EUGENOL
Explanation
This substance was evaluated for acceptable daily intake for man
by the Joint FAO/WHO Expert Committees on Food Additives in 1967, and
1979 (see Annex I, Refs. 14 and 51). A toxicological monograph was
issued in 1980 (see Annex I, Ref. 52).
Additional data have become available and are summarized and
discussed in the following monograph. The previous monograph has been
expanded and is reproduced in its entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Absorption, metabolism and distribution
Intraperitoneal injection of a single 450 mg/kg dose of 14C
methoxy labelled eugenol resulted in rapid distribution to all organs.
Both ether- and water-soluble materials were recovered from most
tissues and excretions. Only 0.2-1.0% of the dose was eliminated as
expired 14CO2 (Weinberg et al., 1972). Over 70% of a lethal dose of
eugenol was recovered on death, from the urine of rabbits (Schroder &
Vollmer, 1932).
Administration of single 200 mg doses to rats led to increased
urinary output of ethereal glucuronides of 33-35 mg/rat in 12 hours
compared to a control value of 4 mg/rat. Ester glucuronide values were
unchanged (Yuasa, 1974).
Studies carried out in liver microsomal preparations from male
and female Fischer rats and CD-1 mice showed that formation of eugenol
2',3-epoxide from eugenol occurred in just detectable amounts (Swanson
et al., 1981).
Epoxidization of eugenol by rat liver cell cultures has been
reported. The dihydrodiol metabolite of eugenol has been isolated from
liver homogenates and urine of rats pretreated with eugenol. These
metabolites may arise from the action of epoxide hydrase on the
eugenol epoxide (Delaforge et al., 1980).
Incubation of eugenol with rat liver epithelial cells resulted in
production of 4-(2'-3'-dihydroxy)propyl-2-methoxyphenol (Janiaud,
1936).
Effects on enzymes and other biochemical parameters
The pharmacological effects of eugenol include the previously
reviewed inhibition of ß-D-glucosiduronic acid conjugation in rats
receiving 150 mg/animal (Hartiala et al., 1966).
Hydroxylating activity of liver homogenate on dimethylaminopyrine
or hexobarbital was depressed in tissue from mice dosed with 160 mg/kg
of eugenol and sacrificed after 1 hour (Jaffe et al., 1968).
Eugenol had no effect on aminopyrine-N-demethylation activity in
the liver of rats given about 10% of the LD50 3 times daily for 2-3
days. There was a slight decrease in hexobarbital lateral deflection
time and in urinary ascorbic acid content (Gruebner, 1972).
Eugenol was reported to inhibit respiration in vitro in
mitochondria isolated from the liver of adult male, Charles River
rats. Concentrations of from 0.11 to 3.50 mM of eugenol were present
in the suspension medium; inhibition of respiration began at
concentrations of 0.88 mM (Cotmore et al., 1979).
At 1 mM concentration, eugenol was reported to cause a 61%
inhibition of noradrenaline induced oxidative metabolism in isolated
brown fat cells from adult hamsters (Peterson et al., 1980).
Intraperitoneal injection of 200 mg/kg eugenol induced
anaesthesia in male Swiss albino mice; the mean sleeping time in a
group of 10 dosed animals was 17 minutes. Two of the animals died
within 24 hours of treatment (Sell & Carlini, 1976). Intraperitoneal
administration of eugenol is also associated with hypothermia in rats
and myorelaxtion and anticonvulsant effects in mice (Dallmeier &
Carlini, 1981).
Eugenol is used as a dental analgesic (Tyler et al., 1977); the
compound relieves pain from irritated or diseased tooth pulp, but is
not a true local anaesthetic (Sticht & Smith, 1971).
Subcutaneous injection of 50 mg of eugenol daily for 7 days
(total dose 1365 mg/kg bw) to partially hepatectomized male, Charles
River rats had no effect on rate of liver regeneration (Gershbein,
1977).
Subcutaneous injection of 0.1 ml of purified eugenol in adult
Walter Reed white rats caused necrosis and inflammation at the
injection site (Webb & Bussel, 1981).
Other effects include:
Animal Dose Route Effect Reference
Dog ca. 50 mg/kg i.v. Choleresis Chabrol, 1931
Mice 100-340 mg/kg i.p. Reduction in rectal Caujolle &
temperature Meynier, 1960
50 mg/kg i.p. Increase in sleeping Seto & Kemp,
time 1969
pentobarbital - 131%
ethanol - 120%
Rat 100 mg/kg i.p. No effects on deMello
spontaneous motor et al.,
activity 1973
160 mg/kg i.p. Severe depression and
paralysis of hind
quarters
200 mg/kg i.p. Catatonia
Frog 0.1-100% Direct Blockage of Kozam, 1977
exposure transmission of
of nerve evoked impulses
in exposed
sciatic nerve
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
Groups of about 30 young adult female CD-1 mice were fed 0 or
0.5% eugenol in the diet for 12 months. Other groups received 0.05%
phenobarbitol or 0.5% dietary eugenol in addition to 0.05%
phenobarbitol in the drinking-water. The animals received control
diets for an additional 6 months following administration of the test
diets. In the groups given only phenobarbitol, 3/29 mice developed
liver tumours, while no liver tumours were found in the other 3
groups. The liver was the only organ examined for the occurrence of
tumours. A second experiment was conducted in which groups of 40 to 60
male and 40 to 60 female CD-1 mice were given 0 or 2.5 µmol of eugenol
twice weekly by gavage starting at 4 days of age and continuing until
35 days of age. The animals were then maintained without dosing until
the experiment was terminated at 14 months. No effect of treatment
upon the incidence of liver tumours was noted in either sex. In a
third study, groups of 40 to 50 male CD-1 mice were injected i.p. at
1, 8, 15 and 22 days of age with 0.63, 1.26, 2.52 and 5.04 µmol,
respectively, of eugenol or eugenol 2',3'-epoxide. As compared to
concurrent controls receiving the trioctanoin solvent only, neither
treatment group had an increased incidence of liver tumours (Miller et
al., 1979).
The ability of eugenol to promote skin tumours was studied using
groups of 20 female ICR/HA Swiss. One group was given a single
cutaneous initiating dose of 7,12-dimethylbenz(a)anthrecene (DMBA) to
the back. This group and another group not initiated with DMBA
received 3 times weekly cutaneous applications of 5 mg of eugenol for
63 weeks. No carcinomas were found in either group and no papillomas
were found in the animals receiving only eugenol. Three animals
developed papillomas in the group initiated with DMBA and also treated
with eugenol. Two papillomas and 1 carcinoma developed in control
animals initiated with DMBA and then treated 3 times weekly with DMSO,
the solvent control (Van Duuren et al., 1966). In another study
carried out in the same laboratory, eugenol was reported to have a
partial inhibitory action on the carcinogenicity of benzo(a)pyrene
when the compounds were applied together in a carcinogenic skin
painting study (Van Duuren & Goldschmidt, 1976).
In a limited study in mice, eugenol did not potentiate the
tumorigenic effects of methylcholanthrene (Hitchcock, 1952).
Groups of 50 male F-344 rats were given 3000 or 6000 ppm (0.3 or
0.6%) of eugenol in the diet for 103 weeks. The animals were given
diets without eugenol for an additional week prior to the terminal
sacrifice. Groups of 50 female F-344 rats were fed diets containing
6000 or 12 500 ppm (0.6 or 1.25%) of eugenol in the diet for 103
weeks. The animals were given diets without eugenol for an additional
1 or 2 weeks prior to the terminal sacrifice. Concurrent control
groups of 40 males and 40 females were maintained on diets without
eugenol for 105 weeks. There appeared to be a dose-related effect on
weight gain, especially in the females. Small decreases in feed
consumption were also noted in the dosed animals. There was no
significant compound-related effect on survival. Endometrial stromal
polyps of the uterus were found in increased incidence in female rats.
The incidence was 6/40, 6/50 and 16/50 in the controls, low and high
doses, respectively. Incidence of alveolar-bronchiolar adenomas of the
lung in males was 0/40, 5/49 and 2/50 in the controls, low dose and
high dose, respectively. The historical incidence of this tumour in
male F-344 control rats at the performing laboratory is 6/299 (2%). No
statistically significant increase in this tumour was observed in high
dose males or in any of the female groups. C-cell adenomas of the
thyroid gland were observed at the following incidence in females:
3/40, 11/49 and 2/50 in the controls, low dose and controls,
respectively. The increase was statistically significant in the low
dose animals. There was no increased incidence of this tumour at
either dose in the males. The conclusion of the report stated that
eugenol was not carcinogenic to rats (NTP, 1980).
Groups of 50 male and 50 female B6C3F1 mice were given 0, 3000 or
6000 ppm (0, 0.3 or 0.6%) of eugenol in the diet for 103 weeks.
Animals were maintained on a control diet for another 2 weeks prior to
terminal sacrifice. A small dose-related decrease in weight gain was
noted for both males and females throughout the study. No compound-
related clinical signs were reported; however, survival was somewhat
lower in high dose males and low dose females but the effect was not
statistically significant. The incidence of hepatocellular tumours
(carcinoma, adenoma) was 14/50, 39/45 and 19/49 in the controls, low
dose and high dose males. The corresponding incidence in females was
2/50, 7/49 and 9/49. In the males, the low dose, but not the high
dose, had a statistically significant increase in hepatocellular
tumours. The conclusion of the report stated that there was evidence
that eugenol increased liver tumours in B6C3F1 mice; however, the
results were judged to be equivocal because of the limited weight of
this evidence (NTP, 1981).
Special studies on mutagenicity
Eugenol was negative in a Salmonella assay employing 4 mutant
strains (TA-1530, TA-1531, TA-1532, TA-1964) both directly and after
the use of mouse liver postmitochondrial fraction for activation. It
was also inactive in a host-mediated assay (Green & Savage, 1978).
Eugenol was also reported not to be mutagenic in Salmonella
TA-100 in a liquid suspension assay with or without an S-9 fraction
from Aroclor induced rat liver (Eder et al., 1980). Negative results
were also reported with eugenol using Salmonella strains TA-98,
TA-1535 and TA-100 in the plate assay system with or without liver
activation from Aroclor or 3-methyl cholanthrene induced rats. The
2',3'-epoxide of eugenol was mutagenic for strain TA-1535 in the
absence of a liver activation system (Swanson et al., 1979).
Eugenol was reported not to be mutagenic to Salmonella strains
1535, 1537 or 1538 with or without a rat liver activation system. The
2',3'-epoxy derivative of eugenol was mutagenic to strain TA-1535 with
or without liver activation (Delaforge, 1977).
The acute toxic effects include desquamation of the gastric
mucosa (Hitchcock, 1952), and punctate haemorrhages in dogs (Hartiala
et al., 1966), gastric inflammation and depression of secretory
capacity (Sober, 1950), liver discoloration and mottling in rats
(Taylor et al., 1964) liver congestion in dogs (Lauber & Hollander,
1950).
Acute toxicity
LD50
Animal Route (mg/kg bw) Reference
Mouse Oral 3 000 Jenner et al., 1964
i.p. 500 Caujolle & Meynier, 1960
i.p. 630 Fujii et al., 1970
Rat Oral 1 930 Sober et al., 1950
Oral 2 680 Taylor et al., 1964
Guinea-pig Oral 2 130 Jenner et al., 1964
Subchronic studies
Ten male and 10 female rats given 89.7 mg/kg eugenol for 12 weeks
showed no adverse effects (Trubek Laboratories, 1958).
Twenty male rats were given increasing doses from 1400 to
4000 mg/kg bw for 34 days. There was considerable mortality, slight
liver enlargement and adrenal enlargement. Histology showed enlarged
liver cells. The forestomach showed moderately severe hyperplasia and
hyperkeratosis of the stratified squamous epithelium with focal
ulceration (Hagan et al., 1965).
In another study, groups of 10 males and 10 females were fed
diets containing 0, 0.1 and 1.0% eugenol for 19 weeks without any
adverse effect on growth rate, haematology, organ weights and
histology of major tissues (Hagan et al., 1967).
Groups of 5 male and 5 female F-344 rats were fed 6000, 12 500,
25 000, 50 000 or 100 000 ppm (0.6, 1.25, 2.5, 5 or 10%) of eugenol in
the diet for 14 days. There were no concurrent controls. One high dose
male and all high dose females died during the study. There appeared
to be a dose-related reduction in weight gain (NTP, 1981).
Groups of 10 male and 10 female F-344 rats received 0, 800, 1500,
3000, 6000 or 12 500 ppm (0, 0.08, 0.15, 0.3, 0.6 or 1.25%) of eugenol
in the diet for 90 days. There was no compound-related effect reported
on mortality or gross or microscopic pathology. Relative to controls,
weight gain was reduced 12% in the high dose males (NTP, 1981).
Groups of 5 female and 5 male B6C3F1 mice were given 6000,
12 500, 25 000, 50 000 or 100 000 ppm (0.6, 1.25, 2.5, 5, or 10%) of
eugenol in the diet for 14 days. There were no concurrent controls.
There was a dose-related decrease in weight gain in both males and
females. All 5 of the males in the 100 000 ppm (10%) group died before
the end of the study. In the females, all of the 100 000 ppm (10%)
group died before the end of the study (NTP, 1981).
Groups of 10 male and 10 female B6C3F1 mice were given 0, 400,
800, 1500, 3000 or 6000 ppm (0, 0.04, 0.08, 0.15, 0.3 or 0.6%) of
eugenol in the diet for 13 weeks. There was no mortality, compound-
related gross or microscopic pathology (NTP, 1981).
Comments
Acutely, high doses of eugenol are hepatoxic to dogs and rats.
Metabolic data is limited. Formation of small amounts of eugenol
2',3-epoxide from eugenol using in vitro systems has been reported.
Mutagenicity tests using Salmonella strains with and without
activation gave negative results for eugenol, although the
2',3-epoxide compound was active in these systems. Eugenol was not
carcinogenic in lifetime study in the rat. In a lifetime study with
B6C3F1 mice, there was evidence of an increased incidence of liver
tumours. Statistical analysis of the results suggested a positive
trend in male mice. Although the incidence of tumours in the female
mice was increased at the low dose level, the effect was not dose
related. The significance of this result is difficult to interpret and
has been judged to be equivocal. In this respect, it is important to
note that in studies in CDI mice which compared the carcinogenic
potential of eugenol with that of structurally-related compounds,
namely estrogal and suffrole, eugenol was negative, whereas suffrole
was positive. Thus most of the available evidence indicates that
eugenol is not carcinogenic.
No studies on teratology or reproduction are available.
The lifetime feeding study in the rat provides additional
information for evaluating an acceptable daily intake for man. This
data supports converting the previous temporary acceptable daily
intake to an acceptable daily intake.
EVALUATION
Level causing no toxicological effect
Rat: 250 mg/kg bw in the diet.
Estimates of acceptable daily intake for man
0-2.5 mg/kg bw
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See Also:
Toxicological Abbreviations
Eugenol (FAO Nutrition Meetings Report Series 44a)
Eugenol (WHO Food Additives Series 14)
EUGENOL (JECFA Evaluation)
Eugenol (IARC Summary & Evaluation, Volume 36, 1985)