ß-ASARONE
Explanation
ß-asarone and calamus have not previously been evaluated by the
Joint FAO/WHO Expert Committee on Food Additives.
ß-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 ß-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% ß-asarone; Acorus calamus from Kashmir is hexaploid
and yields an oil containing approximately 5% ß-asarone (Vashist &
Handa, 1964). The European variety of the plant is diploid and also
yields an oil with approximately 5% ß-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 ß-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). ß-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
ß-asarone, at concentrations of 2-200 µg/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
ß-asarone, ß-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, ß-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). ß-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 ß-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
ß-asarone alone blocked conditioned avoidance behaviour in some rats.
Asarone suppressed fighting behaviour; ß-asarone did not. Asarone
potentiated the action of reserpine and chlorpromazine on conditioned
avoidance behaviour and fighting behaviour; ß-asarone did not. Asarone
protected against electroshock convulsions, but ß-asarone increased
duration of spasm. Asarone potentiated the lethal effect of
chlorpromazine during electroshock convulsions; ß-asarone did not.
Pretreatment with Acorus oil, asarone, or ß-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 ß-asarone, and ß-asarone or 0.04-4.0 mg of alpha- or
ß-asarone. There was an absence of teratogenic effects due to the
calamus oil and alpha-asarone. In the case of ß-asarone at the
0.04 mg/egg, 43% of the embryo survived. At 4.00 mg/egg of ß-asarone,
there was 100% lethality (Yabiku, 1980).
Acute toxicity
LD50
Material Animal Route (mg/kg bw) Reference
ß-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
ß-asarone)
alpha-asarone Mouse i.p. 225.5 ± 1.1 Yabiku, 1980
ß-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 ß-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%) ß-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%) ß-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%) ß-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%) ß-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 ß-asarone dose. Both sexes on 800 and 2000 ppm (0.08 and
0.2%) ß-asarone and 2500 ppm (0.25%) JOC had slightly depressed body
weights throughout the study; the males on 400 ppm (0.04%) ß-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
ß-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
ß-asarone in the rat, and none for man. Short-term administration of
ß-asarone to rats resulted in both hepatic and cardiac damage. These
changes were much more severe following long-term administration of
ß-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
ß-asarone content. Tumours were also reported in the liver.
Low levels of ß-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,
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Hagan, E. C. et al. (1967) Food flavourings and compounds of related
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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