Document NezQJ7qmMbRYLOjNwVq6yVGLg

f.Y. feetlng. Loon, ;1961). *' U74 f r a!aehem*ul IWJ. VoU 12* PP WJ-tOJ. Perj*mn Prm Ltd** Printed In Great BHuin. V /" i ,-r? a--. -VA- THE METABOLISM OF "Cl-LABELLED TRICHLOROETHYLENE AND TETRACHLOROETHYLENE IN THE RAT . i J. W. Daniel *. Imperial Chemical Industries Ltd,, Industrial Hygiene Research Laboratories, The Fr^thc, Welwyn, Hertfordshire (Received 22 February 1963: accepted l May 1963) Abstract--`'O-labclled tri- and tetrachloroetiiylcne were synthesized and fed to rats. The partition of radioactivity in urine, faeces and expired air was subsequently measured and the nature and amount of individual metabolites determined. The specific activities of metabolic trichloroacetic acid and trichlontethanol were shown to be the same_as that ot the administered trichloroethylene thus demonstrating an intramolecular re arrangement of chloride. About 13 per cent of the dose ot trtchlo'octhylcnc was exCfetclT'in tne unneTTctrachlorocthylene underwent little metabolism ar.d only 2 per cent or the radioactivity was excreted in the urine Both trichiQi'oeihv ant snd :ci.'. chloroethylenc are largely excreted through th^Tiung? altnoupn there is an .-'nin-.-m c7r(trenec oetween the rates of excretion of the two compounds, rctmctiloroethylcr..has no cfTcct on the liver-lipid content of rats exposed to high concentrations (tOOO p.p.m.) of the vapour, A scheme is presented which would account for the formation of the known metabolites of both tri- and tctrnchloroethylcne v \ <C- -- <-' ^ *-/ (_ |-- ' r =r t i* r,t ,`p c. / ok The chlorinated hydrocarbons trichloroethylene and tetrachioroethylene are impor tant industrial solvents especially for dry-cleaning and degreasing. They possess the advantage of non-inflammability and low toxicity. The metabolism of trichloroethy lene has been studied both in man and in a variety of experimental animals while the metabolism of tetrachioroethylene has received but little attention and published results are conflicting. It is well known that trichloroethylene is excreted in the urine as trichloroacetic acid and trichlorocthanol in all species of experimental animals studied while, in addition to these compounds, monochluroaceoe acid is also reported to be a unitary metabolite in man This information has been tdcquaieiy reviewed by Defalque.1 Dc'pitc the large amount of published work no adequate balance sheet has been presented for the excretion of trichloroethylene and its Metabolites ir urine, faeces and expired air. A. further point of major importance which has not been elucidated is the nature of the re-arrangement which results in the formation of 2.2,2-trichIoroethanol and trichloroacetic aci'd from 1,1,2-trichloroethylcnc This may involve an exchange of a chlondc-ion with the body ehioride-pool or mav be an intra-molecular re arrangement. I .ahum,1 using a combination of gas- and papoi-cnromulograpiiie techniques, identified di- and tri- chloroacctic acids and the corresponding alcohols in the urine of rats dosed orally with tetrachioroethylene (I g/kgl. YUner1 exposed mice to the vapour of nC-tctrachloroethyjene and subsequently recognised trichloroacetic acid and oxalic 795 ' 1 t !*- -u-^ A V , . . . I iul ; i , h j ,1 f J rL iJ / t k ti -lj\: k L 1 1 V i, ic L ^ ix - i 796 J. W. Daniel acid in the urine. These conflicting observations may in'part be due to the different methods of dosage employed. The metabolism of trichloroethylene and tctrachloroethylcnc, both uniformly labelled with "Cl, have been studied in the rat in an attempt to resolve some of the outstanding problems. In addition the effect of exposure to the vapour of tctrachlorocthylene'on the lipids of rat liver has been investigated following a report* that expo sure of mice to tctrachloroethylcnc vapour (400 p.p.m.) produced fatty-infiltration of the liver. MATERIALS AND METHODS Synthesis of3iCl labelled trichloroethylene and tetrachloroethylene "Cl- Hydrochloric acid (100 /tc) was converted into AgCl which was subsequently oxidized by heating with a sulphuric acid-phosphoric acid-Cr03 mixture.5 The "Cl- chlorine gas so produced was allowed to react" with an excess of acetylene for 60 min at 20 at 0-5 atm and the tctrachloroethane collected by vacuum distillation. The tctrachloroethane (1-47 mmole) was dchydrochlorinatcd with alcoholic-KOH1 and the product of 1,1,2-trichloroethylene was separated and purified by gas-chro- matography at 50 on a 2-m column of Cclite (60-80 mesh; Johns-Manviile Ltd). containing 25% (w/w) silicone grease (Dow-Corning Ltd.) as liquid-phase. The pro duct was diluted with inactive trichloroethlene to a specific activity of 0-27 ;tc/mmole. "Cl- trichloroethylene (15 mmpie) was allowed to react for 36 hr at 20' with an excess of "Cl- chlorine gas in the presence of anhydrous FeCI3 (0-2 mmole). The pentachloroethane was collected, dehydrochlorinated and the product of J5CJtetrachloroethylenc purified by gas-chromatography as described above p^CI-CjClJ requires C, 14-3; Cl, S5-7; found C, 14-4; Cl, 85-4]. It was diluted with inactive tetrachloroethylene to a specific activity of 1-3 yic/mmole. t t [! II lI ii { \t b {. i ii i t absor distil! (60-8 Kath: Qtiar. . Ra at 42 gena! Estin Tr Fujh acid The i 1 fc funn colic 40 rr ,2 2,2 docs acid colo Preparation of intermediates 2,2,2-TrichIoroethyl-fJ-D-glucosiduronic acid (Na+ salt) was isolated from the urine < of dogs dosed with 2,2.2-trichloroethanol (700 mg/kg) (Imperial Chemical Industries ? heat Ltd.; General Chemicals Division, Widnes) as described by KiilzT Methyl 2,2,2- ' hyd: trichioro-(tri-o-acetyl-^-D-glucosid)-uronate (m.p. 154-5) was prepared according to 18 ; the method of Smith and Williams/ J pH 4 10 n Animal studies tub*, Wistar rats from a closed colony, randomly mated, (180-220 g) were dosed with esti: trichloroethylene and tetrachloroethylene respectively by stomach-tube, Amimals A were then transferred to individual glass metabolism cages which permitted collection : [ of urine, faeces and expired air/ In order to collect the radioactivity in the expired air Qw the metabolism cage was vented by a current of air (500 ml/min). The air then passed .; 7 through a vertical glass column (30 X 300 mm) which was loosely packed with j (4-6 stainlcss-stccl rings through which toluene percolated from a reservoir at 30 ml/hr. : Ter Chlorinated material was readily extracted into the toluene which was collected in fro; fractions each of I hr until no further radioactivity was detected. While this was a sob convenient procedure for extracting the radioactivity from the expired air it was not ; the possible to concentrate the fractions so as to allow analysis by gas chromatography. Fu To achieve this purpose the expired air from dosed animals was drawn through two A& tt t. - a' i i O -/ < * -- ' h I- -z -iL l j-! vj\ J I> : T"""'......... "............ ....................' l :rcnt rmls rthl or<> xpoin or rntly The for . ion. . 0H` f hro- * .td). . aroole, i an The *acij tive me ties U2to' ith 3 Is on air ed ith hr. in a ot 7vo Tlic mctabvtiMn of 'Q.TIahclIed Iriclilitroetliylcnc ;iiuJ IctnidiU'fiKjtliylcng in the rat 7V7 absorbers containing anhydrous CaCI2 and soda-lime respectively. It was then drawn through two traps each containing -ft in. Lcssing-rings kept at the temperature of liquid-air. The chlorinated metabolites were retained in the traps under these con ditions and were subsequently recovered from the traps by vacuum distillation. The distillate was then examined by gas chromatography at 50 on 1-m column of Celite (6G-S0 mesh) containing 28-4 per cent (w/w) Reoplex 400 as liquid-phase and a Katharometer as detector. Quantitative analysis Radioactivity was measured in a liquid Geiger-Mtiller tube (Mullard MX 124) at 420 V. Urine and expired air fractions were counted directly. A 30% (w/v) homo genate of faeces was prepared in water and the homogenate counted directly. Estimation of trichloroacetic acid Trichloroacetic acid was determined in the urine using a modilication or the Fujiwara procedure. Urine (I ml), containing not more than 200 /ig trichloroacetic acid was added to 25% NaOH (16 ml) in a test-tube fitted with a ground-glass stopper. The contents of the tube were heated, with frequent shaking, on a water-bath at SO3 ;- l8 for 15 min. When cool the contents of the tube were transferred to a separating funnel and the lower aqueous layer discarded. The red-coloured pyridine layer was collected, diluted to 10 ml with ethanol and the extinction measured in cither 10 mm or 40 mm glass cells at 520 m^t in a suitable spcctrophometer. Under these conditions 2,2,2-trichloroethanol gives a yellow colour (Amj^ 412 m/t; 8000). This colour does not interfere with the procedure described for the estimation of trichloroacetic acid. A standard of trichloroacetic acid was included with each estimation since the colour developed varies appreciably with the temperature of the water-bath. Trichloroethy/ghtcosiduronic acid. This was estimated as trichloroethanol after hydrolysis of the urine either with mineral acid or with /J-glucronidase, Optimum conditions for acid hydrolysis were 15 min at I008 with 3 N HC1. Since prolonged heating with acid results in destruction of the liberated trichloroethanol enzymic hydrolysis is to be preferred for quantitative work. Urine (1 ml) was incubated for IS hr at 37 with a ^-glucuronidase preparation10 in 0-2 M acetate buffer (1 ml; pH 4-5). The liberated trichloroethanol was separated by steam-distillation until a 10 ml-vol. of distillate had been collected. The distillate (2 ml) was heated in astoppered- tubc at 100 for 2 min with NaOH (0-33 N: 1 ml) and the liberated formaldehyde estimated using the acetylacetone reagent described by Nash.11 Alerrnpturie aetd-csiruricrnT estimated using the iodometric procedu' QiialiTutive analysis Trichloroethylene. The combined urines (48 hr) of 6 rats given ^Cl-trichloroethylenc (4-6 /ic) were pooled, acidified with HC1 and extracted continuously with ether for 168 hr. During the first 4 hr Fujiwara-positive reacting material was extracted from the urine (Fraction A). At this stage therefore the solvent was separated, fresh solvent was added and the extraction continued until the bulk of the radioactivity in the urine had been removed (168 hr). This second fraction (Fraction B) was free of Fujiwara reacting material. A portion (20 ml) of the residual urine was treated with N .AgNO, and the precipitate collected, washed with water and dissolved in ammonia urlkT,U 1Z / l! ' *3 1 T- R&S 136073 7<J8 J. W. Daniu, fsn. cr. 0-90), No radioactivity wrts detected in this ammoniricnl solution. Fraction B was the jvas concentrated in vacuo and the residue rcdissolved in 0-5 U acetate buffer (pH 4-5). A saturated solution of normal lead acetate (0-5 vol.) was jiddcd and the precipitate The ctk *is separated on the centrifuge. The residue was washed twice with water on the centri m fuge and the washings added to the supernatant which wa^s then adjusted to pH S by and ret the drop-wise addition of ammonia (sp. gr. 0-90). The precipitate that formed was point (` collected on the centrifuge, washed three times with wa|er, suspended in water and chloroc gassed with H,S. The PbS was removed by filtration and the filtrate concentrated This in vacuo. The residue was dissolved in methanol and filtc/ed free of insoluble material. and die The filtrate was then treated with an excess of diazomethane in ether and allowed to stand overnight at room temperature. The solvent was distilled and the process of methylation repeated. The gummy residue of the methyl-ester was dissolved in NaC: a few d collecte pyridine (5 ml) and acetic anhydride (5 ml) added. Af{cr standing for IS hr at roo^? temperature the solution was poured into water and he solid which separabxTwas c_o__lle__c_te_d__a_n_d__re_c_r_y_s_t_a_ll_iz_e_d_, to constant _s5pye,ciffriocmacatiqvui eousjrthanol. The I j j then di was rep materi: material was identified as <jfnetfm 2.2,2-trichloroethyl-{tri-fVttcetyf^-D-glucosid)- "(juronate (m.p, and mixed m.p/T54^155: [o]d 38-6 (c. M mrcTuH) [Sm.ith and d' i V. illiams,* quote m.p, 158 [a]o -- 37' (c. 1-0 in CHCl3): Seto and Schultze13 quote -- acid is in 01 was ad ft-aluCC m.p. 157-158 [a]D - 40-7 (c. H in CHCI,)] ClsHsOin :,CJ3 requires ^Cl 23-11; found ^CI 22-7. The specific activity of dm material was 0-31 ^c/mmo[e. // \ <3 [ / Amoun Fraction A contained both trichloroacetic acid and the normal urinary ether- 7'^' obtain-. I fuble acids.' It was shaken with Na,.COa (2 N) and "the ether layer discarded. The ! The i aqueous solution was adjusted to pH 4 with 70% H3P04 and extracted for 18 hr ; vapour with ether. This procedure removes a considerable amount of non-radioactive ether- Anima soluble material which would otherwise intercferc with subsequent procedures. No Atmos significant amount of radioactivity was extracted. The aqueous solution was then livers r :5I' acidified with cone, HC1 and extracted for 4 hr with ether. The ethereal extract was The dr I 1;' concentrated to a small volume (5 ml) and treated with excess of an ethereal solution ic. of diazomethane. Gas chromatography of the mixture of the methyl-esters thus ob tained was carried out on a 2-m column of Celitc (60-80 mesh) containing 30% by weight of di-n-decylphthalate as liquid phase operating at 105 with nitrogen (70 ml/min) as carrier gas. Two peaks were obtained which were separately collected in traps cooled in liquid-N.. The contents of both traps were weighed, dissolved in 10 ml toluene and counted. Only one fraction was radioactive and corresponded to that peak given to methyl trichloroacetate. The materia! isolated (7 mg) had a specific activity of 0-29 ^c/mmolc. wCl-Trichloroethy!ene (0-6 ^c) was administered to a rat and urine was collected for { : j : The with tables excreti time o; of exp 72 hr. To the urine (10 ml) was added trichloroacetic acid (533 mg) and cone. HCl ; f , (5 ml). The solution was refluxed for 30 min and the urine was extracted with ether. ' The residue, after evaporation of the solvent, was treated with p-chlorobenzyl- 1 pseudothiuronium chloride.14 The resulting solid, p-chlorobenzylpseudothiuronium trichloroacetate (m.p. 150) was rccrystallized from dioxane to constant specific ; ;> D< activity and corresponded to 1-7 per cent of the dose of administered trichloro ethylene. No activity was found when mono- and di-ehloroacctic acids were separately added to urine (10 ml) and the above procedure carried out. j uC!-Tetrach!oroeihylene. Trichloroacetic acid (530 mg) was added to urine (20 ml) : of rats which had received 3,Cl-tetrachloroethylene (2 /ic) by stomach tube. The urine j The metabolism of MCl-labcllcd tridilori'clhylene and tctradiluruelhylcnc in the rat 7'J9 was then acidified with cone. HCl and extracted continuously with ether for 4 hr. The ether was separated and distilled in a current of nitrogen. The residue (1-02 g) was dissolved in ethanol (5 ml) and treated with a solution of p-chlorobenzylpscudothiuronium chloride (1 g) in ethanol (5 ml). The solid which separated was collected and recrystallizcd from aqueous ethanol to constant specific activity and melting point (ISCn. and corresponded to 0-6 per cent of the dose of the administered tetrachlorocthylenc. This procedure was subsequently repeated after the addition of monochloroacctic and dichloroacctic acid respectively. The products were not radioactive. NaCI (20 mg) was added to a portion of the urine (20 ml), the solution acidified with 5S a few drops of cone. HN03 and treated with 0-1 N AgNOj (3 ml). The precipitate was in ' collected on the centrifuge, washed three times with water, dried and weighed. It was :tt then dissolved in ammonia (sp. gr. 0-90) and the radioactivity determined. The AgCl was reprecipitated by the addition of acid and the cycle repeated until the' precipitated material was of constant activity. It had previously been shown that trichloroacetic acid is not precipitated from urine under these conditions' In order to determine whether any trichloroethanol was present the urine (100 ml) was adjusted to pH 4-5 with acetate buffer (0-1 M) and incubated for 18 hr with ^glucuronidase. Volatile metabolites were separated by steam-distillation and the amount of radioactivity in the distillate determined. Only background activity was obtained indicating the absence of chlorinated alcohols. The effect of tetrachloroethylene vapour on liver lipids. Animals were exposed to the vapour of tetrachloroethylene (1000 p.p.m.) for three successive periods each of 6 hr. Animals had ready access to food and water during pre- and post-exposure periods. Atmospheres were prepared as described by Gage,11 The animals were killed, the livers removed and extracted for 6 hr with 25 ml of a mixture of alcohol-ether (3:1). The dried, defatted tissue was then separated and weighed. The solvent mixture was distilled, the residue weighed and the results expressed as mg lipid per 100 mg dry weight of liver. RESULTS AND DISCUSSION The distribution of radioactivity in the urine, faeces and expired air of rats dosed with ^Cl-trichloroethylene and 3SCl-tetrachloroethy!ene is shown in the accompanying tables (Tables I and 2). Both compounds arc largely excreted through the lungs, the excretion following an approximately exponential course. Trichloroethylene (half time of expiration 5 hr) is more rapidly eliminated than tetrachloroethylene (haif-time of expiration 8 hr). There was no evidence from analysis of the expired air that any Table 1. The excretion of radioactivity in expired air, urine and FAECES AFTER THE ORAL ADMINISTRATION OF 3,Cl-TRICHLOROETHYLENE Dose fpc) 8-6 7-5 3-7 4-0 Expired air 84-8 82-3 -- 72-1 Radioactivity (%) in' Urine 10-7 13-8 n-3 ' 20-6 1 Faeces 0-5 0 0 0 Total Radioactivity (%) 96-0 96-1 [ll-3| 92-7 ,**.! ir? 4 r;- tj i!;- n r !i v il' i ;i >i 1 j!;! V< > :f J- * I; t t-` v *- if-* ii i i->1i1. If 4i>lk !<I.; *rf i:i 33 99 03 CT> O -4 CJ1 -T- a 800 J. W. Daniel Tabu- 2. The excretion of radioactivity in expired air, urine and faeces after the administration of 3"C1-tetrachloroethylf.ne Dose (pc) ' 1-75 13 No of animals 1 A Radioactivity (%) in Expired atr Urine 97-9* -- 2* It I-6f Faeces 0 0 43 hr. f 18 days. metabolite of either compound was present, although the presence of chloroform in the expired air of men exposed to the vapour of trichloroethylene1* has been reported. In a single experiment a small amount of (1-2%) of irons 1,2-dichloroethylene was identified in the expired air of a rat dosed with trichloroethylene. This observation could not be confirmed in subsequent experiments and the result was thought to be due to traces of impurities in the trichloroethylene. Quantitatively the urinary meta bolites of trichloroethylene (10-20%) in the rat are due to trichloroacetic acid (1-5%) and trichloroethanol (10-15%). No monochloroacetic add was detected in the urine. Tctrachloroethylene undergoes little metabolism and only about 2 per cent of the radioactivity is excreted in the urine. Trichloroacetic acid (0-6%) and inorganic chloride were the only metabolites detected. About 25 percent of the total radioactivity in the urine was precipitated as chloride on the addition of silver nitrate solution. The radioactivity remaining in the urine could be accounted for as trichloroacetic acid. Little oxalic acid could, therefore, be present since there was a 1 :l ratio between trichloroacetic acid and chloride ion. These results are in partial accord with those described by Yllner for the metabolism of ^C-tetrachloroethylene in mice. Radioactivity is excreted in the urine for upwards of IS days after the administra tion of both trichloroethylene and tetrachloroethylene. It is well known that trichloro acetic acid is slowly eliminated from the body17 after exposure to trichloroethylene. The excretion of radioactivity after dosing with trichloroethylene corresponds closely to the excretion of trichloroacetic acid in rats dosed orally with sodium trichloroace- tate (unpublished experiments). It may be assumed that the radioactivity after dosing with tetrachloroethylene is due to the excretion of both trichloroacetic acid and of chloride ion since the latter has a biologic half-life of 29 days. It is certain that 70-80 per cent of the radioactivity excreted during the third week is precipitated as inorganic chloride. The specific activities of both trichloroacetic acid (0-29 ^c/mmole) and trichloro ethanol (0-31 jic/mmole) isolated from the urine after dosing with trichloroethylene were approximately equal to that of the administered trichloroethylene (0-27 [tcj mmole). This indicates that there is an intra-molecular rearrangement of trichloro ethylene and no exchange of chloride with the body chloride pooL A reaction mech anism which may reasonably be expected to operate in vivo and which would account for the known metabolites of trichloroethylene would be, Cl Cl \c=c/ Cl O Cl \c--/\c/ ^CCU-COOH^S 7 \J /V \ H :/ Cl . \ H Cl \ CClj.CH^OH J - o) c1 . o I 1 ,,C / <0 n o fc JPrc genate ment < CXpOSt appear metab enzyrr chlora ethane that li admin oxide Tcisir. tetraeincrca howe>. metab in the The Ci Cl The r. m. admir Th. (Tab! .toxici Acknc Pham techm 2. S. ID--B R&S 136076 The metabolism of "Cl-labelled trichloroethylene anil (ctrachlorocthylcnc in the rat 801 The formation of an hypothetical oxide was originally postulated by Powell.13 Tri chloroethylene oxide is thought to be formed in vitro when trichloroethylene is oxy genated in the presence of actinic radiation. (British Patent 523, 55; 1940). Rearrange, ment of the oxide would mvr* fnrhlnrn v..ta&ch/dc--fe-forrnation of which, in men frpnsrrl Tn trichla-nr'hvlnm* v-^pno-, hmt h-"n qiinntirnfivnfy_jjtcrmincd.10 Chloral appeared in the blood within 30 min of exposure but subsequently underwent rapid . metabolism. The oxidation of chloral to trichloroacetic acid may be carried out by an enzyme present in the liver of a variety of experimental animals.30 Reduction of chloral to trichloroethanol would involve alcohol dehydrogenase. That trichloroethanol is not.thc-prccursor of trichloroacetic acid follows from thE oble=Tva-TtioTMn - that little trichloroacetic acid (0-3%) informed'when trichloroethanol (300 mg/kg) is admfmsiered orally to rats (unpublished results). The formation of trichloroethylene oxide in vivo is probably due to a non-specific enzyme system. Bartonicek and Teisinger11 have shown that the oxidation of trichloroethylene in man is inhibited by tftraethylthhjmm disulphideJDisuifiramT-This was demonstrated by a considerably increased excretion of trichloroethylene through the lungs. The oxidation is not : however inhibited by SKF-acid (50 mg/kg) since the excretion of~radioactivity and of. metabolites uf uluhloroethylTifein the urine is the same both in control animals. anH in those animals treated wun stiri-acid 60 min before dosing. | The metabolism of tetrachloroethylene may involve the following series of reactions, a ci a o ci \C=c/ -> \cy--\c/ CljC.COOH /\ Cl Cl / -\ Cl Cl The acid chloride would be rapidly hydrolysed to trichloroacetic acid13 and neither trichloroethanol or oxalic acid would be formed. There was no increase in'the urinary excretion of mercapturic acid following the administration of either trichloroethylene or tetrachloroethylene. The absence of any effect of tetrachloroethylene on the liver-lipid content in rats (Table 3) is contrary to that reported in mice1 but is consistent with the low mammalian toxicity of tetrachloroethylene. \i ' & P' J Table 3. The effect of exposure of rats to the vapour of TETRACHLOROETHYLENE (1000 p.p.m.) ON THE LIVER-LIPID CONTENT No. of animals 7 7 7 7 Sex male male female female Scries control test control test mg lipid/100 mg dry wt. liver 11-2 i H 11-3 2-2 10*7 2*2 8-0 i 1-5 AcIcnnwUi/ifcntfnii--The author wishes to thank Dr. A. R. Somerville and Mr. A. F. Henson (I.C.I. Pharmaceutical Division) for cooperation in the early stages of the work and Mr. D. I. Jones for technical assistance. REFERENCES 1, R- J. Defalque, Ctin. Pharmacol. Thcrcp. 2, 665 (1961). 2, S. Laham, Free. Canad. Physiol. Soc. 36 (1957). zb-- B.r. r J7? r-."~ R&S 136077 302 J. W, Daniil BloctKirnoJ PT 2. S. Yllner. Nature, Land. 191, 320 (1961). 4. B. Kylin. H, Reiciiard, I. Sunfgi and S. Yllner, Natttre, Land, 193, 395 (1962). 5. D. D. van Slyke and J. Fot.cn. J. hint. Chem. 136, 509 (1940), 6. W. Taylor and A. H. Ward. A Chem. Sue. 2003 (1934). 7. E. KUlz, PflSg. Arch. ges. Physiol. ZS, 506 (1SS2). 8. J. N. Smith and R. T. Williams, Biachem. J. 56. 613 (1954). 9. J. C. Gage. Brit. J. industr. Med. In press (1963). T 10. E. H. La8ros.se, C. S. Hill and D. R. Parker, Arch, Biachem. Biophys, 46, 432 (1953). 11. T. Nash, Biachem. J. 55. 416 (1953). 12. J. A. Stekol. J. biot. Chem. 113, 279 (1936). 13. T. A. Seto and M. O. Schultzi, J. Amer. Chem. Soc. 78, J6I6 (1956). 14. F. Wild, Characterisation of Organic Compounds p, 152, Cambridge University Press (1947). 15. J. C. Gage, Brit.J, industr. Med. 16. 11 (1959), Department 16. B, Soucex., D. VlachovX, J. Hurych and S. CrKal, Pracovni lekartsvn, 143 (1955). 17. V. O. Paykoc and J. F. Powell, J. Pharmacol. 82, 289 (1945). 18. J. F. Powell, Brit. J. industr. Med. 2, 142 (1945). 19. G. Scansrm, G. F. Rubino and G. Tro.mpeo, Med. d. lauoro 50[ 743 (1959). 20. J. R. Cooper and P. J. Friedman, Biachem. Pharmacol. 1, 76 (1958). 21. V. Bartoni^EX and J. Teisinger, Brit. J. industr\ Med. 19, 216 (1962). i> Z2. E. H. Huntress, Organic Chlorine Compounds. John Wiley & Sons, Inc., 680 (1948). Abstr ' amph tract! ease by w phosj t \ In ADDrnc clotting, v; of other e slices,' wh inhibit vit coronary : ' In view be respor. processes coagulant oral anticc on metab' Such effee view of th test this i< effects we phosphat; depressed creatine p' 1I | Male \V was rapic * This re w I