Document DdBJpkk1LLMjXrdERXxj3MGq5

FDA 72-116 Fourth Quarterly Report ' June 1, 1973 Further refinements in analytical chemistry have been pursued and analytical capability is being expanded. No further progress has been made on the svine chronic toxicity study as analytical efforts have been directed'toward the kinetic studies. Some significant species variations in the kinetics of Aroclor 1254 are becoming apparent in swine and sheep. 2he pilot swine reproduction study is near completion and the contract modification involving blood and backfat kinetics has been initiated. I. Analytical Chemistry A. Extraction technique Extraction of all tissues with hexane-saturated acetonitrile ap pears to be more efficient than the hexane extraction originally pro posed. Recovery from fat samples has been demonstrated to be more complete using a method similar to the blood extractions reported in the Ibird Quarterly Report. B. Column Chromatographic Evaluation Silica gel (grade 950> Davison Chemical) has 6hown promise in sample# clean-up. Separation of chlorinated* pesticides and detector contaminants in feces, fat and feed is improved by using 12 cm Eilica gel in a 6 mm i.d. column; an elution volume of JO ml hexane is ade quate to remove all PCBs and leave most contaminants behind. Alumina is still used for blood clean up, but silica gel will probably replace alumina in the processing of feed, feces and tissue samples. OSW 037516 STLCOPCB4021340 -2- C. Gas-Liquid Chromatographic Quantitation Quantitation by digital integration has been somewhat inconsistent for low-response peaks when concentrations fell below 0.3 ppm Aroclor. Separate standard curves have demonstrated that, at low concentrations of the smaller peaks, peak height/amount injected is a more consistent parameter than integrated area/amount injected; at higher concentrations and for larger peaks (e.g. n, o, p) the integrated area remains the most satisfactory measurement. In addition, peak-height measurements allow quantitation of the later peaks ( q, r, s end t) which wbb not practical in the earlier studies. II. Pilot Swine Reproduction Study. Although the number of animals was too small to warrant definitive conclusions, the study does show some trends. Residue end histopathology data are not complete, but the performance data, while of a pilot nature only, do show some apparent detrimental effects of low level feeding of Aroclor 1242. 5he treated sows were bred in the order of presentation (numbers 2 through 6). Sows 2, 3 and 4 received treated feed for 4-5 weeks before breeding; sow 5 was mated after eight weeks on the diet but came into heat and was ma#ted again three weeks later; sow 6 was unsuccessfully' mated after 7 weeks on the diet and subsequently confined with the boar for 3 weeks, isolated for 2 months, then returned to the boar for an additional 3 weeks. Ihis sow never farrowed end was terminated, excluding her data from the experimental group; the lack of fertility may have been due to PCB'e, how ever, since she had farrowed three times prior to the experiment. Close DSU 037517 STLCOPCB4021341 Table II. 1. Farrowing Records of Sows Receiving Control Rations . or Rations Containing 20 ppm Aroclor 1242. Sow Treatment 2. 1242 3. 1242 4. 1242 5. 6.3) 1242 1242 Means Weight3-) (lbs.) 590-547 580-513 595-495 635-550 630-660 No. No'. Matings Pigs No. Stillborn 1 13 2 1 11 1 17 25 30 1 0 -- 9*o 3.7 1.0 Mummified2) Fetuses 0 1 8 1 Number and Mean Weight of Live Pigs 11 2.6 lbs. 10 3.3 ' 6 3.0 5 3.4 mm 2.5 8.0 2.9 7. 8. 9* 10. u. Means Control Control Control Control Control 590-445 610-446 585-450 570-433 580-444 1 18 1 12 1 11 1 11 1 12 12.8 3.0 51*) 0 1 0 1 1.4 0 0 0 0 1 0.20 13 '2.4 12 3.3 10 3.6 11 3.2 11 3.3 11.4 1.1 1) Farrowing weight; second number, when present, is weaning weight of sow. 2) Hot included in number of pigs born. 3; Rot included'in means. 4) Last five in litter; delivered 28-36 hours after first pig. DSW 037518 STLCOPCB4021342 Table II. 2. Performance Records of Pigs From Pilot Reproduction Study Treatment 1242 1242 1242 1242 Means Number of live pigs and mean weiffots (lbs) 1 week 3 weeks 7 weeks ti weeks*7 No. Wt. Mo. Wt. No. Wt. No. Wt. 7 6.1 7 11.1 6 31*6 6 36.5 7 5*5 6 7.4 7 11.8 6 14.8 7 33.9 6 38.0 7 39.4 6 42.2 5 7.3 5 15.2 5 37.8 5 42.2 39.9 - 5.6 Total Pounds sow 219 276 253 211 240 Control 7 5-6 6 15.8 6 32.7 6 41.2 * 247 Control 10 5.6 10 15.6 10 37.5 10 44.8 . 448 Control 62) 6.5 6 15.I 6 39.7 6 43.8 263 Control 11 6.4 11 16.9 11 35.8 11 4o.l 44l o Control 10 5.1 ..... 10 10.2 ........ 9 32.6 9 37.2......... 335 C/5 sc Means 41.3 - 6.6 347 oGUJij 1) Mean and standard deviation calculated from individual weights for each litter. >0 2) Sow 9 only six functional teats. STLCOPCB4021343 Hfeble II. 3* Grgan/Body Ratios for Sows and Pigs Organ Weight/Body Weight (mean x 10^) GroupLiverKidneySpleenHeart.___________ BrainThyroidAdrenal Pigs 1242 Control 25.20 l.TO 24.23 .97 4.39 *70 5.08 .35 1.27 .13 1.72 ,2k k.ko .35 4.82 .51 3-TO .J>k 3.55 .44 .076 .008 .111 .015 .068 .012 .080 i .010 Sows 1242 Control 14.50 1.68 11.37 .08 2.02 .40 1.71 -33 1.19 .19 1.09 .05 2.53 .22 2.64 .40 57 .06 70 .40 .050 .003 .055 .056 i .008 .070 i .002 STLCOPCB4021344 (GA st OfUvUoM1ii Group 1242 Control Illinois Average n 8 15 Crushed 25* 40* 30.9* liable II. 4 Causes of Death of Live-Born Pigs Starvation 0 33-3* Cause of Death and Occurence Born Weak Diarrhea Septicemia 37.5# 6.6* 12.5* 0 12.5* 13.3* 17.6 14.7 12.9 Congenital 12.5* ' 6.6* -3- observation in confinement revealed no evidence of abortion and post mortem examination revealed enlarged ovaries vith cystic follicles. Treated sows had somewhat larger livers than control sows (Table II. J). Stable II. 1. indicates that the number of pigs farrowed decreases in the treatment group with length of exposure to PCB. Although the compari sons between treated and control litters may not be highly significant, longer exposure may exaggerate these differences. The number of mummified fetuses 1b quite large for the treated group, and, since the sows were con fined within close proximity, factors such as brucellosis and other communic able diseases can be discounted. After the first week, few pigs were lost and the mean weights of the surviving pigs did not vary to any great extent between groups (Table II. 2) nevertheless, if all these subtle differences are summated, the treated group produced on average of 2^0 pounds of eight-week pigs per sow while the control group performed nearly 50$ better with 3^7 Poun<ls of pig per sow. Thus It appears that reproductive efficiency is decreased in swine ae well as mink and chickens with exposure to polychlorinated biphenyls. Ik-om a limited number of milk samples, it is estimated that the sows generally excreted about 2 ppm PCB in the milk, a level not approaching the 50 ppm in cow's milk detected after a similar feeding level. This indicates a definite species variation and perhaps should be pursued further. III. Swine Chronic Toxicity Study. Performance data have been reported previously, indicating that both Arodors exert a detrimental effect on swine production; the difference van greatest in the groups receiving a normal ration and somewhat reduced DSW 037522 STLCOPCB4021346 lbble III. Organ-Body Ratios for Swine Chronic Toxicity Study _________________ Organ Weight/Body Weight (mean x 10^) Diet Normal Normal Normal Treatment Control 1242 123b Liver 16.4 1.24 15.2 1.1 15.3 2.0 Spleen 1.43 .43 I.63 .38 I.56 .36 Brain 1.15 .08 1.19 .12 1.05 .12 Heart 3.99 -40 3.94 .29 3.84 .14 Kidneys 3.63 .45 3.50 .33 3.45 .51 Thyroid .063 .016 .054 .007 .058 ,0o6 Adrenals .037 .009 .052 .013 .055 .009 m fat Hi fat Hi fat Control 1242 1254 14.4 .66 15.8 1.09 16.5 1.12 1.55 .30 1.8l .35 1.54 .25 1.09 .40 1.06 .15 0.97 .12 3-97 .23 3.83 .39 3.45 .23 3.21 .41 3.63 .38 3.47 .65 .064 (n=*l) .063 .016 .063 .008 ND ND .052 .012 .047 .008 Lo Pro Lo' Pro Lo Pro Control 1242 1254 17.1 1.2 17.7 i 1.6 17.7 1.2 1.57 .32 I.67------1.75 *20 1.48 .25 1.36 .35 1.15 .24 3.82 .26 4.17 .31 3.73 .49 2.97 .33 3-41 .50 3.13 .60 ND ND .061 .014 .063 .008 .062 .010 .056 .009 .043 .010 (oS) ar oU) \lU\XJ>\ STLCOPCB4021347 -4- in the groupB receiving a high fat ration, vhile the control plge on a low-protein ration performed more poorly than those on low protein treat ed rations. Organ to body ratios have been calculated (Tfeble III). There appear to be no significant variations in any of the groups. This Is an unex pected result, especially for the livers since PCB's have been shown to cause hepatic enlargement in other species and in the sows from the re productive study described previously. The lack of significant variation may be due to the relatively low dose (20 ppm) which the animals received. IV. Pharmacokinetics of Aroclor 1254 after Intravenous Administration in Sheep and Pigs. Three cross-bred sheep and three cross-bred pigs were administered in travenous doses of Aroclor 125k In propylene glycol. The dosage in both species was 5 mg/kg given as an I.v. bolus. Blood samples were collected for 24 hours following injection. In the sheep, injections were made by needle and syringe into a Jugular vein and samples taken from the opposite Jugular vein. The pigs were fitted with indwelling venous cannulas for injection as well as sampling. The three sheep (5-8 year old ewes) weighed JO, 58, and 8l kg and the average dose was 208 mg. The three pigs (2 gilts and 1 barrow 10-12 weeks old) weighed 29, 29, and 58 kg and the average dose was 96 mg. Blood samples were extracted with hexane-saturated acetonitrile and analyzed for PCB with electron capture gas chromatography as previously described. Individual PCB components were quantitated on the basis of the relative anounts normally present in the commercial mixture (ie. component concentration is given as "1.0 ppm" at level present in 1 ppm Aroclor 1254); DSW 037524 STLCOPCB4021348 -5- components were also quantitated on the basis of actual component concen tration by utilizing the percentage contributed to the commercial mixture by that component. The relative concentrations of components (ie. as Aroclor) can be used for estimation of all rate constants and for comparing relative accumulation or depletion of components. The i.v. data was fit to a two-compartment open model by a high speed computer program for solution of the general least squares problem (Moore and Zeigler, 1959). The general two-compartment open model: hB Central Peripheral h V elimination metabolism h The mathematical two-compartment open model: dA * KgB - K^A - K^A dB * KjA - KgB dt The equation for the open two-compartment system, derived by inte grating the model differential equations: I Dose (a. + j)ose (Kg - p)e"a^ WA (aT-p) VA (p - a) or CA - Ae"at + Be" ^ A or C. log A -at + log B - p t A 2.3 2.3 DSW 037525 STLCOPCB4021349 -6- vhere: CA * Blood concentration at time t. A Bloodconcentration contributed by compartment A at t=0. B Bloodconcentration contributed by compartment B at t0. Of First order rate constant of elimination from compartment A. 0 First order rate constant of elimination from compartment B. V. * Volume of the central compartment. ii ' Ihe following relationships are used to calculate pharmacokinetic parameters for an open two-compartment system. ; Co (Blood concentration at t*0) = A + B t^A (central compartment) ** .693 Ct t^B (peripheral compartment) .693 .0 - a+0-Kg-Kj Kg * ap/K^ K, C0/A + B p a0 VA " Dose/A + B Vd (apparent volume of distribution) **. Dose B TBC (total body clearance) 0Dose B 9. The values for A, B, cct and 0 are generated by the computer program. Tables IV.l. and IV.2. contain calculated pharmacokinetic parameters for the designated components of Aroclor 125** in sheep and pigs. The var ious components have previously been designated by letter and character ized by mass spectrometry and GC fractionation to provide information on percent chlorination and percent of each component to the total PCB in QSW 037526 STLCOPCB4021350 -7- the standard solution* Pharmacokinetic information was not obtained for components m, q, end r in sheep because of insufficient data. Component m was not seen in sheep blood three hours after injection. ^ Components q and r, relatively small fractions of Aroclor 1254, were not detected with enough consistency in sheep blood to permit proper data analysis. She values for apparent volume of distribution (Vd) and total body clearance (TBC) are based on component values relative to the standard Aroclor 1254. Tables IV.J. and IV.4. contain the blood concentrations of each com ponent of Aroclor 1254 for 24 hours in sheep and pigs. Total PCB concen trations for each sampling time are obtained by summing values on indivi dual components. Figures IV.l. through IV.4. are representative semi-log plots of data for two components in sheep and pigs. Solid dots represent the actual mean values. Circles represent the calculated functions. Bars represent stan dard deviations of actual means. Solid lines represent the fitted curve and broken lines represent extrapolations of calculated functions. The slopes, representing elimination from compartments A and B are indicated by - Q/2.5 and - P/2.5, respectively. Figures IV.5* and IV.6. contain the semi-log plots of fitted curves for each component relative to the standard Aroclor 1254 in sheep and pigs. Blood concentrations are given as ppm. Figures IV.7* and IV. 8. contain the semi-log plots of fitted curves for each component of Aroclor 1254. Blood concentrations are given as ppB. The slopes for each component remain the same whether it is shown in OSW 037527 STLCOPCB4021351 -8terms of 1254 or actual concentration, but the positioning on the graph relative to the other components varies because the components are pre sent at different percentages in the standard mixture and they are eli minated at different rates. For example, at 24 hours in sheep, there is more of components o and p relative to the amounts injected, but there is more of component K in absolute quantity. Figure IV.9 shows the disappearance of total PCB (summation of all components) from blood for 24 hours in sheep and pigs. In the sheep, the concentrations of components m, q, and r were used where available for total PCB concentration, even though these components were not fit to the model. DSW 037528 STLCOPCB4021352 Table IV 1. Estimated Pharmacokinetic Parameters for Aroclor 125*+ Components in Sheep (3.0 mg/kg IV) A ppm . a Hr-1 B ppm t^A Hr-1 Hr. t|B Hr. Vd Li. TBC Li/Hr. J .647 .584 .103 .107 1.188 6.492 2019.42 216.08 k .664 .455 .129 .028 1.524 25.106 1612.42 45.15 1 .794 .731 .077 .050 .948 13.767 ; 2701.30 135.07 n .612 .394 .134 .045 1.758 15.399 ; 1552.24 69.85 0 .628 .494 .202 .042 1.404 16,400 1029.70 43.25 P .638 ..253 .142 .029 2.733 23.970 1464.79 42.48 Table IV. 2. Estimated Pharmacokinetic Parameters for Aroclor 1254 Components in Pigs (3.0 mg/Kg IV) A aB 8 . t^A t^B ppm Hr-1 ppm Hr"1 Hr. Hr. Vd Li. i 1.284 .768 .189 .058 .903 ' 11.991 508.47 k 1.136 .400 .188 .029 1.732 23.561 511.17 1 1.123 .425 .110 .033 1.630 21.178 873.64 XU n 1.438 o 1.4o8 p 1.309 4 1.230 r 1.818 .534 .583 .521 .402 .805 .287 .344 .489 .370 .806 .054 .044 .037 ;028 .072 1.298 1.189 1.329 1.725 .861 12.731 15.886 18.582 24.517 9.668 334.84 279.36 196.52 259.73 119.23 TBC Li/Hr. 24.49 14.82 28.83 18.08 12.29 7.27 7.27 8.58 DSW 037529 STLCOPCB4021353 Table IV. 3. Concentration of Individual Components of Aroclor 1254 in Sheep Blood After 3*0 mg/Kg IV Mean ppb each Component Time (hours) .i k 1 m n o p q r 1 72 90 44 36 94 79 54 16 17 2 ' 48 66 27 19 69 60 4l 11 12 3 27 45 l4 12 45 4o 35 8 11 4 24 4l 13 -- 45 39 30 7 10 6 . 12 25 6 27 28 21 5-- 8 7 20 6 20 24 17 3-- 12 5 15 4 -- 14 16 11 1-- 24 11 2 ---- 8 11 6 ... mmm Table IV, 4, Concentration of Individual Components of Aroclor 1254 in Pig Blood After 3*0 mg/Kg IV __________ Mean pob each Component Time (hours) -d ... k l m n o J> _ ..q r 1 125 165 89 91 185 152 102 27 32 2 69 . no 57 54 127 104 72 17 20 3 48 83 4o 39 87 73 60 l4 16 4 34* 66 32 30 70 58 49 13 14 6 26 45 20 20 46 47 34 9 11 8 17 33 n 8 32 32 30 6 8 12 17 22 10 5 27 31 29 6 7 24 8 15 5 4 13 16 16 4 3 Total PCB (pnm) .502 .353 .237 .209 .124 .097 .066 .038 Total PCB (nnra) .968 .630 .460 .366 .258 .177 .154 .084 DSU 037530 STLCOPCB4021354 SCMl-LOGAnUUMtC 4G 4 9 7 0 2 CYCL.ES X 7 0 O lV lC tO H S K IM m * % .*. K E U FFC L ft ES- ER CO* SEM I-LO G AR ITH M I c v' / sv or- c--o/<- t -7-- /<2O/vJ /3 0 /^ aS7- a 4 $ /JZi> < / SEMl-LOCARITHMtC 4 6 4 9 7 0 U: ^ c /v/ ' ^ Wf ' t-/n, r~. Cor,Jro7~J or CT O & /Jt. V- rt/?0CCo/2 STLCOPCB4021360 SEM I-LO G ARITHM ! c.:c> "'/A'a'^y <-- ^ / t vr f-TA_ CJCL-C. cjxlt -.* v <20'77/9>aS~*S7~ /^ /9 STLCOPCB4021363 C 6LM I.LOGARITHMIC 4G 4970 9Em Z CYCLES X 70 DIVISIONS uKIl *.*.. K KCUFFEL ft KSCR CO. ' / r>07.o 0,7/v Projected Progress Most of the samples and data from the 1254 kinetic studies have heen collected and the data are being analyzed statistically for presentation. Information has been accumulated which should permit more rapid assess ment of the kinetics of Aroclor 1242. We have expanded our analytical capability and soon should be able to analyze tissue samples from the chronic and reproduction studies along vith the weekly influx of blood samples. Hie contract modification funded by the Bureau of Foods has been initiated and data should be complete by the next reporting period. Chicken and catfish studies are to be initiated during this quarter and the synthesis of purified PCB analogs will begin. DStt 037540 STLCOPCB4021364