928. Anethole, trans- (WHO Food Additives Series 42)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

WORLD HEALTH ORGANIZATION

SAFETY EVALUATION OF CERTAIN
FOOD ADDITIVES

WHO FOOD ADDITIVES SERIES: 42

Prepared by the Fifty-first meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)

World Health Organization, Geneva, 1999
IPCS - International Programme on Chemical Safety

trans-ANETHOLE (addendum)

First draft prepared by
E.J. Vavasour
Chemical Hazard Assessment Division, Bureau of Chemical Safety, Food
Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario,
Canada

Explanation
Biological data
Biochemical aspects
Biotransformation
Effects on enzymes and other biochemical parameters
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Long-term studies of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Special studies on immunotoxicity
Observations in humans
Comments
Evaluation
References

1. EXPLANATION

trans-Anethole was first evaluated by the Committee at its
eleventh meeting (Annex 1, reference 14); after re-evaluation at the
twenty-third meeting (Annex 1, reference 50), it was allocated a
temporary ADI of 0-2.5 mg/kg bw. At the thirty-third meeting (Annex 1,
reference 83), the temporary ADI was reduced to 0-1.2 mg/kg bw on
account of the tumorigenic effects observed in the livers of female
rats in a 27-month study. The Committee requested further details of
the long-term study in rats and metabolic and mechanistic information,
and further noted that a long-term study in mice and an
epidemiological study might be required. At the thirty-seventh meeting
(Annex 1, reference 94), the temporary ADI for trans-anethole was
reduced to 0-0.6 mg/kg bw after consideration of the results of three
independent reviews of the histological changes in the livers of rats
in the long-term study. At that time, a recommendation was made for
further metabolic and pharmacokinetic studies, tests for chromosomal
aberration, and a test for gene mutations in mammalian cells
in vitro; depending on the results of these studies, a long-term
dietary study in mice and a study of reproductive toxicity or
teratogenicity might be required. At the thirty-ninth and forty-ninth
meetings (Annex 1, references 101 and 131), the Committee further
extended the temporary ADI of 0-0.6 mg/kg bw, pending completion of
the recommended studies.

At the present meeting, the results of new 90-day studies were
evaluated, together with those of studies on the metabolism of
trans-anethole in mice and rats, studies on the effects of
short-term dietary administration of trans-anethole on hepatic
enzyme induction in these species, cytotoxicity and genotoxicity
in vitro, and some epidemiological data that had become available
since the thirty-seventh meeting. These studies provided information
on the significance to humans of the results of the long-term study in
rats. In addition, studies on reproductive toxicity and immunotoxicity
were reviewed. The results of a long-term study in mice, which had
been requested at the thirty-ninth meeting, were not available.

These data were reviewed and are summarized in the following
monograph addendum.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.2 Biotransformation

The effect of dose, sex, and pre-feeding of trans-anethole on
its metabolic disposition was determined in Sprague-Dawley rats and
CD-1 mice. Groups of six animals received trans-anethole in the diet
for three weeks at concentrations of 0.05, 0.1, 0.25, or 0.5% (mouse)
and 0.1, 0.25, 0.5, or 1.0% (rat). Control groups for each treated
group received untreated diet. Each animal was then given a single
oral dose of [¹⁴C- side chain-1]- trans-anethole (purity 97%) which
was equal to the daily intake from consuming treated diet: 62, 140,
300 and 430 mg/kg bw, respectively, in mice and 100, 250, 520, and
1000 mg/kg bw, respectively, in rats. The control animals received the
same dose of ¹⁴C- trans-anethole as the corresponding treated group.
After the oral dosing, the rodents were placed in metabolism cages for
four days for the collection of urine and faeces. The amount of
radioactive label excreted was quantified, and individual urinary
metabolites were quantified and identified by high-performance liquid
chromatography (HPLC)-mass spectrometry.

Pretreatment with trans-anethole resulted in persistently lower
body weights in both male and female mice receiving the two highest
dietary concentrations and a transient reduction in body-weight gain
in the males receiving 0.1% for the first week. In rats, the body
weights of males at the three highest dietary concentrations and of
females at the two highest concentrations were significantly lower
than those of controls, and the body weights of females fed the next
lowest dose (0.25%) were significantly lower during the initial week
of the feeding period. In all groups of both species, recovery of
radiolabel was essentially quantitative, indicating that
trans-anethole was completely absorbed, metabolized, and excreted.
The main route of excretion was the urine, comprising > 90% of the
administered dose in rats; recovery of trans-anethole in the urine
of mice represented 70-90% of the total dose, but some faecal

absorption of urine was suspected. Most of the radiolabel was excreted
within the first 24 h. Fifteen urinary metabolites were identified and
quantified.

In mice, no statistically significant change was observed in the
rate of ¹⁴C elimination over the dose range of 62-430 mg/kg bw per
day, and there was no significant metabolic shift from
O-demethylation to side-chain oxidation/epoxidation, although the
metabolic profiles within pathways varied conside-rably with
increasing dose. After pre-feeding, a greater proportion was
metabolized through the O-demethylation pathway than in controls
(20-25% for controls, 24-33% for pre-fed animals). The proportion of
trans-anethole metabolized by side-chain epoxidation was increased
in pre-fed mice of each sex receiving the highest dose in comparison
with lower doses (9 and 12% at 430 mg/kg bw versus 7 and 7% at 62
mg/kg bw for pre-fed males and females, respectively). This was
attributed to an increase in the amount of metabolite derived from the
glutathione conjugate of the diol,
S-[1-(4'-methoxyphenyl)-2-hydroxypropane]- N-acetylcysteine (7 and
3% versus 1 and 1% in pre-fed females and males receiving the highest
and lowest doses, respectively). The amount of this metabolite was
also increased in the pre-fed mice receiving the high dose over that
in their controls. The amounts of metabolites derived from
omega-side-chain oxidation did not vary significantly with dose
(67-74% of total urinary metabolites), although there was a tendency
for increased excretion of 4-methoxybenzoic acid and decreased
excretion of 4-methoxycinnamoyl-glycine with increasing dose (except
in male control mice), showing a decreasing tendency for glycine
conjugation with dose. In this regard, the females had a reduced
capacity for glycine conjugation in comparison with males.

The overall rate of ¹⁴C excretion decreased significantly with
increasing dose in the control rats. This was attributed by the author
to overload of the mechanisms involved in the metabolism and excretion
of trans-anethole and implied that pre-feeding increased the
capacity of the rats to eliminate high doses. In both pre-fed and
control rats, the metabolism of trans-anethole shifted from
O-demethylation to side-chain oxidation and epoxidation with
increasing dose. The proportion of metabolites arising from
O-demethylation was significantly decreased, from 39-43% at 100
mg/kg bw to 22-32% at 1000 mg/kg bw. The overall decrease was due in
large part to a decrease in the amount of 4-hydroxypropenylbenzene
metabolites, suggesting saturation of the phase-I enzymes responsible
for the initial demethylation step. There was a significant increase
with increasing dose in the proportion of the dose excreted as
metabolites of side-chain epoxidation (12-18% in groups receiving 100
mg/kg bw versus 20-23% in groups receiving 1000 mg/kg bw). This
increase was the result of increased production of trans-anethole
diol stereoisomers and 4-methoxyphenylacetone, the immediate products
of epoxide hydration. The proportion of the glutathione conjugate,
S-[1-(4'-methoxyphenyl)-2-hydroxypropane]- N- acetylcysteine, did
not change with increasing dose. The increase in the amounts of diols
produced from the cytosolic epoxide hydrolase pathway at the highest

dose suggests that the glutathione S-transferase capacity was
overwhelmed. Female rats in the control groups formed more epoxidation
metabolites than did control males at the three lower doses, which was
reflected in greater amounts of mercapturic acid metabolites derived
from glutathione conjugation. This sex-related difference was less
apparent in the pre-fed animals, suggesting an induction of the
capacity for glutathione conjugation. Side-chain oxidation increased
with increasing dose (42-45% of metabolites at 100 mg/kg bw to 47-56%
at 1000 mg/kg bw), although the amount of the major metabolite,
4-methoxy-hippuric acid, was significantly decreased. The increase was
due mainly to increased production of 4-methoxybenzoic acid,
4-methoxycinnamic acid, and, to a lesser extent,
4-methoxycinnamoylglycine, suggesting saturation of both glycine
conjugation and the ß-oxidation pathways. The increased excretion of
4-methoxycinnamic acid and the decreased excretion of
4-methoxyhippuric acid were more marked in pre-fed animals than in
their respective controls. Both pre-fed and control female rats
excreted significantly less 4-methoxy-hippuric acid than males in the
corresponding groups at all doses, suggesting a sex-related difference
in the capacity for glycine conjugation (Bounds, 1994).

Racemic trans-anethole epoxide was incubated with water,
buffers, and rat liver microsomes to determine the relative
proportions of stereoisomers of anethole diol produced as compared
with those formed in vivo in rats given trans-anethole. The
formation of four stereoisomeric anethole diols was estimated from
HPLC peak areas. Some stereoselectivity was observed in the formation
of the diols from the epoxide, both in vivo and in vitro, which
favoured threo over erythro formation. The authors concluded that
further studies were required to determine whether greater influence
over stereochemical selectivity was exerted at the epoxidation or
hydration step of metabolism of trans-anethole (Ishida et al.,
1995).

The metabolic fate of [1'-¹⁴C]- trans-anethole was determined
in Sprague-Dawley rats and CD-1 mice given single doses of 250 mg/kg
bw by gavage. Faeces and urine were collected from groups of six
animals of each sex for four days. Urine samples for 24-h periods were
pooled for each species and their profiles determined by
reversed-phase HPLC before and after incubation with ß-glucuronidase
and/or sulfatase. In addition, hepatocytes isolated from male
Sprague-Dawley rats were incubated with 0.2-1.0 × 10^-3 mol/L
14C- trans-anethole, and samples of supernatant were collected at
regular intervals for 6 h and analysed by HPLC.

Radiolabel was recovered essentially quantitatively in both rats
and mice, most being excreted in the 24-h urine. trans-Anethole was
completely metabolized by animals of each species, and no unchanged
compound was excreted. Fifteen metabolites were detected in urine,
their structures elucidated, and their relative abundance estimated.
The authors proposed a metabolic pathway to explain the formation of
these metabolites.

Anethole undergoes three primary oxidation pathways:
O-demethylation, omega-side-chain oxidation, and side-chain
epoxidation, which accounted for 32, 28, and 41% of the metabolites,
respectively, in mice, and 37, 13, and 49%, respectively, in rats.
These initial steps are followed by a variety of secondary pathways of
oxidation and hydration, the products of which are extensively
conjugated with sulfate, glucuronic acid, glycine, and glutathione.
These metabolites included one that had not been identified
previously: an S-substituted cysteine conjugate, which was probably
formed through the epoxidation pathway by conjugation of the diol with
glutathione at the 1'-hydroxyl group. This metabolite comprised 16% of
the metabolites in 24-h urine from rats and only 3% of metabolites
from mice. Species differences were observed in the amounts of
trans-anethole metabolized via the three major pathways. Thus, the
O-demethylation and epoxidation routes were marginally more
prevalent in rats than in mice (37 and 49% versus 32 and 41%,
respectively), while proportionally more trans-anethole was
metabolized by omega-side-chain oxidation in mice (28% versus 13%).
Since O-demethylation is a deactivation pathway, the authors
suggested that the species differences in the toxicity of anethole
might be related to differences in the types and/or amounts of
metabolites generated through the omega-oxidation and epoxidation
pathways. They suggested that the epoxide was responsible, by
depleting glutathione and exerting cytotoxic activity at high
concentrations.

In isolated rat hepatocytes, 82% of trans-anethole was
metabolized within 6 h, and three major metabolites were identified:
4-methoxycinnamyl alcohol (1.6%), 4-methoxycinnamic acid (7%), and
4-methoxybenzoic acid (33%). These are all products of the
omega-oxidation pathway (Bounds & Caldwell, 1996).

2.1.3 Effects on enzymes and other biochemical parameters

Mice

Hexobarbital sleeping time was measured in mice which had been
pretreated with trans-anethole as a means of measuring its effect on
mixed-function oxidase activity. Groups of five male and five female
CD-1 mice were dosed by gavage with 60 mg/kg bw phenobarbital or 875
mg/kg bw anethole, daily for four days. Five animals of each sex
served as vehicle controls. On day 5, each animal was given 90 mg/kg
sodium hexobarbital intraperitoneally, and the time elapsing from loss
of the righting reflex until the animal could successfully right
itself from the supine position twice within 30 s was measured. Male
and female mice treated with phenobarbital had significantly shorter
sleeping times than mice treated with the vehicle. Treatment with
anethole had no effect on sleeping times in either male or female
mice, suggesting that trans-anethole does not induce the hepatic
microsomal enzymes responsible for metabolism of phenobarbital in mice
(Borriston Laboratories, 1982a).

The effects of dietary administration of trans-anethole on
end-points related to induction of hepatic enzymes were investigated
in CD-1 mice. Groups of 24 male and female mice received 0, 0.1, 0.25,
0.5, or 1% trans-anethole in the diet (equivalent to 190-1500 mg/kg
bw per day) for 22 days. After treatment, the mice were sacrificed,
and the livers were excised. Microsomal protein and cytochrome P450
content were assessed in hepatic microsomal preparations. Animals
given 1.0% trans-anethole were sacrificed prematurely because of
extreme body-weight loss; large dose-related body-weight decrements
(20-30% of control values) were also noted in male and female mice at
0.25 and 0.5%. The absolute liver weights were increased over those of
controls in mice at 0.1% and were decreased in those at 0.25 and 0.5%.
The relative liver weights were statistically significantly increased
over those of controls in mice of each sex receiving 0.1 or 0.25%. The
hepatic microsomal protein content, expressed as milligrams of protein
per gram of liver, was statistically significantly increased in males
at 0.25 and 0.5%, while the cytochrome P450 content (in nanomoles per
milligram protein) was increased significantly in males at the high
dose (110% of control value) and in females at the intermediate and
high doses (110 and 120% of control values, respectively).

In a subsequent study, pair-fed controls were added to exclude
the effects of caloric restriction on cytochrome P450 content. Groups
of 12 female mice received a diet containing 0.5% trans-anethole,
control diet, or appropriately restricted diet for 22 days. The
cytochrome P450 content of hepatic microsomes from mice receiving
trans-anethole in the diet was increased 33% over that of pair-fed
controls. The authors concluded that trans-anethole has a moderate
inductive effect on the cytochrome P450 content of mouse liver (Reed &
Caldwell, 1993; Reed, 1994).

Rats

The effect of anethole on the induction of hepatic microsomal
enzyme activity for O-demethylation of para-nitroanisole and
hydroxylation of aniline was determined in Sprague-Dawley rats. Groups
of five male and five female rats received the vehicle, 60 mg/kg bw
per day phenobarbital, or 875 mg/kg bw per day trans-anethole by
gavage for four days. The animals were then sacrificed, and the livers
excised and weighed. Microsomal suspensions were prepared from the
livers, and assays conducted to determine enzyme activities. Both the
absolute and relative liver weights of phenobarbital- and
anethole-treated rats were increased over those of controls, although
the difference was significant only for the relative weights.
Treatment with anethole resulted in the deaths of three male rats. At
necropsy, the livers of these animals were found to be rough and
pitted; the livers of two female rats were found to be discoloured.
The activity of aniline hydroxylase was elevated in the remaining male
rats treated with anethole; neither enzyme activity was elevated in
the female rats. Both O-demethylation and hydroxylation were
increased in male and female rats treated with phenobarbital
(Borriston Laboratories, 1982a).

trans-Anethole was administered to Sprague-Dawley rats either
intraperitoneally or in the diet in order to determine the effect on
hepatic cytochrome P450 content and related activities. When
administered intraperitoneally in trioctanoin at a dose of 300 mg/kg
bw per day for seven days, trans-anethole induced statistically
significant increases in relative liver weight (8%), microsomal
protein content (18%), and microsomal cytochrome P450 content (45%) in
comparison with vehicle controls. Intraperitoneal administration of
ß-naphthoflavone (50 mg/kg bw per day in trioctanoin for three days),
phenobarbital (80 mg/kg bw per day in saline for four days), or
isosafrole (150 mg/kg bw per day in trioctanoin for three days)
resulted in increased cytochrome P450 contents that were 96, 120, and
130% those of vehicle controls, respectively. The cytochrome
P450-dependent activity of 7-ethoxycoumarin O-deethylase was
increased by 69% in trans-anethole-treated rats in comparison with
vehicle controls and by 1600, 290, and 590% in the positive controls.

In a second study, trans-anethole was administered to groups of
eight male and eight female rats in the diet at concentrations of 0,
0.25, 0.5, or 1% (equivalent to 125-500 mg/kg bw per day) for 21 days.
The hepatic microsomal protein content (as milligrams of protein per
gram of liver) was increased over that in male (13, 17, and 29%,
respectively) and female (8, 15, and 27%, respectively) controls; and
the cytochrome P450 content (nanomoles per milligram protein) was
increased by 5, 23, and 28%, respectively, in males and 20, 42, and
69%, respectively, in females. The relative liver weights were also
increased over those of controls at the intermediate and high doses in
males (13 and 27%, respectively) and females (16 and 42%,
respectively). No data were supplied on absolute liver weights or body
weights, so that the relative weights could not be put in perspective.
The differences were statistically significant in all groups of
females mentioned and in males at the intermediate and high doses. The
authors concluded that the results showed moderate inducing activity
of anethole in rat liver, which was more marked in females for given
dietary concentrations (Reed & Caldwell, 1992a,b; Reed, 1994).

In order to ascertain whether the changes described above were
associated with cell proliferation, groups of three male and three
female rats receiving trans-anethole in the diet for 21 days were
given 20 µg/h of 5-bromo-2'-deoxyuridine subcutaneously via osmotic
minipumps for the last three days of treatment. The initial results
suggested an increased number of 5-bromo-2'-deoxyuridine-labelled
nuclei, indicating an increased number of cells passing though S phase
(Reed & Caldwell 1992b; Reed 1994).

The effects of trans-anethole on the activity of
drug-metabolizing enzymes was studied in vivo in male Wistar rats.
Groups of three rats received doses of 0 (vehicle control), 125, 250,
or 500 mg/kg bw per day for 10 days in corn oil by gavage. Because of
the deaths of two rats at the highest dose after two days, this group
was excluded from the study. At the end of treatment, blood samples
were collected for measurement of haematological parameters and some
clinical chemical parameters (alanine and aspartate aminotransferase

activities), livers were weighed, and cytosolic and microsomal
preparations were made from liver tissue. The microsomal content of
cytochrome P450 was determined, as were the activities of
7-ethoxyresorufin dealkylase, 7-pentoxyresorufin dealkylase,
UDP-transferase, glutathione- S-transferase, and DT-diaphorase.
Treatment of male rats with trans-anethole resulted in an apparently
dose-related decrease in body-weight gain when compared with controls,
although the difference was not statistically significant. Liver
weights were increased relative to body weight over those of controls
in a dose-related manner, which was significant for animals at 250
mg/kg bw per day, but this may have reflected the treatment-related
body-weight decrement. There was no effect on haematological
parameters or on alanine or aspartate aminotransferase activities. The
total cytochrome P450 content of the liver was also not affected by
treatment. The activities of the microsomal enzymes 7-pentoxyresorufin
dealkylase and ethoxyresorufin O-deethylase were slightly but not
significantly increased (less than twofold). Induction of some
cytosolic metabolizing enzymes was noted. Significant increases in
UDP-glucuronyl transferase activity were seen with the substrate
4-hydroxybiphenyl at the high dose (250 mg/kg bw per day) and with
4-chlorophenol at both doses. The activities of
glutathione- S-transferase and DT-diaphorase were also increased
after treatment with trans-anethole; the increases were significant
at both doses and at the high dose, respectively. The lack of effect
on induction of hepatic cytochrome P450 content in this study was
attributed by the authors to factors such as differences in route of
administration (diet versus gavage), strain, and sex (Rompelberg et
al., 1993).

trans-Anethole was tested for its ability to induce hepatic
microsomal enzyme activity in female Sprague-Dawley rats. Groups of
seven rats received doses of 0 (vehicle control), 75, or 300 mg/kg bw
per day of trans-anethole for four consecutive days in corn oil by
gavage. Homogenates were prepared from the liver of each animal and
assayed for the activity of the microsomal enzymes para-nitroanisole
demethylase, 7-ethoxycoumarin O-deethylase, and ethoxyresorufin
O-deethylase. Treatment had no effect on absolute or relative liver
weights; however, a twofold increase in the activities of
para-nitroanisole demethylase and ethoxyresorufin O-deethylase was
noted in microsomal preparations from rats at the high dose;
7-ethoxycoumarin O-deethylase activity was not increased in either
treated group when compared with controls. The authors concluded that
trans-anethole induced cytochrome P450 and P448 in female
Sprague-Dawley rats (Wenk, 1994).

2.2 Toxicological studies

2.2.1 Acute toxicity

After a single intraperitoneal administration of anethole to
Sprague-Dawley rats, the LD₅₀ was determined to be 738 mg/kg bw for
males and 703 mg/kg bw for females (Borriston Laboratories, 1984).

2.2.2 Short-term studies of toxicity

Mice

A range-finding study was conducted with CD-1 mice. Groups of
five male and five female mice were fed trans-anethole (purity,
> 99%) for an unspecified period (probably 28 days) at concentrations
in the diet intended to deliver doses of 60, 120, 240, 360, or 500
mg/kg bw per day. Control animals received untreated diet. The three
highest doses were achieved gradually over two weeks. The actual
intake was calculated to be 58, 120, 220, 290, and 440 mg/kg bw per
day in males and 59, 110, 240, 350, and 460 mg/kg bw per day in
females. The mice were observed twice daily, body weights twice
weekly, and food consumption daily. Blood samples were taken before
sacrifice on day 33 for determination of a standard range of
haematological and a limited number of clinical chemical parameters.
Gross necropsy was performed on all animals, including those which
died on test, and the weights of the brain, kidney, thymus, adrenal
gland, and liver/gall-bladder were determined at terminal sacrifice. A
range of tissues and organs were preserved for histopathological
examination, but only slides from the liver were examined.

Deaths were observed among 2/5, 3/5, and 2/5 male mice at the
three highest doses, respectively, and among 2/5 females at the
highest dose. Body-weight decrements exceeding 10% of control values
were noted in the same groups. Food consumption was reduced in all
groups except that at the lowest dose and was most pronounced in male
mice. The lower body weights and reduced food consumption were
probably the result of the unpalatability of the diet. A
treatment-related decrease in total leukocyte count, which was
statistically significant at the two highest doses, was noted in male
mice; although the total leukocyte counts were also lower in the
treated females, no treatment-related response was evident. As
differential leukocyte counts were not made, it was not possible to
identify the affected fraction. An increase in liver weight was seen
at doses just below those at which mortality and drastically reduced
body weights were observed. Histopathological examination of the
livers revealed no changes that could be attributed to treatment. The
results of this study indicate that the maximum doses that did not
compromise food intake to such an extent as to affect the health
status of the animals were 110 mg/kg bw per day for male mice and 350
mg/kg bw per day for female mice (Minnema, 1997a).

On the basis of the results of the above study, a 90-day feeding
study was conducted in mice at target doses for trans-anethole of
30, 60, 120, and 240 mg/kg bw per day. Groups of 20 male and female
mice received each of these doses in the diet; control groups received
untreated diet. Because of the known problems of unpalatability, the
two higher doses were achieved over two weeks. The actual doses
achieved during weeks 3-13 were within 2% of the target doses. The
animals were observed twice daily, and a thorough physical examination
was conducted weekly. Body weights were recorded twice weekly for five
weeks and weekly thereafter. Food consumption was recorded daily and

reported weekly. An ophthalmoscopic examination was conducted before
treatment and during week 12. Blood samples were collected for
haematological determinations at week 13 and for clinical chemical
parameters at week 14, after sacrifice. All animals were subjected to
gross necropsy. At terminal necropsy, the weights of 16 organs,
including the liver, were recorded, and 43 tissues and organs from
each animal were preserved for histopathological examination. The
lung, liver, kidney, and tissues with gross lesions from mice at all
doses were examined microscopically. The remaining tissues were
examined only for animals in the control and high-dose groups.

Several deaths occurred which appeared to be related to
treatment; 2/19, 1/19, and 2/19 males at 60, 120, and 240 mg/kg bw per
day, respectively, and 1/20 and 2/20 females at 120 and 240 mg/kg bw
per day dose, respectively, died, probably as a result of reduced food
consumption and dehydration. A number of clinical observations
associated with a starvation and dehydration (hunched posture, pale
body, hypoactivity, no or few faeces) were noted in animals of each
sex at the two highest doses. The body weights of males at the
intermediate and high doses and of females at the high dose were
statistically significantly lower than those of controls once the
target doses had been achieved. The difference exceeded 10% only in
males at the high dose. Food consumption (expressed as grams per
animal per day) was consistently, statistically significantly lower
than control values except in males at 120 and 240 mg/kg bw per day
and females at 240 mg/kg bw per day. Ophthalmoscopic examinations
revealed no treatment-related effects. An apparently treatment-related
decrease in the values for calculated haematological parameters, mean
cell volume and mean cellular haemoglobin in male mice, was probably
due to a dose-related increase in erythrocyte count, which was not
statistically significant. A nonsignificant reduction in leukocyte
counts was seen in treated male mice, which was reflected in the
lymphocyte counts. There were no effects of treatment on the
haematological parameters in female mice. Statistically significant
increases were noted in alkaline phosphatase activity in males at 120
and 240 mg/kg bw per day but not in treated females. The serum
albumin:globulin ratio showed a dose-related increase in both male and
female mice, which was statistically significant in all treated males
and in females at the two highest doses. The albumin concentrations
tended to be higher and the globulin concentrations lower than in the
corresponding controls for these groups.

Gross examination after sacrifice revealed enlarged livers in
many treated males (0, 9, 7, 11, and 9, in order of ascending dose)
and in several females at higher doses (0, 0, 1, 1, and 2).
Statistically significant changes in absolute and relative organ
weights were seen in males only, which were treatment-related
decreases affecting the spleen (60, 120, and 240 mg/kg bw per day) and
kidney (120 and 240 mg/kg bw per day) and increases affecting the
liver (all treated groups) and adrenal glands (60, 120, and 240 mg/kg
bw per day). Histopathological examination showed an increased
incidence of delayed development of the kidneys and reduced
cellularity of the spleen in males at the high dose. An increased

incidence of glycogen depletion of the liver was noted in all treated
male groups and females at the two highest doses when compared with
controls. The incidence of centrilobular hepatocyte hypertrophy was
increased in all treated males, primarily at the two highest doses;
none of the female mice were reported to be affected. No unusual
changes were noted in the adrenal cortex or medulla of the treated
male mice. Although deaths occurred at all but the lowest dose, the
incidence was not dose-related and was probably associated with the
poor condition due to rejection of the treated feed. The NOEL was 120
mg/kg bw per day on the basis of body-weight decrements < 10% in male
mice (Minnema, 1997b).

Slides of the livers from all of the mice in the 28- and 90-day
feeding studies (Minnema, 1997a,b) were re-examined by an independent
pathologist. With respect to the 28-day study, the independent
observer agreed with the conclusions of the pathologist who initially
reviewed the slides, that no treatment-related effects were present in
the liver. In the slides from the 90-day study, the occurrence of
enlarged hepatocytes in centrilobular to lobular areas was noted with
increased frequency and degree of involvement in all treated males and
females (in 10, 17, 20, 19, and 20 males and 6, 16, 12, 11, and 16
females). The increase was statistically significant in comparison
with controls for males at the three highest doses and for females at
the low and high doses. Decreased glycogen content was observed in all
treated groups, and the difference from controls was statistically
significant, except in females at the lowest dose. The glycogen
depletion was thought to be due to an 'inanition syndrome' resulting
from reduced food consumption and body weight as a consequence of the
unpalatability of the feed. The centrilobular hypertrophy was
consistent with induction of hepatic drug metabolizing enzymes,
especially when considered together with the increased liver weights
in the males. As neither the glycogen depletion nor the centrilobular
hypertrophy was considered to be a direct toxic effect of the compound
on the liver, the NOEL in the 90-day study was 240 mg/kg bw per day,
the highest dose tested (Newberne, 1997a).

Rats

Five weanling Osborne-Mendel rats of each sex received 10 000 ppm
anethole (purity not indicated) in the diet for 15 weeks or 2500 ppm
in the diet for one year. Ten control rats of each sex for each study
received untreated diet. Body weights, food intake, and general
condition were assessed weekly and haematological parameters
(leukocytes, erythrocytes, haemoglobin, and haematocrit) were assessed
at termination of the study. The tissues of all rats were examined
macroscopically at termination, and the liver, heart, spleen, kidneys,
and testes were weighed. The thoracic and abdominal viscera and bone
marrow, bone, and muscle from three or four animals of each sex from
the control and treated groups were examined histopathologically. It
was noted that 7% of the test material was lost from the treated diet
remaining in the cages at room temperature for seven days. The only
result reported in the 15-week study was slight hydropic changes in
hepatocytes of males. No effects were noted in the one-year study
(Hagan et al., 1967).

A 28-day range-finding study was conducted in Sprague-Dawley
rats. Groups of five rats of each sex received target doses of 0, 150,
300, 600, 900, or 1200 mg/kg bw per day trans-anethole (purity,
> 99%) in the diet for 28 days. Owing to the effect of the test
material on palatability, higher doses were achieved by stepwise
increases during the first week of the study. The rats were observed
for signs of morbidity or mortality twice daily, and a thorough
physical examination was performed weekly. Body weights were recorded
twice weekly, and food consumption was measured and recorded daily.
Before sacrifice on day 30, blood samples were collected from each
animal for determination of haematological and clinical chemical
parameters. At sacrifice, all animals were necropsied. The weights of
the adrenal glands, brain with brainstem, liver, kidneys, and thymus
were recorded. Samples of 48 tissues and organs were preserved,
although only the results of histopathological examination of the
liver were reported.

No deaths occurred during the study, and none of the clinical
observations was related to treatment. The body weights of male rats
at 900 and 1200 mg/kg bw per day were statistically significantly
lower than those of controls starting from the second week (when the
target dose was achieved) and throughout the rest of the study. The
body weights of females at the same doses were slightly lower than
those of the respective controls, without statistical significance. At
the end of the study, the body-weight decrements of males at 900 or
1200 mg/kg bw per day and females at 900 mg/kg bw per day exceeded 10%
of the control values. Marked, sustained reductions in food
consumption were noted in males at 1200 mg/kg bw per day throughout
the study, starting at day 8 when the target doses were achieved.
Reduced food consumption was also noted, mostly during week 2, in
males receiving 900 mg/kg bw per day and in females receiving 900 or
1200 mg/kg bw per day. Compound consumption remained within 9% of
nominal values and within 2.5% in most groups. Slight but significant
decreases were noted in mean cell volume and mean cell haemoglobin in
males at 1200 mg/kg bw per day and females at 900 or 1200 mg/kg bw per
day when compared with controls. The lower values in these groups were
within the reference ranges for these values at the testing laboratory
but may also have been due to the nonsignificant but dose-related
increase in erythrocyte count.

A number of clinical chemical parameters were altered by
treatment with anethole. gamma-Glutamyl transferase activity was
significantly greater than the control values in animals of each sex
at the two highest doses. Alanine aminotransferase activity in males
at the highest dose was significantly higher than that of controls,
and the values were outside the reference ranges for this laboratory.
The total serum cholesterol concentration was significantly higher in
females at the two highest doses than in controls, and the serum
concentration of triglycerides was significantly lower in males at the
three highest doses. The serum inorganic phosphorus concentrations
were significantly lower than those in controls in males at the two
highest doses, but accompanying alterations in the serum calcium ion
concentration were not observed. At necropsy, no unusual observations

associated with treatment were seen. The absolute and relative weights
of the kidney, liver, adrenal, and thymus in males and of the adrenal
in females and those relative to the weight of the brain showed
treatment-related decreases which reached statistical significance
mostly at the highest dose but also at the intermediate and next
highest doses for some of the organs. Histopathological examination of
the liver revealed cytoplasmic clearing in hepatocytes of the
centrilobular to midzonal regions in three rats of each sex at 900
mg/kg bw per day and five at the high dose. No explanation of the
significance of this finding was offered. The independent review of
the slides from the 28-day study (Newberne, 1997b) did not mention
this observation, but the description was consistent with the signs
associated with glycogen depletion in the 90-day study (Minnema,
1997c).

In a 90-day study based on the results of the range-finding
study, 20 Sprague-Dawley rats of each sex received trans-anethole
(purity, > 99%) in their diet at concentrations designed to deliver a
target dose of 0, 150, 300, 600, or 900 mg/kg bw per day. The two
highest doses were achieved in a stepwise manner during the first two
weeks of the study in order to reduce the impact of diminished
palatability of the diet containing the test material. Body weights
were recorded twice weekly for the first four weeks of the study and
weekly thereafter. Food consumption was measured and recorded daily.
An indirect ophthalmoscopic examination was performed on each animal
before initiation of the study and at week 12. The animals were
sacrificed after 13 weeks of treatment. Blood samples were taken just
before sacrifice for determination of haematological and clinical
chemical parameters. All animals underwent gross necropsy, and the
adrenals, brain with brainstem, heart, kidneys, liver, lungs, ovaries,
pituitary, prostate, spleen, testes with epididymides, thymus,
thyroid/parathyroid, and uterus were removed and weighed. A total of
46 tissues and organs, including the liver, from animals in the
control and high-dose groups were preserved for histopathological
examination, and the lungs, liver, and kidneys of animals at the three
lowest doses were examined.

No deaths occurred during the study, and none of the clinical
observations could be attributed to treatment. A consistent,
statistically significant decrement in body weight was noted in males
at the two highest doses and in females at the high dose starting at
two weeks, when the target dose was achieved. These differences
exceeded the control values by more than 10% throughout the study (14
and 25% for males and 16% for females, respectively, at the end of the
study). Statistically significant differences in body weight were also
noted in males at 300 mg/kg bw per day and in females at 600 mg/kg bw
per day, which commenced later in the study and were about 8% by the
end of the study. Reductions in food consumption followed a similar
pattern, statistically significant differences for the entire study
period affecting males at the three higher doses and females at the
two higher doses, animals at the highest dose being the most
consistently affected. Consumption of the test material was within 1%
of the target doses for all treated groups. The platelet count of

males and females at the high dose was statistically significantly
lower than that of controls, and mean cell volume and mean cell
haemoglobin were significantly lower in females at the high dose.
Neither observation was considered to be toxicologically significant,
as the changes in the measured parameters were slight.

A number of statistically significant differences in clinical
chemical parameters was noted. gamma-Glutamyltransferase activity was
greater than control values in males and females at the two higher
doses. Alkaline phosphatase activity was significantly elevated only
in males at 600 mg/kg bw per day, although there was a tendency to
increased activity at the intermediate and high doses. Small but
significant increases in the activities of alanine and aspartate
aminotrasferase were noted in females at the high dose but in none of
the other groups. Significant decreases in total serum concentrations
of protein and albumin were seen in males and females at the high
dose. Other significant changes of small magnitude were: decreased
serum calcium concentrations at the high dose, without accompanying
changes in inorganic phosphorus concentrations; dose-related decreases
in serum glucose in female rats at the two higher doses; and decreased
blood urea nitrogen in female rats at the high dose. No unusual
changes that could be related to treatment were seen at necropsy. The
absolute weights of all organs measured and those relative to the
brain were decreased in a dose-related manner; male rats were more
often affected than females. Significant differences in the weights of
the spleen, kidney, and heart were seen in males at all doses above
300 mg/kg bw per day. Histopathological examination revealed a number
of treatment-related effects in the liver, which included
centrilobular to diffuse hepatocellular hypertrophy, individual cell
necrosis, decreased glycogen content, cytoplasmic clearing, and
pigment deposition. None of the other organs showed effects that could
be related to treatment. The NOEL was 300 mg/kg bw per day on the
basis of increased serum gamma-glutamyltransferase activity in animals
of each sex and body-weight decrements exceeding 10% in males at the
next highest dose (Minnema, 1997d).

The livers of all rats in the 28- and 90-day feeding studies
(Minnema 1997c,d) were examined histopathologically by an independent
pathologist. None of the histopathological changes in the liver seen
in the 28-day study was considered to be dose-related. In the slides
from the 90-day study, the pathologist observed three categories of
lesion in the liver: hepatocellular hypertrophy, defined as
centrilobular to diffuse lobular enlargement of hepatocytes with
poorly defined cell walls and variable amounts of eosinophilic
cytoplasm; hepatocellular necrosis, defined as occasional focal and
single-cell necrosis of hepatocytes, usually associated with small,
predominantly mononuclear-cell infiltrates and an occasional pigmented
macrophage; and decreased hepatocyte glycogen content, which was
assessed indirectly from the pattern and extent of fine to coarsely
vacuolated perinuclear cytoplasmic clearing in haematoxylin and eosin-
stained sections. The only findings that were considered to be related
to treatment were the increased incidence and grade of hepatocellular
hypertrophy and the decreased hepatocellular glycogen; neither was

considered to be pathologically relevant. In the absence of
histological evidence of hepatocellular injury, the increase in serum
gamma-gluta-myltransferase activity was considered to be of little
biological significance (Newberne, 1997b).

2.2.3 Long-term studies of toxicity and carcinogenicity

No new information was available.

2.2.4 Genotoxicity

The results of studies of the genotoxicity of trans-anethole
are shown in Table 1.

A modified ³²P-post-labelling assay was used to investigate the
DNA binding of a series of alkenyl benzenes, including
trans-anethole, to the liver DNA of adult female CD-1 mice. The mice
were given single intraperitoneal injections of 10 mg of the test
compound, and their livers were removed 24 h later. Compounds shown to
be hepatocarcinogenic in mice (i.e. safrole, estragole, and
methyleugenol) bound most strongly to hepatic DNA, with 200-300 pmol
adduct per mg DNA. Anethole, which was predicted not to be
carcinogenic in this assay (Miller et al., 1983), resulted in a very
low level of adduct formation, about 1 pmol adduct per mg DNA. Prior
administration of pentachlorophenol, a potent, specific inhibitor of
hepatic sulfotransferases, strongly inhibited DNA adduct formation by
safrole, one of the hepatocarcino-genic compounds. The authors
concluded that a major fraction of DNA binding with safrole proceeds
through an activated sulfate ester (Randerath et al., 1984).

The modified ³²P-post-labelling assay was used to test a series
of nine alkenylbenzenes after administration to pre-weanling male
mice. C57Bl × C3H/HeF₁ mice were injected with 0.25, 0.5, 1, or
3 µmol of a test compound on day 1, 8, 15, or 22 after birth,
respectively. Groups of mice were killed on days 23, 29, and 43, and
their hepatic DNA was isolated. As in the previous assay with adult
female mice, the highest level of adducts was detected after treatment
with methyleugenol, estragole, or safrole (approximately 70, 30, and
20 pmol/mg DNA, respectively). In comparison, very low levels of
binding were detected with anethole (< 1 pmol/mg DNA). While all but
one of the alkenylbenzenes tested formed DNA adducts in newborn mouse
liver, the carcinogenic compounds were associated with higher levels
and a greater persistence of adducts than were the non-carcinogenic
compounds (Phillips et al., 1984).

An assay for unscheduled DNA synthesis was used to compare the
relative importance of epoxidation and hydroxylation of the
alkenylbenzene side-chains of estragole and anethole in their
genotoxicity. Maximal positive responses were obtained with estragole
and its 1-hydroxy derivative in the assay in freshly isolated rat
hepatocytes. The response was prevented by the sulfotransferase
inhibitor pentachlorophenol, showing the importance of sulfation in

the genotoxicity of estragole. Anethole did not induce unscheduled DNA
synthesis, even when cells were treated with buthionine sulfoximine,
thus reducing the amount of glutathione available for conjugation with
anethole epoxide; both this pretreatment and inhibition of cytosolic
epoxide hydrolase were found to markedly enhance cytotoxicity. Neither
3'-hydroxyanethole nor the epoxides anethole-1,2-oxide and
estragole-2,3-oxide induced unscheduled DNA synthesis. The authors
concluded that the genotoxicity of simple alkenylbenzenes is a
consequence of 1'-hydroxylation followed by sulfation of the
1'-hydroxy group, and that epoxidation, while enhancing cytotoxicity,
does not play a role in the induction of unscheduled DNA synthesis
(Caldwell et al., 1992).

Quantum mechanics were calculated for a series of allylbenzenes
and propenylbenzenes and the results compared with the known activity
of the same compounds in the unscheduled DNA synthesis assay.
Carbonium ions formed from genotoxic congeners such as estragole,
methyleugenol, and safrole were more stable than those formed from
inactive compounds such as trans-anethole and iso-safrole. In
addition, the calculations were consistent with the carbonium ion
being the actual genotoxic species. The identical geometry and heat of
formation of the radical species formed from the isomeric pairs
estragole/ trans-anethole and safrole/ iso-safrole were postulated
by the authors to be due to delocalization of the unpaired electron.
Double-bond migration on the alkenyl chain was either nonexistent or
negligible in the metabolites of propenylbenzenes such as
trans-anethole and iso-safrole, suggesting that formation of the
1'-hydroxy metabolite is unlikely. The authors suggested that the lack
of activity of the 3'-hydroxy metabolites of propenylbenzenes in the
unscheduled DNA synthesis assay might also be due to their rapid
subsequent bio-oxidation, resulting in the formation of cinnamic and
benzoic acids (Tsai et al., 1994).

2.2.5 Reproductive toxicity

Rats

A four-generation study of reproductive toxicity was conducted in
rats given a single dietary concentration of 1% trans-anethole
(purity, 98%). Groups of 20 four-week old Wistar rats of each sex were
fed either treated or control diet for 70 days. The animals were then
mated on a one-to-one basis for a maximum of 15 days, with nine pairs
of rats fed control diet (group I), nine pairs fed treated diet (group
IV), 10 pairs of males fed control diet and females fed treated diet
(group II), and 10 pairs of males fed treated diet and females fed
control diet (group III). During the mating period, only animals in
group IV were fed treated diet. After the mating period, the females
were housed individually and were fed control or treated diet as
established during the pre-mating period. The dams were allowed to
litter and nurse the pups to weaning (three weeks). After weaning, the
offspring received the same dietary treatment as both of their
parents. Feeding of the appropriate diet was maintained during
pre-mating, mating, gestation, and lactation for 18/15 F₁, 30/26 F₂,

and 16/14 F₃ control/treated pairs, respectively. The actual dose of
trans-anethole varied from 1400 mg/kg bw per day in the earlier
weeks of treatment to 700 mg/kg bw per day at the end of the
pre-mating period. In addition, a cross-fostering experiment was
conducted by mating six control and six treated females from the F₁
generation with an equal number of F₁ control males. At birth, the
litters of control and treated dams were exchanged, and the litters
were reared by a dam of the other group.

The body weights of all animals were recorded daily for the
duration of the 70-day and two-week mating periods; the body weights
of the females were recorded until delivery. Food consumption for each
cage was recorded daily during the pre-mating period. Pups were
examined for viability and any external abnormalities daily, starting
from birth. Litters were weighed one and two weeks post partum. Pups
were weighed individually from weaning at three weeks of age. The
reproductive parameters assessed were: fertility index, gestation
index, number of dams with stillborn pups, pup survival (viability
index and lactation index; litter size, total and liveborn, percent
male pups, average number of live pups per litter on days 1, 4, 14,
and 21 of lactation), pup growth (average pup body weight per live
litter at weeks 1-4), and clinical findings in the offspring.

All groups of rats treated with trans-anethole had reduced
body-weight gain. Treated rats from the F₁, F₂, and F₃ generations
started the pre-mating period with lower body weights (approximately
70% of that of controls), and the decrement decreased to 75-85% of
control weights during the pre-mating period. The body-weight
decrements of treated rats of the F₀ generation were 80-90% during
the pre-mating period. Food consumption was reduced in the treated
rats during the initial weeks of the study but was mostly comparable
to that of the control group for the remainder of the treatment
period, except in F₂ males and females in which the food consumption
was significantly lower than that of controls throughout the
pre-mating period. The lower food consumption was attributed by the
authors to reduced palatability of the diet.

There was no difference in the percentage of successful matings
or in the number of dams that brought litters to term (fertility index
and gestation index, respectively). There was no treatment-related
effect on the number of dams with stillborn pups or on pup viability,
survival through lactation, or litter size. The viability of the F₂
cross-fostered litters born to treated dams and reared by control dams
and of cross-fostered pups born to control dams and reared by treated
dams was reduced in comparison with both control and treated groups.
This finding was not considered to be toxicologically significant,
since reduced viability was not observed for pups born to and reared
by treated dams. There were no unusual clinical findings that could be
attributed to treatment. The average pup body weights per litter were
significantly reduced for all pups reared by treated dams, regardless
of the diet fed to the males or to the dams during gestation. Thus,
postnatal growth was influenced by exposure of the dams to
trans-anethole during lactation, but not gestation; however, the

author suggested that the test material may be directly toxic via the
milk rather than by an effect on the quality of nutrition (LeBourhis,
1973).

An abbreviated one-generation study of reproductive toxicity was
conducted in Sprague-Dawley-derived rats. Groups of 10 female rats
received daily doses of 0, 35, 175, or 350 mg/kg bw trans-anethole
in corn oil by gavage for seven days and then during a seven-day
mating period with untreated male rats and during gestation and
parturition, up to day 4 of lactation. Rats that did not show evidence
of mating received the test material for 25 days after the
cohabitation period. The female rats were examined daily for evidence
of effects of the test material, death, and delivery of litters. Body
weights were recorded daily throughout the study except during
cohabitation, and food consumption was measured at regular intervals.
The reproductive parameters evaluated were: length of gestation,
litter size, and pup viability at parturition. After parturition, pup
viability was assessed at least twice daily. Pup body weights and sex
and maternal litter interactions were recorded on days 1 and 4 of
lactation. All animals were sacrificed at day 4 of lactation or at the
end of administration of trans-anethole for rats that did not show
signs of mating. Adult animals underwent gross necropsy, and the pups
were examined externally.

The body weights of females at the high dose were significantly
lower than those of controls during the pre-mating, gestation, and
lactation periods. Animals at the intermediate dose also had lower
body weights throughout the study, without statistical significance
except at several intervals during gestation. Food consumption was
significantly reduced during the pre-mating period in animals at the
two higher doses when compared with controls; lowered food consumption
was also seen in rats at the high dose at the end of gestation. During
lactation, some of the animals at the high dose appeared to be in poor
condition, as indicated by clinical observations such as emaciation,
pale, ungroomed coat, and stained fur. Treatment did not affect mating
performance or fertility; however, a number of the other reproductive
parameters were affected significantly in animals receiving the high
dose of trans-anethole, including an increased number of dams with
stillborn pups and with total loss of litters by day 4, an increased
number of stillborn pups, a decreased number of liveborn pups
surviving to day 4 (viability index), and decreased pup weight at day
1. No effect on reproductive parameters was noted at the low and
intermediate doses. The effects on reproductive parameters were
considered to be secondary to the maternal toxicity observed at 350
mg/kg bw per day. The NOEL was 175 mg/kg bw per day (Argus Research
Laboratories, Inc., 1992).

Table 1. Results of assays for the genotoxicity of trans-anethole

End-point Test object Concentration Result Reference

In vitro
Reverse mutation S. typhimurium TA98,TA100, TA1535, 2-200 µg/plate^a Negative Hsia et al. (1979)
TA1537, TA1538
Reverse mutation S. typhimurium TA98, TA100 < 3000 µg/plate^b Positive^c (with Swanson et al. (1979)
S13 in TA100)
Reverse mutation S. typhimurium TA98, TA100, TA1535, < 200 µg/plate^a Negative Nestmann et al. (1980)
TA1537, TA1538
Reverse mutation S. typhimurium TA98, TA100 Up to level of toxicity^b Positive^d (with Marcus & Lichtenstein
(no other details provided) S13 in TA100) (1982)
Reverse mutation S. typhimurium TA98, TA100, TA1535, 60-600 µg/plate^a Negative Sekizawa & Shibamoto
TA1537, TA1538; E. coli WP2 uvrA (1982)
Reverse mutation S. typhimurium TA98, TA100, TA1535, 0.05-50 µg/plate^a Positive (with S9 To et al. (1982)
TA1537, TA1538 and PAPS in TA1535^e)
Reverse mutation S. typhimurium TA98, TA100, TA1535, 1-280 µg/plate^f Negative Mortelmans et al. (1986)
TA1537
Reverse mutation S. typhimurium TA98, TA100, TA1535, < 25 000 µg/plate^a Negative Heck et al. (1989)
TA1537, TA1538
Reverse mutation S. typhimurium TA100 25-500 µg/plate^g Negativeⁱ Gorelick (1995)
100-750 µg/plate^h
Reverse mutation Saccharomyces cerevisiae D7 Not reported Negative Nestmann & Lee (1983)
and XV185-14C
Gene mutation^a L5178Y mouse lymphoma cells, tk^+/- < 62.5 µg/ml (-S9); Positive with S9 Heck et al. (1989)
< 7.8 µg/ml (+S9)
Gene mutation^a L5178Y mouse lymphoma cells, tk^+/- 20-84 µg/ml Positive with S9 Gorelick (1995)

Chromosomal aberration Chinese hamster ovary cells in vitro 13-200 µg/ml Negative Gorelick (1995)

Unscheduled DNA Rat hepatocytes (male Fischer or < 30 µg/ml Negative Heck et al. (1989)
synthesis Sprague-Dawley)
Unscheduled DNA Rat hepatocytes (Fischer or 10^-6-10^-2 mol/L Negative Marshall et al. (1989)
synthesis Sprague-Dawley)

Table 1. (continued)

End-point Test object Concentration Result Reference

Unscheduled DNA Rat hepatocytes (male Fischer) 10^-6-10^-2 mol/L Negative Howes et al. (1990)
synthesis
Unscheduled DNA Rat hepatocytes (male and < 10^-2 mol/L Negative Caldwell & Marshall
synthesis female Sprague-Dawley) (1990)
Unscheduled DNA Rat hepatocytes (Wistar, sex 10^-5-10^-2 mol/L Negative Muller et al. (1994)
synthesis not specified)
Unscheduled DNA Rat hepatocytes (male and 10^-6-10^-2 mol/L Negative^j Marshall & Caldwell
synthesis female Sprague-Dawley)

In vivo
Micronucleus formation Mouse (no other details available) 2025 mg/kg bw × 2, orally^k Negative Siou et al. (1984)
Micronucleus formation Mouse (no other details available) 250 or 500 mg/kg bw × 2, Negative Marzin (1979)
intraperitoneally
Unscheduled DNA Rat (female Sprague-Dawley) 0,1, 125, 500 mg/kg bw Negative Marshall & Caldwell
synthesis by gavage (1996)

S9, hepatic microsomal fraction; S13, hepatic microsome and cytosol fraction
^a In the absence and presence of S9 from Aroclor 1254-induced rats
^b In the absence and presence of S13 from Aroclor 1254-induced rats and an enhanced NADPH-generating system (high microsomal protein)
^c More strongly positive results obtained with 3-hydroxyanethole
^d Positive results also obtained with fennel oil (70% trans-anethole) and anise oil (90% trans-anethole) under the same conditions
^e Only strain tested with 3'-phosphoadenosine-5'-phosphosulfate (PAPS)
^f In the absence and presence of hepatic S9 fraction from Aroclor 1254-induced rats or hamsters
^g In the absence and presence of hepatic S9 fraction from Aroclor 1254-induced rats with an enhanced NADPH-generating system (high microsomal
protein)
^h In the absence and presence of hepatic S9 fraction from Aroclor 1254-induced rats and PAPS
ⁱ Positive results were obtained in studies with a higher dose (in the presence of the enhanced NADPH-generating system) or a different strain
(PAPS)
^j Anethole 1,2-oxide and anethole 1,2-diol also produced negative results in this assay.
^k Close to LD₅₀, significant mortality observed

2.2.6 Special studies on immunotoxicity

Mice

trans-Anethole was tested for its ability to suppress the
production of antibodies in the sheep red blood cell assay. Groups of
eight male B6C3F₁ mice received vehicle, 85 mg/kg bw per day
mercaptopurine (positive control), or 875 mg/kg bw per day anethole by
gavage for 11 days. On day 3, all animals were injected
iintraperitoneally with sheep red blood cells. Body weights were
recorded on days 1, 8, and 12. The mice were sacrificed on day 12,
blood was collected for the assay, and the spleen, thymus, and
adrenals were removed and weighed. Serum was separated from the blood
samples and inactivated by heating to destroy complement activity;
sheep red blood cells were added to the serum samples, which were
incubated at 37°C and then scored for agglutinating activity. The
antibody index scores were similar for anethole-treated mice and
vehicle controls, which also had similar body and organ weights. In
the positive controls, the antibody index scores were markedly
reduced, and the weights of the spleen and thymus were lower than
those of the vehicle controls (Borriston Laboratories, 1982b).

The immunomodulatory properties of trans-anethole in female
B6C3F₁ mice were tested in two types of assay: the ability to
withstand infection with Listeria monocytogenes and the ability to
generate antibody plaque-forming cells after immunization with sheep
red blood cells. Effects on body weight, thymus and spleen weights,
spleen cellularity, and spleen cell viability were also measured. In
an initial range-finding study to determine the dose to be used in the
assays, 1500 mg/kg bw per day resulted in the deaths of all animals
within five days, while 750 mg/kg bw per day had no noticeable effect
when administered over five days. For the Listeria challenge, groups
of 20 mice received vehicle or 188, 375, or 750 mg/kg bw per day
trans-anethole for five days. On day 3 of treatment, all mice
received an intravenous injection of sufficient colony-forming units
of L. monocytogenes to produce an LD_5-35 in the control mice. The
numbers of mice surviving in each group was not related to treatment.

For the sheep red blood cell plaque-forming assay, groups of 10
mice received the vehicle or the doses of test material used for the
infectious challenge assay for five days; 10 mice served as untreated
controls and five mice as positive controls (cyclophosphamide,
80 mg/kg bw intraperitoneally 24 h before assay). Mice were injected
with sheep red blood cells after five days of treatment (four days
before the assay); untreated and positive controls were inoculated at
the same time. Body weights were measured at the beginning and end of
treatment and at sacrifice. The spleens and thymuses were removed and
weighed, and spleen-cell suspensions were prepared and cell viability
was determined. The spleen cells were incubated with equal volumes of
80% guinea-pig complement and 16% sheep red blood cells, and the
resulting immunoglobulin M anti-sheep red blood cell plaques were
evaluated.

The final body weights of treated and control animals were
similar. No difference was seen in spleen weights, numbers or overall
proportion of viable spleen cells, number of plaque-forming cells as a
proportion of viable cells, or total cells isolated from the spleens
of mice treated with trans-anethole. In the positive control group,
the absolute and relative spleen weights were significantly lower than
those of untreated controls. A large reduction in the number of
plaque-forming cells relative to viable spleen cells was noted, as
well as in the number of plaque-forming cells relative to total cells
collected from the spleen. The number of viable cells collected from
the spleen was also lower in the positive controls, although the
percentage of viable cells was similar to that in all other groups
(IIT Research Institute, 1995).

2.3 Observations in humans

In a review of epidemiological data sets from a number of studies
which provided information on exposure to trans-anethole-containing
alcoholic beverages and the incidence of cancer, including
hepatocellular carcinoma, the authors found that it was virtually
impossible to distinguish the effect of a putative liver carcinogen
against the background of a very low occurrence of hepatocellular
carcinoma, for which alcoholic or non-alcoholic cirrhosis and/or
hepatitis B are the major etiological factors. One data set allowed a
comparison of patients with alcoholic cirrhosis (indicating high
consumption of alcohol) with and without hepatocellular carcinoma.
There was no statistical difference in the prevalence of habitual
anise liquor consumption between the two groups. Using an ecological
approach, the incidence of hepatocellular carcinoma in a number of
western countries was compared; no relationship with anethole intake
was indicated. Of the countries considered, France had a consumption
that exceeded that in the other countries by a minimum of 10-fold
(Caldwell & LeBourhis, 1997).

3. COMMENTS

The NOEL from the previously reviewed long-term study in rats was
0.25% in the diet (equal to 100 mg/kg bw per day), which is based on
an increased incidence of hepatic focal nodular hyperplasia in animals
of each sex at the two highest doses. Administration of
trans-anethole to 30 female CD-1 mice for 12 months at 0.46% of the
diet did not result in the induction of hepatomas in any of the
animals 18 months after feeding had been initiated; the related
alkenylbenzenes, safrole and estragole, induced hepatomas in the same
assay.

Most of the studies on genotoxicity and DNA interactions of
trans-anethole reviewed at the present meeting suggest that it is
not genotoxic. Under standard assay conditions, trans-anethole was
not mutagenic to Salmonella typhimurium, did not induce chromosomal
aberrations in Chinese hamster ovary cells in vitro, did not induce
micronuclei in mice in vivo, and did not cause unscheduled DNA
synthesis in vitro or in vivo. The only positive results were

mutation at the tk locus in mouse lymphoma cells in two studies and
in three assays in S. typhimurium when a microsomal activation
system with enhanced protein content or the co-factor
3'-phosphoadenosine-5'-phosphosulfate was added. The rate of formation
of adducts in hepatic DNA from adult and weanling mice exposed to
trans-anethole was much lower than that after exposure to the
hepato-carcinogenic alkylbenzenes safrole, estragole, and
methyleugenol.

The potential of metabolites of trans-anethole to induce
unscheduled DNA synthesis in rat hepatocytes in vitro was also
investigated. Neither the initial omega-oxidation product,
3'-hydroxyanethole nor the epoxide products anethole 1,2-oxide and its
metabolite anethole 1,2-diol induced unscheduled DNA synthesis.
Anethole 1,2-oxide itself was cytotoxic, while the diol exhibited
little cytotoxicity. Agents that prevented further metabolism of the
epoxide markedly enhanced the cytotoxicity of trans-anethole.

The metabolism of trans-anethole proceeds via
O-demethylation, side-chain omega-oxidation, and side-chain
epoxidation. These pathways of metabolism occur in mice, rats, and
humans, but the proportion of the dose metabolized by the various
routes depends on species and dose. O-Demethylation and omega-
oxidation are major pathways in rats and mice receiving doses of
200-300 mg/kg bw (similar to those administered in the studies of
toxicity) and in humans receiving a dose of 1 mg (equivalent to 0.015
mg/kg bw). The formation of the cytotoxic epoxide metabolite was
assessed from urinary elimination of the isomeric diols and the
N-acetylcysteine metabolite derived from glutathione conjugation.
The total amounts of these metabolites excreted by rats (about 15-20%)
were higher than those formed by mice (5-10%) or humans (about 3%);
however, only two subjects given ¹⁴C- trans-anethole have been
described adequately in this respect. The available data indicate that
rats given doses of 200-300 mg/kg bw metabolize proportionally more
trans-anethole to the cytotoxic metabolite than do humans given a
dose of 1 mg. The data on human metabolism were considered by the
Committee to be too limited to permit accurate quantitative
comparisons of exposure to anethole epoxide in rats and humans.

Recently conducted 90-day feeding studies in CD-1 mice and
Sprague-Dawley rats were reviewed, together with the results of
preliminary 28-day range-finding studies. Most of the observed effects
were related to an 'inanition syndrome' resulting from decreased food
consumption by the treated animals. The effects of diet rejection were
much more apparent in mice and were reflected in the much lower
dietary levels tolerated. In contrast, in studies in which single
doses were given by gavage, mice appeared to tolerate higher doses of
trans-anethole than did rats. The doses used in the 90-day studies
were 30-240 mg/kg bw per day for mice and 150-900 mg/kg bw per day for
rats. In both species, the major indications of inanition were large
decrements in body weight, hepatic glycogen depletion, and lower organ
weights, while enlarged livers and centrilobular hepatocellular
hypertrophy were suggestive of hepatic enzyme induction. The NOEL in

the study in mice was 120 mg/kg bw per day on the basis of a body
weight decrement greater than 10% in male mice at the next highest
dose.

Toxicologically significant effects were apparent in the 28- and
90-day studies in rats. In both male and female rats, a hepatotoxic
effect of trans-anethole was suggested by elevated serum activity of
gamma-glutamyl transferase in the 90-day study at doses of 600 or 900
mg/kg bw per day and by a reduction in serum protein concentrations at
900 mg/kg bw per day; however, no proliferative or other
toxicologically significant lesions were apparent on histopathological
examination of the livers. There was no evidence of a similar effect
in mice over the lower dose range necessitated by their lower
tolerance to the treated diet. The NOEL was 300 mg/kg bw per day on
the basis of elevated gamma-glutamyl transferase activity in rats of
each sex and body weight decrements exceeding 10% in males at the next
highest dose.

Studies of reproductive toxicity with trans-anethole were
conducted in rats over one or four generations. In the one-generation
study, litter effects (reduced viability of pups, pup survival, and
pup body weights) were noted only at doses that were toxic to the
maternal animals. The NOEL for maternal toxicity was 175 mg/kg bw per
day, on the basis of reduced food consumption and body-weight gain and
overall poor condition of the maternal animals. In the four-generation
study, in which treated groups received 1% trans-anethole in the
diet (equal to 700-1400 mg/kg bw per day), the only effect seen in the
pups was reduced body-weight gain. A cross-fostering experiment
conducted as part of the four-generation study indicated that the
reduced pup body-weight gain observed in the treated groups was
related to exposure during lactation rather than the gestation period.

trans-Anethole was not immunotoxic when tested in the sheep red
blood cell plaque-forming assay or the Listeria challenge assay.

4. EVALUATION

The data reviewed at the present meeting indicate that
trans-anethole and its metabolites are unlikely to be genotoxic
in vivo and suggest that a cytotoxic metabolite, anethole epoxide,
is the possible causative agent of the hepatotoxic effects in rats.
Since trans-anethole is metabolized along the same three major
pathways in mice, rats, and humans, hepatotoxicity in rats was
considered an appropriate end-point on which to base the ADI. Because
the 90-day study in rats was well conducted by current standards and
the hepatic effects observed were consistent with the hepatoxicity
observed in the long-term study, it was used to derive the ADI. The
NOEL was 300 mg/kg bw per day on the basis of alterations in serum
parameters considered to be indicators of hepatotoxicity. Because of
the low tolerance of mice for trans-anethole-treated diets, they
could not be fed doses comparable to those that were toxic to rats. It
is therefore unlikely that a new long-term study in mice could include
sufficiently high doses to induce effects in the liver, uncomplicated
by inanition.

Because of limitations in the long-term studies in rats and mice,
the Committee concluded that the recent 90-day study in rats provides
the most reliable basis for determination of the NOEL for adverse
effects in the liver. Application of the usual safety factor of 100 to
the NOEL from the 90-day study was considered inappropriate because of
the limitations of the available long-term studies. An overall safety
factor of 200 was considered adequate to allow for deficiencies in the
long-term studies and to provide a suitable safety margin for the
maternal toxicity found in studies of reproductive toxicity and for
the effects seen in the 90-day study in mice.

The Committee allocated an ADI of 0-2 mg/kg bw on the basis of
the NOEL of 300 mg/kg bw per day in the 90-day study in rats, to which
a 200-fold safety factor was applied, with the value rounded to one
significant figure.

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    See Also:
       Toxicological Abbreviations
       Anethole, trans- (FAO Nutrition Meetings Report Series 44a)