Document erJXarGOaaLZBQwzYVOYp1k9

cc: C.F. Reinhardt E, 1. d u p o n t d e Ne mo u r s a n d Co mp a n y Haskell Laboratory for Toxicology TECHNICAL FILE and Industrial Medicine P.O. Box 50, Eikton Road Newark, Delaware 19714-0050 ^ ~ )<s DU PONT CENTRAL RESEARCH AND DEVELOPMENT August 9,1903 Dr. Kent Hatfield Document Development Branch Division of Standards Development and Technology Transfer Centers for Disease Control NiOSH Robert A. Taft Laboratories 4676 Columbia Parkway Cincinnati, OH 45226-1998 TOXICITY INFORMATION ON LEAD ACETATE FEEDING STUDIES (Ref: Letter from K. Hatfield to HJT, 4-26-93; Telephone Conversations of 7-93) In response to your detailed inquiry on our two-year feeding studies with lead acetate in rats, I offer the following information: 1. Two chronic lead feeding studies were conducted in rats, in Study I, rats were fed diets to which lead acetate was added at concentrations of 0, 10, 50,100, or 500 ppm (as lead). The experimental phase of this study began on 4-14-65 and the last sacrifices took place between 5-67 and 867. In Study II, rats were fed diets to which lead acetate was added at concentrations of 0; 1000, or 2000 ppm (as lead). The study began on 5-24-66 and the last sacrifice occurred on 5-2-68.. 2. In the preceding studies, when weekly mean dietary lead intakes were reported, the values did include background dietary lead. For example, the average lead intake for rats ingesting a diet to which 500 ppm lead was added was 548 pom (over a two-year period). a.) Blood, urine, and tissue were ultimately analyzed for lead content by atomic absorption spectrophometry using a method described by E. Berman (1964) for blood and urine or by a method reported by Perkin-Elmer Corporation (1966) for tissues (See Attachment 1). b,) No differentiation between bound and unbound forms of lead was attempted. N 27672 Dr. Kent Hatfield NIOSH Page 2 August 9,1998 3. in Study I, body weights, food consumption, and mean lead intakes were monitored weekly during the two-year feeding period. In the lead intake tables (VII and VIII) I previously sent, dietary lead intake on a mg/kg/day basis was calculated from the body weight and food consumption data. Therefore, control diets to which no lead acetate was added would have no numerical value for lead intake. On the other hand, lead measurements in the feed were periodically made but I could find no details (How many measurements over two years? How often were diets analyzed?, etc.). In Study II, body weights and food consumption were monitored weekly only for the first 90 days of the two-year study. Those data, in tabular and graphic form, are included here (Attachment 2). The last graph of this attachment shows the calculated average daily intake of lead (mg/kg/day) for 0, 1000, and 2000 ppm over a 90-day period, in addition, similar to my response for Study 1,1 could not locate specific data on periodic lead measurements in diet, 4. For information on pathology diagnoses, I am enclosing a table entitled "Incidences of Microscopic Observations in Rats" for rats sacrificed from one to two years. These tables, labeled as Attachment 3, covers rats from both Study I and Study II. Relative to statistical analysis in general, the preceding data were subjected to an analysis of variance to determine the significant effect of three variables (dietary lead, sex, and time) on the various quantitative responses which were measured during the two years of dietary lead administration. Statistical comparisons to historical controls (as opposed to concurrent controls) were not made. However, such comparisons were conducted in a publication (under preparation) by Dr, E-. Stuia, the original pathologist for the preceding studies. When a pre publication draft is available, I will send it to you. 5. Only internal pathology personnel were originally used to assess the tissues from these studies. However, for the publication cited above, kidney lesions were sent to an NTP pathologist and others, and such Information will be included in the paper. Archived pathology records (slides, notebooks) do exist at this time and are not siated to be destroyed. DUP040Q10924 Dr. Kent Hatfield NIOSH Page 3 August 9, 1993 6. During the time span of the two lead acetate studies, no specific reference historical histopathology tumor data base existed. However, in the paper in preparation (cited above), Dr. Stula will cite tumor data on 2000 control rats/sex from studies conducted over a 25-year span, 7. The only lead intake data from Study II is given in Attachment 2. No tables similar to Tables VII and VIII (for Study I) were located. Food consumption measurements for Study II were only conducted for the first 90 days of the two-year feeding period. 8. Blood lead levels were serially measured in rats throughout Study I and only terminally in Study 11. For Study I, the table below summarizes those blood lead measurements (as ]ig/mi) from lab notebooks: DOSE LEVEL OF Pb fopm) BLOOD LEADIpg/rpI] f e ma l e s CD p3 0 0 10 50 too BOO: 3 mo. 0.0 0.0 0.0 0.0 0.20' 0.25 6 mo. 12.m,Q,,,. 0.026 0.0 0.006 0.0 0,0 0.0 0.0 0.10 0.06 0.0 0.17 0.09 0.20 0.14 0.22 0,60 0.59 Mim 0.123 0.153 0.121 0.203 0.400 0.790 M.A L,_E $ 0 0.0 0.025 0.03 0.05 0.102 0 0.25 0.0 0.03 0.03 0.121 10 0.0 0.063 0.10 0.06 0.98 50 0.0 0,029 0.14 0.09 0.168 too 0.0 0.0 0,17 0.24 0.302 500 0.18 0.38 0.62 0.53 0.761 Zero levels were below limits of detection (0.01 jig/ml) For Study II, blood lead levels were measured ip rats at the final, sacrifice (24 months) only: DUP040010925 N 27672.01 Code: n =* c a lc u la te d on n basis of a c tu a l lead ( 3 6 , 6 /S o f lead a c e ta te ) b - see Table 4 fo r various te s ts performed ' .pM M 33 r ft rr H* r- c5 -rr r* -rr rr :rr . < M >M > rr SS s 5 : :; s _ f3rrt3.: pm Ui ^ *<573 rVr? O .59 n>rffbtt- >.es<ft >4 W 0 uSt' l H* VX 5> X3- H 33,. fcD< -Sn3 A. 3 nr 03 3 .KM >o >a >p W3 H* OO pop V* rr pop uoi oo o -- o oWl tsSl >r--r k* > VJ >O U3 M J> ^ C?V I C9 ^ ft V WiIU1 M* ..p\ *S3 : * p :S S 5 -5 2 Vi p S3 5 2 S 3 5 s sp VI ,_3W3_S > Orr ch? .fgCl>Ul (rfa0t*. ' a ft 5* -* H- > ** ">f3Ht "3ft 3 333 f0t3 1S3 3 0 >- P sx r * 3f3t' ft r? 03 Z H* O ft 0ft CCrQO*oIH -*3 ft 3a ZO r-* ft cr3 X rr N N3 U>. 55. 5 5 5 S 2 r- Cv S $G Pr N> : x r4 ft Zc s * ftS 5aS Oa f-t< fZt -O2'' 0Vf3ti -rf3tr ca rt 2 W HfHt N3 Q ft .3 ft >r :w - 'f3t C/5 O55 CrSrU-pf3tg ft P ft -nf>t frtr frtr fS3t fT fftt c- JOof&tB <*> N3 cb ^ N422* ' 03 SvjN (S3 .PO .-ft* -J> Ni pm* Vfi >" CO V 40SSS P^-Ui *NJ U>*! 3^ cs *3 fsa .a * o-2O' *=- H- *O-** ft- 3 ft ft & - > ft a* 3 .f3t* \T 2 rfftt* -?Hi* o ft- o r> " > JDUP040010927 I r' ft 2_7 67Z.0 ROUGH DRAFT April 9,1963 /tnifcmeAJT i RAT TISSUE. LEAD the tissues were plaped ip individual plastic bags and frozen on solid carbon dioxide immediately following removal from the animal and then stored at -4 C. At the time of analysis j the samples were brought to room ' ......... temperature j, weighed into 35 milliliter Kjeldahl flasks and digested with 5 milliliters of a concentrated sulfuric-nitric acid solution (1:4) followed by 2 milliliters of a sulfntic-perchloric acid solution (1:1).. After the digestion was completed,: all nitrate and perchlorate were driven off -by heating. The samples were diluted: and an aliquot was taken for analysis by atomic absorption spectroscopy. The lead was extracted into an organic solvent with a chelating agent without pH adjustment and, analyzed according to the. methods recommended by. the Perkin-Elmer Corporation (1)Tissues taken at interim periods were pooled by groups; sample size and dilution were chpsea' to obtain one to ten micrograms of lead in the final aliquot. Livers. from the animals remaining at the end of the experiment were analyzed - individually and the kidneys were combined within groups in order to obtain, an adequate sample. Bones were stripped of soft tissue (cartilage caps remained), crushed, extracted; in. a Soxhlet; with an ethyl alcohol-diethyl ether solution (1:1), dried and weighed into flasks. Digestion and analysis were similar ' '""7" ' N 27672.02 .................. 2.747-2;0z DUP040010928 -2~ to soft tissue. Blood and urine were analyzed By the method described by Berman (2). Samples were pooled by groups at the interim sacrifices and analyzed individually at the end of the experiment. Feed, and feces were dried, ground through a 20 mesh screen . % and analyzed by the method described for soft tissue. Shir was'washed"'""1'"'""" with acetone, water, then acetone again, dried, ground through a 20 megh screen and treated the same as soft tissue. Standards containing 1 to 10 micragrams of lead per milliliter were prepared from lead nitrate in 1 normal sulfuric acid. One milliliter VA S of each ^chelated and extracted into methyl-isobutyl Ketone without pH adjust ment and aspirated into the flame. These standards were used for all digested tissue as there was- no appreciable difference between standards prepared in 1 normal sulfuric acid and those prepared in 0,1 normal sulfuric aeid. Standards for blood and urine samples were prepared according to Berman (2), The limit of measurability for digested samples was approximately 0,1 microgram per gram while that of blood and urine was 0.1 mierogram per ' > 10 milliliters. Only a single analysis of each sample was performed, but samples containing added lead were included with each batch of tissues Iteea - recovery of added lead was less than 85% the entire batch was re-analyzed, .-i - ~yQ- ] _ (c*S( ^ in. priff)56ri/tt<3r\ y a 7 )4 M *S IS f<L 49 4-6 6S 70 77 si 7! /) : ; '' ~ MU/Ol&yt. Siud^ yf^U^kld asr'-fS s/a,cj,kf^ Ty-aA a*uy <M&rr<vu& /&& sfed C/itsUirusi <zpr ,**4 X XL ZZ7 Qirff.hdi O-J'OnTb 0, 3%Pb X 3P MM Qjrr&sU! eJ%Ph 6-,77j t V7 tbS 37/ 3/A. 344 37? ioi 433 SS7 'ib'L<#4? SOj Sih t Z 'iA' A' SU/fr.......T..L..&..t.&. t^fi. JtoMyklt t/7 /S3 AOS asy 4*7 34,/ 33-4> f47 y^v 4 Si 4fe Ht> m if? S47 /<? 3 S3"? 777 go-s 344 364 3Bit 4ok 4o4 42>% 4$S flpb 7or /3b /ho m /?? u$ . 3a. 743, osz Asy a<ei 171 33 3sa soy /3z. /T8 /SO. 200 z/f 33o 0.41 7.S0 3.S2 ata 373 aM AS3 /07 /Zi (4 7 /b% /H /}$ . W? 21(5 4*7 5 3a 337 347 3*^ ASb KTWmr 6-7 7-/# /4-2J %/-M 4s 35" 3$ 43* Z'7f Y7--5& Sri}& 63-70 70-77 77-84 H'p (tosOi-L^C Hf 31 <rs Hi SZ 3S 7(7 3 3*. 3.4 5 33 7.4 7 f< 47 4? fi zz 4? -?r qi n 7 aC 20 3 30 Mb 46 3f Z7 37 HO ` /* 3 a3 a3 n *-&<2Mrv C'f^ey) if / xf 0J* x& OJb if 0J /3 /$ 13 /f If (Z, 79 16 iS SO It j z 70 ? 7 7d 6 X 4' 4 nn 16 '! a i -t 0 N 27672.03 *L7 &7Z.( ts \ ^l^^^#i^W S!a^l^frl^rt!^fiS^5 ^e^^3*'^^ t^ ,|{M^,!rss^ff e .^c^r/^`''i'!v ^ >-v*-i.,.>S li'rt f t g h M r t & M f e t '<~> * * f c A < * * w * m * MklMf- ^(ro-gf j^f '7s7*%s& Of^a' &+U? 'Jfes/ sC%s& *-e*s/ CfJaU-vcU 'TOaJUn X ;n in jh*. (Urnpit? C-J%f% o.Sc]/6 JUS X it at Ol%Po 0-Z%Pa &Hh/ <9-7 7-if U+ai 3J-2.2 n-$s is-z t-? 4?-s$ s 4-&>3 &*>- 70 70-77 77-37 S4-J/ 17.1. AO.S 22-h A,}* a3-7 .23.3 M. r 34.7 a 4-s a3 .y Z3-7 A i-7 sA.% /fc.3 i&.4 Sz-r 33-b Sit Sb-(? 4i 0.4-0 034 03-Z2f.) a 3-2 /S'. 7 I?.? Ai.f 33.3 33`7 34.4 23.? 33.3 -J3-S 33 -O A%* i^.a. zf.%. J.f Z+.9 IS.6> /b-O ile-t 16.* /7.y >7-? 174 n.p /(,.? it.. V >4.0 l4~? I$.b h.i n.i it.4 17.3 Ik-9 lb? 17.O Ib-b 16.0 /i"7 13-1 iS.sr IS- 3 /v<a* /3~ V /f.i IS-S IS. =u /o:aIt.,3, li'0 IS-O iS-f 0-7 7-if if-SJ 31 -33 32-3 3S-4a. Ya-y? 4?-so St,-hi 4.3-70 70-77 77-sv W-Ji ,v 3b .2.7 .is . .36 33 .3? .32 3 J zr . 25* ai n If .lb .V . 0 3J -n .<5 -.IX <1 4. 'f ., H 3o .11 0<t 04 . a a. 'If '.if .IH }tf 1 3 > if oy " O S' <3 ^ ..as 20 ay Y2 .13- lb .0% . 0$ <36 0 2. .10 0 2sr ,ar . 2a >/Sis. i* .0? > 09 07 . o3 .0? So 0 . If zo 0 .If ` If /I II Ob , * -id . p$ 0 f ' GROWTH RESPONSE OF RATS FED LEAD ACETATE 2_ 117 2 ,oM DUP040010932 N 27672.04 b .wo b j j mi j i & i I&u--.. jJ . LU __i ^ ' <qg::ixJ--I C3 -uj..-aEie.iJi' .<*: c k : u j _i u j i <r 3~ t~ __J <XZ Lx. ZEZ. IxJ 3ZS'4p'*** IX- Caras: m- tLJ' a>$'cfi 'imC >*l .OSC i x j 4-i -- x|: .CD z: 3 ' q$ gfe ff*ri !6*3 RATS FED VARIOUS LEVELS OF Pb AVERAGEW ffW J P dail y f ood consumpt ion of wal e and f emal e CO ixi -C3 cn -. <r M3E C3 CK <E 3lr CT3 UL O O C3 CJ O Z CD 3 3C LU O I DUP040010933 ' f= ; UEplsi* 'Jrt'i .o-Pup o_ ;CS3' tar HHt.-e* C3 feS.lfst LU I Lli ._J UJ l3'3d: _^i'4aS.3E5 t# *jc 3d: uu s* DUP040010934 i TABLE B STUDY I : IHGI DEUCES OF MICROSCOPI C OBSERVATIONS I N MALE RATS j'uijn' n oii it : .48 OU O--H U *un*r ait a < * * * * Otco-*-< *-h i *jn*ir*--i*TinCMP?^<3,t*P-CQ * <a-i r** I | K3" I I 04 1 I S t l I I I I I .2-1 <r pj i i i mi i *--ii/t0r*vI"** i i cuusws^f CT*>.< >ojjac m cn h r**Ui>-i op i i Ma-iv-*** i mj ^->^->4.-- i i. ir>sr s j >- O< >~J ^p it oM H- i(iI m CO on C iMt --* 0**3* H g It Ii It ,11 CM U *--<r n it to c. it j) it it it o 2 O II n UJ >- A. tl 11 -v i i---* >i uo si Z a.&r " i zO^Vu5jdSa aii PS.=3 If 0 0 2 It O! i i * evil i 0004 ro i i ih .o O'H CTy N.'^ I co| .1 I I r-t | HH I I f * I I I .1 I I -f* fdt OYt III II CVt:*^*:P4O0 t I I CO<3"LO CO--*-<rCMSO I -r~* CM 4 ro*-^ *-- J 1 I I I UJ --1 Cu a. >>- I--I UJ OS . S 1 gg >-.<Qg UJO*^w Jz,uo aOa'UIO<SP<Sc =>.<oo 2ECQC0 GO U3'3'3 Ul -JLUUi =3--I--I UJ >03 CO d-UJ^OC. co --J F-< 23'=>*-/ O 1-- GO g _j ^ UJ --11----3C Z co< ><*-< =3 S -J o X JS u -*-i <q :o 3: u-xza-aso *-*0*""* .-Q-.CQ d cs x co - - -J -fii--i-<UI <ua>M<.ooo. ca<c -co ts 001X13 gs tD o. a-o'Q o: 3-- '-* --j :oUJUJ >-->S!5C:HO--UaJC vZo-.Js>i s-b: tuoj CO 3 .a. g 2fc LMX CUJ Z<L? j-- > > * C < < < >-^>- >- >^ >r > fl.WJ<SX ==-===5 COOVJM H*4-- si COCO*-* OK KtOCEJ --40UJCaS0<COfi-p0023SfiS ,fa UJ'SUIUJCCXXXXXX JC5Z O h6Q..0.h^hfl*fi>0>Q..P> UJC< X* C<C_J.>-S>-=ZH<C,.U!JiiUSJUZJ2UaJ UZJ U3l o s? UJ oc o .PS CO UOtco o --4 UJ< .H- O' Hr s UJ JO Q < as_tj <=-: --zoaos--: --Jj "<- /J-- UJ,<33 f--`i-P.o-as -sc _jg U--4li=--3 Ccn ,s:g _Ocj -Ji-- zxoauKw UIV>--OI-- 03---vi1=e>ye . _|V1UJUJ0UI 3 Z O ^ ^ Q S ^ irtcj<-ip.vunzu o c<c<<*2 _as atat3C-acs:s:s. 3000.0.0P00 i--a: as seas-at seas UZZJi/) :IotJ*"Oe wjj-OuZoU~OZo JQOOQ 0.0 --UJ LU UJ LU UJ UJ 03 ca___ia.a.awt-zca gi<CU. LU U. _J Q..Q. a. <C CuJ CJ.U. zmaooammomaaz m.ea zzarszjc I. ***!>ie* * *+: ****** >= N 27672.05 DUP040010935 o II --* u til w , <=>*- voj | ^ | I I I i I CM I * .us>Ur mi >-f Tj*-rf ja * i * j i c m t * i ,i *-<m i csim * eg i* j ->l--J | I I *--*--l I *-** * f>.|r^.rrt --iH |H l.]rt mi ^ 'Ti *m il > 1 Iff IW I 1 t ,i 1 *--* H < *vn*r n 3C co m r*- u 5 V) --.-- -- ,11 coj 1 i I J I I J I I Lrti Ififiiij.li "I o j i i 3* c sj I *rj i i i *m :i i i i rr>* ** i i --* j | triJCM c j ~l --lroV)^om i % i * <Tj f-H %i> i n i * i c j :i I g *ow <y V) 5g -MO =*-* O CU OO on coo 'OS U1CJ *mr> rh < <a o - LU *-- <C -- -*r , CM o'*r sj` I *-*.*-* I I (MH I ,1 -*lpv> wpm i COI I H I H I H I I I I sr *er cotcovoyacv iiii.it t i mI J SflH -- I * o P a? o r- (--* 0. VI i-- :=3 Ui .< O o sz OS ss o o | V)S.Z O .3E-,-- .--* as *c OU..JVC1OS as o=a aszs V) 3* -UJ If p Vi II VJ K MU H- H *2 < :s 5> aUsJ oC ss>- *a-s UiO S3 UU - H- H-J S<SZV.iS- ^llll .UJ t-- VI osc l3Eyh.O.O< <- oasvi i~aL< CM i0--.0<*<--C I--j JiO*-SZijJ3 ! o uj QO.UJJ: s-P OUJ O --j oca. u s e q s u j .--o-o-c Ci vs h- --i OSC u Z-IC^pL o , :Vq iUolOu .iOo <j j o -- u o s u iu j <*:oa: X.4DO<OXXS sc.o.o aZHl^JKHHU --:e H<<OH. O >-v>s __: Ui u j *i > u j ui zz <zs^J:'Sa 'osse-sc ars:ac.se ac: I -tt *.*, *1 * * * UJ a V Vtrt QSHasujuj *-' * Vi V>V5 Ui --s -* ctes.cc -JUJUJ ISi < d. o uj uj uj m.s ;-*o.a<uj *----ug:j -uJj o os *o-._.o__o . -2C <C EZ UJ.-- 0.0 0^ ._ cao'l-- 4A .-3E.J-OS "UJ U-i I-- 2= 0.00 ' SS '-----I.OSS35>^.?3 _ 5to,vj2 = c.s:.TMi:s=sos.5:o o, US ss .* .O c< j o e w i-i-o ~t~<~.ceczz:>--c -x.o ujcusiairc +-ao>-<usc -jniuutuuiroKwifla: aoh-^l-ut/isxiLSjiKi-t.aua:iEjsCEO,>-f'x.uaj itu--iiu--iaberi--s o: ' t-- ui,oizw<wS:ci-o.Qy>s-r _ ,oa- ' UJ . .. w(iiei--eo-C^EctV--5.U-J-.ao.Ea-.=S,:uj.ou=i5.=idt--i - u j ss c m co ea v^ *-- u j -- as u j o o rs --* ss > u j ?4 g Mfl^teMa;3;a:SC3=-i--urs zz o-o-o-.ca-.as to < o DUP040010936 u on H* < .05 UJ _J < sr st. CO -32 , H* < > *~*o: *o tu GJ 1/7 .sen e C-* oo. uo on COO ex LUO cos; < ' ^Uu . on LU CJ 2S LxJ .. .M t u . u >* .0 =3 i+on CO m r>. >c .0. .0 h <n U*J rn - s 0 ex u. 0 LU --4 OfS CL. O 05 O LJ .2S on --2 -P 55 n 11 it it w .it u n H Oa ""i It :> to *g- 11 n ;n 11 B C O .8 B | 1 3it p b ** . -- n p ilt *~nrt*T it H M a a a ^-H mh s t !? ft II U II n 11 <c *53- U u 11 a II 11 cur a 1--1 -^r b H a R u a H 55 B *- U O. IT H- H < s= ex 11 -- *-*.--* n onzrss .a LU.O.-4 u O CL U wft H a. l u .it sduj c o n onsc h CKI.OD 11 35 U II II U 15 It u u it u it it 1! II U II U II n .SB ft . ft -- It on u UJ u -u 11 ft UJ ft 11 on ti on 11 *-- H- ft UJ Q5 ,53 .00 .vn cc UJ ex 05 .55 I-* * 30 UJ UJ -O CJ> 1-- -J :S 0 f< on < HUJ 32 u --' 05 O < on -j <: 55 u* E 05 UJ 1-- .* LO O V .0. 1-- on UJ h~ P i-- UJ O 35 .< C X LU . UJ ' Ui on --J -- =c on 05 S LU UJ ; ' II -- < - 1-- on -JX -05 u. 05* 'UJ -T" . ` - .525 ,2S O. >- on os on on=C >-*25 UJ a. U* UJ 0.05 ;^c a. 05 0>-" ro C5 ' H* . ` -35 UJ < 05 0 35 *"' Cu.on25 -UJ <-- LU!'**-! 250 ' -Ji--;iO-<_> 05 < <CH OSU<hS u. .on 0 . II --Th LU 05 -LUOnLL. UJUJC5 J6 LU >- h- s: ; -1-- ,C ;0.,-J 05 xs C LU LU jJ U- ... P%l5..%u onuj-u sr O <C QUQ u o>* 05 --t on on .H* UJ C5 K* U. "O 3=<sr w/i>LU(J on on p*4 --| .u j h > O Li- LU h~.CC cn u. 3; J on Lu ' .35 05 ca; 103 on on . : ' !' UJ5C ft! u 11 IC'JO 1 on LU O O O O H- P y'r DUP040010937 o oo chi ( rt.i I NlhOOlfflO I CurOf-uVOin) CM"-* i "u<vj i eoi"*"* i c m i en <nj- --* --> t i-u.- i i -- .: O >ow c mh-* t r-, i i ojeorovo i i i cK^ corn eg **<"*> <M- ` i i csi i j c m-- i * i i ?* Ji l I ( | I CM I U I I III I II .1 c m --QO * i I winmvo * I i CM I I1I I1 5i c m *-* -*l * I .1^ INI* .1 :-* i I CM * I w * * srjl * CM o Umm i *- CM*r i *~ico Ii -i h i ^c m * i *"* r;U--J1 UH*i O. 3JJ ,-- : >Vi ju j aoscoq:vo VI o >- s <UJ h< in < CCLl J os z- S<UJ COOSO -J Uov4><* JO <3CH--* Duihsa. ca-J h-< =5=3-0 o H-CQC-J uiJ:H3E^<rWz VS)S<JOx.*X-<S LL.~<a50=E u-xx&as.o *0-*x*v- >-o--epW-? -Q-0 3C>-<U4 -t QS.O-J*-0- C*_5Q^0_><SL_iJjr--1Zj;ZWDshU-2O-aJW-.S-Jt-f>it5---f-)oJ-d ogu<--jPU5-lSJu ZCVJ , >--.>- CU-OCOSE3C V a< V) 3 CV C UJ as a- :ui g 5* 'p* SCO o Cd >- -J > c e as u j ocz BS O DS C/5 o<c=> 2S out .fi S~ OOSBSrZOSL-UJO>C!-<C--/)22<=_ V) 0J 2O<_C=_VZ0> OEPWMO.<U3?i ?* --** * in --j'uii-- *-*=3 - . swisuj cE3;s UJ Q Ul =5 o: <CCS OC 4J 3l-V)OOS< S --i " oo-jt--ujsr^-uj -u j rs 33 a: s e SB s:* ss << LfliUisQto---a.t--3d.z>wci--a<o:,o=s<jLuu.uuoyoo.-Jj Ox in ,UJ 5 ISl tUZnJa><c&0-o<C*s*O3r<--*=O.i'.ro>vls*ts^_Uuc*.ooIv>--slx*--.H~ci....Wfltc<--W/ra-c-*1O0--oo9u3jeCjio.WO=cDO>U-Mc -e<>i--cC.'Va-u-s1jt-O-s--uz<iS0etst4uc<oJ>J3jJcSc<C<u<^oO-OjSH.axa=l<oOu-.ZHria>ouS<aOSjZ-?.hou=<eo>ZusOj'.*iX.co><am--SZsO-u->H.czoufa<=*ct.j.-hto>Jurl-ti-Oio,vVPsaoHs-c*ii:hZ*v-wcz<*->c-* a: ~ - - - -u j _ia:x:t--cso:.ccJ3:Q:uj<-s O C.a: a *C 3Q03 S S-< *scw, s3h -x0zs0seOssk -Oassj<O='Ote OasreuOa:*_-i--cij>3E0.<>X--OszjOzyjzuOja-Oc;ooOc:oI--p:Weo3: --I QUJ OUJ OUJ OQ--'-CtfLJOOOLiJ LU CO CO CO S --__f UJ*UJ UoJ 0-3 O"-OUOJ. C--L;. ,c .<c.c *?: <s c u. u. l u u. <s _i o, <e <: < u. u, = --i zmffiffi mmmramofflQ cos: s: ii: cc ccs; SSi ' * * ****.* * ******** : DUP040010938 < .05 < s. LU M- a a K. xn a p 'h- C > -UJ <y o3 m CO X C o o h* ro uo rn x C MO C'*M o a. oo oo ovo . .ft; .UJCU Uh as cC1 V--U. o oo UJ o a UJ Q' --> 0J s -O a u. o UJ n OO a o a O LU 3g OO --J < s a < .* >-- : * OOO ItUsHSt U H.0 5*O<"MST : 1uR1 aM MU --acvi a -MLr>*T a 9 U II --u ** U N H II U u u <U K li II H u s ii ii n Ji u it * ii s it O ii ' CU It a II < O ll a a ii 05 -U3 u it oo s z ii l u a -- it O Q 11 wtf || fi. UJ u a uu m u o vo SI u ao.au ooa u oo ii LU 11 ^ LU II a ii on il CO II ~H- n1<1i :IHI I CCi | I *-* *-i 1 0^^ .Ijcsw'P^r * ro ir>) I I f*U3 I *-tC0 I ` I s V I I l C*M1IH-* * 0 IfM * CulcM !^Pt culcvvoSOp*.| 5Tl ` -M* I. OO3i I I 1 \il i cops-m *srl *- i i *-* <SI P^ICM j ii l I csi i VO*-* .1 i iff'W i> i i -- i .8 8 i *-41> c\J rn * i i i i 8 --ii I I I -H -- cuuovcm i --* i i CTl .-- i < j j co i t * i { ^mit iu d v d i i i --<*o i i,in i i cb i t ;i i 1 Sa 1 f" c Co Oo .u a CO --Of<lCKS0cS'00 u-JsMe* OsCO JQ o LU2SO CJSC *-4 OOS O UJ 0a0 05 5O--' SOO H~J-oJU--oJ as<c<--ac*j -ho* as: oo =c o <svr4 a fifzrz x; l * <K * * OUOJ Ussj 1K J UJ UJ OO <C UJ oa ao < ->< 03 OO y r C . 8 uiujujaas S t o--* oo--t ra- U<J --uJj a--s* Oo LU LuU-U-l-- 02^02 QOO < '.-a: O8 u s; s Ca i -- OO LL> *"* CO C 5 UJ. QOO LU H" .8 Q!tfQ:<J -OO ISO! UUZ as UOUm.G <OflZXU -O SZS (U <C'C OOOO OO UJ - o so ss SS;UpH .00 OH" SSX oo .J J--Z i --<c 8 C_J t OOLUO. 35 -aa>- -C.O UJbJfif-4 <LU J Hoooo.a: <o -J05>- hh<Z 050. J a ass sscoooooooo asa UJ Ui H- O .J-- --J :* u. a o OOU1 U.OOM avK oo*^s UZ-.--c o*-*a -a >- i c mc ecu. u_a sus ZZQ.O. C> h-r*u i O .a o 03 s o>-/ 05 OH a oa<s- <5 0 0 UJ*--'UJ i--JH" oG5s<u< U- O u^ o LU S LU s* a S --Hcao xo a LU*-" OOfJJ__! a<Cm' .oa o a <u a Ot/i * LU u. a O 03 00 00 aui--* LU Q H CO.O SO-J zaz11.eoe.-<~i 03 UJ o o o SIGHIFICAMTLY DIFFERENT FROM THE CONTROL GROUP USING FISHER'S EXACT TEST, p < 0 .0 5 . DUP040010939 .11 o * OlLsCMCT'--ito --:oco u H- <cr i >^if>acsir^'>w3 0%rr.i0:<nOO I H i .oJ .* fX IftlJ > I .w** --4| S cnkn^r *1' *T CMi I % *^ 5g V> it n I! H O ~o! 1 CM B O s to Ln .rn if If li 15 o~ .'-'CL o LUO ___ItO cab :gg S l! H 5 u CO R OMCT*-*LPJCM ~1 COj I I 1 * *** icp^r^.csJOo'icp r*-rvc\icu *--* COh- i l m--i | rl *--* | OM'IT ^ 1 OIO X : =rtr>Lhca i i u3f>*T,ct!Csji-H i oj e|J * ^csi 1 r-4 --* I I eoKryo I-Hl 1 I coj >-- I =C -- LO 0g OS V> ILU o u u JJ * u Si o co Q U CD * CD II .1 U o COSE.3S II jac l u a. *-* is: OCX. II <e wos U C. UJ if 3XLUCQ R a. =s <, OO CQ -J buj pt--# LU CL jjjLULU UJ CO: -i ,bi33 CO S3UJHca-U S3-SD-J Z LU .> OIOS It fXOS . a. C< .<H >~co< 0 ?Z: CO CD OSS U u AC o 52S JE a s oxrW< ,>< --CUS-J H- - 2:2s j 31 R U I II n n u u H a u == IS O It It V> u UJ It -u .it. u UJ IS O I! Vi ,! gg rooo. cc SS-SC2? co: uxsaaoLl <CC03 o(-*z*x-vi*a.*ca- *<;0 oo oc<c<occoc<c <c dcooscos cs.<s; --J -QSO -U.*-< UJ C0< -it/XD-O-*- o:< cuoo ooo UJ UJ UJ LU UJ UJ UJ O U U S3 UJ --J I-- 33 JmJJSO. oo U3Q.&UOZ>Mj . =3- ->-2:SSH-UJZE-J^ > ^--*?**--<>-.cx. <{/j s 2 CQ 02 CO CO CQ G3 +- - --CJ^3S * - CGH- Hhh-JHI- Q.M-<Li.U<ZXX toss CO O toco mJ <. *-- I-- h- h- G< <<< .<< .*- U O OV) < c < <C -a; to to - *CSB zzz<zz HX-J-JDUI lL- oc C- GL ss s C --> CL o CL o CL o fi.CU.OK. OhOM ocac^srviC IZ CC S LJ CC CC QtZ LU oc oi <c O CL oc occs ssss-j.oasiLU :2C LU UJ &Z >- <C XS SC SS H--iO O.CL1--CH>CLCLa- a.o.uia;< S casc-ass : srsqs. C CD,33.3=^ 3 :S3SSS:=5* 4* *c m^'LU >> se C UJ LU LU LU l u .l u >> s Q ZXP-.H.I- s o cc ecco oo ^ -fu- 1,<Oao<0iiv'-MS<7E UCo:-LaJU. lU;LU^CO'3-'=C!CO -ieC .cC.-J <J -J 3E l-- S l t02D^-*0 CCSOS O 4-<"*-J* -L3U= bo s.=E;s:.s:< <C0S3O3K-<oOCcOPUS--3OJJl 3<=OC3- 3000.00 -*OI--3S c j o s c oh-.aESE:^ce :o c c q c l si. i jjj j_y Uj CD cj>u-sr u-U'0,00 0*r '* LU > <G <C <C <C < La. acamajcaaxESx I -K -Js' ^" :"* " *" >c: tiv. tSoSUtoJUvJi O-SCH`- cotovi LU *----i -J K-- Sg OC ; :< LU CU a: ss^ H*.y> c l u =: to cu tO li *- II o < .1-- II 5>< II .31 DUP040010940 0 -* v> '*- . It i .1 *r 1 *-* +-- m-i CUCU MI *-< *-* 1 UJ LU < SE .SE *-* Vi 1--'' .HC 5> ^-saius 55 V> cP3coo ec^ ?> vp rrs > < 1-- m Vi ro s ee W^J .0 Lu .--PLCJ-*_S UiS *t * 1--:xon s G z: .ua?s s V* d s--. 00 n it 11 ,;K u || n --O <1 a u lit .11 i -o2 Ia! itti 1 Q.*_ 11f1U315t H- n h . xn aslss :b LGM&P-~" II SujSi t. .case, ft a a u a O Vi :S 1 i 1 eg cm t * col--1 1 . vs vs 3 . H* < v> 5 S .S .SO CS 2S H- *Z 5* LU --J --1 .< .< J3Q. SS -CK ; 1 gpsgsss *--* P-* h-- ^i t j s z e e : CLO PC- UJ< <VS < UJ LU fiS s:'rua. 1-- & vs gssgggz =b < 1 Li f--- v <p. tv<--sH-~ 1--Vi LU UJ -S--'4l-- H* J-U.O IS SS.LU 1-* VS * <e 5LU UJ S 3S II V*> >~ vs *--seuW-. CPS-.SD :^:S9 O.C&C;O*~' OS , OH3-:-0J =o:s<e'.ef--s CLBSS ULU>-- 1 OLU SSO as UJ.SE U13--K* zf-- s * -LU sca.ec >< SJ3LU LU n--.Lu 35LU VSUj -* soa LS U > eev --J cai-- LU C p* LJ ' VJ--t asLu u. UJQ --* CD o s; S UG .3SS&VS ' uis: h 3: V* UJ 0 0 p DUP040010941 ****** to -oo jcU-4H-M-* I <NJCCI^<TlOO>^OiOOcr:-rH *T .*-*-* SH- "*<\JCSJ Cl evil *-- i~* CM Cyl CM "* Csj.j 3 ^* ten 1 -* 1 cbCNl CMj-t s T*. s5- go 2= O 05 j-*0 I a * * * * cnl 1 1 * 1 esi 1 .* mesJ oi o>r-H 'cr cpa- cvj 1 t 1 1 1 1 cvi*~ 1 eg Ol i CMj sI ~ CMIO 4 OJpt 2;.: si * 1 1 --1 1 r 1 1 cotnoj.*-* 1 *~i 00 rx> t 1 ** 'at .--I O. in CO a? 11 Ui ii ex u a o a ha S; =saa m mm to .00. P- W M .s0.tuo sca wn QJ.ODII ooc Jl HA O a. o ca--1 H-rt --1 H-d3<-3 UJ-4,:h..EH<StZo > --1 s >- < CO CC U>J t3O5 na -s S3 t-<ro < eaxgo - -H-<C <c -o-co a. CO sc -OJ <0 0LU5CO u--ISoS3oS UJ U4 << ^S OcJbmO<cUJ-JJc:3SoUof<iJ.-alh-hX- <l u : ess. ssmea<C=3 S3 Cfl *C mU3Q.O.U 0,05 > *-* > e z .z zz h - u j as --J UIUJ too rhn--C-to-os^m CO O tOtO--J <c "*'* i 35.35 EECSH-H-H-E C c < < 5-^>-, Cu-^*~U4--i<.s: H" I-- 8" O: ' "'.ISi305-eC<C <a- <<- *->o!a.&zx<jboScCU^itUSSJ ss;:E i-s -0.-0.*--j OO-- ^QOJ 05 LU az Ot 8C O CLKa (XPZI/) toes: ............ ---------- scar is at S^U.. <ZOW -*0 --I m=2 JC-1CCn--.e4ae0s--|f--t". M2*5Z0*X3:.U--31 05 SSgg *--35 051- _ Q-uicat/iE o.<c.u-3S--i 4c0o*t*o- -'Ij J0L5l * 0.^050. 35 az -401--05 05 S<oS<U3-JS.O>S* 8CxH135^OfOlH>*--<S*.H3-ShC- QH<U2>CL SE CO O O 05.0 Ou.SbKo.rr-taX.tXfoXi 5-0- u3j5* *ca *m --a_e *c ::05-j ; Q.Q.Q.< UJUtUJGC .05 cam. cam cam sc S Ex .1 **.* 4C * * * * * 005 s mtosc-E o^ UJ *sr *--;-- 4-S-I w mh s-jrz .=> KO DUP040010942 It It II tl i! II A. ft CO cn os > * v> sc cc OS O UJ .IS* 1 < >sc UJ ,C3 Lu o 2 H-- * cn 03 Z '30 S3 --i* MC c . o oc U.UI CO H-O .*>4 .o Ui-* JO. 030 *c o sc o .cc u. o Ui Cm CO CL o oc o UJ Sg GO -J < s </> Z u j <c O z Ui O *-u o z 1 . IS : II .: it 11 w a B ft. II IS :iii, ii It II a ill o% -0 $ --c OO n CVJ <NI: Jil K U .i 4 H 13 6 IS ii it a a Oh *- a * **-*<S| II it it ii a ii a B ft II os a u. o *-i a jt H si ii a * a z it O 11 i-i ft. H l-- s a <c u z cc 11 <*--allfM**,cb aU oo-acz: Ufft.*-- u a. it v'OS. 11 cu u j n :SIU:Q U cose a ccos u OQZII u 11 a ii ii ii H n K u H 11 II II ii it n ii z it ,o a a CO a Ui a _J a ii Ui a O it to ii to a u a ii a a 1 Oil Oil 1 1 1. 1 * r 1; 1 1 l: 1 1 1 f t t > S: I' r-1 S 8, fl a i a i il t * i 1 1 Ol 1 *-!" .1 -- 041 1 1 1 1 1 1 1 1 1 1 19 QSh~NJ 8 j 1 -1 1i 1 1 i i 1 1 1 1 1 I 1 CO * CO t >* 1 J H- 1 s1 <1 .1 o. s .^Ui 1 *ou i -z 4-'< .> z O ..*--1 i 8 : O O H* Ui i a =c co i Q2_jft. i oo<- a. 1 uiuiZ-H < QH<ZLU i UH03E I Ll O *- S -- I o ' .s-sior I H- h- UJiUi t UJ 0>--* Z-H* 1 V3ZO.D.3 I s1 <1 u> | 1 1 1 ci .pc 'M saucroa .Oo V I" < ft H-- oo<h f--cO UI Ui Z-< <X Z uj USi--CO .eC.ftS ZUJ jsj: ii tzo uxu. X.* xi CD 3>-tO CCS 0*C -SO. *n+ S L.ftSO 3C-C5S 3'--'CD ZO PC J2I- CJiO-*Z0 -J C<C__ u O U Q <zz x o UJ Lui**!* Uu I'Z u. co ui--i ZCQ Q <i:' ' cs a >Z ~j OfflH r3#c Z u. <: O CJ to -- Z UJ u. UJO-* moz autf S'! -- zzco i.O UJ Z ii ZS Ki- Ct CO LU O O i-- ' o z DUP04Q010943 < cc ; Ul m O s! O V CO z o >e ,o Oa a ^oo a itte >--5lD*-H U 2> S3 V) #n in n * *** * * ** OIwfnHcooooNO^.oi i i -- oil*^ oi, l i oio :i ' ~r j j i e\ia-O UJOO a UJ '* !* m p a. sm OS g %s -*> >. as m s=UJ Cu*-- o cu .> . m via cso: o = it as H S! in ii uj u -J It *-*. IS UJ I! 3.2 m -- ii &- u It j pOiNfg-i 4 i OI, .Oi UJ 1 UJ UJ >- u *u j ,caaa 1-- u-_j p cp.cn mssuiu-zs, >- Cto--5 >*.qC SS J.5D=3.^J O i:n$s Ui o XSD-QiO O *-* CP u*2KSlS^rt JO.O .=> .as os --j a. u* e.in * .<.< */>00 . .- . DOUOUJ9UJ-JUZ C0 H* i-h -J-JSCL <J5,OUJ'UJ UW0.Q.UO'-h ~J0> --j --j -j >-'s: s h -l u z ~i ^o u i <33hHH>.fi.<V)2SW c j >;1--J 'as; h~ *-- <Te'e >-^.>- o* >* a >* j aj men -dieCw^-KhHH1 aeeeeutt^meeeeem C/3S 2E --3 --l SD LU C'O. O- U O-'CL -* 22 *u uj os uj as ex. as cu ce. as <c s:.~Jo o- cc os, os o os.cs t-- *- mosi- u j u i z >><s .z x s = -J >-o- ^ O.A.H-QS h -* a. a. a. a-a. u j Ui sss >- >-25e UJ UJ UJ UJ UJ UJ >- as* * :=caswacsEjsssteSssso- s UJ h -c /s ssuj UJ }-- mmm ZUJtU PEh 2C J Ui C=t SB LU .. t *,DCQ as O sbl -- osos UJUJUJ H-J--'H- uSieOeS UJ m Ui a ree>1-- V5-.3S c -- LUZCC Ol-O. o's.ai UiCOUJ mu as a e _> m ui . C O UJ UJ ,o m-- seo. .CLCCO 3C.C! O'^-ocs CE <JJ;hU ao astta O'Sir<'Z U3^:0 ' -JU* se CES u. o us o u. JUJ3C aizs.ee X.OLU muj *+ S03 O e.' era u >- OS ~J omi-- * 2K .l u e o o (/)tr OS Ui .U> .UJOHi ffipz soo 3 --< SE'.QSm 1O UiS: !!" m ui . o ,o DUP040010944