Document 3Jm2EgDKQMdXeJR1DenmYwbJ6

V A A A 6 _ U f-U i Distribution H a t EFFECT OF FLUOROCARBON DISPERSING AGENTS ON THE LIVERS OF RATS AND DOQS Medical Research Project Nos. MR-639. 10-B, 10-C and 10-D Report No. 123-65 1, 2 J. A. Zapp Jr. 3, 4 j 6 J. W. Clayton Jr. J. R. Barnes 0. J. Stopps ? P. E. Smith, Jr. 8 E. F. Stula 9 J. F. Morgan 10 Haskell Laboratory File 11 A. J Fleming, M.D. 12 0. G. Mitchell 13, H D. B. Hood lj H. Sherman 16 R. S. W&rltz 17-19 W. J. Brehm . HLAB000003 000Q56 USEPA 16941 EFFECT OF FLUOROCARBON DISPERSING AGENTS ON THE LIVERS OF RATS AND DOGS Medical Research Project Woe. MR-639. 10-B, 10-C and 10-D Report Me. 123-65 Ammonium perfluorocaprylate (Ca APFC), '-hydrohexadecafluorononanoate (Cq AFC) and ammonium 3,6-aioxa-2,5-dl(trlfluoroethyl)undecafluorononanoate (AHT) are powerful surfactant. In acute oral toxicity studies with these compounds, the most striking abnormality In animals surviving a single sublethal dose was gross enlargement of the liver (Haskell Laboratory Report Nos. 5^-61, 55-61 and 70-61). A similar but less marked effect was observed in rabbits that had absorbed AST through the skin. Although liver enlargement Is not an uncommon finding In chemical Intoxications, the effect of AHT was remarkable in that 56 days after a single dose of 12 mg/kg the liver was approximately three times larger than norsial. The experiments reported here were preliminary studies designed to determine more details concerning: (1 ) the histological changes, that occur with increasing periods of time, in the livers of rats after a single oral dose of AHT; (2) the biochemical changes during the period of rapid growth and recovery In the livers of rats treated with AHT; (3 ) the combined effects of oral administration of ethanol and AHT in the rat; (4) the effect of AHT upon the pentobarbital sleeping time in the rat; and (5 ) the effect on liver function In dogs treated with AHT, CaAPFC and Cq AFC In order to find some clin ical laboratory procedure that might be useful In detecting liver Injury In personnel exposed to fluorocarbon dispersing agents. I . HISTOLOGICAL CHANGES DURING THE TIME CP RAPID GROWTH A. Procedure: In order to study the changes In size and structure of the liver following a single oral dose of AHT, 14 male rats were each given a dose of 12 mg/kg. A second group of animals of the same age and weight served as controls. One test and one control animal were killed at Intervals over a period of 56 days and two test and two control animals at 75 and 128 days. The livers were removed, weighed and a section taken for microscopic examination. '!' .0000S 7 USEPA 16942 HLAB000004 2 B. Resulta: The rapid growth of the liver, In cerms of both weight ana per cent of whole body mass, is shown in Figure 1. The increase in size was aost marked during the first ten days, reached a maximum between 40 and 60 days, and then began to decrease In size, although some enlargement was still evident after 126 days. Morphological changes In the liver were evident 24 hours after the single dose. The Initial change was characterized by markedly enhanced mitosis of the hepatocytes. This was less evident by the second day; by the ninth day, the mitotic activity had decreased. After the p6th day, most of the features which characterized the response were less intense. Th structural changes In the cells were quite evident upon microscopic examination and appeared to result principally from an enlargement of the Individual cells. I I . BIOCHEMICAL AMD METABOLIC CHANGES IN RAT LIVER 1. Changes In the Liver of Intact Animals A. Procedure: Thirty male ChR-CD rats were given 12 mg/kg of AMT as a single oral dose. Two to three weeks later, eight of the rats were sacrificed by decapitation, the livers removed, weighed, and analyzed. An equal number of untreated controls wereexamined to establish the normal range of values that might be expected In rats of this age and strain. Two AHT>trested rats and two controls were also sacrificed for microscopic examination of the tissue. Eight to ten weeks after the single oral dose of AHT, a second group of ten rats was sacrificed and the remaining ten rats three months later. Tissue slices were prepared from a portion of the median lobe for respiration and choline oxidase measurements In a Warburg respirometer. One portion of the left lateral lobe was homogenized for measurement of alkaline phos phatase activity (AFase), esterase activity, and the nucleic acids. Another portIon.was used for glycogen determination. The remain ing tissue was used to measure water, protein, fat, ash, chol esterol, phospholipids, and potassium. An average value was calculated f*>r each group of animals and compared with the controls. Differences uetween groups were tested for significance with the "t" test. B. Results: The results of the biochemical measurements are given in Table l. Two weeks after the single dose of AHT, the livers of the treated rats were quite large in size, more than twice that of the controls, and comprised approximately 9%of the body weight. An increase in the alkaline phosphatase activity and phospholipids and a decrease In glycogen were the aost important changes. Small but statistically significant (p^O.Op) increases in oxygen consumption, non-protein nitrogen, water and protein and decreases in the respiratory quotient, choline oxidase, RNA and DNA occurred. Two months after the single oral dose of AHT, the livers were no*longer growing at an accelerated rate, for there was no further increase in the absolute size of the liver and lte portion relative i 000058 USEPA 16943 HLAB000005 i - 3- to the whole body had decreased. The Increase in phospholipids, protein and non-protein nitrogen, and decrease in glycogen respiratory quotient, RNA and ENA again was observed. Five and one-half months after the dose of AHT, the livers of the treated rats were still slightly larger than normal, but the email differences in the biochemical measurements were no longer significant. These results indicate that AST produces significant bio chemical as well as morphological changes in the liver of rats. A number of these changes point to disturbances in the normal meta bolic and synthetic functions of the liver. The lowered concen tration of nucleic acids is consistent with the observed arrested mitotic activity. These effects are most evident when the liver is rapidly increasing in size. Later, when the accelerated growth has slowed down or ceased, a reversal of biochemical changes is apparent. Five or six months after the dose of AHT, when the liver is nearly normal in sice, the recovery of the organ is virtually complete. 2. Biochemical Changes and Liver Enlargement in Hepatectomized Rats A. Procedure: To study the effect of AHT on liver tissue already m a state of rapid growth or regeneration, six male ChR-CD rats were subjected to partial hepatectomy by removing 60 to 70%of the organ. Forty-eight hours after the operation, the rats were given a single oral dose of ABF of 12 mg/kg. The experiment was also conducted reversing the order of treatment by removing 60 to 70% of the liver of rats which had been treated with ABT w hours previously. Sham-operated animals dosed with AHT and hepatectomized animals served as controls. Fourteen days after the single oral dose of AHT, the livers of the rats were removed, weighed and analyzed for glycogen, phospholipids, ENA, RNA and alkaline phos phatase activity. B. Results: The results of these experiments are shown in Table 2 . t w o weeks after the operation, the livers of the partially hepatectomized control animals were normal in size for rats of this age, sex, and strain, but the alkaline phosphatase activity and ENA were higher than in normal livers. When partially hepatectomized rats were treated with AHT, the livers grew very rapidly; two weeks after treatment with the trimer, the livers were more than twice those of the hepatectomized controls. The phos pholipid content and alkaline phosphatase activity were increased while the glycogen content decreased, m e ENA was again decreased as in the livers of intact ABF-treated rats, but the RNA had Increased. Except for this increase in RNA, the results were in agreement with the previous measurements on livers from ABF-treated rats shown in Table 1. The biochemical changes in the liver occurred whether partial hepsteotony preceded or followed treat ment with AHT, although the effects were somewhat less when hepatectomy followed the treatment with AHT. 000059 USEPA 16944 HLAB000006 -4* Effect of Chemical Injury on AHT-treatad Rat Livers A. Procedure: Six M l ChR-CD rats were given a single oral dose or 12 og/icg or AHT and then, 48 hours later, a single sublethal dose of carbon tetrachloride, equivalent to 0 .1 si per 100 gm of body weight, lntraperltoneally. Aniaals which received only carbon tetrachloride served as controls. B. Results: The results of this experlsent are also shown in Table z. O n e o f the six rats dosed with carbon tetrachloride died eight days later, but all six rats treated with A8T prior tc the carbon tetrachloride died within *8 hours. No analysis of the liver was possible, for all of the aniaals were found dead and post-norten changes had begun. When examined grossly, however, the livers were aasslve in site, approxlaately 30 to 40 gw, and yellow in color, probably fron acute fatty infiltration. The rapidly enlarging liver, with a greater.than n o m a l lipid:glycogen ratio, is sore susceptible to the toxic effeot of CC14 than normal liver. Biochemical measurements on the surviving carbon tetrachloride rats showed no appreciable differences fron controls two weeks after the treatment. I I I .__ COMBINED EFFECTS OF ORAL ADMINISTRATION ______ OF ETHYL ALCOHOL AND AHT IN THE RAT A. Procedure: The study was designed as an additional evaluation or tne effects of combining hepatotoxic agents. The objective was to determine, by oral adnlnlstratlon to rats, whether repeated sublethal doses of ethyl alcohol would enhance the effect of a single hepatotoxlc dose of ANT. The outline of the study, in tabular fora, is presented in Table 3. Male ChR-CD rats, weighing between 300-400 gm, were used in the study. They were offered water and Purina Laboratory Chow on an ad libitum basis and were weighed daily. At the tine of the prescribed sacrifices, the animals were subjected to gross pathological evaluation. The liver was removed, weighed, end preserved in appropriate fixatives. B. Results: A stannary of the liver weights obtained at the various scheduled autopsies is given in Table 4. The preliminary results of this study, wherein AST was administered to rats in a single dose of 1 2 ng/kg, simultaneously with, or after, their exposure to ethyl alcohol, indicate that the increase in liver weights observed in this experiment was not any greater than that produced by AKP alone at a dose of lg ng/kg. IV. THE EFFECT OF AHT UPON PMtrOBAMCTAI t x k p t m n T im IN THE RAT A. Procedure: Changes in liver function or liver norphology have served as traoitional Indicators of hepatotoxlelty. More recently, a quick and simple pharmacologic test has been developed 000060 r USEPA 16945 HLAB000007 -5- which has been used to determine liver damage - Fentobaroltal Sleeping Time (PST). Pentobarbital is metabolized in the liver; a change In particular liver enzymes is reflected by a prolongation or shortening, of the sleeping time induced In animals by this com pound's normal anesthetic action. Therefore alteration in the PST has been correlated Kith changes in liver function and liver morphology. When young adult male ChR-CD rats are given 30 mg kg Na pentobarbital in an aqueous solution intraperitoneally their average sleeping time was found to be 58 19 minutes (for 110 rats). In studying the effect of AST administration upon PST groups of ten rats were esployed. A control value for PST was obtained for each group of ten rats, usually 2b hours before the test began. Twenty-four hours later, the rats were given a single oral dose of AHT at the rate of 12 mg/kg. PST was determined on each group of rats four hours, 24 hours, 48 hours, and at other time intervals after the oral administration of the AHT. Control groups were similarly examined for PST at the same time Intervals. The results of two of these tests are summarized in Table 5 . B. Results: The PST of rats that received AHT was first prolonged; it then became shorter and shorter until, at approxi mately six days after dosing, none of the rats could be anesthetized by a dose of Na pentobarbital that still affected untreated rats. Approximately seven weeks later, the PST of AST-treated rats could again be determined, but it was still much shorter than that observed in control animals. The a*me observations were made for the next two weeks, at the end of which time the experiment was terminated. V. EFFECT OF FLUOROCARBON SURFACTANTS ON LIVER POMCTION IN DOGS A. Procedure; Three male beagles from the stock colony were given a single oral dose of AHT, equivalent to 4, 6, or 9 mg/kg. Samples of blood were taken at frequent Intervals for three weeks and then weekly thereafter. The dog that received 4 mg/kg was later given doses of 26 and 60 mg/kg. Pour other male beagles were f iven a single oral dose of either CaAPFC or Cq AFC, equivalent to 30 mg/kg and a similar series of measurementsmade. Since both dogs receiving the CgAFPC died within 48 hours, the experiment was repeated with two otner dogs which were given a 200 mg/kg dose of this dispersing agent. The two dogs that survived the exposure to the CqAFC were given an additional oral dose, equivalent to 670 mg/kg, at this time. The following biochemical measurements were made routinely on the blood: sugar, urea nitrogen, total cholesterol and alkaline phosphatase. When the 60 mg/kg dose of AHT, 470 mg/kg dose of CoAFFC* or the 670 mg/kg dose of Cq AFC were administered, the level or activity of lactic dehydrogenase (LEH), isocitric dehydrogenase (ICZ9H), aldolase, glutamic oxalacetic (OOT) and glutamic pyruvic (OPT) transaminase were also measured. A routine hematological 000061 sMB USEPA16946 HLAB000008 - t- examination and an analyals of a 24-hour urine specimen were made at intervals on these animals. The level of the various components of the blood following the dose of fluorocarbon dispersing agents was compared with an average value observed prior to the exposure and with a similar measurement made at the same time on specimens from stock colony dogs. For the enzyme activities, a normal range was established from measurements made on a number of stock doga. The activity was also measured at least once, prior to treatment, on the dogs dosed with the dispersing agents. B. Results: The significant biochemical and clinical findings are summarized briefly in Table 6. The principal finding indicative of some injury or dysfunction in the dogs that received AHT was a decrease in the plasma chol esterol and an increase in bromsulfalein retention (BSP) and APase activity. The effects on cholesterol and APase are shown in Figures 2 and 3. These began to change within one week and became "abnormal", l.e., exceeded an arbitrary limit of two standard deviations f.om the pre-exposure mean within three weeks after the doBe was administered. The increase in BSP retention occurred during the first ten days and then began to return to normal. The depression of plasma cholesterol in all three dogs occurred earlier and began to return to normal sooner than the rise in plasma APase activity. The dog that received the highest dose of AUT showed the greatest change in plasma APase from the pre-exposure m a n . The activity of a number of enzymes considered to be sensitive indi cators of liver injury was measured in the plasma when a 60 mg/kg dose of AHT was administered. The results of these biochemical measurements are presented in Table 7. All the plasma enzymes measured were elevated within the first three days after the dose of 60 mg/kg of AHT was administered. The greatest increases were in the OPT and APase. Within one week, all but these were within the normal range. The depression of the plasma cholesterol occurred more slowly, but there was no evidence of a return to normal after three weeks aa occurred in the dogs receiving 6 or 9 mg/kg. With the 200 mg/kg dose of CgAPFC, the OPT and GOT were elevated in both dogs within 48 hours. One week later, they were in the normal range. When 450 mg/kg of this compound was admin istered, all of the enzymes measured were markedly elevated 24 to 48 hours later. The greatest change occurred in the OPT and ICDH. Both animals expired within 48 hours after dosing. The 450 mg/kg dose of Cq AFC caused elevated levels of all the enzymes measured within 48 hours in one (No. 2) of the two dogs exposed. On the tenth day after the dose was administered these were within the normal range. The other dog showed no effect from the treatment. At the 670 mg/kg dose of this compound, 000062 USEPA 16947 HLAB000009 -7 - Dog No. 2 showed a rise In GOT OPT and APase during the first 48 hours, and a return to normal within two weeks. There was a slight rise in the APase, OPT and GOT in Dog No. 1. SUMMARY Single oral doses of 12 mg/kgof AHT, administered to rats, cause a rapid growth of the l.'ver that continues for as long as two months after the treatment. Morphologically, the change is characterized by an enlargement of the hepatocyte. Changes in the biochemical composition, enzyme activity and respiration, accompany this enlargement and indicate a disturbance in the normal metabolic functions of the organ. The changes in nucleic a d d content and morphology of the cell suggest an interference in mitotic activity. Reversal of these changes begins in two months and is nearly complete in five to six months. Rats that have been subjected to excision of 60 to 70% of the liver 48 hours before or after a single oral dose of 12 mg/kg of AHT regenerate larger than normal livers. Carbon tetrachloride is more toxic for rats that have been given a single oral dose of 12 mg/kg of AST. This may result from the accumulation and retention of the CCI4 in the enlarging liver which has more fat and less glycogen than normal liver. When AHT is administered to rats in a single dose of 12 mg/kg simultaneously with or after their exposure to repeated sublethal oral doses of ethyl alcohol, the increase in liver weight observed is not any greater than that produced by AMT alone at the same dose. Pollowlng an initial depressant effect by ART on enzymes that metabolize sodium pentobarbital, there is apparently an Increase in the rate of metabolism of sodium pentobarbital so that it cannot exert its anesthetic action at usually anesthetic dose levels. Liver function studies in dogs have shown that ART, CoAPPC and Cq AFC dispersing agents affect the liver. On the basis of mg/kg dose, ART is moat, and Cq AFC least, toxic to the liver. Doses of 26 mg/kg of AHT or^SOO mg/kg of CgAPFC produce elevated plasma levels of enzyme activity indicative of cellular damage. Only ART lowers tha cholesterol level in dogs given 4 mg or more per kg of body weight in a single oral dose. Of all.the measurements made, alkaline phosphatase and QPT are the most sensitive in detecting an effect on the liver in dogs from all three dispersing agents. : ' 000063 USEPA 16948 HLAB000010 8- - EFFECT OF FLUOROCARBON DISPERSING AGENTS ON THE LIVERS OF RATS AND POOS Medical Research Project Noa. MR-639. 10-Bi 10-C and 10-D Report No. 123-65 HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE Report by JRB/HS/ah 8-19-65 Approved by Chit_, . ion HLAB000011 oooo* USEPA 16949 TABLE 1 BIOCHEMICAL CHAMPES IN RAT LIVER 2-3 Weeks Control AHT 2 Months Control AHT Body Weight ga Liver Weight ga Liver %Body 0* fL/hr. COg /*L/hr RQ APase BD/ga Esterase U/ga 'Choline Oxidase Water ft Protein ft Pat ft Olyoogen ft AshjT Cholesterol ft KPN ft Phospholipids ft Potasslua ft RMA * DMA ft Plasaa APase BU IOC nil Plasaa Cholesterol mg 345 13-5 3.94 7.23 5.81 0.81 2.45 245 69 68.8 18.1 .1 * 4.54 1.38 0.43 0.23 3.15 0.34 0.907 0.160 324 28.5 8.80 7.97 5-73 0.72 3.60 180 61 69.8 19.6 4.16 2.60 1.36 0.40 0.26 3.96 0.33 0.872 0.124 546 18 .O 3.30 7.00 5.67 0.81 2.66 210 65 68.0 17.2 5.03 3.86 1.36 0.44 0.21 3.29 0.42 0.834 0.176 51 93 476 27.9 5.86 6.99 5.08 0.72 2.86 2j5 78 68.2 18.3 5.31 2.93 1.32 0.40 0.23 4.17 0.40 0.786 0.158 51 144 5 1/2 Months Control AHT 640 18.8 2.93 602 21.4 3.52 6.10 4.68 0.77 5.93 4.50 0.76 3.41 3.04 74 68.3 16.7 4.87 4.44 1.36 78 68.3 16.9 4.81 4.05 1.37 0.29 3.08 o.\, 0.824 0.185 0.30 3.20 0.42 0.815 0.176 000065 C cn > ! oONONO ^looooaviH TABLE 2 Treatment 1 0 AHr* Hepatectomy 'a AMT cci4b AHT* Treatment 2 No. of Rata Mortality Hepatectomy A 0 Sham Operated 6 0 AHT* 60 Hepatectomy 6 0 0 6 1/6 cci4b 6 6/6 Liver Liver * Qly- Phospho Weight Body cogen APace lipids HNA DNA 12.6 3.15 A .60 3.80 3.3 0.95 0.20 30.5 8 .2A 2.A7 3.9A 3.8 1.15 0 .12 29.2 7.29 2.07 A.55 A.3 1 .0A 0 .12 2A .6 7.18 1.7^ 3.82 A.O 0.93 0.15 13.6 3.71 A .16 2.69 3.0 0.96 0.19 All animals died within A8 hours after receiving AHT a) 12 mg AHT/kg body weight b) 0.1 ml CCI4/IOO gm body weight O O m USEPA 16951 uooooqvih I TABLE 3 DOSINO AMD SACRIFICE SCHEDULE OF ANIMALS QIVEN ETHYL ALCOHOL AND AHT Group Total No of Anlaals A Hrs After 10th Dose Number of Animals Sacrificed 1 A Days After 28 Days After 2 Months 10th Dose 10th Dose After 10th Dose Control (no dosing) 822 2 2 C.H.OH 822 2 2 (2250 ag/kg/day, 5 x seek for 2 weeks) c 8h so h (2250 ag/kg/day, 6 2 2 5 x week for 2 Meeks) + AMT (12 mg/kg on day of first alcohol dose) 2 CaHsOH (2250 ag/kg/day, 5 x Meek for 2 weeks) + AHT (12 ag/kg on day of 10th alcohol dose) 6 222 CbHsOH (2250 ag/kg/day, 5 x week for 2 weeks) AHT (12 ag/kg on 14th 'day after 10th alcohol do8'}) A 22 oo 1If: 0 USEPA 16952 WOOOOaviH TABLE Jl Oroup LIVER WEIOHTS OP ANIMALS RECEIVINO REPEATED DOSES OP ETHYL ALCOHOL AND A SINGLE DOSE OP AHT Liver Weight (ga) of Animals Sacrificed h Hra. After lU Days After 28 Days After 10th Dose 10th Dose 10th Dose After 10th Doae Control (no doelng) CH*OH (2250 mg/kg/day, 5 x week for 2 weeks) C*H8OH (2250 ag/kg/day, 5 x week for 2 weeks) + AHT (12 ag/kg on day of first alcohol dose) C>HsOH (2250 ag/kg/day, 5 x week for 2 weeks) + AHT (12 Mg/kg on day of 10th alcohol dose) CsHsOH (2250 mg/kg/day, 5 x week for 2 weeks) + AHT (12 mg/kg on lAth day after 10th alcohol doae) 16 ; 15 15; -* 25; 30 16; 20 2*; 19 *2 ; 32 39; ** 19; 23 25; 21 36; 33 30; *U 22; 16 16; 22 *0 ; 30 36; 36 37; 36 000068 * I animal died after the fourth doae GO 9> ON NtyOi S10000V1H TABLE 5 AVERAGE SLEEPINQ TIMES OP RATS GIVEN Na PENTOBARBITAL INTRAPERITONEALLY AT A DOSE LEVEL OP 30 w/kg ** Pre-Expoeure 24 Hours Treatment PST t SD R Control 49 t 14 10 10 AMT H7 + 6 10 (12 mg/kg) VS 4 Hours PST 1 35 R 52 t 8 10 VS 121 t 14 10 VS Post-Exposure PST2*1-rHSouDrs H W Hours PST I S C R 56 + 11 10 4o t 9 TO l8 36 8 25 + 6 tJ 8 TO b Days PST t SD R 7 Days PST Su R 41 + 12 8 TO 0 TO Control 50 t 12 10 47 t 8 VS 10 47 + 6 10 46 + 11 8 56 t 12 10 VS TO TO TO AHT 56 + 15 10 143 + 18 10 33 + 12 22 + 6 (12 mg/kg) VS VS ri 0 TO PST t SD - Average Pentobarbital Sleeping Time - Standard Deviation (minutes) R . Number of rate which went to sleep Total number of1rata dosed Oo o 0* 910000SV1H TABLE 5 (Contd) AVERAOE SLEEPING TINES OP RATS OIVEN Na PENTOBARBITAL ____________________________ INTRAPER1TONEALLY AT A DOSE LEVEL OP 30 m&/kg ________________________ 9 Dayc Treatment PST t S D S T Post-Exposure______ 16 Days _ 27 Days *9^76 PST t S D R ^ PST I S D R ^ PST + S D f P _ 56 Days 6k Days PST f S D P S T t S D R Control 1 ART | (12 ag/kg Discontinued after seven days Control 72 t 10 l8 65 - 18 8 13 80 + 25 l8 81 t 20 10 .13 76 * 16 10 13 82 + 17 10 13 ART (12 mg/kg) 0 13 0 13 18 4 13 t J 22 " 6 6 18 + 2 13 iS PST + SD - Average Pentobarbital Sleeping Time + Standard Deviation (minutes) R Number of rats which went to sleep Total number of rats dosed . >1 O iQoooaviH TABLE 6 ' CLINICAL AND HTOCHKMICAL OBSERVATIONS IN D0Q3 ADMINISTERED FLUOROCARBON DISPERSING AOEHTS Compound Mo. Dogs Dose * Rat ALD Clinical Mortality Biochemical 16 1C None 0 Hypocholesterolemia, elevated APase 19 15 None 0 Hypocholesterolemia, elevated APase, BSP retention 14 AKT 26 7 *3 None None 0 Hypocholesterolemia, elevated APase, BSP retention 0 Hypocholesterolemia, elevated APase, OPT, BSP retention 60 Cq PDA 2 200 2 *50 C^FDA i 2 *50 2 670 100 Vomiting, anorexia, polydipsia, weakness. wt. loss, black tarry feces for * days 30 Vomiting, polydipsia 0 0 67 Vomiting, polydipsia, 2 blood, feces and vomttus, tremors, convulsions, anorexia,death 1 - 3 days 30 Vomiting, 2-* hours 0 *5 Vomiting 0 Hypocholesterolemia, elevated APase, OFT, LEH, ICOH, aldolase. Jaundice, billrubinurla Elevated APase (1/2), OOT,QPT (2/2), lowered amylase Elevated APase, OPT, ICDH, LDH, aldolase. Jaundice Normal or slightly elevated cholesterol, APase, OPT, ICOH, LDH (1/2) Normal or al. elevated chol esterol, elevated OCT, OPT (2/2),si. elevated APase(l/2), amylase and sugar (2/2), elevated after 1 week 000071 8100000V I11 TABLE 7 Dose 26** 60 Days After Dose 0* 8 15 22 37 3 7 10 1* 21 LIVER FUNCTION TESTS IN DOGS - AHT Cholesterol 8 * 133 66 49 47 86 APase Bod. Units 2.3 5.5 5.5 7.3 7.2 OPT Units 35 ICDH Units 230 7* 21.7 850 3900 70 15.7 270 145 64 13.4 135 50 13.1 91 290 48 11.4 77 145 LDH Units ljO Aldolaae Units <2C 360 47 60 11 40 10 ICO 13 * Average pre-exposure ** Previously received 4 mg/kg 6l days before a dose of 26 g/kg 000072 Ct/i pi P3 > CT\ 'l_O/l -J 6I0000QV1H TABLE 7 (Cont'd) Dose -(g/kg) CoAPFC P200 CoAPPC 8A50 C q APC <50 CoAFC 670 Days After Dose 0* 1 2 5 7 12 21 27 1 2 0* 2 A 7 10 14 22 2 4 5 7 12 21 27 Cholesterol g * ___ l2 101 112 102 93 109 11 1 126 126 106 112 100 127 92 109 89 106 118 164 145 112 112 126 126 102 124 128 152 100 123 121 135 142 156 142 124 15? 149 144 154 124 102 133 135 127 133 140 136 APase Bod. Units 1 2.8 1.7 2.6 2 .1 2.6 6.9 21 .-85 10.1 8.0 1.4 5.4 1.8 3.7 2.4 3.3 7;3 20.5 38.7 1.2 2.4 1.6 3.8 2.0 4.4 1.7 3.8 1.3 2.3 1.7 2 .1 1 . 1 1.4 1.9 2.4 1.9 2.8 1.7 3.6 1.4 1.2 5-5 2.4 1.0 2.0 1.2 2.1 OPT Units 12 42 36 5? 50 56 845 108 425 56 166 40 70 36 44 42 40 280 8500 8600 25 33 30 116 33 84 27 43 40 25 66 52 70 170 58 190 42 100 34 48 36 50 36 46 * Average pre-exposure GOT Units 12 23 21 20 J 6 38 880 34 52 18 22 12 14 16 30 16 16 14 16 38 22 44 172 18 54 14 22 16 16 18 24 14 16 ICOH Units 1 M M Units 2100 114X100 300 302 3860 1320 153 150 72 43 116 595 74 200 145 189 68 48 C^OOQO OSOOOOUV'IH