(+)-and (-)CARVONE
First draft prepared by Dr J.C. Larsen
Institute of Technology, National Food Agency of Denmark
1. EXPLANATION
(+)-Carvone and (-)carvone were evaluated for acceptable daily
intake for man (ADI) by the Joint FAO/WHO Expert Committee on Food
Additives (JECFA) in 1967, 1979, 1981, 1986 and 1988 (Annex 1,
references 15, 50, 56, 62, 73, 83). Toxicological monographs have
been issued in 1968 and 1980 (Annex 1, references 15 and 51). At
the twenty-third meeting of the committee (Annex 1, reference 50) a
temporary ADI of 0-1 mg/kg of body weight was allocated (as sum of
the isomers), pending submission of further relevant biochemical and
metabolic studies. This temporary ADI was continued by the twenty-
fifth meeting (Annex 1, reference 56). At the twenty-seventh
meeting (Annex 1, reference 62) the committee was informed that
lifetime studies with (+)-carvone in rats and mice were in progress.
The committee therefore extended the temporary ADI, pending
submission of the results from these studies. The temporary ADI was
again extended at the thirtieth and the thirty-third meetings of the
committee still awaiting the results of the long term studies.
Since the previous evaluation a long term study in mice with
(+)-carvone has become available along with other studies. The
existing monograph has been extended and is presented in its
entirety.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Carvone was found to be present in the urine of healthy adult
humans. The authors stated that urine profiles were compared over a
period of two months. The diets consumed by the subjects were not
discussed (Zlatkis & Liebich, 1971; Zlatkis et al., 1973a,b).
2.1.2 Biotransformation
When there is unsaturation in the ring, hydroxylation of that
ring is the primary metabolic pathway (F.E.M.A., 1976). This has
been shown to be true for carvone (Hildebrandt, 1902; Williams,
1959). Carvone was metabolized by the rabbit to 1,5-dimethyl-1,5-
hexadien-1,6-dicarboxylic acid and a carbinol in which one ethylene
linkage was saturated and the keto group was reduced (Fischer &
Bielig, 1940; Opdyke, 1973; Opdyke, 1978).
2.1.3 Effects on enzymes and other biochemical parameters
When (-)carvone was administered orally to male albino rats at
a dose level of 600 mg/kg bw/day for three days no effects were
observed on the levels of either cytochrome P-450 or cytochrome b5
(Moorthy et al., 1989).
Carvone (isomer not specified) was administered to three or
four male Wistar rats for 14 days at a dietary level of 1%,
corresponding to approximately 500 mg/kg bw/day. Increased serum
cholesterol and triacylglycerol levels were seen in the dosed rats
compared to controls. Decreased food consumption and body weight
gain were observed over the 14-day period (Imaizumi et al., 1985).
Groups of ten 6-week old male Wistar rats were administered
(+)-carvone or (-)-carvone at levels of 80 or 160 mg/kg bw/day for
28 days. Plasma creatinine was determined in all rats on days 0, 13
and 28 using a specific HPLC-method. A dose related decrease in
plasma creatinine was observed on day 28 in the rats receiving (+)-
carvone. (-)-Carvone had no effect (Lam, 1988).
2.2 Toxicological Studies
2.2.1 Acute toxicity studies
LD50
Species Route (mg/kg b.w.) References
Mouse s.c. 2675 Wenzel & Ross, 1957
Rat Oral 1640a Jenner et al., 1964
Rat oral 3710b Opdyke, 1978
Guinea-Pig Oral 766c Jenner et al., 1964
Rabbit Dermal 3860 Opdyke, 1978
a Oral LD50 reported on "carvone", identified as
p-mentha-6,8-dien-2-one.
b Oral LD50 reported on (+)-carvone.
c Dermal LD50 reported as 4 ml/kg body weight for
(+)-carvone.
Single intraperitoneal injections of (+)-carvone to male Swiss-
Webster albino mice did not result in any observed hepatotoxic or
pulmonary effects. (+)-Carvone was administered in graded doses
(not given) up to 800 mg/kg body weight at which level significant
convulsive effects occurred (Gordon et al., 1982).
2.2.2 Short-term studies
2.2.2.1 Mouse
Groups of five B6CBF1 mice (7-8 weeks old) of each sex were
administered 0, 150, 328, 723, 1,590 or 3,500 mg/kg body weight (+)-
carvone in corn oil by gavage, 5 days per week, total 12 doses over
16 days. Animals were housed five per cage. Water and feed were
given ad libitum. The mice were observed twice per day and were
weighed before the study and on days 1, 7 and 14 and at the end of
the study. A necropsy was performed on all animals. All mice
receiving 1,590 or 3,500 mg/kg body weight died on day 2 or 3. At
necropsy the mean body weights of the treated animals were similar
to controls. Ataxia, impaired grasping reflex, ocular discharge,
corneal opacity, body tremors, prostration, gasping, clonic or tonic
convulsions, or impaired righting reflex were observed at 1,590 and
3,500 mg/kg body weight; and lacrimation, piloerection,
hypoactivity, bradypnea, or ptosis were observed at 723, 1,590 and
3,500 mg/kg body weight. Relative liver weights were increased in
all groups of dosed male mice and relative thymus weights were
decreased in all groups of dosed female mice. Relative weights of
brain, heart, right kidney, lungs and bronchi were not changed.
Histological examinations were carried out on all animals in the
control group and the highest dosed group. However, the lungs and
livers of all dosed mice were examined. No compound-related changes
were seen (details on pathology not given) (NTP, 1989).
Groups of 10 female and 30 male B6CBF1 mice (5-7 weeks old)
were given 0, 93, 375 or 1,500 mg/kg body weight (+)-carvone in corn
oil by gavage, 5 days per week, for 13 weeks. The 20 extra males
per group were included to be used for other studies, and except the
highest dosed group they are not relevant for this evaluation.
Additional groups of 10 mice of each sex were administered 187 or
750 mg/kg body weight on the same schedule. Animals were housed
five per cage and feed and water were available ad libitum. The
mice were observed twice per day. The animals were weighed at the
beginning of the study and then once per week, and at the end of the
study. A necropsy was performed on all animals. All male and 8/10
female mice that received 1,500 mg/kg body weight and 1/10 males at
93 mg/kg body weight died during the first week of the study. An
additional female mouse died during week 12 of the study. Final
mean body weights of dosed and control animals were similar.
Hypoactivity, ataxia, and hypersensitivity to touch were seen at
1,500 mg/kg body weight. The female that received 1,500 mg/kg body
weight and survived to the end of the study had body tremors,
hypersensitivity to touch, and impaired hind limb function during
the study. The relative liver weights for male and female mice that
received 750 mg/kg body weight were significantly greater than of
vehicle controls. Relative weights of brain, thymus, lungs/bronchi,
heart, right kidney, and right testis (males) were not altered.
Histological examinations were carried out on vehicle controls, mice
receiving 750 mg/kg body weight, and all animals that died or were
killed before the end of the study. No compound-related changes
were seen (details on pathology not given) (NTP, 1989).
2.2.2.2 Rat
Groups of five male and five female Osborne-Mendel rats were
maintained on diets containing carvone (p-mentha-6,8-dien-2-one)
(isomer not specified) at a level of 1000 ppm (approximately
equivalent to 50 mg/kg bw/day) for 27-28 weeks, and at a level of
2500 ppm (approximately equivalent to 125 mg/kg bw/day) for one
year. No adverse effects were observed in these rats as judged by
growth, appearance, food intake, haematology, final body weights, or
gross and microscopic examination of the major organs. Rats
maintained on a diet containing carvone at a level of 10,000 ppm
(approximately equivalent to 500 mg/kg bw/day) for 16 weeks showed
growth depression and testicular atrophy (Hagan et al., 1967).
Total doses of 2450-2950 mg carvone/kg body weight (isomer not
specified) through seven daily subcutaneous injections were not able
to increase liver regeneration in five to 15 partially
hepatectomized Charles River rats (Gershbein, 1977).
Groups of five F344/N rats (6-7 weeks old) of each sex were
administered 0, 150, 328, 723, 1,590 or 3,500 mg/kg body weight (+)-
carvone in corn oil by gavage, 5 days per week, for 12 doses over 16
days. The animals were housed five per cage and water and feed were
given ad libitum. The rats were observed twice per day and were
weighed on days 1, 7 and 14 and at the end of the study. A necropsy
was performed on all animals. All rats that received 1,590 or 3,500
mg/kg body weight died during the first week of the study. The male
rats that received 723 mg/kg body weight had 9% lower mean necropsy
bodyweight than controls. Relative liver weights for male rats in
the group receiving 723 mg/kg body weight and the female mice in the
groups receiving 328 or 723 mg/kg body weigh were significantly
higher than those of the vehicle controls. Relative kidney weights
for rats of either sex that received 328 or 723 mg/kg body weight
were significantly higher than those for the vehicle controls. The
relative weights of the thymus of male rats at 723 mg/kg body weight
and of brain in female rats at 723 mg/kg body weight were decreased.
No histopathology was reported (NTP, 1989).
Groups of 10 female and 30 male F 344/N rats (4-5 weeks old)
were administered 0, 93, 375, or 1,500 mg/kg body weight (+)-carvone
in corn oil by gavage, 5 days per week, for 13 weeks. The 20 extra
males per group were included to be used for other studies, and are,
except those in the high dose group, of no relevance for this study.
Additional groups of 10 rats of each sex were included at dose
levels of 187 or 750 mg/kg body weight on the same schedule.
Animals were housed five per cage and allowed feed and water ad
libitum. The rats were observed twice per day and were weighed at
the beginning of the study, once per week thereafter, and at the end
of the study. A necropsy was performed on all animals. All rats
that received 1,500 mg/kg body weight died during the first weeks
and 9/10 males and 10/10 females that received 70 mg/kg body weight
died before the termination of the study (weeks 7-12). Final mean
body weights of male rats that received 187 or 375 mg/kg body weight
were lower (7% or 11%, respectively) than those of the controls.
The relative liver weights for dosed males and females that received
93, 187 or 375 mg/kg body weight were increased. The relative
kidney weights of males administered 187 or 375 mg/kg, and of
females that received 375 mg/kg body weight, were also increased.
The relative weight of the right testis and lungs/bronchi of males
were increased at dose levels of 187 or 375 mg/kg body weight. In
females receiving 375 mg/kg body weight the relative brain weights
were decreased.
Histopathology was performed on vehicle controls, rats
receiving 375 mg/kg body weight and all animals dying or killed
before the end of the study. Testicular degeneration was seen in
the male rats at 750 mg/kg body weight. This lesion was
characterized by moderate to marked loss of germinal epithelium in
the seminiferous tubules and an absence of sperm in the epididymal
ducts. In some rats, syncytial giant cells of spermatids were
present in seminiferous tubules. Atrophy of the thymic cortex was
present in rats of each sex which received 750 mg/kg body weight.
Hyaline droplets and granular casts commonly associated with the
accumulation of alpha2u-globulin were not observed in the kidney
tissue of dosed rats. No further information was given on
pathology.
A planned 2 year chronic toxicity/carcinogenicity study in rats
using doses of 0, 187 or 375 mg/kg body weight (+)-carvone in corn
oil by gavage, 5 days per week, had to be terminated after 60 weeks
due to low survival of dosed animals (males: vehicle control, 48/50;
low dose, 41/50; high dose, 16/50; females: 50/50; 48/50; 38/50).
No further details are given (NTP, 1989).
2.2.3 Long-term/carcinogenicity studies
2.2.3.1 Mouse
Groups of 50 male and 50 female B6C3F1 (C57BL/6N, female x
C3H/HeN MTV male) mice (7 week old) were administered 0, 375, or 750
mg/kg body weight (+)-carvone in corn oil by gavage, 5 days per week
for 103 weeks. Animals were housed five per cage. Feed and water
were available ad libitum. All animals were observed twice per
day for clinical signs. No clinical signs were observed. Body
weights were recorded once per week for the first 13 weeks of the
study, and then at least once per month thereafter. The mean body
weights of dosed and control male mice were similar throughout most
of the study; the mean body weights of the dosed female mice were
within 7% of those of the vehicle controls throughout most of the
study. The survival of both the low dose (29/50 after week 101) and
the high dose (38/50 after week 92) groups of female mice was
significantly greater than that of the controls (14/50). This was
probably caused by a very high death rate in the control females.
The low survival of female vehicle control mice was related to a
high incidence (52%) of ovarian abscesses similar to those seen in
studies associated with Klebsiella sp. infections. Whether the
lower incidence of ovarian abscesses in dosed female mice was
related to (+)-carvone administration is uncertain. No differences
in survival were observed between any groups of male mice. A
necropsy was performed on all animals, including those found dead,
except for tissues that were autolyzed. All organs and tissues were
examined for grossly visible lesions and subjected to
histopathology.
Several lesions occurred in the nasal cavities of male and
female mice with dose-related increased incidences and/or severity.
Atrophy of the olfactory epithelium and hyperplasia of the
underlying Bowman's glands occurred together with high incidence in
either sex in both dosed groups.
The effect on the nasal mucosa was probably due to a local
effect of (+)-carvone caused by reflux of the gavage material when
the gavage needle was withdrawn, since inflammatory exudate and
evidence of the corn oil vehicle was found in over 50% of all dosed
and control animals. No increases in tumour incidences were seen in
mice administered (+)-carvone. The incidence of female control mice
with primary neoplasms was 9/50 and the total number of primary
neoplasms was 9. These numbers are low compared to 15/50 and 18 in
the low dose female mice and 14/50 and 18 in the high dose mice and
may be related to the early deaths of female control mice. The
incidences of male mice with primary neoplasms (control, 27/50; low
dose, 15/50; high dose, 16/50) and the total numbers of primary
neoplasms (control, 38; low dose, 18; high dose, 20) were
significantly lower in dosed groups than in vehicle controls. The
lowered incidence of tumours in dosed male mice was primarily due to
a lower incidence of subcutaneous fibromas, sarcomas, fibrosarcomas,
or neurofibrosarcomas (NTP, 1989).
2.2.4 Reproduction studies
No information available.
2.2.5 Special studies on in vitro toxicity
The toxicity of carvone (isomer not specified) to Ascites
sarcoma BP8 cells (originating from inoculated C3H mice) cultured in
suspension was examined. Carvone was incubated with the cells at
37°C for 48 hours at levels of 0.1 or 1.0 uM. The percent culture
growth inhibition at these levels was 13 and 100%, respectively
(Pilotti et al., 1975).
The effect of carvone (isomer not specified) on cell metabolism
was investigated by measuring the inhibition of noradrenaline-
induced oxidative metabolism in isolated hamster brown fat cells.
At a level of 1 mM carvone, there was a 42% inhibition of
noradrenaline-induced oxidative metabolism (Petterson et al.,
1980).
Carvone (isomer not specified) was reported to be ciliotoxic at
a concentration of 5 mM in chicken tracheal organ cultures, with
complete cessation of ciliary activity occurring six minutes after
the addition of carvone to the culture (Petterson et al., 1982).
Carvone, (isomer not specified), at a concentration of 25 mM,
was incubated for 30 minutes at 37 °C with labelled human lung
fibroblasts. The degree of membrane damage, as measured by the
release of an intracellular nucleotide marker, was significantly
increased, with 95% of the nucleotides being released in 30 minutes
(Thelestam et al., 1980).
When carvone (isomer not specified) was incubated with
microsomes from phenobarbital pretreated rats a 25% conversion of
cytochrome P-450 to cytochrome P-420 was observed. This conversion
was not observed when rats were dosed (dose not specified) with
carvone in vivo (Madyastha et al., 1985).
The eggs of the insect Earias vittella were arranged in
groups of 20 and exposed in vitro to various terpenoids.
Appropriate controls were conducted and each experiment was
replicated five times. Carvone (isomer not specified) did not
inhibit embryonic development at any of the concentrations (not
given) tested (Metha, 1979).
2.2.6 Special studies on carcinogenicity
Groups of female A/He mice were given intraperitoneal
injections of (+)- or (-)-carvone three times weekly for eight
weeks. The mice were then observed for a further 16 weeks until the
end of the study. The total doses given of each compound were 1200
or 6000 mg/kg body weight. No increases in tumour incidences in the
lung, liver, kidney, spleen, thymus, intestine, salivary or
endocrine glands were observed. Approximately 20 and 40% of the
(+)- and (-)-carvone treated animals, respectively, did not survive
until the end of the study regardless of dose. Approximately 20% of
the control animals died. The vehicle, tricaprylin, used to
administer carvone was found to be impure and resulted in a weight
loss of three to four grams in the control animals during the first
week of the study (Stoner et al., 1973).
A group of approximately 15 female A/J mice was administered
0.2 mmol (approximately 30 mg) (+)-carvone by gavage one hour before
oral administration of 20 mg/N-nitrosodiethylamine (NDEA)/kg body
weight. This dosing schedule was repeated once a week for eight
weeks, and the experiment terminated 26 weeks after the first dose
of NDEA. Forestomach tumour formation was significantly inhibited
in (+)-carvone treated mice, with more than a 63% reduction in the
mean number of papillomas per mouse compared to the controls. A
significant reduction (34%) in the number of pulmonary adenomas per
mouse was also observed in the (+)-carvone treated mice (Wattenberg
et al., 1989).
2.2.7 Special studies on genotoxicity
(+)-Carvone at concentrations up to 333 µg/plate was negative
for induction of gene mutations in Salmonella typhimurium strains
TA 100, TA 1535, TA 1537 and TA 98 when tested in a preincubation
assay in the presence or absence of Aroclor 1254 induced male
Sprague Dawley rat or Syrian hamster liver S9 (Mortelmans et al.,
1986; NTP, 1989).
(+)-Carvone induced slight increases in sister chromatid
exchanges and chromosomal aberrations in Chinese hamster ovary (CHO)
cells with and without addition of Aroclor 1254 induced male Sprague
Dawley rat liver S9. Results were statistically positive but in two
of three sister chromatid exchange trials there were no correlation
of dose with response. This phenomenon of no dose/response
relations also occurred in the second trial of the chromosomal
aberration test conducted without S9. In the chromosomal aberration
test without S9 a chemical-induced delay in cell cycle was observed.
This was not observed in the test for sister chromatid exchanges
(NTP, 1989).
2.3 Observations in humans
No irritation was observed in humans exposed to concentrations
of 1% (-)-carvone in petrolatum in a 48-hour closed patch test
(Opdyke, 1973). A maximization test was carried out on 25
volunteers. (-)-Carvone at a concentration of 1% in a petrolatum
produced no sensitization reactions (Opdyke, 1973).
In one of 80 patients suffering from the condition of orofacial
granulomatosis, characterized by swelling of the lips and lower half
of the face, an association with intolerance to carvone (isomer not
specified) was indicated from patch testing (Patton, et al.,
1985).
Three out of 33 patients tested for contact allergy to
toothpaste flavors, experienced a positive allergic response in a
patch-test using 5% carvone (isomer not specified) in petrolatum
(Anderson, 1978).
3. COMMENTS
At its present meeting, the Committee considered that optical
enantiomers should not per se be regarded as toxicologically
identical compounds, and therefore (+)-carvone and (-)-carvone
should be evaluated separately. Although both compounds were placed
on the agenda, sufficient data for toxicological evaluation had been
submitted only for (+)-carvone.
In a long-term toxicity/carcinogenicity study in mice in which
(+)-carvone was administered by gavage, no evidence of
tumorigenicity was observed. Additional short-term studies in mice
and rats, in which (+)-carvone was administered by gavage, and in
vitro mutagenicity tests, were also considered . Rats appear to
be more susceptible to (+)-carvone than mice. The no-observed-
effect level for (+)-carvone in rats was 93 mg per kg of body weight
based on the study in which the substance was administered by gavage
for 3 months. The Committee noted that, in an earlier 1-year
feeding study in rats in which a no-effect-level of 125 mg per kg of
body weight per day was observed, it was not specified which isomer
had been used.
The Committee noted that only limited data were available on
the metabolism and pharmacokinetics of (+)-carvone.
4. EVALUATION
An ADI of 0-1 mg per kg of body weight per day was established
for (+)-carvone based on a no-observed-effect level of 93 mg per kg
of body weight per day in rats. The temporary ADI for (-)-carvone
was not extended, because insufficient data were available for
toxicological evaluation.
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