First draft prepared by Dr F.S.D. Lin,
    Division of Toxicological Review and Evaluation,
    Center for Food Safety and Applied Nutrition,
    US Food and Drug Administration 


          Trans-anethole is a flavoring agent present in the essential
    oils of anise, fennel and star anise.  Chemically it is an
    alkenylbenzene identified as (1-methoxy-4-(1-propenyl) benzene or
    para-propenylanisole, with the following chemical structure:

    FIGURE 1

          Trans-anethole was first evaluated by the eleventh meeting of
    the Committee when a conditional ADI of 0-1.25 mg per kg of body
    weight was allocated (Annex 1, Ref. 14).  After re-evaluation at the
    twenty-third meeting (Annex 1, reference 50), a temporary ADI of 0-2.5
    mg per kg of body weight was allocated pending submission of the
    results of an adequate long-term study. After further reviews at the
    thirty-first and thirty-third meetings (Annex 1, references 77 and 83,
    the Committee extended the temporary ADI but reduced it to 0-1.2 mg
    per kg of body weight pending further details of the long-term study
    and a review of the detailed study records and of the histological

         Since the last evaluation, the new data have been reviewed,
    together with new data which has become available in the interim, and
    are summarized and discussed in the following monograph.  The
    previously published monograph has been expanded and is reproduced in
    its entirety below.


    2.1  Biochemical aspects

    2.2.1  Absorption, distribution, biotransformation and excretion

         Trans-anethole is rapidly absorbed and distributed in the rat (Le
    Bourhis, 1973; Fritsch  et al., 1975) and in the mouse (Le Bourhis,
    1968; Strolin-Benedetti & Le Bourhis, 1972; Le Bourhis, 1973).  The
    same authors reported rapid metabolism in both species and identified
    the following urinary metabolites:

                                            % dose in urine

         1.   p-hydroxypropenylbenzene         32

         2.   p-hydroxycinnamic acid           15

         3.   p-methoxybenzoic acid             4

         4.   p-methoxyhippuric acid           43

         5.   p-methoxyacetophenone            trace

         Metabolites (1) and (2) appeared largely as the glucuronide or
    sulfate.  Virtually identical results were obtained by the i.p. route
    (Solheim & Scheline, 1973).  Administration to rats of ring or methoxy
    side-chain 14C-labelled  trans-anethole confirmed that virtually all
    ring metabolites were eliminated in urine within 48 hrs with
    substantial demethylation and consequent appearance of the methoxy 14C
    in expired air, and to a small extent the body and faeces (Strolin-
    Benedetti & Le Bourhis, 1972).

          Trans-anethole was hydroxylated at the 3'-carbon by hepatic
    microsomes from rats and mice (Swanson  et al., 1981).

         In five human subjects given a 500 mg dose the 24-hour
    metabolites were anisic acid (52%) and p-hydroxybenzoic acid (5%). 
     Trans-anethole was not detectable in the blood (Le Bourhis, 1973). 
    Metabolism in rabbits appeared to be similar to that in humans
    (Axelrod, 1956; Le Bourhis, 1970).

         More recently, Sangster  et al., have compared the routes of
    metabolic degradation of various anisole derivatives, including
     trans-anethole, in both rat and mouse, and the route of degradation
    was dose dependent (Sangster, 1983; Sangster  et al., 1984a,b). 
    After administration of a single dose of 50 mg  trans-[14C methoxy]-
    anethole/kg b.w. orally to female rats or i.p. to male mice, the major
    routes of excretion were in the urine or as 14CO2 in expired air;
    excretion in the faeces or as volatile metabolites (other than CO2)

    in expired air was low (<2% of the dose).  Eleven urinary metabolites
    were identified in the rat and ten in the mouse, arising from side-
    chain oxidation and cleavage, O-demethylation and conjugation. 
    Approximately 4.4% and 10.6% of the dose was excreted as two
    diastereoisomers of 1-(4'methoxyphenyl)propan-1,2-diol in mice and
    rats respectively; these metabolites had presumably arisen from
    epoxidation of the side-chain double bond (Sangster  et al., 1984a). 
    The pathways of metabolism identified are as shown in Figure 1.

    FIGURE 2

         In studies of the dose dependence of  trans-anethole metabolism,
    single doses of 0.05, 5, 50, or 1500 mg/kg b.w. were administered to
    female rats by gavage; similar doses were given to male mice
    intraperitoneally with an additional dose level of 250 mg/kg b.w. also
    being included.  Dose and species-dependent differences in metabolism
    were observed.  The major route of metabolism was via O-
    demethylation in both species and the significance of this route
    decreased with increase in dose from 56 and 72% of the 0.05 mg/kg dose
    to 32 and 35% of the highest dose in rats and mice respectively.  With
    regard to side-chain oxidation, the rat favoured the epoxidation route

    and elimination of the diol-isomers rose from 2% to 15% of the dose
    over the range of doses studied.  In the mouse,  w-oxidation was
    favoured and the diols were formed in smaller amounts of from 1% to
    4.5% over the same dose range (Sangster  et al., 1984b).  It should
    be noted that the inter-species differences were compounded by the sex
    difference between the species and by the different route of

         Studies of the metabolism of several structurally-related food
    flavours, including  trans-anethole, were carried out in human
    volunteers.  Two male subjects received oral doses of 1 mg
    14C[methoxy]- trans-anethole and the urine was collected over a 48
    hour period subsequently; aliquots of expired air also were collected
    at 30 minute intervals for 8 hours after dosing.  Approximately 65% of
    the activity was excreted in the 48 hour urine, mainly in the first 8
    hours, while an estimated 20% of the dose was excreted as 14CO2 in
    exhaled air over the first 8 hours.  Most of the urinary activity was
    in the form of 4-methoxyhippuric acid (ca 55% of the dose) and 4-
    methoxybenzoic acid (ca 3.5% of the dose); approximately 3% of the
    dose was excreted as the two diastereoisomers of 1-(4'-
    methoxyphenyl)propane-1,2-diol (Sangster  et al., 1987).  

         In a study of the dose-dependence of the metabolism of  trans-
    anethole in human volunteers, 5 male subjects received oral doses of
    1, 50 or 250 mg 14C-[methoxy]- trans-anethole on three separate
    occasions in milk.  14CO2 was estimated in expired air at intervals
    up to 24 hours and radioactivity was measured in urine at intervals up
    to 48 hours.  Over the range of doses studied, dose had no systematic
    effect on the rate or route of excretion, the major routes being urine
    (54-69% of the dose) and expired air (13-17% of the dose).  The
    principal urinary metabolite (>90% of urinary 14C) was
    methoxyhippuric acid with much smaller amounts of 4-methoxy-benzoic
    acid and up to three other metabolites; the pattern of urinary
    metabolites was unaffected by dose size (Caldwell & Sutton, 1988).

         Studies of DNA binding of several alkenylbenzenes, including
     trans-anethole, were carried out following i.p. administration to
    neonatal and adult mice.  Very low levels of adduct formation were
    detected only when the dose (300 mg/kg i.p.) approached the LD50 (400
    mg/kg b.w.).

         Groups of 8 male and 8 female CD Sprague-Dawley rats were
    administered (methoxy-14C)- trans-anethole at gavage doses of 100,
    250, 500 and 1000 mg/kg (16 uCi/kg).  Radioactivities appearing in the
    urine, faeces and exhaled CO2 were determined at 24 hour intervals
    for up to 72 hours, but only the summarized urine and CO2 data were
    provided in the report.  The urinary metabolites of  trans-anethole
    in these animals are said to be still under investigation.  Based on
    the available disposition data, the authors have concluded the

         (1)  Excretion of 14C in the urine and expired air was delayed
              as the dose was increased, with a progressively smaller
              fraction of the total radioactivity excreted during 0-24
              hours but a greater fraction in 24-48 hours. 

         (2)  As the dose was increased, the excretion route was shifted
              from the expired air to the urine.

         (3)  Females consistently excreted less of the given doses in 0-
              24 hours than males, "with recovery of 14C in the urine and
              as CO2 being approximately equally affected" (FEMA, 1989). 
              The Committee noted that the above data were not analyzed
              statistically for intergroup differences.  Also noted is
              that with the possible exception of the dose-dependent delay
              of its excretion, the other dose-dependent effects on the
              disposition of  trans-anethole, as concluded by the
              authors, were not very apparent.  Furthermore, the author's
              conclusion that there is a sex-dependent difference in the
              elimination of  trans-anethole from the test animals can
              not be conclusively supported by the data provided.

    2.2  Toxicological Studies

    2.2.1  Acute toxicity
    Species      Route        LD50          Reference

    Mouse        per os     1820-5000       Levenstein, 
                                            1960, Jenner, 
                                            1964, Boissier
                                            et al., 1967

    Mouse        i.p.       650             Caujolle & 
                                            Meynier, 1958

                            1410            Boissier et al., 

    Rat          per os     2090-3208       Taylor et al., 
                                            1964, Shelanski, 
                                            1958, Boissier 
                                            et al., 1967

    Rat          i.p.       900             Boissier et al., 

                            2670            Caujolle & 
                                            Meynier, 1958

    Guinea-pig   per os     2160            Jenner et al., 


    2.2.2  Short-term studies  Rat

         Rats (number not given) receiving anethole in the diet at 0.25%
    for one year showed no adverse effects while those receiving 1.0% for
    15 weeks showed slight hydropic alterations of hepatic cells (Taylor,
     et al., 1964). 

         Groups of five male and five female rats maintained on diets
    containing  trans-anethole at 0, 0.1, 0.3, 1.0 or 3.0% for 90 days
    showed no effects at 0.1% but dose-related hepatic cell oedema,
    degeneration and regeneration at 0.3% and higher.  In a parallel
    experiment with the  cis-isomer, similar changes were noted at 0.03%
    and higher (Shelanski, 1958).

         Groups of 10 adult male rabbits and adult male and female rats
    (number per group not specified) were given orally once per week in
    their drinking water a quantity of  trans-anethole corresponding to
    7 daily doses of 11.4 mg/kg/day over a period of 90 days.  Another
    group of rats (21 days old), 10 rats per group were treated with
    anethole identically but only for a period of 21 days.  Neither
    measurements of growth, electroencephalograms, electrocardiograms nor
    gross and histopathological examinations revealed any evidence of
    adverse toxic effects in any group treated either for 21 or 90 days
    (Vignoli  et al., 1965).

    2.2.3  Long-term/carcinogenicity studies  Rat

         Groups of 25 male and female rats maintained at 0.2, 0.5, 1.0 or
    2.0%  trans-anethole in the diet for 12-22 months showed no effects
    at any level in clinical chemistry, haematology, histopathology nor
    mortality.  Slower weight gain and decreased fat storage were noted
    only at the 1.0 and 2.0% levels.  In a paired feeding study, trans-
    anethole reduced the rate of weight gain (Le Bourhis, 1973).

          Trans-anethole was administered to Sprague-Dawley rats in the
    diet at concentrations of 0, 0.25, 0.5 or 1% for 117 weeks.  The group
    sizes were 52 males and 52 females in two separate control groups and
    in the intermediate and high dose groups, and 78 males and 78 females
    in the low dose group.  A supplementary group of 26 males and 26
    females received the  trans-anethole at a level of 1% in the diet for
    the first 54 weeks of the study, then 10 animals of each sex were
    placed on the control diet until week 121; the remaining animals of
    each sex continued to receive the diet containing 1%  trans-anethole. 
    Food intake was recorded daily during weeks 1-32 and for one day every
    4 weeks thereafter; body weight gain was monitored at weekly intervals
    up to week 26 and then at monthly intervals.  The animals were
    inspected clinically at frequent intervals during the course of the
    study and the animals were palpated weekly from week 27 and the
    appearance of palpable masses recorded.  Moribund animals were killed
    in the course of the study; these animals and those sacrificed at
    termination were subjected to detailed gross and histopathological
    examination (40 tissues).  Also at termination, haematological
    examinations were performed, including RBC, haemoglobin, MCV,
    haematocrit, MCHC, leucocyte count and differential blood count.

         There was a retardation in body weight gain in all treated
    animals during the first six months of treatment after which the
    weight gain of the 0.25% and 0.5% groups was similar to controls,
    although the weight deficit was not recovered; in the high dose group
    the weight gain remained less than controls.  The deficit of weight
    gain was associated with reduced food intake due to unpalatability of
    the diet and animals removed from the 1% diet after 54 weeks rapidly

    gained weight.  There were no treatment-related effects on mortality
    except for a significantly decreased mortality in the high-dose group
    males.  Haematological examination did not reveal any treatment-
    related differences but comparison of organ weights revealed an
    increase in relative liver weight of all treatment groups of females
    (P<0.001 in the highest dose group).  Treatment-related
    histopathological changes were reported in the liver in the form of
    hepatocytic vacuolation (males, 1% dose group), sinusoidal dilatation
    (males and females in the 1% group, females in the 0.5% group),
    focal/nodular hyperplasia (males and females in the 1% group, males in
    the 0.5% group) and hepatocellular hypertrophy in females of the 0.5%
    and 1% groups.

         There was a reported statistically significant increase in the
    incidence of benign hepatocellular adenomas and hepatocellular
    carcinomas in females in the 1% dose group, but it was pointed out
    that this was within the range of spontaneous incidence of similar
    lesions in historical controls for the strain of rat used compiled
    between 1977 and 1985 (Truhaut  et al., 1988).  It was considered
    that the trend of increase in incidence of benign and malignant liver
    cell tumours represented a secondary response to a non-specific effect
    rather than a direct genotoxic effect.

         In the light of new criteria (Maronpot  et al., 1986) for the
    diagnosis of proliferative hepatic lesions in rats, the findings of
    the above study were reviewed by independent pathologists (Newberne
     et al., 1987; Moch, 1990).  The results of these reviews are
    tabulated below and, in the case of hepatocellular adenomas and
    carcinomas, compared with the original diagnosis:

    Table 2: Hepatic Lesions

    Dose Groups    FCA      FNH        HA        HCA       HCA

    Pathologists                    Male Rats

    0% Moch        33/104   2/104      1/104     4/104     5/104
                   (32%)    (2%)       (1%)      (4%)      (5%)
       Truhaut       -      3/104      3/104     2/104     5/104
                            (3%)       (3%)      (2%)      (5%)
       Newberne    48/104   2/104      1/104     3/104     4/104
                   (46%)    (2%)       (1%)      (3%)      (4%)

       Moch        32/78    6/78       1/78      3/78      4/78
                   (41%)    (8%)       (1%)      (4%)      (5%)
       Truhaut       -      3/78       3/78      1/78      4/78
                            (4%)       (4%)      (1%)      (5%)
       Newberne    34/78    7/78       1/78      3/78      4/78
                   (44%)    (9%)       (1%)      (4%)      (5%)

       Moch        27/52    6/52       1/52      3/52      4/52
                   (52%)    (12%)      (2%)      (6%)      (8%)
       Truhaut       -      8/52       0/52      3/52      3/52
                            (15%)                (6%)      (6%)
       Newborne    28/52    6/52       1/52      3/52      4/52
                   (52%)    (12%)      (2%)      (6%)      (8%)

       Moch        25/52    14/52      3/52      1/52      4/52
                   (48%)    (27%)      (6%)      (2%)      (8%)
       Truhaut       -      14/52      4/52      1/52      5/52
                            (27%)      (8%)      (2%)      (10%)
       Newberne    27/52    13/52      3/52      1/52      4/52
                   (52%)    (25%)      (6%)      (2%)      (8%)

                                       Female Rats

       Moch        47/104   4/104      4/104     0/104     4/104
                   (45%)    (4%)       (4%)                (4%)
       Truhaut       -      11/104     2/104     1/104     3/104
                            (11%)      (2%)      (1%)      (4%)
       Newberne    62/104   6/104      4/104     0/104     4/104
                   (60%)    (6%)       (4%)                (4%)

    Table 2 (contd)
    Dose Groups    FCA      FNH        HA        HCA       HCA

       Moch        42/78    8/78       1/78      0/78      1/78
                   (54%)    (10%)      (1%)                (1%)
       Truhaut       -      2/78       2/78      0/78      2/78
                            (3%)       (3%)                (3%)
       Newberne    53/78    10/78      1/78      0/78      1/78
                   (68%)    (13%)      (1%)                (1%)

       Moch        27/52    7/52       0/52      0/52      0/52
                   (52%)    (14%)
       Truhaut       -      6/52       0/52      0/52      0/52
       Newberne    32/52    10/52      0/52      0/52      0/52
                   (62%)    (19%)

       Moch        16/52    18/52      4/52      6/52      10/52
                   (31%)    (35%)      (8%)      (12%)     (19%)
       Truhaut       -      15/52      6/52      6/52      12/52
                            (29%)      (12%)     (12%)     (23%)
       Newberne    21/52    15/52      4/52      6/52      10/52
                   (40%)    (29%)      (8%)      (12%)     (19%)

    *     Hepatic Lesions: FCA = Focus of Cellular Alteration;
    FNH = Focus of Nodular Hyperplasia
    HA  = Hepatocellular Adenoma
    HCA = Hepatocellular Carcinoma

         As shown above, the findings of the independent pathologists are
    in good agreement, all showing a clear increase in high-dose (1%)
    female rats with hepatocellular adenoma and/or carcinoma.  A similar
    increase was not observed in male rats at any dose levels.  The
    differences observed in the incidence of rats with altered cellular
    foci between the original study pathologist and the others, were
    primarily due to the fact that the original study pathologist had
    diagnosed any vacuolation or clearing of hepatocytes observed as
    hepatocellular vacuolation rather than as foci of cellular alteration
    (Moch, 1990).

         An independent audit of this rat study report showed no
    significant discrepancies (Munro & Brillinger, 1989a,b).  Mouse

         In in the first eight weeks of a 24-week screening test, groups
    of 20 female A/He mice received  a total dose of 2, 4 or 12.0 g
    anethole/kg b.w. in 24 3x/week i.p. injections.  The higher dose had
    previously been calculated to be the maximum tolerated dose.  There
    was no increase in the incidence of tumours of the lung, liver,
    kidney, spleen, thymus, intestine, or salivary or endocrine glands. 
    Survival was reduced to approximately 70% (Stoner  et al., 1973).  It
    should be noted that safrole, which has been shown to be carcinogenic
    in other studies, was negative using this protocol, thus there is some
    doubt about its validity.

         A series of experiments was performed to investigate the
    carcinogenic potential of  trans-anethole in mice.  Groups of male
    (56-76 per group) and female (55-61 per group) 4-day old mice were
    exposed to  trans-anethole by gavage at dose levels of 370 or 740
    mg/kg b.w. twice weekly for 10 weeks, then sacrificed at 11-14 months. 
    In a second experiment, 53 male mice received  trans-anethole by
    intraperitoneal injection on days 1, 8, 15 and 22 after birth.  The
    total dose of anethole that each animal received during 4 treatments
    was 703 or 1399 mg.  Animals from the lower dose group were sacrificed
    at 13-18 months and those from the 1390 mg group at 12 months.  In a
    third experiment, a group of female mice, approximately 8 weeks old,
    average body weight 21 g, were fed anethole in the diet at a dose
    level of 690 mg/kg for a period of 12 months.  After 12 months the
    animals received basal diet only and were sacrificed at 18 months. 
    The fourth experimental group of 17 female mice (8 weeks old) was
    given an injection of anethole twice weekly for 12 weeks at a dose
    level of 148 mg/kg b.w.  The mice were killed 8 months after the first
    injection.  All animals from each experiment were necropsied and the
    various tissues examined histologically.   Trans-anethole produced no
    different results from controls (Miller  et al., 1983).

    2.2.4  Special studies on mutagenicity

         Two strains of  Salmonella typhimurium (TA100 and TA98), with
    and without microsomal activation, were used in a plate test to study
    the effect of  trans-anethole (isolated natural compound), anise oil
    (90%  trans-anethole) and fennel oil (70%  trans-anethole).  Each
    test material was assayed over the dose range from zero (control) to
    the level of cell toxicity.  All test materials increased mutagenic
    activities with TA100 tester strain with implementation of microsomal
    activation (S13) system.  Peak mutagenic activity, 4 and 4.4 times
    that of the background rate, occurred with 2 mg anise oil/plate and
    2.5 mg fennel oil/plate respectively.  Isolated natural  trans-
    anethole was also mutagenic (the dose level and rate of mutagenicity
    were not stated).  The compound was considered not to be mutagenic
    unless it was capable of inducing a mutation (reversion) rate at least
    3 times that of the incident background (Marcus & Liechtenstein,

         The mutagenic activities of anethole and its metabolite 3'-
    hydroxyanethole were studied using three tester strains of  Salmonella
     typhimurium (TA1535, TA100 and TA98).  Addition of an NADPH-
    generating system and liver microsomes and cytosol (S13 fraction) from
    Aroclor-treated rats (6.8 mg liver protein/plate) to the incubation
    mixture of TA100 tester strain increased mutagenic activities. 
    Approximately 45 revertants were obtained per µmole anethole.  Under
    the same conditions, 3'-hydroxyanethole showed no significant
    mutagenic activity with less than 7 µmoles/plate.  Above this
    concentration the S13-mediated mutagenicity increased linearly with
    increased doses up to 15 µmoles/plate (about 1000 revertants with 15
    µmoles/plate) (Swanson  et al., 1979).

         Five strains of  Salmonella typhimurium (TA1535, TA100, TA1537,
    TA1538, TA98), with and without metabolic activation (S9 mix), were
    used to study potential mutagenic effects of  trans-anethole.  The
    lowest overtly toxic concentration for  trans-anethole was 1
    mg/plate.  No mutagenic activity was observed at concentrations of up
    to 50 µg  trans-anethole/plate with or without metabolic activation. 
    However, the addition of 3'-phosphoadenosine-5'phosphosulphate (PAPS)
    to the microsomal assay markedly increased the mutagenicity of  trans-
    anethole in TA1535 tester strain.  The mutation rate observed was
    approximately 4, 5, 10, 11, 9 and 3 times that of the background rate
    at  trans-anethole concentrations of 0.05, 0.20, 1.0, 5.0, 15.0, and
    50.0 ug/plate respectively (To  et al., 1982).

         A further study indicated that  trans-anethole showed mutagenic
    activity in the Ames  S. typhimurium assay but was inactive in a  B.
     subtilis Rec assay and was negative in an  E. coli uvr A reversion
    test (Sekizawa & Shibamoto, 1982).

          Trans-anethole was inactive in a mouse micronucleus assay 24,
    48 and 72 hr after administration by gavage of the second of two daily
    doses of 2 ml/kg b.w. to groups of 10 adult males.  At this dose level
    there was significant mortality (3-4 animals per group) (Siou  et al.,
    1984).  Negative results were also obtained in a mouse micronucleus
    assay after i.p. administration to groups of 5 male and 5 female mice
    in two doses of 0.25 or 0.5 g/kg b.w., 30 and 6 hours before sacrifice
    (Marzin, 1979).

         Genotoxicity of  trans-anethole was tested, along with some
    structurally related compounds (safrole, isosafrole, eugenol,
    estragole, allylbenzene, methyleugenol, and p-propylanisole), for its
    capability of inducing unscheduled DNA synthesis (UDS) in freshly
    isolated rat hepatocytes in primary culture.  Hepatocytes were
    isolated from male Fischer rats following liver perfusion.  After
    overnight incubation of the hepatocytes in the presence of the test
    chemical, the UDS or DNA repair was measured by determining the amount
    of 3H-thymidine incorporated into hepatocyte nuclear DNA during the
    repair process.  Under the test conditions,  trans-anethole did not

    induce UDS over the concentration range of 10-6 and 10-2 M, although
    cytotoxicity, as measured by LDH leakage, was observed at
    concentrations of 10-3 M and above.  Of the remaining chemicals
    tested, allybenzene, p-propylanisole, isosafrole and eugenol were also
    negative in the UDS assay, whereas safrole, methyleugenol and
    estragole as well as the positive control, 2-acetylaminofluorene, all
    induced UDS in a dose-related manner.

         Based on the study results, the authors concluded:  "there was an
    excellent correlation between UDS induction and known rodent hepato-
    carcinogenicity, with safrole, estragole and methyleugenol all
    inducing UDS. Anethole, isosafrole, eugenol and allylbenzene, for
    which evidence of carcinogenicity is equivocal or negative, did not
    induce UDS" (Howes  et al., 1990).

         In another test,  trans-anethole was tested for its ability to
    induce UDS in cultured primary hepatocytes isolated from Sprague-
    Dawley derived CD rats of both sexes.  At concentrations up to 10-2,
    the compound did not induce UDS, as measured by 3H-thymidine
    incorporation into nuclear DNA.  The positive control, 2-
    acetylaminofluorene, gave positive response.  At concentrations of 
    10-3 and above,  trans-anethole was cytotoxic and the UDS response
    was below the control values.  The cytotoxicity of  trans-anethole
    was consistent with the increased leakage of cellular LDH into the
    culture medium.

         To investigate the possible involvement of a reactive epoxide
    intermediate in the observed cytotoxicity, the effect of  trans-
    anethole on intracellular glutathione levels was studied.  The results
    showed that anethole caused a dose-related depletion of glutathione in
    freshly isolated hepatocytes in suspension.  However, when hepatocytes
    were pretreated with the non-cytotoxic glutathione depleting agent
    dimethyl maleate, there was no enhancement of anethole cytotoxicity,
    suggesting that the putative epoxide intermediate may not be of any
    toxicological significance (Caldwell & Marshall, 1990).

    2.2.5  Special studies on pharmacological effects

         The pharmacologic effects of  trans-anethole most often noted
    are reduction in motor activity, lowering of body temperature and
    hypnotic, analgesic and anticonvulsant effects.  By either the oral or
    i.p. route, administration of more than 10% of the LD50 by that route
    appears necessary for significant effects (Boissier  et al., 1961;
    Seto, 1969; Gruebner, 1972; Le Bourhis & Soene, 1973).


         The Committee considered the results of three independent reviews
    of the liver histology in the long-term study in rats.  It concluded
    that there is a clear increase in the incidences of hepatocellular
    adenomas and of hepatocellular carcinomas in female rats at 10 mg/kg
    in the diet but not at lower doses.  In male animals a slight increase
    in the incidence of hepatocellular adenomas but not carcinomas was
    seen at 10 mg/kg in the diet only.  There is also evidence of an
    increase in the incidence of non-neoplastic proliferative lesions in
    the liver at all dose levels in both sexes.

         A review of the report of the long-term rat study showed no
    significant discrepancies.  The Committee also noted that differences
    in survival between treated and control animals could not account for
    the increased incidence of liver tumours seen in the study.

         The results of two new studies on genotoxicity (unscheduled DNA
    synthesis) were negative.

         The Committee concluded that insufficient data were available to
    permit a final evaluation of the significance of the malignant liver
    tumours observed in female rats with respect to the use of  trans-
    anethole as a food additive.  Further metabolic and especially
    pharmacokinetic studies in mice, rats, and humans are required for
    evaluation in 1992.  In addition, a long-term dietary study in mice
    may be needed, although the design of such a study will depend upon
    the results of the metabolic and pharmacokinetic studies.  In view of
    the positive results that were obtained in  in vitro bacterial gene
    mutation tests, the Committee also concluded that chromosome
    aberration studies and  in vitro tests for gene mutation in mammalian
    cells were desirable.


         The temporary ADI was extended until 1992, but reduced to 0-0.6
    mg per kg of body weight on the basis of the minimal effect level of
    2.5 mg/kg in the diet (equivalent to a dose of 125 mg per kg of body
    weight per day) for non-neoplastic proliferative changes in the liver
    of rats, adjusted using a safety factor of 200.

         The need for a reproduction/teratogenicity study will be
    considered by the Committee when further relevant data from the above
    studies have been reviewed.  The Committee also reiterated its
    recommendation that an epidemiological study of the effects of
    consuming high dietary levels of  trans-anethole would be desirable.


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    See Also:
       Toxicological Abbreviations
       Trans-anethole (WHO Food Additives Series 14)
       trans-ANETHOLE (JECFA Evaluation)