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    -ASARONE

    Explanation

         -asarone and calamus have not previously been evaluated by the
    Joint FAO/WHO Expert Committee on Food Additives.

         -asarone (cis-isomer of 2,4,5-trimethoxy-l-propenylbenzene) is a
    constituent of oil of calamus, a flavouring agent derived from the
    dried rhizome of Acorus calamus Linn. The -asarone content of
    calamus oils varies with source of the plant. Indian Acorus calamus
    from the Jammu area is tetraploid and yields an oil containing
    approximately 75% -asarone; Acorus calamus from Kashmir is hexaploid
    and yields an oil containing approximately 5% -asarone (Vashist &
    Handa, 1964). The European variety of the plant is diploid and also
    yields an oil with approximately 5% -asarone (Larry, 1973). Normally,
    only the oil of the diploid variety is used for flavouring aromatic
    alcoholic beverages (Usseglio-Tomasset, cited in Larry, 1973). The
    roots and rhizomes of Acorus calamus have been used in the Ayurvedic
    system of medicine for treating a variety of diseases such as epilepsy
    add hysteria (Madan et al., 1960).

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

         A small amount of ninhydrin-positive material was excreted in the
    urine of rats following administration of -asarone in doses of
    75-300 mg/kg bw i.p. Trans-asarone yielded 10-15 times the amount
    found with the cis-isomer in the same doses. The substances were not
    identified but were postulated to require the presence of the allyl or
    propenyl double bond for formation (Oswald et al., 1969). Similar
    ninhydrin-positive materials excreted after the administration of
    safrole were later identified as tertiary amino propiophenones (Oswald
    et al., 1971).

    TOXICOLOGICAL STUDIES

    Special studies on microbes and insects

         An essential oil of Acorus calamus, with an asarone content of
    approximately 80%, showed an in vitro antitubercular action and
    inhibited the growth of gram-negative organisms. The oil was also
    toxic to flies (Chapra et al., 1957). -asarone had an anti-gonadal
    action on the insect Dysdereus koenigii; it did not act as a
    juvenile hormone nor as an antiallatotropic compound (Saxena et al.,
    1977).

    Special studies on mutagenicity

         -asarone, at concentrations of 2-200 g/plate, was not mutagenic
    in the Ames test with Salmonella typhimurium strains TA-98, TA-100,
    TA-1535, TA-1537, and TA-1538 with metabolic activation. Activity
    without metabolic activation was apparently not tested (Hsia et al.,
    1979).

         alpha-asarone was inactive in the Ames test (Salmonella
    typhimurium TA) with and without activation at concentrations of
    50 ppm and 5000 ppm (0.005 and 0.5%). In a similar study with
    -asarone, -asarone was inactive at the 50 ppm (0.005%) level, but
    was active at the 5000 ppm (0.5%) level with activation (Yabiku,
    1980),

    Special studies on pharmacology

         The distilled volatile oil of roots and rhizomes of an Indian
    variety of Acorus calamus, in doses of 20-100 mg/kg bw, (1)
    prolonged sleeping time with pentobarbital, hexabarbital and ethanol
    in mice, (2) lowered body temperature in mice, (3) increased the
    toxicity of Metrazole in rats, and (4) had no effect on amphetamine
    toxicity in aggregated mice but potentiated the action of reserpine in
    reducing amphetamine toxicity in this circumstance. In anaesthetized
    cats, doses of 1-32 mg/kg bw decreased blood pressure and increased
    heart rate. The blood pressure was not affected by carotid occlusion,
    atropinization, adrenergic or ganglionic blockage or spinal
    preparation. Treatment with the oil dilated blood vessels of the
    splanchnic area in cats and constricted the vessels of the frogs hind
    leg. The oil prevented the action of acetylcholine, histamine, and
    barium chloride on isolated guinea-pig ileum (Dandiya & Cullumbine,
    1959). The hypnotic-potentiating action of Indian Acorus oil was only
    partially blocked by LSD in contrast to the potentiating action of
    reserpine which was completely blocked. Mice treated with Acorus oil,
    50 mg/kg i.p., become ataractic (sedated without any marked decrease
    in awareness); combined treatment with iproniazed and Acorus oil
    resulted in excitation (Dandiya et al., 1959b). The volatile oil of
    European Acorus calamus had a similar hypnotic-potentiating effect
    to that of the Indian oil (Dandiya et al., 1959a).

         The essential oil of Indian Acorus calamus had a quinidine-like
    action in combating auricular fibrillation and flutter and ventricular
    fibrillation in anaesthetized dogs and in preventing or abolishing
    veratrine action on isolated frog muscle. It protected against
    electrically induced seizures but not Metrazol seizures in the rat
    (Madan et al., 1960).

         Pretreatment with Indian Acorus oil had a reserpine-like action
    in depleting rat brain of noradrenaline and 5-hydroxy-tryptamine
    (Malhotra et al., 1961). Incubation of rat brain homogenates with
    Acorus oil inhibited oxygen uptake; LSD partially blocked the in
    inhibition whereas 5-hydroxy-tryptamine potentiated the inhibition
    (Dhalla et al., 1961). The hypnotic potentiating action of Indian
    Acorus oil was reduced by pretreatment with LSD or dibenzyline (DBZ)
    in mice. A combination of LSD and DBZ potentiated barbiturate sleeping
    time; this action was not affected by Acorus oil which suggests the
    action of Acorus oil may be mediated through serotonin and catechol
    amines as the action of reserpine is. Acorus oil also significantly
    decreased the disappearance of pentobarbital from the blood in dogs
    (Malhotra et al., 1962).

         Indian Acorus oil, in doses of 10-100 mg/kg bw i.p. had a
    sedative-tranquillizing action in rats, mice, cats, dogs, and monkeys.
    Doses of 25 and 50 mg/kg bw produced vomiting in cats, dogs, and
    monkeys. Doses of 10-150 mg/kg bw i.p. depressed spontaneous and
    forced muscle activity in mice, with the greater depression in
    spontaneous activity. Acorus oil inhibited overt somatic reflexes but
    did not appreciably affect the neuromuscular function in the
    anaesthetized cat since stimulation of the reticular formation
    overcame the patellar reflex inhibition. These actions suggest an
    action of Acorus oil at the spinal cord and subcortical levels of the
    CNS. Acorus oil in vitro inhibited monoamine oxidase activity and
    1- and d-amino acid oxidase activity of rat liver and kidney (Dhalla &
    Bhattacharya, 1968).

         Dandiya et al. (1959a) considered the hypnotic-potentiating
    activity of the Indian oil to be in the hydrocarbon fraction or in an
    oxygenated fraction not removed by the methods used in fractionating
    the oil. Asarone, -asarone, and a third substance, not identified,
    were considered to be responsible for the hypnotic-potentiating action
    of the Indian oil (Baxter et al., 1960). -asarone doubled sleeping
    time with sodium pentobarbital in mice at a dose of 50 mg/kg bw i.p.
    and doubled the sleeping time with ethanol at a dose of 75 mg/kg (Seto
    & Keup, 1969). The action of asarone and -asarone, alone or in
    combination with either reserpine or chloropromazine, on conditioned
    avoidance behaviour in rats, fighting behaviour in mice, and
    electroshock convulsions in rats was determined. Both asarone and
    -asarone alone blocked conditioned avoidance behaviour in some rats.
    Asarone suppressed fighting behaviour; -asarone did not. Asarone
    potentiated the action of reserpine and chlorpromazine on conditioned
    avoidance behaviour and fighting behaviour; -asarone did not. Asarone
    protected against electroshock convulsions, but -asarone increased
    duration of spasm. Asarone potentiated the lethal effect of
    chlorpromazine during electroshock convulsions; -asarone did not.
    Pretreatment with Acorus oil, asarone, or -asarone did not increase
    the concentration of 5-hydroxytryptamine in rat brain (Dandiya &
    Menon, 1963).

    Special studies on teratogenicity

    Chicken embyro

         Eggs were inoculated in the vitelinum sac with 0.2 ml of a
    solution containing 0.15 to 15 mg of European oil or Indian oil, or
    oil deprived of -asarone, and -asarone or 0.04-4.0 mg of alpha- or
    -asarone. There was an absence of teratogenic effects due to the
    calamus oil and alpha-asarone. In the case of -asarone at the
    0.04 mg/egg, 43% of the embryo survived. At 4.00 mg/egg of -asarone,
    there was 100% lethality (Yabiku, 1980).

    Acute toxicity
                                                                        

                                             LD50
      Material     Animal        Route    (mg/kg bw)      Reference
                                                                        

    -asarone       Rat          Oral       1 010      Taylor, 1981

    Acorus oil      Rat          i.p.         221      Dandiya &
    (Indian)                                           Cullumbine, 1959

                    Mouse        i.p.         177      Dandiya et al.,
                                                       1959a

                    Guinea-pig   i.p.           2.75   Chopra et al.,
                                                       1957

    Calamus oil     Rat          Oral       8 880      von Skramlik, 
                                                       1959 (cited in 
                                                       Opdyke, 1977)

    Calamus oil     Rat          Oral         777      Jenner et al., 
    (Jammu)                                            1964

    Calamus oil     Rat          Oral       4 331      Taylor, 1981
    (Kashmir)

    Calamus oil     Rat          Oral       3 497      Taylor, 1981
    (European)

    Calamus oil     Mouse        i.p.     154.5  1.1  Yabiku, 1980
    (Indian)

    Calamus oil     Mouse        i.p.       1 139      Yabiku, 1980
    (European)
                                                                        

                                                                        

                                             LD50
      Material     Animal        Route    (mg/kg bw)      Reference
                                                                        

    Calamus oil     Mouse        i.p.       1 709      Yabiku, 1980
    (European
    free of and
    -asarone)

    alpha-asarone   Mouse        i.p.     225.5  1.1  Yabiku, 1980

    -asarone       Mouse        i.p.     184.2  1.0  Yabiku, 1980
                                                                        

    Short-term studies

    Rat

         Jammu oil of calamus (JOC) was fed in the diet to groups of 10
    male and 10 female rats at 0, 0.1, 0.25, 0.5, and 1.0% for 18 weeks.
    Growth was depressed at all levels and mortality increased at levels
    of 0.25% and greater; gross liver changes and fluid in the abdominal
    and/or pleural cavities were observed at necropsy in rats fed these
    levels. These levels also produced dose-related microscopic pathology
    in the liver and heart. The hepatic changes consisted of variation in
    hepatic cell size with distortion of architecture, capsular thickening
    proliferation of bile duct epithelium, and portal area fibrosis with
    haemosiderin deposition. The heart changes consisted of degeneration
    characterized by slow necrosis of muscle fibres, early fibrosis, and
    infiltration with mononuclear cells (Hagan et al., 1967; Taylor et
    al., 1967).

         Groups of 11 male and 11 female rats received dietary levels of
    0, 0.27, 1.67 and 5.3% of a hydroalcoholic extract of the rhizome of
    Acorus calamus (European variety) for 13 weeks. An additional
    group of the same size received 0.1% Jammu oil of calamus (JOC) in the
    diet. The -asarone content of the diet was 0, 30, 184 and 583 ppm
    (0, 0.003, 0.0184 and 0.0583%) respectively, for the hydroalcoholic
    extracts, and 710 ppm (0.071%) for the JOC diet, on the basis of
    analytical data in the report. Growth depression was noted in the
    JOC-treated group. No gross or microscopic effects or effects on
    haematology, clinical chemistry, urinalysis, or organ weights was
    found in any of the test groups (Weinberg, 1969).

         The short-term toxicity of JOC, European oil of calamus (EOC) and
    Kashmir oil of calamus (KOC) administered in the diet or by gavage was
    compared in rats. Groups of 10 male and 10 female rats received 0,
    1.0% JOC, 1.0% EOC, or 1.0% Koc in the diet or 0 (7 ml corn
    oil/kg bw), 250 mg JOC/kg, 847 mg EOC, or 1082 mg Koc/kg by gavage
    daily, seven days a week for 9-14 weeks. In the feeding study, atrophy
    of cardiac muscle cells (JOC, EOC, Koc), fatty infiltration of the
    myocardium (JOC only) and cardiac fibrosis (JOC, EOC) were observed.
    Hepatic damage was also produced by dietary administration with the
    JOC rats showing the most serious effects. Fatty degeneration occurred
    in the centro-lobular region indicating its association with chronic
    passive hyperaemia. Hepatic passive hyperaemia was observed in all
    groups, including the controls, but was most prominent in the JOC rats
    and was considered indicative of cardiac insufficiency. Coagulative
    necrosis in the centro-lobular region, hepatic fibrosis, and bile duct
    hyperplasia were also observed in the JOC rats. Heart and liver damage
    of the same types seen in the feeding study were observed in the
    gavage study. The severity of heart damage and liver damage was in the
    order JOC, EOC, Koc (Taylor, 1981).

    Guinea-pig

         Groups of four male and two female guinea-pigs were treated with
    0 (untreated control), 0 (solvent (olive oil) control), or 0.01 ml
    Indian Acorus oil/100 g bw i.p. daily, six days a week, for six weeks.
    No effects on physical condition or gross pathology were observed.
    Hard nodules, considered due to local irritation, formed at the site
    of the injections, but these disappeared in a few days (Chopra et al.,
    1957).

    Long-term studies

    Rat

         Groups of 25 male and 25 female rats were fed diets containing 0,
    400, 800, or 2000 ppm (0, 0.04, 0.08, or 0.2%) -asarone for two
    years. A positive control group of the same size received 2500 ppm
    (0.25%) JOC. None of the group receiving 2000 ppm (0.2%) -asarone or
    2500 ppm (0.25%) JOC survived more than 84 weeks; mortality was also
    increased on the 800 ppm (0.08%) dose. Median survival time in the
    females was less than in the males in these groups. The gross
    pathological changes observed were serous fluid in the abdominal and
    pleural cavities, liver and kidney changes, and tumourous masses in
    the intestinal tract.  The tumours were identified as leiomyosarcomas
    of the small intestine and were found in one rat on 400 ppm (0.04%),
    six on 800 ppm (0.08%), nine on 2000 ppm (0.2%) and one on 2500 ppm
    (0.25%) JOC. All the test rats with leiomyosarcomas were males. The
    earliest tumour was observed in a rat 52 weeks old on the 2000 ppm
    (0.2%) dose. Two leiomyosarcomas were also noted in control females

    but the location differed from that of the tumours in the treated
    animals. Atrophy of cardiac muscle cells occurred in controls and all
    test groups but was most prominent in the 2000 ppm (0.2%) -asarone
    and 2500 ppm (0.25%) JOC groups. Cardiac fibrosis generally paralleled
    the incidence of cardiac atrophy. Fatty degeneration and fatty
    infiltration were also in the heart more severe in the treated groups.
    Thrombosis within the chambers of the heart was observed in the 800
    and 2000 ppm (0.08 and 0.27%) -asarone and 2500 ppm (0.25%) JOC
    treated groups. Passive hyperaemia of the lung, kidneys and liver was
    more prominent in the test than control animals indicating faulty
    cardiac function in the test animals. Incidence of hepatic angiectasis
    and hepatic coagulative necrosis also tended to increase with
    increasing -asarone dose. Both sexes on 800 and 2000 ppm (0.08 and
    0.2%) -asarone and 2500 ppm (0.25%) JOC had slightly depressed body
    weights throughout the study; the males on 400 ppm (0.04%) -asarone
    also had depressed body weights at termination but the number of
    survivors was small. Haemoglobin, haematocrit, and red and white cell
    counts were normal except that when the test animals became sick they
    became anaemic with lowered haemoglobin, haematocrit, and red cell
    count (Taylor, 1981).

         Groups of 25 male and 25 female rats were fed diets containing 0,
    500, 1000, 2500 and 5000 ppm (0, 0.05, 0.1, 0.25 and 0.5%) JOC for two
    years. Mortality was increased in relation to dose with females dying
    earlier than males, in all the treated groups. All the 5000 ppm (0.5%)
    group were dead by 45 weeks, all the 2500 ppm (0.25%) group by 68
    weeks and all the 1000 ppm (0.1%) group by 104 weeks. Three major
    gross abnormalities were observed: liver damage, fluid in the pleural
    and/or peritoneal cavity, and tumourous masses in the intestines. The
    intestinal tumours were leiomyosarcomas, which were malignant, highly
    pleomorphic, and highly anaplastic. These tumours occurred most
    frequently in the duodenum and appeared to have arisen from the
    musculature of the tunica propria of the mucosa. The incidence was 0
    in the controls, three in females on 500 ppm (0.05%), five in males on
    1000 ppm (0.1%), two in males on 2500 ppm (0.25%) and 0 on 5000 ppm
    (0.5%). The histopathological changes observed in the heart and liver
    with chronic administration of JOC were similar to those observed with
    -asarone. Cardiac atrophy was observed in both test and controls but
    was more severe in test animals. The severity in all four test groups
    was similar. Fatty degeneration and fatty infiltration were increased
    with doses of 1000 ppm (0.1%) and greater and cardiac thrombosis was
    observed only with these levels. Passive hyperaemia of the liver
    generally increased with dose for all four treatment levels. Hepatic
    nodular hyperplasia was more severe in the treated groups but the
    incidence was not dose-related. In males, the 2500 and 5000 ppm (0.25
    and 0.5%) levels caused marked growth depression. With the lower doses
    weight gain was normal for the first 26 weeks, then decreased. After
    68 weeks, there appeared to be a slight weight loss. In the females,

    growth depression was dose-related and considerable on the 1000, 2500
    and 5000 ppm (0.1, 0.25 and 0.5%) levels. On the 500 ppm (0.05%)
    level, weight gain was normal during the first year and slightly
    depressed thereafter. Haematological values were similar in control
    and test animals (Taylor et al., 1967; Taylor, 1981).

         Groups of 25 male and 25 female rats were fed levels of 0, 50,
    100 and 5000 ppm (0, 0.005, 0.01 and 0.5%) JOC for two years. The rats
    on the 5000 ppm (0.5%) level showed the elevated and early mortality,
    the heart and liver lesions observed with this level in the study
    described above. One leiomyosarcoma was also observed in a male on
    this level. Heart and liver changes with the 50 and 100 ppm (0.005 and
    0.01%) levels were similar in severity to those observed in the
    controls and consistent with geriatric changes. No leiomyosarcomas
    were observed in the control, 50 or 100 ppm (0.005 and 0.01%) levels
    (Taylor, 1981).

         Groups of 25 male and 25 female rats were fed diets containing
    EOC at levels of 0.1, 0.5, 1.0 and 2.0% for two years. This study was
    evaluated independently by two pathologists. Both reported the finding
    of leiomyosarcomas and hepatocellular adenomas and adenocarcinomas on
    the 1.0 and 2.0% dose levels. Liver changes, identified by one
    pathologist as hepatocellular adenomas or adenomatoid hyperplasia and
    by the other as nodular hyperplasia also occurred on the 0.1 and 0.5%
    levels. Other hepatic changes observed were hyperaemia, necrosis,
    hepatocellular vacuolation, and biliary duct proliferation. In
    general, these changes increased in severity and incidence with dose,
    with the changes at 0.1% being similar to those in the controls or
    slightly increased. Heart changes, consisting of myocardial atrophy,
    fibrosis, fatty degeneration and fatty infiltration, increased with
    increasing dose. The myocardial atrophy and damage was considered
    sufficient to account for the passive hyperaemia and congestion seen
    in the liver and other organs (Taylor, 1981).

         The toxicity of calamus oil has been reviewed by Opdyke, 1977.

    Comments

         Only limited information is available on the metabolism of
    -asarone in the rat, and none for man. Short-term administration of
    -asarone to rats resulted in both hepatic and cardiac damage. These
    changes were much more severe following long-term administration of
    -asarone. In addition, tumourous masses were observed in the
    intestinal tract. The tumours were identified as leiomyosarcomas.
    Similar effects were observed following administration of oil of
    calamus, the severity of the effect being directly related to the
    -asarone content. Tumours were also reported in the liver.

         Low levels of -asarone are not mutagenic in the Ames test with
    and without activation, but high concentrations (5000 ppm (0.5%)) have
    been shown to be positive in this system following activation.

    EVALUATION

         No ADI allocated.

    REFERENCES

    Baxter, R. M., et al. (1960) Separation of the hypnotic-potentiating
         principles from the essential oil of Acorus calamus L. of
         Indian origin by liquid-gas chromatograph, Nature, 185, 466-467

    Chopra, I. C., Khajuria, B. N. & Chopra, C. L. (1957) Antibacterial
         properties of volatile principles from Alpinia galanga and
         Acorus calamus, Antibiot. Chemotherap., 1, 378-383

    Dandiya, P. C. & Cullumbine, H. (1959) Studies on Acorus calamus.
         III. Some pharmacological actions of the volatile oil,
         J. Pharmacol.  Exptl. Therap., 125, 353-359

    Dandiya, P. C., Cullumbine, H. & Sellers, E. A. (1959b) Studies on
         Acorns calamus. IV. Investigations on mechanism of action in
         mice, J. Pharmacol. Exptl. Therap., 126, 334-337

    Dandiya, P. C. & Menon, M. K. (1963) Effects of asarone and
         beta-asarone on conditioned responses, fighting behavior and
         convulsions, Brit. J. Pharmacol., 29, 436-442

    Dandiya, P. C. et al. (1959a) Studies on Acorns calamus. II.
         Investigation of volatile oil, J. Pharm. Pharmacol., 11,
         163-168

    Dhalla, N. S. & Bhattacharya, I. C. (1968) Further studies on
         neuro-pharmacological actions of acorns oil, Arch. Int.
         Pharmacodyn., 172, 356-365

    Dhalla, N. S., Malhotra, C. L. & Sastry, M. S. (1961) Effects of
         Acorns oil in vitro on the respiration of rat brain,
         J. Pharm. Science, 50, 580-582

    Hagan, E. C. et al. (1967) Food flavourings and compounds of related
         structure. II. Subacute and chronic toxicity, Fd. Cosmet.
         Toxicol.,  5, 141-157

    Hsia, M. R. S., Adamovics, J. A. & Kreamer, B. L. (1979) Microbial
         mutagenicity studies of insect growth regulators and other
         potential in Salmonella typhimurium, Chemosphere, 8,
         521-529

    Jenner, P.M. et al. (1964) Food flavouring and compounds of related
         structure. I. Acute oral toxicity, Fd. Cosmet. Toxicol., 2
         327-343

    Larry, D. (1973) Gas-liquid chromatographic determination of
         beta-asarone, a component of oil of calamus, in flavors and
         beverages, Journal of the AOAC, 56, 1281-1283

    Madan, B. R., Arora, R. B. & Kapila, K. (1960) Anticonvulsant,
         anti-veratrinic and antiarrhythmic actions of Acorus calamus
         Linn - an Indian indigenous drug, Arch. Int. Pharmacodyn.,
         124, 201-211

    Malhotra, C. L., Das, P. K. & Dhalla, N. S. (1962) Investigations on
         the mechanism of potentiation of barbiturate hypnosis by
         hersaponin, acorus oil, reserpine and chlorpromazine, Arch.
         Int. Pharmacodyn., 138, 537-547

    Malhotra, C. L. et al. (1961) Effect of hersaponin and acorus oil on
         noradrenaline and 5-hydroxytryptamine content of rat brain,
         J. Pharm. Pharmacol., 13, 447

    Opdyke, D. L. J. (1977) Fragrance raw materials monographs: calamus
         oil, Fd. Cosmet. Toxicol., 15, 623-626

    Oswald, O. E., Fishbein, L. & Corbett, B. J. (1969) Metabolism of
         naturally occurring propenylbenzene derivatives. I.
         Chromatographic separation of ninhydrin-positive materials of rat
         urine, J. Chromatog., 45, 437-445

    Oswald, E. O. et al. (1971) Identification of tertiary
         aminomethylenedioxy-propiophenones as urinary metabolites of
         safrole in the rat, Biochim. Biophys. Acta., 230, 230-247

    Sexana, B. P. et al. (1977) A new insect chemosterilant isolated from
         Acorus calamus L., Nature, 270, 512-513

    Seto, T. A. & Keup, W. (1969) Effects of alkylmethoxybenzene and
         alkylmethylenedioxybenzene essential oils on pentobarbital and
         ethanol sleeping time, Arch. Int. Pharmacodyn., 180, 232-240

    Taylor, J. M. (1981) (Food and Drug Administration) Personal
         communication to the World Health Organization concerning
         unpublished studies on beta-asarone and calamus oils.

    (1)  Short-term study comparing toxicity of Jammu, European and
    Kashimir calamus oils. Performed 1966-1967. Pathology reports by
    William S. Monlux (May 1978) entitled "Comparison of microscopic
    lesions occurring in rats gavaged with Jammu, European and Kashmir
    varieties of oil of calamus" and "Comparison of microscopic lesions
    occurring in rats fed Jammu, European and Kashmir varieties of oil of
    calamus".

    (2)  Long-term study Of beta-asarone. Performed 1967-1969.

         (a) Pathology report by William S. Monlux (August 1978) entitled
         "Microscopic lesions occurring in one hundred and ninety-four
         rats fed beta-asarone in their diet for twenty-four months" (This
         report indicates that a level of 2500 ppm beta-asarone was fed in
         the study; this group received Jammu oil of calamus not
         beta-asarone.)

         (b) Memo from Robert T. Habermann to Jean Taylor (16 June 1971)
         entitled "Carcinogenicity of beta-asarone in rats in a two-year
         feeding study". (Histopathological findings on 0, 400, 800, and
         2000 ppm of beta-asarone in a two-year feeding study with
         Osborne-Mendel rats and an additional group given a diet
         containing 2500 ppm Jammu Oil of Calamus.)

         (c) Memo from Jean M. Taylor to Damon Larry (15 October 1976)
         entitled "Chronic rat feeding studies on beta-asarone and
         European oil of calamus".

    (3)  Long-term study of low levels of Jammu oil of calamus. Performed
    1965-1967.

         (a) Pathology report by William S. Monlux (May 1978) entitled
         "Microscopic lesions occurring in fifty rats fed oil of calamus
         (Jammu) for twenty-four months".

         (b) Pathology report by William S. Monlux (May 1978) entitled
         "Lesions occurring in one hundred and forty-eight rats fed oil of
         calamus (Jammu) in their diet for twenty-four months". (The rats
         receiving 50 and 100 ppm levels described in this report and the
         rats receiving 0 and 5000 ppm oil of calamus (Jammu) described in
         the report 3(a) are from the low dose Jammu oil of calamus study.
         The rats receiving 0, 500, 1000, and 2500 ppm Jammu oil of
         calamus described in this report are from the high dose level
         study reported in Taylor et al., 1967).

    (4)  Long-term study of European oil of calamus. Performed 1967-1969.

         (a) Pathology report by Donald A. Willigan (22 October 1971)
         entitled "Project 602 WR 1932: Histopathologic evaluation of
         tissue from rats following continuous dietary intake for 104
         weeks of oil of calamus".

         (b) Pathology report by William S. Monlux (June 1978) entitled
         "Lesions occurring in one hundred and ninety-four rats fed oil of
         calamus (European) in their diet for twenty-four months".

         (c) Memo from Jean M. Taylor, to Damon Larry (15 October 1976)
         entitled "Chronic rat feeding studies on beta-asarone and
         European oil of calamus".

    Taylor, J. M. et al. (1967) Toxicity of oil of calamus (Jammus
         variety), Toxicol. Exptl. Pharmacol., 10, 405

    Usseglio-Tomasset, L. (1966) Estratta Ind. Agr., 4, 3-13. (Cited in
         Larry, 1973), Journal of the AOAC, 56, 1281-1283)

    Vashist, V. N. & Handa, K. L. (1964) A chromatographic investigation
         of Indian calamus oils, Soap, Perfumery & Cosmetics, 37,
         135-139

    Von Shramlik, E. (1959) Uber die Giftigkeit und Vertraglichkeit von
         atherischen Olen, Pharmazie, 14, 435. (Cited in Opdyke, D. L. J.
         (1977), Fd. Cosmet. Toxicol., 15, 623-626

    Weinberg, M. (1969) Studies conducted with Calamus. Unpublished report
         from Foster D. Snell, Inc. submitted to the World Health
         Organization by Comitato Per Lo Studio Delle Bevande Alcooliche
         Aromatizzate

    Yabiku, H. K. (1980) "Calamus oil - Toxicological aspects and their
         control in alcoholic beverages", M. S. Thesis, Sao Paulo, Brazil,
         Submitted to FAO/WHO
    


    See Also:
       Toxicological Abbreviations