IPCS INCHEM Home


    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

    SAFETY EVALUATIONS OF GROUPS OF RELATED SUBSTANCES BY THE PROCEDURE 
    FOR THE SAFETY EVALUATION OF FLAVOURING AGENTS

    INTRODUCTION

         Seven groups of flavouring agents were evaluated by the Procedure
    for the Safety Evaluation of Flavouring Agents outlined in Figure 1
    (Annex 1, references 116, 122, and 131).

         The Committee noted that in applying the Procedure, the substance
    is first assigned to one of the structural classes identified at the
    forty-sixth meeting of the Committee (Annex 1, reference 122):

    *    Class I. Substances that have simple chemical structures and
         efficient modes of metabolism that would suggest a low order of
         toxicity when given by the oral route

    *    Class II. Substances that have structural features that are less
         innocuous than those of substances in Class I but are not
         suggestive of toxicity. Substances in this class may contain
         reactive functional groups.

    *    Class III. Substances that have structural features that permit
         no strong initial presumption of safety or may even suggest
         significant toxicity.

    A key element of the Procedure involves determining whether a
    flavouring agent and the product(s) of its metabolism are innocuous
    and/or endogenous. For the purposes of evaluation, the Committee used
    the following definitions, adapted from the report of its forty-sixth
    meeting:

          Innocuous metabolic products are defined as products that are
    known or readily predicted to be harmless to humans at the estimated
    intake of the flavouring agent.

          Endogenous substances are intermediary metabolites normally
    present in human tissues and fluids, whether free or conjugated;
    hormones and other substances with biochemical or physiological
    regulatory functions are not included. The estimated intake of a
    flavouring agent that is, or is metabolized to, an endogenous
    substance should not give rise to perturbations outside the
    physiological range.

    Intake

         Intakes were estimated from surveys carried out in Europe and the
    United States. Estimates of the intake of flavouring agents by
    populations typically involve the acquisition of data on the amounts
    used in food. In Europe, a survey was conducted in 1995 by the
    International Organization of the Flavor Industry, in which flavour
    manufacturers reported the total amount of each flavouring agent
    incorporated into food sold in the European Union during the previous

    year. Manufacturers were requested to exclude use of flavouring agents
    in pharmaceutical, tobacco, and cosmetic products. In the United
    States, a series of surveys was conducted between 1970 and 1987 by the
    National Academy of Sciences National Research Council (under contract
    to the Food and Drug Administration) in which information was obtained
    from ingredient manufacturers and food processers on the amount of
    each substance destined for addition to the food supply and on the
    usual and maximal levels at which each substance was added to a number
    of broad food categories. 

         In using data from these surveys to estimate the intakes of
    flavouring agents, it was assumed that only 60% of the total amount
    used is reported and that the total amount used in food is consumed by
    only 10% of the population.

       Intake            annual volume of production (kg)  109 (g/kg)
                       =                                                
    (g/person per day)   population of consumers  0.6  365 days


    The population of consumers was assumed to be 31  106 in Europe and
    24  106 in the United States.

    General aspects of metabolism

         The flavouring agents evaluated at the present meeting share a
    number of functional groups, e.g. linear, branched, alicyclic, and
    unsaturated alkyl chains and alcohol, ester, and ketone groups.
    Therefore, they have a number of common and inter-related metabolic
    features, and data on one or more members of a group being evaluated
    can be used to predict the metabolic fate of analogues for which there
    were no data. Metabolic pathways common to the groups of flavouring
    agents evaluated at the present meeting are:

            (i)    hydrolysis of esters;
            (ii)   oxidation of alcohols and aldehydes;
            (iii)  conjugation of alcohols;
            (iv)   reduction of ketones;
            (v)    reduction of double bonds;
            (vi)   oxidation of side-chains ;
            (vii)  oxidation of alicyclics; and
            (vii)  conjugation with glutathione.

    (i)   Ester hydrolysis

         The hydrolysis of ethyl, isoamyl, and allyl esters was considered
    at the forty-sixth meeting of the Committee (Annex 1, reference 122).
    Most esters are hydrolysed rapidly by enzymes present in the gut
    lumen, intestinal wall, and liver to yield the corresponding acids and
    alcohols.

    FIGURE 1

         A wide range of enzymes (carboxyesterases and carboxylic acid
    hydrolases) can hydrolyse esters into their constituent alcohols and
    acids. Carboxyesterases are found in many tissues, and there is strong
    esterase activity in the intestinal tract, blood, liver, kidney, lung,
    brain, and pancreas. The multiplicity of esterases and their wide
    tissue distribution result in rapid hydrolysis of esters  in vivo, 
    and this extensive metabolic activity is the basis for the development
    by the pharmaceutical industry of pro-drugs, which are carboxylic acid
    esters of therapeutically active agents. The esters considered at the
    present meeting contain larger, more complex alkyl constituents in
    both the alcohol and the acid moieties. There was only limited
    information on the hydrolysis of these esters  in vitro, but the data
    were comparable to those considered in the forty-sixth report of the
    Committee. Isopropyl phenylacetate and isopropyl butyrate underwent
    40% hydrolysis after 2 h with pancreatin (Grundschober, 1977), a value
    similar to that for some of the esters considered at the previous
    meeting. Essentially complete hydrolysis of isopropyl palmitate,
    isopropyl oleate, and isopropyl stearate radiolabelled in the fatty
    acid moiety was demonstrated  in vivo by the detection of > 95% of
    the radiolabel in the triglyceride fraction of rat blood (Savary &
    Constantin, 1970).  n-Octyl esters would be expected to undergo very
    rapid hydrolysis on the basis of the relationships between increased
    chain length (in the range C1-C8), increased Vmax, and decreased Km
    (considered at the forty sixth meeting). The hydrolysis of branched-
    chain octyl and larger esters would probably be slower than that of
    the corresponding  n-alkyl analogues, and absorption of the intact
    ester is possible (see below).

         The hydrolysis of esters with larger alcohol substituents, such
    as are present in the linalyl, terpinyl, carvyl, and menthyl esters
    evaluated at the present meeting, has been the subject of only limited
    study. Linalyl acetate was hydrolysed rapidly in water and simulated
    gastric and pancreatic fluids: the mean half-lives for hydrolysis were
    5.5 min in gastric fluid and 52.5 min in pancreatic fluid (R.L. Hall,
    personal communication to the Flavor and Extract Manufacturers'
    Association of the United States), values comparable to those
    considered at the forty-sixth meeting. Allyl tiglate and benzyl
    tiglate were not hydrolysed by these simulated fluids but were
    hydrolysed extensively on incubation with intestinal tissue
    (Grundschober, 1977) and would be predicted to be completely
    hydrolysed by the liver, both  in vitro and  in vivo. (-)-Menthol
    ethylene glycol carbonate and (-)-menthol propylene glycol carbonate
    were hydrolysed slowly on incubation with rat liver homogenate for 4
    h, with 85% hydrolysis to menthol for (-)-menthol ethylene glycol
    carbonate and 75% for (-)-menthol propylene glycol carbonate
    (Emberger, personal communication to the Flavor and Extract
    Manufacturers' Association of the United States). The structurally
    related agent bornyl acetate was excreted largely as the glucuronic
    acid conjugate of borneol after oral administration to rabbits
    (Williams, 1959), consistent with extensive hydrolysis  in vivo. More
    than 80% of radiolabelled cyclandelate, a trimethyl cyclohexyl ester,
    was hydrolysed after 20 min of incubation with rat hepatic microsomes
    (White et al., 1990). Methyl cinnamate was hydrolysed only slowly in

    the rat intestinal lumen  in vivo after oral administration but
    underwent essentially complete first-pass hydrolysis, because none of
    the parent compound could be detected in the systemic circulation
    (Fahelbum & James, 1977). In contrast, propyl anthranilate was not
    completely hydrolysed during absorption, and the intact ester was
    detected in peripheral blood and urine (Fahelbum & James, 1979). These
    findings are similar to those concerning hydrolysis considered at the
    forty-sixth meeting.

         The results indicate that hydrolysis is the major metabolic
    pathway for all of the esters evaluated at the present meeting;
    however, some of the esters in some groups may undergo a low rate of
    hydrolysis, resulting in incomplete pre-systemic metabolism to the
    corresponding alcohol and acid. In general, esters that reach the
    general circulation intact would be expected to be hydrolysed
    extensively by tissue esterases into the acid and alcohol
    constituents.

    (ii)  Oxidation of alcohols and aldehydes

    (a)   Primary alcohols and aldehydes

         Primary aliphatic alcohols (attached to linear, branched, or
    unsaturated alkyl chains) and the corresponding aldehydes are
    efficiently oxidized to the corresponding carboxylic acids by
    high-capacity enzyme pathways. NAD+/NADH-dependent alcohol
    dehydrogenase catalyses the oxidation of primary aliphatic saturated
    and unsaturated alcohols to the corresponding aldehyde (Pietruszko et
    al., 1973). A comparison of the alcohol structure with the enzyme
    binding affinity of alcohol dehydrogenase indicates that increased
    binding (lower Km) occurs with increasing chain length (C1-C6) of
    the substrate and the presence of unsaturation. The maximum rates of
    oxidation were essentially constant, regardless of the alcohol
    structure, suggesting that alcohol-enzyme binding is not the
    rate-limiting step for oxidation; the activity of the enzyme appears
    to be dependent on the lipophilic character of the alcohol substrate
    (Klesov et al., 1977). The three classes of alcohol dehydrogenase
    present in human liver (Eklund et al., 1990) show decreased Km with
    increasing chain length from C2 to C8; the alcohol groups of
    12-hydroxydodecanoic and 16-hydroxyhexadecanoic acid show Km values
    similar to that of hexanol, indicating rapid oxidation of alcohols
    arising from omega-oxidation reactions (see later).

         Similarly, aldehyde dehydrogenase, which is present predominantly
    in hepatic cytosol, has a broad substrate specificity for the
    oxidation of aldehydes to carboxylic acids (Feldman & Weiner, 1972).
    This enzyme is more active on aldehydes of higher relative molecular
    mass (Nakayasu et al., 1978). Three isoenzymes of aldehyde
    dehydrogenase, with overlapping substrate specificities, are present
    in human liver, which oxidize a range of naturally occurring
    aldehydes, such as gamma-butyraldehyde, 3,4-dihydroxyphenyl-
    acetaldehyde, 5-hydroxyindoleacetaldehyde, and acrolein. Alkyl

    aldehydes (C1-C6) have Km values similar to those of these natural
    substrates, and the Km decreases with increasing chain length
    (Ambroziak & Pietruszko, 1991). Xanthine oxidase and aldehyde oxidase
    also catalyse the oxidation of a wide range of aldehydes to the
    corresponding unsaturated acids (Beedham, 1988). Before oxidation to
    the corresponding acid, the aldehyde may be conjugated with sulfhydryl
    groups such as glutathione to yield thiohemiacetals. Oxidation of
    aldehydes with low relative molecular masses requires glutathione
    (Eckfeldt & Yonetani, 1982), which implies that the substrate for
    aldehyde dehydroge-nase-mediated oxidation may be the thiohemiacetal
    (Brabec, 1993).

         Branched-chain aliphatic primary alcohols and aldehydes are
    substrates for alcohol dehydrogenase and aldehyde dehydrogenase,
    although the rates of oxidation are lower than those for linear
    primary alcohols and aldehydes (Blair & Bodley, 1969; Hedlund &
    Kiessling, 1969; Albro, 1975; Saito, 1975; Ambroziak & Pietruszko,
    1991). Unsaturated, linear and branched-chain primary alcohols are
    better substrates than the saturated analogues, but the reverse is
    true of aldehydes (Pietruszko et al., 1973). Citral, a mixture of two
    unsaturated branched-chain aldehydes (neral and geranial), was not
    oxidized to the corresponding acid by aldehyde dehydrogenase in rat
    liver preparations  in vitro but was reduced by alcohol dehydrogenase
    in the presence of NADH (Boyer & Petersen, 1990); oxidation is a major
    metabolic pathway  in vivo, resulting in acid metabolites.

    (b)   Secondary and tertiary alcohols

         Secondary alcohol groups, such as those present in carveol and
    -ionol, undergo reversible oxidation to the corresponding ketone (see
    'ketone reduction', below). The alcohol is the more important form
     in vivo because of its removal from the equilibrium by conjugation
    with glucuronic acid. Tertiary alcohol groups, such as those present
    in linalool and terpineol, do not undergo oxidative metabolism, and
    tertiary alcohols are eliminated by conjugation.

    (iii)  Conjugation of alcohols 

         Conjugation represents the major pathway of metabolism for both
    secondary and tertiary alcohols (Kamil et al., 1953). Extensive
    conjugation with glucuronic acid is followed by excretion primarily in
    the urine or bile, depending on the relative molecular mass and the
    animal species. Glucuronic acid conjugation is a major phase-2
    metabolic reaction, and both the liver and intestinal wall have a high
    capacity for glucuronidation of a wide range of substrates. Sulfate
    conjugation of alcohols occurs in many tissues, especially the liver,
    and results in the formation of highly polar, water-soluble excretory
    products. The urine is the main route of elimination of sulfate
    conjugates of alcohols resulting from the metabolism of the flavouring
    agents evaluated at the present meeting.

         Glucuronic acid conjugation and excretion represent the primary
    route of elimination of linalool, and conjugation with glucuronic acid
    is the major pathway of metabolism of menthol. In rodents, the
    glucuronic acid conjugates of linalool (Parke et al., 1974) and
    menthol (Yamaguchi et al., 1994) are excreted primarily via the bile
    into the intestine, where they may be hydrolysed to yield the free
    alcohol, which undergoes reabsorption and subsequent oxidative
    metabolism. The metabolites of menthol are eliminated in the urine and
    faeces either unchanged or conjugated with glucuronic acid (Yamaguchi
    et al., 1994). Bicyclic tertiary alcohols are relatively stable
     in vivo, and in rabbits thujyl alcohol and -santenol
    (2,3,7-trimethylbicyclo[2.2.1]heptan-2-ol) are extensively conjugated
    with glucuronic acid (Williams, 1938).

    (iv)  Reduction of ketones

         Aliphatic ketones are metabolized primarily  via reduction to
    the corresponding secondary alcohol (Leibman, 1971; Felsted & Bachur,
    1980; Bosron & Li, 1980). Reduction of aliphatic ketones is mediated
    by alcohol dehydrogenases and NADH/NADPH-dependent cytosolic carbonyl
    reductases (Bosron & Li, 1980). The reaction catalysed by carbonyl
    reductase is stereoselective and favours formation of the S enantiomer
    of the alcohol. The reaction is reversible under physiological
    conditions, and oxidation of the secondary alcohol may lead to the
    formation of the corresponding ketone  in vivo (Felsted & Bachur,
    1980). Ketones have been shown to undergo reduction to alcohols in
    human hepatic microsomes (Leibman, 1971). Three reductases have been
    purified from human liver cytosol: two aldehyde reductases, which can
    reduce aliphatic aldehydes, alicyclic ketones, and alpha-diketones,
    and a carbonyl reductase which is active with a broad range of
    aldehyde and ketone substrates (Nakayama et al., 1985). Carbonyl
    reductase activity in human liver shows limited interindividual
    variability, and the enzyme reduces both 4-nitrobenzaldehyde and
    4-nitroacetophenone (Iwata et al., 1993).

         Alicyclic monoketones, such as cyclohexanone, are reduced to the
    corresponding alcohols or undergo alpha-hydroxylation and reduction to
    yield diols, which are excreted as the glucuronic acid conjugates.

         Unsaturated ketones, such as menthone, dihydrocarvone, and
    carvone, undergo extensive reduction of the ketone function to yield
    the corresponding secondary alcohols (pulegone, dihydrocarveol, and
    carveol), which are conjugated mainly with glucuronic acid and then
    excreted in the urine (Hamalainen, 1912; Duterte-Catella et al., 1978;
    Madyastha & Raj, 1993) or bile. In rodents, but probably not in
    humans, the conjugate is excreted primarily into the bile and may then
    be hydrolysed to yield the free alcohol (Matthews, 1994), which may
    undergo enterohepatic recirculation and finally be excreted by the
    kidney (Tamura et al., 1962). Conjugates with relative molecular
    masses below about 500, such as the glucuronides of menthan-2-ol,
    dihydrocarveol, and carveol, would be eliminated by humans in the
    urine; the absence of significant biliary excretion would reduce the
    extent of secondary oxidative metabolism.

         In general, alicyclic alpha-diketones are metabolized  via the
    reduction pathway. Methyl-substituted diketones may be reduced to the
    corresponding hydroxyketones and diols, which are excreted in the
    urine as glucuronic acid conjugates. alpha-Hydroxyketones and their
    diol metabolites may be excreted as glucuronic acid conjugates. This
    pathway is favoured at high concentrations  in vivo, especially for
    longer-chain-length ketones. If the carbonyl function is located
    elsewhere on the chain, detoxification occurs predominantly by 
    reduction. 

    (v)   Reduction of double bonds

         Reduction of endocyclic and exocyclic double bonds occurs in some
    of the flavouring agents evaluated at the present meeting. For
    example, the endocyclic double bond of carvone is reduced to form
    dihydrocarvone and dihydrocarveol, and the exocyclic double bond in
    -ionone is reduced to give dihydro--ionol (Ide & Toki, 1970). The
    enzymes involved in these reactions have not been characterized; the
    intestinal microflora may be involved, either before absorption or
    after biliary excretion.

    (vi)  Oxidation of side-chains

         The position and size of substituents in carboxylic acids
    influences the route of metabolism. As chain length and lipophilicity
    increase, omega-oxidation competes favourably with -oxidative
    cleavage.

    (a)   -Oxidation

         Linear and branched-chain saturated carboxylic acids are
    substrates for  cleavage to yield acetyl coenzyme A (acetyl CoA) and
    a new CoA thioester of the carboxylic acid that has been reduced by
    two carbon atoms. This process continues, to give complete oxidation
    or until a branch point is reached. Acids with an even number of
    carbon atoms continue to be cleaved to acetyl CoA, while acids with an
    odd number of carbon atoms yield acetyl CoA and propionyl CoA. Acetyl
    CoA enters the citric acid cycle directly, whereas propionyl CoA is
    transformed into succinyl CoA which then enters the citric acid cycle.
    Acids with a methyl substituent located at an even-numbered carbon
    (e.g. 2-methylpentanoic acid and 4-methyldecanoic acid) are
    metabolized to carbon dioxide  via -oxidative cleavage in the fatty
    acid pathway. If the methyl group is located at the 3 position,
    -oxidation is inhibited and omega-oxidation predominates, leading
    primarily to polar diacid metabolites that are excreted in the urine.
    Ethyl or propyl substituents located at the alpha or  position in
    relation to the carboxylic acid group inhibit metabolism to carbon
    dioxide (Deuel, 1957), in which case there is direct conjugation of
    the acid with glucuronic acid or omega-oxidation followed by
    conjugation. 

         Linear unsaturated carboxylic acids participate in normal fatty
    acid metabolism. In this pathway, the saturated fatty acid is
    condensed with CoA, which then undergoes catalytic dehydrogenation
    mediated by acyl CoA dehydrogenase (Voet & Voet, 1990). The resulting
     trans-2,3-unsaturated ester  (trans-Delta2-enoyl CoA) is converted
    to the 3-keto-thioester, which undergoes  cleavage to yield an acetyl
    CoA fragment and a new thioester reduced by two carbons. Cleavage of
    acetyl CoA units continues along the carbon chain until the position
    of unsaturation is reached. If unsaturation begins at an even-numbered
    carbon, acetyl CoA fragmentation yields a Delta2-enoyl CoA product
    which is a substrate for further fatty acid oxidation. If unsaturation
    begins at an odd-numbered carbon, acetyl CoA fragmentation will
    eventually yield Delta3-enoyl CoA, which cannot enter the fatty acid
    cycle until it is isomerized to the  trans-Delta2-enoyl CoA by enoyl
    CoA isomerase. If the stereochemistry of the double bond is  cis, it
    is isomerized to the  trans double bond by the action of
    3-hydroxyacyl CoA epimerase before entering the fatty acid oxidation
    pathway. Short-chain acids containing terminal unsaturation may be
    metabolized  via desaturation to yield a substrate which may
    participate in the fatty acid pathway. For example, 4-pentenoic acid
    is converted into the CoA thioester; this is dehydrogenated to yield
     trans-2,4-pentadienoyl CoA, which undergoes NADPH-dependent
    enzyme-catalysed reduction of the Delta2-alkene to
     trans-2-pentenoic acid, which then participates in normal fatty acid
    oxidation (Schulz, 1983). A second, more minor pathway involves
    -oxidation to yield 3-keto-4-pentenoyl CoA.

    (b)   omega- and (omega-1)-Oxidation

         omega- and (omega-1)-Oxidation reactions are important in the
    elimination of compounds with long and/or complex alkyl chains,
    because they eventually yield polar acid metabolites that are
    eliminated in the urine (Horning et al., 1976; Ventura et al., 1985;
    Madyastha & Srivatsan, 1988a). omega-Oxidation results in the
    formation of a primary alcohol, which may undergo further oxidation to
    the corresponding carboxylic acid, which may then be excreted in the
    urine. For example, linalool undergoes cytochrome P450-mediated
    oxidation to form 8-hydroxylinalool and 8-carboxylinalool, whereas
    geraniol is oxidized to 8-hydroxygeraniol, 8-carboxygeraniol, and the
    dibasic acid, Hildebrandt's acid (Chadha & Madyastha, 1984). Citral,
    the aldehyde analogue of geraniol, undergoes oxidation of the aldehyde
    group  in vivo followed by omega-oxidation to yield a mixture of
    diacids and hydroxy acids (Diliberto et al., 1990). Menthol undergoes
    omega-oxidation of the side-chain substituents to yield various
    polyols and hydroxy acids (Madyastha & Srivatsan, 1988b; Yamaguchi et
    al., 1994); the (-) isomer undergoes more extensive oxidation than the
    (+) isomer (Williams, 1939).

         Short-chain ketones (< C4, e.g. butanone) that contain a
    carbonyl function at the 2 position, and alpha-diketones, such as
    acetoin, may undergo omega-oxidation of the terminal methyl group to
    give a diketocarboxylic acid, which would undergo decarboxylation to
    yield an alpha-ketocarboxylic acid. As intermediary metabolites,

    alpha-ketoacids undergo oxidative decarboxylation to yield carbon
    dioxide and a simple aliphatic carboxylic acid, which may be
    completely metabolized  via the fatty acid pathway and citric acid
    cycle. Almost one-half of a dose of radiolabelled acetoin,
    administered by intravenous injection to rats, was eliminated as
    carbon dioxide in expired air (Gabriel et al., 1972); about 20% was
    eliminated as unidentified urinary metabolites.  trans-Methyl styryl
    ketone undergoes essentially complete metabolism of the methyl group
    after oral administration to rats; the glycine conjugates of
    phenylacetic and benzoic acid were the major metabolites in the urine,
    while the intermediate methyl styryl carbinol was detected in plasma
    (Sauer et al., 1997). 

         omega-Oxidation of ketones yields hydroxyketones which may be
    further reduced to diols and excreted in the urine as glucuronic acid
    conjugates. Participation in these pathways depends on chain length,
    the position of the carbonyl function, and the dose (Dietz et al.,
    1981; Topping et al., 1994). Longer-chain aliphatic ketones (i.e.
    carbon chain length > C5) are metabolized primarily  via
    reduction, but omega- and/or (omega-1)-oxidation are competing
    pathways at high concentrations. At high doses, aliphatic ketones may
    undergo omega- and/or (omega-1)-oxidation to yield ketoacids and
    diketones, respectively. 

         A number of the flavouring agents evaluated at the present
    meeting are alicyclic ketones containing an alkyl or alkenyl side
    chain; these undergo oxidation of the side chain (probably by
    cytochrome P450: Madyastha & Raj, 1990) to form polar metabolites that
    are excreted as the glucuronic acid or sulfate conjugates in the urine
    and, to a lesser extent, in the faeces (Williams, 1959; Ishida et al.,
    1989). 

    (c)   Oxidation of double bonds

         Oxidation of double bonds  via an epoxide intermediate to form
    diols is probably only a minor pathway of metabolism for a few of the
    flavouring agents considered at the present meeting. For example,
    alpha-terpineol is oxidized by cytochrome P450 in rats, via an epoxide
    intermediate, to a 1,2-dihydroxy metabolite (Madyastha & Srivatsan,
    1988a); this diol metabolite has been detected in humans after
    ingestion of alpha-terpineol (Horning et al., 1976).

    (vii)  Oxidation of alicyclics

         Cyclohexane and cyclohexene rings, which are present in the
    carvone and ionone groups considered at the present meeting, are
    rapidly oxidized  in vivo by ring hydroxylation, even when other
    pathways of elimination are present. For example, the main pathways of
    elimination of cyclohexane are exhalation unchanged in expired air and
    excretion in urine as glucuronic acid conjugates of cyclohexanol and
    cyclohexane-1,2-diol (Elliott et al., 1959). Cyclohexylamine is highly
    water-soluble and is excreted in the urine as both the parent compound
    and the glucuronides of ring-hydroxylated metabolites, together with

    small amounts of cyclohexane-1,2-diol formed  via deamination and
    ring hydroxylation (Renwick & Williams, 1972). Ring oxidation, which
    is catalysed by cytochrome P450 (CYP3A4), is a major pathway of
    metabolism for the substituted cyclohexene ring systems that are
    present in vitamin A and its metabolites, such as 13- cis- and
    all- trans-retinoic acid, as well as the ionone group of flavouring
    agents. Oxidation of the substituted cyclohexene rings present in
    retinoids and ionones occurs on the carbon adjacent to the double
    bond.

    (vii)  Conjugation with glutathione

         The vast majority of the flavouring agents evaluated at the
    present meeting are metabolized by the pathways described above.
    Possible exceptions are carvone, carveol (and its esters), and some
    members of the ionone group in which a ketone group, or a precursor
    secondary alcohol group, is located adjacent to a double bond or
    conjugated double bonds (alpha,-unsaturated ketone). Glutathione
    conjugation is an important detoxification pathway, especially for
    reactive compounds. The products of conjugation with glutathione are
    usually eliminated in the bile as the glutathione conjugates  per se 
    and in the urine as the  N-acetylcysteine metabolites produced from
    the glutathione conjugates. Glutathione conjugates may also be split
    by C-S lyase (-lyase) in the kidney and intestinal microflora, to
    give a thiol compound; this reaction represents a bioactivation
    process for some chemicals. Only limited data were available on the
    metabolism of the ionone group of flavouring agents, and glutathione
    conjugation products have not been reported as urinary metabolites of
    ionones or of carvone. Oral administration of 20 mg carvone, by gavage
    in corn oil, to mice daily for three days caused a reduction in the
    concentration of glutathione in the liver and an increase in
    glutathione transferase activity (Zheng et al., 1992); however, these
    effects may not have been due to the alpha,-unsaturated ketone
    moiety, because the decrease in glutathione was not found with
    alpha,-unsaturated carvone analogues lacking the exocyclic allyl
    group, while the increase in glutathione transferase activity was
    detected with compounds such as  para-menthan-2-one which contain
    neither an alpha,-unsaturated ketone moiety nor an exocyclic allyl
    group.

         The potential for non-enzymatic interaction with glutathione was
    shown  in vitro (Portoghese et al., 1989). The greatest reactivity
    with glutathione was found with the simplest alpha,-unsaturated
    ketone (butenone), which had a rate of reaction about 1000 times
    greater than that of oct-2-ene-4-one. Damascenone, the 1,3-endocyclic
    diene analogue of damascone, and carvone, which were evaluated at the
    present meeting, showed rates of reaction that were 17 000, 11 000,
    and 320 000 times slower than that of butenone, which indicates low
    reactivity. 

         Overall, the flavouring agents with an alpha,-unsaturated ketone
    moiety that were evaluated at the present meeting have low reactivity,
    and conjugation with glutathione would not be a major route of
    metabolism. The low predicted reactivity is supported by the
    toxicological data for the ionones and carvone.


    REFERENCES

    Albro, P.W. (1975) The metabolism of 2-ethylhexanol in rats.
     Xenobiotica, 5, 625-636.

    Ambroziak, W. & Pietruszko, R. (1991) Human aldehyde dehydrogenase:
    Activity with aldehyde metabolites of monoamines, diamines, and
    polyamines.  J. Biol. Chem., 20, 13011-13018.

    Beedham, C. (1988) Molybdenum hydroxylases. In: Gorrod, J.W.,
    Oelschlager, H. & Caldwell, J., eds,  Metabolism of Xenobiotics, 
    London, Taylor & Francis, pp. 51-58.

    Blair, A.H. & Bodley, F.H. (1969) Human liver aldehyde dehydrogenase:
    Partial purification and properties.  Can. J. Biochem., 47, 265-272.

    Bosron, W.F. & Li, T.-K. (1980) Alcohol dehydrogenase. In: Jacoby, W.,
    ed.,  Enzymatic Basis of Detoxification, Vol. 1, New York, Academic
    Press, p. 231.

    Boyer, C.S. & Petersen, D.R. (1990) The metabolism of
    3,7-dimethyl-2,6-octadienal (citral) in rat hepatic mitochondrial and
    cytosolic fractions.  Drug Metab. Disposition, 16, 81-86.

    Brabec, M.J. (1993) Aldehydes and acetals. In: Clayton, G.D. &
    Clayton, F.E., eds,  Patty's Industrial Hygiene and Toxicology, 4th
    Ed., New York, John Wiley & Sons, Vol. IIA, pp. 283-327.

    Chadha, A. & Madyastha, K.M. (1984) Metabolism of geraniol and
    linalool in the rat and effects on liver and lung microsomal enzymes.
     Xenobiotica, 14, 365-374.

    Deuel, H.J., ed. (1957)  The Lipids, Theit Chemistry and 
     Biochemistry, New York, Wiley Interscience, Vol. III.

    Dietz, F.K., Rodriguez-Giaxola, M., Traiger, G.J., Stella, V.J. &
    Himmelstein, K.J. (1981) Pharmacokinetics of 2-butanol and its
    metabolites in the rat.  J. Pharmacokinet. Biopharm., 9, 553-576.

    Diliberto, J.J., Usha, G. & Birnbaum, L.S. (1990) Disposition of
    citral in male Fischer rats.  Drug Metab. Disposition, 16, 721-727.

    Duterte-Catella, H., Nguyen, P., Dang, Q. & Truhaut, R. (1978)
    Metabolic transformations of the trimethyl-3,5,5, cycloxene-2, one-1
    (isophorone).  Toxicol. Eur. Res., 1, 209-216.

    Eckfeldt, J.H. & Yonetani, Y. (1982) Isoenzymes of aldehyde
    dehydrogeanse from horse liver. In: Wood, W.A., ed.,  Methods in 
     Enzymology, New York, Academic Press, pp. 474-479.

    Eklund, H., Mller-Wille, P., Horjales, E., Futer, O., Holmquist, B.,
    Vallee, B.L., Hg, J.-O., Kaiser, R. & Jrnvall, H. (1990) Comparison
    of three classes of human liver alcohol dehydrogenase: Emphasis on
    different substrate binding pockets.  Eur. J. Biochem., 193, 303-310.

    Elliott, T.H., Parke, D.V. & Williams, R.T. (1959) Studies in
    detoxication: 79. The metabolism of  cyclo[14C]hexane and its
    derivatives.  Biochem. J., 72, 193-200.

    Fahelbum, I.M.S. & James, S.P. (1977) The absorption and metabolism of
    methyl cinnamate.  Toxicology, 7, 123-132.

    Fahelbum, I.M.S. & James, S.P. (1979) Absorption, distribution and
    metabolism of propyl anthranilate.  Toxicology, 12, 75-87.

    Feldman, R.I. & Weiner, H. (1972) Horse liver aldehyde dehydrogenase.
    I. Purification and characterization.  J. Biol. Chem., 247, 260.

    Felsted, R.L. & Bachur, N.R. (1980) Ketone reductases. In: Jacoby,
    W.B., ed.,  Enzymatic Basis of Detoxification, Vol. 1, New York,
    Academic Press, pp. 281-293.

    Gabriel, M.A., Ilbawi, M. & Al-Khalidi, U.A.S. (1972) The oxidation of
    acetoin to CO2 in intact animals and in liver mince preparation.
     Comp. Biochem. Physiol., 41B, 493-502.

    Grundschober, F. (1977) Toxicological assessment of flavouring esters.
     Toxicology, 8, 387-390.

    Hamalainen, J. (1912) [Concerning the behaviour of alicyclic compounds
    with glucuronic acid in organisms.]  Skand. Arch. Physiol., 27,
    141-226 (in German).

    Hedlund, S.G. & Kiessling, K.H. (1969) The physiological mechanism
    involved in hangover. 1. The oxidation of some liver aliphatic fusel
    alcohols and their effect on the mitochondrial oxidation of various
    substrates.  Acta Pharmacol. Toxicol., 27, 381-396.

    Horning, M.G., Butler, C.M., Stafford, M., Stillwell, R.N., Hill,
    R.M., Zion, T.E., Harvey, D.T. & Stillwell, W.G. (1976) Metabolism of
    drugs by the epoxidediol pathway. In: Frigerio, A. & Catagnoli, N.,
    eds,  Advances in Mass Spectrscopy in Biochemistry and Medicine, New
    York, Spectrum Publications, Vol. I, pp. 91-108.

    Ide, H. & Toki, S. (1970) Metabolism of -ionone. Isolation,
    characterization and identification of the metabolites in the urine of
    rabbits.  Biochem. J., 119, 281-287.

    Ishida, T., Toyota, M. & Asakawa, Y. (1989) Terpenoid
    biotransformation in mammals. V. Metabolism of (+)-citronellal,
    ()-7-hydroxycitronellal, (-)-perillaldehyde, (-)-myrtenal,
    cuminaldehyde, thujone, and ()-carvone in rabbits.  Xenobiotica, 19,
    843-855.

    Iwata, N., Inazu, N., Hara, S., Yanase, T., Kano, S., Endo, T.,
    Kuriiwa, F., Sato, Y. & Satoh, T. (1993) Interindividual variability
    of carbonyl reductase levels in human livers.  Biochem. Pharmacol., 
    45, 1711-1714.

    Kamil, A.I., Smith, J.N. & Williams, R.T. (1953) Studies in
    detoxication. 46. The metabolism of aliphatic alcohols: The glucuronic
    acid conjugation of acyclic aliphatic alcohols.  Biochemistry, 53,
    129-136.

    Klesov, A.A., Lange, L.G., Sytkowski, A.J. & Vallee, B.L. (1977)
    [Unusual nature of the substrate specificity of alcohol dehydrogenase
    of different origins.]  Biorganich. Khim., 3, 1141-1144 (in Russian).

    Leibman, K.C. (1971) Reduction of ketones in liver cytosol.
     Xenobiotica, 1, 97-104.

    Madyastha, K.M. & Raj, C.P. (1990) Biotransformations of
    R-(+)-pulegone and menthofuran  in vitro: Chemical basis for
    toxicity.  Biochem. Biophys. Res. Commun., 3, 1086-1092.

    Madyastha, K.M. & Raj, C.P. (1993) Studies on the metabolism of a
    monoterpene ketone, R-(+)-pulegone -- a hepatotoxin in rat: Isolation
    and characterization of new metabolites.  Xenobiotica, 23, 509-518.

    Madyastha, K.M. & Srivatsan, V. (1988a) Biotransformations of
    alpha-terpineol in the rat: Its effects on the liver microsomal
    cytochrome P-450 system.  Bull. Environ. Contam. Toxicol., 41, 17-25.

    Madyastha, K.M. & Srivatsan, V. (1988b) Studies on the metabolism of
     l-menthol in rats.  Drug Metab. Disposition, 16, 765-772.

    Matthews, H.B. (1994) Excretion and elimination of toxicants and their
    metabolites. In: Hodgson, E. & Levi, P., eds,  Introduction to 
     Biochemical Toxicology, 2nd Ed., Norwalk, Connecticut, Appleton &
    Lange, pp. 177-192.

    Nakayama, T., Hara, A., Yashiro, K. & Sawada, H. (1985) Reductases for
    carbonyl compounds in human liver.  Biochem. Pharmacol., 34, 107-117.

    Nakayasu, H., Mihara, K. & Sato, R. (1978) Purification and properties
    of membrane-bound aldehyde dehydrogenase from rat liver microsomes.
     Biochem. Biophys. Res. Commun., 83, 697-703.

    Parke, D.V., Rahman, K.H.M.Q. & Walker, R. (1974) The absorption,
    distribution and excretion of linalool in the rat.  Biochem. Soc. 
     Trans., 2, 612-615.

    Pietruszko, R., Crawford, K. & Lester, D. (1973) Comparison of
    substrate specificity of alcohol dehydrogenases from human liver,
    horse liver and yeast towards saturated and 2-enoic alcohols and
    aldehydes.  Arch. Biochem. Biophys., 159, 50-60.

    Portoghese, P.S., Kedziora, G.S., Larson, D.L., Bernard, B.K. & Hall,
    R.L. (1989) Reactivity of glutathione with alpha,-unsaturated ketone
    flavoring substances.  Food Chem. Toxicol., 27, 773-776.

    Renwick, A.G. & Williams, R.T. (1972) The metabolites of
    cyclohexylamine in man and certain animals.  Biochem. J., 129,
    857-867.

    Saito, M. (1975) Metabolism of lower alcohols.  Nichidai Igaku 
     Zasshi, 34, 569-585.

    Sauer, J.-M., Smith, R.L., Bao, J., Kattnig, M.J., Kuester, R.K.,
    McClure, T.D., Mayersohn, M. & Sipes, I.G. (1997) Oral and topical
    absorption, disposition kinetics, and the metabolic fate of
     trans-methyl styryl ketone in the male Fischer 344 rat.  Drug 
     Metab. Disposition, 25, 732-739.

    Savary, P. & Constantin, M.J. (1970) Intestinal hydrolysis and
    lymphatic absorption of isopropyl asters of long chain fatty acids in
    the rat.  Biochem. Biophys. Acta, 218, 195.

    Schulz, H. (1983) Metabolism of 4-pentenoic acid and inhibition of
    thiolase by metabolites of 4-pentenoic acid.  Biochemistry, 22,
    1827-1832.

    Tamura, S., Tsutsumi, S. & Kizu, K. (1962) Studies on glucuronic acid
    metabolism. I. The influence of borneol, ionone and carvone on the
    urinary excretion of glucuronic acid and ascorbic acid.  Fol. 
     Pharmacol. Jpn., 58, 323-336.

    Topping, D.C., Morgott, D.A., David, R.M. & O'Donoghue, J.L. (1994)
    Ketones. In: Clayton, G.D. & Clayton, F.E., eds,  Patty's Industrial 
     Hygiene and Toxicology, 4th Ed., New York, John Wiley & Sons, pp.
    1739-1878.

    Ventura, P., Schiavi, M., Serafini, S. & Selva, S. (1985) Further
    studies of  trans-sobrerol metabolism: Rat, dog, and human urine.
     Xenobiotica, 15, 317-325.

    Voet, D. & Voet, J.G. (1990)  Biochemistry, New York, John Wiley &
    Sons

    White, D.A., Heffron, A., Miciak, B., Middleton, B., Knight, S. &
    Knight, D. (1990) Chemical synthesis of dual radiolabelled
    cyclandelate and its metabolism in rat hepatocytes and mouse J774
    cells.  Xenobiotica, 20, 71-79.

    Williams, R.T. (1938) Studies in detoxication. II. a) The conjugation
    of isomeric 3-menthanols with glucuronic acid and the asymmetric
    conjugation of dl-menthol in the rabbit. b) d-iso-Menthylglucuronide,
    a new conjugated glucuronic acid.  Biochem. J., 32, 1849-1855.

    Williams, R.T. (1939) Studies in detoxication. III. The use of the
    glucuronic acid detoxication mechanism of the rabbit for the
    resolution of dl-menthol.  Biochem. J., 33, 1519-1524.

    Williams, R.T. (1959)  Detoxication Mechanisms: The Metabolism and 
     Detoxication of Drugs, Toxic Substances and Other Organic 
     Compounds, 2nd Ed., London, Chapman & Hall

    Yamaguchi, T., Caldwell, J. & Farmer, P.B. (1994) Metabolic fate of
    [3H]- l-menthol in the rat.  Drug Metab. Disposition, 22, 616-624.

    Zheng, G.-Q., Kenney, P.M. & Lam, K.L.T. (1992) Effects of carvone
    compounds on glutathione S-transferase activity in A/J mice.
     J. Agric. Food Chem., 40, 751-755.
    


    See Also:
       Toxicological Abbreviations