Document g2OgpkLvKKdO8LRkmER3oz8qe

R&S 026853 ~ i'..* ladenti by 265 surgeons, Phn I,I, n --- < - - VT3 File: K-1711--(100) fHTtii] K-2516-(100) C^ld K-1712K-1716- rwojai K-2520K-2521- ClQ'lU'J Cl K-1717- K-2522- K-2197- K-2525- Cl'iHlO K-2511- K-9387- K-2515- K-9388- Itt. Special Technical Pub!. Nu. |j.y. Thorac. Cardiovasc. Sutg I froe. R. Soc. Med., 63, .11)5, icicn bodies in the lung, Br. / J 9, 1956. Ic implantation of vitallium in lu. C., Eds., American Elscvici lit * ease arising in conjunction I fixation of the tibia, Cancer l'f.,4, 88, 1951. 1 I-opr. Onkol, 9, 95, 1963. I-'.. 95, 579, 1952. I'.Mis by fibres, Lancet, 1, 807, CHEMICAL REACTIVITY, BIOTRANSFORMATION. AND TOXICITY OF POLYCHLORINATED ALIPHATIC COMPOUNDS Authors: . G. Bouse D. Ilcitschlcr Institute of Toxicology University ofWury.burg Wurzburg, Germany Referee: Perry J. C.chring Toxicology Research Labor,ttury Dow Chemical Company Midland, Michigan i of asbestos, Ann, N. Y. Acad. issnii, in Biological Effects o] pr, J. C., Eds., WHO IJtos, IARC Sci. Pub!. No, 8, r.-ncy for Research on Cancer, j"/ Effects ofAsbestos, IARC l.iO International Agency for l.asia JAMA, 204, 106, 1968. l-i asbestos-associated disease Ivski, P., Timbrel!, V., Gilson, I. 1973, 312. dials for Use in Components I institute, National Institutes luella, M. E,, Rapid in vitro lasadena, California, 1971. Inacrylatcs) by the organism, Itfafer-. Res., 6. 173, 1972. Iition of polymers for tissue Inn assessment of lysosomal Itimrm. (Baltimore), 7, 108, J 151, 1973. I.sessment of methods, Food rophage lysosomal enzyme 1. 1973. | us, 1974. tool. Dig, 9, 59, 1970. INTRODUCTION Chlorinated aliphatic compounds are released, in increasing number and quantity, into the environment. They are used on a large-scale basis as industrial solvents, in textile cleaning shops, and as plastic monomers, pesticides, and drugs. Some of the technically important products have long been known as hcpatotoxic agents, and single members of the family (like vinyl chloride and vinylidene chloride) have recently been claimed to be carcinogenic; at present, trichloroethylene is also suspected as a potential carcinogen. In addition, several polychlorinated compounds possess a marked persistence in biological and abiotic systems and may accumulate to healththreatening concentrations. The means and degree of metabolic transforma tion of chlorinated compounds, wliich may vary considerably, are influenced to a great extent by the nature of the chlorine substitution of differ ently hybridized C-atoms. The tendency of the behavior of the compounds under consideration with respect to metabolic and nonmeiabolic reactivity can be particularly well demonstrated with poly- and perchlorosubstitutions. This report describes some aspects of the interrelationship among polychlorinated aliphatics, chlorine substi tution, chemical reactivity, and metabolic fate in biological systems, as well as their toxic properties. The molecular context is emphasized, whereas details of enzyme mechanisms and pathophysio logical aspects of toxic reactions are not dealt with, GENERAL CONSIDERATIONS ON CHLORINE SUBSTITUTION IN ALIPHATIC HYDROCARBONS In general, chlorine substitution provides a decrease in the electron density of the involved C-atoms by their electron-attracting inductive effect (-1 effect) which dominates the mesomeric donator effect. In nuclear magnetic resonance spectroscopy, the frequency of the resonance of an atomic nucleus depends on the electron density of its environment. The diamagnetic protection by the electrons diminishes the effective magnetic field at the nucleus. Thus, the shift of the resonance absorption may serve as a criterion for the electronic influence of substituents.1 Recent publications2"5 reveal the possibility of a linear correlation between 13C-NMR shifts and the total electron density of the corresponding C-atoms. October 1976 395 Viwn """HI 1,1111)1 - - ^ ^-v^CK.v.iT^rr;^ jt'*TR Tji^trrtR'r'f* iF-irj tt^faliM !--3ll m \. .>.. ...---- -- <, j" Thus. the position of the l3C-NMR signals is a measure of the electron-attracting effect of chlorine substitutions in organic molecules. The electronic shifts indicated in Table 1 clearly illustrate that an increase in chlorine substitution induces a decrease in electron density at the substituted C-atom; this correlates with a shift of resonance signals to lower fields. Substitution of hydrogen by chlorine in alkcncs results in a smaller shift of 1 JC-signals than it docs in alkanes. This is probably due to the interaction of the chlorine with the 7r-bond. The inductive effect may be dominant in chlorinated alkanes, but in alkcncs another effect (magnetic anisotropy, resonance effect) may contribute to the shift.* The mesomeric substitution effect encountered with chlorine substituents may also be demon strated separately by microwave and radiowave spectroscopy.9'10 One criterion of the mesomeric effect is the bond extension C-Cl which will be submitted in part to some kind of "double-bond character" and thus to a contraction by the participation of zwittcr ionic structures. Thus, for example, the bond extension C-Cl is 1.76 A with alkanes, 1.69 A with alkcncs (e.g,, vinyl chloride), and 1.63 A with alkyncs (e.g., chloroacctylcnc). The proportion of the double-bond character of the C-C) bond, according to nuclear quadrupole measurements, amounts to 5 to 6% in the cise of vinyl chloride. An enforced electron-attracting effect in connection with stcric factors resulting from poiychloro- or perchlorosubstitution induces decisive changes in reactivity as compared to TABLE 1 1 'C-NMR Shifts of Chlorinated Aliphatic Hydrocarbons (ppm Kcicrnng to CS, = 0 ppm)`"a C, C, c, c, CH, CH,C1 Cll,Cl, ciici, CCl. 194.9 167.7 138.6 115.6 96.8 CM, -CH, Cll, CI-CH, CIICI,-CH, CCl, -CM, CCl, -CCl, CH,=CH, CHC1=CH, CCI,=C11, trans-CItCI--CIICI cis-CHCI=CHCl CCl, "CIICI . cci,=ca, 186.9 152.9 123.6 96.9 87.3 70.0 66.7 64.1 71.7 73.5 67.8 72.1 186.9 174.1 160.3 146.5 87.3 70.0 75.4 77.3 71.7 73.5 75.3 72.1 396 CRC Critical RiT/i-n i in Tnxiciilnyy uMsubstitutcd hydrocarbons. In the scries of alkanes, an increasing chlorine substitution With an elongation of the carbon chain results in an enhanced tendency toward destabilization, which leads to C-C fissions or eliminations of HC1 or chlorine with consequent formation of alkcncs. On the other hand, alkcncs arc stabilized against electrophilic agents by increasing chlorine substitu tion; chlorinated olefines arc characterized by an elevated thermal stability. Alkyncs, unlike alkcncs, are considerably destabilized by chlorine substitu tion. ALKANES ct II --c --c -- II The destabilizing effect in chlorinated alkanes is observed with an increase in the number of chlorine and carbon atoms. The stcric hindrance of accumulated chlorine substituents, as well as their electron-attracting effect, favors C-C fissions (a, structure below! and eliminations of Cla or HCI with the formation of more stable ulcfinic compounds (b, structure below). Cl 1 C" 1 (a) Cl --c c-- \c c/ /\ For example, thermal decomposition of dccachlorobulanc (1) renders, by C--C fission, thetrachlorocthylcnc (2) and hcxachloroethane (3). Under dechlorirtating conditions, hexachlorobutadienc -(4) may easily be obtained from dccachlorobutane (1).' 1 CCl, -CCI, -CCl, -CCl, U) CCI,=CCl, + CCl,-CCl, (2) (3) CCl, =CCI - CC1-CCI, U> C-A Fission Up to now, investigations on metabolic convcr- w iaBhfias'1 R&S 026855 ins. In the series of tine substitution with an n chain results in an a destabilization, which eliminations of MCI or Donation of alkcncs. On are stabilized against tnasing'chlorine substituare characterized by an Alkyncs, unlike alkcncs, id by chlorine substitu- in chlorinated alkanes is :sc in the number of . The steric hindrance of ;utucnts, as well as their favors C--C fissions (a, ^kons of Cl? or HCI ^^^e stable olcfinic clow). / "=C /\ decomposition of de fers, by C--C fission, and hexachloroethane : conditions, hcxaehloro.tiy be obtained front , cci,=cci, +cct,-cct, (2) (3) cct,=cci-cci=cct, (4) ons on metabolic conver- sions of chlorinated aliphatic hydrocarbons have focused on C) and C2 compounds; higher hotnologues of C, and C* units have occasionally been included. Regarding the C( scries (the chlorinated methanes), carbon tetrachloride is converted mclabolically to chloroform and CO?,11 chloroform to CO?,'3 and dichloromcthanc to CO.14 A deeper insight into the mechanism of life hepatotoxicity of carbon tetrachloride was provid ed in 19GI by Butler1 s who suggested a homolytic C-Cl fission with the formation of trichlororncthyl and chlorine radicals. At present, the most probable subsequent pathochcmieal reaction mechanism is the interaction of the trichloromethyl radical with unsaturated fatty acid chains, with the formation of chloroform and a fatty acid radical. The latter reacts with oxygen and forms peroxides and hydroperoxides which, in turn, induce the decomposition of the fatty acid chains.16 At the same time, it was recently demonstrated that the hepatotoxic activity of Cl Ct- \ Cl Cl / C1=C \ Cl Carbon tetrachloride forms an enzymesubstrate complex with cytochrome P45o of liver microsomcs30 in which the following sequence ot reactions takes place.31 CCI, + e,81 . CCI, + c -CCI, + |C:< -ICCI, The complex cytochrome P4so-ICCl,d may then decompose in the presence of protons, releasing chloroform as the stable final product. The work of Heppcl and Porterfield" has well established that enzymes catalyze the oxidative C--Cl fission of chlorinated organic compounds in the organism. According to Van Dyke 23 and Van Dyke and Chcnowcth,34 the enzyme system involved shares all relevant properties with mixed function oxidases. It is inducible by phenobarbital and bcnzo-a-pyrcnc and requires oxygen and NADPll.3* High rates of dechlorination have been found with this enzyme system in the series of chlorinated ethanes; 1,1-dichloroethancs (C MCI ? -Clij), 1 . I ,2-trichlorocthane (CHClj-CM.Cl), and 1,1,2,2-tcirachlorocth.ine (CHClj-CHCli). Ethanes with a trichloromclhyl bromolriehloromcthane (BrCCI,) is considerably greater than that of CCI4 or ClICli.11 There is a correlation betwc'n the cytotoxic effects produc ed in vitro and in vivo and the bond-dissociation energies for the fission of the three methane derivatives (CHC1,, CC14, and BrCCI,); ll-CCI,. 95.7 keal/mol; C1-CC1,, 73 keal/mol; Br-CCI,. 54 keal/mol.1 *'*9 A low dissociation energy corresponds to an increased tendency for a homolytic fission to the trichloromcthyl radical. Correspondingly. BrCCI, is considerably more effective than CCI4 in producing a peroxidaitve breakdown of the microsomal lipids in rat liver.11 In principle, chlorine-substituted alkanes tend to have radical reaction mechanisms because chlorine substituents are capable of delocalizing the unpaired electron via empty d orbitals of low energy. Consequently, the resonance energy of the CCI, radical amounts to 8.3 keal/mol (as compar ed to the CM, radical = 0). Possible resonance structures of the trichloromcthyl radical arc shown below. Cl Cl c/ \ Cl Cl / Cl--c V Cl. (CCI,-) group (for example. 1.1,1-trichloroethane (CCI,--CH,), 1,1,1,2-tctraehloroethane (CClj-CHjCI), and 1.1,1.2,2-pentachloroethane (CCI,-CHClj)] are characterized by a low ten dency towards oxidative dechlorination; the same is true for tetrachloroethylene (CCI;=CC1?). Accordingly, acute and chronic hepatotoxicity arc high in the former and low in the latter group of chlorinated aliphatics.36"3 8 Elimination of Chlorine The elimination of chlorine with formation of more stable alkcncs represents the second type of metabolic conversion of chlorinated alkanes. This is the main pathway and the first step in the metabolic conversion of pentachlorocthane (5124 and hexachloroethane (6 ).J 0 leading to the formation of trichloroethylene (7) or tetrachloroethylenc (2), respectively. CHCI,-CCI, (5) CI1CI=CC1, (7) cct,-cct, ~t:1' cci.^cci, (*>) (2) October 1976 397 'TPJ'-t..". t YJm. r,MIT^ I However, an analogous mechanism with which is then conjugated alter aromatic hydroxyl- 1.1.2.2-tetrachloroeihane ^8) which would yield alion.3 s 1.2- dichlorocthylene (9) could not be demon strated up to now. The predominant pathway in the metabolism of 1.1.2.2-tctrachlorocthanc (8) goes through the sequence dichluroaccialdchydcdichloroacetic acid (10-i I). while the product of noncnzyniatie dchydrocholorination (b) is found only in minor proportion; this reaction is already found in neutral phosphate buffer.31 In addition, traces of oxidatively formed tctraehloroethylene (2) arc found. ALKENES CHCI=CIIC1 M*> Cl cct.-cct, (D CHC1, -CHCI, t -* ictici, -ciioi ,00) T CHC1=CC1, id C1ICI, -COOH 1 (ID CO, Elimination of MCI The elimination of hydrogen chloride from polychlorinated alkanes, leading to olefinic structures, also represents a major pathway in the metabolism of p.p'-dichlorodiphenyltrichloroethanc (DDT) (12J. with p,/)'-dichlorodiphenyldichlorocthylenc (DDE) (13) as the first metabo lite,3 2 This conversion is additionally catalyzed by the enzyme "DDT-dchydrochlorinase" (E.C. 4.5. 1.1,) in the presence of glutathione.33 Cl I -- CCI, -net c=cct, (ID (ID Dehydmchloriuations arc also discussed as primary metabolic pathways of polychlorinated cyclic alkanes, lot example as in the case of 1 .2,3,4.5 ,(l-hcxachloroeyelohcxane.:,`, '3 s The 7-isomer nl (',,11,,Cl,, (14) is converted, by successive dchydioehloriuation. via a 7-pcnticlilorocycloliexene to 1,2,4-trichlorubcn/cne (15), ()ilh til AYWrwv m To.xivuluxy Considerations on Stability Poly- and pcrchlorosubstitution at sp2-hybrid ized C-atoms in unsaturated systems increases the thermal and chemical stability of these molecules. The -1 effect dominates the +M effect of the chlorine substituents, thus resulting in a "depriva tion" of the electron density of the double-bond system. This provides, in combination with a steric protective effect of the bulky chlorine substitu ents, an increased stability against electrophilic attack. A good example of tins effect is the reaction of chlorinated ctliylcncs with ozone. The relative rates of ozonization in the scries ethylcnc:vinylchlondc; ttichlorocthy lone: let raehloroethylcnc arc 2S00:1180:3.6; l.36 CH, =CH-CH~CH, CCl, ^CCI-CCl^CCl, (16) (17) Liquid butadiene (16) (bp760 *5.6C) poly merizes when 'eft open to air for months after some period of induction; light irradiation acceler ates the reaction. On the other hand, hexachlorobutadicnc (17) (bp76o 2!3C) is stable up to 500C and cannot be brought to polymerization by pressure up to 100 atm (17), in addition, is characterized by a remarkable chemical stability, similar to the' higher homologous stcreoisomeric percitlorohexairiciies.3 7 They arc fairly stable against strong mineral acids and aqueous alkali even at high temperatures, but arc converted to highly chlorinated vinyl ethers by alcoholic alkali at 80"C.3" At least the action of fuming nitric acid at 130" and additional treatment with concentrated sulfuric acid at 170 are necessary to convert hexaclilorobuc maleic acid anhydride.3 * 11 H Cyclooctotetrcne representative of cyci^ naiing double bonds, oxygen. After an exter heating, it converts to resinous substances, the molecule is again addition of halogens.] (0:, KMnC>4, and Cr( with ring contradict chlorocylooctatctrene. octaictrcne (20) cot thermal and chemica the pressure tube for cyclooctatctrene (19)i With elemental bromn the composition CgHj octatctrcne (20) doe aqueous KMnO* under varying conditJ stable up to 180C.l remarkable inertness be determined in thej latcd voluminous cl render an electrophilic their -1 effect. The va of an extraordinary systems by poly- and be further demonstruf (Table 2). Metabolism Chlorinated et converted to prcdoml A significant interrej st rated between the j with increasing nun and the metabolic with all chlorinated Cfl 026857 .iter aromatic hydroxyl- ci ct (IS) ES n Jtution at sp2-hybrid systems increases the tv of these molecules, ne +M effect of the esuliing in a "deprivav of the double-bond ination with a steric y chlorine substituagainst electrophilic ttect is the reaction of ozone. The relative series ethylene:vinyi- trachloroethylcnc arc CC1, =CCt-CCi=CCI, (17) op-j6o ~5.6C) polyatr for months after uit irradiation accclerrer hand, hexachloro'C) is stable up to iti to polymerization (17), in addition, is :e chemical stability, ogous stcreoisomcrie v arc fairly stable and aqueous alkali >ul arc converted to :s by alcoholic alkali on of fuming nitric -al treatment with 70 are necessary to convert hexaclilorobutadieiie (17) to dichloro* maleic acid anhydride.'9 II II ci II ci ci it n nti a ii im ci ci iro Cyclooctotctrcnc (18) (bp7to 141*0. a representative of cyclic polyolefines with alter nating double bonds, is sensitive against light and oxygen. After an extended stay, or acceleration by heating, it converts to a mixture of dimers and resinous substances. The enormous reactivity ol the molecule is again demonstrated by the rap id addition of halogens.40'''1 Attack of oxidan's (02, KMn04, and CrOj) easily results in pmducii with ring contraction. The transition to penta chlorocylooctatctrcnc (19) and octachlorocyclooctatctrene (20) coincides with an increase in thermal and chemical stability. After heating in the pressure tube for 6 hr at 250C, pentachlorocyciooctatctrcnc ( 19) can be isolated unchanged. With elemental bromine it reacts to a product of the composition Cs 113CI<,lir>.4 2 Oetachlorucyclooctatctrenc (20) does not icact with ozone or aqueous KMn04 and cannot be brominated under varying conditions. It remains thermically stable up to 180C.4'! ,44 The reason for this remarkable inertness of the double bonds should be determined in the protection by the accumu lated voluminous chlorine atoms which, per sc. render an electrophilic attack more dillicult due to their -1 effect. The validity of the general principle of an extraordinary stabilization of olcfinic systems by poly- and pcrchlorosubstitution may be further demonstrated by some paired examples (Table 2). Metabolism Chlorinated cthylenes are metabolically converted to predominantly C: alcohols and acids. A significant interrelationship has been demon strated between the enhanced stability associated with increasing numbers ol chlorine substitutions and the metabolic behavior in biologic systems with all chlorinated cthylenes in die isolated perfused rat liver pieparation.'1''2 At a giver prehcpatic concentration d the cthylenes, a significant rise in the proportion mclaholi/cd was found in the series tetra-, tri-, and cis-1,2-dichloio- cthylcnc vinylidcnc chloride. The metabolic changes ol chlorinated cthylenes arc initiated with the oxidation to corresponding oxiranes5J'it' by monooxygenases. Up (o now this class of compounds has only found limited interest. The principal reactions of oxiranes m biological systems are summarized in figure I. The main toxic effects, acute as well as chronic, have been associated with electrophilic reactions with essential cellular components (alkylation), whereas the other pathways (reduction, hydrolysis, and conjugation, both enzymatically and nonen/.ymatically) and rearrangements of earbonyhc com pounds are considered to be detoxication mechanisms. The type of rearrangement in chlori nated cthylenes depends on the number and position of chlorine substitutionts); if goes to either chlorinated aldehydes or acyl chlorides. R \ c=c / /\ Cl R.-H.CJ VA / > c--c /\ Cl -- cI -- Cl o Cl R further metabolic steps with the rearrangement products are oxidations or reductions of aldehydes to carboxylic acids of ethanol derivatives or hydrolyses of acyl chlorides to corresponding adds. With (eirachloroetltyh'iic (2). the metabolic formation of trichloroacetic acid (23) can plausibly be explained by the primary formation of an oxirane (21) and subsequent rearrangement to trichloroacctyl chloride (22) and its hydroly sis.5; 5 3 The transition of the oxirane to trichloiuacetyl chloride (21 to 22) is, in analogy, found with the (eirachlomcthylcue oxide (2) which can be synthesized by photooxidation and which rearranges in vitro in different solvents to trichloroacctyl chloride.5 7 October 1976 399 R&S 026859 / CC1, -- c \ OH (23) olefines ndency to italic cont-8) 1 WktfMt i TAULE 2 (continued) Comparison of (he Stability of Unsubslitutcd and Pcrchlorosubslnutcd Cyclic Polyolefines HH Cl Cl (49) HH Hcptafulvcnc; polymerization in solution already at -80C (SO) reaction with cellular macromolecules (alkylation) conjugation (glutathione) / oxidation C \ reduction /\ rearrangement ,0 --c --c s hydrolysis OH OH iI --c-- c -- FIGURE 1. Synopsis of metabolic conversions and biological consequences of tovication (alkylation) and detoxication (conjugations, hydrolysis, and rearrangement) of jlkcncs. Trichloroethylene (7) is mctabolically convert ed to trichloroethylene oxide (24) and further converted to chloral.5 3"s s ,s s,s 9 [n subsequent metabolic reactions, chloral (25) is in part reduced to trichlorocthanol (26) or oxidized to trichloro acetic acid (23).60 The real occurrence of the oxiranc (24) as a metabolic intermediate could be substantiated by spectroscopic investigations with the catalytic hemoprotcin Paso of the converting enzyme monooxygenasc.6 1 After administration of 36CI-labcled trichloro ethylene to experimental animals, the specific activity remains unchanged in the recovered metabolites tricbiorocthanol and tridiloroacetic acid; no exchange of 36 Cl with the chlorine pool of the organism is observed.53 Again, this is indicative of an intramolecular chlorine migration in the course of the transition of trichloroethylene oxide to chloral (24 10 2S). Surprisingly, at first glance, the trichloroethylene oxide (24) which can be synthesized from trichloroethylene by photo chemical oxidation63 thermally rearranges (in vitro) mainly to dichloroacctyl chloride (27).6 3 In vivo, however, no dichloroacetic acid is found as a metabolite.52,64 This remarkable difference has important practical implications. In principle, there are three possible ways of rearranging the oxiranc of trichloroethylene (24). n |iiWPH|lm'Hamtt &>* October 1976 401 zircmttwrr&zrM 0 ^/'` (i) Cl CI1CI,-- c \ Cl n /o f 11' \IU\\/ J C-- C "' / Cl V Cl Cl ^ H \r\/ C--C -- s\ iW Cl Cl ' (3) CClj-- c \ Regarding the two rearrangements leading to dichJoroacetvichJoride, the hydride shift (2) has low probability, according to investigations by McDonald and Schwab.65 If one postulates that a-ketocarbonium ions or ion pairs66*68 are transi tion forms of the rearrangement (a synchronous process cannot, however, positively be ruled '-ut6,)1 the carbonium ion (which after C-0 hetcrolysis is solely destabilised by one direct, liganded Cl atom) should be indicative of a favored rearrangement. Way (3) would involve a carbonium ion with two direct liganded Cl atoms, consequently giving a lower degree of probability. Experiments on the behavior of the oxirane are in accordance with this assumption; rearrangement to dichloroacctyl chloride is accelerated by tertiary amines10 which, after addition of methanol, render dichloroacctic acid methyl ester.71 Upon heating to 100 to I40C for 4 hr in a glass tube, the oxirane is converted in high yields (88%) into dichioroacctylchloride, and only a small propor tion of chloral can be detected.63 The rearrangement of trichloroethylene oxirane to chloral in vitro can only be elicited by Lewis acids such as AJCh or FeClj,53 Thus, it has been suggest ed liiat in the living organism the ``environment"at the site of formation of the oxirane (24) might poss ess electron acceptor properties and induce the re arrangement in the direction of chloral.61 '6 3 The most plausible mechanism for the influence of Lewis acid C -- CCI, / Lewis acids is an interaction with the oxirane (24) at the sue where there is a steric opening of the molecule with the oxirane oxygen, with the nearest chlorine atom, or with both, inducing the final rearrangement according to shift (3) to chloral (25). Cl (28} (29) Crs- and trans-I. 29) both form thej (32) and dichloroace identified in perfu preparation.61 ,S1 `lined by the pr (33) The isomeric I,i chloride) (33) is m/ acid (36).64 '13 If 11 with wt-chloroperbe^ chloride (35) (the eij of the oxirane) ca seems to be extred 402 CMC Cmit a! Ki vii ws in Toxico/ogy (40) .Doth, inducing the U *o shift (3) to and 31) and subsequent rearrangement to didiloroacctaldeliydc (10), which is then oxidized to dicliloroacetic acid (I 1) or reduced to dichloroethanol (32). The thermal rearrangement of the oxirancs that occurs with chlorine migration to dichloroacctaldehydc (10) has been demonstrated.46 CH.CI CH,C1 The isomeric I,1 -dichloroeihytenc (vinylidenc chloride) (33) is metabolized to monochloroacctic acid (36).4,1'72 If 1,1 -dichloroethylene is oxidized with rw-chloroperbcnzoic acid, only chloroacetyl chloride (35) (the expected rearrangement product of the oxirane) can be isolated. The oxtrane itself seems to be extremely unstable and resistive to /Cl synthesis under varying conditions.13 However, from these findings it can be speculated that the oxirane (34) is formed as a metabolic intermediate and rearranges spontaneously to chloroacetyl chloride (35), which is subse quently hvdrolyzcd lo monoehloroaeeiie acid (36). \(37) C1 ,0. H (38) / CH,C1 -- C: \ (39) / CH,C1 -- C \ OH (36) R&S 026861 JS&3i Monochloroacetic acid (.16) is likewise formed as a metabolite of inonticlilorovihylcnc (vinyl chloride) (37).14'16 One possible explanation of the formation of monochloroacctic acid (36) is the primary oxidation of monochlorocthylcnc (37) to vinyl chloride oxirane (38), which rearranges to chloroacetaldehydc14 (33) followed by further metabolic oxidation to chloroacelic acid (36). In analogy, monochloroacetaldchyde (39) is formed as a rearrangement product of the oxirane of vinyl chloride, which can be synthesized by chlorination , of ethylene oxide (40) in the gaseous phase.17,18 The further metabolic fate of vinyl chloride oxirane has been elucidated in part. A very small proportion of metabolized vinyl chloride is bound covalently to tissue components, as has been shown in vitro1* and in vivo.80 Major metabolites in vivo are 2-hydroxycthylcysteine81 (41), 2-hydroxycihyl-N-acctylcy$icinc81 (42), car- boxymelhylcystcinc8 5 (43), and thiodiacetic acid81,82 (44). The mechanism of the formation of these conjugates can easily be explained by the previous rearrangement of the oxirane to chloroacetaldehyde (39) and oxidation to chloroacetic acid (36) which reacts with glutathione; carboxymethylglutathione is converted to carboxymcthylcysteine (43), and the latter, by further oxi dations, is converted to thiodiacetic acid (44). Yllncr has identified carboxymethylcysteine (43) and thiodiacetic acid (44) as the main meta bolites81 in the urine of mice after dosing with chloroacelic acid. The formation of S-(2-chloroethylj-cysteine and its N-acetyl conjugate76 as products of simple addition reactions has been questioned;81 m fact, this hypothesis is incon sistent with an enzymatic mechanism postulated according to the results of experiments in vivo with enzyme inhibitors.14'80 The formation of a cyclic vinyl chloride peroxide, in equilibrium with an oxirane-singlet oxygen complex, might account for the observed formation of formaldehyde as a decomposition product and oxidation to carbon dioxide, which has been identified in part, in vivo, alter metabolic incorporation into urea or methionine and serine.16 Another interesting hypothesis is the addition of vinyl chloride to hydrogen sulfide under formation of bis-(2-chlorocthyl)-sulfide. which then might account for the formation of thiodiacetic acid16 (44); the alky lating intermediate could be suspected as one of the carcinogenic metabolites oT vinyl chloride. However, further experiments, particularly in iso- latcd biological systems, arc necessary to confirm or reject these assumptions. NH, /0 CH, OH -- CH, -- S -- CH, -- CH -- C (41) H / NH --C CH,OH-- CH, -- S-- CM, --CH -- C \ OH (42) O NH, q \ C-- CH, -- s-- CH, i --CH -- y C /\ HO (43) OH \ CC -- CH, -- S -- CH, / HO (44) / \ OH Mutagenicity and Carcinogenicity Three chlorinated ethylenes exert mutagenic effects: vinyl chloride,84'86 vinylidene chloride,86,81 and trichloroethylene86 (sec Figure 2), The common molecular feature of these compounds is the formation of unsymmetric oxiranes,86,6S the stability of which (as tested in non polar solvents) is far less61,71,71 than that of the others which form symmetric oxiranes61'66 and are not mutagenic. Oxiranes may react in vivo enzymatically or noncuzymalically with SHcompounds to form nonactive conjugates.89 On the other hand, the highly electrophilic oxiranes may react directly with nucleophilic constituents of the animal cell as a first step in a gcnctoxic effect. If this mechanism is taken to be essential for the mutagenic effects (as has been elucidated in the case of vinyl chloride),86 the interre lationship between sytnmctry/asymmclry and stability/instability of the oxiranes might offer an important rule88 and possibly a useful criterion which may be used to measure chemical reactivity and predict biologic activity (sec Figure 3). Vinyl chloride has been demonstrated to be carcinogenic 404 ; Cmifdl Kerwws in Tuximbwy Cl Cl 200 FIGURE the compoc activating i different ed methyltryp- / / FIGURE 2, Mutagenicity of chlorinated ethylene* in an in vitro test system. Incubation of the compounds tn concentrations (aqueous phase) from 0.9 to 10.9 inM in a metabolic activating microsomal system. Results as percent of spontaneous (s 1007?) mutation rate m different operons of the test germ t\ colt K,a: gal*, galactose; arg*. arginine; MTR*, mcthyltryptophane; nad*, NAD.14 tetrachloroethylene cis-1,2* dichloroethylene trichloro* ethylene 0 Q H \/\/ 1,1-dichlorO' ethylene trans`1,2' dichloroethylene vinyl chloride iymmetric reL stable not mutagenic asymmetric unstable mutagenic FIGURE 3. Molecular features of oxidative metabolic intermediates (oxiranes) of chlorinated cthylcncs in relation to their mutagenic potential in vitro (see Figure 2), October 1976 405 ii ' 4i, m' l in humans'10 and experimental animals.91 Iji high oral doses, trichloroethylene produces malig nancies in mice.91 Vinylidenc chloride also has been reported as an animal carcinogen.9 3 ALKYNES -C^C-Cl In general, chlorine substitution of sphybridi/.cd C-atoms in allenes and acetylenes results in a destabilization of the molecules. This has been demonstrated with pcrchloroallcne (45), which is stable only below -50C and starts dimerization from -30C upward.94'95 Similar behavior is encountered with tribromoallcne, pcrbromoallcne, and trichloroallencs with electronegative sub stituents.96'99 Cl xct c=c=c Cl''' Xci Cl-C=C-CI C1,C=CC1-C~CC1 (4S) (46) (47) Dichloroacctylenc (46) is also extremely reactive (explosive) and decomposes immediately, in the presence of air, to phosgene and carbon monoxide.1 00 The destabilizing effect of chlorine substitutions at sp-hybridi/ed bonds has also been demonstrated (though in a less intensity) with pcrchlorobutenync101 (47); this thermally un stable compound forms (slowly above 25C) a dimer, CC1. The metabolic fate of chlorinated acetyl enes and allenes has not yet been investi gated. 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