National Toxicology Program

CAS NO. 607-91-0

EVIDENCE FOR POSSIBLE CARCINOGENIC ACTIVITY

Human Data:

Acute Toxicity.Ingestion of five or more gramsof nutmeg causes acute nutmeg poisoning, which includes giddiness,hallucinations, and feelings of depersonalization. Symptoms usuallyappear three to six hours after ingestion of 1-3 whole nutmegsor 5-15 gm of the grated spice. Recovery usually occurs within24 hours. Nevertheless, duration of action may extend beyond severaldays and even include death (Painter et al., 1971; Green,1959).

Subacute/Subchronic Toxicity.A central problem in the pharmacologyof nutmeg was identification of the active compound (Truitt, 1979).This is now believed to be myristicin, but the myristicin fractionof nutmeg oil lacks adequate potency to explain nutmeg intoxicationcompletely.

Interest in the pharmacologic action of myristicinand nutmeg grew in the late 1960s and 1970s when nutmeg becamea drug of abuse. Recognition that myristicin has a structure resemblingmescaline and hallucinogenic amphetamines prompted investigationssuch as the behavioral studies described in the animal studiessection.

Chronic Studies.No epidemiological studies or casereports investigating the association of exposure to myristicinand cancer risk in humans were identified in the available literature.

Animal Data:

Acute.Acutely toxic doses of myristicincan cause organ damage. Cats were especially sensitive to myristicin,exhibiting central excitation followed by coma. Oral administrationof myristicin at 400 mg/kg to cats caused fatty degeneration ofthe liver. Rabbits and guinea pigs administered a subcutaneous(sc) dose of myristicin developed brain and liver lesions andmetabolic derangement (NLM, 1997).

Acute toxicity data reported for myristicinare summarized below (NLM, 1997).

rat oral LD₅₀ = 4260 mg/kg

cat oral LD_Lo = 400 mg/kg

rabbit sc LD_Lo = 900 mg/kg

guinea pig LD_Lo = 2 gm/kg

Subacute/Subchronic Studies.Ozaki and coworkers (1989) fractionatedthe ingredients in mace and administered them orally to rats witha carrageenin-induced edema. One fraction produced a lasting anti-inflammatoryactivity. It contained myristicin, suggesting the analgesic ingredientin mace is myristicin.

Myristicin and elemicin ^a are structurally similarto the hallucinogen, mescaline (Buchanan, 1978). All three compoundsimpaired the rope-climbing and bar-pressing performance of rats(Cesario de Mello & Carlini, 1973).

Several tests have shown myristicin to bea weak monamine oxidase (MAO) inhibitor (Truitt, 1979). Syntheticmyristicin showed considerable activity when samples were fresh.The distilled concentrate of oil of nutmeg was much less activethan synthetic myristicin.

Myristicin can alter the toxicity of othercompounds. For example, myristicin caused a change in the pathwayof parathion degradation when fed to rats (Marcus & Lichtenstein,1982). Myristicin also completely prevented carbon tetrachloride-inducedhepatotoxicity in mice (Zhao & O'Brien, 1996).

Chronic/Carcinogenicity Studies.No 2-year carcinogenicity studiesof myristicin in animals were identified in the available literature.

Myristicin was one of 23 naturally occurringand synthetic alkenylbenzene derivatives assayed for hepatocarcinogenicityin mice (Miller et al., 1983). Specifically, myristicinwas administered intraperitoneally (ip) to male B6C3F₁mice 24 hours after birth and at days 8, 15, and 22 for a totaldose of 4.75 µmol. At 13 months, 10% of the mice had hepatomas;at 18 months (end of experiment), 31% bore hepatomas (0.4 tumors/mouse)but the findings were not statistically significant compared withvehicle or untreated controls.

Myristicin was tested as an inhibitor of benzo[a]pyrene(B[a]P)-induced tumors in female A/J strain mice (Zheng etal., 1992). B[a]P was given orally twice a week for four weeksat 1 mg per dose. Myristicin was given orally, 10 mg per dose,three times a week on days other than the B[a]P treatment andfor one week before the first administration of B[a]P. Eighteenweeks after the first dose of B[a]P, the mice were killed forexamination of lung and forestomach tumors. Myristicin significantlyinhibited lung tumor multiplicity; the mean number of tumors intumor-bearing animals was reduced by 65%. Myristicin slightlyinhibited forestomach tumor multiplicity; the mean number of tumorsin tumor-bearing animals was reduced by 31%. ^aElemicin is present in the same nutmeg fraction as myristicin and probably accountsfor some of the activity observed with nutmeg (Braun & Kalbhen, 1973).

Short-Term Tests:Few studies of the potential mutagenic effects of myristicin wereidentified in the available literature, perhaps because myristicinhas bacteriocidal properties. Marcus and Lichtenstein (1982) mentionedthat nutmeg oil and myristicin showed no mutagenic activity inSalmonella typhimurium TA100 and TA98 at up to cytotoxicdoses with and without S13 metabolic activation.

Damhoeri and coworkers (1985) conducted specializedmutagenicity tests on the oleoresins of pepper, shallot, and nutmegfruit and related compounds. Streptomycin-dependent strains SD1018and SD7823 isolated from S. typhimurium TA100 and TA98,respectively, were prepared for spot and plate incorporation tests.All assays were conducted without metabolic activation. In spottests, nutmeg oleoresin and myristicin were positive in both SD1018and SD7823 although the oleoresin tested negative in TA98, TA100,and Escherichia coli WP2. In plate incorporation assays,oleoresin of nutmeg kernels prepared from raw seeds produced adose-responsive mutagenic effect. Oleoresin from dried and storednutmeg was not active, suggesting the mutagenic substance is volatile.

Myristicin did not induce unscheduled DNAsynthesis (UDS) in hepatocytes derived from male Fischer 344 ratsin doses up to 0.01 molar, ten times the dose where cytotoxicitywas noted (Hasheminejad & Caldwell, 1994).

Metabolism:There are several possible pathways for myristicin metabolism.Limited data for myristicin suggest the relative importance ofthese pathways may be dependent on dose, species, sex, lengthof exposure, and the like.

Kamienski and Casida (1970) demonstrated thatone metabolic pathway for myristicin is cleavage of the methylenedioxyphenyl moiety through 4-oxidation, producing a polar productcontaining two hydroxy substituents on the benzene ring. Suchmolecules undergo glucuronidation and excretion. The methylenegroup on the five-member ring is exhaled as carbon dioxide.

Methylenedioxyphenyl compounds such as myristicinalso interact with liver enzymes (Delaforge et al., 1985).Mixed function oxidation is briefly inhibited, but this is followedby a period in which cytochrome P450 enzymes are induced. Bothphenobarbital-induced cytochrome P450 and 3-methylcholanthrene-inducedcytochrome P450 can metabolically convert methylenedioxybenzenecompounds to their reactive "carbenes" and then interactwith them to produce ligand complexes. The oxidation of the allylicchain to epoxy- or hydroxy-derivatives enhances the affinity ofthese compounds for cytochrome P448, but not for cytochrome P450,as demonstrated for safrole and its metabolites.

Delaforge's study suggests that the rate andextent of oxidation of the allyl side chain on myristicin maybe an important factor in whether or not myristicin is a carcinogenin chronic studies. Miller and coworkers (1983) have suggestedthat the ultimate carcinogen would be the sulfuric acid esterof the 1'-hydroxy derivative. Miller did not believe that epoxidemetabolites were the major contributors to carcinogenic activity.

Because myristicin has hallucinogenic properties,Braun and Kalbhen (1973) suspected that the allyl group on myristicinmight undergo amination. Using isolated perfused rat liver andrat liver homogenate, these authors reported detecting 3-methoxy-4,5-methylene dioxyamphetamine. These results seem speculative, butanother research group reported finding 3-piperidyl-1,3'-methoxy-4',5'-methylenedioxyphenyl-1-propanonein rat urine and 3-pyrrolidinyl-1(3'-methoxy-4',5'-methylenedioxy-phenyl)1-propanone in guinea pig urine following administration of myristicin(Oswald et al., 1971).

To date, studies of each metabolic pathwayfor myristicin appear to have been conducted in isolation, withno intent to examine the importance of that pathway relative toother pathways. No available studies addressed which metabolicpathway predominates or whether this depends on dosing patternsor length of exposure.

Other Biological Effects:The correlation between induction of glutathione S-transferase(GST) and inhibition of tumorigenesis by anticarcinogens is welldocumented (Zheng et al., 1992). On the other hand, theability to form DNA adducts is a common property of most chemicalcarcinogens or their metabolites (Gupta et al., 1993).Myristicin has induced GST and formed DNA adducts in rodent livers.Myristicin can also induce several families of rat liver P450,including the P450 1A enzyme which is also induced by PAHs, dioxins,and polychlorinated biphenyls.

When 10 mg of myristicin was given to femaleA/J mice every two days for a total of three doses, GST activityin the liver increased over 4-fold and in the small intestineover 3-fold (Zheng et al., 1992). One of two µ class GSTsubunits was selectively induced, while GST-p activity wasunchanged and GST-a was moderately increased. These results suggestedthat myristicin might be a chemopreventive agent (Tobola etal., 1996).

Jeong and Yun (1995) examined the effectsof myristicin on rat liver P450 enzymes. Myristicin was administeredip to groups of four male Sprague-Dawley rats at doses of 10 to5000 µmol/kg. After 24 hours, the activities of P450 1A1/2, P4502B1/2, and P450 2E1 were determined. Myristicin caused a dose-dependentincrease of P450 activities, reaching a significant maximum at500 µmol/kg. The maximum activity was attained for P450 1A1/2and P450 2B1/2 at 24 hours; for P450 2E1 the maximum activityoccurred at 12 hours. The increase in P450 enzyme activities wasaccompanied by increases in P450 apoprotein content. Inductionof P450 1A1/2 and P450 2B1/2 by myristicin was accompanied bya corresponding increase in mRNA encoding these proteins. In contrast,no significant change in P450 2E1 mRNA was observed.

In an earlier study, Iwasaki and coworkers(1986) examined species effects on cytochrome P-448 (P450 1A)induction by myristicin and safrole. Wistar rats, CD1 mice, andSyrian golden hamsters received ip injections of inducing agentfor three days when the P-448 activity in liver, kidneys, andlungs was measured. Safrole, a liver carcinogen, produced a 9-foldincrease in liver ethoxyresorufin O-deethylation (EROD) in rats,a 1-fold increase in hamsters, and a 3-fold decrease in mice.Myristicin caused a 4-fold increase in EROD activity in rat liver,nearly half the increase seen for safrole. Neither myristicinnor safrole caused an increase in EROD activity in the lung orkidney.

Randerath and coworkers (1984) examined DNAadduct formation in the livers of mice administered myristicinand other alkenylbenzenes. Randerath's studies were meant to complementcarcinogenicity studies by Miller and coworkers (1983). FemaleCD-1 mice were given an ip injection of test compound (2 or 10mg/mouse), and 24 hours later alkenylbenzene-DNA adducts in theliver were isolated. The mouse liver carcinogens safrole, estragol,and methyleugenol exhibited the strongest binding to mouse-liverDNA, followed by myristicin, which was three to four times weaker.The adduct present in highest concentration was thought to bethe 3',5'-bisphosphate of an N²-(trans- propenylbenzene-3'-yl)deoxyguanosineadduct. The other DNA adduct was thought to be the correspondingderivative of an N²-(allylbenzen-1'-yl)deoxyguanosineadduct.

The same research group analyzed DNA adductsformed in the livers of newborn mice given alkenylbenzenes, includingmyristicin (Phillips et al., 1984). Male B6C3F₁mice were given ip injections of test compound on days 1, 8, 15,and 22 after birth. Groups of three mice were killed on days 23,29, and 43 to detect liver adducts. The highest levels of adductswere detected with the liver carcinogens, methyleugenol (72.7pmol/mg DNA), estragole (30.0), and safrole (17.5), but significantDNA binding by myristicin was also found (7.8 pmol/mg DNA). Onlylow levels of adducts were detected with dill apiol and parsleyapiol; no DNA binding was detected with eugenol.

More recently, Randeranth and coworkers (1993)examined DNA adduct formation in the livers of ICR mice drinkinga widely used non-diet cola instead of water for up to eight weeks.Adducts chromatographically identical to those induced by coladrinks were detected in mice treated with myristicin, extractsof nutmeg, mace or spices from the nutmeg tree. The predominantmyristicin adduct accounted for about 80% of the total adducts.When pregnant mice were intubated with myristicin, myristicinadducts were also found in fetal liver which was of particularconcern since the rapid division of fetal cells facilitates expressionof such lesions. Adducts were not detected in hepatic DNA of micegiven one of three different brands of a non-cola beverage.

Structure/Activity Relationships:Four chemicals structurally similar to myristicin were screenedfor relevant information on carcinogenicity and genotoxicity,including in vitro tests for mutagenicity and informationon DNA adduct formation (see Table 2 for summary information onmyristicin and structurally similar compounds). Three (estragole,eugenol, and safrole) are found in spices, but dihydromyristicinwas also examined because it has a saturated side chain. Threeother compounds (methyleugenol, isoeugenol, and vinyl benzene)are discussed briefly. All compounds but dihydromyristicin containan alkenyl side chain, believed by many to be the active sitefor carcinogenic activity.

A significant structural difference amongthe selected compounds is the number of substituents other thanthe alkenyl side chain. Vinyl benzene, the simplest member ofthe structural group has a covalent binding index 100 times lowerthan estragole, methyleugenol, and safrole, which are mouse livercarcinogens (Randerath et al., 1984). Structural differenceson the molecules at sites other than the alkenyl side chain mayalso influence metabolic pathways and alter carcinogenic potency.

Methyleugenol is on test at the National ToxicologyProgram (NTP), presently at the pathology assessment stage (NTP,1997). Isoeugenol was nominated for testing in January 1997. Methyleugenoland its metabolite 1'-hydroxymethyleugenol were tested in weanlingmale B6C3F₁ mice. At 18 months, 96% of the methyleugenolgroup and 93% of the 1'-hydroxymethyleugenol had liver tumorscompared with 41% of the controls. The number of tumors per tumor-bearingmouse was 3.2, 3.5, and 0.5, respectively. These results weresignificant at P<0.001 (Miller et al., 1983).

Table 2. Summary of informationon myristicin and structurally related compounds