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| CH3 CH2--CM-CH2 EUGBNOL Synonyms 4 allyl-2-methoxyphenol 4 allylgualacol 2 methoxy-4-prop-2-anyl phenol 1-hydroxy-2-methoxy-4-allylbenzene k Physical propertiesz colourless liquid strong odour of clove, pungent taste Melting point -9.1oc Boiling point 127Oc OccurEence: Main constituent o£ several important essential oils such as 0tl of clove, clove stem and leaf, pimenta berEy and leaf, bay and cinnamon leaf, cinnamon bark, oananga, calamus and ylang ylang Use : FlavouEs, fragrance, disinfectant, local anodyne in dentistry. ACUTBTOXZCITY The acute oral dose required to kill 18:- 2.68 g/kg rat 3.00 g/kg mouse 2.13 g/kg guinea pig (Hagan et al, 1965). 50t of t:eated animals Oral doses o~ 150 mg eugenol to rats and guinea pigs produced histologLcaldamage consisting of desquaaatation of the epithelium and punctate haemor,hages in the pyloric and glandular regions of the stomach (Hartiala et a....__~l, 1966). Degenerative and reparative changes were observed in the gastric mucous ha=clef after application of a 5t eugenol emulsion to the mu~osa ..- O o£ Heidenhain's pouches in dogs (Hollander and Goldfischer • __. 1949; discussed by Opdyke, 1975). When the dose o£ eugenol r~J qiven orally to cats was gradually increased to 4.00 g/kg - ~O

-2- there was considerable mortality. Histological examination of the forestomach revealed moderately severe hyperplasia and hyperke[atosis of the strat£fied focal ulceration (Hagan et al, 196S; Part of the effects In the stomach squamous epithelium with 19671 see Opdyke, 1975). may bave been due to the irritant action of eugenol or Impurities found in commercial prepauations (Webb and Busselle 1981}. Rata treated with 4.00 g eugenol/kg (Bagan et al, 1967; see Opdykee 1975) exhibited a small degree of osteopoEosise there was also enlargement of the liver~ liver cell size and the adrenal glands. Four daily doses of 900 mg eugenol/kg to rats produced liver damage consisting of dtscolocatlone mottling and blunting of the lobe edges (Taylor et al, 1964! see Opdyke, 1975). Bugenol thus has some bepatotoxlc activity. eugenol had no adverse effects on growth ratee organ weight, histology o£ any of the major tissues o~ haeat, ology in rats fed it eugenol in the diet (Bagan et al, 1967; see Opdyke, 1975). P~COLOG IC~ efFECTS Cardiovascular System. Bugenol affects the peripheral aspects of the ca~diovascula~ system. The heart is not the principal site of action since eugenol has little effect on the electrical activity and only slightly ~educes the contractile force unless a near fatal dose is used (Sticht and Smithe 1971). The intravenous injection of eugenol (up to 0.5 ml) to mongrel dogs caused a drop in arterial blood pressure. Increased blood flow observed after int~a- arte£ial and intravenous injections suggests that the blood vessels are the ,~n site of action withfn the cardiovascular system. Large doses of eu~enol (0.033-0.06 ml/kg) may damaga t~4

-3- capillaEy aenbcanes as suggested by the release of a bloody ezudate from "the respIEatory tract. Stiffening of the limbs J has been observed after large lntca-a=tecial doses of eugenol to dogs (Sticht and Smith, 1971). Nervous System All concentrations between 0.1t and 100t eugenol blocks the transmission of evoked impulses in frog sciatic nerve tissue (Zosama 1977). Bugenol affects the central ne=vous system. Tt iS an anaesthetic In mice and dogs st doses In excess of 300 mg/kg (Sticht and Smlthe 19?lp Dallamter and Calinl, 1981). Larger doses lengthen the sleeping time (Dallmeler and Carlini, 1981). Barbiturate sleeping time is increased by eugenol (see Sell and Carlini, 1975). vomitting as long as two hours after the oral administration of eugenol to dogs (Lauber et alw 1950e see Sttoht and Smithe 1971) may have been the result of local irritation in the stomach or an effect on the vomltttng centre in the brain. Activity and Behaviour Spontaneous motor activity was not affected in mice at a dose of 100 ~/eugenol/kg (de Mello et el, 1973). Eugenol has a myo:elaxant activity in mice at doses exceeding 50 mg/kg (Dallmeter and Carlini, 1981). Rope climbing time was depressed by doses in excess of 160 mg/kg in rats. Some of these animals also showed paralysis of hindquarters which also impaired climbing pe=foE.mance (de Hello et a_.___~l, 1973). Motor co-oEdination Is lost in dogs treated with 0.033-0.06 ml/kg (Sticht and Smiths 1971). In mice eugenol (200 mg/kg) protects against qbo.nvulsion and has a catatonic e~fect althou.gh the latter response may have been O mamdb O%

--4-- non-specific because the animals were very depressed (de Mello et al, 1973; Dallmelec and Carlini# 1981). • . Other aspects eugeno$ reduces body temperature in rats at doses exceeding 50 ng/kg (Dallmeter and Carltn£, 1981)..~n increase in salivary flow was observed in dogs injected wth 0.033-0.06 ml eugenol/kg (Sticht and Smith, 1971). From quantum chemical calculations, the ability of halluc- inogenic amines to donate electrons has been related to their activity. No such relat£0nship has been seen with eugenol or its analogues (discussed by de Mello et a_.__~le 1973). THE MUT&GENICITY AND CARCINOOBNICITY OF EUGF£qOL The potential mutagenicity and carc£nogenicity of both eugenol and its metabol£tes are of particular lnte~e8t and importance to the tobacco industry following studies on a chemically similar compound called 8afEole. Saf~ole is not mutagenic in the Ames Test using a wide range of strains of S. typhimu£1um in the absence of metabolic activation (Dorange et a.__~le 1977; Swanson et ale 1979). Some mutagentc activity was observed. £n the presence of metabolic activation (Green and Savage, 1978). There is good evidence that safrole is a weak hepatocarctnogen (Miller et al, 1979). In contrast eugenol i8 non-mutagenic in several strains of S. typhimurium with or without the presence oF metabolic activation (Dorange et al 1977; Swanson et a__.~lw 1979). To date there has been no evidence to demonstrate a significant carcinogenic effect of eugenol in any species. Eugenol may have some activity to p~omote skin tumou=s £n mice p[etcea~ed with the inihia~o~ 7,12-dimethylbenz (a) anthracene CD

-5- (Van Duu~en et al 19G61'see Opdyke 1975). The co-administration # of eugenol and henzo(a)pyrene to the skin pf mice partially inhibited benso(a}pyrene carcinogenicity (Van DuuEen and Goldschm£dt, 1976). This may be due to the metabolism of eugenol by, and thus reducing the availability of enzymes required fort the activation of benzo(a|pyrene to the active carcinogen. In a limited study in mice eugenol did not potentiate the tumorogsnic effects of a known carcinogen ~-methylchol- anthEene (Hitchcock, 1952~ see Ol~yke, 1975). THB METABOLISM OF EUGENOL Eugenol and other related allyl benzenes can be metabolised in the mathoxy group or the allyl side chain The methylene dioxy group of safrole can be cleaved with loss of the carbon atom. The ma~or metabolite is allylcatechol or its isomer propen¥1catechol. Eugenol is a secondary metabolite (Ioannides et a__.~l, 1981). Demethylation of eugenol can also occur in rats (Weinberg et a____~l, 1972) p~obably also resulting in the formation of allyloatechol. Little is known about the mutagenic or carcinogenic potential of these compounds. The~e are some similarities between the metabolism of the allyl side chains of eugenol and safrole. Both compounds can be converted to the 'respective 2',3e-epoxide and dihydrod£ol (see Figure 1) by rat liver cells and both rat and mouse liver microsomas and are found in the urine and liver homogenates of rats pretceated with eugenol oc saf~ole (Delaforge etal, 1980). The epoxides of both compounds are mutagenic in the Ames Test (Swanson et al, 1979). The 2',3e~epoxides react non-enzymically with guanosine at neutral pH (Swanson et a.__~l, O O~

-6- 1981). Guanosine Is a tlboslde found in DN&, the genetic material of the cell, and leNA. The 2' ' ,3 -epozIde of safcole but not eugenol had DNA repair Inducing act£vity. DN& repair inducing activity is greatest following the addition to the tn._n_ vitro assay system of a di=ect acting carcinogen. However, a negative result does not Ran that the compound Is not carcin- ogenic oc mutagenic (Francis et alw 1981). Fornatton of 2',3'-epoxy euganol is greater in male than female rats. Felale mice exhibited a higher rate of formation of el)oxide than stole mice. The rate of epozidatlon of eugenol In both species was much slower than that of estcagole oc safcole (Swanson et el, 1981). The metabolism of estragole and | aafcole to the 2',3'-epoxtdes and 1 hydroxides depends on cytochroma P450 and the presence of N&DPB (Swanson et a__~l, 1981). This has not been determined for eugenol. The 2t,3'-epoxldes of eugene1, estcegole and safrole were all very susceptible to the action of epoxlde hydrase in cat and house liver aicrosomes resulting in the formation o£. d£hydcodtols. The reactions could be Inh£bLted by the presence of a cytochcome P450 inhibitor tcichloropropylene oxide (Swanson et el, 1981). 8afrole and estcagole can be netabolized to a lt-hydcozy metabolite, lt-Hydcoxysafrole forms ¢ovalently bound adduces with hepatic DNA, ~RN& and protein in wive in the rat and mouse. The l'hydroxy metabolites ace weakly mutagentc, cacc£nogenict can be metabolized to the l'-hydroxy,2e,3'-epoxides which ace also weakly autagentc and carcinogenic (Hiller et a__._~l, 1979). The l'-hydrox~,2'r3'-epoxides are more resistant to the action of epoxide hydrase than the simple 2',3'-epoxides. The hydroxyl O O

-7- viclnal to the epozlde ring confers :esiEtance of the epoxlde to hydrolysis by alc:osomtl epoztde hydrase, reduces the activity as direct acting autagens, but retains or enhances the activity ot the conpounds to tnLttate paplllona fo:zatton in meuse skin ('Swanson et al, 1981). Bsterificatton of lehydrozy-safrole and estragola by :at and house liver cytosol yields electrophtltc esters which can form hepatic DNA adducts (Swanson et al, 1981) and could be carcinogenic. Comparable metabolism of eugenol has not been established but could possibly occur. Any metabolttes so formed might reflect the mutagenic and carcin- ogenic activities of the safrole and est:agole analogues. On the other hand, an absence of metabolism of eugenol at the l' position of the allyl side chain might be associated with its lack of carclnogenlcity In rats and mice. After a single o:al administration of 200 aKj of eugenol to rats# a large amount of ether-type glucu=onide was found in the uEine up to 24 hou=~ late=. In contrast there was little change in urfna=y content of the ester type glucu,onide. Eugenol increased the specific activity of UDP glucu:onyltransferase (the enzyme which catalyses the conjugation of a hydroxyl emtabolite of a drug with glucu~onic acid). The time course of induction was simila, to that of cytochcone P450 oE any other drug inducible metabolizing enzymes in liver nic=osomes (¥uasa, 1974). Thus after t=eataent with eugenol the body inc:eases its ability to metabolize the compound to a pola~ conjugate which can be mole readily, excreted. THE EFFECT OF EUGENOL ON NETAROLISN Eugenol inhibited mouse llver nlc=osomal hydroxylation CD CD C~ ~O

-8- of dinmt:hylgminopyrene and hexobscbital" (Jaffa etal, 2968). Thin uy have bean due to the preferential° use of available enzysm8 to metabolize augenol. ltesplration of isolated mitocbond~la pcepaced f~om rat llve~8 18 inhibited by low concentration8 of eugenol. High concentrations of eugenol uncoupled oxidation and pbospbo~y- lation (Cot~oce et al, 1979). Tbesm actions nay contcibute to the hepat~toxicity of eugenol. No=adcenaline induced oxidative Mtabolima in hamsters isolated bcown fat cells i8 also inhibited by eugenol (Pette~8on et alw 1980). There have been no studies investigating the metabolisn o£ eugenol administered by inhalation. Similarly the~e ace no studies on the effects of inhaled eugenol on metabolism. r~ 0~ ~0 CO

FIGURE I -9- IHE ~ OF BF.~KIL ~ SAFam~ 0--- ester sa£role 0 (7", ~D