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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

    CARVONE AND STRUCTURALLY RELATED SUBSTANCES 

    First draft prepared by 
    Dr P.J. Abbott
    Australia New Zealand Food Authority, Canberra, Australia 

          Evaluation 
              Introduction
              Estimated daily  per capita intake
              Absorption, metabolism, and elimination
              Application of the Procedure for the Safety Evaluation 
                   of Flavouring Agents
              Consideration of combined intakes from use as
                   flavouring agents
              Conclusions
          Relevant background information
              Biological data
                   Absorption and metabolism
              Toxicological studies
                   Acute toxicity
                   Short-term and long-term studies of toxicity and 
                        carcinogenicity
                   Genotoxicity
                   Other relevant studies
          References


    1.  EVALUATION

    1.1  Introduction

         The Committee evaluated the safety of carvone and eight related
    substances using the Procedure for the Safety Evaluation of Flavouring
    Agents (Figure 1, p. 222, and Annex 1, reference 131). The substances
    in this group are terpenoid ketones, secondary alcohols, and related
    esters containing a 2-menthyl carbon skeleton (Table 1).

         The Committee previously evaluated carvone on several occasions.
    A conditional ADI of 0-1.25 mg/kg bw for the (+) and (-) isomers was
    established at the eleventh meeting (Annex 1, reference 14). A
    temporary ADI of 0-1 mg/kg bw was established for (+)- and (-)-carvone
    at the twenty-third meeting (Annex 1, reference 50), which was
    extended at the twenty-fifth, twenty-seventh, thirtieth, and
    thirty-third meetings (Annex 1, references 55, 62, 73, and 83). At its
    thirty-seventh meeting, the Committee determined that the (+) and (-)
    enantiomers should be evaluated separately. Owing to lack of data on
    (-)-carvone  per se, the temporary ADI for (-)-carvone was not
    extended. In its review of (+)-carvone, the Committee considered a
    long-term study of toxicity and carcinogenicity in mice, short-term
    studies of toxicity in mice and rats, and tests for mutagenicity
     in vitro. On the basis of a NOEL of 93 mg/kg bw per day in a
    three-month study of toxicity in rats, the Committee established an
    ADI for (+)-carvone of 0-1 mg/kg bw per day.


        Table 1.  Summary of the safety evaluation of carvone and structurally related substances used as flavouring agents

                                                                                                                                              

    Substance            No.  CAS No.       Estimated per   Step 1       Step 2        Step A3        Step A4      Step A5       Conclusion
                                            capita intake,  Structural   Metabolized   Intake exceed  Endogenous?  Adequate      based on 
                                            Europe/USA      class        to innocuous  threshold of                NOEL for      current 
                                            (g/day)                     products?     concern?a                   substance or  levels of
                                                                                                                   related       intake
                                                                                                                   substance?
                                                                                                                                              

    para-Menthan-2-one   375  499-70-7      0.01/1          II           Yes           No             -            -             No safety 
                                                                                                                                 concern
    CHEMICAL STRUCTURE 

    para-Menthan-2-ol    376  499-69-4      0.01/7          I            Yes           No             -            -             No safety 
                                                                                                                                 concern
    CHEMICAL STRUCTURE 

    Dihydrocarvone       377  7764-50-3     0.02/180        II           Yes           No             -            -             No safety 
                                                                                                                                 concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                              
    Substance            No.  CAS No.       Estimated per   Step 1       Step 2        Step A3        Step A4      Step A5       Conclusion
                                            capita intake,  Structural   Metabolized   Intake exceed  Endogenous?  Adequate      based on 
                                            Europe/USA      class        to innocuous  threshold of                NOEL for      current 
                                            (g/day)                     products?     concern?a                   substance or  levels of
                                                                                                                   related       intake
                                                                                                                   substance?
                                                                                                                                              


    Dihydrocarveol       378  619-01-2      3/320           I            Yes           No             -            -             No safety 
                                                                                                                                 concern
    CHEMICAL STRUCTURE 

    Dihydrocarvyl        379  20777-49-5    15/0.1          I            Yes           No             -            -             No safety 
    acetate                                                                                                                      concern
    CHEMICAL STRUCTURE 

    Carvoneb             380  2244-16-8(+)  2800/9900       II           Yes           Yes            No           Yes           No safety 
                              6485-40-1(-)                                                                                       concern
    CHEMICAL STRUCTURE 


    Table 1.  (continued)
                                                                                                                                              
    Substance            No.  CAS No.       Estimated per   Step 1       Step 2        Step A3        Step A4      Step A5       Conclusion
                                            capita intake,  Structural   Metabolized   Intake exceed  Endogenous?  Adequate      based on 
                                            Europe/USA      class        to innocuous  threshold of                NOEL for      current 
                                            (g/day)                     products?     concern?a                   substance or  levels of
                                                                                                                   related       intake
                                                                                                                   substance?
                                                                                                                                              

    Carveol              381  99-48-9       15/140          I            Yes           No             -            -             No safety 
                                                                                                                                 concern
    CHEMICAL STRUCTURE 

    Carvyl acetate       382  97-42-7       6/36            I            Yes           No             -            -             No safety 
                                                                                                                                 concern
    CHEMICAL STRUCTURE 

    Carvyl propionate    383  97-45-0       N/D/0.04        I            Yes           No             -            -             No safety 
                                                                                                                                 concern 
    CHEMICAL STRUCTURE 
                                                                                                                                              

    a  Threshold of concern is 1800 g/day for class I and 540 g/day for class II.
    b  The ADI of 0-1 mg/kg bw previously established for (+) carvone at the thirty-seventh meeting was maintained.
    

    1.2  Estimated daily per capita intake

          Per capita intake was estimated from data derived from surveys
    in Europe (International Organization of the Flavor Industry, 1995)
    and the United States (US National Academy of Sciences, 1987) (see
    Table 2). The estimated total daily  per capita intake of carvone
    (No. 380, stereochemistry unspecified) and related substances from use
    as flavouring agents is 2.8 mg/person in Europe and 10 mg/person in
    the United States. Carvone accounts for approximately 99% of the total
    annual  per capita intake of this group of substances when used as
    flavouring agents in Europe and 96% in the United States. 

         Seven of the substances in this group have been reported to occur
    naturally in foods, including fruits, spices, and berries. (-)-Carvone
    (55-75%) has been reported in the oils of  Mentha (spearmint).
    (+)-Carvone (20-75%) has been reported in  Carum (caraway) and
     Anethum (dill) (Maarse et al., 1994). Quantitative data on the
    natural occurrence and consumption ratios have been reported for five
    of these substances (Nos 377, 379, 380, 381, and 382), which indicate
    that they are consumed predominantly in traditional foods (i.e.
    consumption ratio > 1) (Stofberg & Kirschner, 1985; Stofberg &
    Grundschober, 1987).

    1.3  Absorption, metabolism, and elimination

         The substances in this group are terpenoid ketones, secondary
    alcohols, and related esters containing a 2-menthyl carbon skeleton.
    The group consists of three ketones (Nos 375, 377, and 380), three
    secondary alcohols (Nos 376, 378, and 381), and three esters (Nos 379,
    382, and 383). Their structures are shown in Table 1.

     Terpenoid esters

         Each of the tthree esters would be expected to be hydrolysed to
    its corresponding alcohol and carboxylic acid by carboxylesterases,
    which predominate in hepatocytes. Esters of carveol (Nos 382 and 383)
    and dihydrocarveol (No. 379) would be expected to be hydrolysed to
    yield carveol (No. 381) and dihydrocarveol (No. 378), respectively,
    and the corresponding saturated aliphatic carboxylic acids.

     Terpenoid alcohols and ketones

         The terpenoid alcohols resulting from ester hydrolysis and their
    corresponding ketones are metabolized like other alicyclic terpenoid
    ketones and secondary alcohols. Five detoxification pathways have been
    identified:

    *    reduction of the ketone, followed by conjugation of the resulting
         alcohol with glucuronic acid;

    *    side-chain oxidation yielding polar metabolites, which may be
         conjugated and excreted;

    Table 2.  Most recent annual usage of carvone and structurally related 
    substances as flavouring agents in Europe and the United States

                                                                             

    Substance (No.)                    Most recent      Per capita intakea
                                       annual
                                       volume (kg)      g/day     g/kg bw
                                                                   per day
                                                                             

    p-Menthan-2-one (375)
      Europe                           0.1              0.01       0.0002
      United States                    3                1          0.01
    p-Menthan-2-ol (376)
      Europe                           0.1              0.01       0.0002
      United States                    37               7          0.1
    Dihydrocarvone (377)
      Europe                           0.1              0.02       0.0003
      United States                    920              180        3
    Dihydrocarveol (378)
      Europe                           3                1          0.01
      United States                    320              61         1
    Dihydrocarvyl acetate (379)
      Europe                           80               15         0.3
      United States                    0.3              0.1        0.001
    Carvone (380)
      Europe                           15 000           2800       46
      United States                    52 000           9900       170
    Carveol (381)
      Europe                           78               15         0.2
      United States                    740              140        2
    Carvyl acetate (382)
      Europe                           33               6          0.1
      United States                    190              36         1
    Carvyl propionate (383)
      Europe                           -                -          -
      United States                    0.2              0.04       0.001
                                                                             

    a National Academy of Sciences (1987); International Organization of the 
      Flavouring Industry (1995)



    *    glutathione conjugation of ketones, followed by excretion;

    *    hydrogenation of the endocyclic double bond of carveol; and

    *    excretion of unchanged parent compound.

    1.4  Application of the Procedure for the Safety Evaluation of 
         Flavouring Agents

    Step 1.   According to the decision-tree structural class
              classification (Cramer et al., 1978), six members of this
              group are in class I, namely,  para-menthan-2-ol (No. 376),
              dihydrocarveol (No. 378), dihydrocarvyl acetate (No. 379),
              carveol (No. 381), carvyl acetate (No. 382), and carvyl
              propionate (No. 383), while three members are in class II
              because they contain an alpha,-unsaturated ketone, namely,
               para-menthan-2-one (No. 375),  para-menth-8-en-2-one (No.
              377), and carvone (No. 380). 

    Step 2.   Sufficient data were available on carvone to define the
              major pathways of metabolism. This compound contains all of
              the key structural elements and potential sites of
              metabolism of all other members in the group. Better data
              were available on the metabolism of isophorone
              (3,4,5-trimethyl-cyclohex-2-en-1-one), which is not a member
              of this group of flavouring agents but which shows close
              structural similarities to carvone and uses the same routes
              of metabolism (reduction of the carbonyl group, conjugation
              of the resulting alcohol, and side-chain oxidation). It is
              predicted that carvone and other alpha,-unsaturated ketones
              in the group would undergo glutathione conjugation, but this
              would be expected to be a minor route because of the low
              reactivity  in vitro, a conclusion supported by the
              toxicological data.

                   For the three terpenoid esters in class I, the most
              likely route of metabolism is hydrolysis to carveol or
              dihydrocarveol. For the three terpenoid alcohols, carveol,
              dihydrocarveol, and  para-menthan-2-ol, which are also in
              class I, the available data indicate that the most likely
              route of metabolism is conjugation with glucuronic acid,
              followed by excretion; however, other routes of metabolism
              such as side-chain oxidation followed by conjugation and
              excretion may also occur. In each case, metabolism yields
              innocuous metabolites, and the evaluation should proceed via
              the 'A' side of the scheme. 

                   For the three terpenoid ketones in class II, the
              available data indicate that the most likely route of
              metabolism is reduction of the ketone to the corresponding
              alcohol, followed by conjugation with glutathione and
              excretion. Other routes of metabolism may occur, such as
              side-chain oxidation followed by conjugation and direct
              conjugation of the ketone with glutathione. In all cases,
              metabolism yields innocuous metabolites, and the evaluation
              should proceed via the 'A' side of the scheme.

    Step A3.  The intakes of the six terpenoid esters and alcohols in
              class I are below the threshold of concern for this class
              (1800 g/person per day), and they would not be expected to
              be of safety concern. The intake of two of the three
              terpenoid ketones in class II,  para-menthan-2-one and
               para-menth-8-en-2-one, is below the threshold of concern
              for this class (540 g/person per day), and these substances
              would not be expected to be of safety concern. The intakes
              of carvone in both Europe (2800 g/person per day;
              International Organization of the Flavor Industry, 1994) and
              the United States (9900 g/person per day; US Academy of
              Sciences, 1987) are above the threshold of concern. This
              substance therefore proceeds to step A4. 

    Step A4.  Carvone is not endogenous in humans. Its safety evaluation
              therefore proceeds to step A5. 

    Step A5.  A NOEL for (+)-carvone of 93 mg/kg bw per day (three-month
              study in rats) was identified by the Committee at its
              thirty-seventh meeting (Annex 1, reference 94). If it is
              assumed that all of the carvone consumed was (+)-carvone, a
              margin of safety of > 500 exists between this NOEL and the
               per capita daily intake for the (+) isomer of carvone.
              Therefore, this substance would not be expected to be of
              safety concern. The (-) isomer of carvone would be expected
              to share a common metabolic pathway with (+)-carvone. A NOEL
              of 125 mg/kg bw per day for carvone (isomer unspecified) was
              identified at the eleventh meeting (Annex 1, reference 14),
              and the only material in commerce at that time was
              (-)-carvone. This NOEL is considered to apply to (-)-carvone
              and, if it is assumed that all of the carvone consumed was
              (-)-carvone, a margin of safety of > 750 exists between
              this NOEL and the  per capita daily intake for the (-)
              isomer of carvone. Therefore, this substance would not be
              expected to be of safety concern.

         Table 1 summarizes the evaluations of the nine substances in this
    group. 

    1.5  Consideration of combined intakes from use as flavouring agents

         All nine substances are expected to be efficiently metabolized to
    innocuous substances. In the unlikely event that carvone, which
    accounts for > 95% of the total estimated dietary intake of the
    group, was consumed concomitantly with the eight related substances,
    the estimated combined intake would exceed the human intake threshold
    for structural classes I and II, but, in the opinion of the Committee,
    this would not give rise to perturbations outside the physiological
    range. 

    1.6  Conclusions

         The results of the evaluations of carvone and related substances
    indicate that these substances would not present safety concerns at
    the current estimated intake. In using the procedure, the Committee
    noted that all of the available data on toxicity are consistent with
    the results of the safety evaluation. The ADI established previously
    for (+)-carvone was maintained. 


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Biological data

    2.1.1  Absorption and metabolism

     Terpenoid esters

         The three esters would be expected to be hydrolysed to their
    corresponding alcohol and carboxylic acid by carboxylesterases, which
    predominate in hepatocytes (Heymann, 1980). Esters of carveol (Nos 382
    and 383) and dihydrocarveol (No. 379) would be expected to be
    hydrolysed to yield carveol and dihydrocarveol, respectively, and the
    corresponding saturated aliphatic carboxylic acids. Evidence that this
    metabolic route is used for these esters comes from studies of related
    compounds, namely, the (-)-menthol esters, (-)-menthol ethylene glycol
    carbonate and (-)-menthol propylene glycol carbonate, which were
    completely hydrolysed after incubation with a rat liver homogenate
    (Anon., 1994). Similarly, more than 80% of radiolabelled cyclandelate,
    a structurally related ester, was hydrolysed after 20 min of
    incubation with rat hepatic microsomes (White et al., 1990), and
    rabbits given bornyl acetate orally excreted borneol as the glucuronic
    acid conjugate (Williams, 1959).

     Terpenoid alcohols and ketones

         The terpenoid alcohols resulting from ester hydrolysis and their
    corresponding ketones are metabolized like other alicyclic terpenoid
    ketones and secondary alcohols. Five detoxification pathways have been
    identified: 

         (i)   reduction of the ketone followed by conjugation of the
               resulting alcohol with glucuronic acid;
         (ii)  side-chain oxidation, yielding polar metabolites which may
               be conjugated and excreted;
         (iii) glutathione conjugation of ketones, followed by excretion; 
         (iv)  hydrogenation of the endocyclic double bond of carveol; and
         (v)   excretion of unchanged carvone.

    Evidence for each of these pathway is given below:

     (i)  Reduction of the ketone followed by conjugation of the 
          resulting alcohol with glucuronic acid

         There is considerable evidence that ketones are reduced to the
    corresponding secondary alcohol and conjugated mainly with glucuronic
    acid (Fischer & Bielig, 1940). In rodents, but probably not in humans,
    the conjugate is excreted primarily into the bile, where it may be
    hydrolysed to yield the free alcohol (Matthews, 1994). The alcohol may
    then enter enterohepatic circulation and be excreted by the kidney
    (Hamalainen, 1912; Tamura et al., 1962). If a double bond is present
    in the molecule, the metabolite may be hydrogenated to the dihydro
    derivative (Fischer & Bielig, 1940; Madyastha & Raj, 1993). 

         Ketones are reduced primarily by cytosolic carbonyl reductase,
    and the reaction is stereoselective to yield a mixture of
    diastereomeric alcohols (Leibman & Ortiz, 1973). In experiments in
    rabbits, carvone was reduced to carveol, which was converted to the
    glucuronic acid conjugate and excreted in urine (Fischer & Bielig,
    1940). The glucuronic acid conjugate of dihydrocarveol has been
    detected in the urine of rabbits (Hamalainen, 1912). 

         Groups of five rats (species and sex not specified) received
    carvone at 5, 25, 50, 100, or 1000 mg/kg bw in olive oil daily by
    gavage for 10 days. Daily urinary elimination of glucuronic acid,
     ortho-glucuronide and ascorbic acid increased at daily doses of 100
    mg/kg bw (Tamura et al., 1962). 

     (ii)  Side-chain oxidation yielding polar metabolites which may 
          be conjugated and excreted

         Alicyclic ketones containing an alkyl or alkenyl side-chain may
    undergo oxidation of the side-chain to form polar metabolites, which
    are excreted as the glucuronic acid or sulfate conjugates in the urine
    and, to a lesser extent, in the faeces (Ishida et al., 1989; Williams,
    1959).

         A racemic mixture of carvone was reported to undergo side-chain
    oxidation in rabbits. Each of six male rabbits was given approximately
    2 g ()-carvone orally in water. Urine collected over three days was
    separated into neutral, acidic, and phenolic fractions. After
    hydrolysis of the glucuronic acid and sulfate conjugates,
    chromatographic analysis of the neutral fraction revealed the presence
    of (+)-9-hydroxycarvone (10%) (Williams, 1959; Ishida et al., 1989). 

         Oxidation of the side-chain has been observed for other acyclic
    (Nishizawa et al., 1987) and alicyclic ketones (Williams, 1940; Nelson
    et al., 1992), with cytochrome P450 acting as a catalyst  in vitro 
    (Madyastha & Raj, 1990); however, no effects were observed on the
    activity of either cytochrome P450 or cytochrome b5 in male albino
    rats given (-)-carvone at 600 mg/kg bw per day orally for three days
    (Moorthy et al., 1989).

     (iii)  Glutathione conjugation of ketones followed by excretion

         In a study to examine the ability of carvone and carvyl
    derivatives to induce enzymes for the detoxification of carcinogens
    (Wattenberg et al., 1989; Lam & Zheng, 1991, 1992), glutathione
     S-transferase activity increased and glutathione content decreased
    in various tissues of groups of A/J mice given repeated oral doses of
    carvone and 10 carvyl derivatives. Groups of four female A/J mice were
    given three oral doses, each containing 20 mg of a carvyl derivative
    in cottonseed oil, by gavage over two days. The test substances
    included alpha,-unsaturated ketones, (+)-carvone,
    8,9-dihydro-carvone, 9-hydroxycarvone, 9-acetoxycarvone, the
    unconjugated ketones 2,3-dihydrocarvone and carvomenthone, and five
    carvyl alcohol derivatives. Cytosolic glutathione  S-transferase
    activity and acid-soluble sulfhydryl levels were measured in mouse
    liver, forestomach, lung, and small and large bowel mucosa. Treatment
    with carvone and, to a lesser degree, other alpha,-unsaturated
    ketones increased the activity of glutathione  S-transferase in all
    tissues by two to four times over that in controls and in animals
    treated with other carvyl derivatives. Carvone intake was associated
    with a decrease in glutathione content in the liver, lung, and
    large-bowel mucosa (Zheng et al., 1992). Carvone rapidly conjugated
    with glutathione in the absence of glutathione  S-transferase
    (Portoghese et al., 1989). 

     (iv)  Hydrogenation of the endocyclic double bond of carveol

         Carvone is also metabolized in rabbits by hydrogenation of the
    endocyclic double bond to yield 8-menthen-2-ol (dihydrocarveol), which
    is excreted unchanged (Fischer & Bielig, 1940).

     (v)  Excretion of unchanged carvone 

         Carvone has been detected unchanged in the urine of humans,
    presumably arising from its dietary intake (Zlatkis et al., 1973).

    2.1.2  Toxicological studies

    2.1.2.1  Acute toxicity

         The results of studies of the acute toxicity of carvone and
    related substances are shown in Table 3.

    2.1.2.2  Short-term and long-term studies of toxicity and
             carcinogenicity

         The results of all short-term and long-term studies of carvone
    are shown in Table 4. Details of the studies that were critical to the
    safety evaluation of carvone and related substances are given below. 

        Table  3.  Acute toxicity of carvone and related substances tested by gavage 

                                                                                                

    Substance                  No.    Species        Sex     LD50        Reference
                                                             (mg/kg bw) 
                                                                                                

    Dihydrocarvone             377    Rat            NR       > 5000      Moreno (1977)
    Dihydrocarveol             378    Rat            NR       > 5000      Moreno (1977)
    Dihydrocarvyl acetate      379    Rat            NR       > 5000      Moreno (1980)
    Carvone                    380    Rat            M/F      1640        Jenner et al. (1964)  
    Carvone                    380    Rat            NR       3710        Levenstein (1976)
    Carvone                    380    Guinea-pig     M/F      766         Jenner et al. (1964) 
    Carveol                    381    Rat            NR       3000        Keating (1972)
    Carvyl acetate             382    Rat            NR       > 5000      Levenstein (1976) 
    Carvyl propionate          383    Rat            NR       > 5000      Levenstein (1976)
                                                                                                

    NR, not reported; M/F, male and female


    Table 4.  Short-term and long-term studies of the toxicity of carvone 

                                                                                                  

    Species          Sex    No. groups/      Route    Duration      NOEL           Reference
                            no. per group                           (mg/kg bw
                                                                    per day)
                                                                                                  

    Carvone
    Rats             M/F    3/10             Oral     16 weeks      ND             Hagan et al. 
                                                      to 1 year                    (1967)
    (-)-Carvone
    Mice             M/F    5/10             Gavage   16 days       328            US National 
                                                                                   Toxicology 
                                                                                   Program (1990)
    Mice             M/F    3/40             Gavage   13 weeks      375            US National 
                            2/20                                                   Toxicology 
                                                                                   Program (1990)
    Rats                    5/5              Gavage   16 days       150            US National 
                                                                                   Toxicology 
                                                                                   Program (1990)
    Rats                    5/10             Gavage   13 weeks      93             US National 
                                                                                   Toxicology 
                                                                                   Program (1990)
                                                                                                  

    ND, not determined; M/F, male and female
    
     Carvone

         Carvone (unspecified stereochemistry) was administered to four
    male Wistar rats for 14 days at a dietary level of 0 or 1% (equivalent
    to 500 mg/kg bw per day). Significant increases in serum cholesterol
    and triacylglycerol concentrations were reported in rats given carvone
    when compared with the controls. Significant decreases in food
    consumption and body weights were also reported in treated animals
    (Imaizumi et al., 1985). 

         In a study to examine the toxicity of a large number of
    flavouring agents, groups of five male and five female Osborne-Mendel
    weanling rats were fed a diet containing carvone at a concentration of
    1000 mg/kg (equivalent to 50 mg/kg bw per day) for 27-28 weeks, 2500
    mg/kg (equivalent to125 mg/kg bw per day) for one year, or 10 000
    mg/kg (equivalent to 750 mg/kg bw per day) for 16 weeks. Although the
    stereochemistry of the test material was unspecified, a survey of
    industrial producers of carvone who actively marketed carvone during
    the period of the study (1960-70) indicated that the material in
    commerce in the United States at that time was the (-) isomer (Bauer,
    1991; Flynn, 1991; Wrigley Co., 1991). Body weights and food intake
    were measured weekly and haematological examinations performed at 3,
    6, 12, and 22 months. Although no effects were noted, only a very
    limited report of the results of this study was provided. Depressed
    body-weight gain and testicular atrophy at a dose of 750 mg/kg bw per
    day were the only reported effects. The NOEL was 125 mg/kg bw per day
    (Hagan et al., 1967). 

     (-)-Carvone

         Studies on this enantiomer were evaluated at the thirty-seventh
    meeting (Annex 1. reference 94)

    2.1.2.3  Genotoxicity

         The results of studies of the genotoxicity of these substances
    are shown in Table 5. 

    2.1.2.4  Other relevant studies

         In a study to examine the anti-carcinogenic properties of
    carvone, groups of 15 female A/J mice were given 0.2 mmol (30 mg)
    (+)-carvone by intubation I h before administration of
     N-nitrosodiethylamine at a dose of 20 mg/kg bw once a week for eight
    weeks by intubation. The animals were necropsied 26 weeks after the
    initial dose of nitrosamine. (+)-Carvone inhibited forestomach tumour
    formation, with a > 63% reduction in the mean number of papillomas
    per mouse when compared with the controls. The number of pulmonary
    adenomas in mice given (+)-carvone was significantly less (34%) than
    in control animals (Wattenberg et al., 1989). 


        Table 5.  Results of assays for the genotoxicity of carvone and related substances

                                                                                                                                           

    Substance      No.     End-point              Test object                        Dose            Result         Reference
                                                                                                                                           

    Carvone        380     Gene mutation          S. typhimurium TA1535,             3 mol/plate    Negative       Florin et al. (1980)
                                                  TA1537, TA98, TA100
    (-)-Carvone    380     Gene mutation          S. typhimurium TA1535,             333 g/plate    Negative       Mortelmans et al. 
                           (preincubation)        TA98, TA100, TA1537                                               (1986)
    (-)-Carvone    380     Gene mutation          S. typhimurium TA1535,             333 g/plate    Negative       National Toxicology 
                                                  TA98, TA100, TA1537                                               Program (1990)
    Carvone        380     rec assay              Bacillus subtilis H17 (rec+)       0.6 ml/disc     Negative       Matsui et al. (1989)
                                                  and M45 (rec-)
    (-)-Carvone    380     Sister chromatid       Chinese hamster ovary cells        502 g/ml       Equivocal      National Toxicology
                                                                                                                    Program (1990)
                           exchange
    (-)-Carvone    380     Chromosomal            Chinese hamster ovary cells        400 g/ml       Equivocal      National Toxicology
                           aberration                                                                               Program (1990)
    Carveol        381     Gene mutation          S. typhimurium TA1535,             560 g/plate    Negative       Mortelmans et al. 
                           (preincubation)        TA98, TA100, TA1537                                               (1986)
    Carvyl acetate 382     Gene mutation          S. typhimurium TA1535,             333 mg/plate    Negativea      Mortelmans et al. 
                           (preincubation)        TA98, TA100, TA1537                                               (1986)
                                                                                                                                           

    a  With and without metabolic activation
    

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    See Also:
       Toxicological Abbreviations