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BASIS OF NOMINATION TO THE CSWG
Thujone is presented to the CSWG for review
because of its potential for widespread human exposure through
spices, herbs, and essential oils containing thujone. This compound
is also presented to the CSWG as a representative of a structurally
related group of natural compounds that contain six-member rings
constrained by a bridge across the 2 and 4 positions. This bridge
has profound conformational effects on the cyclohexane ring that
would be expected to influence toxicity of the molecule. Other
ingredients in spices and herbs with this structure include sabinene,
umbellulone, and thujene.
Thujone was identified through a review of
direct food additives given "GRAS" status by the Food
and Drug Administration (FDA). Although thujone has known toxicity
that has caused it to be banned from some products, 24 direct
food additives in the FDA Priority-Based Assessment of Food Additives
(PAFA) database contain thujone. Tens of thousands of workers
are also potentially exposed to Dalmatian sage oil and cedarleaf
oil, both of which contain a high concentration of thujone.
SELECTION STATUS
ACTION BY CSWG: 12/10/97
Studies requested:
- In vitro cytogenetics
- In vivo micronucleus
- Neurotoxicity studies
- Mouse lymphoma assay (NCI Short-Term Test Program)
Priority: Moderately high
Rationale/Remarks:
- Although thujone is banned in some food additives, potential for widespread consumer and worker exposure remains.
- Confer with FDA/NCTR regarding neurotoxicity
studies.
INPUT FROM GOVERNMENT AGENCIES/INDUSTRY
Dr. Dan Benz, Center for Food Safety and Applied
Nutrition (CFSAN), Food and Drug Administration (FDA) and Dr.
Ed Matthews (formerly with CFSAN) provided information on
alpha-thujone from the FDA PAFA database.
Ms. Joellen Putnam, Scientific Project Manager, Flavor and Extract Manufacturers' Association (FEMA) provided a copy of the FEMA monograph on alpha-thujone.
CHEMICAL IDENTIFICATION
CAS Registry Number:
546-80-5
Chemical Abstract Service Name:
Bicyclo(3.1.0)hexan-3-one, 4-methyl-1-(1- methylethyl)-, (1S-(1-,
4-, 5-alpha))- (9CI)
Synonyms and Trade Names:
(-)-Isothujone; (1S, 4R, 5R)-(-)-3-thujanone; thujon; thujone;
(-)-thujone
Structural Class:
Monoterpene ketone
Structure, Molecular Formula and Molecular Weight:
C10H16O Mol. wt.: 152.24
Chemical and Physical Properties:
Description:
Colorless or almost colorless liquid with a menthol- like odor
(Albert-Puleo, 1978; Budavari, 1996)
Boiling Point:
201°C (FEMA, 1997)
Flash Point:
148°F, CC (FEMA, 1997)
Density:
0.9109 at 25°C/4°C (Budavari, 1996)
Solubility:
Insoluble in water; soluble in ethanol, diethyl ether and chloroform
(Albert-Puleo, 1978; Lewis, 1993)
Vapor Pressure:
~0.2 mm Hg @ 20°C (FEMA, 1997)
Technical Products and Impurities:
Alpha-Thujone is available at a purity of ~99% from Fluka. It
is also available as a technical grade product containing 17.8%
fenchone, 10.5% bornyl acetate, 3.1% camphor, 2% p-cymene and
1.2% limonene from Aldrich. Mixtures of alpha and beta-thujone
isomers are available from TCI America and Fluka (Aldrich Chemical
Company, Inc., 1996; TCI America, 1996; Fluka Chemical Corp.,
1997).
EXPOSURE INFORMATION
Production and Producers:
Thujone occurs in nature as a mixture of alpha- and beta-isomers
(Micali & Lanuzza, 1995). The equilibrium mixture consists
of 33% alpha-thujone, 67% beta-thujone (Budavari, 1996). Alpha-Thujone
can be isolated from natural oils with bisulfite, or via fractional
distillation and crystallization, and it has been synthesized
(Albert-Puleo, 1978). According to recent chemical catalogs and
directories, alpha-thujone is manufactured and/or distributed
by Aldrich and Fluka (Aldrich Chemical Company, Inc., 1996; Fluka
Chemical Corp., 1997).
No data were reported for alpha-thujone by
the US International Trade Commission (USITC) in the ten most
recent volumes of Synthetic Organic Chemicals, US Production
and Sales, for the years 1983-1993 and no other quantitative
information on annual production was found in the available literature.
Alpha-thujone is listed in the EPA's Toxic
Substances Control Act (TSCA) Inventory (NLM, 1997a).
Use Pattern:
Thujone is the primary constituent of essential oils derived from
a variety of plants including wormwood (Artemisia absinthium),
mugwort (Artemisia vulgaris), sage (Salvia officinalis),
clary (Salvia sclarea), tansy (Tanacetum vulgare),
and yellow cedar (cedarleaf oil) (Thuja occidentalis) (Albert-Puleo,
1978). Thujone itself has a limited use pattern; Lewis (1993)
cites a solvent use only. However, the essential oils in which
thujone occurs are used in herbal medicine and as flavorings,
fragrances, and rodent and mite repellants.
Herbal medicine:
Essential oils containing thujone have been used in traditional
medicine for the following purposes: abortifacient, female hormone
activity, and emmenagogue (sage, cedar, tansy, mugwort); digestive
problems and carminative (sage, cedar, tansy, mugwort); anthelmintic
(tansy, wormwood, mugwort); and corns, warts, venereal warts,
acne, fever, cough, rheumatism, scurvy, dropsy (cedar) (Albert-Puleo,
1978; Millet et al., 1981; Ishida et al., 1989;
Kim et al., 1992; Hui et al., 1994; Dupont, 1995;
Anon., 1997a; Grieve, 1997). Cedarleaf oil has been used for decades
for treating the common cold in products such as Vick's Vaporub™
(Anon., 1997b).
Flavorings:
Thujone is banned as a food additive in the US and its presence
in foods and beverages is regulated in several countries. However,
many of the thujone-containing plant oils are used as flavoring
substances in the alcoholic drink industry. Absinthe, made from
wormwood, is available in Spain, Denmark and Portugal. Vermouth,
chartreuse, and benedictine all contain small amounts of thujone
and wormwood is popular as a flavoring for vodka in Sweden (Galli
et al., 1984; Baggott, 1993; Micali & Lanuzza, 1995).
Sage oil is an important food flavor, especially in sausages,
meats, condiments, and sauces (Rogers, 1981).
Fragrances:
Cedarleaf oil is used for the scenting of various technical preparations
such as shoe polish (Rogers, 1981). It is also used in the men's
fragrances Ralph Lauren Safari™ and Hugo Boss (Anon., 1997b).
Europeans have used the essential oil of mugwort in perfumery
(Kim et al., 1992).
Pest repellants:
A paint containing 25-31% of cedarleaf oil has been patented as
a rodent repellant (Harding, 1987). A new OFF™ product, based
on cedarleaf oil and used to repel mites in closets, is soon to
be marketed (Anon., 1997b).
Human Exposure:
There is potential for exposures to alpha-thujone in occupational,
consumer, and environmental settings by inhalation, ingestion,
and dermal contact. Thujone (alpha- and/or beta-) was found in
41 direct food additive substances listed in the Priority-Based
Assessment of Food Additives (PAFA) database (FDA, 1994). In an
updated analysis of PAFA, the number of thujone occurrences reported
was reduced to 24 (FDA, 1997a). No listing was found for thujone
in the National Occupational Exposure Survey (NOES).
Dalmation sage oil and cedarleaf oil contain
approximately 40-60% and 60% thujone (isomer not specified), respectively
(Rogers, 1981). NOES, which was conducted by the National Institute
for Occupational Safety and Health (NIOSH) between 1981 and 1983,
estimated that 10,944 workers, including 6,777 female workers,
were potentially exposed to Dalmation sage oil in the workplace
and that 38,433 workers, including 10,450 female workers, were
potentially exposed to cedarleaf oil in the workplace. The NOES
database does not contain information on the frequency, level
or duration of exposure to workers of any chemical listed therein
(NLM, 1997b,c).
Environmental Occurrence:
Thujone is found in nature as a constituent of essential oils
where it exists as a mixture of the alpha- and beta-isomers (Micali
& Lanuzza, 1995). Thujone levels in various essential oils
are shown in Table 1.
| Cedar leaf | Pinto-Scognamiglio, 1967 | |||
| Sage |
|
|
| Pinto-Scognamiglio, 1967
Farag et al., 1986 |
| Tansy | Pinto-Scognamiglio, 1967 | |||
| Wormwood | Lawrence, 1995 | |||
| Thyme | Farag et al., 1986 | |||
| Rosemary | Farag et al., 1986 | |||
Thujone has been identified as a volatile
organic compound emitted by vegetation to the atmosphere (Guenther
et al., 1994). Arey and coworkers (1995) identified alpha-
and beta- thujone in the emissions of California sagebrush. Alpha-thujone
has been reported in emissions from kitchen and garden waste (Wilkins
& Larsen, 1995).
Regulatory Status:
No standards or guidelines have been set by NIOSH or OSHA for
occupational exposure to or workplace allowable levels of alpha-thujone.
Thujone was not on the American Conference of Governmental Industrial
Hygienists (ACGIH) list of compounds for which recommendations
for a threshold limit value (TLV) or biological exposure index
(BEI) are made. Thujone is potentially toxic and the presence
of alpha- and beta-thujone in food and beverages is regulated
by law in several countries (Micali & Lanuzza, 1995). The
use of thujone as a food additive has been banned in the United
States (Rogers, 1981; Galli et al., 1984). The natural
flavoring substances wormwood, white cedar, and oak moss may be
used in food under the condition that the finished food is thujone-free.
Tansy may only be used in alcoholic beverages provided that the
finished product is thujone-free, and yarrow use is specified
as beverages only, thujone-free (FDA, 1997b).
In 1979, the FAO/WHO Codex Committee on Food additives restricted the use of apha- and beta-thujones to the following maximum levels in final products ready for consumption: 0.5 ppm in food and beverages, 10 ppm in alcoholic beverages containing more than 25% alcohol, 5 ppm in alcoholic beverages containing less than 25% alcohol, and 35 ppm in bitters (Galli et al., 1984).
EVIDENCE FOR POSSIBLE CARCINOGENIC ACTIVITY
Human Data:
No epidemiological studies or case reports investigating the association
of exposure to alpha-thujone and cancer risks in humans were identified
in the available literature.
At a concentration of 4%, alpha-thujone was
negative in dermal irritation and maximization tests in humans
(FEMA, 1997).
Commercial preparations of essential oils
of sage, thuja, and cedar which contain thujone have caused central
nervous system effects. The following reported poisonings occurred
in France and all victims had taken large doses of these thujone-containing
oils or hyssop oil for their therapeutic benefits. The clinical
intoxications were characterized by tonico-clonic or solely clonic
convulsions. The outcomes were satisfactory and electro-encephalograms
made after the crisis were all normal. The cumulative effect of
treatment was shown in the case of a 50-year-old woman who took
20 drops of undiluted thuja oil twice a day for 5 days uneventfully,
but 30 minutes after the tenth dose suffered a tonic seizure (Millet
et al., 1981).
Thujone has been suggested as the neurotoxic
cause of absinthism. This syndrome characterized by addiction,
hyperexcitability, and hallucinations was caused by chronic use
of absinthe, an alcoholic drink made with an extract from wormwood;
thujone makes up 40-90% (by weight) of the essence of wormwood.
The debilitating illness suffered by Vincent Van Gogh during the
last decade of his life has been linked to absinthism. The toxicity
of thujone was responsible for the eventual ban of absinthe at
the beginning of this century (Bonkovsky et al., 1992;
Baggott, 1993).
In a recent case, a male subject drank about
10 ml of essential oil of wormwood under the impression that it
was in fact absinthe which he had learned of on the Internet/World
Wide Web. He was found several hours later agitated, incoherent,
and disoriented. He improved with treatment but subsequently acute
renal failure developed (Reese, 1997).
Animal Data: No 2-year carcinogenicity studies of alpha-thujone in animals were identified in the available literature. Toxicity information was limited to acute data. Acute toxicity values are shown in Table 2.
| Oral | NLM, 1997a | ||
| Intravenous | NLM, 1997a | ||
| Subcutaneous |
|
| NLM, 1997a
Rice & Wilson, 1976 NLM, 1997a |
| Dermal | FEMA, 1997 | ||
In rats, intraperitoneal injections of thujone
induced electro-cortical seizures associated with myoclonic activity.
The convulsant and lethal effects appeared together at a low dose
(0.2 ml/kg) (Millet et al., 1981).
Short-Term Tests:
Thujone was tested at 1.5 and 3% in DMSO for its effect on the
mutagenicity of aflatoxin B1 in Salmonella typhimurium
strain TA100. The plates treated with thujone showed evidence
of colony damage which the researchers felt indicated some mutagenic
activity on the part of thujone (Kim et al., 1992).
Thujone concentrations of 1, 10, and 100 &g/ml
were not cytotoxic to HeLa cells (Zolotovich et al., 1967).
Metabolism: After oral administration of a mixture of alpha- and beta-thujone (9:2), two neutral metabolites were identified in the urine of rabbits, beta-hydroxy-beta-thujane (neoisothujanol) and beta-hydroxy-beta -thujane (thujanol). As shown below, in the metabolism of alpha- and beta-thujone, the carbonyl group was reduced to yield secondary alcohols. The configurations of these reduced hydroxyl groups in both metabolites are beta-orientation in spite of the different configurations of the methyl group (Ishida et al., 1989).
Other Biological Effects:
Thujone (97% alpha-, 3% beta-) produced an increase in porphyrin
production in primary cultures of chick embryo liver cells. At
the highest concentration tested (1 mM), thujone led to 80-100
pmol total porphyrin/mg protein (control=19-50). The accumulated
porphyrins were predominantly copro- and protoporphyrins. Thujone
produced inductions of 5-aminolevulinic acid synthase and benzphetamine
demethylase. It also induced heme oxygenase by a heme-dependent
mechanism. The results indicated that thujone would pose a threat
to individuals with underlying defects in hepatic heme synthesis
(Bonkovsky et al., 1992).
The antinociceptive (pain-blocking) activity
of alpha-thujone in mice was evaluated using the hot-plate and
Nilsen Tests. In the hot-plate test, alpha-thujone (ED50
= 6.5 mg/kg) was found to be codeine-like and equipotent with
(delta)- 9-tetrahydrocannabinol, the major psychoactive
component of marijuana. Less antinociceptive activity was observed
in the Nilsen test (ED50 = 14.1 mg/kg) (Rice &
Wilson, 1976).
Structure Activity Relationships: The unsymmetric bicyclic ring structure of thujone is characteristic of a group of natural products found widely in spices and herbs. Other examples include sabinene and umbellulone. None of these compounds appear to have any toxicity data relevant to a structure-activity analysis.
Albert-Puleo, M. (1978) Mythobotany, pharmacology,
and chemistry of thujone-containing plants and derivatives. Econ.
Bot., 32(1), 65-74
Aldrich Chemical Co., Inc. (1996) Aldrich
Catalog/Handbook of Fine Chemicals 1996-1997, Milwaukee, WI,
p. 1421
Anon. (1997a) Arsenic and Old Lace. Our
featured herb. Brookline, MA, Arsenic & Old Lace [http://www.arsenic.com/prevherbs/tansy.html]
Anon. (1997b) Specifications Data Sheet
for Cedarleaf Oil (Thuja occidentalis), Cédres
Recyclés de l'Outaouais, Inc., Canada [http://www.aei,
ca/~belletuf/specs.htm]
Arey, J., Crowley, D.E., Crowley, M., Resketo,
M. & Lester, J. (1995) Hydrocarbon emissions from natural
vegetation in California's south coast air basin. Atmospher.
Environ., 29(21), 2977-2988
Baggott, M. (1993) Absinthe FAQ [http://realbeer.com/brewery/library/absfaq.html]
Bonkovsky, H.L., Cable, E.E., Cable, J.W.,
Donohue, S.E., White, E.C., Greene, Y.J., Lambrecht, R.W., Srivastava,
K.K. & Arnold, W.N. (1992) Porphyrogenic properties of the
terpenes camphor, pinene, and thujone. Biochem. Pharmacol.,
43(11), 2359-2368
Budavari, S., ed. (1996) The Merck Index,
12 ed., Whitehouse Station, NJ, Merck & Co., Inc., p. 1603
Dupont, P. (1995) Cosmetic Compositions
for the Treatment of Corns and Warts Comprising Plant Tinctures
and Essential Oils. (Patent No. Fr. 2710266) [Abstract: CA123-017496X]
Farag, R.S., Salem, H., Badei, A.Z.M.A. &
Hassanein, D.E. (1986) Biochemical studies on the essential oils
of some medicinal plants. Fette, Seifen, Anstrichm., 88(2),
69-72
FDA (1994) Priority-based Assessment of
Food Additives (PAFA) Database: Thujone, Center for Food Safety
and Applied Nutrition, U.S. Food and Drug Administration, p. 64
FDA (1997a) Priority-based Assessment of
Food Additives (PAFA) Database: Thujone, Center for Food Safety
and Applied Nutrition, U.S. Food and Drug Administration
FDA (1997b) Flavoring Agents and Related Substance.
US Code Fed. Regul. Title 21, Part 172. 510, pp. 49-52
FEMA (1997) FEMA Database: Thujone,
Washington, DC, Flavor and Extract Manufacturer's Association,
12 pp.
Fluka Chemical Corp. (1997) Fluka Chemika
Biochemika Analytica, Milwaukee, WI, p. 1456
Galli, C.L., Galli, G., Tragni, E. & Caruso,
D. (1984) Quantitative analysis of alpha, beta-thujone, pulegone,
safrole, coumarin and beta-asarone in alcoholic beverages by selected-ion
monitoring. J. Appl. Toxicol., 4(5), 273-276
Grieve, M. (1997) A Modern Herbal. Cedar,
Yellow [http://www.botanical.com/botanical/mgmh/c/ cedye141.html]
Guenther, A., Zimmerman, P. & Wildermuth,
M. (1994) Natural volatile organic compound emission rate estimates
for U.S. woodland landscapes. Atmospher. Environ., 28(6),
1197-1210
Harding, N.T., Jr (1987) Rodent Repellent
Paints and Bars Containing Thujone Oil. (Jpn. Kokai Tokkyo
Koho Patent No. 87181212) [Abstract: CA107-20496K]
Hui, Y.H., Gorham, J.R., Murrell, K.D. &
Cliver, O.D., eds. (1994) Foodborne Disease Handbook, vol.
3, New York, Marcel Dekker, Inc., p. 48
Ishida, T., Toyota, M. & Asakawa, Y. (1989)
Terpenoid biotransformation in mammals. V. Metabolism of (+)-citronellal,
(Å)-7-hydroxycitronellal, citral, (-)-perillaldehyde, (-)-myrtenal,
cuminaldehyde, thujone, and (Å)-carvone in rabbits. Xenobiotica,
19(8), 843-855
Kim, J.O., Kim, Y.S., Lee, J.H., Kim, M.N.,
Rhee, S.H., Moon, S.H. & Park, K.Y. (1992) Antimutagenic effect
of the major volatile compounds identified from mugwort (Artemisia
asictica nakai) leaves. J. Korean Soc. Food Nutr.,
21(3), 308-313
Lawrence, B.M., ed. (1995) Natural Flavor
and Fragrance Materials "Perfumer & Flavorist."
Essential Oils 1992-1994, Carol Stream, IL., Allured Publishing
Corporation, pp. 11-14
Lewis, R.J., Sr. (1993) Hawley's Condensed
Chemical Dictionary, 12th ed., New York, Van Nostrand Reinhold
Company, p. 1149
Micali, G. & Lanuzza, F. (1995) HPLC determination
of alpha- and beta-thujone, potentially toxic components of natural
flavourings, in alcoholic beverages. Flavour and Fragrance
J., 10(5), 329- 333
Millet, Y., Jouglard, J., Steinmetz, M.D.,
Tognetti, P., Joanny, P. & Arditti, J. (1981) Toxicity of
some essential plant oils. Clinical and experimental study. Clin.
Toxicol., 18(12), 1485-1498
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Effects of Chemical Substances), Bethesda, MD, National Library
of Medicine, searched September, 1997 [Record No. 84457]
NLM (1997b) RTECS (Registry of Toxic Effects
of Chemical Substances), Bethesda, MD, National Library of
Medicine, searched October, 1997 [Record No. 78462]
NLM (1997c) RTECS (Registry of Toxic Effects
of Chemical Substances), Bethesda, MD, National Library of
Medicine, searched October, 1997 [Record No. 25939]
Pinto-Scognamiglio, W. (1967) Current knowledge
on the pharmacodynamic activity of the prolonged administration
of thujone, a natural flavoring agent. Boll. Chim. Farm.,
106(5), 292-300
Reese, K.M. (1997) Internet, World Wide Web
put absinthe within reach. Chem. Eng. News, 75(43),
80
Rice, K.C. & Wilson, R.S. (1976) (-)-3-Isothujone,
a small nonnitrogenous molecule with antinociceptive activity
in mice. J. Med. Chem., 19(8), 1054-1057
Rogers, J.A. (1981) Essential oils. In: Mark,
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M., eds., Kirk-Othmer Encyclopedia of Chemical Technology,
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Portland, OR, p. 1349
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& Stoichev, S. (1967) Cytotoxic effect of carbonyl compounds
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1213-1216
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