-9 0/1 1/2 11/11 25/25 3-0 0/0 2/2 9/9 18/18 Al te rm in al kill 6/6 9/9 4/4 14/16 36/36 19/19 11/11 21/21 T o tal4 76/36(33/.) 44/50(33% ) 40/50(30% ) 44/50(33% ) 83/86(97% ) 48/50(96% ) 48/50 (96%) 50/50(100% ) r ' Ip .T ' 'l'- - i t - :l I'' .} .L i1 t. V .| '1 . - J I. 1 [' .1 . V alues for treated groups show ed no statistically significant differences from the control values w hen analysed by F i s h e r 's E x a c t P r o b a b i l i t y T e s t ( P < 0 0 5 ) . . in creased incidence o f p erip o rtal inflam m ation, focal b ilia ry h y p e rp la sia a r.d in fla m m a to ry cell ag g re g a tio n s adjacent to d eg en erate hepatocvtes; these find ings w ere acc o m p a n ie d b y a decreased incidence of m u ltip le foci o f sw ollen h ep atccy tes and focal c y to p la sm ic v acuolization. L u n g s of rats given 30 m g 2,4 ,5 -T /k g /d ay s h o w e d indications o f focal p u lm o n a ry in flam m a tio n , including focal acc u m u la tio n s o f alveo la r m a c ro p h a g e s a n d c h o lestero l clefts. O n ly m in im a l toxicity w as asso ciated w ith the in te rm e d ia te dose level (10 m g/kg/day), prim arily an in c r e a s e d in c id e n c e o f m in e ra liz e d d e p o sits in the. r e n a l p e lv is - A n in c re a se in th e u rin a ry e x c re tio n o f c o p ro p o r p h y r in w a s n o te d in m ales o n this d o se level o n ly d u rin g the early p h ase o f the study. At th e low er d o s e level (3 m g /k g /d a y ) th e re w ere n o effects a ttr ib u tab le to 2,4,5-7 treatm en t th ro u g h o u t the 2-yr study. T h e absence o f an y tu m o u rs considered to b e the re su lt o f tre a tm e n t w ith 2,4,5 -T at a n y o f th e th ree d o s e levels c o n c u rs w ith th e results o f m o st o f the o n c o g e n ic stu d ie s o n 2,4,5 -T prev io u sly re p o rte d , [rm es e t al. (1969). fo u n d n o o n c o g e n ic re s p o n s e in e ith e r o f tw o strain s o f m ice given the m a x im u m to le ra te d d o se o f 21-5 m g 2 ,4,5-T /kg/day, a n d th e B ionetics R esearch L a b o ra to ry (1963) noted no in crease in tu m o u rs in m ice o f tw o strains given a single . ' sc injection o f 215 m g 2,4,5-T/kg. T h e only published o n c o g e n ic s tu d y re p o rtin g a n in crease in tu m o u rs a lle g e d ly d u e to tr e a tm e n t w ith 2 ,4,5-T is o n e o f th e f o u r s tu d ie s c o n d u c t e d b y N f u r a n y i- K o v a c s e: al. ( 1 9 7 6 Sc. 1 9 7 7 ) . I n t h e s e s t u d i e s , m i c e o f t h e C 3 H f stra in given a single d o se level o f 80 p p m (ap p ro x im a tc ly 12 m g /k g /d a y ) o f 2,4,5-T h a d a statistically sig n i f i c a n t i n c r e a s e o f t h e s o - c a i ' e d `n o n - i n c i d e n t a l ' t y p e s o f tu m o u rs, b u t m ice o f the X V IIG strain given iden tical 2,4,5-T tre a tm e n t sh o w e d no increase in tu m o u r i n c i d e n c e . W h e n M u r a n y i - r C o v a c s zc a l. (1 9 7 7 ) g a v e n e o n atal m ice, of these sam e tw o strains a series of s c i n j c c : :i o n : i o f 2, 4 , 5 - ' i t h e r e v / 2 s n o r e s u i t a n t i n c r c r . s e I n t u r n o u r i fiCVUw r . c e i n e i t h e r s t r a i n . C is ir.t s r e s t i n i ; t o n o t e t h a t i n t h e o n e s t u d y m w h i c h N I u r a r*v *.- Dvncs e: cl. re p o r w d a n on c o g en ic resp ^nse to 2 , 4 , 5 - T , n o s p e d C.Wi. ,t a r g e t t i s s u e s h o w e d ;. h e a l l e g e d r e s p o n s e . T h i s is c o n t r a r y to th e v a s t m a j o r i t y o f cases, in w h ich a p o sitiv e o n c o g en ic resp o n se typically affects specific ta rg e t tissue(s), fro m w h ic h th e re su lt ing increase in tu m o u rs originates. O verall, therefore, the results of this study c o n cu r w ith th o se o f all b u t o n e o f the p rev io u sly re p o rte d o n c o g e n ic s tu d ie s o n 2,4,5-T . R a ts g iv e n 10 o r 30 m g 2,4,5-T /kg/day for 2 y r sh o w ed som e d o se-related to x icity b u t no o n co g en ic response, w hile in [hose g iven 3 m g /k g /d a y th ere w ere no adverse effects related to treatm ent. REFEREN CES B ionetics R esearch L a b o ra to rie s (1963). E v a lu a tio n of C a r cinogenic, T eratogenic and M utagenic Activities of Selected Pesticides and Industrial Chem icals. R eport N o. N C I-D C C P -C G -1 9 7 3 -M , issued A ugust 1968; Bethesda, M D. Drill. V. A. & H ira tz k a , T. (1953). T oxicity o f 2,4-dichIoro- phenoxyacetic acid and 2,4,5-trichlorophenoxyaceiic acid. A re p o rt o n their acu te a n d c h ro n ic toxicity in dogs. A rchs ind. rly g . occup. M ed . 7, 61. G e a r in g . P; J , K r a m e r , C. G , Schweiz,- B7 A / T f c s e , J. Q . Sc. R o w e , V. K . (1 9 7 3 ) . T h e f a t e o f 2 , 4 , 5 - t r i c h l c r o p h e n - oxyacetic acid (2.4,5-T) follow ing o ral a d m in is tra tio n to m a n . T o xic, appl. P harm ac. 26, 1:2. H i g h m a n , B , G a i n e s , T . B. Sc. S c h u m a c h e r , K . 0 7 ( 1 9 7 6 ) . Sequential histopathologic, hem atologic, and blood ch em istry c h an g e s in d u ced in mice by a tech n ical an d a purified' preparation of 2,4,5-trichlorophenoxyacetic acid. J . T o xica !, e m ir. H ltli 1, 469. Innes, J. R. M . UUand, B. M , Vaierio, M . G ,, Petrucclli, L , F ish b ein , L,, H a rt, E. R,, P allo tta, A. J , Sates, R. R_ F a lk . H . i _ G a r t, J. J . K lein, M , M itc h e ll, I. & Feters, J. (1969). B ioassay o f pesticides a n d in d u strial chem icals for tu m o rig e n ic ity in m ic e : A p re lim in a ry note. J . nnln. C ancer [nst. 42, 1101. L una, L. G. (195$). M anual o f H istologic S ta in in g -M eth o d s o f the A rm ed Forces In stitu te o f P athology. 3rd Ed. M cG raw -H ill Book C om pany, Inc, New York. M u ran v i-K o v acs, [, Rudali, G. A Im bert, J. (976). Bio- assay of 2,4,5-irichloropiienoxyacetic acid for carcin o genicity in mice. Br. J. C areer 33, 26. M u r a n y i - K o v a c s , i , R u d a l i , G . -ic I m b e r t , J. (1 9 7 7 ) . Stud y- on the c arcin o g en icity of 2,4,5-T in mice. A bstract, F o u r t h Nice t i n g o f t h e Eui r e p c a n 1 A SSOCtl'. i o n f o r C w e t : R e s e a r c h , V D iv e r s i t y o f Ly o n . S.-ml o c r L97 7. '. ocr, W. N.. R o se, j. Q . Ler.g. ' & Gehrinz, p. j . (1973). Ttte fate of 2,4 ,5-trich :or oprisn o x y acetic acid (2,4.5-T) foil o w in g o ra l acm ir.ist rat io n to ra ts a n d dogs. T oxic, i ph.arm .ac. 26, 339. 4 ... I . . _: .v. ' '. - u 7../T -7 ft t. ' ' : 1 . v .c t- >` { t- :.: 7 I, J*: r I V ", ..2- il i Chronic toxicity of 2.4,5-T in rats 22! Rowe. V. K. & Hymas. T. A. (1954). Summary of toxico logical information on 2,4-D and 2.4,5-T type herbicides ar.d an evaluation of the hazards to livestock associated with their use. Am. J. ref. Res. 15, 622. Siegel. S. (1956). Non-parametric Statistics for the Behav ioral Sciences. McGraw-Hill Book Company, Inc, New fork. Smith, F. A , Scnwetz. B. A , Murray. F. J , Crawford, A. A, John, J. A, Kociba. R. J. Sc Humiston, C. G. (1993). Three-generation reproductive study in rats ingesting 2,4,5-trichlorophenoxyacctic acid in the diet. Abstract, 17th Annual Meeting of Society of Toxicology, March 1973, Toxic, appl. Pharmac. 45, 293. Steel, R. G. Si Torne, H. H. (1960). Principles anti Pro cedures o f Statistics. McGraw-Hill Book Company, Inc, New York. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY BEFORE THE ADMINISTRATOR In re: FIFRA Docket Nos. 415, et al. The Dow Chemical Company, et al ) */ DIRECT TESTIMONY OF DR. ROY E. ALBERT Scheduled Date of Testimony: September 23, 1980 Dorothy E. Patton Kevin M. Lee Patricia A. Roberts Richard P. Bozof Timothy D. Backstrom Andrew G. Gordon Karl 0. Bayer John W. O'Donnell Counsel for Respondent U.S. Environmental Protection Agency 401 M Street, S.W. Washington, D.C. 20460 ^_/ EPA Exhibit No. 32A. Dr. Albert's direct testimony is a revision of his statement for his appearance in this proceeding on March 18, 1980. Dr. Albert's original statement was assigned Exhibit 32, and this revised statement is designated as Exhibit 32A. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY BEFORE THE ADMINISTRATOR In re: ) ) ) FIFRA Docket No. 415, et al. The Dow Chemical Co., et al.) ) __________________________________________________ ) */ DIRECT TESTIMONY OF DR. ROY E. ALBERT (UPDATE) INTRODUCTION My name is Roy Albert. I am Deputy Director of the Institute of Environmental Medicine, New York University Medical Center, and Chairman of EPA's Carcinogen Assessment Group (CAG). CAG is responsible for reviewing data relating to the possible carcino genicity of compounds which EPA has authority to regulate and for assessing the risk of cancer these compounds pose to the human population. During the past three years, as Chairman of CAG, I have participated in the evaluation of approximately 100 compounds, including 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenoxypropionic acid (silvex), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an unavoidable contaminant of commercial 2,4,5-T and silvex, for possible carcinogenic potential. */ EPA Exhibit No.32A. - 2- CAG has evaluated several laboratory animal studies of the */ carcinogenicity of TCDD, 2,4,5-T and silvex and several epidemiological studies of the association between the risk of cancer among humans and exposure to 2,4,5-T, other chemically related chemicals, and TCDD. CAG has concluded that this body of information provides substantial evidence that TCDD, and there fore commercial 2,4,5-T and commercial silvex, pose a carcinogenic risk to exposed humans. CAG has also concluded that this body of information provides highly suggestive evidence that the pure 2,4,5-T component of commercial 2,4,5-T may pose a carcinogenic risk to exposed humans. In addition, based on the laboratory animal studies, CAG has concluded that TCDD appears to be one of the most potent known carcinogens. In this testimony, I first describe some of the policies and principles which EPA (and CAG) follow in determining whether chemical substances pose human cancer risks. I then provide an analysis of the carcinogenicity of TCDD, 2,4,5-T, and silvex based on CAG's review of the relevant laboratory animal studies and epidemiological studies. Finally, I summarize CAG's quantitative assessment of part of the cancer risk posed by some routes of exposure to 2,4,5-T, silvex, and TCDD. I. EPA's Cancer Policy The Agency's assessment of the carcinogenic risk associated with the uses of 2,4,5-T and silvex is founded on principles */ CAG1s updated "Risk Assessment on 2,4,5-T, Silvex, and TCDD," Tthe CAG Report) is appended as Exhibit 33a. r-S t) QA* - 3- developed and followed by EPA as well as other federal regulatory agencies and research institutions charged with measuring the risks associated with toxic chemicals in the environment. The Agency analyzes carcinogenic risks in accordance with its "Interim Guideline for Carcinogen Risk Assessment" (Interim Guideline). [Exhibits 34. and 35]. The policies described in the Interim Guideline are consistent with a recent report of the Interagency Regulatory Liaison Group (IRLG) entitled "Scientific Basis for Identification of Potential Carcinogens and Estimation of Risks" (IRLG Report). [Exhibit 36], The IRLG report reflects the consensus of scientists and policymakers of four major federal regulatory agencies, including EPA, which regulate carcinogenic substances as well as senior scientists V from two major federal agencies involved in cancer research. According to the Agency's cancer principles, the best evidence that a chemical substance is a human carcinogen comes from a combination of epidemiological studies and experimental animal data. Substantial evidence is provided ^_/ The four agencies comprising the IRLG at the time the IRLG Report was written were the Consumer Product Safety Commission (CPSC), Environmental Protection Agency (EPA), Food and Drug Administration (FDA), and Occupational Safety and Health Administration (OSHA). The IRLG Report was written by scientists and policymakers from all these Agencies with the assistance of senior scientists from the National Cancer Institute (NCI) and the National Institute of Environmental Health Sciences (NIEHS). Scientists from the Food Safety and Quality Service of the Department of Agriculture, which subse quently joined the IRLG, reviewed and concurred in the report after it was completed. f - 4- by animal tests which demonstrate that the substance induces, in one or more animal species, malignant tumors or benign tumors, which are generally recognized as precursors of malignant tumors. Suggestive evidence includes positive short term in vitro tests such as mutagenicity and cell transformation tests. While epidemiological cancer studies (i.e., studies of cancer incidences in human populations) can provide the most direct evidence that a chemical poses a cancer risk to humans, such evidence is frequently not available because studies have not been done. Furthermore, human populations do not live in controlled environments and, therefore, are subject to many factors which may affect cancer incidence. Thus, the force of epidemiological evidence increases by the repetition of similar findings under different circumstances, all of which involve exposure to the agent being investigated. Another limitation of epidemiological cancer studies lies in the fact that development of cancer requires a long period, often 5-40 years after initial exposure to carcinogens. This latency period makes it nearly impossible to promptly detect by epidemiologic studies the carcinogenicity of substances newly introduced into the environment . Because it is often difficult to determine the carcino genicity of a substance in humans by epidemiological means alone, cancer tests on laboratory animals are heavily relied t - 5- upon by regulatory agencies and the scientific community to make this determination. A close qualitative similarity has been demonstrated in the nature of the response of laboratory animals and humans to carcinogenic substances. Currently available evidence indicates that almost all agents known to cause cancer in humans also induce oncogenic effects in laboratory animals [Exhibit 36]. Therefore, the induction of tumors in laboratory animals by a substance is regarded as substantial evidence that the substance is a human carcinogen. The Agency has adopted the no-threshold concept for cancer induction by complete carcinogens (i.e., substances which themselves alone cause cancer). According to this con cept, any exposure to a complete carcinogen, however small, will confer some risk of cancer on the exposed population. Thus, if epidemiolgic studies or laboratory animal studies indicate that a chemical substance is a likely human carcinogen, the Agency considers that substance to pose a carcinogenic risk to exposed humans at any level of exposure, no matter how small. The no-threshold concept is recommended in the IRLG Report as an appropriate regulatory policy. II. Carcinogenicity of TCDD, 2,4,5-T, and silvex in Laboratory Animals Several cancer bioassay studies have been conducted on the effects of TCDD using rats and mice. These studies provide substantial evidence that TCDD administered alone causes cancer in rats and mice at exceedingly low dose levels. - 6- Therefore, in view of the no-threshold concept for cancer induc tion, these studies provide substantial evidence that TCDD is a complete carcinogen and is likely to pose a risk of cancer to humans at any level of human exposure. There is evidence that TCDD is both a promoting agent and a cocarcinogen. A cancer promoter is an agent which, when */ applied after a complete carcinogen or a cancer initiator, increases the incidence of tumors and shortens the latency period for tumor development. This enhancement can occur even if the exposure to the cancer promoter occurs long after the exposure to the complete carcinogen or initiator. A chemical is a cocar cinogen if the incidence of tumors induced by that compound and another carcinogen to which an animal is simultaneously exposed is greater than what would be expected if the effects of the two compounds were only additive. TCDD has been shown to be a power ful promoting agent in the rat liver and a cocarcinogen in the mouse skin. It is possible that the promoting and cocarcinogenic effects may extend to other organs in other species including man. ^_! A cancer initiator is an agent which causes irreversible transformation of a cell into a latent tumor cell (i.e., a cell capable of developing into a tumor). ^ < 3 ''V I Since commercial 2,4,5-T and silvex are unavoidably con taminated with low levels of TCDD, the laboratory animal studies on TCDD provide substantial evidence that commercial 2,4,5-T and silvex are likely to pose a risk of cancer to humans at any level of exposure. One study in which rats were administered specially purified 2.4.5- T containing no detectable TCDD provides highly suggestive evidence that pure 2,4,5-T, independent of any TCDD contamination, is carcinogenic in rats. Thus, commercial 2,4,5-T may pose a risk of cancer to exposed humans, which is in addition to the risk of cancer due to its TCDD contaminant. Several other chronic laboratory animal studies have been conducted on 2,4,5-T and a few have been conducted on silvex. Because of deficiencies in design or conduct as well as other factors, none of these studies provides significant evidence of either the oncogenicity or non-oncogenicity of commercial 2,4,5-T and silvex. In summary, data from the animal cancer studies on TCDD, 2.4.5- T, and silvex, considered as a whole, provide substantial evidence that TCDD, commercial 2,4,5-T, and commercial silvex pose a carcinogenic risk to exposed humans at any level of exposure. A. Laboratory Animal Studies on TCDD 1. Rat Studies CAG reviewed two chronic toxicity studies involving - 8- the administration of TCDD to rats. In these studies, exceedingly low dose levels of TCDD induced a carcinogenic effect. 1/ Kociba et al_. (scientists at Dow Chemical Company) adminis tered TCDD mixed with food to groups of male and female Sprague- Dawley rats (Spartan substrain) at TCDD doses of 0.1 ug, 0.01 ** j ug, and 0.001 ug/kg body weight per day) per kilogram of rat body weight per day for up to two years. At the highest dose level (0.1 ug/kg/day), TCDD induced statistically significant increases of several types of cancers in both male and female rats. In high dose female rats, statistically significant increases in liver (hepatocellular) carcinomas, carcinomas of the lung, and carcinomas of the hard palate and/or nasal turbinates were observed. The increases in the liver tumors and lung carcinomas were very highly statistically signi ficant (p < .001 for both types of tumors). In high dose male rats, statistically significant increases in carcinomas of the hard palate and/or nasal turbinates and in carcinomas of the tongue were observed. In addition, TCDD induced a very highly significant increase (p < .001) in the combined incidence of The published report of this study, a letter with an attachment providing supplemental data, and some additional unpublished data are appended as Exhibits 13, 14, and 15, respectively. **/ Doses of 0.1 ug, 0.01 ug, and 0.001 ug/kg body weight per day were administered by feeding the rats diets containing 2.2 ppb, 0.22 ppb, and 0.022 ppb TCDD (1 ppb = 1 part per billion or 10"^ gram TCDD/gram of food). fl 1/ liver carcinomas and neoplastic nodules of the liver in the middle dose group (0.01 ug/kg/day). Thus, the results of this study provide very strong evidence that TCDD is a carcinogen in ** j rats at exceedingly low doses. The National Cancer Institute (NCI) recently released a camera ready report of its cancer bioassay of TCDD (Exhibit 577). In this bioassay, male and female Osborne-Mendel rats were administered TCDD by oral intubation at doses of 0, 0.01, 0.05, and 0.5 ug/kg/week (equivalent to 0, 0.001, 0.007, and 0.07 ug/kg/day, respectively). The results from this study indicate that TCDD induced statistically significant increases in tumors of the thyroid gland (follicular cell adenomas or carcinomas) at the high (p<.001), middle (P<.01), and low (p=.042) doses in the male rats. The results also indicate that TCDD induced in female rats a statistically significant increase of each of the following tumors in the high dose group: carcinomas and neoplastic nodules of the liver (p=0.001), subcutaneous fibrosarcomas (a 37 It is appropriate to combine the incidences of all neoplasms arising from the same type of cell for purposes of evaluating the carcinogenicity of a test compound in a laboratory animal study. It is especially appropriate to combine hepatocellular carcinomas and neoplastic nodules of the liver because neoplastic nodules have clearly been shown to be precursors of hepatocellular carcinomas. Squire and Levitt (1975) [Exhibit 16]; Hirota and Williams (1979) [Exhibit 18]; Farber (1976) [Exhibit 37]. **/ At CAG's request, Dr. Robert A. Squire, a pathologist at Johns Hopkins School of Medicine, evaluated all liver tissue sections from all dose groups and all other tissue sections from the control and high dose groups. His diagnoses are summarized in Exhibit 760 and in Appendix B to Exhibit 33A. The discussion in the text regarding the results of the Kociba TCDD study are correct whether we take into account Dr. Kociba's or Dr. Squire's diagnoses. i -10- cancer of the fibrous tissue beneath the skin) (p=0.023), and adrenal cortical adenomas (p=0.039). These results confirm the findings of the Kociba study that TCDD induces observable carcino genic effects in rats at exceedingly low doses. 2. Mouse Studies on TCDD CAG has reviewed three studies involving the adminis tration of TCDD to mice. In two companion studies to the NCI cancer bioassay on rats described above, mice were administered low doses of TCDD by oral intubation and by skin application. In the oral bioassay (Exhibit 577) male B6C3F1 mice were administered TCDD by oral intubation at doses of 0.001, 0.05, and 0.5 ug/kg/week (equivalent to 0, 0.001, 0.007, and 0.07 ug/kg/ day, respectively) and to femal e mice at doses of 0,0.04, 0.2, and 2.0 ug/kg/week (equivalent to 0,0.006, 0.03, and 0.3 ug/kg/day) for up to two years. The results indicate that among female mice TCDD induced statistically significant increases in the incidence of hepatocellular carcinomas (p=0.014), thyroid tumors (follicular cell adenomas), fibrosarcomas and histiocytic lymphomas. The results indicate that among the male mice TCDD induced a statistically significant increase in the incidence of hepatocellular carcinomas (p = 0.002) in the high dose group. In the skin application bioassay (Exhibit 578) male and female Swiss Webster mice were administered TCDD in acetone by application to the skin three times a week at doses of 0.001 and 0.005 ug per male and female mouse, respectively (equivalent to approximately 0.01 and 0.07 ug/kg/day, respectively) for up 1 -11to two years. A statistically significant increase in fibrosarcomas of the skin was found among treated female mice compared to controls. In summary, both the oral and skinpainting NCI studies on mice provide substantial evidence that TCDD is carcinogenic in mice. In the third study, conducted by Toth et al., [Exhibit 20] TCDD was administered to outbred Swiss male mice by gavage (feeding through a tube which extends from the mouth to the stomach) in a sunflower oil vehicle at doses of 7.0 ug, 0.7 ug, and 0.007 ug TCDD/kg body weight per week over a period of a year. A matched control group of mice, which were identical to the treated animals except for the administration of TCDD were administered the sunflower oil vehicle. In conjunction with a companion study on another compound, three other control groups were also maintained. A statistically significant increase in the incidence of liver tumors was observed in the middle dose group (0.7 ug TCDD/ kg/week) when compared to the matched sunflower oil control group. The incidence of liver tumors in the middle dose group was also significantly greater than the incidence of these tumors in the pooled controls (all four control groups combined). The high dose group (7.0 ug TCDD/kg/week) had an increased incidence of liver tumors compared to the matched control group, but this increase was not statistically significant. Discrepancies in survival between the treated groups and the control groups may have tended to increase the tumor -12- incidence in the middle dose group in comparison to the controls and decreased the tumor incidence in the high dose group in comparison to the controls. Therefore, CAG has concluded that the results of this study provide suggestive evidence of an oncogenic effect. B. Promotion and Cocarcinogenicity Studies On TCDD In Laboratory Animals A promotion study by Pitot et al. (Exhibit 574) indicates that TCDD is a very potent liver cancer promoter. In this study, some groups of rats received a single dose of diethylnitrosamine (DEN), a known complete carcinogen, for purposes of cancer initiation. All groups of rats had partial hepatectomies (removal of part of the liver) to cause qell proliferation. To test the promoting effects of TCDD, beginning seven days after treatment with DEN, two groups of DEN-treated rats received biweekly subcutaneous injection of TCDD at doses of 0.14 and 1.4 ug/kg body weight, respectively (equivalent to 0.01 and 0.1 ug/kg/day, respectively) for 28 weeks. Different control groups received no compound, DEN alone, or TCDD alone at the above doses. Compared */ to the control groups, which developed no liver tumors, a highly statistically significant increase in liver cancers (hepatocellular While TCDD administered alone induced observable liver tumors at exceedingly low doses in the Kociba and NCI studies, the fact that no liver tumors were found among the control groups receiving TCDD alone in the Pitot study is not unexpected. This follows from the fact that TCDD was administered in the Pitot study for 28 weeks (rather than for approximately two years as in the Kociba and NCI studies) and from the fact that only 5 animals were used in each TCDD control group. 1 i- -13carcinomas) was found in the group receiving DEN and the higher TCDD dose, providing strong evidence of a promoting effect by TCDD. In addition, the presence of liver tumors (hepatocellular neoplastic nodules) among three of five animals in the group receiving DEN and the lower TCDD dose in comparison to none in the control groups is indicative of a promoting effect. Thus, TCDD has liver tumor promoting effects down to the lowest dose tested (0.01 ug/kg/day). A study conducted by Kouri et al^. (Exhibit 575) provides substantial evidence that TCDD acts as a cocarcinogen in mice when administered to mice simultaneously with 3-methylcholanthrene (MCA) (a carcinogen). In this study, a single dose of MCA was administered subcutaneously to DBA/2 Cum mice. A single dose of TCDD was administered either subcutaneously or intraperitoneally (into the abdomen) either simultaneously or 2 days before the administration of the MCA. Two dose levels of TCDD were administered ( 1 ug/kg and 100 ug/kg). The incidence of fibrosarcomas at the site of MCA inoculation when TCDD was administered simultaneously with MCA at the higher dose (100 ug/kg) was significantly greater than the incidence of fibrosarcomas which occurred when MCA was admin istered alone. Since TCDD administered alone as a single dose in controls did not result in an increase of fibrosarcomas, the study provides evidence that TCDD acts as a cocarcinogen. That is, it acts synergistically with another carcinogen to produce a carcinogenic effect greater than the carcinogenic effects of the two carcinogens added together. 4- -14- D. Potency of TCDD The carcinogenic potency of a chemical refers to the strength of the carcinogenic effect induced by that chemical. TCDD appears to be a more potent carcinogen than aflatoxin Bj which was previously considered to be the most potent carcinogen known. This conclusion follows from a comparison of the carcinogenic potency of TCDD in the Kociba et al. study (Exhibits 13, 14, and 15) to the carcinogenic potency of aflatoxin in a chronic study by Wogan et al. (Exhibit 40) in which aflatoxin Bj induced hepatocellular carcinomas in male Fisher rats at an oral dose of 50 ppb in the diet. CAG estimated the respective potencies of TCDD and aflatoxin Bi in these two studies by calculating the v slope (B) of the linear one-hit model for the two compounds. Potency (B) was calculated for each compound according to the following formula: 1 1 - Pc B = In d 1 - Pt d = dose inducing a significant increase in carcinomas in the respective studies on TCDD and aflatoxin; Pc = tumor incidence in control animals in the respective studies; Pt = tumor incidence in treated animals in the respective studies at dose d. The linear one-hit model is a mathematical model which was previously used by CAG for purposes of estimating the probability that an observable case of cancer will be induced by doses of a carcinogen which are lower than doses which have empirically been demonstrated to cause carcinogenic responses. While CAG now uses a different mathematical model for estimating cancer risks (see discussion below), it is appropriate to use the linear one-hit model for estimating potency. % -15- This calculation was made on the basis of the lowest dose level at which TCDD or aflatoxin Bj caused a significant increase in hepatocellular carcinomas, the incidence of hepatocellular carcinomas at the respective dose levels, and the spontaneous incidence of this type of cancer in the control animals of each study. The following table (copied from Exhibit 33) shows that TCDD is approximately three times more potent a carcinogen than aflatoxin 1/ (0.110/0.032 = 3.45). TABLE 23. COMPARISON OF CARCINOGENIC POTENCY OF TCDD WITH AFLATOXIN TCDD Aflatoxin Author Species Sex Kociba et al. Sprague-Dawley rats Female Wogan et al. Fischer rats Male Tumor incidence in controls (Pc) 1/86 0/18 Dose(d), Tumor incidence in treated animals (Pt) 2.2 ppb, 11/49 50 ppb, 20/25 Carcinogenic Potency (B) 0.110 (ppb)"l 0.032 (ppb)"l ^/ The calculation of relative potency was based only on the incidence of hepatocellular carcinomas in both studies because the classification of other liver lesions in the Wogan study was ambiguous. 1 -16D. Laboratory Animal Studies on the Carcinogenicity of 2,4,5-T 1. Rat Studies CAG has reviewed two chronic toxicity studies involving the administration of 2,4,5-T to rats. The results of one of these studies provides highly suggestive evidence that 2,4,5-T itself (i.e., aside from any TCDD contamination) is carcinogenic in rats. The results of the other rat study do not appear to provide evidence of a carcinogenic response. Aspects of both studies limited their sensitivity for purposes of detect ing an oncogenic response. The study providing suggestive evidence that 2,4,5-T is carcinogenic in rats was conducted by scientists at Dow (Kociba */ et al_.) [Exhibits 21 and 22] These scientists administered specially purified 2,4,5-T containing no detectable TCDD (level of detection = 0 . 3 3 parts per billion TCDD) in the diet to groups of male and female Sprague-Dawley rats (Spartan substrain) at doses of 30, 10, and 3 mg of 2,4,5-T/kg body weight per day for up to two years. The authors concluded that no carcinogenic response was observed. However, the incidence of squamous cell carcinomas on the tongue which they observed in the high dose group males was marginally statistically significant compared to the incidence of this tumor in the controls (p = 0.063). In addition, the dose-related trend for the incidence of tongue 37 The published report of this study and certain unpublished data from this study are appended as Exhibits 21 and 22, respectively. 1 -17- carcinomas in males diagnosed by the authors is statistically 1/ significant by the Cochran Armitage test (p<0.03). Dr. Robert A. Squire, evaluated tongue tissues from male rats in the Kociba 2,4,5-T study at CAG's request. Dr. Squire diagnosed one more high dose male rat animal with a squamous cell carcinoma of the tongue than the author reported. (Exhibit 760 and Appendix B, Exhibit 33A). When Dr. Squire's diagnoses are considered, the incidence of squamous cell carcinomas in the high dose male rats is statistically significant compared to the incidence in the controls (p = .025). In addition, when the incidence of carcinomas of the tongue in the high dose males (using either Squire's or Kociba's diagnoses) is compared to the incidence of these tumors in historical control Sprague-Dawley male rats from the Dow laboratories, the incidence is highly statistically significant (p <0.001). (Exhibit 576). These results provide highly suggestive evidence that specially purified 2,4,5-T induced a carcinogenic effect in rats. The highly suggestive evidence provided by this study is particularly noteworthy in light of the fact that this study was relatively insensitive for purposes of detecting a carcinogenic effect. This insensitivity resulted from the very early mortality *_/ It is assumed that the incidence of tumors induced by a carcinogen increases as exposure to the carcinogen increases. Such an effect in a cancer study is known as a dose-related trend. Observation of a dose-related trend in a cancer study, by itself, may provide suggestive evidence of a carcinogenic effect. a iws V among all groups of males and females which reduced the number of animals at risk for late developing tumors. The insensitivity of the study was further increased by the relatively high incidence of spontaneous tumors in some organs and tissues in the control animals (e.g., hepatocellular carcinomas and hepatocellular neoplastic nodules). A high incidence of spontaneous tumors in controls makes it difficult to detect small increases in tumor incidence in the treated groups. In the other chronic rat study on 2,4,5-T [Exhibit 23], Leuschner et al. fed commercial grade 2,4,5-T (containing 0.05 ppm TCDD) dissolved in acetone and mixed with the diet to SpragueDawley (SIV 50 substrain) rats at doses of 30, 10, and 3 mg 2,4,5-T/kg body weight/day for up to 130 weeks. The authors concluded that no carcinogenic effect was observed. However, a statistically significant increase in interstitial cell tumors of the testes in the high dose group as well as a significant dose-related trend for these tumors were observed when comparison is made to the matched controls, which were bred at the same laboratory as the treated animals and which were also fed the acetone vehicle. The results are not statistically significant when the inci dence of testicular tumors in the high dose group is compared to the incidence of this tumor in a non-matched control group of animals discussed in the study as well as in historical controls (provided to EPA by Leuschner, attachment to Exhibit 33A). In each of these control groups, the incidence of testicular tumors is comparable to that in the high dose group. However, the reported non-matching $ ,i (b 0 k * - 19 controls as well as the historical controls were untreated controls, rather than vehicle (acetone) treated controls, and therefore may not be appropriate for comparison. Thus, the results of this study are equivocal. It should be noted that the highest dose of 2,4,5-T administered in this study was apparently less than the maximum tolerated dose (MTD). Therefore, the sensitivity of this study for detecting an oncogenic effect was less than prescribed by standard protocols. Although the adequacy of the histopathological examina tion of the tongues in the Leuschner study is not clear at the 1/ present time, the unusual site of tumor formation in the tongue in the Kociba study and the apparent lack of reproducibility of this tongue tumor response in the Leuschner study, reduced our judg ment of the strength of evidence of the carcinogenicity of pure 2,4,5-T provided by the Kociba study from substantial to highly suggestive. 2. Mouse Studies Several chronic mouse studies have been conducted on 2,4,5-T contaminated with low levels of TCDD. No significant increases of any specific tumor type were reported in any of these studies. However, because of deficiencies in their design, the reporting of their results, and other factors, these studies fail to demon- EPA recently requested Dr. Leuschner to cut cross-sections from the tongues of control and high dose male rats in his study which had been preserved. No sections had been made previously. The request was made by overseas telephone conversations. Due to some misunderstanding, longitudinal sections rather than crosssections were cut. s[kt>7 -20strate that commercial 2,4,5-T is not likely to be oncogenic in mice. Bionetics Research Laboratories conducted, as part of a large-scale cancer screening test on 140 chemicals, two sets of studies in which 2,4,5-T contaminated with TCDD was administered in daily oral doses to two strains of mice and in a single sub cutaneous dose to two strains of mice. [Exhibits 24 and 25] The use of very small groups of animals in both sets of studies made the studies very insensitive for purposes of detecting an oncogenic effect. This insensitivity was increased still further by the termination of the studies after only 18 months, which is short of the two years or greater life-expectancy of mice. Moreover, the subcutaneous study was extremely insensitive since the test compound (i.e., the 2,4,5-T) was administered only once. In a properly designed oncogenicity study, the test compound is ordinarily administered every day for nearly the entire lifetime of the animals tested. In other chronic mouse studies on commercial 2,4,5-T, MuranyiKovacs et al. administered 2,4,5-T orally to mice from two different strains [Exhibit 26] and subcutaneously to mice from the same two strains [Exhibit 27]. No significant increases in tumors at any specific sites were observed. However, these studies fail to provide significant evidence of the noncarcino genicity of 2,4,5-T in mice. This conclusion for the oral study is based on the following reasons: 1) the study was very insensitive for the purpose of detecting an oncogenic effect because insufficient numbers of animals were used in the treatment groups and because the maximum tolerated dose was apparently not 2-1 hO$ -21used; 2) since only "macroscopically altered" tissues were pre served for histopathologic evaluation, many microscopic tumors may not have been detected? 3) some mice were arbitrarily excluded from the calculation of tumor incidence; and 4) the histology data on all animals were not available. The subcutaneous study cannot be considered as evidence of the lack of carcinogenicity of 2,4,5-T because the study was so incompletely reported that the details of its methodology are unclear. In addition, this study was very insensitive because insufficient numbers of animals were used in the treatment groups, and only a few subcutaneous doses were administered. C. Laboratory Animal Studies on The Carcinogenicity of Silvex The very few chronic animal toxicity studies on silvex which have been conducted have not demonstrated a carcinogenic response. However, because of their deficiencies, these studies do not constitute significant evidence of the non-carcinogenicity of silvex. Oral and subcutaneous mouse studies on silvex were conducted by Bionetics Research Laboratories as part of the same largescale screening test of which the Bionetics 2,4,5-T studies described above were a part. [Exhibits 24 and 25]. While no significant increase in the incidence of any type of tumor was observed in either the oral or subcutaneous study, these studies do not provide significant evidence of the non-oncogenicity of silvex because they suffered from the deficiencies described above for the Bionetics study on 2,4,5-T. t* -22- Dow conducted a study (Exhibits 28, 29 and 30) in which groups of 30 male and 30 female Dow-Wistar rats were fed diets containing Kurosal SL (potassium salt of silvex) for up to 24 months. Some animals were intentionally killed before the con clusion of the experiment. Therefore, at most, only about 21 or 22 animals of each sex in each group were maintained until the */ end of the experiment. No significant increases in tumors were reported. However, because very small groups of animals were used and the maximum tolerated dose apparently was not used in females, this study was a very insensitive screen for carcinogenicity. Therefore, this study cannot be considered as decisive evidence of the non carcinogenicity of silvex in rats. Dow also conducted a study (Exhibits 29, 30, and 31) in which groups of three beagle dogs of each sex (initially four dogs) were fed silvex for two years. No increases in tumors were observed. However, this study does not constitute a valid cancer study because its duration was far less than the life expectancy of a beagle dog and because the sizes of the animal groups were exceedingly small. 37 Because of the inadequate reporting of the results, the extent to which additional animals died before the end of the experiment cannot be determined. It is possible that considerably fewer than 21 or 22 animals survived. 9 - 23 - 1/ III. Epidemiologic Studies of the Carcinogenicity of 2,4,5-T and TCDD The evidence of carcinogenicity of commercial 2,4,5-T and silvex and their TCDD contaminant provided by studies on labora tory animals is supported by epidemiologic studies of people exposed to 2,4,5-T, other chemically related herbicides (known collectively, as phenoxyacetic acids), and TCDD. Two Swedish epidemiologic studies reporting highly significant associations between a certain type of cancer and exposure to phenoxyacetic acids provide strongly suggestive evidence for the carcinogenicity of 2,4,5-T and/or TCDD. Suggestive, but weaker, evidence is provided by other epidemiologic studies. The two Swedish epidemiologic studies providing strongly suggestive evidence of the carcinogenicity of 2,4,5-T and TCDD are both case-control studies. A case-control study is one in which a subject group (or group of cases) who share a common disease is selected at the beginning of the study. The purpose of a case-control study is to compare the cases to a control group (i.e., a group without the disease but similar to the cases with respect to factors other than the factors being studied) to determine if the cases share exposure patterns not seen in the controls. This type of study is well suited for rare diseases with long periods of induction. ^_/ An epidemiological study is a study of the patterns and causes of disease in human populations. `X l & t t 9 -24In one of the Swedish case-control studies, Hardell and Sandstrom (Ref. 1) investigated the possible association in northern Sweden between sarcoma of soft connective tissue (i.e., cancer of such tissues as smooth muscle and fat) and exposure to phenoxyacetic acids and chlorophenols, whether considered separately or together. In northern Sweden, occupational exposure to phenoxyacetic acids took place in both forestry and agricultural work. Occupational exposure to chlorophenols took place mostly in sawmill work and paper V pulp production. In the second Swedish case-control study, the same group of epidemiologists (Eriksson et al. 1979, Ref. 2) investigated in southern Sweden the possible association between soft-tissue sarcomas and exposure to phenoxyacetic acids and chlorophenols. Occupational exposure to phenoxyacetic acids in southern Sweden was primarily through agricultural use. In both studies, controls (people without soft tissue sar coma) were selected to match the cases with respect to age, sex, vital status, and place of residence in order to eliminate the possibility that these factors would affect the results of the studies. In both studies, information regarding occupational exposure to phenoxyacetic acids and chlorophenols was obtained primarily through questionnaires and interviews. The investigators The principal phenoxyacetic acids used as herbicides were 2,4,5-T and 2,4-D, only the former of which is known to be contaminated with TCDD. Chlorophenols are also known to to contain chlorinated dibenzodioxin. 25 took great care to minimize the possibility that people with sarcoma would overreport exposure to these chemicals and therefore bias the results. The two studies indicated approximately fivefold to seven fold increases in the risk of developing soft-tissue sarcoma among people occupationally exposed to phenoxyacetic acids and chlorophenols, whether considered singly or together, in comparison to people not exposed to these chemicals. The estimated increases in risk were highly unlikely to have resulted solely by chance. A third case-control study by Hardell et al. (1980)(Ref. 3) provides suggestive evidence of an association between another * type of cancer and exposure to 2,4,5-T and/or TCDD. In this study, the investigators found a fivefold to sixfold increase in the risk of developing malignant lymphoma (cancer of lymphatic tissue) among people exposed to phenoxyacetic acids and chlorophenols, whether considered separately or together. The reliance which can be placed on this study at this time is limited somewhat by the fact that the methods and results were incompletely documented in the published report of the_study. However, the researchers have indicated that an additional report of this study is in preparation. Two cohort studies of people occupationally exposed to phenoxyacetic acids and/or to TCDD provide suggestive evidence that 2,4,5-T and/or TCDD may cause stomach cancer in humans. A * - 26 cohort study is one in which a group of individuals with a known history of exposure to the factor being investigated (phenoxyacetic acids in one study and TCDD in the other) is followed over time to see if any adverse health effects develop in comparison to a control group of unexposed or less highly exposed persons. In one of these cohort studies (Axelson, et al. 1980, Ref.4), 348 Swedish railroad workers with at least 46 days of herbicide exposure (greater than one spray season) between 1955 and 1972 were followed through October 1978. The workers were grouped on the basis of their primary herbicide exposure; those previously exposed to phenoxyacetic acids (2,4,5-T and 2,4-D) only, to amitrole (aminotriazole) only, and to both types of herbicides. Workers with primary exposure to phenoxyacetic acids only showed a mortality rate from stomach cancer that was six times higher than expected from Swedish national mortality rates specific for age, sex, and calendar year. Even though this was not likely to be a chance finding (p= 0.044), firm conclusions about the carcinogenicity of 2,4,5-T and TCDD cannot be drawn from this study because the number of stomach cancer deaths observed was small. In the second cohort study, Thiess and Frentzel-Beyme (1977) (Ref. 5) followed 75 workers exposed to TCDD during and after a 1953 runaway reaction at a trichlorophenol manufacturing facility in Ludwigshafen, West Germany. These workers had a 27 sevenfold to eightfold increase in the rate of stomach cancer mortality in comparison to the expected rate (p<0.05). Again, however, firm conclusions cannot be drawn from this study because the number of stomach cancer deaths observed was relatively small. In another cohort study (the "Nitro study"), workers exposed to TCDD in a trichlorophenol processing accident in Nitro, W.Va. (Zack and Suskind, 1980, Ref. 6) were followed. A threefold increase in deaths from cancers of the lymphatic and hematopoietic system was found in comparison to the expected rate (p = 0.06), but this result also was based on a small number of observed deaths. In addition, single deaths from soft-tissue sarcoma were found in the Nitro study and in a Midland, Michigan study on workers exposed to TCDD in the manufacture of trichlorophenol (Cook, et al. 1980, Ref. 7). The authors of the Nitro study noted this type of cancer as rare. These findings are consistent with a carcinogenic effect from the TCDD exposures in both studies. These, as well as two additional cohort studies, reported results that did not show increased stomach cancer mortality rates in groups of workers exposed to phenoxyacetic acids and/or TCDD. The additional two studies were of 2,4,5-T herbicide production workers in Midland, Michigan (Ott, et al. 1980, Ref. 8) and Finnish phenoxyacetic acid herbicide applicators (Riihimaki, et al. 1978, Ref. 9). Because the Nitro study and 4 - 28 - the two Midland studies had insufficient statistical power for purposes of detecting the increased stomach cancer risks suggested by the Swedish and West German cohort studies discussed above, they should not be considered to be inconsistent with those studies. The Finnish study reported a larger number of expected cancer deaths than the Nitro or the two Midland studies and therefore was more powerful than those studies for purposes of detecting small increases in risk. However, the Finnish study was designed to include subjects who were exposed to herbicides for a considerably shorter period of time than the Swedish cohort study discussed above (Ref. 4). In addition, certain incon sistencies in the data from the Finnish`study will require clari fication in order for the results to be adequately interpreted. IV. Conclusions and Qualitative Risk Assessment of the Carcinogenicity of TCDD, 2,4,5-T and Silvex The laboratory animal data provide substantial evidence that TCDD is likely to pose a carcinogenic risk to exposed humans as a very potent carcinogen and as a very potent cancer promoter, and also provide highly suggestive evidence that essentially pure 2,4,5-T is a carcinogen. Evidence that TCDD is a potent carcinogen is provided by the Kociba study on rats, and the NCI studies on both rats and mice. These studies indicate that TCDD administered alone at exceedingly low doses induces a carcinogenic response in several organs. On the basis of the Kociba study, it appears that TCDD is one of the most potent carcinogens known. 2.|6>|fe ^ - 29 The highly suggestive evidence that essentially pure 2,4,5-T is carcinogenic is provided by the Kociba 2,4,5-T study. Strongly suggestive human evidence that 2,4,5-T and/or TCDD pose a carcinogenic risk is provided by the two Swedish epidemiolo gical studies demonstrating a strong association between increased risk of developing soft tissue sarcoma and exposure to 2,4,5-T and other phenoxyacetic acids. Suggestive but weaker evidence of increased cancer risk is provided by other epidemiological studies. The laboratory animal data and the epidemiological data con sidered as a whole provide very substantial evidence that TCDD is carcinogenic in human and strongly suggestive evidence that essentially pure 2,4,5-T is carcinogenic in humans. In accordance with the no-threshold concept, TCDD, as a carcinogen, poses a human cancer risk at any level of exposure no matter how small; the degree of cancer risk posed by TCDD is directly proportionate to the level of exposure to TCDD. The evidence that TCDD is a potent cancer promoter and is a cocarcinogen is provided by the Pitot and Kouri studies, respec tively. These studies demonstrated that very low doses of TCDD significantly enhanced the induction of liver and skin tumors by other carcinogens in rats. The human population is exposed to a large number of carcinogens in the environment. Therefore, it is possible that exposure to a potent promoter such as TCDD could increase the number of cancers induced by environmental carcinogens and shorten the latency period for the development of cancer. This enhanced ' Z l f c t *7 4 - 30 induction of tumors by TCDD acting as a promoter would be in addition to the tumor induction by TCDD as a carcinogen. There is no theoretical basis for making even ballpark estimates of the risk posed by promoters and cocarcinogens to exposed persons because the mechanism for promotion is not well understood and because the degree of total exposure of the human population to the numerous carcinogens in the environment cannot be well quanti fied. However, it is possible that TCDD could significantly increase human cancer as a promoter or cocarcinogen at exceedingly low levels of TCDD exposure. The question arises as to whether the carcinogenic action of TCDD by itself such as exhibited in the studies of Kociba and the NCI studies on rats and mice could be due to the action of TCDD as a carcinogen and/or a promoting agent. There is evidence that TCDD can be metabolized to an electrophilic metabolite (Ref. 10) which could react with DNA and thereby produce genetic damage of the sort that is associated with the induction of cancer. However, the react ivity of this metabolite with cellular protein is extremely high and, to date, the degree of interaction with DNA that has been demonstrated is low. This may be peculiar to the tissues that have been examined for this reaction so far but may not be generally applicable to the reaction of TCDD with DNA in the body. Furthermore, TCDD has a chemical structure which makes it likely that it could inter calate into DNA and also act as a genotoxic carcinogen (Exhibit 33A). Promoting agents, when administered alone, characteristically produce a relatively small increase in the occurrence of tumors 5U - 31 and those tumors that are increased seem to be of the type that would be expected to occur spontaneously as the result. This is not characteristic of TCDD, particularly in relation to its ability to induce squamous carcinomas of the lung and of the hard palate and turbinates. Squamous carcinomas of the lung are exceedingly uncommon in the rat in contrast to adenomas of the lung. For these reasons the CAG believes that it is prudent, given the present state of knowledge, to regard TCDD as a complete carcinogen as well as a promoting and cocarcinogenic agent. Commercial 2,4,5-T and silvex pose a carcinogenic risk to exposed humans because they contain low levels of TCDD as a contaminant. In addition, based on the highly suggestive evidence of the Kociba study on specially purified 2,4,5-T, CAG concludes that the pure 2,4,5-T component of commercial 2,4,5-T may pose a cancer risk to humans at any level of exposure. V. Quantitative Risk Assessment of TCDD and 2,4,5-T A. General In this part of my testimony, I summarize CAG's quantitative estimates of the carcinogenic risks posed to humans by certain sources of exposure to TCDD from the use of commercial 2,4,5-T or silvex in the U.S. CAG asessed the risks posed by TCDD only as a complete carcinogen, not as a promoter or cocarcinogen, because as discussed above there is no basis for quantitatively estimating the risks posed by cancer promoters or cocarcinogens. 9.1 >1^ 32 However, it should be noted that the risks posed by TCDD as a promoter or cocarcinogen may be higher than those risks which have been calculated by CAG. I also summarize CAG's quantitative estimates of the carcino genic risks which may be posed by certain sources of exposure to the pure 2,4,5-T component of commercial 2,4,5-T. It should be kept in mind that the evidence for the carcinogenicity of essentially pure 2,4,5-T is only highly suggestive, not substantial. CAG's quantitative estimate of human cancer risks posed by TCDD and 2,4,5-T were derived from dose-response data from the laboratory animal cancer studies on these compounds and from estimates of human exposure to these compounds. CAG used Dr. Squire's diagnoses of tumor incidences in the rats from Dr. Kociba's TCDD and 2,4,5-T studies in making its risk estimates. Exposure estimates used by CAG in its risk assessment were those provided by the Agency's Hazard Evaluation Division (HED) (See, Appendix F to Exhibit 33A). In making quantitative estimates of cancer risks from human exposure information and from dose-response data provided by animal cancer studies, CAG uses a mathematical risk extrapolation model. The mathematical model used by CAG in its risk assessment of 2,4,5-T and TCDD was the linearized multistage extrapolation model, which was recently adopted by CAG in response to public comments expressing dissatisfaction with the "one-hit" extrapo- q.\<c 7-0 - 33 - V lation model previously used by CAG. The linearized multistage model has also been adopted by the National Academy of Sciences Drinking Water Panel. The linearized multistage model is based in the assumption of a linear non-threshold dose response relationship at low doses. Further details describing this model are provided in the CAG Report (Exhibit 33A) and in the document, "Procedure for Calculating Air Quality Criteria" which was prepared by CAG and is attached as Appendix G to the CAG Report (Exhibit 33A). It should be noted that CAG made estimates of risk for only some routes of exposure to TCDD and 2,4,5-T. CAG's anaysis does not attempt to assess cancer risks resulting from all routes of exposure to these compounds because, as explained in H E D 's exposure analysis quantitative exposure estimates could be made for only some potential exposure routes. It should also be noted that HED expressed in its exposure analysis document certain qualifications and reservations concerning the quantified exposure analyses contained in that document. CAG's quantitative risk estimates should be viewed in light of these qualifications and reservations. ^_/ The risk estimates calculated by CAG for TCDD, 2,4,5-T, and silvex by the linearized multistage model are approximately the same as those which would be calculated by the use of the one-hit model. However, the linearized multistage model is methodologically more sound than the one-hit model because it gives a more systematic way of utilizing all the available data. 34 B. Quantitative Risk Assessment in General Before I summarize CAG's quantitative risk assessment with respect to 2,4,5-T and silvex, I would like to discuss the functions and limitations of quantitative risk assessments in general. The purpose of a quantitative risk assessment is to provide Agency decisionmakers with a rough approximation of the degree of risk posed by exposure to a carcinogenic compound. It will be clear from the discussion below that one cannot hope to generate an estimate of the degree of risk posed by a compound that is more accurate than a crude, "ball-park" figure. A quantitative risk assessment can give a decisionmaker a feel for the degree of risk posed by one compound in comparison to the risks posed by other compounds. Agency decisionmakers are aware that, although CAG expresses its risk assessments in terms of specific numbers, those numbers probably do not describe with precision what will happen in the real world. The principal reasons why quantitative risk assessments can be only rough approximations of risk are the following: 1. Mathematical models for such assessments involve extra polating from results observed in laboratory experiments in which animals are exposed to a compound to situations in which people will be exposed to the compound. This procedure introduces a number of variables whose impact can be substantial. First, there are uncertainties about the relative sensitivities of 35 different species (e.g.f rats and people) to carcinogenic compounds. Moreover, there are variations in sensitivity to carcinogens within each species. 2. Extrapolation from high doses administered to experi mental animals to relatively low exposures likely to be experienced by people may be inaccurate because of uncertainties about the shape of the dose-response relationship. The mathematical model adopted by CAG for risk extrapolation is intended to provide a reasonably conservative assessment of risk. 3. Human exposure to the compound, an essential part of a quantitative risk assessment, often can be estimated only roughly. 4. Quantitative risk assessments based solely on animal data relate to individual compounds and do not take into account the fact that these compounds may act as promoters or have synergistic effects. This is an especially serious limitation when strong evidence exists, as in the case of TCDD, that the chemical under consideration is a potent cancer promoter and is a cocarcinogen. B. Summary of CAG's Quantitative Risk Estimates The quantitative risk assessment for lifetime cancer risks from exposure to the TCDD contaminant of commercial 2,4,5-T and silvex and to 2,4,5-T itself have been estimated for worker exposure and to a limited extent, for food contamination. In estimating lifetime cancer risks from the linearized multistage 2. l 4>3-^ l' - 36 model, CAG assumed that the general population and local popula tions would be exposed to dietary sources of TCDD over a 70 year lifetime and that applicators of 2,4,5-T or silvex would be occupationally exposed over a 40 year period. Although the risk estimates are expressed as numbers, they should be considered to be only very rough estimates of risk and subject to the qualifi cations and limitations discussed above. It should be noted that CAG's risk estimates took into account the percentage of total acres for each use which were in fact treated according to recent useage patterns. For almost all uses, only a very small percentage of the total acreage which could potentially be treated consistently with the pesticide labeling have in fact been treated. If the percentage of acreage treated were to increase in the future, the estimated cancer risks to the general population posed by food contaminated with TCDD or 2,4,5-T would increase proportionately. Similarly, the number of workers applying 2,4,5-T and silvex and therefore exposed to these compounds as well as TCDD would increase propor tionately. The individual lifetime risks of developing cancer which are projected for workers involved in the application of 2,4,5-T or silvex in forestry, range, rice, and rights-of-way uses are provided in Tables QA-19 and QA-20 in the CAG Report (Exhibit 33A). These estimates are based on the assumption that the workers do not generally wear special protective clothing. i 37 Workers involved in different uses may have different risks because the average application rates and therefore average exposures may differ. In addition, different categories of workers within each use may have different risks. Workers involved in forestry application of 2,4,5-T are projected to have individual lifetime risks of developing cancer ranging from 1 0 "4 Up to 3x10 "3, depending on the category of worker. Workers involved in the application of 2,4,5-T to rightsof-way are projected to have individual lifetime risks of develop ing cancer ranging from 2xl0"4 to 5xl0"3. Most categories of workers for this use have risks greater than 1Q"3. Workers involved in the .application of 2,4,5-T to rangelands have projected risks ranging from 7x10" for flaggers to 2xl0"4 for mixer-loaders. Projected risks for workers involved in 2,4,5-T application on rice fields range from 7x10" to 8x10". Each of the projected risks described above take into account the risk posed by TCDD as well as the possible risk posed by the pure 2,4,5-T component of commercial 2,4,5-T. The contribution of each of the two components to the total risk are approximately equal. Since pure silvex has not been demonstrated to be carcin ogenic, the risks to workers who apply silvex are estimated only on the basis of the TCDD component of silvex. Thus, the projected individual lifetime risks of developing cancer for silvex applicators are about half of those for 2,4,5-T applicators. > - 38 The number of applicators in each category for each use are provided in Tables QA-19 and QA-20 of Exhibit 33A. TCDD contamination of beef resulting from the use of 2,4,5-T and silvex on rangeland and directly treated pasture is projected to result in individual lifetime risks of developing cancer of approximately 2xl0"4 in local populations which exclusively consume beef produced locally on grazing land treated every five years with silvex or 2,4,5-T. It is estimated that combined size of such local populations could be as high as 12,000 persons. Individual lifetime cancer risks in the general population of the U.S. resulting from consumption of beef contaminated with TCDD beef is projected to be approximately 2x10"^. This route of exposure is projected to result in approximately 8 cases of cancer per year among the general population. It is estimated that TCDD contamination of dairy products resulting from the use of 2,4,5-T or silvex on pastures may result in individual lifetime risks of developing cancer as high as 5xl0"4. The percentage of dairy cattle grazing on 2,4,5-T or silvex treated pasture could not be estimated. There fore, risks to the general population resulting from consumption of dairy products contaminated with TCDD could not be estimated. > - 39 It is estimated that TCDD contamination of deer or elk meat as a result of the use of 2,4,5-T or silvex on forests may result in individual lifetime risks of developing cancer as high as 1 0 "4 and 3x 1 0 " 5 if twelve meals of deer meat and elk meat, respectively, are consumed each year for a lifetime. More or less consumption would lead to proportionate increases or decreases. A 4& Z -4 RoyE. Albert, M.d / ,0, \(o3- V EXHIBITS IHIBIT N O . *13 - Kociba, R.J., Keyes, D.G., Carreon, R.M., Wade, C.E., Dittenber, D., Kalnins, R., Frauson, L., Park, C.N., Hummel, R., and Humiston, C.G. 1978a. Results of a Two-year Chronic Toxicity and Oncogenicity Study of 2.3.7.8- Tetrachlorodibenzo-p-dioxin in Rats. Toxicol. Appl. Pharmacol. 46:279-303 *14 - Kociba, R.J. 1979a. Letter and three tables sent to H. Warnick, Special Pesticide Review Division, U.S. Environmental Protection Agency, from R.J. Kociba, Toxicology Research Laboratory, Health and Environmental Sciences, Dow Chemical U.S.A., Midland, Mich. February 20, 1979 *15 - Kociba, R.J., Keyes, D.G., Carreon, R.M., Wade, C.E., Dittenber, D., Kalnins, R., Frauson, L., Park, C.N., Hummel, R., and Humiston, C.G. 1977. Results of a Two-year Chronic Toxicity and Oncogenicity Study of 2.3.7.8- Tetrachlorodibenzo-p-dioxin (TCDD) in Rats. Unpublished study. Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical, U.S.A., Midland, Mich. September 28, 1977 *16 - Squire, R.A., and Levitt, M.H. 1975. Report of.a work shop on classification of specific hepatocellular lesions in rats. Cancer Res. 35:3214-3215 *18 - Hirota, N., and Williams, G.M. 1979. Persistence and growth of rat liver neoplastic nodules following cessation of carcinogen exposure. J. Natl. Cancer Inst. 63:1257-1265 *20 - Toth, K . , Sorafai-Relle, S., Sugar, J., and Bence, J. 1979. Carcinogenicity testing of herbicide 2,4,5-trichlorophenoxy' ethanol containing dioxin and of pure dioxin in Swiss mice. Nature 278:548-549 *21 - Kociba, R.J., Keyes, D.G., Lisowe, R.W., Kalnins, R.P., Dittenber, D.D., Wade, C.E., Gorzinski, S.J., Mahle, N.H., and Schwetz, B.A. 1979b. Results of a two-year chronic toxocity and oncogenic study of rats ingesting diets containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Food Cosmet. Toxicol. 17:205-221 *22 - Kociba, R.J., et al. 1978b. Results of a Two-Year Chronic Toxicity and Oncogenesis Study of Rats Ingesting Diets Containing 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T). Unpublished study. September 27, 1978 Asterisks (*) indicate Exhibits which have been previously transmitted with the testimony of Dr. Dawn G. Goodman. f EXHIBIT N O . *23 - Leuschner, F., Leuschner, A., Hubscher, F., Dontenwill, W., and Rogulja, P.V. 1979. Chronic Oral Toxicity of 2,4,5-T, Batch No. 503, Control No. 153574 b-- Called for Short '2,4,5-T' -- in Sprague-Dawley (SIV 50) rats. Unpublished study. Laboratorium fur Pharmakologie and Toxikologie, Hamburg. April 9, 1979. Pp 1-29, 39-41 *24 - Bionetics Research Labs, Inc. 1968. Evaluation of Carcinogenic, Teratogenic, and Mutagenic Activities of Selected Pesticides and Industrial Chemicals. Vol. 1. Pp 1-53, 57, 67, 75, 97-110, 225-228 *25 - Innes, J.R.M., Ulland, B.M., Valerio, M.G., Petrucelli, L., Fisbein, L., Hart, E.R., Pallotta, A.J., Bates, R.R., Falk, H.L., Gart, J.J., Klein, M., Mitchell, I., and Peters, J. 1969. Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. Natl. Cancer Inst. 42:1101-1114 *26 - Muranyi-Kovacs, I., Rudali, G., and Imbert, J. 1976. Bioassay of 2,4,5-trichlorophenoxyacetic acid for carcinogenicity in mice. Br. J. Cancer 33:626-633 *27 - Muranyi-Kovacs, I., et al. 1977. Study of the Carcino genicity of 2,4,5-T in Mice. Paper presented to the Fourth Meeting of the European Association for Cancer Research. September 13-15, 1977 *28 - Dow Chemical Company. 1965. Results of Two-Year Dietary Feeding Study in Rats of Kurosal SL (Potassium Salt of Silvex) Unpublished study. Pp 1-14, 38, 43-46, 73-95. *29 - Gehring, P.J., and Betso, J.E. 1978. Phenoxy acids: Effects and fate in mammals. In Ramel, C., ed. Chlorinated phenoxy acids and their dioxins. Ecol. Bull. (Stockholm) 27:122-133 *30 - Mullison, W.R. 1966. Some toxicological aspects of silvex. Presentation at the Southern Weed Conference, Jacksonville, Fla., January 18, 1966 *31 - Dow Chemical Company. 1965. Results of a Two Year Dietary Feeding Study in Dogs of Kurosal SL (Potassium Salt of Silvex). Unpublished study. Pp 1-5. Asterisks (*) indicate Exhibits which have been previously transmitted with the testimony of Dr. Dawn G. Goodman. gL[ 'HIBIT NO. 33A - Carcinogen Assessment Group. EPA. Risk Assessments on 2,4,5-T, silvex, and TCDD. June 16, 1980. 34 - U.S. Environmental Protection Agency. Interim procedures and guidelines for health risk and economic impact assessments of suspected carcinogens. 41 F.R. 21402-21405 (May 25, 1976). 35 - Albert, R.E., R.E. Train, and E. Anderson 1977. Rationale Developed by the Environmental Protection Agency for the Assessment of Carcinogenic Risk. J. Natl. Cancer Inst. 58(5) 1537-1541. 36 - Work Group on Risk Assessment of the Interagency Regulatory Liaison Group (IRLG). 1979. Scientific Bases for Identi fication of Potential Carcinogens and Estimation of Risks. 44 F.R. 39858-39879 (July 6, 1979). 37 - Farber, E. 1976. Putative precursor lesions: summary and some analytical considerations. Cancer Research 36:2703-2705 40 - Wogan, G. et al. 1974. Carcinogenic effects of low dietary levels of aflatoxin B^ in rats. Food Cosmet. Toxicol. 12: 6 81-6 85 574 - Pitot, H.C., T. Goldsworthy, and A. Poland. Promotion by 2,3,7,8-Tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine. Submitted for publication to Cancer Research. 575 - Kouri, R.E., et al., 1978. 2,3,7,8-tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-methylchloronthrene-initiated Subcutaneous Tumors in Mice Genetically "Nonresponsive" at Ah Locus. Cancer Research 38: 2777-2783. 576 - Historical Control Data on Incidence of Squamous Cell Carcinomas of the Tongue in Male Sprague-Dawley Rats from Dow Laboratories. 577 - National Cancer Institute. Bioassay of 2,3,7,8-Tetrachlorodibenzo-p-dioxin for Possible Carcinogenicity (Gavage Study). U.S. Dept, of Health and Human Services Publication No. (NIH) 80-1765. 1980. 578 - National Cancer Institute. Bioassay of 2,3,7-8-tetrachlorodibenzo-p-dioxin for possible carcinogenicity (dermal study) U.S. Dept, of Health and Human Services Publication No. (NIH) 80-175 7. 1980. al3o # * REFERENCES 1. Hardell, L. and Sandstrom, A. 1979. Case-control study: softtissue sarcomas and exposure to phenoxyacetic acids and chlorophenols. Br. J. Cancer 39: 711-717. 2. Eriksson, M. Hardell, L., Berg, N.O., Moller, T. and Axelson, 0. 1979. Case-control study on malignant mesenchymal tumors of the soft tissue and exposure to chemical substances. Lakartidningen 76: 3872-3875 (Translation). 3. Hardell, L., Eriksson, M. and Lenner, P. 1980. Malignant lymphoma and exposure to chemical substances, especially organic solvents, chlorophenols and phenoxy acids. Lakartidningen 77: 208-210 (Translation). 4. Axelson, 0., Sundell, L., Andersson, K. Edling, C., Hogstedt, C. and Kling, H. 1980. Herbicide exposure and tumor mortality: an updated epidemiological investigation on Swedish railroad workers. Sc. and J. Work Environ. Health (in press). 5. Thiess, A.M. and Frentzel-Beyme, R. Mortality study of persons exposed to dioxin following an accident which occurred in the BASF on 13 November 1955. Proceedings of MEDICHEM Congress V, San Francisco, September 5-9, 1977 (in press). 6. Zack, J .A. and Suskind, R.R. 1980. The mortality experience of workers exposed to tetrachlorodibenzodioxin in a trichlorophenol process accident. J. Occup. Med. 22: 11-14. 7. Cook, R.R., Townsend, J.C., Ott, M.G., and Silverstein, "Mortality Experience of Employees Exposed to 2,3,7,8-Tetra chlorodibenzo-p-Dioxin (TCDD)," (Pre-publication copy) (1980). 8. Ott, M.G., Holder, B.B. and Olson, R.D. 1980. A mortality analysis of employees engaged in the manufacture of 2,4,5trichlorophenoxyacetic acid. J. Occup. Med. 22: 47-50. 9. Riihimaki, V., Asp, S., Seppalainen, A.M. and Hernberg, S. 1978. Symptomatology, morbidity and mortality experience of chlorinated phenoxy acid herbicide (2,4-D; 2,4,5-T) sprayers in Finland. A clinical and epidemiological study. Working paper for an IARC working group meeting on coordination of epidemiological studies on the long-term hazards of chlorinated dibenzodioxins and chlorinated dibenzofurans, Lyon, France, January 10-11, 1978. 10. Guenther, T., Fysh, J. and Nebert, D.W. 1979. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: covalent binding of reactive metabolic intermediates principally to protein JLn vitro. Pharmacology 19: 12-22. aits' f '* i *11. Bionetics Research Labs, Inc. 1968. Evaluation of carcinogenic, teratogenic, and mutagenic activities of selected pesticides and industrial chemicals. Vol. I. *12. Leuschner, F., Leuschner, A., Hubscher, F., Dontenwill, W. and Rogulja, P.V. 1979. Chronic oral toxicity of 2,4,5-T, Batch No. 503, Control No. 153574 b - called for short '2,4,5-T' in Sprague-Dawley (SIV 50) rats. Unpublished study. Laboratorium fur Pharmakologie and Toxikologie, Hamburg. April 9, 1979. jVThese references were used as references 1 and 6 in the direct testimony of Dr. Dawn G. Goodman. t tl t CERTIFICATE OF SERVICE I hereby certify that copies of the foregoing DIRECT TESTIMONY (AND EXHIBIT*) DR. ROY E. ALBERT were hand delivered or mailed first class postage prepaid on September 1980 to 15 the following persons. *Edward W. Warren L. Mark Wine Richard L. McConnell/ Jr. Kirkland & Ellis Counsel for Dow Chemical Company 1776 K Street, N.W., 12th Floor Washington, D.C. 20006 *William A. Butler, Esq. Jacqueline M. Warren, Esq. Counsel for Environmental Defense Fund, Inc. 1525 - 18th Street, N.W. Washington, D.C. 20036 *Margaret M. Breinholt Judith A. Wenker Terrence G. Jackson Room 2036, South Ag. Bldg. Office of the General Counsel U.S. Department of Agriculture Washington, D.C. 20250 *Robert S. Kirk, Jr., Esq. Counsel for Vertac, Inc. 2414 Clark Tower 5100 Poplar Avenue Memphis, Tennessee 38137 Richard J. Wertheimer, Esq. Arnold & Porter Counsel for National Forest Products Association 1200 New Hampshire Avenue, N.W. Washington, D.C. 20036 Stephen W. Jacobson Joseph E. Stevens, Jr. William Ray Price, Jr. Lathrop, Koontz, Righter, Clagett, Parker & Norquist 2600 Mutual Benefit Life Building Post Office Box 1200 2345 Grand Avenue Kansas City, Missouri 64108 Judy Kahle Northwest Coalition for Alternatives to Pesticides, Inc. P.O. Box 375 454 Willamette Street Eugene, Oregon 97401 *Sonia G. Anderson Hearing Clerk (A-110) U.S. Environmental Protection Agency 401 M Street, S.W. Washington, D.C. 20460 Allen A. Lauterbach John J. Rademacher American Farm Bureau Federation 425 - 13th Street, N.W. Washington, D.C. 20004 Elizabeth M. Whelan, Sc.D., M.P.H. Executive Director American Counsel on Science & Health 1995 Broadway New York, New York 10023 September 12, 1980 Richard P. Bozof^/ t Jk 1, 'v i M UNITED STATES ENVIRONMENTAL PROTECTION AGENCY il . ^ ?| BEFORE THE ADMINISTRATOR 'l MAR 2 1980 In re: The Dow Chemical Company, et a l . ) ) ) ) FIFRA Docket Nos. 415, et al. */ DIRECT TESTIMONY OF MR. RONALD THOMAS February 26, 1980 Dorothy E. Patton Kevin M. Lee Patricia A. Roberts Richard P. Bosof Timothy D. Backstrom Andrew G. Gordon Cara S. Jablon Counsel for Respondents U. S. Environmental Protection Agency 401 M Street, S.W. Washington, D.C. 20460 */ This is the first of two documents containing Mr. Thomas' direct testimony. A portion of Mr. Thomas' testimony contains data which Dow claims to be confidential. This testimony is presented in a second document which has been filed UNDER SEAL for use i_n camera, and served only on the Administrative Law Judge, the Hearing Clerk, and the Dow Chemical Company. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY BEFORE THE ADMINISTRATOR In re: ) ) ) The Dow Chemical Company, et al. ) ) ______________________________) FIFRA Docket Nos. 415, et a l . DIRECT TESTIMONY OF MR. RONALD THOMAS INTRODUCTION . My name is Ronald Thomas. I am currently employed as a supervisory chemist in the Residue & Special Projects Unit, Chemistry Section, Chemical & Biological Investigations Branch, Benefits & Field Studies Division, Office of Pesticide Programs, Environmental Protection Agency. Since 1956 I have been employed as an analytical chemist by federal regulatory agencies, namely the Food and Drug Administration, the U.S. Department of Agriculture, and the Environmental Protection Agency. Much of my career as a chemist has been in the field of pesticide residue analysis, and I currently specialize in that field. My curriculum vitae with a list of some of my publications is attached to the end of this statement. My testimony is intended to describe the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in commercial samples of 2,4,5-T and silvex products. Based on the work - ^4<o 3?3 -2done in my own laboratory, EPA contract laboratories, and others, I have concluded that commercial 2,4,5-T and silvex products manufactured in the United States in 1977-78 routinely contain TCDD at levels in the 10-30 ppb range. Dow discovery documents demonstrate the presence of TCDD in some batches of 2.4.5- T at levels higher than 30 ppb during this time period. HISTORICAL BACKGROUND My laboratory is responsible for investigating and attempting to resolve various analytical problems for the Office of Pesticide Programs, including analysis of air, soil, water and commodity samples for pesticide residues, and evaluation or development of analytical methodologies for detection and quantification of pesticide residues. From 1970 to the present, we have been involved in the analysis of the dioxin content of pesticide products, including the analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 2.4.5- T and silvex products. The work in my laboratory has entailed the clean-up of samples by chromatographic means and the use of gas chroma tography (GC) and low-resolution mass spectrometry (MS) for dioxin 1/ detection. The limit of detection for dioxin achieved by the equipment in my laboratory is 0.1 ppm, with a margin of error of approximately 20% in the determinations. Between 1970 and 1978, my laboratory screened several hundred pesticide products for dioxin content. Dioxin levels greater than 0.1 ppm 1 / A short discussion of the theoretical basis of mass spectrometry is provided in Appendix A. a\(&3^ 3 were detected in pre-1975 samples; however, for pesticide products manufactured after 1975, we did not find any samples with a dioxin content exceeding 0.1 ppm (100 p p b ) . The work performed in my laboratory from 1970-1972 is detailed in a paper published in 1972 (Exhibit A). In 1977-78, the methodology for dioxin detection improved, and equipment became available which allowed-u limit of detection of 0.01 ppm (10 ppb) for low-resolution GS/MS. In 1978, the Office of Pesticide Programs (OPP) undertook a prpject to determine the levels of dioxin in technical grade pesticide products likely to contain TCDD, including 2,4,5-T and silvex. Since my laboratory did not have the equipment to detect dioxin below the 0.1 ppm level, OPP entered into a contract with Gulf South Research Institute (GSRI), which had the equipment and the technical expertise to detect dioxin at a 0.01 ppm limit of detection level. My laboratory conducted an on-going quality control program for the GSRI contract throughout the duration of the contract. In this capacity, we had the responsibility of advising the contractor on the appropriate methodology, techniques and reporting methods; transmitting coded pesticide samples and reference samples for analysis; reviewing the results of the analytical determi nations (for the reference samples and duplicates of the samples) while the study was in progress to assure that quality control was maintained; and providing any needed assistance and advice for the project. -4- On the basis of this monitoring program, we were able to conclude with great assurance that the contract and the analytical work had been carried out in a proper scientific manner commensurate with the state of the art of low resolution dioxin analysis. The results of the study, which will be discussed in greater detail in my ensuing testimony, establish that TCDD is present in commercial technical grade 2,4,5-T \ and silvex manufactured from 1977-78 at levels ranging from 0.01-0.03 ppm (10-30 pp b ) . GULF SOUTH RESEARCH INSTITUTE (GSRI) STUDY ON TCDD LEVELS IN TECHNICAL GRADE 2,4,5-T AND SILVEX AT THE PPB LEVEL In 1978-1979, Gulf South Research Institute (GSRI), New Orleans, Louisiana, analyzed technical grade 2,4,5-T and 2/ silvex for TCDD at the 10 ppb level under an EPA contract. The project was carried out under the direction of Joseph G. Montalvo, Jr., James F. Ryan, III, and Roy Flagg.; the EPA Project Officer for the contract was Patricia Ott, Environmental Fate Branch of the Hazard Evaluation Division. The quality control for the contract was monitored by the EPA Residue & Special Projects Unit, under my supervision. The samples for the analysis were procured by the Enforcement Division of the Environmental Protection Agency from the manufacturing plants of the major manufacturers of the pesticide products in 1977-1978; 27 In addition to various 2,4,5-T and silvex products, the study included the analysis of 2,4,5-trichlorophenol, an organophosphate, and a chlorinated phenolic antiseptic for TCDD content. a 1&3 -5- all the samples under study were taken from production tanks, bins or drums. The samples were coded by EPA in order to keep the sample contents anonymous; decoding information was provided to the Gulf South Research Institute only after completion of the assays and reporting of the TCDD data. GSRI used low resolution gas chromatography/mass spectrometry (GC/MS) to,determine the total TCDD content in 4 technical grade samples of 2,4,5-T, the isooctyl ester of 2,4,5-T, the butoxyethyl ester of 2,4,5-T, the butoxypropyl ester of silvex and the isooctyl ester of silvex. A copy of the final contract report submitted by GSRI is attached (Exhibit B ) . The analytical method which was used to determine the levels of TCDD in the pesticide samples is described in detail in Exhibit B. In order to assess dioxin recovery, an aliquot of a standardized radioactive solution of ^Zl-2, 3,7,8-TCDD was added to each sample prior to the sample clean-up procedure. The samples were prepared for analysis by caustic potassium hydroxide saponification of the pesticide followed by extraction of the neutral TCDD into hexane. The hexane solution was cleaned-up by extracting several times with sodium hydroxide solution and sulfuric acid, and was then dried, concentrated and chromatographed on alumina. The eluate was concentrated under nitrogen for GC/MS analysis. The total TCDD in the technical grade samples was determined by low resolution gas chromatography/mass spectrometry -6using a Hewlett-Packard 5985 GC/MC in the electron ionization source configuration. The standard addition injection 3/ technique was used and selective ion monitoring at 320/322 m/e was performed for all samples. Complete mass spectra over the 150-350 range were obtained for all samples which gave a positive selective ion monitoring (SIM) scan. Six qualitative criteria for TCDD detection-were developed`to delineate the. likelihood of TCDD detection in the analytical measurement system; these criteria are summarized in Table I of Exhibit B. For positive TCDD detection the criteria included (1) a recovery of 37ci-TCDD between 50 and 120 percent, inclusive; (2) both 320 and 322 m/e peaks at the TCDD retention time region; (3) well-defined 320 and 322 m/e peaks whose retention time exactly equals that of TCDD standards; (4) a signal-to-noise ratio of at least 2.5 times background at m/e 322; (5) a ratio of 320/322 m/e peak areas in the proper isotopic proportion arid (6) TCDD confirmation by molecular ion spectra. The confidence in the TCDD detection was highest if all six criteria were met, and decreased as fewer criteria were met. Samples were processed in sets which included internal 4/ standards and EPA supplied quality controls. When the recovery J7 TCDD gives a characteristic 320/322 m/e ratio; the presence of the expected 320/322 ratio provides a strong indication of the presence of TCDD, if other necessary criteria are met. 4/ TCDD standards in benzene were supplied by Aubry Dupuy, Toxicant Analysis Center, EPA. a 14 ^ 0 -7fell within the specified contract range of 50-120% for a given sample, a duplicate assay was not performed; in cases where the recovery fell outside the specified range, a duplicate assay on an aliquot of the extract was performed. Contract requirements were satisfied if the average of the two recovery values fell within the specified range. Where the average recovery fell outside the'specification range for the two aliquots, the original sample was reextracted and assayed again. The submission of at least two coded duplicates by my laboratory for each sample assured that the TCDD determinations were each based on a minimum of two duplicate analyses. The contract did not require GSRI to report detectable values that fell below 10 ppb. Thus, although the mean detection limit achieved by GSRI was 4.7 + 3.7 ppb, any result below 10 ppb was reported as non-detectable. In some samples, however, the presence of TCDD was indicated at levels below 10 ppb even though these samples were reported as nondetectable for TCDD at the 10 ppb level. The results of the study are summarized in Table I. RiG^( -8 - TABLE I SUMMARY OF GSRI STUDY PESTICIDE NO. OF SAMPLES (PERCENT) */ NO. OF SAMPLES POSITIVE DOUBTFUL NON-DETECTABLE 2,4,5-T 3 1(33.3%) 1(33.3%) 1(33.3%) butoxyethyl ester of 2,4,5-T 10 9(90%) 1(10%) 0 isooctyl ester of 2,4,5-T '3 0 ,3(100%) ` 0 butoxyethyl ester of silvex 7 6(86%) 1(14%) 0 isooctyl ester of silvex 1 1(100%) 0 0 *7 A sample was Judged doubtful if it did not meet all the six criteria for positive TCDD detection. TCDD was detected at levels ranging from 10 to 30 ppb in 9 of the 10 samples of the butoxyethyl ester of 2,4,5-T, in 1 of the 3 samples of 2,4,5-T, and was not detectable in three samples of the isooctyl ester of 2,4,5-T. For the silvex samples, TCDD was detected in 6 of 7 samples of the butoxypropyl ester and in the one sample of the isooctyl ester of silvex at levels of 10-30 ppb. The results of the GSRI study are consistent with studies on 2,4,5-T and its derivatives performed, using methods similar to those employed by GSRI, by Buser and Bosshardt in 1974 and by Dow (Ramstad, Mahle, and Matalon) in 1977. The Li? -9- Buser and Bosshardt study, which describes a gas chromatographic method for TCDD determination in technical 2,4,5-T (with con firmation by mass spectrometry of samples with a TCDD content greater than 100 ppb) and a gas chromatography/mass spectrometry method for TCDD determination in a 2,4,5-T alkyl ester and a 2,4,5-T amine salt, is attached as Exhibit C. Table II contains a summary of the. r.esialts of the Buser and Eosshardt study. The ' TCDD content of these 2,4,5-T products ranged from 2 ppb to approximately 2000 ppb (or 2 pp m ) . TABLE II SUMMARY OF BUSER AND BOSSHARDT STUDY Pesticide a/ No. of Samples TCDD Content (PPb) 2.4.5- T (obtained from a German manufacturer prior to 1971) 3 300-600 2.4.5- T (obtained from a German manufacturer- after 1971) 4 40-80 2#4,5-T (obtained from an American manufacturer prior to 1971) 1 2000 2.4.5- T (obtained from an American manufacturer after 1971) 1 2 2.4.5- T alkyl ester (obtained from commercial sources in Switzerland in June, 1973) 2 20-50 2.4.5- T amine salt (obtained from commercial sources in Switzerland in June, 1973) 3 20-70 a7 The paper does not specify the number of duplicate determinations performed. 3464-3 -1 0 - The Dow study, which describes an automated adsorption chromatographic system for the clean-up of commercial samples of 2,4,5-T ester for TCDD analysis by gas chromatography/mass spectrometry, is detailed in Exhibit D. The results of this study are summarized in Table III. The TCDD content of these 2,4,5-T samples ranged from 8 ppb to 26 ppb. . TABLE III Pesticide SUMMARY OF DOW STUDY N o . of Samples a/ TCDD Content (ppb) l-isobutoxy-2-propyl ester of 2,4,5-T butoxypropyl ester of 2,4,5-T b/ 1 d/ 1 c/ 26 8 a7 The detection limit for TCDD was 5 ppb. b/ The isobutoxypropyl ester was analyzed 18 times. oj The standard deviation was + 2 ppb. d/. .The butoxypropyl ester sample was analyzed at least in duplicate. Exhibit E details a Dow method for the determination of TCDD in 2,4,5-T and related materials which is typical of the current state of the art in dioxin analysis. Appendix B contains a summary of confidential Dow documents obtained in discovery which report the presence of dioxin in commercial samples of 2,4,5-T and related products. Because Dow claims that this information is confidential, this testimony has been filed UNDER SEAL for use in camera. APPENDIX A Mass spectrometry is one of the most important tools available to the analytical chemist for the analysis, identifi cation, and confirmation of organic compounds. Gas chromatography (GC) is often used in conjunction with mass spectrometry (MS) to achieve a separation of the components that contributes to 1/ the overall sensitivity of the analytical determination. There are many variations in the instrumentation of modern GC/MS equipment, but basically all GC/MS systems contain (1) an inlet system for the test substance; (2) an ion source which bombards the test substance to produce ion fragments; (3) a mass analyzer which accelerates the ions at rates which depend on the mass/charge (m/e) ratio for the individual ions; (4) a collector on which ions of different m/e values are individually focused; and (5) a data processing system which gathers the spectral information and prints this information out on request. The two most widely used mass analyzers are (1) the magnetic analyzer, which usually achieves the acceleration of the ions by varying the magnetic field at a constant electric potential and (2) the quadrupole system, which depends on the oscillation of the ions at varying radio frequency levels in a constant electrostatic field to achieve different acceleration rates for the ions generated in the source. T 7 I n gas chromatography, the components of a sample travel through a chromatographic column by means of a carrier gas with retention times that depend on the physical and chemical characteristics of the components, the type of column used, and the running conditions for the instrument. ai.feit.5i 2 The most widely used technique for producing fragments in mass spectrometry is electron impact ionization, which produces a mass spectrum that is distinctive for the compound undergoing ionization. In electron impact ionization mass spectrometry, a substance is bombarded with an electron beam (usually 70. electron volts) to produce positive ion fragments, sometimes including a "parent ion", which gives the exact molecular weight of the compound. The largest fragmentation peak in the spectrum is called the "base peak" and is assigned a value of 100%; the other peaks, including the parent peak, are usually given as percentages of the base peak. To identify and/or quantify the molecular species under investigation, the analytical determination focuses on the fragmentation pattern, i.e. the m/e peaks, the parent peak and its isotope contributions, and the intervals between the peaks, as well as the abundance of the m/e species in the spectrum. Of particular importance are the isotope contributions to the parent peak (P), which are represented by the P + 1 and P + 2 peaks; reference to tables allows the determination of the molcular formula for a test compound on the basis of the P, P + 1, and P + 2 peaks. Compounds containing halogens (chlorine, fluorine, bromine and iodine) display a very distinctive set of peaks representing the isotopic contribution of the heavy halogen isotopes; for each halogen containing species, these -3- peaks appear in characteristic ratios which can be found in 2/ standard reference tables . The other prevalent method for generating ions is the 3/ chemical ionization method. In chemical ionization mass spectrometry, electrons are used to ionize a reagent gas which subsequently reacts with the sample molecules to produce ions. Since the ions produced by chemical ionization have a much lower internal energy than those produced by electron impact ionization, chemical ionization mass spectra usually exhibit intense ions in the molecular weight region, while electron impact ionization spectra contain a preponderance of highly fragmented ions. Chemical ionization mass spectrometry has a comparable sensitivity to electron ionization mass spectrometry, as well as the advantage of allowing a choice of reagent gases to best achieve the characterization of the sample. The mass spectra obtained by chemical ionization are often quite different from those obtained using electron impact ionization. Sample preparation, which usually entails such procedures as solvent extraction and chromatography, is similar for the two methods. ~2] The most abundant ions in the electron impact ionization mass spectrum of TCDD are those of the molecular ion chlorine isotope cluster at m/e 320, 322, 324, and 326. The ions at m/e 320 and 322 are the most intense. 3J In recent years, other methods such as field ionization and field desorption have received some attention, but these methods are not widely used. -4- For quantitative purposes, a technique known as selective ion monitoring (SIM) can be used to increase the sensitivity of the analytical determination. In this method, the instrument is set to detect only selected key ions, and a lower limit of detection is achieved for the ions of interest. Ronald F.Thomas 5U4.4 EXHIBIT LIST Exhibit A Woolson, E.A., Thomas, R.F. and Ensor, P.D.J. 1972. Survey of Polychlorodibenzo-p-dioxin Content in Selected Pesticides. J. Ag. Fd. Chem. 20: 2. Exhibit B Monalvo, J.G., Ryan, J.F. and Flagg, R. Analysis of Technical Grade Pesticides for TCDD at the ppb Level. EPA Project No. 68-01-3981. Physical Engineering Sciences Division, Gulf South Research Institute, New Orleans, Louisiana. Exhibit C Buser, H. and Bosshardt, H. 1974. Determination of 2,3,7,8TCDD at ppb Levels in Technical Grade 2,4,5-T in 2,4,5-T Alkylester and 2,4,5-T Amine Salt Herbicide Formulations by Quadrapole Massfragmentography. J. Chrom. 90: 71-77. Exhibit D Tore-Ramstad, Mahle, N.H. and Matalon, R. 1977. Automated Cleanup of Herbicides by Adsorption Chromatography for Determination of 2,3,7,8-TCDD. An. Chem. 49: Exhibit E Dow Chemical Company. Method ML-AM-75-34. Determination of TCDD in 2,4,5-T and Related Materials. Applicable to 2,4,5-T, Silvex and Chlorinated Phenols (unpublished). Exhibit F Documents which Dow claims contain confidential data on Do w 1s analysis of TCDD content in commerical 2,4,5-T produced between 1975 and 1979. (Filed Under Seal for use in in camera). certificate of service I hereby certify that copies of the foregoing DIRECT TESTIMONY OF MR. RONALD THOMAS were hand delivered or mailed first class postage prepaid on February 26, 1980 to the persons on the attached list. Cara S//Jablon February 26, 1980 ACTIVE SERVICE LIST FOR RISKS Edward W. Warren L. Mark Wine Richard L. McConnell, Jr. Kirkland & Ellis Counsel for Dow Chemical Company 1776 K Street, N.W., 12th Floor Washington, D.C. 20006 William A. Butler, Esq. Jacqueline M. Warren, Esq. Counsel for Environmental Defense Fund, Inc. 1525 - 18th Street, N.W. Washington, D.C. 20036 Margaret M. Breinholt Judith A. Wenker Terrence G. Jackson Room 2036, South Ag. Bldg. Office of the General Counsel U .S . Department of Agriculture Washington, D.C. 20250 Robert S. Kirk, Jr., Esq. Counsel for Vertac, Inc. 2414 Clark Tower 5100 Poplar Avenue Memphis, Tennessee 38137 Richard J. Wertheimer, Esq. Arnold & Porter Counsel for National Forest Products Association 1229 Nineteenth Street, N.W. Washington, D.C. 20036 Stephen W. Jacobson Joseph E. Stevens, Jr. William Ray Price, Jr. Lathrop, Koontz, Righter, Clagett, Parker & Norquist 2600 Mutual Benefit Life Building Post Office Box 1200 2345 Grand Avenue Kansas City, Missouri 64108 Marla GiIlham Northwest Coalition for Alternatives to Pesticides, 454 Willamette Street Eugene, Oregon 97401 Inc. Allen A. Lauterbach John J. Rademacher American Farm Bureau Federation 425 - 13th Street, N.W. Washington, D.C. 20004 Sonia G. Anderson Hearing Clerk (A-110) U.S. Environmental Protection Agency 401 M Street, S.W. Washington, D.C. 20460 Elizabeth M. Whelan, Sc. D., M.P.H. Executive Director American Counsel on Science & Health 1995 Broadway New York, New York 10023 U>-L EXHIBIT " \- Reprinted from A G R I C U L T U R A L A N O F O O D C H E M I S T R Y . Voi. 20, No. 2, Page351, March/April 1972 Copyright 1972 by the American Chemical Society and reprinted by permission of the copyright owner. Survey of Poly.chlorodibenzo-p-dioxin Content in Selected Pesticides MAR 2 1980 Edwin A. Woolson,* Ronald F. Thomas, *1and Peter D. J. Ensor One-hundred-twenty-nine samples of 17 different pesticides derived from chlorophenols were ex amined for polychlorinated dibenzo-p-dioxins by electron capture gas chromatography (ec-gc). The method of cleanup involved a concentrated sulfuric acid extraction of impurities from hexane and a mild nitration of the chlorophenol extracts. Seventy-six percent of the samples analyzed contained less than 0.1 pg/g of 2,3,7,8-tctrachlorodibenzo-p-dioxin (TCDD) in the technical material, whereas 7 % con tained between 0.1 to 1.0 /g/g, and 9% contained greater than 10 tg/g TCDD. N o TC D D was de fected in the 20 tri-, tetra-, or pentachlorophenol samples examined. However, high levels of other dioxins were found. All samples which contained more than 1.0 g/g of dioxin by ec-gc were confirmed by gas chromatography using a flame ionization de tector (fid-gc), a microcoulometric detector (mc-gc), p values, uv irradiation, and/or gas chromatography/ mass spectrometry (gc-ms). Samples of phenoxy herbicides from current production contained lessthan 0.5 ig/g TCDD. Polychlorodibenzo-p-dioxins may be contaminants in chlorophenols or in pesticides which use chlorophenols in the manufacturing process. Chlorinated dibenzo- o f dioxins in the 17 different pesticides. The method developed and results o f the survey are reported in this paper. p-dioxins are hazardous materials and may cause skin erupP ROCEDURE tions, teratogenesis, and are toxic to animals at low levels. The dioxins are formed usually when the reaction tempera ture for making o-chlorophenol by hydrolysis exceeds 160C under pressure. The reaction may be an alkaline hydrolysis of polychlorobenzene or a chlorination of phenol to form a polychlorophenol. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, Figure 1) is the dioxin that may be formed when the reaction tempera ture for making 2,4,5-trichlorophenol from tetrachlorobenzene exceeds 160C (Schultz, 1968). In chlorinating phenol lo make tetra- or pentachlorophenol, heat must be supplied to the reaction mixture, in order to maintain the mix as a melt. If too much heat is supplied, hexa-, hepta-, and/or octa:hlorodibenzo-p-dioxins may be formed. Since little or no neat is required to form di- and trichlorophenols by chlorina tion, there is little likelihood that di-, tri-, or tetrachlorodibenzo-p-dioxins will be formed in this process. Interest in dioxins originated in 1957 from outbreaks o f "chick edema (CE) disease." The disease was characterized by hydroparicardium in chickens. In 1958 a toxic substance, isolated from an unsaponifiable fraction pf feed fats which caused CE, was identified (Cantrell et ai., 1969) by single crystal X-ray crystallography as 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (HCDD). In 1970, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was implicated as a potential teratogen in pregnant rats (Courtney et al., ' 1970). Later tests indi cated that the teratogenesis may have been caused by 27 8 ppm of TCDD present as contaminant in the 2,4,5-T. In order to assess possible environmental contamination with TCDD or higher chlorinated dioxins through pesticidal materials, samples of 17 pesticides based on use o f chloro phenols in their manufacturing process were collected from U.S. Department o f Agriculture's former Pesticide Regula tion Division (PRD) laboratories. Additionally, samples of 2,4,5-T from one manufacturer, covering a 3-year period, were collected. A method was developed for the analysis Materials. N o attempt should be made to analyze for dioxins without proper safety procedures. The following solvents were distilled in glass or were o f pesticide grade quality: hexane, methanol, petroleum ether, acetonitrile, benzene, and diethyl ether. The following apparatuses were required: chromatography columns (450 X 19 mm i.d.) with Teflon stopcock, gas chromatograph (gc), preferably equipped with a N i63 detector (flame ionization or microcoulometric detectors also desirable for confirmation steps), glass gc columns (1.83m X 4-mm), a sun lamp with maximum output at 310 nm, and thin-layer chromatography (tic) apparatus. The following compounds were examined for polychlorodibenzo-p-dioxins: (2,4-dichlorophenoxy)acetic acid, 2,4-D; 4-(2,4-dichiorophenoxy)butyric acid, 2,4-DB; 2-(2,4-dichlcrophenoxy)propionic acid, 2 ,4 -D P (2 ,4 ,5 -tric h lo r o phenoxy)acetic, 2,4,5-T; 2-(2,4,5-trichlorophenoxy)propionic acid, silvex; 2-(2,4-dichlorophenoxy)ethyl sodium sulfate, sesone; 3,6-dichloro-o-anisic acid, dicamba; pen tachlorophenol, PCP; tetrachlorophenol; trichlorophenol; 2,4-dichlorophenyl p-nitrophenyl ether, nitrofen; 0-(2,4dichlorophenyl) O-methyl. isopropylphosphoramidothioate, DMPA; 2-(2,4,5-trichlorophenoxy)ethyl 2,2-dichloropropionate, erbon; tris[2-(2,4-dichlorophenoxy)ethyl]phosphite, 2,4DEP; 0,0-dim ethyl 0-2,4,5-trichlorophenyl phosphorothionate, ronnel; 2,4,5,4'-tetrachiorodiphenyl sulfone, tetradifon; and 0-2,4-dichlorophenyl 0,0-diethyl phosphorothicate, VC13, Nemacide. The cleanup procedure consisted o f three steps which were dependent on the nature of impurities found in and the com plexity o f the sample. Step A. T echnical Salts and Acids of Phenoxy Acids. One-hundred milliliters of MeOH was added to 3.00 g of herbicide and 10 ml of 5 N KOH contained in a 1000-ml separatory funnel. Two-hundred milliliters of H 20 and 100 ml o f hexane were added after 20 min and shaken. The aqueous phase was reextracted with 100 ml of hexane and the Plant Science Research Division, Agriculture Research Service, U.S. Department o f Agriculture, Beltsviile, Maryland 20705. 1 Present address: Pesticide Regulation Divison, Environ mental Protection Agency, Beltsville, Maryland 20705. extracts were combined. The hexane extracts were washed twice with 100 ml o f 1 /V NaOH, twice with 1 N HC1, and twice with 100 ml of HsO. The extracts were dried over anhydrous NajSCL, transferred to a 250-ml beaker, and evap orated to ca. 10 ml.. The sample was transferred to a pre- . J. A G R . F O O D C H E M ., V O L . 20, N O . 2, 1972 3 5 1 ` ; ' j : 1 | ; j j ; ! J j j (sj53 WOOLSON, THOMAS, ENSOR . Table I. Relative Retention Times of Several Chlorinated Hydrocarbon Insecticides and Polychlorodibcnzo-p-dioxins on Five Gas Chromatography Columns'* 5% OV-225. Relative retention times* on columns 1.5% OV-17 + 2.0% QF-1 (1:1) 5% OV-17 5% UCW-98 15% QF-l + 10% DC-200 (1:F Aldrin p.p'-'DDE p,p'-TDE p,p'-DDT Endrin Endrin, A keto 2,7-CDD* 2,3,7-CDD tri-CDD tetra-CDD' tetra-CDD' 2,3,7,8-CDD penta-CDD hexa-CDD (1) hexa-CDD (2) hexa-CDD (3) hepta-CDD (1) hepta-CDD (1) octa-CDD 1.00 (2.17)' 2.63 5.84 5.62 3.69 15.76 1.29 1.94 2.30 2.86 3.27 4.33 8.29 11.61 13.13 16.45 23.32 26.96 46.31 1.00 (1.80) 2.50 3.62 4.17 2.78 6.89 1.00 1.67 1.83 2.50 2.52 3.44 6.56 9.28 10.56 12.44 18.39 21.17 36.28 1.00 (0.91) 1.98 - 3.19 d 3.08 8.57 1.21 2.09 7.10 2.31 2.33 3.41 7.18 8.24 11.10 13.19 15.82 23.41 1.00 (1.26) 1.67 2.14 2.70 1.90 3.25 0.83 1.51 1.57 2.22 2.24 2.865.08 7.06 7.94 9.97 13.09 14.68 24.52 1.00 (4.2) 1.90 2.64 3.14 2.64 5.48 1-.02 1.64 1.83 2.38 2.52 3.19 5.48 7.26 8.09 9.45 13.26 14.88 24.05 C o lu m n co n d itio n s: length, 1.8-m X 4 -m m glass; solid su p p o rt, C h ro m o so rb W , 8 0 -1 0 0 m esh ; in jecto r, 2 4 0 C ; co lu m n , 2 2 0 * 0 ; d etecto r, Ni**, 3 1 0 C ; flow ra te , 8 0 -1 0 0 m l/m in . * 1 % , T im e in m in u te s in p a re n th e se s. d N o t re so lv ed fro m T D E . C h lo ro d ib e n z o -p -d io x in GDI). / M o re th a n o n e iso m e r w as p re s e n t in th e tri-, te tra -, h e x a-, a n d h e p ta c h lo ro d ib e n z o -p -d io x in s . K n o w n iso m e rs h a v e th e p o s itio n s n u m bered. '\ ; Figure 1. Structure of dibenzo-p-dioxin. Chlorines may be at tached at the 1,2,3,'4,6.1,8, and/or 9 positions to yield chlorodibenzop-dioxins washed (petroleum ether) 15-g AI20 3 column (450 X 19 mm i.d.) and eluted with 100 ml of petroleum ether followed by 50 ml of 5% (v/v) diethyl ether in petroleum ether, which were discarded. One-hundred milliliters o f 50% (v/v) di ethyl ether in petroleum ether was collected, evaporated just to dryness, and.adjusted to volume with hexane. The amount of each dioxin present was determined. Step B. L ow Organic M atter Sorts, P henol-Based I nsecticides, Oil, and E ster F ormulations o f Phenoxy H erbicides. The following steps were performed prior to the A12O j column in Step A. The extract was shaken with concentrated H-SCL until the acid was clear (not yellow or cloudy after standing ca. 10 min), washed with H20 , and passed through a N aH C O j-N a^O , column (1 cm o f each layered in a 19 mm i.d. column). The eluant was evaporated to ca: 10 ml and continued with the A1>03 column in Step A. Step C. Chlorophenols, H igh O rganic M atter Soils, and Other Samples D ifficult to Clean U p. After com pleting parts A and B of the cleanup procedure, the eluant from the A120 3column was evaporated just to dryness, cooled to ca. 0C on an ice bath, and 10 ml of 1 :1 (v/v) H N 0 3:H2SO< mixture was added. The mix was gradually warmed to room temperature and added to 50 ml o f ice water in a sep aratory funnel after 15 min. The beaker was rinsed with 5 X 10* ml o f hexane. The combined hexane rinses were 3 5 2 J. A G R . F O O D C H E M ., V O L . 20, N O . 2, 1972 shaken for 1 min, washed with H20 , and the hexane was drained through a N a H C 0 3-N a 2S 0 4 column. The volume was adjusted for analysis. Quantitation was made on a 5% OV-225 column using electron-capture gas chromatography (ec-gc). Analysis was made with temperature programming from 230 to.260C at 4C/min and a 16-min hold at the final temperature. Confirmation Techniques for Dioxins. I f a positive re sponse was found by ec-gc, the p value was determined for the dioxin between hexane and acetonitrile on a portion of the extract. The p value equals final concentration in hexane, initial concentration in hexane (Beroza and Bowman, 1965). Another .portion of the extract was irradiated for 16 hr under a sun lamp (maximum output, 310 nm) at a distance o f 30 cm since T C D D and the lower dioxins are destroyed under these conditions. The remainder o f the extract was condensed for confirma tion on different gc columns using different detectors. The columns preferred were 1 :1 (w/w) mix o f 1.5% OV-17 plus 2.0% QF-1 and 5% UCW-98, both on Chromosorb W, 80100 mesh. Flamfe- ionization and microcoulometric detec tion were with these detectors. The ultimate confirmation was provided by a Perkin-Elmer Model 270 gc-mass spectrometer (gc-ms). There was very little fragmentation since the parent peak (m/e 322) was the parent ion and m/e 257 and 194 were the only fragments over 20% in relative abundance for the tetradloxin. Two-dimensional tic was used for cleanup and partial con firmation occasionally. The plate was developed first in acetonitrile followed by benzene. The spots were visualized under a uv lamp and were scraped off, extracted, and assayed by gc i f desired. Infrared spectrometry was also used to confirm the identity o f various chlorodioxins. ) R ESU L TS A N D D ISC U SS IO N . Relative rtention times o f 13 dioxin isomers and a few selected chlorinated hydrocarbon insecticides are presented in Table I. , The values were determined isothermally at ^ ' - j j j ; 3-i ' POLYCHLORODIBENZO-p-DIOXIN 220C. Because of the large value (rt. = 46.31) for the octaCDD, the 5% OV-225 column was normally used with tem perature programming from 230 to 260C at a rate o f 4C / min and a final temperature hold. Under these conditions, malysis was completed in about 40 min. The use o f the tree recommended columns (5% OV-225, 1.5% OV-17 plus 2% QF-1, and 5% UCW-98) prevented confusion o f some chlorinated hydrocarbon insecticides in environmental sarn ies with dioxins because relative retention times were suffi.tently different. p Values for dioxins are presented in Table II. Values for four specific isomers (2,7-, 2,3,7-, 2,3,7,8-, and 1,2,3,4,6,7,8,9chlorodibenzo-p-dioxins) are given. Other values are for mixtures of isomers and were determined using total peak area or peak height. R t values for all dioxin isomers-by tic in acetonitrile were 0.82 0.08 and in benzene were 0.80 0.02. The di-, tri-, tetra-, and hexadioxin were not separated from each other under our test conditions, but were separated from most interfering impurities in formulations. Care must be taken not to overload the spots or the material appears to stay on the origin. This behavior is presumably due to the low solubility.of dioxins in most solvents. In a further confirmatory step, dioxins were destroyed by uv light. The lower isomers (< 4 Cl) were completely de stroyed in 16 hr by irradiation (Crosby etal., 1971) at concen trations less than 0.2 ppm in hexane. Nondestruction o f the suspected di-, tri-, or tetrachlorodioxin is sufficient evidence that the peak is not a chlorodioxin. The higher the degree of chlorination, the more resistant the dioxins were to irradi ation. Only 20% o f the octa-CDD was destroyed in 16 hr at concentrations o f about 0.3 ppm. A total o f 129 pesticide samples were analyzed for dioxins. Table III presents the occurrence and content of the dioxins in all samples. TCDD (tetra-) was found primarily in 2,4,5-T samples. Only one sample o f silvex contained more than 0.5 ppm o f TCDD. Detectable amounts o f hexa-CDD were found in four 2,4,5-T samples, one 2,4-D, and two other sam ples other than the chlorophenols. Hepta- and octa-CDD also occurred in four other samples apiece in addition to their presence in chlorophenols. Most samples were made before 1970 and may not represent current production materials. The TCDD concentration in selected pesticides is also pre sented in Table III. Twenty-two o f 42 2,4,5-T samples col lected contained less than 0.5 ppm of TCDD. 'O f the 20 samples containing more than 0.5 ppm of TCDD, 15 were obtained for the yearly .survey o f one manufacturer. The samples were from 1966-1970, with four.sam ples usually collected each year. There was a tenfold drop in TCDD Table II. p Values for Chlorodibcnzo-p-dioxins in a 1:1 (v/v) Acetonitrile :Hcxanc Solvent System " Isomer p Value std dev 2,7-CDD 2,3,7-CDD 2,3,7,S-CDD hexa-CDD hepta-CDD octa-CDD 0.76 0.86 0.51 0.94 0.90 0.90 0.03 ' 0.03 0.06 0.03 0.05 0.05 " R a tio o f.p e a k h eig h t in h ex an e so lu tio n b efo re e q u ilib ra tio n w ith h e x an e-satu rated aceto n itrile to p eak height in hexane a fte r eq u ilib ra tio n . Figure 2. Infrared spectra of authentic octachlorodibenzo-pdioxin and a cleaned-up pentachlorophenol extract. The material was incorporated in KBr and the spectra made on a Perkin-EImer 625 infrared spectrometer content by this manfacturer between 1968 and 1969. How ever, their technical 2,4,5-T still contained 2-3 ppm o f TCDD in 1970. Four o f the five remaining samples containing over 0.5 ppm of TCDD also came from this same manufacturer and were received from the PRD laboratories. Recent (1970) samples from another manufacturer contained <0.5 ppm of TCDD. N o other samples tested had over 0.5 ppm of TCDD. Any sample could contain one or more different chlorodioxins. The higher polychlorodibenzo-p-d ioxin content o f the same selected pesticides is also presented in Table III. Four sam ples o f 2,4,5-T contained greater than 0.5 ppm of the hexa isomers. N o higher dioxins were found in silvex or dicamba Pesticide Table III. Number and Content of Pol ychIorod ibenzo-p-d iox ins in Selected Pesticides _____________________ ppm of -chlorodibcnzo-p-dioxin___________________________ tetra hexa hepta octa <10 <100 <10 <100 <1000 <10 <100 <1000 <10 <100 <1000 No. of Total samples no. of contam- samples inated tested 2,4,5-T Silvex 2,4-D (-DB.-DP) Dicamba Chlorophenol tri- tetrapenta- Others 7" 1 ND ND ND ND ND ND 13 3 0 ND 1 ND 4 1 0 1 1 . 0 ND6 ND 0 ND ND ND ND ND ND 0 01 1 01 7 00 0 13 0 0 2 2 01 4 60 0 13 00 20 46 10 Any sample may contain one or more different dioxins. 4ND = <0.5 ppm ofany one chlorodioxin. 23 42 17 1 28 08 46 33 10 11 7 24 J. A G R . F O O D C H E M ., V O L . 20, N O . 2, 1972 3 5 3 SLIC SA C H ER . O LIN and only one sample of 2,4-D contained measurable amounts o f the hexa isomers. The hexa isomers present presumably were there because o f tetrachlorophenol impurities which condensed with each other. Higher dioxins were found in some samples of several other pesticides, including erbon, tetradifon, ronnel, sesone, and DMPA. The dioxin content o f the 20 chlorophenol samples is also presented in Table III. N o TCDD was detected in any sam ple at levels above 0.5 ppm. However, the higher dioxins were plentiful. Trichlorophenol contained only small amounts of hexachlorodibenzo-p-dioxin and no sample con tained over 10 ppm. Tetrachlorophenol contained less than 100 ppm of hexa-, hepta-, and octachlorodibenzo-^-dioxins, while six o f 20 pentachlorophenol samples contained over 100 ppm of the hepta- and oetachlorodibenzo-/?-dioxin iso mers. An infrared spectrum o f a pentachlorophenol ex tract is presented in Figure 2. It is quite obvious that the extract contains octachlorodibenzo-p-dioxin when the spec trum is compared to the standard octachlorodibenzo-p-dioxin spectrum. Analysis by ec-gc indicated both hepta- and octachlorodioxins were present in the sample. This may account for the peak broadening. The reason for the high amounts of dioxin in chlorophenol is probably due to heat treatment during synthesis. As chlorination o f phenol pro ceeds past the dichlorophenol stage, heat must be supplied in order to keep the reaction mixture in a melt condition. Since heat is being supplied in the presence o f chlorophenols, the formation o f higher chlorinated dibenzo-p-dioxins might be expected. The rate and time of melt heating probably governs the formation and amounts of the various dioxin isomers. If the chlorination temperature is raised too high too quickly, the lower chlorinated dioxins may be formed since the lower chlorophenols would be present and availab' for reaction with each other. If the temperature is raised toe high after nearly all chlorophenol is in the penta form, octachlorodibenzo-p-dioxin would be the predominant impurit formed. In conclusion, TC D D has been present at levels above 0.5 ppm in the past, but was less than 0.5 ppm in the current pro duction samples examined. Higher chlorodioxins are pres ent predominantly in chlorophenols with the highest amounts present in pentachlorophenol. Thirty-eight percent o f all samples examined contained at least one chlorodioxin, with many containing more than one. LITERATURE CITED Beroza, M., Bowman, M. C., J. Ass. Offic. Anal. Chem. 48, 358 (1965). Cantrell, J. S., Webb, N. C., Mabis, A. J., Acta Crystallogr. B25, 150 (1969). Courtney, K. D., Gaylor, D. W., Hogan, M. D., Falk, H. L:, Bates, R. R., Mitchell, I., Science 196,864 (1970). Crosby, D. G., Wong, A. S., Plimmer, J. R., Woolson, E. A., Science 173, 748 (1971). Schultz, K. H., Arbeitsmedizin-Socia/medizin-Arbeitshygiene 3, 25 (1968). Receivedfor review August 19,1971. Accepted November 15,1971. EXHIBIT B ! SU&ST7 ANALYSIS OF TECHNICAL GRADE PESTICIDES FOR TCDD AT PARTS-PER-BILLION LEVEL by Joseph G. M ontalvo, J r . , James F. Ryan, I I I , and Roy Flagg P hysical and Engineering Sciences D ivision Gulf South Research I n s titu te New O rle a n s, L o u isia n a 70186 Number 68-01-3981 Project O fficer P a tric ia Ott Environmental Fate Branch of the Hazard E valuation D ivision O ffice of P e s tic id e Programs W ashington, D.C. 20460 OFFICE OF PESTICIDE PROGRAMS OFFICE OF TOXIC SUBSTANCES U.S. ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 -DISCLAIMER This re p o rt has been reviewed by the Environmental Fate Branch of the Hazard E valuation D iv isio n , U.S. Environm ental P ro te c tio n Agency, and not approved for p u b lic a tio n . Approval does not sig n ify th a t the contents n e c e s s a rily r e fle c t, th e views and p o lic ie s of th e U.S. Environm ental P ro te c tio n Agency, nor does mention o f tra d e names o r commercial products c o n s ti tu te endorsement or recommendation f o r use. ' t ii .ABSTRACT Gulf South R esearch I n s t i t u t e (GSRI) analyzed 69 te c h n ic a l grade p e stic id e batch samples (Silvex; 2 ,4 ,5 -trich lo ro p h en o x y acetic acid ; an organophosphate; 2 ,4 ,5 -trich lo ro p h en o l; and'a chlorinated phenolic a n tis e p tic ), 31 r e p l i c a t e sam ples o f th e se b a tc h sam ples, and 41 q u a lity c o n tr o l sam ples f o r t o t a l te tra c h lo ro d ib e n z o -p -d io x in (TCDD). The samples were e x tra c te d w ith hexane and the e x tra c ts were d rie d , co n cen trated , and chromatographed on alum ina. Samples were q u a lita tiv e ly and q u a n tita tiv e ly analyzed by gas chrom atography/m ass sp ec tro m e try (GS/MS) in th e s e le c tiv e io n m o n ito rin g (SIM) mode. C e rta in ty o f TCDD d e te c tio n was a ss e ss e d v ia summation o f s ix q u a lita tiv e c r i t e r i a . Based on th is assessm ent, the d a ta were ra te d p o s itiv e , d o u b tfu l, or no n d etectab le. For those a u th e n tic samples in which d e te c tio n was Judged p o s itiv e , le v e ls o f t o t a l TCDD ranged from n o n d e te c ta b le to 30 ppb; q u a lity c o n tr o l sam ples c o n tain ed up to 3984 ppb. The u se of h ig h e f f ic ie n c y c a p i l l a r y column GC/MS i s recommended f o r fu tu r e TCDD i n v e s t i gations. t iii Q-i (o4>0 CONTENTS A b s t r a c t.......... F ig u r e s ............. T a b le s ............... Acknowledgmen t iii v vi v ii 1. 2. 3. 4. **^5. In tr o d u c tio n ..................................... . C o n clu sio n s............................................. .. Recommendations............................ ............... E xperim ental P ro c e d u re s.......................... Safe Handling In the Laboratory General Procedures. R e s u lts and D is c u s sio n ............... .. Q u a lity A ss u ra n c e ........................... 1 4 5 6 6 6 11 11 R e fe re n c e s............................................... Appendices 20 A. D ata s h e e ts f o r sample 148919-5-2.............................. ....................... . . . . 38 B. M aster sample s ta t u s form ................................................................................. 41 C. : Cum ulative ta b u la te d a n a l y t i c a l d a ta form ............. 43 D. GC/MS perform ance io n c u r r e n t c h ro m a to g ra m s .* ................... 45 ^ E. Io n c u rre n t chromatograms f o r TCDD d e te c te d in a p e s t i c i d e sam ple.................................................................... 50 F. Mass spectrum f o r TCDD d e te c te d in a p e s ti c id e s a m p l e . . , ............. 53 G. Mass abundance ta b u la tio n f o r TCDD d e te c te d in a p e s t i c i d e sam ple............................................................................................... 56 H. Ion c u rre n t chromatograms f o r n o n d e te c ta b le TCDD in a p e s t i c i d e sam ple............................................................................................................... 58 I . Ion c u r r e n t chromatograms f o r q u e s tio n a b le TCDD d e te c tio n in a p e s ti c id e sam ple.......................................................................................... 63 iv s u .4 r ________ FIGURES Number Page 1 S tr u c tu r e of 2,3,7,8-TCD D ......... ..................... .......... ................................ .. 2 2 Q u a lity c o n tro l check f o r GC/MS p e rfo rm a n c e ..................................................12 3 Method b lan k q u a lity c o n tr o l c h a r t ....................................................................... 13 4 Accuracy q u a lity c o n tr o l c h a r t . . . . ........................................................................14 V 1 i ; ___ T A B L E S Number [ Page 1 Q u a lita tiv e C r i t e r i a E x p la n a tio n ........... ......... ...................................................... 21 2 Dependence o f C r ite r io n #6 O v e ra ll B inary C h a r a c te r is tic on S u b c r ite r ia R e s u l t s : .......................................................................................... 22 3 TCDD D e te c tio n Judgement Based on Q u a lita tiv e C r i t e r i a C h a r a c t e r i s t i c s ...................................................................................................... 23 **`4 ^V arious EPA Samples A n a ly z e d 'fo r T o ta l T C D D . r . . T . 24 5 TCDD Data f o r A u th e n tic 2 , 4 , 5 -T ric h lo ro p h e n o l............................. .................. 25 6 TCDD Data f o r A u th e n tic 2 ,4 ,5 -T (2 ,4 ,5 -T ric h lo ro p h e n o x y a c e tic Acid) . .........................................................1.............................................................. 26 7 TCDD D ata f o r A u th e n tic B utoxyethyl E s te r of 2 ,4 ,5 - T .................................... 27 . i 8 TCDD Data f o r A u th e n tic I s o o c ty l E s te r o f 2 ,4 ,5 - T ..... 28 - * _* 9 TCDD Data f o r A u th e n tic B utoxypropyl E s te r o f S ilv e x ................................. 10 TCDD. D ata f o r A u th e n tic I s o o c ty l E s te r of S i l v e x . . .................'. .................. 30 11 TCDD Data f o r A u th e n tic C h lo rin a te d P h en o lic A n t i s e p t i c . . . . T................... 31 12 TCDD Data f o r A u th e n tic O rganophosphate............ ................................................. 32 13 TCDD D ata f o r Q u a lity C o n tro ls .................................................................................... 33 14 V a ria tio n o fTCDDD e te c tio n Judgement V ersus EPA Sample Type........................ 34 15 V a ria tio n o f T o ta l TCDD Level V ersus Sample Type f o r P o s itiv e Sam ples............................................. 35 16 Comparison of R e p lic a te R e s u lts f o r EPA Q u a lity C o n tro l Sam ples............... 36 `17 Comparison of R e p lic a te R e s u lts f o r A u th en tic P e s tic id e Batch Sam ples....................................................................................................................... 37 vi . ...... - . .................... ........... ACKNOWLEDGMENT -:---------------------------- ------------------- The c o o p e ra tio n o f Mr. Ronald F. Thomas, S u p erv iso ry C hem ist, B e n e fits and F ield S tudies D ivision, Environm ental P ro tec tio n Agency, in sampling design and shipment of the samples to Gulf South Research I n s titu te is a p p re c ia te d . The a s s is ta n c e of Ms. P a t r i c i a O tt, P r o je c t O f f ic e r , i s g r a t e f u l l y acknowledged. The a s s is ta n c e of Dr. Mary M. McKown, GSRI s e n io r a n a ly tic a l chem ist, throughout the p ro je c t performance is ap p reciated . J .. v i i ' I .SECTION 1 INTRODUCTION P o ly c h lo rin a te d d ib e n z o -p -d io x in s (PCDDs) may be formed a s u n d e s ira b le tr a c e im p u r itie s in th e p ro d u c tio n of te c h n ic a l grade c h lo ro p h e n o ls . The l a t t e r a re employed e x te n s iv e ly as p e s t i c i d e s , a s wood p r e s e r v a tiv e s and as s ta r tin g m a te ria ls fo r a s e rie s of o th e r products. Through c a re fu l co n tro l over conditions in the p e stic id e m anufacturing p ro cess, only minimal q u a n titie s of PCDDs a re form ed, b u t a t te m p e ratu res above 200C a condensa tio n o f c h lo ro p h e n o ls may occur w ith fo rm atio n o f in c r e a s in g q u a n titie s of PCDDs (1 ) . _ . _ ____ _ " S e v e ra l PCDDs have been found to be h ig h ly to x ic and p o s s ib ly c a rc in o genic and are s ta b le in b io lo g ic a l systems (2 ). T h e o re tic a lly , a to ta l of 75 d i f f e r e n t PCDDs e x i s t , in c lu d in g 22 te tra c h lo ro d ib e n z o - p -d io x in s . The to x ic o lo g ic a l p r o p e r tie s o f p o s itio n a l isom ers of th e same d io x in may v ary s ig n if ic a n tly . Since th e 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n z o -p -d io x in (Figure 1) is ex trem ely to x ic ( 3 ) , TCDDs d e se rv e s p e c ia l c o n s id e r a tio n . A lthough th e s y n th e tic ro u te fo r TCDD fo rm atio n d u rin g ch lo ro p h en o l m an u factu re fa v o rs 2,3,7,8-TCDD, o th e r te tra c h lo ro d ib e n z o - p -d io x in iso m ers may a ls o be formed .(A).- .. ' Under the aeg is of the F ederal In s e c tic id e , F ungicide, and R odenticide Act (FIFRA), th e U.S. E nvironm ental P r o te c tio n Agency (EPA) has s e t maximum TCDD co n tam in atio n l i m i t s in te c h n ic a l grade h ex achlorophene a t 100 ppb (0 .1 ppm) in o rd e r to reduce human and en v iro n m en tal e x p o su re. The purpose of th e GSRI re s e a rc h e f f o r t was to d eterm ine le v e l s o f TCDD p re s e n t in te c h n ic a l grade p e s t i c i d e sam ples su b m itted by EPA. In view of th e in h e re n t t o x i c i t y o f TCDD, s p e c ia l p re c a u tio n s in handling are required during a n a ly sis. C oncentration by sev eral orders of magnitude i s sometimes n e c e ssa ry , and an u ltr a s e n s itiv e d e te c tio n method i s p re fe rre d . A com bination of gas chromatography and mass spectrom etry has been shown to be an e x c e lle n t method f o r d e te c tin g TCDDs a t v e ry low le v e l s ( 5 ) . I d e n t i f i c a t i o n o f in d iv id u a l TCDD iso m ers r e q u ir e s an i n i t i a l , h ig h e f f ic ie n c y s e p a ra tio n such as g la s s c a p i l l a r y gas chrom atography (GC) p r io r to mass s p e c tro m e tric (MS) a n a ly s is . Low r e s o lu tio n GC colum ns, i . e . , packed columns, p ro v id e a co n v en ien t means f o r TCDD isom er summation ( t o t a l TCDD) by GC/MS. The p re s e n t stu d y has focused on d e te rm in a tio n of t o t a l TCDD in te c h n ic a l grade samples by low re s o lu tio n gas chrom atography/m ass s p e c tro m etry. Sample p re p a ra tio n in clu d e d a d d itio n of Cl 2,3,7,8-TCDD to a s s e s s 1' I Figure 1. S tru c tu re of 2,3,7,8-TCDD. L I 2 j; c-lQ>(oG d io x iir re c o v e ry ,, s o lv e n t e x tr a c ti o n , and cleanup on alum ina. GC/MS a n a ly se s were c a rrie d out using a packed column; the standard a d d itio n in je c tio n te ch n iq u e was used. S e le c tiv e io n m o n ito rin g (SIM) and com plete mass s p e c tr a l d ata' were o b ta in e d . S ix q u a l i t a t i v e c r i t e r i a f o r TCDD d e te c tio n were developed in co rp o ratin g the p re c isio n of the a n a ly tic a l measurement system . The p r o je c t q u a lity a ssu ra n c e p ro to c o l included i n t e r n a l q u a lity c o n tro l samples (p rec isio n and accuracy) and q u a lity c o n tro l c h a rtin g . \ 1 I cR. I Q <9 ' N ....... ...... . _______ ... SECTION' 2 _________ ________ __ ________________ CONCLUSIONS ` ! The r e s u lts of low re s o lu tio n gas chrom atographic/m ass sp ec tro m e tric : a n a ly s is o f 141 samples (in c lu d in g q u a lity c o n tro ls and a u th e n tic p e s tic id e sam ples c o n ta in in g unknown q u a n t i t i e s of TCDD) fo r t o t a l te tr a c h lo r o d ib e n z o - ! * p -d io x in have in d ic a te d l e v e l s ra n g in g from n o n d e te c ta b le to 30 ppb f o r 69 a u th e n tic p e s tic id e b atch and 31 r e p lic a t e samples and n o n d e te c ta b le to ; 3984 ppb f o r 41 q u a lity c o n tr o l sam ples. TCDD was n o t d e te c te d (d e te c tio n l i m i t 10 ppb) in 66 p e rc e n t o f th e a u th e n tic sam ples, d e te c tio n was d o u b tfu l in 9 p e rc e n t and p o s itiv e in 25 p e rc e n t. Whereas TCDD could n o t be d e te c te d - in a m a jo rity of a u th e n tic sam ples, p o s itiv e samples co n tain ed 10-30 ppb t o t a l TCDD. 4 .. SECTION 3 RECOMMENDATIONS The in v e s tig a tio n of t o t a l te tra c h lo ro d ib e n z o -p -d io x in le v e ls in compounds fo r whose s y n th e s is 2 ,4 ,5 -tr ic h lo ro p h e n a l i s used should be c o n tin u e d . In view of th e known t o x i c i t y o f th e 2,3,7,8-TCDD isom er and incom plete sep aratio n of the isom ers using packed columns, fu tu re in v e stig a tio n s should be performed using high e ffic ie n c y gas chromatography (c a p il la ry column)/mass spectrom etry. These data are_of major, im portance from th e p o in t of view of environm ental p ro te c tio n . . In s p i t e o f th e em inent need f o r s ta n d a rd iz e d methods f o r TCDD a n a ly s is in te c h n ic a l grade p e s tic id e form ulations and environm ental sam ples, such p ro ced u res a re s t i l l la c k in g . The e s ta b lis h m e n t of s ta n d a rd iz e d methods would perm it s u b s ta n tia l p ro g re ss in b a s e lin e and on-going s tu d ie s . Such methods should be d ire c te d toward improved sample p re p a ra tio n (to reduce in te rfe re n c e s ) and improved re s o lu tio n /d e te c tio n of the 2,3,7,8-TCDD isom er, w ith th e c o n s id e ra tio n in mind th at, such methods should be s u ita b le fo r use by m oderately w ell equipped la b o ra to rie s. ..... .. .. SECTION 4 ............. EXPERIMENTAL PROCEDURES SAFE HANDLING IN THE LABORATORY D isposable rubber g loves were worn w hile h an d lin g samples and s o lu tio n s , and a l l ev ap o ratio n s were performed in a hood. Waste m a te ria ls were sto re d in a capped s t e e l drum la b e le d TCDD WASTE. P e rso n n e l a s s o c ia te d w ith th e p ro je c t had p e rio d ic p hysical exam inations. GENERAL PROCEDURES . ....... ........... _____ _ l ________ ___________ _ _ A fte r ta k in g custody of samples fu rn ish e d by th e EPA, TCDD re s id u e s were id e n tifie d and q u a n tifie d using a th re e -s ta g e a n a ly tic a l p ro to co l in clu d in g sample p re p a ra tio n , mass s p e c tra l d a ta a q u is itio n , and d a ta a n a ly sis. TCDD S tan d ard s ; TCDD s ta n d a rd s in benzene were su p p lie d by Dr. Aubry Dupuy, P e s tic id e M onitoring L a b o ra to ry , EPA. The 2 , 3 , 7 , 8-TCDD s ta n d a rd c o n c e n tra tio n was 500 picograms (p g ) /m ic ro lite r ( p i ) . C o n cen tratio n of th e Cl la b e le d 2^.3,7,8-TCDD sta n d a rd was 480 p g /y l. Labeled TCDD was used to d eterm in e dioxin recovery through the e n tire ex tractio n /clean u p procedure. Nonlabeled TCDD was used to q u a n tify d io x in le v e l s in th e sam ples. '. Sample P re p a ra tio n ! E thanol, hexane, benzene, carbon te tra c h lo rid e , and methylene c h lo rid e were nanograde (p e s tic id e ) q u a lity ; in o rg an ic s a l t s , a c id s and bases were ACS g ra d e . D i s t i l l e d w ater was p rep ared by f i l t e r i n g through a mixed bed ion exchanger, a c tiv a te d carbon, and d i s t i l l i n g in an a l l g la ss s t i l l . Aluminum oxide Woelm n e u tr a l, a c t i v i t y grade I , was used fo r column chroma tography. ,\ ; I The pro ced u re used in th e se a n a ly se s i s a m o d ific a tio n of t h a t pro v id ed by EPA in th e c o n tr a c t. In g e n e ra l, i t in v o lv ed c a u s tic KOH s a p o n if ic a tio n o f th e p e s tic id e follow ed by e x tr a c tio n of th e n e u tr a l TCDD in to hexane. The hexane s o lu tio n was cleaned up by e x tr a c tin g s e v e r a l tim es w ith sodium hydroxide so lu tio n and s u lf u r ic a c id , follow ed by chromatography on alum ina. The e lu a te was c o n ce n tra te d fo r GC/MS a n a ly s is . D e ta ils o f th e p ro c e d u res fo llo w . A 1 g q u a n tity o f sample was tr a n s f e r r e d q u a n t i t a t i v e l y to a 500 ml " s e p a ra to ry fu n n el c o n ta in in g 60 ml o f p e s tic id e grade e th a n o l. E x actly 48 ng o f Cl la b e le d 2,3,7,8-TCDD was added w ith a m ic ro sy rin g e and th e m ix tu re s w irle d . Four m i l l i l i t e r s o f 40 p e rc e n t KOH was added and the m ix tu re im m ediately shaken. The room tem p eratu re d ig e s tio n was allow ed to proceed fo r 30 m in u tes, w ith o c c a s io n a l s w irlin g . Two-hundred m i l l i l i t e r s of d i s t i l l e d w ater was added to d is s o lv e .th e p r e c i p i t a t e formed upon base a d d itio n , and th e r e s u l t i n g s o lu tio n was e x tra c te d w ith th re e 100' ml p o rtio n s of nanograde hexane. The combined hexane e x tr a c ts were washed s e q u e n tia lly w ith th re e 100-ml volumes of IN NaOH, one 80-ml volume of d i s t i l l e d w a te r, and 95 p e rc e n t H^SO, u n t i l no f u r th e r c o lo r appeared in l^SO ^. A f i n a l w ashing w ith 80 ml d i s t i l l e d w ater com pleted th e e x tr a c ti o n . The hexane e x tr a c t was f i l t e r e d through a 15 mm x 20 cm column, o f *' g ra n u la r anhydrous Na2C0, follow ed by c o n c e n tra tio n in a K uderna-D anish e v a p o ra to r to a volume or about 4 ml. . The anhydrous Na CO. had been p u rif ie d by p re -e x tra c tio n w ith nanograde C ^ C lj in a Soxhlet e x tra c to r and d ry in g a t 100C in a vacuum oven. The c o n c e n tra te was reduced to 0 .5 ml under a stream o f UHP o r u l t r a high p u r ity n itr o g e n (M atheson, R u th e rfo rd , J}_.J.). The re s id u e was r e c o n s titu te d in 2 ml nano grade hexane and chromato graphed through a 4 .5 cm column. The column c o n s is te d o f a c tiv a te d alum ina over which a 1/4 in c h la y e r o f p u r if ie d g ra n u la r Na_S0, was p la c e d . The column was p re c o n d itio n e d by washing w ith 5 ml of CH CI^, follow ed by rem oval o f r e s id u a l CH^Cl_ by blow ing d ry N^ th ro u g h th e column, and h e a tin g f o r 48 hours a t 240C. Tne sample was e lu te d from th e column w ith 6 ml CCl^, follow ed by 4 ml o f C ^ C lg . The carbon t e t r a c h l o r i d e la y e r was d isc a rd e d and th e C ^ C ^ f r a c tio n ev ap o rated under UHP to 20 y l . The c o n c e n tra te was r e c o n s titu te d to 2 ml w ith hexane, ev ap o rated to 20 y l , and r e d ilu te d w ith hexane to 2 ml. The f i n a l e x tr a c t volume was c o n c e n tra te d to 200, y l. The th re e e v ap o ratio n s were n e ce ssa ry to remove r e s id u a l m; The e x tr a c t was d iv id e d e q u a lly in to two g la s s am pules. Ampules were flam e se a le d , and th e e x tra c t volume noted by marking th e li q u i d 'l e v e l . A fte r coding th e ampules were s to re d in a f r e e z e r a t - 1 0 C. Samples were processed in " s e ts " . Each s e t of samples c o n siste d of i n t e r n a l and EPA s u p p lie d q u a lity c o n tr o l (QC) and p e s t i c i d e sam ples. G e n era lly each s e t of sam ples co n tain ed two q u a lity c o n tr o l sam ples and fo u r p e stic id e samples processed from l e f t to r ig h t as in d ic a te d below: iI . . . A3.. Xj, Xj.+ 1 X1.+2 XJ. +3 Bk.' ; .. 'i : where A. = i t h o b s e rv a tio n of th e accu racy 1 QC sample ( f o r example, p e s tic id e sample #0093, fu rn is h e d by EPA, acc e p ted v a lu e fo r TCDD = 2.3 ppm). X. *= th ej t h EPA p e s ti c id e sample (unknownTCDD le v e l ) , and = k th o b s e rv a tio n of th e zero le v e l TCDD QCsample(s o lv e n t blan k spiked o n ly w ith Cl TCDD, 48 n g ) . ; 5 i SU71 QC and EPA sample numbers fo r each p a ire d e x tr a c t were coded w ith a num erical s u f f ix : one ampule, - 2 (f o r immediate GC/MS assay ) and the o th e r, -1 (f o r backup a n a ly s is ) . EPA sample numbers ranged from 2 to 9 d i g i t s . R e p lic a te sam ples in th is re p o rt a re denoted by an " I" ( i n i t i a l ) , "D" ( d u p lic a te ) , "T" ( t r i p l i c a t e ) , or "Q" (q u a d ru p lic a te ) a f t e r the sample b atch number. The s e r ie s sample number ( e .g ., 148920) re p re se n ts one source of tech n ical grade m aterial; the -1 , -2 , -3 , e tc ., following the s e rie s sample numbers each re p re se n ts a d if f e r e n t b atch sampled from th a t source. TCDD and 37 Cl TCDD s ta n d a rd s were tr a n s f e r r e d to M in in e rt (Supelco) valve v ia ls . A back-up s e a l in the T eflon v alve had been removed and the assembly prewashed. Standards were coded and frozen u n til u tiliz e d . Mass S p e c tra l Data A q u is itio n The low r e s o lu tio n gas chrom atograph/m ass s p e c tro m e te r used was a H ew lett-P ackard (HP) 5985 GC/MS in th e e le c tr o n io n iz a tio n (E l) so u rce c o n fig u ra tio n . The c o n tr o l u n it d isp la y e d th e io n c u rre n t (peak a re a ) on a T ek tro n ix 4012 CRT computer te rm in a l, and hard c o p ie s were pro v id ed by the T ektronix 4631 hard copy u n it. G enerally sp ectro m eter o p e ra tin g c o n d itio n s were as follow s: column 3 f t x 2 mm ID g la s s ; packing 3 p e rc e n t OV-11 on 100/120 mesh S u p elco p o rt; column tem periture 200C f o r 1 m inute to 240C a t 6C p e r m inute; in je c tio n h e a te r tem perature 250C; j e t se p a ra to r in te r f a c e tem perature 260C; ion source te m p eratu re 150C; c a r r i e r g a s, helium 32 m l/m in; e le c tr o n im pact source 70 eV; em ission 0 .3 mA; e le c tr o n m u l t i p l i e r 2 .6 KV; mass range 150330 amu; scan tim e 3 ms/amu. The mass sp e c tro m e te r was tuned to m onitor th re e m olecular ions a t m/e v alu es o f 320, 322, and 328. R eten tio n tim e of TCDD was approxim ately 3 .6 m inutes under th e above c o n d itio n s . The GC/MS p ro to c o l f o r TCD^measurement was d esig n ed to , p erm it sim u l taneous d a ta a c q u is itio n f o r : Cl TCDD re c o v e ry , TCDD d e te c tio n 'l i m i t , TCDD q u a n tif ic a tio n , and c o n firm a tio n v ia mass s p e c tr a (m olecular io n abundances). In t h i s p ro ced u re, sample e x tr a c ts and TCDD s ta n d a rd s were thawed to room te m p e ra tu re . Ampules c o n ta in in g sample e x tr a c t were broken above th e liq u id le v e l and firm ly p o s itio n e d in a p l a s t i c b lo c k . TCDD so lu tio n s were always handled under a hood, and liq u id and s o lid w astes were tr a n s f e r r e d d a ily to th e TCDD w aste drum. A 10 y l H am ilton 701N m icro sy rin g e was used to i n j e c t sample and sta n d a rd a liq u o ts in to th e GC/MS. GC column perform ance and MS l i n e a r i t y were checked by c o -in je c tin g th e follow ing m ixtures of stan d ard s, one in je c tio n per m ixture in the sequence tabulated: 1 y l n a tiv e TCDD + 1 y l 37C1 TCDD 2 y l n a tiv e TCDD + 2 y l 37C1 TCDD 1 y l n a tiv e TCDD + 1 y l 37C1 TCDD 2 y l n a tiv e TCDD + 2 y l 37Cl TCDD 8 ^/4>7c2- Data fo r each i n j e c t i o n were d isp la y e d on th e CRT computer te rm in a l. Peaks (m/e = 320, 322 and 328) co rresp o n d in g ' to th e TCDD r e te n tio n time re g io n were in te g r a te d u sin g a HP-21MXE d a ta system and r e s u l t s d is p la y e d . C o lle c te d d a ta f o r each i n j e c t i o n were reco rd ed on a T e k tro n ix 4610 hard copy u n it. Q u a lity c o n tro l and p e s ti c id e samples were run in th e same o rd e r in which the e x tra c ts were prepared. C o -in jectio n of m ix tu res, one in je c tio n p er m ixture, was as fo llo w s: COMPOSITION OF ANALYSIS SET _____ ( p i c o -in je c te d )_______ In je c tio n Number Sample Volume (pl) TCDD 37 Cl TCDD N ative Standard Standard 1 2+ 0 +0 2 2+ 1 +2 3 2+ 0 +0 -2~ - + 2 During th e TCDD r e te n tio n tim e-sp an th e m/e 320, 322 and 328 peak areas were in te g ra te d , and a photocopy prepared fo r each in je c tio n (see sample hard copies in A ppendices). Background n o ise le v e l was in te g ra te d from th e b a s e lin e a d ja c e n t to th e m/e 322 peak. The io n c u r r e n t chrom ato gram from in je c tio n 1 was used f o r n o is e d e te rm in a tio n i f th e 322 peak was p re s e n t; th e chromatogram from i n je c tio n 2 was used i f th e 322 peak; was n o t found. A photocopy o f .the n o is e le v e l was p re p a re d . \ Hard copies from in je c tio n s 1 and 3 were in sp ec te d to determ ine i f bo th m/e 320 and 322 peaks were found f o r b o th i n j e c t i o n s . A sample m eeting th e se c r i t e r i a was s u b je c te d to GC/MS mass fra g m e n ta tio n confirm a tio n by e v a p o ra tin g th e e x tr a c t under N,, to about 10 p i. Two m ic r o lit e r s of th e co n cen trated e x tr a c t was in je c te d . Both mass spectrum and a b so lu te abundances for m/e values were obtained and hard copies prepared (see sample copies in A ppendices). ; Data A nalysis . In te g ra te d peak a re a s f o r TCDD sta n d a rd s o lu tio n s were s u b je c te d to lin e a r regression an aly sis (y = peak area; X = p i in je c ted ) . C orrelation c o e f fic ie n t ( r ) , slo p e ( s ) , in te rc e p t ( i ) , and the r a t i o i / s were computed f o r m/e = 320, 322, and 328. In stru m e n ta l s e n s i t i v i t y , expressed as a re a /p g , was computed fo r m/e 320 and 328 by d iv id in g " s" by 500 (TCDD sta n d a rd c o n c e n tra tio n in p g /p l) and 480 ("* Cl TCDD sta n d a rd c o n c e n tra tio n in p g / p l ) . . An average 320/322 r a t i o was computed from th e ( 2 + 0 + 0 ) i n j e c t i o n s . P e r tin e n t d a ta were p lo tte d on a QC c h a rt to m onitor in stru m e n t perform ance. Expressed as p e rc en t, i / s x 100, average values were < + 5 p e rc e n t. Large n e g a tiv e v a lu e s , i . e . , 20 p e rc e n t, could be in d ic a tiv e o f TCDD a d so rp tio n o r d e g ra d a tio n in the system . In any c a se , t h i s problem was u s u a lly r e c t i f ie d by changing th e GC column- 9 to, 73 Peak a re a s fo r th e method blan k (QC), accuracy (QC) , and EPA samples were ta b u la te d on d a ta s h e e ts , designed e x c lu siv e ly fo r th is p r o je c t, along with other p ertin en t inform ation. d a ta s h e e t is- shown in Appendix A; the method fo r computing p e rc e n t Cl TCDD re c o v ery , TCDD sample concen tr a tio n , d e te c tio n lim it, and re s u lts of the confirm ation a n a ly s is are also i l l u s t r a t e d . Note th a t re p o rte d TCDD le v e ls a re c o rre c te d f o r re c o v e ry . The c o n tra c t s p e c if ic a tio n f o r 37 Cl TCDD re c o v ery in th e p e s tic id e samples was 50 to 120 p e rc e n t, in c lu s iv e , i f th e i n i t i a l GC/MS reco v ery assay was o u tsid e th e s p e c ifie d ran g e, th e backup a liq u o t of th e o r ig in a l e x tr a c t was assay ed . I f th e average o f th e two reco v ery v a lu e s was w ith in the s p e c ific a tio n range, c o n tra c t requirem ents were s a ti s if ie d . I f the average reco v ery was o u tsid e th e s p e c if ic a tio n ran g e, th e sample was re e x tra c te d and assayed one a d d itio n a l tim e. C onfirm ation o f TCDD d e te c tio n by re c o rd in g com plete mass s p e c tra was o p tio n a l a t TCDD co n tam in atio n le v e ls <10 ppb and a c o n tr a c t req u ire m e n t a t >10 ppb. The sta n d a rd a d d itio n tech n iq u e was n o t s u c c e s s fu l w ith sam ples conta in in g la rg e q u a n titie s ( p a r ts - p e r - m illio n ) of TCDD because th e amount of TCDD stan d a rd c o - in je c te d was in s i g n i f i c a n t compared to th e TCDD-.level.--in.the p e stic id e e x tra c t. D eterm ination of reg ressio n slope and in te rc e p t fo r TCDD q u a n tif ic a tio n s w ith such samples was v a r ia b le due to poor c o r r e la ti o n s . An a l t e r n a t i v e GC/MS p ro to c o l was used f o r th e s e sam ples. An e x te rn a l c a lib r a tio n curve was c o n stru c te d by in je c tin g v a ry in g amounts of th e TCDD sta n d a rd in to th e GC/MS. The r e s u l t a n t l i n e a r re g re s s io n eq u atio n fo r the standard, in terms of picograms ra th e r than m ic ro lite rs in je c te d was: ' y Xs + i (D- where y = peak area (m/e = 322) s - slo p e (a re a /p g TCDD) i = intercept X = pg TCDD in je c te d t Rearranging Eqv (1): (2) ' where X = pg TCDD in th e p e s ti c id e sample e x tr a c t and y = av erag e m/e 322 a re a f o r - a 2 p i sample e x t r a c t . TCDD c o n c e n tra tio n (ppm? in th e sample i s . equal to: , ppm (200 Xp ^ 2 pi sample in je c te e d x tr in ac to t volume) GC/MS) 7 (R sample w eight 4 10 ) where R i s th e p e rcen tag e re c o v ery of 37Cl TCDD. 10 SECTION 5 RESULTS AND DISCUSSION QUALITY ASSURANCE . Several asp ects of th is p ro je c t were monitored fo r q u a lity assurance. These included (1) tak in g custody of standards and sam ples, (2) m a te ria ls and re a g e n ts used in th e a n a ly s is , (3) stan d ard p ro c e d u re s , (4.) c a l i b r a t i o n s , (5) record keeping, and (6) q u a lity c o n tro l c h a rts . Chain o f custody re c o rd s were tra n s m itte d to GSRI alo n g w ith sam ples and s ta n d a r d s . Sample r e c e ip t was acknowledged by d a tin g , sig n in g , and r e tu rn in g a copy o f the c h a in o f tr a n s f e r re c o rd to EPA. D isc re p a n c ie s in la b e lin g o r rep lacem en t re q u e sts were noted on th e tr a n s f e r re c o rd . M a te ria ls and re a g e n ts used in th e a n a ly s is in g e n e r a l were ACS or p e s ti c id e grade. New l o t s o f m a te r ia l were te s te d f o r TCDD in te r f e r e n c e by s u b je c tin g them to th e a n a l y t i c a l p ro to c o l. The sample e x tr a c ti o n and c le a n -u p procedure d e sc rib e d in S e c tio n 4 was used th ro u g h o u t th e p r o je c t, w ithout m odification, and th e re fo re c o n stitu ted a standard o p eratin g proce d u re . G e n e ra lly , th e GC/MS p ro to c o l follow ed th e s ta n d a rd p ro c e d u re o u tlin e d i n th e previous s e c tio n ; c e r ta in m o d ific a tio n s, i . e . , changes in column tem perature programming, were n ecessary to overcome in te rfe re n c e s and r e f in e th e method. The GC/MS in stru m e n t was tuned d a ily p r io r to a n a ly s e s . GC/MS p erfo rm - . ance and lin e a r ity stan d ard s bracketed 5-6 su ccessive a n a ly se s. Record keep in g included an in s tru m e n ta l p re v e n tiv e m aintenance lo g . GC/MS d a ta and computer f i l e re fe re n ce number, date of e x tra c tio n , and th e sample . w eight were recorded in th is log fo r each sample assayed. j Sample p ro c e ssin g a c c o u n ta b ility was an im p o rtan t p a r t o f re c o rd k eep in g . R e su lts were com piled on a m aster sample s ta t u s form (se e Appendix B) in th e o rd e r sam ples were re c e iv e d . R e su lts were a ls o com piled on a cum ulative d a ta form (Appendix C) in th e o rd e r sam ples were a ssa y e d . ; Cumulative q u a lity c o n tro l c h a rts were u sefu l in d ic a to rs of whether or not th e measurement p ro c e ss was in c o n tr o l. The c h a r ts showed tre n d s o r ru n s , sudden s h if ts in the mean, increased v a r ia b ility and o fte n in d ic a te d the n atu re of a problem. t i F ig u re s 2 through 4 show q u a lity c o n tro l d a ta f o r GC/MS p erfo rm an ce, method b la n k , and accu racy o f TCDD d e te rm in a tio n s . C o n tro l c h a r t a n a ly s is of graphs A, B, C, and D (F ig u re 2) shows sy ste m a tic v a r i a t i o n s w ith probable causes. A s e r ie s of in c re a sin g and d ecreasin g v a lu e s (graphs B and D, o b se rv a tio n numbers 1-6 and 29-34) was o b ta in e d a f t e r th e in stru m e n t 11 .9 i*-' 03 CO o o OJ ro u i_ it o N0 3 O 1.00 no # L0.98 60 t> <K j= O o 0- CDQ t_ O 03 CL H o O 0L). h- < K) 40 20 0 .......... * o < 03 I.OOpo <3 o I " * * . o Oo CVJ fO II e> CL 0) s . L- 03 CL 0..998R *-- 30 20 **<*<**<< ><<>*<<i <<$ oa Q 0L3. O< 1- 10 0L *****# m7 5 to OJ CJ 0.95 to s in 0.75 o .o-.-- -a__ f-,........... .?..ft,* a 9t _____________J__ ^ _____ _ * OJ X+3s 9 . 9 ro 0.55 - n=4|,X =0.78'4 Theoretical = 0 .7 6 6 , s =0.042 CV = 5 . 3 % X- 3s X XX 10 15 20 25 30 35 4 0 OBSERVATION NUMBER F igure 2. Q u a lity c o n tro l c h a r t fo r GC/MS perform ance (Sample: 37Cl TCDD and 35Cl TCDD s ta n d a rd s ). 45 'i 140 o o a) 100 *< a 80 o t-- 60 1 ro 4 0 L- xx U) Q. 4 Q. 6c o 2 O CO aa 0 o h* ND & to L 0 Ail Observations 7 n =16 EPA X = 109% X = 8 I% Specification s =*49% Range CV =4 5 % ____________ i _L 10 15 20 25 30 35 4 0 45 OBSERVATION NUMBER . F igure 3. Method blan k q u a lity c o n tro l c h a r t (Sample: s o lv e n t b la n k w ith added 37,Cl TCDD). V < fr-- ; * ; F ig u re 4. Accuracy q u a lity c o n tro l c h a rt (Sample: EPA //0093) * i 4 :5 was shut down and th e ion source c le a n e d . O b se rv a tio n numbers 18-29 and 35-39 show d ecreased s e n s i t i v i t y p r io r to so u rce m aintenance. Ion c u rre n t chromatograms f o r o b s e rv a tio n 21 a re shown in Appendix D. Concom itant w ith low er s e n s i t i v i t y , runs o f 2 to A o b s e rv a tio n s (graphs A and C) were observed w ith reg ressio n c o rre la tio n c o e ffic ie n ts <0.980. N onetheless, graph E shows th a t only 1 o b s e rv a tio n ou t o f 41 was o u ts id e th e a r b i t r a r i l y chosen +3 sta n d a rd d e v ia tio n l im it s f o r 320/322 ion c u r r e n t peak a re a s . The t h e o r e t i c a l r a t i o o f 0.766 (6) compares fa v o ra b ly w ith th e observed mean v a lu e (X = 0 .7 8 4 , p e rc e n t d if f e r e n c e = + 2 .3 p e r c e n t) . The c o n c lu sio n was th a t the measurement process (320/322 r a t i o ) was in c o n tro l fo r 97.6 p e rc e n t of the o b se rv a tio n s i s based on D uncan's (7) c r i t e r i a th a t d e fin e s t a t i s t i c a l c o n tro l to be any p o in t w ith in 3 s ta n d a rd d e v ia tio n l i m i t s . No l i m i t s were p laced on A-D g rap h s. * F ig u re s 3 and 4 show th e method b la n k (s o lv e n t sp ik ed only w ith 37 Cl la b e le d TCDD) and accu racy (EPA r e fe re n c e p e s ti c id e sample #0093) q u a lity c o n tro l c h a r ts w ith EPA reco v ery s p e c i f i c a t i o n ra n g e . Mean re c o v ery o b se rv a - i tio n s f a l l i n g w ith in EPA s p e c if ic a tio n s were 81 and 86 p e rc e n t, r e s p e c tiv e ly . The EPA a cc e p ted v a lu e f o r t o t a l TCDD c o n c e n tra tio n in th e p e s ti c id e ------ re fe re n c e sample i s 2 .3 ppm (8 ) . A mean o f 2.25 + 0.3 8 ppm was o b ta in e d , w ith "a c o e f f i c i e n t of v a r ia tio n o f 17 p e rc e n t. No TCDD was ob serv ed in any method b lan k . _ Several recovery observations were ou tsid e the sp e c ific a tio n range. A ru n o f o u t- o f - s p e c if ic a tio n v a lu e s (F ig u re 3, curve A, o b s e rv a tio n s 6-8) was due in p a rt to a p a r t i a l l y clogged j e t s e p a ra to r. Cleaning th e j e t s e p a r a to r r e c t i f i e d th e problem . Random o b s e rv a tio n s o u ts id e s p e c i f i c a t i o n lim its probably re fle c t o v e ra ll variance of the to ta l procedure. A m a jo rity o f o u t-o f -s p e c ific a tio n recovery v alu es were on th e high . s id e , i . e . >120 p e rc e n t, pro b ab ly b ecau se th e upper o n e -sid ed to le r a n c e o f th e a n a l y t i c a l p ro c e ss i s only 20 p e rc e n t above th e ^ 'c h e o re tic a l re c o v e ry v a lu e o f 100 p e rc e n t; th e c o rresp o n d in g low er o n e -sid e d t o l e r a n c e 'i s 2.5 tim es as g r e a t, i . e . , 50 p e rc e n t. ; C r i t e r i a f o r a P o s itiv e Sample Q u a lita tiv e c r i t e r i a developed f o r th e d e t e c t ^ n of TCDD in th e p e s ti c id e e x t r a c t m a trix were: (1) a re c o v ery o f 1 C l TCDD betw een 50 and 120 p e rc e n t, in c lu s iv e ; (2) b o th m/e 320 and 322 peaks e x is t in g a t th e TCDD r e te n tio n tim e re g io n ;- (3) w e ll d e fin e d m/e 320 and 322 peaks whose r e te n tio n tim e e x a c tly e q u a ls t h a t o f TCDD s ta n d a rd s ; (4) s ig n a l - to - n o is e r a t i o o f a t le a s t 2 .5 /1 a t m/e 322; (5) r a tio of m/e 320/322 peak are as in th e proper is o to p ic p ro p o rtio n ; and (6) TCDD c o n firm a tio n by m o le c u lar io n s p e c tr a . Data fo r th e f i r s t fiv e requirem ents were obtained by s e le c tiv e ion m onitor in g a t m/e 328, 322, and 320; d a ta f o r TCDD c o n firm a tio n were g e n e ra te d by reco rd in g s p e c tra in the m/e range 150-350 and computer ta b u la tio n of a b s o lu te abundances o f 119-131 m/e. The range of i n t e r e s t was m/e 320-328; the wide range f a c ilita te d background measurement. P e rtin e n t inform ation e x p lain in g th ese b in ary (p o s itiv e /n e g a tiv e ) d e c isio n making p ro cesses is shown in T able 1. 15 C r ite r io n /Fl was based s o le ly on reco v ery s p e c if ic a tio n s in th e c o n tra c t. C r ite r ia 5 and 6 in c o rp o ra te v a ria n c e a sso c ia te d w ith th e measurement system . S t a t i s t i c a l l i m i t s imposed a re shown in Table 1. The t h e o r e t i c a l is o to p ic abundance r a t i o of m/e 320/322/324/326 r e l a t i v e to 322 i s 0 .7 6 6 /1 .0 0 0 /0 .4 8 9 /0 .0 0 1 (6 ) . C r ite r io n #6 was d iv id ed in to th re e s u b c rite ria (Table 1); eight binary perm utations of the s u b c rite ria are p o s s ib le as shown in Table 2 \ O v e rall c r i t e r i o n #6 t e s t c h a r a c t e r i s t i c was a r b i t r a r i l y chosen from th e th re e s u b c r i t e r i a . The r a t i o n a l fo r s u b c r i t e r i a d e c isio n making c o rre la te s w ith is o to p ic abundance r a tio s . G reater emphasis was given to 6a (m/e 320/322) s in c e th e s e two m/e v a lu e s a re th e l a r g e s t . More p re c is e r a t i o measurements a re a n tic ip a te d w ith 6a r a th e r th an th e s m a lle r 324/322 r a t i o f o r 6b o r 6c. Table 3 shows th e e v a lu a tio n o f th e c e r t a i n t y o f TCDD d e te c tio n based on summation o f q u a l i t a t i v e c r i t e r i a ; c h a r a c te r is tic s . C rite ria 1 through 4 were ab so lu te or n ecessary fo r d eterm in in g i f TCDD was d e te c te d except f o r th e D ( d u p li c a te ) , T ( t r i p l i c a t e ) and Q (q u a d ru p lic a te ) reru n s o f o r ig in a l sam ples. D etection iim its and mass confirm ation sp e c tra were not req u ired fo r the l a t t e r . ' ;i i Appendices D -I in c lu d e r e p r e s e n ta tiv e h ard copy d a ta f o r GC/MS i n s t r u - ' m ental perform ance and TCDD d e t e c t i o n . .............. -- -- -- -- --- -- ------- -- -- - Sample Data A wide v a r i e t y of p e s tic id e sam ples were analyzed f o r t o t a l TCDD r e s id u e s . Subm itted EPA sam ples (unknowns and q u a lity c o n tr o l sam ples) were analyzed on a b lin d b a s is . The two sample ty p es were v i s u a l l y and e x p e rim e n ta lly in d is tin g u is h a b le . EPA provided decoding in fo rm a tio n only a f t e r com pletion o f th e a ssa y s and r e p o r tin g o f th e TCDD d a ta . T ab le 4 summarizes the decoding inform ation. G enerally, a 6 -d ig it sample code .r e p r e s e n ts a s e r i e s of p e s tic id e sam ples of th e same g e n e ric c l a s s ; i n d i v i - ' u a l samples were denoted by a hyphenated (in some c a se s) s u f f ix made up of 'numbers and/or l e t t e r s . For example, 137832-5D r e la te s .to th e d u p lic a te determ ination of the f i f t h subsample in the ch lo rin ated phenolic a n tis e p tic 137832 s e r ie s . Each subsample re p re se n ts a d if f e r e n t b atch of th e manufac tured technical grade p esticid es. \ ; i , Based on EPA decoding in fo rm a tio n , r e p l i c a t e sample d a ta , i f a v a il a b le , ! were c o lla te d according to sample batch number. A fter f i r s t combining ; ; r e p l i c a t e in fo rm a tio n w ith th e a p p ro p ria te f i r s t - r u n r e s u l t s , a TCDD d e te c - . tio n judgement was made fo r each b a tch sam ple, r e g a r d le s s o f th e number of ; r e p lic a te s assayed. These judgements were e ith e r p o s itiv e , n e g ativ e, or j d o u b tfu l. For example, i f th e f i r s t ru n (I) sample of b a tc h number 1234 i 'w as p o s itiv e ( e . g . , 12341 i s +) , th e d u p lic a te (D) run n e g a tiv e (1234D i s - ) , . and th e t r i p l i c a t e (T) run q u e stio n a b le (1234T i s d o u b tf u l ) , th en th e judgement was th a t TCDD d e te c tio n was d o u b tfu l in sample 1234. On th e * o th e r hand, i f a sample b atch were judged TCDD p o s itiv e , r e g a r d le s s of how many r e p l i c a t e s were ru n , t o t a l TCDD le v e l found was r e p o r te d , in c lu d in g r e p lic a te d ata and the average v a lu e, i f a p p ro p ria te . A t o t a l of 69 a u th e n tic p e s tic id e b a tch sam ples were a ssa y e d . TCDD was d e te c te d in 17 batch samples (25 p e r c e n t) ; TCDD d e te c tio n was d o u b tfu l in 6 samples (9 p e r c e n t) ; and- TCDD was n o t-d e te c te d in 46 b a tc h sam ples (66 16. ai6g i 'r ! p e r c e iit) . T ables 5-12 summarize d a ta f o r TCDD in v a rio u s ty p es o f a u th e n tic p e s tic id e sam ples. In those in sta n ce s where a l l or most batch samples were p o s itiv e fo r a given s e r i e s , ta b u la tio n o f mean v a lu e , s ta n d a rd d e v ia tio n , and c o e f f ic ie n t of v a r ia tio n i s based on p o s itiv e sample d a ta . NA (not applicable) re fe rs to inform ation not av ailab le or not requested. For exam ple, q u a l i t a t i v e d e te c tio n c r i t e r i a //4 and 6 were no t re q u ire d f o r D, -T , o r -Q sam ples. Data f o r EPA' q u a lity c o n tro ls a re shown in Table 13. V a ria tio n of TCDD d e te c tio n judgem ents v e rsu s EPA sam ple type i s shown in Table 14. For th e a u th e n tic b a tch sam ples TCDD was n o n d e te c ta b le (d e te c tio n l i m i t 10 ppb) in 100 p e rc e n t of th e 2 ,4 ,5 - tr ic h lo r o p h e n o l samples analyzed (n, number of batch samples assayed = 16). For compara tiv e purposes, batch samples of 2 ,4 ,5 -T , the butoxyethyl e s te r of 2 ,4 ,5 -T , * and th e is o o c ty l e s t e r o f 2 ,4 ,5 -T a re grouped under 2 ,4 ,5 -T as shown below. Type 2 ,4 ,5 -T butoxyethyl e ste r of 2,4,5-T isooctyl ester "of 2,4,5-T 2 ,4 ,5 -T SAMPLE TYPE (16 BATCHES) Number of Batches Number of B a tc h e s /(p e rc e n t) P o sitiv e Doubtful N ondetectable 3 1/(33.3) 1/(33.3) 1 /0 3 .3 ) 10 : 9/(90) 1/(10) 0 _3 _0 3/(100) 1 16 10/(63) 5/(31) 1(6) O v e ra ll, d e te c tio n o f TCDD was p o s itiv e in 63 p e rc e n t (n=10) o f th e b a tc h sam ples, d o u b tfu l in 31 p e rc e n t (n=5), and n o n d e te c ta b le in 6 p e rc e n t ( n = l) . S p e c if ic a lly , d e te c tio n o f t o t a l TCDD was p o s itiv e in 90 p e rc e n t (n=9) of the butoxyethyl e s te r of 2 ,4 ,5 -T batch sam ples. In c o n tra s t, d e te c tio n of TCDD was judged e q u a lly p o s i t i v e , d o u b tfu l and n o n d e te c ta b le i n a l i (n=3) of th e 2 ,4 ,5 -T b a tc h sam ples, and TCDD d e te c tio n was q u e s tio n a b le in a l l (n=3) of th e is o o c ty l e s te r of 2 ,4 ,5 -T b atch sam ples. E ight S ilv e x type b a tc h sam ples were a ssa y ed . TCDD d e te c tio n was p o s itiv e in 86 p e rc e n t (n=6) o f th e butoxypropyl e s t e r o f S ilv e x and q u e s tio n a b le in the rem aining sample (n=l) of th is type. Only one b a tch of is o o c ty l e s t e r of S ilv ex was assa y ed ; th e TCDD d e te c tio n judgem ent was p o s i t i v e . One-hundred p ercen t of both the c h lo rin a te d p h en o lic a n t i s e p t i c (n=22) and th e organophosphate (n=7) b a tc h samples gave n e g a tiv e TCDD d e te c tio n c h a ra c te ris tic s . 17 Summarizing th e TCDD d e te c tio n judgem ents f o r a u th e n tic p e s tic id e b atch samples in term s o f d e c re a sin g p e rc e n ta g e s, 66 p e rc e n t were n o n d e te c ta b le , 25 p e rc e n t p o s itiv e , and 9 p e rc en t d o u b tfu l. L ikew ise,, judgem ents fo r the 20 d i f f e r e n t EPA QC sam ples an aly zed were 40 p e rc e n t n o n d e te c ta b le , 30 p e rc en t d o u b tfu l, and 30 p e rc e n t p o s itiv e . V a ria tio n of t o t a l TCDD le v e l found v e rsu s sample type f o r p o s itiv e samples i s shown in T able 15. A ll au th en tic- p e s ti c id e b a tc h sam ples fo r which TCDD d e te c tio n was judged p o s itiv e co n tain ed t o t a l TCDD in th e range 10-30 ppb. By c o n tr a s t, t o t a l TCDD c o n te n t of EPA q u a lity c o n tr o ls v a rie d from a low range of 31-99 ppb to a high o f 3000-3499 ppb. P re c is io n o f TCDD d e te rm in a tio n s in th e EPA q u a lity c o n tro ls i s shown in Table 16. R e la tiv e s ta n d a rd d e v ia tio n ranged from 2 .6 p e rc e n t to 83 p e rc e n t. S tandard d e v ia tio n ra n g e -fo r a u th e n tic unknown r e p l i c a t i o n s was .3 .1 p e rc e n t to 52 p e r c e n t, a s shown in T able 17. 1 The p re v a le n c e o f d o u b tfu l TCDD d e te c tio n judgem ents in a u th e n tic p e s tic id e samples and EPA q u a lity c o n tro ls s u g g e sts th e need f o r improvements in the measurement system . A s ig n if ic a n t number of d o u b tfu l d e te c tio n in s ta n c e s a re r e l a t e d to a p p a re n t TCDD le v e ls <10 ppb; in a d d itio n , TCDD co n firm a tio n was n o t a c o n tr a c t req u irem en t a t th e s e low l e v e l s . Mean d e te c tio n l i m i t was 4 .7 ppb + 3 .5 ppb (s ta n d a rd d e v ia tio n ) . Mean v a lu e p lu s two sta n d a rd d e v ia tio n s = 11 .7 ppb; th u s q u e s tio n a b le TCDD d e te c tio n a t th e 10 ppb l e v e l can be e x p ec te d . T h e re fo re , any r e s u l t found below 10 ppb in a u th e n tic samples was re p o rte d as n o n d e te c ta b le (ND). A ctu al v alu es below 10 ppb in th e q u a lity c o n tro ls a re ta b u la te d in T able 13 to d em o n strate th e inaccuracy in re p o rtin g such d a ta . The q u a l i t a t i v e c r i t e r i a were used to d e fin e th e judgem ent below 10 ppb ( i . e . , p o s i t i v e , d o u b tfu l, o r n e g a tiv e ) . Problems encountered in the p ro je c t - low and h ig h re c o v e rie s , u n r e lia b i l i t y of stan d ard s, in a b ility to d istin g u ish m atrix problems from instrum en t a l ones, v a r ia b ility in machine s e n s itiv ity , changes in chromatography, and in terferen ces - contributed to observable v a ria tio n s in re la tiv e standard d e v ia tio n and d e te c tio n lim it s . E x tra c ts y ie ld e d in te rfe re n c e s of varying s e v e rity from o th e r substances depending upon the e ffic ie n c y and re p ro d u c i b i l i t y of the cleanup procedure and sample type. Varying in te rfe re n c e s a ls o a ffe c te d ' C l TCDD re c o v e ry . For exam ple, e x c e lle n t re c o v e rie s were obtained fo r samples 148921-3, 148921-3D, and 148921-4D. Zero percen t re c o v eries were obtained w ith 148921-3T and 148921-4T. I n te rn a l q u a lity c o n tro l samples in th e same sample s e ts (p e rta in in g to e x tr a c tio n ) gave excellent recoveries. Low re c o v e rie s r e s u l t i n g from in te r f e r e n c e s m ight be p re v e n te d by employing a second Al^O cleanup column o r o th e r m o d ific a tio n in th e e x tra c tio n procedure. R ecoveries higher than t^e th e o r e tic a l v alu e were a n tic ip a te d when m/e 32S peaks c o -e lu te d w ith Cl-TCDD. I n te r f e r i n g m/e 320 and 322 peaks c o - e lu tin g w ith TCDD c o n trib u te d to v a r i a b i l i t y in c a lc u la te d TCDD le v e l s . In some in s ta n c e s peaks e lu tin g p r io r to or im m ediately a f t e r TCDD p rev en ted a c c u ra te peak i n t e r p r e t a t i o n . 18 A p p licatio n of high re s o lu tio n gas chromatography should help to overcome problems with in te rfe re n c e s. _ Problems w ith standard r e l i a b i l i t y included irr e p r o d u c ib ility of lo ts and c o n c e n tra tio n upon s ta n d in g . TCDD sta n d a rd s were p ro v id ed by EPA and l o t s were checked a g a in s t each o th e r to id e n tif y su sp e c t s o lu tio n s . TCDD stan d a rd s were provided in benzene s o lv e n t. ' A liq u o ts of the sta n d a rd s were tra n sfe rre d to small v ia ls sealed w ith T eflon-lined rubber septums. S ta n d a rd s were withdrawn w ith a 10 p i m ic ro sy rin g e . R epeated i n s e r t i o n s of th e m icrosyringe needle (fo u r in s e r tio n s were req u ired per sample assay) re s u lte d in form ation of holes in the septum. Benzene evaporated through th e porous septum and c o n c e n tra tio n o f th e TCDD sta n d a rd in c r e a s e d . An a tte m p t was made to so lv e th e problem by adding a sm all amount o f m ercury to th e standard v ia ls to s e a l punctures in the septum. Between in je c tio n s , * v ia ls were sto red in an in v e rte d p o s itio n which prevented benzene ev ap o ratio n . However, a f te r a few days th e s o lu tio n turned c lo u d ly , in d ic a tin g i n s t a b i l i t y o f th e sta n d a rd s o lu tio n s . The problem was r e c t i f i e d by u s in g M in in e rt v ia ls as outlined in the experim ental section. ^ V a r ia b ility in machine s e n s i t i v i t y c o n trib u te d to v a r ia b le TCDD .. ... d e te c tio n lim its and re g re ssio n c o rre la tio n c o e f fic ie n ts . Approximately one hour was re q u ire d to com plete th e fo u r GC/MS ru n s needed to c o n s tr u c t th e r e g re s s io n l i n e . D r i f t in GC/MS s e n s i t i v i t y and s p e c i f i c i t y o v er t h i s period contributed to a poorer co rre la tio n c o effic ien t w ith increased v a r i a b i l i t y in re p o rte d TCDD re c o v e ry and q u a n tif ic a tio n . D istin g u ish in g m atrix problems from in stru m e n ta l ones was d i f f i c u l t . R e p lic a te re c o v ery a ssa y s o f th e same e x tr a c t pro v id ed d a ta to e lu c id a te problem s. Poor p re c isio n was in d ic a tiv e of in stru m e n ta l problem s. High p re c is io n combined w ith re c o v e rie s o u tsid e th e s p e c if ic a tio n range suggested m atrix or ex tractio n problems. T o ta l TCDD was judged n o n d e te c ta b le in over 50 p e rc e n t o f th e a u th e n tic p e stic id e batch samples assayed. Extending the lower working range of the measurement system seems d e s ir a b le . This ta sk might be accom plished by fu rth e r cleanup of the e x tra c t, evaporation of the e x tra c t to a sm aller volume, and employing h ig h -e ffic ie n c y gas chromatography/mass spectrom etry . A p p licatio n of th e more te d io u s stan d ard a d d itio n tech n iq u e to th e se analyses appears ju s tifie d over external c alib ratio n . V ariations in the l i n e a r re g re s s io n slo p e were observed f o r some sam p les, which may be in d ic a tiv e of m a trix e f f e c t s . The stan d ard a d d itio n technique a ls o tends to compensate fo r instrum ent d r i f t . T h erefo re, more a c c u ra te d a ta would be a n tic ip a te d w ith the standard a d d itio n approach than w ith e x te rn a l working curve techniques. 19 REFERENCES B la ir , E.H. C hlorodoxins-O rigin and F a te . P re fa c e , Advances in Chemistry S eries #120, American Chemical S o ciety , W ashington, D .C ., 1973. p. 141. Schwetz, B .A ., J.M . N o rris , G.L. Sparschu, V.K. Rowe, P.J-. G ehring, J .L . Emerson, and C.G. G erbig. Toxicology, of C h lo rin a te d D ibenzoPara-D ioxins. Environ. H ealth P erspect. 5:87-99, 1973. IARC (WHO). Some Fum igants, th e H e rb ic id es 2,4-D and 2 ,4 ,5 -T , C h lo rin a te d D ibenzodioxins and M iscellan eo u s I n d u s t r i a l C hem icals. IARC - Monographs on the E v alu atio n of the C arcinogenic R isk of Chemicals to Man, 15, 1977. 354 pp. Rappe, C ., H.R. B user, and H.P. B osshardt. I d e n tif i c a tio n and Q u a n tifi c a tio n of P o ly -C h lo rin a te d D ibenzo-p-D ioxins (PCDDs) and D ibenzoFerans (PCDFs) in 2 ,4 ,5 -T -E s te r F o rm u latio n s and H e rb ic id e Orangei Chemosphere, 7 (5 ): 431-438, 1978. F ir e s to n e , D. The 2 , 3 , 7 , 8 -T e tra c h lo ro d ib e n z o -p -D io x in Problem : A Review. In : E co lo g ical B u lle tin s (Stockholm ), 27:39-52, 1978. Beynon, J.H . Mass Spectrom etry and i t s A p p lic a tio n s to O rganic Chemis t r y . D. Van N ostrand Co-., P rin c e to n , N .J. 1960. 640 pp. Duncan, A .J. Q uality C ontrol and I n d u s tria l S t a t i s t i c s , 4th e d ., R ichard D. Irw in , I n c . , Homewood, 1 1 1 ., 1974. p. 207. '* P e rso n a l Communication, Mr. Ronald Thomas, E nvironm ental P r o te c tio n Agency, B e lts v ille , Md., 1978. T A B L E 1. Q U A L I T A T I V E C R I T E R I A E X P L A N A T I O N No. C r i t e r i a E x p lan a tio n 1 50%<R<120% I f i n i t i a l R value exceeds sp ecificatio n s backup e x tra c t is assayed. I f R s t i l l - . o u t-o f-s p e c ific a tio n , sample is e x tra c te d and assayed one a d d itio n a l tim e. 2 existence o f both I f only one of two peaks e x i s t s , sample m/e 320 and m/e 322 l i s t e d as ND, n o t d e te c te d . w ell defin ed m/e 320 and 322 peaks s t TCDD r e te n tio n tim e ............A change in slo p e from n e g a tiv e to p o s i t i v e i f th e TCDD peak i s a sh o u ld er on a con tam inating peak, 4 m/e 322 in te g r a te d I f l e s s th an 2 .5 , sample l i s t e d a s ND, peak area a t le a st not detected 2.5 tim es g reater than baseline integ rated noise .5*. m/e 320/322 iso to p ic C o n tro l l i m i t s s e t by mean v a lu e + 3 ra tio , selected ion standard d e v ia tio n s as determ ined by m onitoring q u a lity c o n tr o l d a ta o b ta in e d w ith EPA referen ce p e s tic id e sample i f 0093. 6 m/e range 320-326,. Divided in to th ree s u b c r ite r ia : a b so lu te abundances, (a) m/e 320/322 c o n tro l lim its s e t by m o le c u lar io n s p e c tra mean v a lu e + 3 s ta n d a rd d e v ia tio n s as determined by q u a lity c o n tro l data o b tain ed w ith EPA re fe re n c e p e s ti c id e sample #0093. (b) m/e 324/322 c o n tro l lim its s e t by mean v a lu e + 3 sta n d a rd d e v ia tio n s as determined by q u a lity co n tro l data o b tain ed w ith EPA re fe re n c e p e s ti c id e sample #0093. (c) E xistence of m/e 320, 322, 324, and 326 in a b s o lu te abundance ta b le . 21 3.l>$5 TABLE 2. DEPENDENCE OF CRITERION If 6 OVERALL BINARY CHARACTERISTIC ON __ _______________________ SUBCRITERIA RESULTS O verall C riterion If6 C haracteristic: _____ P erm u tatio n ______ 6 a 6b 6c (negative) 4* (p o s itiv e ) + + ++ + +......................- ......... -- + ;+ + - + -+ + -- 22 ' T A B L E 3. T C D D D E T E C T I O N J U D G E M E N T B A S E D O N QUALITATIVE CRITERIA CHARACTERISTICS Q u a lita tiv e C r i t e r i a No 123456 Judgement For F irst-R un Samples: ++ +. + + .+ - +- + ++- + ++++ -- + + + ' + - 4* - Any o r a l l n e g a tiv e 3 NAb NA - TCDD d e te c te d TCDD d e te c tio n - - questionable TCDD n o t d e te c te d For D u p lic a te (- D ), T r i p l i c a t e (-T ), or Q u a d ru p lic a te (-Q) Sam ples: + .+ + NA + NA TCDD d e te c te d if- a ls o detected in first-ru n sample TCDD d e te c tio n q u e s tio n able i f firs t-ru n sample also questionable or not detected. + + + NA NA TCDD d e te c tio n q u e stio n able regardless of f ir s t- r u n judgement Any o r a l l n e g a tiv e C-) NA + NA TCDD n o t d e te c te d regardless of f ir s trun judgement 3 Q u alitativ e c r ite r i a 1 through 4 a l l ab so lu te or necessary f o r TCDD d e te c te d ( p o s itiv e ) judgem ent. bNA = n o t a p p lic a b le . 23 TABLE 4.__ VARIOUS EPA SAMPLES ANALYZED FOR TOTAL TCDD Sample S e rie s Number of Batch Samples P e s tic id e Sample Type (G eneric Name) 137401 137402 137403 137832 137834 137833 137835 148917 148918 141996 141998 141997 148919 148920 148921 229617 226617 225511 222841 222455 220777 93-01 14-01 0093 1-01 305 304 303 90 11 4 3W 3X 3Y 3Z . 1 1 1 14 8 2 4 5 5 1 2 ... 1 10 7 7 : AUTHENTIC PESTICIDES 2,4,5-T a - ........................ chlorinated phenolic a n tis e p tic 2 ,4 ,5-trichlorophenol isooctyL e s te r of 2 ,4 ,5-T iso o c ty l e s te r of Silvex*5 butoxyethyl e ste r of 2 ,4 ,5-T butoxypropyl e s te r of Silvex organophosphate in se c tic id e QUALITY CONTROLS cl2 ,4 ,5-T i s 2 ,4 ,5 -tric h lo ro p h e n o x y a c e tic a c id . ^Silvex is 2,4,5-TP 24 SL\>? !$ & EPA S e rie s Number EPA Batch Sample Number 137833 137835 148917 148918 -1 -2 -1 -2 -3 -4 -1 -2 -3 -4 -5 -1 -2 -3 -4 -5 3D tf l. *= D oubtful TABLE 5. TCDD DATA..FOR AUTHENTIC 2,4,5-TRICHLOROPHENOL _____________ ______ (6 B atch Samples) . ____________ Percent Recovery C137TCDD 95 79 102 104 82 89 89 69 100 80 86 51 93 96 95 61 Limit of D etection (ppb) Q ualitative C riteria 123456 4.6 + 3.5 j. + NA NA NA NA NA NA NA NA 3.7 + -- NA NA NA NA 5 .0 ' + - NA NA NA NA 9.3 ! + - NA NA NA NA 2.1 ; + NA NA NA NA 2.6 + NA NA NA NA 1.9 + + + + + NA 2.5 + - NA NA NA NA 8.8 + - NA NA NA NA 2.5 + - NA NA NA NA 1 .8 + + + + + NA 5 .6 + - NA NA NA NA 2.6 + - NA NA NA NA 5 .0 + - ' NA NA NA NA 1.7 + NA NA NA NA Judgement Pos. D t f l . a ND / / / / / / / / / / / / / / / / i Total TCDD (ppb) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND iA^is J 'i - .. . .......... -1 - EPA S e rie s Number TABLE 6. TCDD DATA FOR AUTHENTIC 2 ,4 ,5 -T ( 2 ,4 .S-folCHLOROPHENOXYACETIC ACID) ________________________________ (3 Batch Samples) t_______________________________ EPA Batch Sample Number Perdent Recovery C137TCDD D etection (ppb) Q ualitative C riteria 1 2 3 4 5 6: Judgement T o tal TCDD Pos. D tf l. ND (ppb) Average T o tal TCDD (Ppb) 137400 -137401 -137402 -1374021 -137402D -137403 -1374031 -137403D 71 68 105 71 58 2 .4 + + + + 4* NA / ND / 18 5.2 + + + + - + / : 17 NA + + + NA + NA / 18 / D tfl. 4 .2 + + 4* + + + / a 14 NA + - NA NA NA NA / ND aP robably f a ls e p o s itiv e sin c e th e d u p lic a te sample (137403D) was ND. o\N> . . . . . ,. ? /, ' . * I; il I \ EPA Series Number TABLE 7. TCDD DATA FOR AUTHENTIC BUTOXYETHYL ESTER' OF 2,4,5-T ______________________ (IQ Batch Samples)_________ _____________ EPA Batch Sample Number Percent Recovery C137TCDD Detection Q ualitative C riteria (ppb) 1 2 3 4. 5 6 Judgement Pos. D tfl. ND Average Total Total TCDD . TCDD (ppb) ' (PPb) 148919 1 -1 -li -ID -2 -21 -2D -3 -4 -5 -51 -5D -6 -61 -6D -6T -7 -71 -7D -8 -81 -8D -9 -91 -9D -9T -10 -101 -lOD 80 114 67 99 ' 104 82 89 114 108 78 72 53 111 117 . 116 97 70 99 61 104 6.8 NA 2.5 NA 8.0 6.4 4.3 NA 3.0 NA 1.1 2.0 NA 1.8 NA 3.1 NA 4.5 1 3.5 ` NA ++++++ + + + NA + NA + ; .+ + + + + + + + NA + NA ++f +++ ++++ + + + + + + -+ + + + NA + NA + + '+ + + + + t + NA + NA ++++++ ++ ++ - + + . NA + NA + +++ + t 4- NA + NA ++t +++ + + NA + NA ++++++ ++++++ + + + NA + NA / / / / / / / / / / / / / / / / / / / / / / / 23 19 25 21 24 29 22 23 28 22 21 33 19 14 30 23 23 19 . 27 19 S t a t i s t i c a l inform ation fo r TCDD p o sitiv e batch samples: n mean to ta l TCDD (ppb) standard d ev iatio n (ppb) coefficient of variation (%) 7 22.7 1.3 5.7 21 23 23 25 D tfl. 22 22 23 'A * \ l EPA S e rie s Number EPA Batch Sample Number TABLE 8. TCDD DATA'FOR AUTHENTIC ISOOCTyt ESTER OF 2 ,4 ,5 -T (3 Batch Samples) Percent Recovery C137TCDD D etection (ppb) Q ualitative C riteria 123456 Judgement Pos. D tf1. ND T o tal TCDD (ppb) Average T o tal TCDD (ppb) 141996 141998 -1419961 -141996D -141996T -141996Q -1 -11 -ID -2 -21 -2D 103 '61 78 103 110 73 108 79 2.0 t + + + + NA + : + + NA + NA 5 .9 + .j; + + + + + NA + 1: 4* H" NA - NA V / / Dtf 1 15 30 21 27 / D tfl 2.5 + ! t + + + + / NA + j + H- NA - . NA / 28 13 / Dtf 1 2 .2 + " - NA NA NA NA / ND 1.5 + ! + + + + + 27 3' Other re p lic a te (s ) doubtful or n o n d etectab le, th e re fo re , probably not dioxin-contam inant or in te r ferences present. I *, EPA S e rie s Number EPA Batch Sample Number TABLE 9. TCDD DATA FOR AUTHENTIC BUTOXYPRO/PYL ESTER OF SILVEX (7 Batch Samples) * _______ Percent Recovery C137TCDD D e te c tio n -Q1--u--a--l-i-t-a--t-i-v--e----C--r--i-t-e--r-i--a--- . (ppb) 12 3 4 5 6 T o tal . Judgem ent TCDD P o s. D tf1. ND (ppb) Average T o tal TCDD (ppb) 148920 -1 71 4.4 + + + + + +' / -2 / -21 83 . 2.3 ++++++ / -2D 115 NA + + f NA + NA / -2T 83 6.3 + + + + + + -3 /. -31 66 5.5 + + + + + + / -3D 77 NA + NA + NA / -4 / -41 67 1 .0 + + + + - NA / -4D 113 NA + + + NA + NA J '-5 t / -51 97 5 .2 + 4* + + + + / -5D 79 NA + + NA + NA / -5T 66 7.2 + + + + - + / -6 103 0 .9 4* + + 4* + + / -7 / -71 74 5.6 + + + + + + / -7D 111 NA ` + + + NA + NA / 21 19 17 22 21 17 20 20 24 17 23 12 22 21 S t a t i s t i c a l in fo rm a tio n f o r TCDD p o s itiv e , b a tc h sam ples: 1 n4 Mean t o t a l TCDD (ppb) 20.3 Standard d e v ia tio n (ppb) 1.5 C o e f fic ie n t o f v a r ia tio n (%) 7.4 19 19 Dtf 1. 21 22 P 6s -0 I EPA S e rie s Number 141997 EPA Batch Sample Number 141997 ,i ! TABLE 10. TCDD DATA FOR AUTHENTIC ISOO,CTYL ESTER OF SILVEX _______________________ (1 Batch Sample)________________________ *I Percent Recovery C137TCDD 81 Lim it of D etection (ppb) 9.7 Q ualitative C riteria 12 345 6 ++++++ Judgement Pos. D tf1. 'ND T o tal TCDD (ppb) / 20 ou> I - 912 EPA S e rie s Number 137832 137834 i TABLE 11. TCDD DATA FOR AUTHENTIC CHLORINATED PHENOLIC ANTISEPTIC ________________________ (22 B atch Samples) ` 1______________ _ -------------------- * 1' 11 ....... . EPA Batch Sample Number Percent ' Recovery C137TCDD Lim it of D etection (ppb) Q ualitative C riteria 12 3 456 Judgement Pos. D tfl. ND -1 -2 -3 -31 -3D -4 -41 -4D -5 -51 -5D -6 -7 -8 -9 -10 -11 -12 -13 -14 -1 -2 -3 -4 -5 -6 -7 -8 83 77 102 68 50 61 91 79 90 84 93 68 64 78 74 96 68 93 111 91 78 100 93 117 102 16 12 8.9 NA 148 NA 4.0 NA 3.9 9.4 2.2 8.4 3.6 2.3 4.4 2.7 2.6 7.1 2.4 1 1.9 3.0 1.7 2.3 4.3 1.9 + + -+ + + + + + + t + + t + + + + + + + + + ;+ + - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - - - - NA - NA NA NA NA NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - NA NA NA NA - .NA NA . NA NA - NA NA NA NA NA NA NA NA / / / / / / / / / / / / / / / / / / / / / / / / / / / / T o tal TCDD (ppb) ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND EPA S e rie s Number EPA Batch Sample Number TABLE 12. TCDD' DATA FOR AUTHENTIC ORGANOPHOSPHATE _________ _________ (7 Batch Samples) , ____________ Percent Recovery C137TCDD Lim it of D etection (ppb) Q ualitative C riteria 123456 Judgement ' Pos. D tf l. ND T o tal TCDD (ppb) 148921 -1 -2 -3 -31 -3D -3T -4 -41 -4D3 -4T -5 -6 -7 51 110 100 100 0 111 95 0 78 . 69 93 9 .9 ;.v + 2.9 5.2 t NA + NA - 2.2 ' NA + + ' NA ....... Y-i ' ~ 1.5 4 + 4.5 + 9.3 + - NA NA NA NA - NA NA NA NA NA NA NA NA - NA NA NA NA - NA NA NA NA NA NA NA NA + + NA + NA - NA NA NA NA - NA NA NA NA - . NA NA NA NA - NA NA NA NA / ND / ND / ND / / / ND / / / ND / ' ND / ND aProbably f a ls e p o s itiv e due to c o n tam in atio n o r in te r f e r e n c e s p r e s e n t, s in c e r e p l i c a t e sam ples were ND. QC Sample Number Percent Recovery C137TCDD 222455 2224551 222455D 220777 2207771 220777D 226617 2266171 226617D 225511 2255111 225511D 229617 2296171 229617D 1-01 1-011 1-01D ^IT 11 111 11D 11T 3W 3Y 3YI 3YD 3X 3X1 3XD 3XT 93-01 4_ 41 ' 4D 14-01 14-011 14-OID 14-01T 3Z 3ZI 3ZD 222841 2228411 22284ID 0093 90 901 90D 303 3031 303D 304 3041 304D 305 3051 305D 92 76 71 119 95 116 76 77 58 82 101 61 94 80 84 100 77 59 83 83 92 89 115 113 90 83 107 89 68 106 ' 89 76 97 88 60 79 81 98 72 116 94 TABLE 13. TCDD DATA FOR QUALITY CONTROLS (20 Different QC Samples; 41 Assays)____ Limit of Detection (ppb) Qualitative Criteria 123456 Judgement Pos. Dtfla ND ToCal TCDD (ppb) 3.1 NA 2.5 NA 4.9 NA 5.9 NA 3.6 NA 12.7 NA NA 0.7 ' NA NA 9.9 + + + + ++ + - NA NA NA NA ++++++ + + NA + NA ++++++ + + + NA + NA + + + + _ + + + + NA + NA +- + + NA + - NA NA NA NA ++ ++++ + + . + NA + NA + + + NA - NA + + ++ ++ +- ++ NA + NA NA NA ++ + NA + NA NA NA Z3 Z z* z1 Z Z Z Z Z* z / Z Z Z Z Z Z Z 4.9 NA 2.4 NA NA 5.5 13 NA + + *+ -+ +- ..- NA NA NA NA + + + +- -++ + + NA NA ++ -- +' + NA + ' NA ++ ++++++ + + NA + NA Z Z -- '-- . z z z z z z 3.1 NA NA . 2.5 NA + + + + + ' NA + - NA NA NA NA + + + NA + NA - NA NA NA NA + - NA NA NA NA z z z 2.0 NA82 2.8 NA 2.1 NA ++ + - +- + + + + NA NA NA NA NA + + + + + + + + + +- ++ NA - NA + ++++ + + + NA + NA z /* z z z zz z 1.2 NA 14 NA + + + + + + Z" + + + NA + NA z + + + ++ + z1 + + + NA + NA z* z 3.$ z ND 39 A3 23 6 74 43 Z' z 7 ND 236 126 274 80 83 z z 79 ND z z ' ND ND ' 56 124 - 82 1620 3270 z 3984 z ' 5.7 ND z 9.6 z ND z ND z 2.2 ND 2250 85 57 16 z 18 9.6 7.1 1610 1732 Average Total TCDD ' (ppb) ND ^ i ! Dcf1 .i i Dtfl. ! i Dtfl.j ND 212 81 ND 87 . 3627 ND ND ND Dtfl. Dcfl. ND Dtfl. 8Replicate doubtful or ND or ocher reruns suggesc probably not dioxin-contaniinanc or Interferences present. 33 .....................* ........................................' I i TABLE 14. VARIATION OF TCDD DETECTION. JUDGEMENT VERSJJS EPA SAMPLE TYPE Sample Type Number Batch Samples Analyzed * N um ber/(Percent) of Batch Samples Versus D etection Judgement P o sitiv e Doubtful Nondetectable 2 ,4 ,5-Trichlorophenol 2,4,5-T Butoxyethyl E ster of 2,4,5-T Isooctyl Ester of 2,4,5-T Butoxypropyl E ster of Silvex LO Is o o c ty l E s te r o f S ilv e x C hlorinated Phenolic A ntiseptic Organophosphate TOTALS Twenty d i f f e r e n t QC Samples Assayed I . 16 3 10 3 7 1 22 7 69 AUTHENTIC PESTICIDE BATCH SAMPLES 16/100 1 /(3 3 .3 ) 1 /(3 3 .3 ) 1 /(3 3 .3 ) 9/(90) 1/(10) 3/(100) . 6/(86) 1/(14) 1/(100) 22/(100) 7/(100) 17(25) 6/(9) 46/66 QUALITY CONTROLS 6/(30) 6/(30) 8/(40) TABLE 15, Sample Type ! VARIATION OF TOTAL TCDD LEVEL VERSUS SAMPLE TYPE FOR POSITIVE SAMPLES TCDD Range (ppb) Number Batch Samples ^ ^ P o s itiv e f o r TCDD 100- 500- 1000- 1500- 2000- 2500- 3000- 350031-99 499 999 1499 1999 2499 2999 3499 3999 Number Batch S a m p le s/(P e rc en t) 2,4,5-T Butoxyethyl E ster of 2,4,5-T Butoxypropyl E ster of S ilv e x u> '-n I s o o c ty l E s te r o f Silvex 1 9 6 1 Six d i f f e r e n t QC Samples P o s itiv e fo r TCDD ` 1/(18) AUTHENTIC PESTICIDE BATCH SAMPLES 9/(100) 6/(100) 1/(100) QUALITY CONTROLS 2/(32.8) 1/(16.8) 1/(16.8) 1/(16.8) 1/(16.8) I b^re *$ Sample 1-01 11 3Y 3X 4 14-01 3Z 222841 222455 220777 226617 225511 229617 90 303 304 305 TABLE 16. COMPARISON OF REPLICATE RESULTS FOR EP'A QUALITY CONTROL SAMPLES I T o ta l TCDD C o n ce n tra tio n (ppb) Run No. 1 Run No. 2 (-D) Run No. 3(-T ) 236 80 ND ' 56 3270 5.7a ND 2.2a 3.6 39 23a 74 7a 85 16a 9.6 1610 126 83 ND 124 3984 ND ND ND ND 45 6a 43 ND 57a 18a 17.1 1732 274 79 NA 82 NA 9 .6 a NA NA NA NA v NA NA $ : NA NA NA NA ; NA S ta tis tic a l Analysis l Vverage (ppb) / 212 81 NA 87 3627 . Standard R elative Standard D eviation . De\f i a t ion (ppb) (%) 7-7 2.1 NA 34 504 36 . 2.6 NA 39 14 . 42 15 59 : 71 17 ! 8.4. 1671 4.2 12 22 20 1.4 1.8 86 9.4 83 37 28 8.3 21 5.2 Q u a lita tiv e c r i t e r i a judgem ent: TCDD d e te c tio n d o u b tfu l. . ) H $ re I TABLE 17. COMPARISON OF REPLICATE RESULTS FOR AUTHENTIC PESTICIDE BATCH SAMPLES Type Sample Batch Sample T o ta l TCDD C o n ce n tra tio n (ppb) Run No,. Run No. Run No. Run No. 1 2 (-D) 3(-T ) 4(-Q) S ta tis tic a l A nalysis Average Cppb) Standard D eviation (ppb) R elative Standard D eviation (%) 2 ,4 ,5 -T 137402 137403 17 14 18 ND 18 .0 .7 1 4 C hlorinated Phenolic ' 137832-3 A ntiseptic 137832-4 137832-5 ND ND ND ND ND ND Isooctyl Ester of 2,4,5-T 141996s 141998-1 15 28 30 21 13S 27 23 7 21 11 30 52 Butoxyethyl E ster of 2,4,5-T 148919-1 148919-2 148919-5 . 148919-6 .148919-7 148919-8 148919-9 138919-10 23 25 22 28 33S 14 23 27 19 21 23 22 21 19S 30 23 19 19 21 23 23 24 26 ` 22 22 23 2 .8 2.8 0.71 3.8 9.9 11 2.3 5.7 14 13 . 3.1 15.8 38 51 10.3 25 Butoxypropyl E ster of Silvex 148920-2 148920-3 148920-4 148920-5 148920-7 19 21 20s 24 22 17 22 17 20s 17 23 21 19 ' 2 .5 19 2.8 20 - 21 3 .8 . 22 0.71 13.2 15 - 18.1 3.3 Organophosphat^ F orm ulations 148921-3 148921-4 ' ND' ND ' ND n 14 Q u a l i t a t i v e c r i t e r i a judgem ent: TCDD d e te c tio n d o u b tfu l. Q th ir d sample o f 148921-3 and 148921-4 was a ls o run b u t n o t re p o rte d due to a 0 p e rc e n t re c o v e ry . io/, n s Appendix A Data S h eets fo r Sample 148919-5-2 ! 38 A N A L Y S IS O F T E C H N IC A L G R A D E P E S T IC ID E S FO R T C D D P aid Sheet I Sam ple No. 198319-5-2 Date Received 2 / 8 /7 3 r Date and Type of Extraction /Cleanup _7//a/7a ;Sam ple W eight / O Q 8j Sample Volume _ Date of G C /M S A nalysis s / e / 7 9 / ______ Total Extract _ 2 0 0 ^ ______ ; GC Conditions Column 3 O/-//^ 3 % Injector Temperature 2 ^ 0 <Z Column Temperature 2 /o (d & 1 Carrier Gas and Flow Rate O '///* //<? / *32 m S/sy/Us (0 )2 6 O t: G C /M S Data fi\ Co-Injected Sample 35c i-t c d d 37c i -TCDD 320 Peak Area m/e 322 328 Ratio 320/322 20 21 2 0. 22 0 2 0 I 209 2& 2/S /36 333 /o&o 2 83 /9oe // 337 7023 237/ O. 790 X 0.738 X Corr.(r)= 0.9933 0.9991 0.997/ 1Slope.(s)= SS- / 73/. 7 / 2 S lntcp.(i)= 2/. 30. ///a i/s = O. 0 9 0.383 0.933 Avg. Ratio ~ O. 0 2 7 A N A L Y S IS O F T E C H N IC A L G R A D E P E S T IC ID E S /V 3 3 /3 -S & -Z TCDD R e su lts FOR ; iI RECOVERY ' ` 37C1-TCDD Std.Conc.(dq/^ D s V 8 % Recovery(R) Added to SQmple(nq) = 8 ( I) R =_2_x |oo= S S % Recovered (ng) : I. i/s x tys - ^ 2 . 8 a n ; QUANTIFICATION 35 CI-TCDD Std. Conc.(pg//Al) = S O O Amount in Extracting) =i/s xmO? Amount in Sample (ng) =I E / R Sample Conc.(ppb)= I E / g Sample 320 20.3 2.2-8 22. C 322 /S.3 ( m i 2/.y U 2 ) 2/. 5" Avg.Conc. (ppb) = 22./ 0 .7 3 LIMIT OF DETECTION N /A _______ A^ m/e = 32.Z________ _ L.D..2.5(S/N) = CONFIRMATION ANALYSIS ( Fragmentation Pattern Recorded) N /A ___ __ A Result Positive Result Negative ^ _____ _______ 40 Appendix B. . M aster Sample S ta tu s Form Al ai7a? ! Sample .i Master Sample Status For 328-884-11 Recovery,% TCDD Level Screening Repeat Average (ppb) Full Scale Calculated Configurations LD Sample Needed Done (ppt) Completed i page <3.170* Appendix C. Cumulative T ab u lated A n a ly tic a l Data Form 43 "El 1. ai77 ...CUMULATIVE / TABULATED ANALYTICAL DATA' FOR 328-884-11 Recovery, % TCDD Level Calcuiated Sample LD Completed Sample Screening Repeat Average Ippb) Confirmation (ppb) GSRI EPA \ J i Code: N A -not applicable _______ sample completed * N D -not detectable _ __ 4 - task performed ______ page Appendix D. GC/MS Perform ance Ion C u rren t Chromatograms (O bservation #21) OBSERVATION #21 DATA pi co-iniected 35C1 TCDD 37C1 TCDD m/e/Peak area / 320 322 328 2 + .2 1+ 1 .. 2+ 2 ............... 1+ 1 1257 583 1203 " 576 1560 735 1612 722 2198 1073 " 2317 1176 Corr (r) Slope (s) Intercept (i) s/5 0 0 o r 480 0.9999 680.5 -101 1.36 0.9990 857.5 -129 1.71 0.9952 1133 -8 .5 2.36 Avg. 320/322 = 0.795 45 M 7 o 2J 2-1 I t -ell ix e JL, H ? ..Appendix E. Ion C u rrre n t Chromatograms fo r TCDD D etected in a P e s tic id e Sample (EPA Sample #4) _____________QUALITATIVE CRITERIA__________ _ 1 2. 3 4 5 6 '+ + + ;+ + + JUDGEMENT: TCDD d e te c te d in th e sample because a l l s ix c r i t e r i a were p o s itiv e . i 50 } t pll'jW / l -A Vr/ot 1 irti*l / A p p e n d i x F. M a s s S p e c t r u m f o r T C D D D e t e c t e d in a Pesticide Sample (EPA Sample #148919-5) NOTE: E x iste n c e of m/e lin e s a t 320, 322, 324 and 326 i n d i c a t i v e o f l i n e a t m/e 328 due to 37Cl TCDD. i 53 2 U J \ V . .. WSZ---- -imr 392 mi.miliii n u lm i lmt.4inliiix.imlm>. nnl "9F2 -mzr wr '0 '02 i i Qtr 09 08 09T 0 H 05T 00T . 08 09 0t- 05 I'III iiiilmi i i i i I i i h iinlini m u I i ii i niiliiu iiiiliin iliiliiil iiiiliin imliiii liulm i iinlini imliiii iiiilun iiiilim iiiilim mi 001 0 02 in 0t- 09 OS OOTJ TSI '6` B2 0001'6 2 `62 ' 0 '2 6 2 2 > 0 '6 0 0 'T - A T 30Ud ~* 2C 6TC ; B S - CS + S ir - 0'91 'S'S2 0 ' T2 ' 6 C 2 C -1- S t H s etr ' 8 i s e o -0 0 1 '6 'se >tr i s o a u i S 6 TT2 fiad w n a i0 3 d s u3au >iaon WF3 -- :-- m z ----~ w s ------ 0 2 S------ m s ------ I n n -U t l l l l l l 1 1 1 l i m i m i l i i i i . m i l i m . I i l i l m i i t u l n n i i i i l m i m i l i m m i l i i n i m l i i i i n u l i n i M u l i n i n n l i u i n i i l i i i i i L i i l i i i i - i l i i '0 02 0tr 09 08t" 09b" <VV 02^ 00{r 088 Linimilmi i i l l l i t n l i i i i i n i m i t i l i I t n l n i i i i i i I i i i i i i i i I i i i i m i l n n m i m i i l i i l i m n n l i m 090 0tr n n l i m i m t u n i m l i u i n t iliiii imi 00T 0 02 ITI TSI '6828 0 `0 0 T ' 6 `28 2 '6 2 '0 2 6 2 2 > 8 '66T0 C O 'I - A 2 3 0 U d 09 08 O0 T S S - 0 S + Sfr- 0 9T '8 '3 2 8 0 T2 '6 8 2 8 ir J.Stn 8 8tr '8 'T2S 0 '00T '6 ` 2 8 fr I S O d b H 261 t s i-rdd u n a io 3 d s U3dy xdon <3ci n.w ifvum ilPqiM ' A p p e n d i x G. M a s s A b u n d a n c e T a b u l a t i o n for T C D D Detected in a Pesticide Sample (EPA Sample #90) NOTE: Mass abundances a t m/e 320, 322, 324 and 326 in d ic a tiv e o f TCDD. 56 `J .1 rRN 21643 SPECTRUM 47 RET. TIME >PAUSE HASS AUND MASS ABUND 247 43 249 250 251 251 252 253 254 255 256 257 259 260 261 262 263 264 264 264 265 266 267 268 269 270 15. 11. 19. 11. 15. 15. 8. 45. 22. 40, 15. 59. 473. 130. 41, 21. 28. 8. 7. 7. 9. 8. 19. 10. 15. 7. 271 272 273 274 275 276 277 278 279 280 281 282 283 284 2 8 5 287 288 289 290 291 292 293 294 295 296 8 11 14 135 81 23 11 8 8 6 40 13 15 6 .12 12 26 11 26 15 11 21 8 7 4 ; 4.5 MASS ABUND 297 . 4 . 297 3. 298 4* 299 7. 300 3. 3O0 3. 301 5* 302 5. 303 7 304 5 1 305 4 * 306 8 307 11. 307 10 308 9* 309 13 310 5 311 7, 312 4 313 9 314 6. 315 45. 316 19. 317 53. 318 18. MASS r 319 319 320 321 322 324 ,325 l?26 328 329 330 331 332 333 334 334 336 336 337 337 338 339 340 341 ABUND 7. 7. 22. 10. 29. 14. 9. 7. 38. 10. 5. 15. 11. 9. 5. 4. 4. 4. 6. 6. 6 4. 2. 7. 342 <C0NT> 4. Appendix H. Io n .C u rre n t Chromatograms fo r N o n d etectab le TCDD in a P e s tic id e Sample (EPA Samples //137834-3 and 3YD) ____________ QUALITATIVE CRITERIA___________ Sample 137834-3 3YD 12 +-- -+ - 34 56 NA NA NA NA NA NA NA NA JUDGEMENT: TCDD n o t d e te c te d in th e sample because c r i t e r i a 1 1 2 was n e g a tiv e due to absence of both m/e 320 and 322 p eak s. 58 i i /SCLIX ~L)'yC o I / / * Appendix I . Ion C u rren t Chromatograms f o r Q u estio n a b le TCDD D e te c tio n in a P e s tic id e Sample (EPA Sample i?90D) ________ QUALITATIVE CRITERIA___________ ' 1 2 3 4 56 + ++ NA - NA JUDGEMENT: TCDD d e te c tio n q u e s tio n a b le because c r i t e r i a $ 5 was n e g a tiv e due to m/e r a tio o u t-o f -s p e c ific a tio n . 63 f! ; I * i EXHIBIT C r SU130 i Reprinted from l: .f'-: H Jo u rn a l o f C hrom atography^ 90 (1974) 7 1 -7 7 tlsc v ic r Scientific P ublishing C o m p a n y , A m s te rd a m -- P rinted in T h e N e th erla n d s Ex.C ..;1 % i DETERMINATION OF 2,3,7,8-TETRACHLORODIBENZa-i,^-blOXIN AT 3 S PARTS PER BILLION* LEVELS IN TECHNICAL-GRADE 2,4,5-TRI- "iV, 'YV CHLOROPHENOXYACET1C ACID, IN 2,4,5-TALKYL ESTER AND 2,4,5-T AMINE SALT HERBICIDE FORMULATIONS BY QUADRUPOLE MASS -i FRAGMENTOGRAPHY %; H A N S -R U D O L F BU SER and H A N S-PA U L B O SSH A R D T : / A S w iss F ederal R esearch S ta tio n fo r A rb o ricu ltu re, V iticulture a n d H o rticu ltu re, C H -8 8 2 0 W a cd en sw il 7 Y Y (S w itzerla n d ) (R eceiv ed N o v e m b e r 1st, 1973) l [ . ;\- i SUM M ARY 7J j A fast, highly selective gas chromatographic method was developed for the -i J: determination of 2,3,7,8-tetrachIorodibenzo-l,4-dioxin (TCDD) in technical 2,4,5trichlorophcnoxyacctic acid (2,4,5-T) and also in 2,4,5-T alkyl ester and amine salt herbicide formulations using quadrupolc mass fragmentography. The method is faster, more sensitive and has a higher specificity than previously reported methods using conventional gas chromatography. It requires minimal or nc sample clean-up. The content of TCDD found in batches of 2,4,5-T produced from 1967 to 1973 by one manufacturer showed a sharp decrease after 1970 from 500 to 50 ppb.* -I .3 . IN T R O D U C T IO N Polychlorinated dibenzo-l,4-dioxins, especially 2,3,7,8-tetrachlorodibenzo1,4-dioxin (TCDD), are extremely toxic1,2, teratogenic3 and mutagenic4 compounds. Owing to their stability in biological systems5,6 and their potential for accumulation through food chains7, they present a threat to man, animals and the environment. In order to prevent environmental contamination with TCDD and thus to prevent irreparable biological damage, the further use of 2,4,5-trichlorophcnoxyacetic acid (2,4,5-T) and other chemically related pesticides possibly contaminated with TCDD must therefore depend on the purity of the active ingredient. The Swiss Federal 3 Research Station at Wacdenswil, as the authority for the licencing of agricultural pesticides in Switzerland, established a specification for a maximum content of V- "rt 50 ppb* of TCDD in 2,4,5-T. Similar specifications for TCDD in 2,4,5-T have been adopted in other countries. There has been a considerable international effort in the development of methods1,6,8-11 to enable manufacturers and government laboratories to control the purity of these products. As a contribution, this paper d/; describes a simple method that permits the rapid and accurate determination of YJ ` T h r o u g h o u t (h is a r tic le (h e A m e r i c a n (10) b illio n is m e a n t . A 3 9 '2 6 6 /4 3 1 .\1 3 M S Lo-lyv't; 7 2 H.-R. BUSER, 1I.-I\ BOSSHARDT TCDD at the required parts per billion level in technical-grade 2,4,5-T and its formulations. Mass spectrometry is widely used for pesticide analysis17'. But mass fragmentography so far has been used mainly for the analysis of drugs and their metabolites in biological systems13"15. We assume that this technique will also gain more and more acceptance in the fields of pesticide residue analysis and formulation control. EXPERIMENTAL Reagents Methanol, diethyl ether and ethyl acetate were obtained in puriss quality and light petroleum (b.p. 40-65) and lithium hydroxide in purum quality from Fluka (Buchs, Switzerland). Aluminium oxide (basic, cationotropic) from Woclm (Eschwege, G.F.R.) was used as received. Reference compound 2,3,7,8-Tetracldorodibenzo-1,4-dioxin (TCDD) was obtained from Stickstoff- werke Linz (Linz, Austria) and its purity was checked by gas chromatographic (GC), mass spectrometric (MS) and micro-elemental analysis. It was found to be suitable for use as a standard without further purification. Standard solutions of TCDD in ethyl acetate were prepared at concentrations of 1 and 10 /zg/ml. The safety precautions to be taken when handling this highly toxic compound have been stressed elsewhere11. Instrumentation A Finnigan Model 1015D quadrupole gas chromatograph-mass spectrometer was used to obtain conventional mass spectra on the reference compound. A GC column was interfaced with the mass spectrometer via a venting system and a glass jet separator. The glass column (1.5 m x 2 mm I.D.) was packed with 3% silicone OV-225 on Chromosorb W AW-DMCS. The column had been previously treated with silylation reagent. The helium carrier gas flow-rate was adjusted to 25 ml/min. The injection port temperature was 250, the column temperature 230 and the separator temperature 250. Mass spectra were recorded at 70 cV. The ion energy used was approximately 10 V and the electron multiplier voltage 3 kV. A scan time of 2 sec w-as found to be suitable for an m/e range of 35-550. For mass fragmentography, the instrument was operated in a similar fashion. With the instrument set at unit resolution, the ion intensities at m/e 320 for TCDD were monitored (single ion detection). This adjustment was made using the precision mass meter of the instrument. The signals were recorded on a regular 10-mV recorder using an additional 0.16-Hz filter. Asecond.similarlytreated glasscolumn with an auxiliaryT-port at approximate ly one third of the column length from the inlet was used for back-flushing possible late-eluting, interfering substances. This column was packed in the first portion (50 cm) with 3% Carbowax 20M on Chromosorb W AW-DMCS*, and in the second portion (100 cm) with 3% silicone OV-225; The column was installed in the gas chromatograph with helium carrier gas lines connected to both the inlet port and the auxiliary port. Additionally, venting lines were mounted for the inlet port and jj QUADRUPOLE MASS FRAGMENTOGRAPHY OF TCDD IN 2,4,5-T 73 auxiliary port. The carrier gas and venting lines could be individually switched on ; and of! by toggle valves outside the chromatograph. Operation of this column at 240 was done with constant pressure (2.0 atm) rather than constant flow-rate in order to achieve efficient back-flushing. The total retention time for TCDD on m both this composite column and on the first conventional column was between 5 and 6 min. 4- Sample preparation A sample preparation based on the work of Edmunds et at.11 was used. Twenty grams of sample were dissolved or suspended in 150 ml methanol and 25 ml of 5 TVlithium hydroxide solution were added. The mixture was refluxed for 20 min and then cooled to room temperature, and 500 ml of water and 200 ml of light petroleum were added and the mixture was vigorously shaken. After separation of the phases, the organic phase was dried over anhydrous sodium sulphate. A ; -U portion of about 100 ml was concentrated under vacuum on a rotary evaporator :>* /-t (maximum temperature 50) to about 10 ml. An aliquot was usually taken because some formulations formed emulsions that were difficult to separate. The concentrate was transferred to an alumina column (45 ml of basic aluminium oxide in a 50 cm x20 mm l.D. column). The column was eluted with 100 ml of light petroleum, 50 ml of 5% diethyl ether and then by 100 ml of 25% :n diethyl ether in light petroleum. These eluates were discarded. TCDD was eluted with 150 ml of diethyl ether and the solution was concentrated to approximately 'i 10 ml. After transfer to a suitable receiver, it was further concentrated to about 0.2 ml with a gentle stream of nitrogen. Care was taken never to allow the sample to be evaporated completely to dryness. The samples, made up to 1.0 ml with ethyl acetate, were now ready for GC-MS analysis. A shortened procedure was used for technical 2.4,5-T or 2 ,4,5-trichIorophenol by omitting the refluxing and the clean-up steps. To a 20-g sample, dissolved in 150 ml of methanol, 500 ml of water and 25 ml of 5 N lithium hydroxide solution were added. After addition of 200 ml of light petroleum and vigorous shaking, the organic phase was separated, dried and an aliquot concentrated on the rotary evaporator. Again, final concentration was carried out with a gentic stream of nitrogen. The samples, made up to volume (1.0 ml) with ethyl acetate, were then analyzed by GC-MS. Samples that contained TCDD at levels above 100 ppb were re-analyzed by mass fragmentography after being methylated with diazomethane according to the micro-method described by Schlcnk and Gcllerman'6. Melhylation was carried out in 10% methanol-diethyl ether solution. Recovery Recovery experiments were carried out by adding differing amounts ofTCDD, ranging from 0.1 to 10 /<g, to tire hydrolysis reagents, to technical 2,4,5-T, 2,4,5,1richlorophcnol or to formulations. These fortification levels correspond to 5-500 ppb for technical materials and 50-5000 ppb for 10% 2,4,5-T formulations. Determination For quadrupolc mass fragmentography, the mass spectrometer was used to S " i 74 H.-R. RUSER, H.-P. BOSSHARDT monitor at unit resolution the molecular ion for TCDD at m/e 320. The precision mass meter was adjusted by slowly evaporating a few nanograms of TCDD from the direct insertion probe. A standard curve was prepared by injecting differing amounts of TCDD (0.5-100 ng) on the GC column and plotting peak heights versus amount injected. Quantitation of TCDD in samples was then achieved by comparing their peak heights with the standard curve. In order to allow injection of up to 50 /A of solution, a venting period of 1 min was used to prevent overloading the MS vacuum system with solvent vapour. Samples prepared by the shortened procedure were analyzed using the dual-column system. An initial venting period of 1 min was used and back-flushing initiated after 4 min, which prevented early- and late-eluting components from entering and contaminating the separator and ion source. This timing period was previously determined by a few standard injections so as to ensure that none of the TCDD is lost through venting and back-flushing. Confirmation of the identity of TCDD in samples with a suspected content higher than 100 ppb was carried out by MS. A larger aliquot, corresponding to approximately 100 ng of TCDD, was injected and complete mass spectra were recorded. Five mass spectra taken at 15-sec intervals brackciting the retention time of TCDD were sufficient. The spectra were evaluated _by_comparison of the mass peaks in the region m/e 200-500 of scans obtained before, during and alter ^lutTon of"suspectec! TCDD. The km intensities of m/e 320, 322 an <1*324 ( M ^ J o u r clilorm<raroms)^nd^57Tahcr259 (M.+ --COCTTTlire^liloiTne atomsj"wcrc compared with liiuforarTCDD'stanHard. Iri acBItTbruTIte speCtTS'WrCTheckc'OTgrthcmbScffCt omilorinc-cxmtainiiTgMiTglicr molecular*"1ons liiat ~b'uTd'~pb^'ibly"'rorii~rraginents at IhcsejmJFvttcs.^ *~ ~ "in addition, samples containing large amounts of TCDD were reassessed by mass fragmentography after methylation in order to observe any reduction of the TCDD content caused by this treatment. RESULTS AND DISCUSSION The MS data for TCDD indicate intense molecular ions'with the characteristic clustering due to chlorine isotopes at m/e 320, 322 and 324. The only other ions of some intensity in the higher mass range are at 257 and 259 (M +-COCI). Quadrupole mass fragmentography was carried out by single ion detection of the ion at m/e 320. This ion was used rather than the more intense ion at m/e 322 because of possible interference of PCBs. In addition, column bleeding from silicone OV-225 showed a minor signal at m/e 322. The response of the Finnigan 1015D quadrupole mass spectrometer proved to be sufficiently stable over a suitable period of time to allow the use of an external standard for quantitation with acceptable precision. The peak height of the response was found to be linear in the range 0.5-100 ng of TCDD injected. A detection limit of better than 0.5 ng could easily be achieved without any attempt to maximize the response. This corresponds to a level of approximately 1 ppb based on a 20-g sample of technical material. This detection limit was found to be sufficient for ,1 V wrrr QUADRUPOLE MASS FRAGMENTOGRAPHY OF TCDD IN 2,4,5-T 75 the present work. For the samples investigated, a single GC peak was usually ob tained. Back-flushing and venting the dual-c ,Iumn system was achieved by switching the carrier gas stream and vent by toggle valves outside the heated zones. Such a system has been described by Deans17 as early as 1965 and more recently by Warner cl a!.18. The system allowed the back-flushing of unwanted late-ciuting components and prevented them from entering the second, final GC column. With the same system, early-eluting components are vented and do not contaminate the separator and ion source. It is of importance in pesticide analysis that the com ponents of interest are in contact only with glass and not with any metallic parts such as valves. Only components with retention times in a narrow range arc deter mined. Of course, this system is applicable only when a few components are of interest. The use of a first column to cut out unwanted components results in an efficient clcan-up and simplifies sample preparations prior to GC analysis. Such systems arc easily built, easy to operate and suitable for routine application and automation. In combination with single ion detection, this leads to a high specificity of detection. We are sure that such techniques will gain an increasingly wider application in pesticide analysis. >0 & S f >/ i>/ i L Recovcry experiments for TCDD added to/fne reagents and carried through all the steps of the procedure ranged from 40%` at the 0.1 //g level to 80-100% at the 1-10 /tg level. Addition of similar amounts of TCDD to 2,4,5-T and 2,4,5- trichlorophcnol led to recoveries of 80-100% at levels corresponding to 50-500 ppb. Recoveries in both 2,4,5-T ester and amine salt formulations were lower (60-80%). Some results for the content of TCDD in 2,4,5-T are reported in Table I. The samples listed icpresent the production of a German manufacturer over a. period of 6 years. We were told after this investigation that the sample with code 12.8.70 and a content of ca. 2000 ppb TCDD was not of German but of American origin. TABLE I T C D D C O N T E N T IN S A M P L E S O F 2 ,4 ,5-T O B T A IN E D F R O M A G E R M A N M A N U F A C TURER S a m p le code T C D D co n ten (ppb) C o m p lete procedure Sim p lified procedure 7.8.67 J 7.7.70 12.8.70 12.11.70 12.12.70 14.9.71 22.9.72 18.9.73 D ow icide 2 (2 ,4 ,5-tricliloropheno!) 300 390,490 1840 420 50 80 n.a. * n.a. * 2 240 520 1950 5S0, 640 60 60, 70 40 80 <3 ' N ot analysed. 76 H.-R. BUSER, H.-P. BOSSHARDT The first column in Tabic I lists the results obtained according to the original procedure, while the second column lists the results obtained by the shortened "'Gccdurc. The results indicate acceptable agreement for duplicate samples and also for the two procedures. The results show that the content of TCDD in technical 2,4,5-T for this manufacturer ranged from 300 to 600 ppb prior to 1971. Since then, the TCDD content decreased to 40-S0 ppb, which is about compatible with the 50 ppb specification valid in Switzerland. As a comparison, a single result for a sample of 2,4,5-trichlorophenol (Dowicide 2) is included, which shows a content of TCDD of about 2 ppb. In addition, a comparison of the results listed in Table I indicates that refluxing samples with methanolic lithium hydroxide docs not increase the TCDD content. The simplified method results in a very fast procedure for the analysis of TCDD in technical 2,4,5-T and 2,4,5-trichlorophenol and is suitable for routine analysis in quality control. Table II lists some typical results obtained by analyzing 2,4,5-T formulations taken from commercial sources in Switzerland in June, 1973. These formulations were analyzed using the complete procedure with refluxing and alumina column clean-up. The results indicate TCDD contents of 20-70 ppb relative to 2,4,5-T. Again, the results are acceptable with o.' specification. TCDD in samples with a suspected content above 100 ppb.could be confirmed in all instances by MS. Confirmation of TCDD by complete MS analysis on these samples was preferred to multiple ion detection on three or four prominent ions. Scanning the whole mass range from mfe 35 to 550 in fixed intervals before, during and after elution of the suspected TCDD allowed the confirmation and identification of TCDD in all those samples, although no GC peak could usually be observed on the total ion monitor, because of a multitude of other components that were eluted from the column at about the same retention time as TCDD. These components TABLE II TCDD CONTENT IN 2,4,5-T FORMULATIONS TAKEN SO URCES IN S W ITZER LAN D IN JU N E , 1973 Results are given in ppb relative to 2,4,5-T. FROM COMMERCIAL F o rm u la tio n S a m p le TC D D c o n te n t Ester Amine salt Manufacturer 1 (15% 2,4,5-T) Manufacturer II (45% 2,4,5-T) Manufacturer III (12% 2,4,5-T) Manufacturer IV (9% 2,4,5-T) Manufacturer V (9% 2,4,5-T) 50 20 10,20 50,70 20, 50 :/I .3; .5 -K n4 ' v '';4; 4 QUADRUPOLE MASS FRAGMENTOGRAl'HY OF TCDD IN 2,4,5-T 77 could be identified by MS as polychlorinated biphenyls, dibenzofurans and diphenyl oxides. The presence of polychlorohydroxydiphenyl ethers as possible precursors of TCDD could not be substantiated. This was observed by Rappe and Nilsson19 for octachlorodibenzo-1,4-dioxin. Our finding is based on the fact that with the samples examined in this study, no significant reduction of the apparent TCDD content occurred after mcthylation of the neutral part. R EFER EN C ES 1 N .I.E .H .S . C onference on C h lo rin a ted D ib en zo d io xin s a n d D ib en zo fu ra n s, A p ril 2 -3 , 1973, R esea rch T riangle P a rk , N .C ., U .S .A ., E nviron. H ea lth P ersp ecl., 5 (1973). 2 R e p o rt o f the A d v iso ry C o m m itte e on 2 ,4 ,5 - T to the A d m in istra to r o f the E n v iro n m e n ta l P ro tectio n A g en cy, G o v e rn m e n t P rin tin g Office, W a sh in g to n , D .C ., M ay, 197!. 3 K . D . C o u rtn e y , D . W . G a y lo r, M . D . H o g a n , H . L. F a lk , R . R . B ates an d I. M itchell, S cien ce, 168 (1970) 864. 4 J. P. Seiler, E xp cricn tia , 29 (1973) 622. 5 R. H . S lchl, R. R. Papenfuss, R . A . Bredevvcg a n d R. W. R o b erts, u n p u b lish e d results. 6 P. C . K e a rn e y , Jo in t M eetin g on P esticides, U nited K in g d o m , C anada a n d U .S .A ., W a shington, D .C ., N o vem b er, 1970. 7 3. V c rrc tl, S ta te m e n t b efo re th e U .S . S e n a te S u b -C o m m itte e on E n e rg y , N a tu r a l R e so u rc e s a n d the E n v iro n m en t, A p ril 15, 1970. 8 D . F ir e s to n e , J. R ess, N . L. B ro w n , R . P. B a r ro n a n d J. N . D a m ic o , J. A ss. O ffic. A n a l. C lient, 55 (1972) 85. 9 W . B. C r u m m c t t a n d H . R . S te h l, E n viro n . H e a lth P e r sp c c t., 5 (1973) 15. 10 K . S. B re n n e r , K . M u lle r a n d P. S attcl, J. C h ro m a to g r., 64 (1972) 39. 11 J . W . E d m u n d s , D . F . L e e a n d C . M . L . N i c k e l , P e s t i c . S c i . , 4 ( 1 9 7 3 ) 1 0 1 . 12 F . J . B i r o s , R e s id u e R e v ., 4 0 ( 1 9 7 3 ) 1. 13 C .-G . H a rn m a r, B. H o im s tc d t a n d R. R y h ag c , A nal. B io ch e m ., 25 (1968) 532. 14 C .-G . H a rn m a r, B. H o im s tc d t, J.-E . L in d g rc n a n d R . T h a m , A d va n . P h a rm a co l. C h em o th cr., 7 (1969) 53. 15 J. M . S tr o n g a n d A . J. A tk in s o n , J r ., A n a l. C lien t., 49 (1972) 2287. 16 H . S ch lcn k a n d J. J. G c lle rm a n , A nal. C h em ., 32 (1960) 1412. 17 D . R . D e a n s , J. C h ro m a to g r., 18 (1965) 477. 18 C . R . W a r n e r , M . C . J o h n s o n , D . G . P r u e an d B. T. K h o , J. C h ro m a to g r., 82 (1973) 263. 19 C . R a p p e a n d C . A . N ilsso n , /. C h ro m a to g r., 67 (1972) 247. .f ii. H EXHIBIT D (29) S. J. W. Price and A. F. Trotman-Oichenson, Trans. Faraday Soc., 54. 1630 (1958). 130) J. W. Robinson, R. Garcia. G. Hindman, and P. Slevin, Anal. Chim. Acta, 69, 203(1974). (31) G. E. Parris and F. E. Brinckman, Environ. Scl. Techno!., 10, 1128 (1976). t32) J. J. Ritter and N. K. Adams. Anal. Chem.. 48, 612 (1976). (33) G. L Everett. T. S. West and R. W. Williams. Anal. Chim. Acta, 70, 291 (1974). (34) D. J. Johnson. B. L. Sharp. T. S. West, and R. M. Oagnall, Anal. Chem., 47, 1234(1975). (35) G. Torsi and G. Tessari, Anal. Cham., 48, 1318 (1976). R e c e i v e for review September 10, 1976. Accepted N o vember 17,1976. Contributions from the National Bureau of Standards are not subject to copyright. G.E.P. was supported by a-National Research Council Post-doctoral Research Associateship at NBS during the completion of this work. We acknowledge with thanks the continued encouragement and partial financial support of the U.S. Naval Ship Research and Development Laboratory (Annapolis), Maryland and the NBS Office of Air and Water Measurement. Certain commercial equipment, instruments, or materials are identified in this paper in order to adequately specify the experimental pro cedure. In no case does such identification imply recommen dation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose. Automated Cleanup of Herbicides by Adsorption Chromatography for the Determination of 2,3,7,8-Tetrachiorodibenzo-p-dioxin Tore Ramsiad,* N. H. Mahle, and R. Mataln Analytical Laboratories, Dow Chemical U.S.A., Midland, Mich. 48640 An autom ated system is described w hich repeatedly u s e s a iow -cost silica column tor ch em ical cleanup. Its u se In se p a rating and collecting trace lev els of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDO) from esters of 2,4,5-lrichlorophenoxyacetic acid (2,4,5-T ) in com m ercial herbicide prep arations is demonstrated. Previously, chem ical cleanup by column adsorption chromatography required a new ly-packed colum n for ea c h sam p le. More than 4 0 0 runs h ave b een achieved on a single colum n to d ate. S y stem a ccu ra cy , a s determined by checking against identical sam ples manually preftared, is excellen t. The standard deviation for the sa m e ester) analyzed 36 tim es, w as 0.002 ppm at an average TCDD concentration of 0 .0 2 6 ppm. There Is no m easu rab le c r o ss contamination from sam ple to sam ple. Chemical cleanup by column adsorption chromatography has become a widely used technique, particularly in the area of pesticide residue analysis. Many different adsorbents have found application, notably Florisil, magnesium oxide, alu mina, charcoal, and silica gel (1). T he procedure with such columns has been to discard the packing after a single use. In order to reuse an adsorption column for chemical cleanup, the surface must be "renewed" by removing the polar molecules which comprise the bulk of the sample. While manual regen eration of an adsorption column would be impracticably slow, automatic regeneration makes repeated use of an adsorption column possible. Automation is well-known in liquid column chromatogra phy, particularly in amino acid analysis by ion exchange chromatography (2-4). Perhaps less well-known are auto mated peptide (5) and sugar (6) chromatography systems. More recently Small et al. have devised schemes for auto mated ion exchange chromatography utilizing a novel tech nique which allows for universal ion detection conductimetrically (7). While a number of advances have occurred in the devel opment of automated ion-exchange chromatography employing "clean" samples, development of automated cleanup systems employing reusable columns has lagged. A semi-automatic apparatus for purifying amino acids from non-amino acid components such as salts and protein pre- cipitants has been developed recently by Blanshard e t al. (8). The purification, which takes place on a cation exchange column, precedes normal automated amino acid determina tion. Their two-hour procedure includes a regeneration of the resin with HCI to remove bound cations prior to introduction of a new sample. The use of automated gel permeation chromatography for the separation of pesticides from lipids and oils has been successfully applied by Tindle and Stalling (9, 10). Gel per meation can effect a satisfactory separation for these samplesbecause many pesticides have molecular weights in the range of 200-400, while most lipids fall between 600 and 1500. An evaluation of their GPC unit was conducted by Griffitt and Craun (11). They reported that cleanup efficiency was supe rior to that obtained with acetonitrile partitioning, although in some cases an ensuing cleanup on Florisil was necessary prior to gas chromatographic determination. This instrument is now available in commercial form (12). Stalling et al. have recently expanded their system to permit combined exclusion/carbon bed cleanup (13,14). Three recent review articles on automated pesticide residue analysis describe no additional automated cleanup units utilizing column chromatography (15-17). However, since'the completion of the present work, one paper has appeared which describes the regeneration of a liquid-liquid chromatographic column (18). The authors report that an analytical column packed with Bio-Sil A coated with sulfuric acid is made re usable by successively passing 1:1 chloroform-cyclohexane, 0.1 N sulfuric acid in 50% ethanol, and chloroform-cyclo hexane between analyses. It is necessary that a new precolumn be packed for each analysis, but the analytical column lasts from 10-30 runs. We sought to automate the separation and collection of trace levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a toxic impurity formed in the manufacture of esters of 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), from commercial herbicide preparations. As the molecular weight of the ester used m ost in this study is 370 and that of TCDD 320, GPC would not be expected to resolve these two compounds satis factorily. Reported here is an automated adsorption"chro matographic system which successfully effects the separa tion. P rin cip le o f the M ethod. The ester of 2,4,5-T used 386 A N A LY T IC A l C H F M IST R Y VOi. 49. NO 3. M A R C H 1977 0-175? friction Collector Figure 1. Schematic of automated system. See text for details . throughout most of this study was the l-lsobutoxy-2 -propyl (designated PiB-propylene glycol isobutyl). To separate TCDD from the PiB ester of 2,4,5-T on silica gel, a solution of 1:4 (v/v) benzene-hexane may be used {19). Under these conditions the TCDD passes through the column essentially unretained while the more polar ester is held up on the packing. To prepare the column for another sample, the ad sorbed molecules must be removed from the silica surface. This is accomplished by eluting with a more polar solvent combination-- 15:85 (v/v) tetrahydrofuran (THF)-benzene. The TH F is then removed with benzene followed by a short re-equilibraLion with 1:4 benzene-hexane prior to application of the next sample. EXPERIMENTAL D e s c r ip tio n o f S y s te m . T h e sy ste m is c o n s tru c te d o f co m m e rc ially A vailable c o m p o n e n ts e x c e p t for a few e a sily -m a c h in e d p a rts. A s c h e m a tic d ia g r a m is s h o w n in F ig u r e 1. (A ls o s e e F ig u r e 2 .) T h e c o lu m n is c o n s tru c te d o f 316 s ta in le s s s te e l tu b in g a n d h a s a sin g le tu rn ; it m e a su re s 1 m X 5 m m . I t is p a c k e d d ry w ith 1 0 0 -2 0 0 m esh " h ig h -p u rity " silica gel (C u rtin S c ie n tific , N o. 531-178). As o u tlin e d in th e previous sectio n , th re e d iffe re n t so lv en t co m b in atio n s a re p u m p e d th ro u g h th e c o lu m n . T h e ir d e liv e ry is c o n tro lle d by 2-w uy valv es A , H , a n d C (A ltex, 201-00; p n e u m a tic a c tu a to rs, A ltex, 201-12). A f t e r s a m p l e a p p l i c a t i o n , t h e 1:4 b e n z e n e - h e x a n e e l u t e s T C D D w h ile t h e e s t e r is r e t a i n e d o n t h e c o l u m n . A p p r o x i m a t e l y 2 0 m l o f t h e 1:4 b e n z e n e -h e x a n e e lu e n t is p u m p e d a t a flo w ra te o f 1 m l/m in (S lo w P u m p , M ilto n H oy, M odel 396-31) to m axim ize th e sep a ra tio n o f T C D D a n d e ste r, a n d o c cu rs in th e fo rw a rd d ire c tio n th ro u g h th e co lu m n , as show n by th e arro w . T o rem o v e th e e ste r from th e silica s u rfa c e c a. 100 m l o f 15:85 T H F - b e n z e n e is p a ss e d th ro u g h th e c o lu m n in th e re v e rse d ire c tio n a t a flow ra te o f 4 m l/m in (F a s t P u m p , M ilto n R oy, M o d el 396-89). S w itc h in g b e tw e e n th e fo rw a rd a n d re v e rse flow d ire c tio n s is a c c o m p lish e d w ith th e a id o f 3 -w ay v a lv e s D (A lte x , 2 0 1 -0 1 ) a n d E (V a)co , A C V -3 H P ). B a c k flu s h in g is u s e d to p re v e n t e ste r from co n ta ctin g th e low er p o rtio n o f th e co lum n. T h is serv es tw o p u rp o se s: 1) m in im ize s c o e lu tio n o f c o m p o n e n ts w h ich m a y in te rfe re in th e e n su in g d e te rm in a tio n o f T C D D by gas c h ro m a to g ra p h y -m a ss sp e c tro m e try (G C -M S ), a n d 2) e x te n d s th e c o lu m n life by p re v e n tin g th e low er p o rtio n o f th e co lu m n from c o n ta ctin g th e b u lk o f th e sa m p le . A s th e o b je c tiv e o f th is w o rk is to re u se th e sa m e c o lu m n fo r a n e x te n d e d p e rio d o f tim e , th e la tte r c o n s id e ra tio n is o f p rim e im p o rta n c e . O u r e x p e rim e n ts h a v e sh o w n th a t .th e e s te r is re ta in e d on th e to p th ird o f a fresh ly -p ack ed co lum n. U se o f a long colum n sh o u ld h e lp p ro lo n g c o lu m n life. A s a fin al p re c a u tio n to e x te n d c o lu m n life. Valve A Valve B (Open) (Closed) (Open) (Closed) - Valve C (Open) (Closed) - Vrivtt 0 (FF) (BF) - Valve E (FF) (BF) - Valve F (Sii) (Slo) - Sampling AssemblyfActuated) i I Peristaltic (On) Pump (Off) Slow Pump (On) (Off) Fast Pump (On) (Off) Fraction (Advance)______________________ Collector I II I I I I I I l I 0 2 4 6 8 10 n Figure 2. Timing diagram "FT 96$ __ n J ________!__________ t 25 30 35 Drum Position On Timer I 42 FF = Switched for forward flow, BF = Switched for backflushing, Sll = Sam ple loop switched In, and Slo -- Sample loop switched out r L _r _j_i 59 60 ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977 387 -_-W XT Figur J.Schematic of start/stop circuitry Figure 4. Typical mass chromatogram T H F is s h a k e n w ith 5- m o le c u lar siev e (L in d e ) to m in im ize su rfa c e d e activ atio n d u e to th e in tro d u ctio n o f w ater. A fte r th p a ssa g e o f 15:85 T H F - b e n z e n e , 110 m l o f b e n z e n e a re p a sse d . T h e p u rp o se o f b en zen e is to s tr ip o ff th e T H F p rio r to in tr o d u c t i o n o f th e n e x t s a m p le . F o llo w in g a r e e q u i l i b r a t i o n w ith 1:4 b en zen e-h ex an e a t 4 m l/m in , the n e x t sam p le m ay b e a p p lie d to th e co lu m n . T h e sam pling sy stem consists o f a p lu n g er fitted w ith a hy p o d erm ic n eed le, in co m b in a tio n w ith a B u ch ler p e rista ltic p u m p (N u c le arC hicago) em p lo y in g A u to A n aly zer tu b in g (T ec h n ic o n In s tru m e n t C o rp .). S a m p le in tr o d u c tio n is a c c o m p lis h e d w ith a V a le o a u to m a tic sam p lin g valve (A V S V -6 -H P ) w ith a 5 .0 -in l e x te rn a l lo o p a tta c h e d . S a m p le a n d co llection vials a re su ita b ly p laced o n a fractio n co llecto r ( I S C O , M o d e l 2 7 8 ) a s s h o w n in F ig u r e 1. T h e s o lv e n t s a n d v o lu m e s u sed in th e elu tio n sch em e p re sen te d h ere w ere a rriv e d a t u sin g T L C , co lu m n ch ro m ato g rap h y , an d u ltrav io le t an d N M R sp ec tro m etry . T h e e n tire sy stem , cap ab le o f seq u e n tia lly pro cessin g ten sam p les, is u n d e r th e co n tro l o f a m u ltic h a n n e l, m e c h a n ic a lly -p ro g ra m m a b le tim e r (S ealectro, 092-286-5501-000). E ach ch an n el of th e tim er d ru m h a s 60 p o sitio n s. In th is s y ste m th e d ru m is s te p p e d o n c e e v e ry 9 6 s b y a y.., r p m s y n c h r o n o u s m o t o r ( S e a l e c t r o , 0 9 5 - 0 2 6 - 6 5 2 5 - 0 0 0 ) s o t h a t an e n tire cycle (one sam p le p ro cessed ) la k e s 96 m in. S everal featu res o f th e tim ing d iag ram (F ig u re 2) a re a p p a re n t from th e prev io u s d iscussion. T h e e x tra sam p lin g tim e (p o sitio n s 28-8 3 ) is fo r flu sh in g o u t th e viscous sam p le w ith so lv en t to p re v e n t plugging Table I. TCDD C oncentration vs. Run No. for PiB Ester o f 2,4,5-T Run No. ppm TCDD Run No. ppm TCDD 21 24 25 27 29 31 33 - 34 35 36 37 38 39 41 43 44 48 49 : 0.026 0.028 0.030 0.024 0.020 0.026 0.029 0.028 0.034 ' 0.028 0.026 0.028 0.026 0.026 0.026 0.023 0.024 0.026 50 51 52 53 . 55 57 59 60 61 62 63 64 65 67 72 73 79 90 0.025 0.024 0.025 0.026 0.025 0.024 0.024 0.025 0.024 0.024 0.025 0.026 .024 0.025 0.023 0.026 0.024 0.024 o f th e sam p le line a n d e n su res a g a in st c ro ss-c o n ta m in a tio n from one s a m p le to th e n e x t. D u rin g p o s itio n s 2 7 -5 9 , th e c o lle c te d T C D D is p o sitio n ed u n d e r an a ir je t to e v ap o rate th e b e n zen e-h ex an e so l v e n t. A p ro te c tiv e fe a tu re o f th e a p p a r a tu s is a s im p le s ta r t/s to p c irc u it sh o w n in F ig u re 3. T h e flip -flo p sh o w n is in te rp o s e d b e tw e e n a m ic ro sw itch a n d th e p o w er re la y , a n d serv es as n b u ffe r in case o f m e ch an ica l slip p a g e o f th e s h u t-o ff asse m b ly . T h e m icro sw itch is closed by a p in located on th e fractio n co llector reel w hen th e tim er reaches position 59 a fte r th e la st sam p le h as been processed. A fter loading th e n e x t b a tc h o f sa m p le s, on e n e ed s o n ly to p ress th e clear b u tto n to re s ta rt th e sy stem . A L ig h t E m ittin g D iode (L E D ) in d icates w hen sam p les a re being p ro cessed . P ro p e r c irc u it lay o u t, ro u tin g o f critical le a d -in w ire, a n d c irc u it sh ie ld in g a re e sse n tia l to p re v e n t false trig g e rin g o f th e d ig ita l c irc u itry . A n a d d itio n a l p ro te c tiv e fe a tu re is th e in clu sio n o f a p re ssu re sw itch (B a rk sd a le V alves, D 1S-A 80) w h ich a u to m a tic a lly s h u ts o ff p o w er to th e sy ste m in th e e v e n t o f a sev ere a ir leak. T h e tim er, s ta rt/s to p circ u it, air so len o id s, term in al strip s, an d m a n ifo ld fo r th e p n e u m a tic lin e s a re all lo c a te d in a c o n tro l m o d u le . B ecause o f th e hig h fla m m a b ility o f th e so lv en ts, th e so lv en t con- 3 M ANAI Y T I r t - F M I S T P Y . VOI 4P. NO. 3. M A R C H 1977 Table II. Correlations of Automated and Manual Cleanups Run No. Sample 40 PiB ester of 2,4,5-T 42 PiB ester of 2,4,5-T 45 PiB ester of 2,4,5-T 46 PiB ester of 2,4,5-T 47 PiB ester of 2,4,5-T 68 PiB ester of 2,4,5-T 69 PiB ester of 2,4,5-T 75 PiB ester of 2,4,5-T 76 PiB ester of 2,4,5-T 77 PiB ester of 2,4,5-T 80 PiB ester of silvex 81 PiB ester of 2,4,5-T 82,91 PiB ester of 2,4,5-T 83 PiB ester of 2,4,5-T 84 PiB ester of 2',4,5-T 85 Butoxyprpyi ester of 2,4,5-T 96 PiB ester of 2,4,5-T a Difference = Automated --Manual. Automated 0.017 0.030 0.020 0.032 0.027 0.028 0.023 0.028 0.030 0.056 0.036 0.050 0.092 0.020 0.048 0.008 0.114 ppm 2,3,7,8-TCDD Manual 0.014 0.029 0.021 0.032 0.027 0.029 0.023 0.029 0.029 0.056 0.034 0.049 0.091 0.022 0.050 0.008 0.119 Difference0 0.003 0.001 - 0.001 0.000 0.000 -0.001 0.000 -0.001 0.001 0.000 0.002 0.001 0.001 - 0.002 - 0.002 0.000 -0.005 ta in e rs a re 0.5 -g allon " sa fe ty " can s w ith flam e a rre s to rs . A s a p re c a u tio n a g ain st a c c u m u la tio n o f fla m m a b le v a p o rs in th e e v e n t o f a leak , p o in ts o f sp ark in g are pu rg ed w ith a lig h t stre a m o f air. F in ally , sin ce th e co llected T C D D is c o n c e n tra te d u n d e r a c u rre n t o f a ir, all c o m p o n en ts o th e r th a n th e control m o d u le a re lo cated in sid e a hood. O p e ra tio n o f th e S y ste m . F ive-gram sam p les a re w eighed in to N o. 7 d r a m v ia ls a n d d ilu te d to 25 m l w ith 1:4 b e n z e n e -h e x a n e . T h e v ia ls a re c a p p e d w ith a lu m in u m foil, sh in y sid e u p , a n d p laced o n th e in sid e tra c k o f th e frac tio n co llecto r reel. U p to te n sa m p le vials m ay be p laced on th e reel. A fter th e m icrosw itch h a s b een m o v ed fo rw ard a n d c la m p e d , th e sy ste m is re a d y to be tu rn e d o n fo r u n a tte n d e d o p e ra tio n . P rio r to d e te rm in a tio n o f T C D D by G C -M S , th e collected dio x in is m a n u a lly tra n s fe rre d fro m th e N o . 7 d ra m v ia l to a N o . 1 d ra m v ial, e v a p o ra te d to d ry n ess, an d th en b ro u g h t to a fin al vo lu m e o f 200 pi w ith o -x y le n e . A s th e sam p le is a p p lie d to th e c o lu m n fro m a 5.0-m I lo o p , th is m a k e s fo r a fiv efo ld c o n c e n tra tio n (1 g -* 200 til). D e te rm in a tio n o f T C D D . T h e ex tracts o b tain ed from th e a u to m a te d u n it w ere a n aly zed fo r T C D D a s follow s. T h e c o n ce n tratio n o f T C D D w as d e te rm in e d by G C -M S u sing a L K B 9000S. In jectio n s o f 3 -5 p i w ere m ad e o n to a 3 ft X 3 m m glass co lu m n p ack ed w ith 3% O V -3 on G as C hrom Q . T h e an aly sis co n d i tio n s w e re a s follow s: c a rrie r gas: h e liu m a t 35 cm 'V m in ; te m p e ra tu re s : c o lu m n , 230 C ; in jecto r, 250 C ; s e p a ra to r, 27 0 C ; ion so u rc e, 270C ; e le c tro n en erg y : 70 eV ; a c c e le ra tin g voltage: 3.5 kV ; tra p c u rre n t: 60 fiA. A ty p ic a l m a ss c h ro m a to g ra m is sh o w n in F ig u re 4. RESULTS AND DISCUSSION Three factors were evaluated: 1) the number of times a column can be reused in a cleanup role; 2 ) system accuracy and precision; and 3) the degree of cross-contamination, if any, from one sample to the next. N inety-six runs were made in our initial study. Samples of the same PiB ester of 2,4,5-T were run interm ittently from runs 21-90. The results are shown in Table I. The average TCDD content for these determinations was 0.026 ppm with a standard deviation of 0.002 ppm. Three separate manual cleanups of the same sample, performed per the method of Crummett and Stehl (19), also yielded an average dioxin concentration of 0.026 ppm. Nine blanks were interspersed among.these runs; no TCDD was detected in the blanks with a detection limit of 0.005 ppm. . Sixteen samples of the PiB ester of 2,4,5-T were cleaned up by both the manual and automated methods and their dioxin levels determined. In addition, one each of a butoxy propyl ester of 2,4,5-T and a PiB ester of 2-(2,4,5-trichlorophenoxy) propionic acid (silvex) were compared. T he TCDD concen trations found are shown in Table II along with the corre sponding run number. The agreement is excellent for more than 50 runs. A t this point there was no indication o f column degradation. Since completing the initial study, we have run several other esters of 2,4,5-T. We have not accumulated the same extensive: correlative data for these esters as reported for the PiB ester of 2,4,5-T. Once the performance of the column was demon strated, esters differing only in the alcohol portion would be expected to chromatograph similarly. F u tu re D evelop m en t. The demonstrated ability to re peatedly use a silica gel column for chemical cleanups pens up other possibilities. Immediately obvious is the substitution of other adsorbents such as alumina and Florisil where ap plicable. As with any analysis, certain advantages attend to its automation. Routine monitoring of production materials is simplified. In contrast to the 3 hours required to work up a sample manually using the procedure of Crummett and Stehl (19), the automated system does its work on up to 10 samples, overnight. Improved accuracy and precision as well as increased reliability result. When working with TCDD, the less chance of human contact with this toxic substance, the better. Also, from the human standpoint, when performing a routine analysis, repeated use of a single column eliminates the tedium of packing a new column for every sample. We wish to emphasize the simplicity of the present system. As sequencing is under the control of a mechanically-pro grammed electromechanical timer, familiarity with computer programming and computer interfacing is not required, as would be the case if a m ini- or microcomputer were used. 1/ it is desired to alter the timing sequence for use with a dif ferent cleanup scheme, only a different timing drum is re quired for a quick changeover, or the actuators are reposi tioned for a slower changeover. ACKNOWLEDGMENT The authors thank Larry McFarland for the construction of the control module and Don Girardin for performing most of the manual cleanups. The authors are also grateful to their colleagues for much stim ulating discussion. LITERATURE CITED (1) W. P. McKinley, D. E. Collin, and K. A. McCully, J. Assoc. Off. Agr. Chem., 47, 863 (1964). (2) D. H. Spackman, W. H. Stein, and S. Moore, Anal. Chem., 30, 1190 (1958). (3) K. Dus, S. Llndroth, R. Pabst. and R. M. Smith, Anal. Blochem., 14, 41 (1966). (4) G. Ertingshausen, H. J. Adler, and A. S. Reichler, J. Chromatogr., 42,355 (1969). ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977 389 (5) D. J. Benrfelt and E. H. Creaser, Anal. Biochom.. 37, 191 (1970). ' (6) A. R. Tschida a n d R Markowitz, Anal. Biochem., 2 6 , 337 (1968). (7) H. Small. T. S. SOvens, and W. C. Bauman. Anal. Cham., 47, 1801 (1975). (8) K. C. Blanshard. H. F. Bradford. P. R. Dodd, and A. J. Thomas. Anal. Bio chem.. 67, 233(1975). (9) D. L. Stalling. R. C. Tindle. and J. L. Johnson, J. Assoc. OH. Anal. Cham., 55, 32(1972). (10) R. C. Tindle and D. L. Stalling. Anal. Chem., 44, 1768 (1972). (11) K. R. Grilfitt and J. C. Craun, J. Assoc. 01f. Anal. Cham.. 57, 168 (1974). (12) GPC 1001-Autoprep. Analytical Biochemistry Laboratories lnc,, Columbia, Mo. 65201. (13) D. L. Stalling, J. Johnson, and J. N. Huckins. "Automated Gel PermeatlorvCarbon Chromatographic Cleanup of Dioxins. PCBs, Pesticides, and Industrial Chemicals", in "Environmental Quality and Safety. Supplement Vol. Ill, Pesticides Lectures of the IUPAC Third International Congress of Pesticide Chem istry" (Helsinki, July 1974), F. Coulslon and F. Korte, Ed., G. Thiomo Publ.. Stuttgart, Germany, 1975. pp 12-18. (14) D. L. Stalling, Proc. Conf. Environ. Sensing Assessment, Las Vegas. Nev., Institute of Electrical and Electronic Engineers, Inc.. U S A Annals No. 75CH1004-1, New York. 1976. Section 7-5. (15) B. A. Karihuber and D. 0. Eberle. Anal. Cham.. 47, 1094 (1975). (16) H. A. McLeod. J. Chromatogr. Set.. 13, 302 (1975). (17) D. E. Ott, Residue Bov.. 55, 1, 1975. (18) J. F. Morot-Gaudry, V. Fiala, J. C. Huet. and E.Jolivet, J. Chromatogr., 117, 279 (1976). (19) W. B. Crummett and R. H. Stehl, Environ. Health Perspectives, 15, Sept., 1973. R E C E I V E D for review October 8,1976. Accepted December 2,1976. Characterization of Coal by Laser Pyrolysis Gas Chromatography R. L. Hanson,*1 N. E. Vanderborgh,2 and D. G. Brookins The University of New Mexico, Albuquerque, N.M. 87131 ' Applications of laser pyrolysis g a s chromatography (LPGC) to studyjhe Influence of composition of co a ls on the distribution of g a seo u s products are p resen ted . LPGC provides a rapid method when used in conjunction with plasm a stoichiom etric analysis for determining the relative concentrations of carbon, hydrogen, and oxygen in co a ls. Pyrogram s and correlations between experimental products and elem ental com positions are presented. Pyrolysis gas chromatography has been used to characterize coals of various rank (1-3).T h e highest pyrolysis temperature used.in these studies was 1273 K, resulting in the evolution of large quantities of low molecular weight gases. Benzene and toluene are the major aromatic products (1). Changes in the chromatograms with coal rank were apparent (1, 2). The volatility of the organic compounds in the coal was enhanced by the addition of water to the coal samples before pyrolysis (J). Coal pyrolysis in nitrogen at temperatures of 623-773 K produced methane and ethane as major products with carbon monoxide, ethylene, and propane also formed (4). An argon plasma torch reactor has also been used to study the rapid devolatilization of small coal particles (5). The weight loss increased with both increased heating rate and increased temperature. Heating rates from 10s-1 0 fi K /s and a final temperature of 2000 K produced a greater loss of vol atile matter than is obtained by proximate analysis. A mi crowave discharge in argon was used to study the gasification reactions of coal (6 ). The major products were H-j and CO with the major hydrocarbon products being CH.t, C2H2, and C2Hj. Also formed in smaller quantities were H20 , C 0 2, HCN, and higher molecular weight hydrocarbons up to C. Rapid pyrolysis of coal has been achieved by having pulsed lasers irradiate the samples and using mass spectrometry for identification and quantitation (7-18). Laser pyrolysis gas chromatography (LPGC) has been used in studies of organic compounds and oil shales (19-21). Because of the rapidity of analysis, this technique was selected to use to characterize coal samples. 1P r e s e n t a d d re s s , I n h a la tio n T o x ic o lo g y R e s e a r c h I n s titu te , L ovelace B io m ed ical a n d E n v iro n m e n ta l R esearch In s titu te , P . 0 . B ox 5890, A lb u q u e rq u e , N .M . 87115. 2P re se n t a d d re ss, Los A lam os S cie n tific L a b o ra to ries, U n iv e rsity o f C alifo rn ia , L os A lam os, N .M . 87044. T he method of free energy minimization was used in a computer program to calculate the gaseous product distri bution for coal samples at temperatures from 2000 to 3500 K at a pressure of 2280 Torr (22). Correlations are presented of the experimental gaseous product compositions to the eler mental composition of the coal. The comparison between the calculated product distributions at 3300 K and the experi mental results agree for H2, CO, and C2H2 to 5% by volume or better for the five coal samples. EXPERIMENTAL T h e coal sam p les w ere received as pow ders from P a u l W eir C om p a n y , C h i c a g o , 111. T a b l e I l i s t s t h e c o m p o s i t i o n o f e a c h s a m p l e . T h e sam p les w ere p elletized a t 20 000 p s i.to form p e llets o f 1 -2 m m th ic k n e ss. T h e sa m p le s w ere se c tio n e d a n d p la c ed in q u a rtz sa m p le tu b e s o f 6 -m m o.d. A P e rk in -E lm e r m odel 3920 gas c h ro m a to g ra p h w as used w ith a b e ta -in d u c ed lu m in escen ce d e te c to r (B IL D ) a n d th e h y d ro g en flam e ionization d e te cto r con n ected in series. T h e B IL D d e te cts co m p o n en ts in th e c o lu m n e fflu e n t by m e a su rin g th e q u e n ch in g o f th e lu m in e s c e n c e fro m tra c e le v els o f n itro g e n in th e h e liu m c a rrie r g a s th a t is e x c i t e d b y b e t a 's f r o m a t r i t i u m fo il. T h e d e s i g n a n d R e s p o n s e o f t h i s d e te c to r h a s b e e n d e s c rib e d (2.7). T h e q u a r tz s a m p le tu b e c o n ta in in g th e p elletized sam p le w as m o u n te d on th e in jectio n p o rt o f th e gas c h ro m ato g rap h . H eliu m c arrie r gas p u rged th e sy stem a n d c arrie d th e p y ro ly sis p ro d u c ts o n to th e a n aly tical colum n. A p u lsed ru b y laser w ith a n o u tp u t o f 2.6 jo u les in th e n o rm a l-m o d e w as used in th ese stu d ie s. T h e b eam w as focused o n th e sam p le su rface in th e q u a rtz tu b e s. O n ly a b o u t 0.01 cm 2 o f th e s a m p le w as irra d ia te d by th e focused laser beam . RESULTS AND DISCUSSION Hydrogen sulfide and hydrogen cyanide have been reported as products from laser pyrolysis of coal (7, 11). Hydrogen sulfide and hydrogen cyanide have retention times similar to the retention times of water and acetylene, respectively, under the experimental conditions. The experimental peaks are reported as water and acetylene with recognition that hy drogen sulfide and hydrogen cyanide may be formed. Equilibrium product distribution calculations were made for five coal samples for the elem ents carbon, oxygen, hydro gen, nitrogen, and sulfur. An initial calculation at 2280 Torr and 2500 K was made for 33 compounds: CH4, C-iH.i, C2H 2, c , h . c h 3, c h .,o , h c o , co, co2, h 2, h , o h , h 2o ; C2N 2i HCN, CS2, HNCO, HNO, N 2, N, NO, N 0 2, N 20 , NH, N H 2, NH.i, SN , SH, S2, SO, S 0 2, H2S, and C(s). Nine species with insignificant concentrations svere excluded from further consideration. They were OH, HNCO, HNO, N, N 0 2, N 20 , 24 7^-3 EXHIBIT E 7 7 MAR 2 1980 METHOD ML-AM 75-34 DETERMINATION OF 2,3,7,3-TETRACHLORODI3ENZ0-P-DIOXIN IN 2,4,5-TRICHLOROPHENOXY ACETIC ACID AND RELATED MATERIALS The method herein described is applicable to: 2,4,5-T Acid, Silvex*Acid, Chlorinated Phenols, and includes Caustic Extractions Silica Gel Cleanup Ion Exchange Cleanup Alumina Cleanup ^Trademark of The Dow Chemical Company. May 2 0 , 1975 ANALYTICAL 1ETH0D ML-AM-75-34 DETERMINATION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN IN 2,4,5-TRICHLOROPHZNOXYACETIC ACID AND RELATED MATERIALS 1. Scope This procedure is applicable to the determination of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) and other.re lated chlorinated materials in 2,4,5-trichlorophenoxyacetic acid (2,4,5-T acid), 2,4-dichlorophenoxyacetic acid, and related acidic and phenolic materials and esters of these acids. 2. Principle The neutral components of the sample are separated from the matrix. This is accomplished by extraction from caustic or by silica gel column chromatography, or by ion exchange column. The residue"is concentrated and a portion examined by gas chromatography-mass spectrometry for species which have ions of the same mass, number of chlorine atoms, and retention time as TCDD. 3. Interferences There are no known interferences when applied to 2,4,5-T acid, 2,4-D acid, 2,4,5-trichlorophenol, tetrachlorophenol, pentachlorophenol, and esters of these acids. Other isomers of tetrachlorodibenzo-p-dioxin are separated from TCDD on the chromatographic column and do not interfere. 4. Safety Precautions 2,4,5-T acid and related materials should be handled with the normal amount of caution. Avoid breathing sample dust or vapors. The preparation of standards of TCDD should be done so as to prevent exposure to the cure material and with extrem 'are to avoid soills. All glassware us nr? should : well scrubbed and rinsed with acetone, benzene, or chi 'O O T u l n o " : ' returning to general laboratory use. Special car should be 1 of 5 p May 2 0 , 1975 2 of 6 Pages ML-AM 7 5 - 3 4 exercised in their disposal. It is advisable to read "Summary of Safe Handling of 2,3,7,8-tetrachlorodibenzo-pdioxin in the Laboratory", available from the Sample Co ordinator, Ag-Organics Department, 9008 Building, The Dow Chemical Company, Midland, MI 48640. Some of the other reagent used can be hazardous to handle also} follow the safe handling precautions recom mended by the supplier. 5. Sampling Samples should be taken and treated to insure homogeniety If the product can be melted easily (not 2,4,5-T acid), minimum temperatures and times should be used. Grinding and blending of large pieces of material is recommended. 6. Apparatus (a) Gas chromatograph-mass spectrometer (GC-MS): LK39000S (LKB Productor, Bromma, Sweden) or equivalent, with multiple ion detector. (b) Gas chromatographic column for LKB: 1 m x 3 mm I.D. (glass) packed with 3% OV-3 on 80/100 Gas Chroin. Z. --- .--(c)- 50 cm x 16 mm I.D... glass chromatography columns with 250 ml reservoir. 7. Reagents (a) 10% aqueous sodium hydroxide: weigh 100 g of ACS reagent grads sodium hydroxide into a 250 ml beaker and pour with swirling into 750 ml of distilled water in a liter volu metric flask. When solution is complete, dilute to volume. (b) 1%' aqueous sodium hydroxide: dilute 100 ml of 10% sodium hydroxide.to one liter with distilled water. (c) Benzene: ACS reagent grade; "distilled in glass" grade is desirable. (d) Methanol: ACS reagent grade. (e) Hexane: ACS reagent grade, "distilled in glass" grade is desirable. (f) Silica Gel: High purity 100-200 mesh, used as re ceived without activation. (g) Ion exchange resin AG-2IK brand chloride form, 50/10 mesh, available from Bio-Rad Laboratories, 32nd and Griffin Avenue, Richmond, CA 94804. (h) Pure TCDD: available from Sample Coordinator, AgOrganics Department, 9003 Building. The Dow Chemical Company Midland, Ml"48540. 8. Calibration (a) Standard TCDD: Weigh 1-2 mg tc :he nearest 0.01 mg in a small weighing cup. Transfer to a 10-ml volumetric and dii ut; to volume r t n cercana. Preoare lower May 2 0 , 1975 3 of 6 Pages ML-AM- 7 5 - 3 4 concentrations by dilation. Make concentrations of approximately 2 ygs/ml to be used for tuning and 0.2 ygs/ml to be used as standard calibration. (b) Inject 5 yl of the strong standard (2 yg/ml) in the GC-MS. As the TCDD elutes, fine tune the magnet and accel erating voltage alternator control to maximize the response at ra/e 320, 322 and 324. (c) Inject 3 yl of the weak standard (0.2 yg/ml in the GC-MS and record the response. Measure the ion intensities at the chromatographic peak maximum using a tangent baseline. 9. Procedure A - Chlorinated Phenoxyacetic acids _ (a) Preparation of Ion-exchange Resin, 21K Hydroxide form. i) Place one liter of ion exchange resin 21K (Cl- form) into a 2000 ml beaker and slurry with 500 ml of distilled water. Transfer the slurry to a 2 inch (I.D.) by 30 inch glass column having a glass wool plug above the stopcock made of Teflon plastic. ii) Pass approximately 3000 ml of 5% sodium hydroxide through the resin at a flow rate of approximately 10 ml/min. iii) Pass distilled water through the column until the eluent is no longer basic (as determined by testing with pHydrion paper). iv) Pass approximately one gallon of methanol through the resin to remove the v/ater. v) Divide the resin into two equal portions; transfer each to a 1 liter amber bottle. vi) Decant the excess methanol from the resin and fill the bottle with benzene. Shake vigorously. If the resin is not used immediately it should be refrigerated to retard decom-position. (b) Transfer eighty ml of the 21K (OH- form) resin to a 16 mm (I.D.) by 60 mm glass column having a glass wool plug above the stopcock made of Teflon plastic. Adjust the solvent level to slightly above the resin bed. (c) Weigh 5.0 gram of sample into a 7-dram vial. Add 20 ml of 1:1 benzene/methanol (V/V) and shake until dissolved. (d) Transfer the solution to the ion exchange column using a minimal amount of solvent for rinsing. (e) Elute the dioxins from the column with 1:1 benzene/ methanol. Discard the first 25 ml eluting from the column. Collect the next 125 ml eluting in a 150 ml beaker. ay 20 1975 4 of 6 Pagas ML-AM-75-34 (f) Evaporate the solvent in the beaker to approximately 15-20 ml at ambient temperature using a stream of nitrogen. Transfer this (with adequate rinsing of the beaker) to a 7-dram vial. (g) Evaporate the solvent in the vial to dryness at ambient temperature using a stream of dry nitrogen. Pipet 1.0 ml of benzene into the vial and cap tightly. th) Inject a l.Oul portion into the G C - M S and monitor the three molecular ions at m/e 320, 322, 324 by either repeti tively scanning over this region or by means of the multipleion detector assembly which will focus these three ions successively at 1/2 second intervals. Measure all three ion intensities at the chromatographic peak maximum using -a tangent baseline. 10. Procedure B - Chlorinated Phenols..... ~ h) Weigh 5.0 g of sample into a 400 ml beaker. . Add 200 ml of methanol, 15 ml of 10% sodium hydroxide, and 200 ml of dis tilled water and stir 'until complete solution is obtained. Transfer to a 500 ml separatory funnel and extract with 50 ml hexane (or benzene)., (Note 12b) . .(b) Transfer the aqueous phase to the 400 ml beaker and the organic layer to a separate 125 ml separatory funnel. Repeat the extraction of the aqueous phase with fresh hexane (or benzene) and add the second organic portion of the first. (c) Wash the combined organic phases with an equal volume of 1% sodium hydroxide followed by washing with an equal volume of water. .(d) Add the hexane (or benzene) in portions to a 5 dram (25 ml) vial and evaporate to near dryness -at room temperature under a gentle stream of air. when, the last portion has been evaporated, rinse the separatory funnel and evaporate just to dryness. CAUTION; Do not evaporate at elevated temperatures or allow the vial to be dry for more than several minutes. (e) Add 0.5 ml benzene to the vial, cap securely, and rotate gently to wet all interior surfaces. (f) Inject a 1.0 yl portion into the GC-MS and monitor the three molecular ions at m/e 320, 322, 324 by either repetitively scanning over this region or by means of the multipie-ion detector accessory which 'will focus -these three ions successively at 1/2 sec. intervals. Measure all three ion intensities at the chromatographic peak maximum using aa tangent baseline. May 20, 1975 5 of 6 Pages ML-AH-- 75-34 11. Operating Conditions Column: (glass) 1 m x 3 mm I.D. .3% OV-3 on 80/100 Gas Chrom Z Temperatures: Column - 240CFlash Heater - 250C Separator - 2S0C Ion Source - 270C Flow Rate: 40 cc/min. helium as read on rotameter Trap Current: 60 yA Accelerating Voltage: 3.5 KV Electron Energy: 70 ev iMultiplier: 4.9 Masses to be monitored. m/e 320, 322 and 324 Oscillograph chart speed: 1 cm/min. - 12. Calculations (a) The concentration of TCDD present is calculated from C = ^n_ x ^a x 1_ x S ' b n x D,b E. vhere C is the concentration of TCDD in the sample; " a = ion intensity as recorded in lOf. for m/e = 320 (sample) b = ion intensity as recorded in 8b for m/e = 320 (standard) D = Oscillograph attenuation range for largest on-scale ion intensity for sample D^ = Oscillograph attenuation range for largest on-scale ion intensity for standard E = ratio of sample weight in grams to final solution volume in ml. (usually 10) S = concentration of the standard in yg/ml (b) The above calculation is repeated for m/e = 322 and for m/e = 324. The- values should agree to 155. If they do not, the value recorded is the lowest concentration calculated. 13. Recovery and Precision The precision of this procedure for duplicate samples should ,e 201 of the amount present. The lower limit of detection of this procedure is 0.01 to 0.02 p o m . 14 Notes (a) For samplas which are non-ionizabla, such as esters, the TCQD can be separatee from the matrix by substituting the folicvine procedure for steps 10 a, b, and c . 1. Weigh a 5.0 gm sample and dissolve/slurry with 25 ml of 20% benzene in hexane. Pour (with rinsing) this sample onto 50 g of silica gel in a 16 mm. i.d. glass column previously equilibrated-with 20% benzene/hexana. Allow 25 ml of solvent to elute. Collect the next 100 ml of effluent, adding 20% benzene/hexane to the column as necessary to main tain liquid above the silica gal bed. Continue as in lOd. (b) The detection of other species present or suspected in the neutral residue can be accomplished by either scanning or focusing on the most characteristic ions formed from these species. Quantitation is achieved as described for TCDD. A**************************************** The analytical procedures given herein nave been adapted from literature sources or developed upon the basis of experi mental data believed to be reliable- In the hands of a quali fied analyst they are expected to yield results of sufficient accuracy for their intended purposes. Users are cautioned to infirm the suitability of the methods by appropriate tests. Anyone wishing to reproduce or publish the material in whole or in part, should request written permission from The Dow Chemical Company. s .' METHOD 375953 ISOOCTYL ESTER OF 2,4,5-T: THE TCDD METHOD HEREIN DESCRIBED IS IDENTICAL TO THAT USED FOR ALL ESTERS 2,4,5-TRICHLOROPHENOXY ACETIC ACID AND 2,4,5-TRICHL PHENOXYRRO?IONIC ACID oo THE DOW C H E M I C A L C O M P A N Y M ID L A N D . M IC H IG A N -SAO February 12, 1976 Method No.-87596b 2,4,5-TRICHL0R0PHEM0XYACETIC ACID ISOOCTYL ESTERS A. Scooe This method is applicable to the analysis of 2,4,5trichlorophenoxyacetic acid, isooctyl esters. A procedure is given for the determination of the assay of 2,4,5-, trichloropnenoxyacetic acid, (2,4,5-T) isooctyl esters, and free acid. The acid equivalent is calculated from the assay. A procedure is also given for the determination of 2,3 ,7,3-tetrachloro-dibenzo-p-dioxin. (TCDD) . B. Safety Precautions The isooctyl esters of 2,4,5-T may cause mild eye and skin irritation but are not readily absorbed through the skin in toxic amounts. Avoid skin and eye contact. Handle with care. Nitric acid is corrosive and a strong oxidizing agent., Wear gloves and goggles when handling nitric acid.. Sodium biphenyl and 30% hydrogen peroxide are strong oxidizers. Avoid eye and skin contact. Follow the safety precautions as recommended by the supplier. Potassium cyanide (KCN) is TOXIC. Handle with care. To dispose of KCN solution flush sink drain with water for 15 minutes before and after KCN is poured down the sink. Some of the other reagents used may also be hazardous to handle. Follow the recommendations given by the supplier for their safe handling. 1. Principle The sample is decomposed using sodium biphenyl. Excess biphenyl reagent is precipitated and the soluble chloride is titrated potentiometrically with standard silver nitrate. 2. Apparatus (a) Metrohm E - 436 Potentiograph, Brinkman Instruments Cantiague Road, Westbury, N.J. 11590, or equivalent. c, fRAG/, 1 of 10 pages '< l a m li iTreoiuarv 12, 1975 2 of 10 races Ms thod N o . 37 59 5b (b) Silver Billet electrode, Beckman 439261, or ecu .valent. Coat silver billet with silver chloride (see Section 4). The Beckman electrode is available from Beckman Lab Instruments 25511 Southfield Road, Southfield, MI 43075. (c) Glass electrode, Sargent S3005-15C, or equivalent non-chloride containing reference electrode. The Sargent electrode is available from Sargent-Welch Scientific Co., 3560 West Chicago Avenue, Detroit, MI 43204. (d) Hot plate. 3. Reagents (a) Sodium biphenyl reagent, available from Southwestern Analytical Chemicals, Inc., ?.0. Box 485, Austin, TX 43757. Reagent comes in 1/2 ounce bottles suitable for a single determination... Store in a refrigerator prior to use. (b) 1,2-Qimethoxyethane (DME), available from Eastman Kodak Company, Rochester, iY 14650. (c) Nitric acid, 1:1 solution. Carefully add 500 ml of concentrated nitric acid to 500 ml of distilled water. (d) Silver nitrate, 0.1 N. Dissolve 17 g of reagent grade silver nitrate- in 1 liter of distilled water. Standardize the silver nitrate solution with dry primary standard sodium chloride. Alternatively, an analytical concentrate "Dilut-it" from J. T. Baker 0.1 N-AcNO3 available from Curtin Scientific, ? .0. Box 1223, Midlanc, MI 43640 can be made to volume. (s) Hydrogen Peroxide, 30% . if) Potassium chloride approx i. 7.5 gm po tassium chloride in L 12.tsr* i l v Q. 1 N . Dissolve water. (g) Potassium cyanide, 5% - Di oive o g potassium cyaniae in 100 ml of water. TOXIC. Handle with care. (Note-DO MOT add KCN to an acid solution). . 4. Preparation of the Silver-Silver Chloride Electrode. (a) Polish the silver portion with fine steel wool until it is shiny and clean. Chemically clean the electrode bv brief 1'/ soaking ic in a oh solution or KCN ana rinsinc with water. The same KCN solution can be used reoaatediv for clear ire -h<a electrode. (b) Coat tne eisc_roce witn silver chloride bv a"--^t^o`position from an approximately 0.1 N s o i u t i S i T o f ~ ? o M m using a 3--voir cry cen, arc a olatinum wire electrode n tsb ru a r v 1_ 2 1976 3 of 10 pages Method Mo. 37596b Connect the silver electrode to the positive pole o~ tne. oai__ery and plate with chloride for 20 seconds. Then, by means or a switch, reverse the current for five seconds to partially remove the chloride. Repeat this operation twice and finally coat the silver electrode with chloride for 20 seconds. Coating must be uniform. (c) In the presence of biphenyl, the silver chloride coating lasts for about 20 determinations. Silver billet should be coated with chloride dailv. 5 . Procedure (a) Accurately weigh, to the nearest 0.1 mg, 100-200 mg of sample or a sufficient quantity to yield about 1.5 milliequivalents of chloride, into a dry 250-ml beaker. (b) Add 10 ml of 1,2-dimethoxyethane (DlME) to the sample Swirl to dissolve. Run a blank on reagents used. (c) Shake the contents of a sodium biphenyl reagent boctle and add in small increments with swirling. The mixture should remain green. If it is not, add more biphenyl reagent until green color persists indicating an excess of the reagent. Let it srand for 5 minutes. (d) """ Carefully' heat the mixture to boiling on the hot plate by-swirling the beaker's contents to avoid any bumping. Cool to* room temperature. (e) Carefully add 50 ml of distilled water with swirling and 5 mi of 30% hydrogen peroxide. Add some glass beads and cover with a watch glass. (f) Heat on the hot plate to incipient boiling and then transfer to a steam bath. 3oiT gently for 10 minutes. (g) Remove from steam bath and let stand for 5 minutes. While stirring solution with a glass rod, add. sufficient water to dilute to 150 ml. Let stand for 10 minutes. (h) After standing, break up soli ied biphenyl with a glass rod. (See Mote 3b) Add 5 ml of 1 nitric acid. (i) Place a magn3etiirring bar in the bea ker . with a standard silver ni ate 30 1U tion to cas serv tion point. The end coin 1 3 3 'T as the mid -coi n c o inflection curve. OC' -- February 12, 1975 of 10 pages Method Mo.3' 5. Calibration (A - 3) x N x 367.5 C x 30' %2,4,5-trichlorcphenoxyacetic acid, isooctvl esters. where : A 3 C N 367 5 ml of silver nitrate used for the sample ml of silver nitrate used for the blank sample weight in grams normality of silver nitrate solution molecular weight of 2,4,5-T isooctyl esters 7. Precision -Data obtained by this procedure indicate a standard devi ation of 0.05%. Any single determination may be expected to differ from the average by not more than 0.1% (95% confidence limits). 3 . Motes (a) It is advisable to run an Analytical Standar or 2,4,5-T with each set of samples. This is available from the Sampie Coordinator, Agricultural Products Department, 9003 Building, The Dow Chemical Company,. Midland, ML 43640. (b) Rapid cooling of solution will cause a thick crust or biphenyl precipitate to form which is difficult to break up. Also, possible occlusion of chloride in biphenyl crust can occu if- solution is allowed to cool rapidly. Slow cooling causes a finer precipitate of biphenyl to form. (c) Presence or b r o m e e and chloride in some samles will ce indicated by two breaks in the titration curve, with the bromide end-point occurrinc first. 9 ., Principle \ d d Equival The acid equivalent is calculated from the assay as determined in Section 5. . 10. Calculation 255.5 = molecular weight of 2,4, 5-trichlcroohenc:<vacatL: F e b r u a r ' / 12, 197 8 5 of 10 oaqe: He thod N o . 37596b 367.5 = m o l e c u l a r w e i g h t o f 2 , 4 , 5- t r i c h l o r o p h e n o x y a c e t i c a c i d , isooctyl esters.. %2,4,5-trichloroohenoxy acid isooctyl esters (as obtained in Section 61 x 255.5 = %2,4,5-T acid equivalent. 367 .5 Or %2,4,5-trichlorophenoxyacetic acid isooctyl esters x 0.6952 = %2,4, 5-T acid equivalent E . Free Acid 1 1 . Princiole The free acid is determined by a potentiometric titration of' an alcoholic solution of the ester with 0.1 N sodium hydroxide solution to an apparent pH of 7.0- (see Note 17a) . The free acid is calculated as 2,4, 5-tr ichloroohe.ooxvace tic acid. 12. Apparatus (a) pH Meter, any commercial instrument which is capabi; of giving1 reproducible results with a precision of 0 .1 units (b) Reference electrode, saturated'calomel. (c) Glass electrode. 13 . Reacents (a) Presare 1 liter of 0.1 N NaOH bv cuantitativel' crans :err m g :n.Ci ontents or an anaivticai concentrate ui^ur-i' tram J. T. Baker to a 1 liter volumetric flask. Dilute to exactlv 1 liter with distilled water. (b) Ethanol, 95% Formula 23 or Formula 30 neutralized to an ancarer.t pH of 7.0. ' (c) Buffer solution, equimolar phosphace, 0.05 M, pH o . a . Dissolve 3.53 3 g of .ACS reagent grade disodium hvcrcgen pncspnare (NajHPCK) and 3.333 c of ACS reacent crade corassium. ; n r7 - ' o p * : :?o.) , -i, :evicusiy r ied a r 12 0 0' ror rwo hours, in .ed water to 10 00 Februarv 12, 1976 * 6 o f 10 pagas Method No. 373960 14. Procedure (a) Standardize the electrode system at 25C against the pH 6.35 aqueous buffer (13c) . (0) Accurately weigh, to the nearest 0.05 g, 10 g of sample into a 150 ml beaker. (c) Add 100 ml of neutral alcohol and titrate the solution potentiometrically to an apparent pH of 7.0 with 0.1 N sodium hydroxide (see Mote 17b). 15. Calculation % Free acid as 2,4,5-trichlorophenoxyacetic acid = ml NaOH x N x 0.2555 x 100 grams of sample 16. 'Precision and Accuracy The standard deviation for both the precision and the accuracy of this method has been determined by the analysis of three series of production and prepared standard samples to be 0.04% absolute. The expected limit of variation at the 95% confidence level is = 0.08% absolute for a range of free acid from 0 to 7%. 17. Notes (a) The oVo tentiometric endpoint for the titration of the pher.oxyacet ic ac ids is an apparent pH of 3 .5 . However, in the presence of tU0 ester it is necessary to stoo the titration at an apparent pH of 7.0 in order to avoid saponification. With a slight excess of alkali, the ester can be completely saponif at room temperature within a few minutes. (b) Methyl red can be used as an indicator instead of the pH meter if the color change can be seen. (c) Since the titration is stopped at pH 7 in order to avoid saponification of the ester, the free acid determination is always biased low. February 12, 1976 7 of 10 pages Method Mo. 37595b F. 2,3,7,8-Tetrachlorodibenzc-p-dioxin (TCDD) 13. Principle The neutral components including any TCDD in the sample are separated from the matrix. This is accomplished by silica gel column chromatography. The residue is concentrated and a portion examined by gas chromatography-mass spectrometry for species which have ions of the same mass, number of chlorine atoms, and retention time as TCDD. 19. Interferences No substances are encountered to an extent sufficient to cause interference down to approximately 10 p p b . 20. Soecificitv and Selectivitv The gas chromatography-mass spectrometry (GC-MS) technique is extremely sensitive and specific for the determination of TCDD. In order to assign a signal to TCDD three things must occur simultaneously: 1) the retention time must be correct (the peak maximum is reproducible to within 0.1 min.); 2) all three m/e 320, 322 and 324 must respond and; 3) they must respond in the proper ratio. 21. Safety Precautions TCDD in amounts handled in the laboratory can produce chloracne if allowed to contact the skin. Extreme safety precautions should be exercised in the preparation and disposal of standard material and solutions. It is advisable to read "Summary of Safe Handling of 2,3,7,8-tetrachlorodibenzop-dioxin in the Laboratory," available from the Sample Coordinator, Agricultural Products Department, 9003 3uilding, The Dow Chemical Companv, Midland, MI 43640. Handle ail solvents in a fume hood and avoid breathing vapors. 2 2.. Apeara tus (a) Gas chromatograph-mass spectrometer: 1K3-9000S (1K3 Productor, 3rcmma, Sweden) or equivalent, with multiple ion detector. (b) (grass. i c x e c w i t n _hie column for 1K3: 1 m x 3 mm i .c . OV-3 on 30/100 Gas Chrom. Z. s l h s "? February 12, 1976 i 3 o f 10 p a c e s Method Mo. 37o9 6b (c) 30 cm :< il mm i.d. glass chromatography columns with 125 mi reservoir. 23. Reagents (a) Benzene: ACS reagent grade; available from Burdick S Jackson Laboratories, Inc., 1953 S. Harvey Street, Muskegon, MI 49442. (b) Hexane: ACS reagent grade; available from Burdick & Jackson Laboraotries, Inc., 19 53 S. Harvey Street, .Muskegon, MI 49442. (c) Silica Gel: High purity 100-200 mesh, Davidson 923 Brand, available from Curtin Scientific, Midland, MI 48640. (d) 20% benzene in hexane: Add 200 ml benzene to 800 ml hexane and mix well. " , (e) Pure TCDD : available from the Sample Coordinator, Agricultural Products Department, 9003 Building, The Dow Chemical Company, Midland, MI 43640. 24. Calibration (a) Standard TCDD: Weigh 1-2 mg to the nearest 0.01 mg in a small weighing cup. Transfer to a 10-ml volumetric flask ana dilute to volume with benzene. Prepare lower concentrations by dilution. Accurately make concentrations of approximately 2 ug/ml to be used for tuning and 0.2 ug/rnl to be used for standard calibration of the GC-MS. 0 n 1 (b) Inject.5 ul of the coneen MS . As the TCDD elutes, fine tu: vo 1tage al ternator control to maxim 322 and 32~Az lancarc (2 ug/ml) in tne ignet and accelerating resocnse at m/e 320, (c) Inject 3 ul of the diluii GC- MS and record the response . Mea the chroma tographic peak maximum us 25 . Opera ting Conditions (a) Column: (glass) 1 m x 3 : Gas Chrom. nj. (b) Temperatures: Column Flash Hearer Separator Ion Source 24 0C 250 C 29 0 3C 27 0C (c) Flow Rate: 40 cc/m in no (d) Trap Current: 60 u 0-- 1*1<o-0 ary 12, 1976 9 of 10 pages Method (a) Accelera ting Voltage: 3 .5 XV (f) Electron Energy: 70 ev Multiplier: 4.9 (q) ( h j Masses to be moni tored: m/e 320, 322 and 324. (i) Oscillograph chart speed : 1 cm/mi n . 26 . Procedure (a) Weigh a 1.00 g sample of isooctyl ester of 2,4,5-T acid into a 5-dram vial and dissolve it with 5 ml of hexane. (b) Prepare a silica gel column in a glass tube (30 cm long x 11 mm i.d. with a 125 ml reservoir) with 14 g of silica gel equilibrated with 20% benzene-hexane. (c) Quantitatively transfer the sample onto the column with three 2-ml portions of 20% benzene-hexane. Add benzene-hexane to the column as necessary to maintain liquid above the silica bed, until 40 ml are collected. (d) Evaporate the column eluate until 1 to 2 ml remain in the beaker. Transfer to 1-dram vial; rinse the beaker twice with 1-ml portions of benzene-hexane, and aad to the vial. (e) Evaporate just to dryness. (f) Add exactly 200 d of benzene to the vial, cap securely and rotate gently to wet all interior surfaces. (g) Inject a 3-ul portion into the C-C-MS and monitor the three molecular ions at n/e 320,322 and 32' jy means or tne multide-ion detector. Measure ail three ion intensities a the chromatographic peak maximum using a tangent baseline. 27. Calculations (a) The concentration o'f TCDD present is calculated from a _n C = :-- X Dn Da --- D, b x --E x wnare: C is the concentration of TCDD in the sample (pem) a.0 = ion intensitv as recorded in Section 26g for m/e = 320 (sample) bn - i.on intensity as recorded in Seccion 24c for m/e = 320 (standard) Da = Osc illcgraph attenuation range for larges; on-scale intensitv for s a m d e D,o = Oscillocraoh atrer.uation ranee for larges' on-scale ion inoensioy for soandard a i7 6/ February 12, 1975 10 of 10 pages Me tried Mo . 3759 6b E = ratio of sample weight in grams to final solution volume (usually 5 ml) S = concennration of the standard in ug/mi (bh The above calculation is repeated for m/e = 322 and for m/e = 324. The values should agree to zi5%. If they do not, che value recorded is the lowest concentration calculated. 23. Recovery and Precision The precision of this procedure is within 20% of the amount present. This is the acceptable range at this level. The detection limit is 0.01 ppm, therefore, it meets the TCDD specification of less than 0.1 ppm based on the 2,4,5-T acid equivalent. it i t ", it it it it it * it it it it it x it it it it i t i t i t i t it it it it it it it The analytical procedures given herein have been adapted from literature sources or developed upon the basis of experimental data believed to be reliable. In the hands of a qualified analyst, chev are expected to yield results of sufficient accuracy for their intended purposes, but recipients are cautioned to confirm the suitability of the methods by appropriate tests. Recipients are also cautionedthat The Dow Chemical. Company makes no representation or warranty that the practice of the method described herein does not infringe third party patents. Anyone wishing to reproduce or publish the materials in whole or in part should request written permission from The Dow Chemical Company. METHOD 238553 ESTEROM* 3RUSH KILLER : rnr_ TCDD METHOD DESCRIBED HEREIN IS IDENTIC.m-\Li THAT USED FOR ESTERON* 245 KURON* VERTON* 2T BRUSH KILLER LV-2-2 3RUSH KILLER TX BRUSH KILLER X ^Trademark of The Dow Chemical Company. QJ FH E D O W C H E M I C A L C O M P A N Y M IO L A N O . .MICHIGAN A 86A 0 ANALYTICAL METHOD December 2, 1976 ESTERON* 3RUSH KILLER Method No. 23855b A. Scope This method is applicable to the analysis of ESTERON 3rush Killer. Procedures are given for the determination of -total acid equivalent and specific gravity. The active ingredients which are the propylene glycol butyl ether esters of 2,4-dichlorophenoxyacetic (2,4-D) acid and 2,4,5trichlorophenoxyacetic (2,4,5-T)- acid, the inert ingredients and pounds of each acid equivalent (2,4-D acid and 2,4,5-T acid) per gallon (US) are calculated. A procedure is also given for the determination of 2,3, 7,3-tetrachlorodibenzop-dioxin (TCDD). The calculations are based on the assumption that equal weights of 2,4-D and 2,4,5-T acid equivalents have been mixed as required by the specification. If necessary, the actual ratio of the 2,4-D and 2,4,5-T acids present can be determined by other appropriate methods. B. Safety Precautions ESTERON Brush Killer may cause mild eye and skin irritation but is not readily absorbed through the Skin in toxic amounts. Avoid skin and eye contact. Handle with care. Nitric acid is corrosive and a strong oxidising agent. Wear gloves and goggles when handling nitric acid. Sodium biphenyl and 30% hydrogen peroxide are .strong oxidizers. Avoid eve and skin contact. 1 of 9 paces 2 J 7G y 7 December 2, 1976 2 of 9 pages Method No. 23855b Potassium cyanide (KCN) in TOXIC. Handle with care. To dispose of KCN solution flush sink drain with water for 15 minutes before and after KCN is poured down the sink. Some of the other reagents used can also be hazardous. Follow the safe handling procedures recommended by the suppliers. C. Assay -1. Principle The sample is decomposed using sodium biphenyl. Excess biphenyl reagent is precipitated and the soluble chloride is titrated potentiometrically with standard silver nitrate. 2. Aooaratus (a) Metrohm E - 436 Potentiograph Brinkman, Instruments ilantiacue Road, Westbury, N.J. 11590, or equivalent. (b) Silver 3illet electrode, Beckman 239261, or equivalent. Coat silver billet with silver chloride (see Section 4). The 3eckman electrode is available from Beckman Lab Instruments 25511 Southfield Road, Southfield, MI 43075. (c) Glass electrode, Sargent S3005-15C, or equivalent non-chloride containing reference electrode. The Sargent electrode is available from Sargent-Welch Scientific Co., 8560 West Chicago Avenue, Detroit, MI 48204. (d) Hot plate. 3 . Reagents (a) Sodium biphenyl reagent, available from Southwestern Analytical Chemicals, Inc., P.O. 3ox 485, Austin, TX 43767. Reagent comes in 1/2 ounce bottles suitable for a single determination. Store in a refrigerator crior to use. (b) 1, 2-Dimsthoxye thane (DME), ava ila 2<* /'*'--n mr>y, Rochester , NY 14 550 . 3 i rerr, (c ) Ni trie acid, 1 :1 SO 1ution . Car eful di../. add concentrated nitric acid to 500 ml of dis ti i_i_rtr~< va d *mn ij_ or 7LI Decenber 2, 1975 3 of S pages Method No. 28355b (d) Silver nitrate, 0.1 N . Dissolve 17 g of reagent grade silver nitrate in 1 liter of distilled water. Standardize the silver nitrate solution with dry orimar" standard sodium chloride. Alternatively, an analytical concentrate (J. T. Baker "Dilut-it" 0.1 N-AgNO3 available from Curtin Scientific, ?.0. Box 1223, Midland, MI 43540) can be made to volume. (e) Hydrogen Peroxide, 30%. (f) Potassium chloride approximately 0.1 N. Dissolve 7.5 gm potassium chloride in 1 liter of distilled water. (g) Potassium cyanide, 5%. Dissolve 5 g potassium cyanide in 100 ml of water. TOXIC. Handle with care. (Note-DO NOT add XCN to an acid solution). 4. Preoaration of the Silver-Silver Chloride Electrode. (a) Polish the silver portion with fine steel wool until it is shiny and clean. Chemically clean the electrode by briefly soaking it in a 5% solution of XCN and rinsing with water. The same KCN solution can be used repeatedly for cleaning the electrode. (b) Coat the electrode with silver chloride by electrodeposition from an approximately 0.1 N solution of potassium chloride using a 3-volt dry cell, and a platinum wire electrode. Connect the silver electrode to the positive pole of the battery and plate with chloride for 20 seconds. Then, by means of a switch, reverse the current for five seconds to partially remove the chloride. Repeat this operation twice and finally coat the silver electrode with chloride for 20 seconds. Coating must be uniform. (c) In the presence of biphenyl, the silver chloride coating lasts for about 20 determinations. Silver billet should be coated with chloride daily. 5. Procedure (a) Accurately weigh, to the nearest 0.1 mg 100-200 mg of sample or a sufficient quantity to yield about .5 miliiequivalents of chloride, into a dry 250-ml beaker Add 10 of 1,2-dimethoxvechans (DME) to the samele. Swi issolve Run a blank on reagents used. (c) Shake the contents of a sodium b L p h e r . y 1 rear en t bottle and add in small increments with swirling. The mix tu should remain green. If it is non , add mere biphenyl ^os until green color persists incicat ir.g an excess of the rsec;e Let it stand for 5 minutes. 2 J 7 fa December 2, 1975 o 9 oaaes Method Mo. 33355 b ( d ) Carefully heat the mixtu: to boiline on the hot plate by swirling the beaker's contents to avoid any burnedng. Cool to room temperature. (e) Carefully add 50 ml of distilled water with swirling and 5 ml of 30% hydrogen peroxide. Add some glass beads and cover with a watch glass. (f) Heat on the hot plate to incipient boiling and then transfer to a steam bath. Boil gently for 10 minutes. (g) Remove from steam bath and let stand for 5 minutes. While stirring solution with a glass rod, add sufficient water to dilute to 150 ml. Let stand for 10 minutes. (h) After standing, break up solidified biphenyl .with a glass rod. (See Note 3b). Add 5 ml of 1:1 nitric acid. (i) Place a magnetic, stirring bar in the beaker. Titrate with a standard silver nitrate solution to past observed inflection point. The end point is ta'''n as the mid-point of the inflection curve. 6. Calculation (A - 3) x M x 374.9 C x 30 =%2,4,5-trichlorophenoxyacetic and 2,4-dichlorophenoxyacetic acid, acid, propylene glycol butyl ether esters Where: A = ml of silver nitrate used for the sample 3 = ml of silver nitrate used for the blank C = sample weight in grams . N = normality of silver nitrate solution 374.9 = weighted average molecular weight o f 2,4,5-T and 2,4-D propylene glycol butyl ether esters 7. Precision Data obtained bv this oroesdure indicate a standard devi ation of 0.05%. Any single de; miration mav oe exoec rom the average by net more than ;0.1% (95% confident: limi ts) . 3. Notes ia i is aevasaore to run or 2,4,5-T and/or 2,4-D wich o b - hi - available from the Sample Coordinator, Ag-Organic Department: 9001 3uilding, The Dow Chemical Comoanv, Midland, MI 43640. December 2, 1975 5 of 9 pages Method No. 23955b (b) Rapid cooling of solution will cause a thick crust of biphenyl recipitate to form which is difficult to break up. Also, possible occlusion of chloride in biphenyl crust can occur if solution is allowed' to cool rapidly. Slow cooling causes a finer precipitate of biphenyl to form. (c) Presence of bromide and chloride in some samples will be indietabe by two breaks in the titration curve, with the bromide end-point occurring first. D. Acid Equivalent 9. Principle. The acid equivalent is calculated from the assay, as determined in Section 6. 10. Calculation -- 238.2 = weighted average molecular weight of 2,4,5trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid. 374.9 = weighted average molecular weight of 2,4,5trichiorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid, propylene glycol butyl ether esters (as obtained in Section 5) x 238.2 = %2,4,5-T and 2,4-D acid ecuivalent. 374 .9 Or - % 2,4 ,5-tr ichlorop'nenoxyacetic acid and 2,4-dichlorophenoxyacetic acid propylene glycol butyl ether esters x 0.5354 = %Total(2,4,5-T acid 2,4-D), acid equivalent based on weighted averages. E. Inert Ingredients 11. Calculation %'Inert Ingredients = 100 - % active ingredient F. Pounds 2,4,5-T Acid Equivalent per Gallon (US) 12. Principle The specific gravity at 20/20C (69/69?) is determined and the pounds of 2,4,5-T,and 2,4,-D acid equivalent per gallon is calculated. 13. Procedure P ) a r o rm i.n0 r he apparent sp 0 C 1 C i c cr avi tv bv wn ni n r j a now n vo 1\ i me of sam --' 1 n 1 m a cvcncrr.eto -- b t a temp "V o 20 r\ * J 0 53C 0 ( 63 ? ) . Thia C S C a ai.s of the me acioc. g a v e.n in " Ma r hoc c11 - -- he sta XAcard meth G G o test for "S O 0 C '* ic G rav ity of I r . C us tr 1. a a ,v" o r r n - -i -- Hyd r ocarsons and R e i0 -- '^atiacrials I " AS T M Designa o n D a1-v -^- -a- / cub t ished bv r-T C S r ic an Society for m es ti -n-5 n "i3 / 191 6 Rac e S t .f ?h.i.Lad el.phia, Pen.nsy Ivani 19103 . .December 2, 197 5 5 of 9 oaaes Method No. 23355b 14. Calculation Total rounds of 2,4,5--T and 2,4-0 acid quivalant/cation (US) = % total acid equivalent x 3.33 x s p . g r . 20/20C. _ __ G . 2,3,7,3-Tetrachlorcdibenso-p-dioxin'(TCDD) 15. Principle The neutral components including any-TCDD in the sample are separated from the matrix. This is accomplished by silica gel column chromatography. The residue is concentrated and a portion examined by gas chromatography-mass spectrometry for species which have ions of the same mass, number of chlorine atoms, and retention time as TCDD. 16. Interferences No substances are encountered to an extent sufficient to caue interference down to approximately 10 p p b . 17. Specificity and Selectivity The gas chromatography-mass spectrometry (GC-MS) technique is extremely sensitive and specific for the determination of TCDD. In order to assign a signal to TCDD three things must occur simul taneously: 1) the retention time must be correct (the peak maximum is reproducible to within 0.1 min.); 2) all three m/e 320, 322 and 324 must respond and 3); they must respond in the proper ratio. 13. Safety Precautions an produc 0 chlor^cns if allowed to contact the skin. Extreme sa.fety precautions should be exercised in the preparation and disposal of s^a''"'ci.ircL material and solutions. It is advisable to read "Summer; of Safe Handling of 2,3,7,3-tetrachlorodibenzo-p-cioxin in the L< core.torv /" available from the Sample Coordinator, Ag-Organics Departme.enntt,, 90033 B3uilIdcing The Dow Chemical Company, Midland, MI 43640. Handle all solvents in a fume hood and avoid breathing vapors. 19. Apparatus (a) Gas chromatograph-mass spectrometer: 1K3-9000S (LN3 Productor, Bromma, Sweden) or equivalent, with multiple ion of Gas m_or _----i.j_ ilumn for LK3: 1 m x 3 mm i.d. (glass) packed with 3% OV-3 on 30/100 Gas Chrom. 2. an t? December 2, 1975 7 of 9 paces Method Mo. 233 55b (c) 50 cm x 11 mm i.d. glass chromatography' columns with 250 ml reservoir. 20. Reegears (a) Benzene: ACS reagent grade; available from Burdick i Jackson Laboratories, Inc., 1953 S. Harvey Street, Muskegon, MI 19442 (b) Hexane: ACS reagent grade; available from Burdick & Jackson Laboraotries, Inc., 1953 S. Harvey Street, Muskegon, MI 49442 (c) Silica Gel: High purity 100-200 mesh, Davidson 923 3r;anc, available from Curtin Scientific, Midland, MI 48640. (d) 20% benzene in hexane: Add 200 ml benzene to 300 ml hexane and mix well. (e) Pure TCDD: available from the Sample Coordinator, AgOrganics Department, 9008 Building, The Dow Chemical Company, Midland, Ml" 48640. 21. Calibration (a) Standard TCDD: Weigh 1-2 mg to the nearest 0.01 mg in a small weighing cup. Transfer to a 10-ml volumetric flask and dilute to volume with benzene. Prepare lower concentrations by dilution. Accurately make concentrations of approximately 2 yg/ml to be used for tuning and 0.2 yg/ml to be used for standard calibration of the GC-MS. (b) Inject 5 pi of the concentrated standard (2 yg/ml) in the GC-MS. As the TCDD elutes, fine tune the magnet and acceleratin g voltage alternator control to maximize the response at m/a 320, 322 and 324. (c) Inject 3 yl of the diluted standard (0.2 yg/ml) in th GC-MS and record the response. Measure the ion intensities at the chromatographic peak maximum using a tangent baseline. 22. Operating Conditions (a) Column: (glass) 1 m x 3 mm i.d. 3% OV-3 on 80/100 Gas Chrom. Z. (b) Temperatures: Column Flash Heater Separator Ion Source 240 or' 250 0 290 - c 270 c (C) Flow Rate: 40 cc/mi.n . hiil ii'in 5 3T35.C, OH rO t.3.rT;3C3IT (d) Trap Current: 60 ya (e) 1C(^p^ 3."^ncf Vo1 3.^3 ` 3 .5 KV 7 52.1770 December 2, 1975 8 of 9 pages Method No. 23355b (g) Multiplier: 4.9 (h) Masses to be monitored: m/e 320, 322 and 324. (i) Oscillograph chart speed: 1 c m / m i n . 23. Procedure (a) Weigh a 5.00_g sample of'ESTERON Brush Killer into a 30-ml beaker and dissolve it with 15 ml of hexane. (b) Prepare a silica gel column in a glass tube (50 cm long x 16 mm i.a. with a 250 ml reservoir) with 50 g of silica gel equilibrated with hexane. (c) Quantitatively transfer the sample onto the column 'with three 5-ml portions of hexane. Add hexane to the column as necessary to miantai.o liquid above the silica bed, until 120 ml are collected. This eluate is discarded. (d) Add 120 ml of 20% benzene in hexane to the column and collect the eluate in a 150-ral beaker. (e) Add the column eluate. in portions to a 5-dram vial (25 ml) and evaporate until all volatile solvents are gone. (Some heavy oily material may remain.) (f) Add exactly 0.5 ml of benzene to the vial,S 8cCaUpT 2 1 V with a Polyseal cap and rotate gently to wet all interio s u r f a c e s . (g) Inject a 3 yl portion into the GC-MS and monitor the three molecular ions at m/e 320,322 and 324 by means of the multiple-ion detector. Measure all three ion intensities at the chromatographic peak maximum using a tangent baseline. (h) With 1-ml syringe measure the volume of liquid in the vial. 24. Calculations ( a ) T h e concentrt on of TCDD presen is calculated D C a X X S m where: C is the concentration of TCDD in the samp le (ppm) ~n = ion 320 b 1r*1 ---- ion 3 20 intensity as (sample) intensitv as (s tandarc) recorded recorded in Section in Section 23g ~t~\-- m/ 22.C r v m/ Ose 110C 3.C ft 1.19^'JS t'G ^ n"C0 ro - 1 rges ^ w";.~ s 'n tier.s ity for sample Ose illccraph attenuation va rCT^ ~ y- rges t ion intensity for sbandar G. il ii <Tj ,o aQ SUT7I December 1975 9 of 9 pages Method No. 23355b E - ratio of sample weight in grams to final solution volume (usually .less than IQ m_; S = concentration of the standard in yg/ml (b) The above calculation is repeated for m/e = 322 and for m/e = 324. The values should agree to 15%. If they do not, the value recorded is the lowest concentration calculated. 25. Recovery and Precision The precision of this procedure is within 20% of the amount present. This is the acceptable range at this-level. The detection limit is 0.01 ppm, therefore, it meets the dioxin specification of less than 0.1 ppm based on __ .. the total acid equivalent. _ The analytical procedures given herein have been adapted from literature sources or developed upon the basis of experimental data believed to be reliable. In the hands of a qualified analyst, they are expected to yield results of sufficient accuracy for their intended purposes, but recipients are cautioned to confirm the suitability of the methods bv appropriate tests. Recipients are also cautioned that The Dow Chemical Companv makes no representation or warranty that the practice of the method described herein does not infrince third oartv patents. Anyone wishinc to reproduce or publish the materials in whole or in part should recuest written permission from The Dow Chemical Companv. sli niv- Exhibit F Exhibit F has been filed with the second portion of Mr. Thomas' testimony which is filed Under Seal for use in camera. SU T7=3 m k .* MB \ CURRICULUM VITAE <31 7 7 ^ Curriculum V itae RONALD F. THOMAS Date of Birth: Place of Birth: Education: Former Employment: Present Position: February 14, 1934 Baltimore, M d . B.S., Washington College Chestertown, M d . 1956 Federal Food and Drug Administration 1956 - 1961 U.S. Department of Agriculture 1961 - 1970 U.S. Environmental Protection Agency 1970 to Present Supervisor, Residue Support Unit CBIB, BFSD, OPP, EPA 34 7 7 ^ Attachment Publications List 1. "Microcoulometric Determination of S,S,S-Tributyl Phosphorotrithioate in Cottonseed." R.F. Thomas, T.H. Harris. JAFC Vi. 13, No. 6, 1965.' 2. "Residues in Fat of Steers Sprayed with a Dieldrin Contami ^ t e d Fly Control Product." Medley, Younger, and Thomas. .m-,Rifi lfiHn of Env. Cont. & Tox., Voi. 12, No. 5, 1974. 3. "Residues of Lindane on Food and Utensils from a Thermal Lindane Vaporizer." R.F. Thomas. JAOAC Voi. 56, No. 2, 1973. 4. "Effects on Eggs of Applications to Poultry of Pesticide Formulations Contaminated with Chlorinated Hydrocarbons." R.F. Thomas, J.G. Medley, JAOAC Voi. 54, No. 3, May 1971. 5. "Survey of Polychlorodibenzo-p-dioxin Content in Selected Pesticides." E. A. Woolson, R.F. Thomas, P.D.J. Ensor. JAFC Voi. 20, No. 2, 1972. 6. "Polychlorinated Biphenyl (PCB) Degrading Bacteria in Estuarine and Marine Environment." Sayler, Thomas, Colwell. Submitted to Estuarine & Coastal Marine Science. 7. "Analytical Properties of Polychlorodibenzo-p-dioxins." Woolson & Thomas. "Methods in Residue Analysis." Proceed ings of the Second International IUPAC Congress on Pesticide Chemistry. 8. "Disposable Inner Liners - Tests for Permeation." Presented at the National Conference on Pesticide Containers, 1972. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY BEFORE THE ADMINISTRATOR ) In re: ) ) FIFRA Docket Nos. 415, et al. The Dow Chemical Company, et al. ) ____________________________________ ) PROPOSED CORRECTIONS TO TRANSCRIPT OF TESTIMONY OF MR. RONALD THOMAS Respondent hereby submits its proposed corrections to the transcript of the testimony of Mr. Ronald Thomas. The proposed corrections are listed in the attached Appendices. Appendix A-- contains the corrections for the public session of Mr. Thomas' testimony; Appendix B contains the corrections for the in camera session. Rule 13 of the Rules of Procedure, December 18, 1979, provide that each party shall submit lists of proposed corrections to the transcript of testimony within four weeks of receipt of each volume of transcripts. We respectfully request that these corrections be accepted notwithstanding the fact that they are being submitted beyond the specified time period. Respectfully submitted, July 16, 1980 Patricia A. Roberts Richard P. Bozof Timothy D. Backstrom Andrew G. Gordon John W. O'Donnell Counsel for Respondent U.S. Environmental Protection Agency 401 M Street, S.W. Washington, D.C. 20460 3JV77 Page 6 7 15 19 26 28 Line 14 8 1 24 6 21 -3APPENDIX B Mr. Ronald Thomas (in camera session) Correction "trade" should read "t r a c e " . "introduction" should read "production". "base" should read "Bates". The line should read "It would not lea d " . The line should read "supplied to Dow b y " . Delete "no". 3,17*0