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    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 -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 -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
    -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 g/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 mol 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 g/platea               Negative               Hsia et al. (1979)
                            TA1537, TA1538
    Reverse mutation        S. typhimurium TA98, TA100             < 3000 g/plateb              Positivec (with        Swanson et al. (1979)
                                                                                                 S13 in TA100)
    Reverse mutation        S. typhimurium TA98, TA100, TA1535,    < 200 g/platea               Negative               Nestmann et al. (1980) 
                            TA1537, TA1538                                                                              
    Reverse mutation        S. typhimurium TA98, TA100             Up to level of toxicityb      Positived (with        Marcus &  Lichtenstein 
                                                                   (no other details provided)   S13 in TA100)          (1982)
    Reverse mutation        S. typhimurium TA98, TA100, TA1535,    60-600 g/platea              Negative               Sekizawa &  Shibamoto 
                            TA1537, TA1538;  E. coli WP2 uvrA                                                           (1982)
    Reverse mutation        S. typhimurium TA98, TA100, TA1535,    0.05-50 g/platea             Positive (with S9      To et al. (1982)
                            TA1537, TA1538                                                       and PAPS in TA1535e)
    Reverse mutation        S. typhimurium TA98, TA100, TA1535,    1-280 g/platef               Negative               Mortelmans et al. (1986)
                            TA1537 
    Reverse mutation        S. typhimurium TA98, TA100, TA1535,    < 25 000 g/platea            Negative               Heck et al. (1989)
                            TA1537, TA1538
    Reverse mutation        S. typhimurium TA100                   25-500 g/plateg              Negativei              Gorelick (1995)
                                                                   100-750 g/plateh
    Reverse mutation        Saccharomyces cerevisiae D7            Not reported                  Negative               Nestmann & Lee (1983)
                            and XV185-14C 
    Gene mutationa          L5178Y mouse lymphoma cells, tk+/-     < 62.5 g/ml (-S9);           Positive with S9       Heck et al. (1989)
                                                                   < 7.8 g/ml (+S9)
    Gene mutationa          L5178Y mouse lymphoma cells, tk+/-     20-84 g/ml                   Positive with S9       Gorelick (1995)

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


    Unscheduled DNA         Rat hepatocytes (male Fischer or       < 30 g/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               Negativej              Marshall & Caldwell
    synthesis               female Sprague-Dawley)

    In vivo
    Micronucleus formation  Mouse (no other details available)     2025 mg/kg bw  2, orallyk    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 37C 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 LD5-35 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)