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 EVALUATION OF ALIPHATIC ACYCLIC AND ALICYCLIC alpha-DIKETONES 
    AND RELATED alpha-HYDROXYKETONES 

    Mrs M.F.A. Wouters and Dr G.J.A. Speijers
    National Institute of Public Health and the Environment, Center of
    Substances and Risk Assessment, Bilthoven, The Netherlands

          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 
              Explanation 
              Intake
              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 a group of 22 flavouring agents (Table 1)
    that includes aliphatic acyclic and alicyclic alpha-diketones and
    related alpha-hydroxyketones, using the Procedure for the Safety
    Evaluation of Flavouring Agents (Figure 1, p. 222, and Annex 1,
    reference 131). One member of the group, diacetyl, was evaluated at
    the eleventh meeting of the Committee; however, because of lack of
    data, no ADI was allocated (Annex 1, reference 14).

    1.2  Estimated daily per capita intake

         In the United States, aliphatic acyclic and alicyclic
    alpha-diketones and alpha-hydroxyketones are generally used as
    flavouring agents up to average maximum levels of 200 ppm. The total
    annual volume of the 22 substances in this group is approximately 44
    000 kg in Europe (International Organization of the Flavor Industry,
    1995) and 56 000 kg in the United States (US National Academy of
    Sciences, 1970, 1982, 1987). In both Europe and the United States,
    more than 95% of the total annual volume is accounted for by three
    substances: acetoin (No. 405: 19 000 kg/year in Europe and 9200

    kg/year in the United States), diacetyl (No. 408: 18 000 kg/year in
    Europe and 42 000 kg/year in the United States), and
    methylcyclopentenolone (No. 418: 4700 kg/year in Europe and 3700
    kg/year in the United States). Two of these, acetoin and diacetyl,
    account for more than 90% of the total annual volume in the United
    States (US National Academy of Sciences, 1987). 

         Nineteen of the 22 aliphatic acyclic and alicyclic
    alpha-diketones and alpha-hydroxyketones have been identified as
    natural components of a variety of foods, including fruits,
    vegetables, cocoa, and coffee (Maarse et al., 1994). Quantitative data
    have been reported for the natural occurrence of six of these
    substances, which indicate that the intake as natural components of
    food is greater than that from their use as flavouring agents
    (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987), with one
    exception (diacetyl).

    1.3  Absorption, metabolism, and elimination

         In rats and mice, orally administered aliphatic alpha-diketones
    are rapidly absorbed from the gastrointestinal tract (Gabriel et al.,
    1972). It is anticipated that at low levels of exposure, humans will
    metabolize aliphatic acyclic alpha-diketone principally by
    alpha-hydroxylation and subsequent oxidation of the terminal methyl
    group to yield the corresponding ketocarboxylic acid. The acid may
    undergo oxidative decarboxylation to yield carbon dioxide and a simple
    aliphatic carboxylic acid, which may be completely metabolized in the
    fatty acid pathway and citric acid cycle. At high concentrations,
    another detoxification pathway is used which involves reduction to the
    diol and subsequent conjugation with glucuronic acid (Westerfeld &
    Berg, 1943; Williams, 1959; Gabriel et al., 1972; Otsuka et al.,
    1996). Acyclic alpha-diketones and alpha-hydroxyketones without a
    terminal methyl group and alicyclic diketones and hydroxyketones are
    mainly metabolized by reduction to the corresponding diol, followed by
    glucuronic acid conjugation and excretion (Mills & Walker, 1990; Ong
    et al., 1991). 

    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), all 22 alpha-diketones
              and related hydroxyketones are in class II.

    Step 2.   Studies  in vitro and  in vivo have demonstrated two major
              routes of metabolism for diacetyl(2,3-butadione) and
              acetoin, involving complete oxidation to carbon dioxide and
              reduction to a diol, but data were not available on the
              higher linear homologues or cyclic analogues. Alcohol
              dehydrogenases, aldehyde reductase, and carbonyl reductase
              are widely distributed enzymes with broad substrate
              specificities; studies conducted  in vitro show that
              1,2-cyclo-hexandione is a better substrate than diacetyl for

              aldehyde reductase and carbonyl reductase. In consequence,
              it was consi-dered that the data on the extensive reduction
              of acetoin can be extrapolated to other members of the
              group. The alpha-diketone group is polar, and it would be
              expected that all members of the group evaluated would be
              eliminated by a combination of oxidation when the carbonyl
              group is adjacent to a methyl group, reduction of the
              carbonyl group, and excretion of the parent compound and
              metabo-lites in urine. The products of these metabolic
              pathways are not of concern.

    Step A3.  The daily  per capita intakes ('eaters only') of the
              substances in Europe and the United States are below the
              human intake threshold for class II (540 g/person per day),
              indicating that they pose no safety concern when used at
              current levels of estimated intake as flavouring agents. The
              intakes of acetoin (2800 g/person per day in Europe; 1800
              g/person per day in the United States), diacetyl (3300
              g/person per day in Europe; 8000 g/person per day in the
              United States), and methylcyclopentenolone (890 g/person
              per day in Europe; 710 g/person per day in the United
              States) are, however, greater than 540 g/person per day.

    Step A4.  Acetoin and diacetyl occur endogenously in humans (Kawano,
              1959; Zlatkis & Sivetz, 1960; Gabriel et al., 1972), but
              methyl-cyclopentenolone does not. 

    Step A5.  A NOEL of 500 mg/kg bw per day was reported for
              methylcyclo-pentenolone in a six-month study of toxicity in
              rats (Dow Chemical Co., 1953). A safety margin of > 10 000
              exists between this NOEL and the estimated daily  per 
               capita intake ('eaters only') of 15 g/kg bw. In addition,
              methylcyclo-pentenolone gave negative results in tests for
              genotoxicity (reverse mutation and unscheduled DNA
              synthesis; Heck et al., 1989). This information indicates
              that methylcyclo-pentenolone would not be expected to be of
              safety concern.

         The stepwise evaluations of the 22 aliphatic acyclic and
    alicyclic alpha-diketones and related alpha-hydroxyketones used as
    flavouring agents are summarized in Table 1.

    1.5  Consideration of combined intakes from use as flavouring agents

         In the unlikely event that all 22 aliphatic acyclic and alicyclic
    alpha-diketones and related alpha-hydroxyketones were consumed
    simultaneously on a daily basis, the estimated combined intake would
    exceed the human intake threshold for class II; however, all 22
    substances are expected to be efficiently metabolized and would not
    saturate the detoxification pathways. On the basis of the evaluation
    of the collective data, there is no safety concern about combined
    intake.


        Table 1. Summary of results of safety evaluations on aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones

    Step 1: All of the substances in the group are in structural class II. The human intake threshold for class II is 540 g/day.
    Step 2: All of the substances in this group are metabolized to innocuous products.

                                                                                                                                            

    Substance                                       No.   CAS No.     Estimated    Step A3      Step A4      Step A5            Conclusion 
                                                                      per capita   Does intake  Endogenous?  Adequate           based on 
                                                                      intake       exceed                    NOAEL for          current 
                                                                      Europe/USA   intake                    substance          intake
                                                                      (g/day)     threshold?                or related 
                                                                                                             substance?
                                                                                                                                            

    Acetoin                                         405   513-86-0    2800/1800    Yes          Yes          N/R1               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    2-Acetoxy-3-butanone                            406   4906-24-5   0.03/23      No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    Butan-3-one-2-yl-butanoate                      407   84642-61-5  0.02/0.95    No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                            

    Substance                                       No.   CAS No.     Estimated    Step A3      Step A4      Step A5            Conclusion 
                                                                      per capita   Does intake  Endogenous?  Adequate           based on 
                                                                      intake       exceed                    NOAEL for          current 
                                                                      Europe/USA   intake                    substance          intake
                                                                      (g/day)     threshold?                or related 
                                                                                                             substance?
                                                                                                                                            

    Diacetyl                                        408   431-03-8    3300/8000    Yes          Yes          N/R1               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    3-Hydroxy-2-pentanone                           409   3142-66-3   ND/0.10      No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    2,3-Pentanedione                                410   600-14-6    220/80       No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    4-Methyl-2,3-pentadione                         411   7493-58-5   0.48/2       No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                            

    Substance                                       No.   CAS No.     Estimated    Step A3      Step A4      Step A5            Conclusion 
                                                                      per capita   Does intake  Endogenous?  Adequate           based on 
                                                                      intake       exceed                    NOAEL for          current 
                                                                      Europe/USA   intake                    substance          intake
                                                                      (g/day)     threshold?                or related 
                                                                                                             substance?
                                                                                                                                            

    2,3-Hexadione                                   412   3848-24-6   13/10        No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    3,4-Hexadione                                   413   4437-51-8   33/0.76      No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    5-Methyl-2,3-hexadione                          414   13706-86-0  2/6          No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    2,3-Heptanedione                                415   96-04-8     2/5          No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                            

    Substance                                       No.   CAS No.     Estimated    Step A3      Step A4      Step A5            Conclusion 
                                                                      per capita   Does intake  Endogenous?  Adequate           based on 
                                                                      intake       exceed                    NOAEL for          current 
                                                                      Europe/USA   intake                    substance          intake
                                                                      (g/day)     threshold?                or related 
                                                                                                             substance?
                                                                                                                                            

    5-Hydroxy-4-octanone                            416   496-77-5    0.02/0.76    No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    2,3-Undecadione                                 417   7493-59-6   0.02/0.01    No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    Methylcyclopentenolone                          418   80-71-7     890/710      Yes          No           Yes. The dose of   No safety 
                                                                                                             500 mg/kg bw per   concern  
                                                                                                             day that had no 
                                                                                                             adverse effects  
                                                                                                             (Dow Chemical Co.,
                                                                                                             1953)  is > 10 000
                                                                                                             times the intake.
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                            

    Substance                                       No.   CAS No.     Estimated    Step A3      Step A4      Step A5            Conclusion 
                                                                      per capita   Does intake  Endogenous?  Adequate           based on 
                                                                      intake       exceed                    NOAEL for          current 
                                                                      Europe/USA   intake                    substance          intake
                                                                      (g/day)     threshold?                or related 
                                                                                                             substance?
                                                                                                                                            

    Ethylcyclopentenolone                           419   21835-01-8  50/23        No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    3,4-Dimethyl-1,2-cyclopentadione                420   13494-06-9  47/2         No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    3,5-Dimethyl-1,2-cyclopentadione                421   13494-07-0  55/29        No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    3-Ethyl-2-hydroxy-4-methylcyclopent-2-en-1-one  422   42348-12-9  ND/0.17      No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    Table 1. (continued)
                                                                                                                                            

    Substance                                       No.   CAS No.     Estimated    Step A3      Step A4      Step A5            Conclusion 
                                                                      per capita   Does intake  Endogenous?  Adequate           based on 
                                                                      intake       exceed                    NOAEL for          current 
                                                                      Europe/USA   intake                    substance          intake
                                                                      (g/day)     threshold?                or related 
                                                                                                             substance?
                                                                                                                                            

    5-Ethyl-2-hydroxy-3-methylcyclopent-2-en-1-one  423   53263-58-4  ND/0.38      No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    2-Hydroxy-2-cyclohexen-1-one                    424   10316-66-2  0.08/0.76    No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    1-Methyl-2,3-cyclohexadione                     425   3008-43-3   2/8          No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

    2-Hydroxy-3,5,5-trimethyl-2-cyclohexen-1-one    426   4883-60-7   2/2          No           N/R2         N/R2               No safety 
                                                                                                                                concern
    CHEMICAL STRUCTURE 

                                                                                                                                            

    N/R1, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A4 of the procedure.
    N/R2, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3 of the procedure.
    N/D, no intake data reported.
    

    1.6  Conclusions

         The 22 aliphatic acyclic and alicyclic alpha-diketones and
    related alpha-hydroxyketones evaluated do not pose a safety concern at
    current levels of intake as flavouring agents. No data on toxicity
    were required for application of the procedure to 21 of the aliphatic
    acyclic and alicyclic alpha-diketones and related
    alpha-hydroxyketones. The Committee noted that the available data on
    toxicity were consistent with the results of the safety evaluations
    using the procedure. 


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

         This group of substances was selected on the basis of the
    criteria that all members of the group are aliphatic and contain
    functional groups (alpha-diketones, alpha-hydroxyketones, and esters
    of a methylcyclopentenolone hydroxyketones) which have similar
    chemical reactivity and participate in common pathways of metabolic
    detoxification. Acyclic and alicyclic alpha-diketones exist to varying
    degrees in equilibrium with unsaturated alpha-hydroxyketones (i.e. the
    ketoenolic form). Therefore, 17 of the 22 substances in this group
    exist as mixtures of alpha-diketone and unsaturated
    alpha-hydroxyketone. Three (Nos 405, 409, and 416) other members of
    the group are aliphatic alpha-hydroxyketones which may be formed
     in vivo by simple reduction of one of the two ketone functions in
    the corresponding alpha-diketone. The two remaining members of the
    group (Nos 406 and 407) are aliphatic esters of the
    alpha-hydroxyketone acetoin (No. 405). Hydrolysis of these esters
     in vivo yields acetoin (No. 405) and simple aliphatic carboxylic
    acids. Consequently, it can be assumed that all of these flavouring
    agents either are, or can readily form, alpha-diketones or
    alpha-hydroxyketones (see Figure 1). In view of the close chemical and
    biochemical relationships between these substances and the consistent
    data on toxicity, they are evaluated here as a group of structurally
    related compounds. 

    2.2  Intake

         The total annual production volume of the 22 substances in this
    group is approximately 44 000 kg in Europe (International Organization
    of the Flavor Industry, 1995) and 56 000 kg in the United States (US
    National Academy of Sciences, 1970, 1982, 1987). Production volumes
    and intake values for each substance are reported in Table 2. On the
    basis of the annual volumes reported in Europe and the United States,
    the total estimated daily  per capita intake ('eaters only') of the
    22 flavouring agents in this group is 120 g/kg bw per day in Europe
    and 180 g/kg bw per day in the United States.

    FIGURE 1


         The vast majority of aliphatic acyclic and alicyclic
    alpha-diketones and related alpha-hydroxyketones have been reported to
    occur in traditional foods (Maarse et al., 1994). Quantitative data on
    the natural occurrence of these substances, with the exception of
    diacetyl, demonstrate that they are consumed predominantly from
    traditional foods (i.e. consumption ratio > 1) (Stofberg & Kirschman,
    1985; Stofberg & Grundschober, 1987).

    2.3  Biological data

    2.3.1  Absorption and metabolism

         Aliphatic acyclic and alicyclic alpha-diketones participate in an
    enol-keto equilibrium with the corresponding ketoenol (see Figure 1).
    The enolic form predominates in alicyclic diketones, especially
    cyclopentyl alpha-diketones (Gordon & Ford, 1972), essentially 100% of
    which exist in the ketoenolic form; 50-80% of cyclohexyl
    alpha-diketones exist in the ketoenolic form at equilibrium.
    Increasing pH would be expected to shift the equilibrium in favour of
    the ketoenolic form (i.e. alpha-hydroxyketone). Therefore, at
    physiological pH, an increase in the alpha-hydroxyketone (i.e. enol)
    form is expected. 

         In rats and mice, orally administered aliphatic alpha-diketones
    are rapidly absorbed from the gastrointestinal tract (Gabriel et al.,
    1972). Carbon dioxide produced primarily from methyl-substituted
    diketones (e.g. diacetyl) is eliminated in expired air. Thus, after
    injection of [2,3-14C]-acetoin to whole albino rats, 14C-carbon

    dioxide appeared in expired air, with an average 12-h production of
    15% (Gabriel et al., 1972). In rats, 54-82% of orally administered
    radiolabelled 2,3-butanedione was excreted as carbon dioxide. At
    increasing doses, the percent of the dose excreted as carbon dioxide
    decreased, while urinary excretion of radiolabel increased, suggesting
    that saturation occurs at higher doses. Chromatographic analysis of
    urine samples yielded three major 14C-labelled components, one of
    which co-eluted with uric acid. Analysis with -glucuronidase or
    sulfatase decreased the amount of radiolabel present in one peak, with
    a corresponding increase in the amount present in another major
    component. Identification of urinary metabolites was not pursued owing
    to the extensive excretion of 14C as carbon dioxide (Dix & Jeffcoat,
    1997). In general, esters are hydrolysed to their corresponding
    alcohols and carboxylic acids. Hydrolysis is catalysed by classes of
    enzymes recognized as carboxylesterases or esterases, the most
    important of which are the B-esterases. Acetyl esters are the
    preferred substrates of C-esterases (Heymann, 1980). These enzymes
    occur in most mammalian tissues (Heymann, 1980; Anders, 1989) but
    predominate in hepatocytes (Heymann, 1980). 2-Acetoxy-3-butanone (No.
    406) and butanon-3-one-2-yl butanoate (No. 407) are expected to be
    metabolized in humans to acetic acid and butanoic acid, respectively,
    and to acetoin.

     Aliphatic acyclic diketones 

         The metabolic fate of acyclic aliphatic diketones depends
    primarily on the position of the carbonyl function and on chain
    length. Aliphatic acyclic diketones and alpha-hydroxyketones that
    contain a carbonyl function at the 2 position (i.e. methyl ketones)
    may undergo alpha-hydroxylation and subsequent oxidation of the
    terminal methyl group to yield corresponding ketocarboxylic acids. The
    ketoacids are intermediary metabolites (e.g. alpha-ketoacids), which
    may undergo oxidative decarboxylation to yield carbon dioxide and a
    simple aliphatic carboxylic acid. The acid may be completely
    metabolized in the fatty acid pathway and citric acid cycle. 

         Alternatively, the methyl-substituted diketones may be
    successively reduced to the corresponding hydroxyketones and diols,
    which are excreted in the urine as glucuronic acid conjugates. This
    pathway is favoured at high concentrations  in vivo, especially for
    longer-chain ketones. If the carbonyl function is located elsewhere on
    the chain, reduction is the predominant detoxification pathway.
    alpha-Hydroxyketones and their diol metabolites may be excreted as
    glucuronic acid conjugates. For example, the glucuronic acid conjugate
    of cyclohexanol was detected in the urine of humans exposed to
    cyclohexanone (Ong et al., 1991), indicating that alpha-hydroxylation
    followed by reduction of the ketone function occurs in humans.

          Acetoin is metabolized primarily  via oxidation at low
    concentrations  in vivo and by reduction to 2,3-butanediol at high
    concentrations. It has been estimated that 1 g of rat liver can
    oxidize 86 g (1 mol) acetoin per day (Gabriel et al., 1972).
    Production of carbon dioxide at low levels and of 2,3-butanediol at

    high levels is associated with the slower rate of ketone reduction
    (Williams, 1959). Oxidation of the terminal methyl group may result in
    formation of an alpha-ketoacid, which undergoes cleavage to yield
    carbon dioxide and a carboxylic acid fragment. Alternatively, methyl
    group oxidation may yield a -ketoacid which undergoes -cleavage to
    yield two-carbon fragments. To a minor extent, these two-carbon
    fragments can act as acetyl donors for acetylation of
     para-aminobenzoic acid (Westerfeld & Berg, 1943).

         A total dose of 78 g of acetoin was administered to a dog over
    two months, both orally in a 3-4% solution and subcutaneously in a 20%
    solution. Urine was collected under toluene from the beginning of
    treatment up to 40 h after the last dose. 2,3-Butanediol was the major
    urinary excretion product, representing 5-25% of the dose. The
    remainder of the dose was completely metabolized (Westerfeld & Berg,
    1943). 

         In liver preparations obtained from rats and rabbits, more than
    95% of the radiolabel of [2,3-14C]-acetoin was detected as a mixture
    of stereoisomers of 2,3-butanediol (Gabriel et al., 1971). Although
    reduction of diacetyl and acetoin has been observed in animals
     in vivo and in animal tissue preparations  in vitro at high
    concentrations, it appears that oxidation of diacetyl is a major
    endogenous metabolic pathway.

         Reduction of ketones is mediated by alcohol dehydrogenase and
    NADPH-dependent cytosolic carbonyl reductases (Bosron & Li, 1980).
    Reduction of the endogenous substances acetoin (No. 405) and diacetyl
    (No. 408) is catalysed by the substrate-specific enzymes diacetyl
    reductase and acetoin reductase, respectively. In rat liver slices,
    diacetyl, acetoin, and 2,3-butanediol are interconvertible (Gabriel et
    al., 1972). 

         In male Wistar albino rats, a single oral dose of diacetyl at  
    5 mmol/kg bw (430 mg/kg bw) was metabolized by reduction to acetoin,
    which was present at high concentrations in major organs 1 h after
    dosing. The subsequent reduction product, 2,3-butanediol, was detected
    in liver, kidney, and brain (Otsuka et al., 1996). Only a 10-min
    incubation is required to convert 10 nmol (9  10-4 mg) diacetyl to
    3.7 nmol (3  10-4 mg) acetoin and 6.3 nmol (6  10-4 mg)
    2,3-butanediol in rat liver homogenate. Diacetyl was reduced by NADH-
    and NADPH-dependent diacetyl reductase isolated from a homogenate of
    male Wistar rat liver. The organ-specific reductase activity was
    greatest in the liver and least in the brain (Otsuka et al., 1996).

         Acetoin was given either orally in a 3-4% solution or
    subcutaneously in a 20% solution to rats, multiple doses being spaced
    equally throughout each day. The acetoin used was obtained as the
    polymer (H, 15), which apparently reverts to an optically inactive
    monomer in aqueous solution. No diacetyl was detected in the urine of
    the rats; acetoin was not excreted to any appreciable extent in the
    urine, and the major excretion product was 2,3-butanediol (Westerfeld
    & Berg, 1943). In dogs, acetoin is excreted in the urine, in part as

    2,3-butanediol. Most of an oral dose disappeared and was presumed to
    be completely metabolized (Westerfeld & Berg, 1943). In another study,
    2,3-butanediol readily conjugated glucuronic acid (Neuberg &
    Gottshchalk, 1971).

         Diacetyl and acetoin are endogenous in humans (Kawano, 1959;
    Zlatkis & Sivetz, 1960). They are formed when pyruvate is converted to
    acetoin and diacetyl by pyruvate decarboxylase (Gabriel  et al., 
    1972). Mean fasting blood concentrations of approximately 100 g
    acetoin per 100 ml blood have been reported (Dawson & Hullin, 1954).
    Pyruvate also forms diacetyl  in vitro in rat liver preparations
    (Jrnefelt, 1955) and in microorganisms (Juni & Heym, 1956). 

     Alicyclic alpha-diketones and alicyclic dicarbonyls

         In general, alicyclic alpha-diketones are metabolized  via a
    reduction pathway (Williams, 1959). In humans, structurally related
    alicyclic monoketones are reduced to the corresponding alcohols or
    undergo alpha-hydroxylation and reduction to yield diols, which are
    excreted as the glucuronic acid conjugates. For example, the
    glucuronic acid conjugate of cyclohexanol is present in the urine of
    humans exposed to atmospheres containing cyclohexanone at
    concentrations of 2-30 ppm (Ong et al., 1991). A mixture of diols,
    including  cis- and  trans-1,2-cyclohexanediol,1,3-cyclohexanediol,
    and 1,4-cyclohexanediol, was detected in the urine of infants exposed
    to cyclohexanone present in dextrose solutions used for intravenous
    feeding. Presumably, the cyclohexanone undergoes alpha-hydroxylation
    and then reduction of the ketone function to yield the corresponding
    alpha-cyclohexanediols (Mills & Walker, 1990). 

         On the basis of the studies described above, it is anticipated
    that humans will metabolize low concentrations of aliphatic acyclic
    methyl ketones principally by oxidation of the terminal methyl group.
    At higher concentrations, reduction to the diol and subsequent
    conjugation with glucuronic acid form a competing detoxification
    pathway. Other aliphatic acyclic alpha-diketones and
    alpha-hydroxyketones and alicyclic diketones and hydroxyketones are
    reduced, conjugated with glucuronic acid, and excreted. 

    2.3.2  Toxicological studies

    2.3.2.1  Acute toxicity

         The results of studies of the acute toxicity of seven of the 22
    diketones, hydroxyketones, and alicyclic dicarbonyls have been
    reported and are summarized in Table 3. The low acute toxicity of the
    group is demonstrated by oral LD50 values in the range 990 to > 8000
    mg/kg bw.

    Table 2. Most recent annual usage of aliphatic acyclic and alicyclic 
    alpha-diketones and related alpha-hydroxyketones as flavouring substances 
    in Europe and the United States

                                                                            

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

    Acetoin (405)
      Europe                          19 000         2800      46
      United States                   9 000          1800      29

    2-Acetoxy-3-butanone (406)
      Europe                          0.2            0.03      0.0005
      United States                   120            23        0.4

    Butan-3-one-2-yl-butanoate (407)
      Europe                          0.1            0.02      0.0003
      United States                   5              0.95      0.02

    Diacetyl (408)
      Europe                          18 000         3300      56
      United States                   42 000         8000      133

    3-Hydroxy-2-pentanone (409)
      Europe                          NR             ND        0
      United States                   0.5            0.10      0.002

    2,3-Pentanedione (410)
      Europe                          1 100          220       4
      United States                   420            80        1

    4-Methyl-2,3-pentadione (411)
      Europe                          2.5            0.48      0.01
      United States                   12             2         0.04

    2,3-Hexanedione (412)
      Europe                          70             13        0.22
      United States                   50             10        0.2

    3,4-Hexanedione (413)
      Europe                          170            33        0.5
      United States                   4              0.76      0.01

    5-Methyl-2,3-hexanedione (414)
      Europe                          9              2         0.03
      United States                   30             6         0.10

    Table 2. (continued)

                                                                            

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

    2,3-Heptanedione (415)
      Europe                          8              2         0.03
      United States                   26             5         0.08

    5-Hydroxy-4-octanone (416)
      Europe                          0.1            0.02      0.0003
      United States                   4              0.76      0.01

    2,3-Undecadione (417)
      Europe                          0.03           0.01      0.0001
      United States                   0.1            0.02      0.0003

    Methylcyclopentenolone (418)
      Europe                          4 700          890       15
      United States                   3 700          710       12

    3-Ethylcyclopentenolone (419)
      Europe                          260            50        0.8
      United States                   120            23        0.4

    3,4-Dimethyl-1,2-cyclopentanedione (420)
      Europe                          250            47        0.8
      United States                   13             2         0.04

    3,5-Dimethyl-1,2-cyclopentanedione (421)
      Europe                          290            55        0.9
      United States                   150            29        0.5

    3-Ethyl-2-hydroxy-4-methylcyclopent-2-en-1-one (422)
      Europe                          NR             ND        ND
      United States                   0.9            0.17      0.003

    5-Ethyl-2-hydroxy-3-methylcyclopent-2-en-1-one (423)
      Europe                          NR             ND        ND
      United States                   2              0.38      0.01

    2-Hydroxy-2-cyclohexen-1-one (424)
      Europe                          0.4            0.08      0.001
      United States                   4              0.76      0.01

    1-Methyl-2,3-cyclohexadione (425)
      Europe                          11             2         0.03
      United States                   44             8         0.1

    Table 2. (continued)
                                                                            

    Substance (No.)                   Most recent    Per capita intakea 
                                      annual  
                                      volume (kg)    g/day    g/kg bw 
                                                               per day
                                                                            
    2-Hydroxy-3,5,5-trimethyl-2-cyclohexenone (426)
      Europe                          10             2         0.03
      United States                   8              2         0.03

    Total
      Europe                          44 000
      United States                   56 000
                                                                            

    NR, not reported; ND, not determined
    a  Intake (g/day) calculated as follows: 
       [(annual volume, kg)  (1  109 g/kg)]/[population  0.6  365 days], 
       where population (10%, 'eaters only') = 32  106 for Europe and 
       32  106 for the United States; 0.6 represents the assumption that 
       only 60% of the flavour volume was reported in the surveys 
       (US National Academy of Sciences, 1970, 1982, 1987; International 
       Organization of the Flavor Industry, 1995).
       Intake (g/kg bw per day) calculated as follows: [(g/day)/body weight], 
       where body weight = 60 kg. Slight variation may occur from rounding off.

        Table 3. Studies of acute toxicity with aliphatic acyclic and alicyclic alpha-diketones and 
    related alpha-hydroxyketones used as flavouring substances
                                                                                                  
    Substance                  No.   Species       Sex   Route    LD50        Reference
                                                                  (mg/kg bw)
                                                                                                  
    Acetoin                    405   Rat           NR    Oral     > 5000      Moreno (1977)
    Butan-3-one-2-yl           407   Mouse, rat    NR    Oral     > 8000      Pellmont (1969)
      butanoate
    Diacetyl                   408   Rat           M     Gavage   3400        Colley et al.(1969)
                                                   F              3000
                                     Rat           NR    Gavage   1580        Jenner et al.(1964)
                                     Guinea-pig    NR    Gavage   990         Jenner et al.(1964)
    2,3-Pentanedione           410   Rat           NR    Oral     3000        Moreno (1977)
    2,3-Hexanedione            412   Rat           NR    Oral     > 5000      Moreno (1977)
    5-Methyl-2,3-hexanedione   414   Rat           NR    Oral     > 5000      Moreno (1979)
    Methylcyclopentenolone     418   Mouse         NR    Gavage   1350        Givadaun Corp.(1952)
                                     Rat           NR    Oral     1850        Moreno (1976)
                                     Guinea-pig    NR    Gavage   1400        Dow Chemical Co. 
                                                                              (1953)
                                     Mouse         NR    Oral     1350        Leberco Labs (1952)
                                                                                                  

    NR, not reported; M, male; F, female
    
    2.3.2.2  Short-term and long-term studies of toxicity and
             carcinogenicity

         The toxicity of nine of the 22 substances has been studied. The
    results are summarized in Table 4 and described below.

     Acetoin (No. 405)

         Groups of 15 male and 15 female CFE rats were given acetoin in
    their drinking-water at concentrations of 0 (control), 750, 3000, or
    12 000 mg/kg (equivalent to 0, 85, 330, or 1300 mg/kg bw per day (US
    Food & Drug Administration, 1993)). No animals died during the study,
    and their condition and appearance were normal. The body weights of
    males at 12 000 mg/kg in drinking-water decreased significantly from
    week 5, and at weeks 2, 6, and 13 the relative weight of the liver was
    statistically significantly greater in these animals than in controls.
    A similar effect was seen in female rats, but only after 13 weeks.
    Haematological examination conducted at 13 weeks showed a small (4-8%)
    but statistically significant  (p < 0.05) decrease in haemoglobin
    concentration and erythrocyte counts in animals of each sex at the
    high dose, but these changes were not accompanied by a decrease in
    haematocrit. Urinalysis and blood chemical determinations performed at
    the end of the study on all animals revealed no statistically
    significant differences between treated and control groups.
    Histopathological examination also revealed no adverse effects. The
    authors suggested that the increased relative liver weights were a
    reaction of the liver to an increased metabolic load resulting from
    the high intake of acetoin. The NOEL was 3000 ppm, equivalent to
    330 mg/kg bw per day (Gaunt et al., 1972), which is greater than
    10 000 the daily  per capita intake ('eaters only') of 46 and 29
    g/kg bw from its use as a flavouring agent in Europe and the United
    States, respectively (see Table 2).

     Diacetyl (No. 408)

         Groups of 15 male and 15 female weanling specific
    pathogen-free-derived CFE rats were given 5 ml/kg bw of an aqueous
    solution containing 0, 0.2, 0.6, 1.8, or 11% diacetyl by gavage for 90
    days, providing daily doses of diacetyl calculated to be 0, 10, 30,
    90, and 540 mg/kg bw. Body weight, food intake, and water consumption
    were recorded weekly. Growth retardation and increased water
    consumption were observed in animals at the high dose, the effects
    being more pronounced in males. Haematological and urinary parameters
    and enzyme activities were measured. Anaemia and increased
    polymorphonuclear leukocytosis were observed in rats at the high dose
    but were attributed to haemorrhage, infections, and ulcers of the
    stomach. The relative weights of the liver, kidney, and pituitary and
    adrenal glands were increased in these animals, and the increases were
    greater than could be accounted for by the reduction in body weight.
    Macroscopic and microscopic examination of all major organs revealed
    ulcers in the squamous and glandular regions of the gastric mucosa.
    The lesions observed in the squamous part of the stomach were
    associated with hypertrophy or intercellular oedema. No histological

    changes were seen in animals treated at lower doses. No other
    significant differences were observed between treated and control
    animals. The NOEL was 90 mg/kg bw per day (Colley et al., 1969), which
    is 1500 times the daily  per capita intake ('eaters only') of 56
    g/kg bw from its use as a flavouring agent in Europe and 500 times
    the intake of 130 g/kg bw from its use as a flavouring agent in the
    United States (see Table 2).

     3,4-Hexanedione (No. 413)

         Groups of 10-16 male and female Charles River CD rats were housed
    in pairs of the same sex and fed for 90 days on a basal diet alone or
    supplemented with 3,4-hexanedione for an average daily intake of
    17 mg/kg bw. Body weight and food consumption were measured daily and
    were considered normal. Haematological examinations and blood urea
    determinations were performed on 50% of the animals at week 7 and at
    the end of treatment: no significant differences were seen between
    treated and control animals. At necropsy, the liver and kidney weights
    were normal, and gross and histological examination performed on a
    wide range of organs revealed no dose-related lesions. The NOEL was 17
    mg/kg bw per day (Posternak et al., 1969), which is more than 30 000
    the daily  per capita intake ('eaters only') of 0.5 and 0.01 g/kg bw
    from its use as a flavouring agent in Europe and the United States,
    respectively (see Table 2).

     Methylcyclopentenolone (No. 418)

         Groups of 15 male and 15 female rats aged four to five weeks
    (strain not specified) were placed on a diet containing 0 or 1%
    methylcyclo-pentenolone, equivalent to a daily intake of 0 or 500
    mg/kg bw, for six months. Each animal was weighed twice weekly and
    observed frequently for gross appearance and behaviour. The tissues of
    most animals that died or were killed during the course of the study
    and of all those kiled at the end of the study were examined grossly.
    Tissues of representative animals from the control and treated groups
    (numbers unspecified) were examined microscopically, and the weights
    of the lungs, heart, liver, kidneys, spleen, and testes were recorded.
    Haematological parameters were measured in representative animals from
    the control and treated groups (numbers not specified) at the end of
    the experiment. There were no statistically significant differences
    between treated and control animals in any of the parameters measured.
    The NOEL was 500 mg/kg bw per day (Dow Chemical Co., 1953), which is
    more than 30 000 the daily  per capita intake ('eaters only') of 15
    and 12 g/kg bw from its use as a flavouring agent in Europe and the
    United States, respectively (see Table 2). 

     3-Ethyl-2-hydroxy-2-cyclopenten-1-one (No. 419)

         Groups of 10 male and 10 female Charles River CD rats were fed
    diets providing 3-ethyl-2-hydroxy-2-cyclopenten-1-one at doses of 0,
    100, 200, or 400 mg/kg bw per day for 91 days. No adverse effects were
    seen on behaviour, body weight, food consumption, haematological,
    urinary, or ophthalmological parameters, or gross or histopathological

    appearance. The NOEL was 400 mg/kg bw per day (King et al., 1979),
    which is more than 50 000 the daily  per capita intake ('eaters
    only') of 0.8 and 0.4 g/kg bw from its use as a flavouring agent in
    Europe and the United States, respectively (see Table 2).

         In a combined study of developmental toxicity and
    carcinogenicity, three successive generations of male and 
    female Charles River CD-COBS rats received 
    3-ethyl-2-hydroxy-2-cyclopenten-1-one in the basal diet at doses of 0
    (untreated control), 0 (propylene glycol control), 30, 80, or       
    200 mg/kg bw per day. The F0 generation was entered into the study
    after weaning and was mated 64 days later. These animals were treated
    for 12 months, during which time the females were given a supplemental
    diet throughout gestation and lactation. The F1 generation was
    initially exposed  in utero, subsequently via the dams' milk until
    weaning, and then treated for two years and bred twice (at days 99 and
    155) to produce F2a and F3a generations. The F0 and F2 generations
    consisted of 40 animals of each sex in the untreated control group and
    20 of each sex in the propylene glycol control and
    3-ethyl-2-hydroxy-2-cyclopenten-1-one-treated groups. In the F1
    generation, there were 100 animals of each sex in the untreated
    control group and 50 of each sex in the propylene glycol control and
    3-ethyl-2-hydroxy-2-cyclopenten-1-one-treated groups.

         Survival, clinical symptoms, food consumption, reproductive
    perfor-mance, and haematological and clinical chemical parameters were
    not adversely affected in the F0 or F1 generations. Slightly
    depressed growth was reported in F1 females receiving the highest
    dose, but the effect was not significant when compared with the growth
    of controls receiving propylene glycol. Gross pathological and
    histopathological examination of animals of these generations revealed
    no significant treatment-related effects. An increased incidence of
    bile-duct hyperplasia observed in these groups was not statistically
    significant when compared with the incidence of in controls. The
    incidence of benign or malignant tumours in treated animals was
    similar to that in controls. The NOEL was 200 mg/kg bw per day (King
    et al., 1979), which is more than 200 000 the daily  per capita 
    intake ('eaters only') of 0.8 and 0.4 g/kg bw from its use as a
    flavouring agent in Europe and the United States, respectively (see
    Table 2). 

         In a three-generation study of toxicity in Charles River 
    CD-COBS rats with 3-ethyl-2-hydroxy-2-cyclopenten-1-one, 120 
    F0 animals were entered into the study just after weaning and were
    mated after 64 days, providing the F1 generation. Animals of the 
    F0 generation were selected at random to be maintained as controls 
    or were given 3-ethyl-2-hydroxy-2-cyclopenten-1-one in the diet. 
    The F1 generation was maintained on the diet for 755 days, during
    which time they were mated twice (at days 99 and 155). The first
    mating provided the rats for the F2 generation, and the second mating
    provided rats which were sacrificed after weaning. The F2 generation
    was treated for 84 days and then mated to produce the F3 generation.
    Rats of all generations were maintained on a basal diet with

    3-ethyl-2-hydroxy-2-cyclopenten-1-one at doses equal to daily intakes
    of 0, 30, 80, or 200 mg/kg bw. Interim clinical chemical analyses were
    performed on the F0 generation at months 4 and 6. Ophthalmological,
    clinical chemical, and haematological examinations were performed on
    the F1 generation at months 6, 12, 18, and 24. At termination, both
    the F0 and the F1 generations were submitted to clinical chemical,
    haematological, and histopathological examinations. F2 adult animals
    were killed without necropsy at the time of weaning of their
    offspring. 

         No evidence of treatment-related effects was found, and the
    general health of the rats was unaffected throughout treatment.
    Mammary swellings in females and other subcutaneous nodules were
    considered not to be related to treatment because they were equally
    distributed among control and treated groups. No effects were seen on
    food consumption, weight gain, or clinical chemical or haematological
    parameters. In the F0 generation, there were no pathological findings
    at the 12-month necropsy or at histopathological examination. In the
    F1 generation, there were no macroscopic observations that could be
    related to the treatment. Histologically, bile-duct hyperplasia was
    seen in all groups, including controls; however, there was no
    statistically significant difference between treated and control
    groups and the findings were considered not to be related to
    treatment. Only tumours usually found in rats of this strain and age
    were observed, indicating the absence of treatment-related effects.
    The NOEL in this study was 200 mg/kg bw per day (King et al., 1979),
    which is more than 100 000 the daily  per capita intake ('eaters
    only') of 0.4 g/kg bw from its use as a flavouring agent in the
    United States (see Table 2).

     3,4-Dimethylcyclopentane-1,2-dione (No. 420)

         Groups of 10 male Charles River CD rats were fed diets containing
    3,4-dimethyl-1,2-cyclopentadione at concentrations of 0, 400, 4000, or
    13 000 mg/kg (equivalent to 0, 20, 200, and 640 mg/kg bw per day) for
    90 days. Food consumption, body-weight gain, haematological
    parameters, organ weights, and gross and microscopic appearance were
    observed throughout the study. Animals at the intermediate and high
    doses showed a moderate but consistent depression in food intake
    throughout the experiment, and their final mean body weights were
    about 10% less than those of controls. There were no consistent
    indications of a profound disturbance of food conversion efficiency,
    although minor increases in the food conversion ratio were seen in
    some animals at the high dose. Haematological parameters, organ
    weights, and gross and microscopic appearance were similar in treated
    and control groups. The authors attributed the decreased body weight
    observed at higher doses to the unpalatability of the test substance.
    The NOEL was 200 mg/kg bw per day (Wheldon & Krajkeman, 1967), which
    is more than 200 000 the daily  per capita intake ('eaters only') of
    0.8 and 0.04 g/kg bw from its use as a flavouring agent in Europe and
    the United States, respectively (see Table 2). 

     3,5-Dimethylcyclopentane-1,2-dione (No. 421)

         Groups of 10 male Charles River CD rats were fed
    3,5-dimethyl-1,2-cyclopenthexadione in the diet for 13 weeks at
    concentrations of 0, 1000, or 10 000 mg/kg diet (equivalent to 50 and
    500 mg/kg bw per day). A fourth group was fed a diet initially
    containing 12 000 ppm (equivalent to 610 mg/kg bw per day), but the
    level was increased to 24 000 mg/kg diet (equivalent to      
    1200 mg/kg bw per day) during week 6 through to the end of experiment.
    Haematological examinations were performed on five animals from each
    group just before the end of the 13-week period; organ weight analysis
    and gross and histopathological examinations at the end of the study
    revealed no significant differences between control and treated
    animals. The food consumption of animals at the two highest doses was
    reduced throughout the experiment, accompanied by decreased weight
    gain, which resulted in mean body weights that were 10-17% lower than
    those of controls at the end of the study. The authors attributed the
    decrease to the unpalatability of the test substance. The NOEL was 50
    mg/kg bw per day (Wheldon & Krajkeman, 1967), which is more than
    50 000 the daily  per capita intake ('eaters only') of 0.9 and 0.5
    g/kg bw from its use as a flavouring agent in Europe and the United
    States, respectively (see Table 2). 

     2-Hydroxy-2-cyclohexen-1-one (No. 424)

         2-Hydroxy-2-cyclohexen-1-one was added to the diet of 15 male and
    15 female albino Wistar rats for 90 days at a concentration calculated
    to result in an average daily intake of 5 mg/kg bw. Detailed
    measurements of haematological parameters, blood chemistry, urine,
    gross and histopathological appearance, organ weights, body weight,
    and food consumption revealed no statistically significant difference
    between treated and control groups (Morgareidge et al., 1974). The
    NOEL was 5 mg/kg bw per day (Cox, 1974), which is more than 500 000
    the daily  per capita intake ('eaters only') of 0.001 and 0.01 g/kg
    bw from its use as a flavouring agent in Europe and the United States,
    respectively (see Table 2). 

     1-Methyl-2,3-cyclohexadione (No. 425)

         Groups of 10 male Charles River CD rats were fed
    1-methyl-2,3-cyclohexadione in the diet at concentrations of 0, 100,
    or 1000 mg/kg (equivalent to 0, 5, and 50 mg/kg bw per day) for 13
    weeks. A fourth group was fed a diet initially containing 13 500 mg/kg
    diet (equivalent to 675 mg/kg bw per day), which was raised to 27 000
    mg/kg bw (equivalent to 1350 mg/kg bw per day) during week
    6. Observation of haematological parameters, organ weights, and gross
    and histopathological appearance revealed no difference between
    treated and control groups. The food consumption of rats at the high
    dose was reduced throughout the experiment, accompanied by decreased
    body-weight gain, and the effect was more marked when the dietary
    concentration was raised. The NOEL was 50 mg/kg bw per day (Wheldon &
    Krajkeman, 1967), which is more than 500 000 the daily  per capita 
    intake ('eaters only') of 0.03 and 0.1 g/kg bw from its use as a

    flavouring agent in Europe and the United States, respectively (see
    Table 2). 

    2.3.2.3  Genotoxicity

         The available studies of genotoxicity are summarized in Table 5.
    Most of the studies were carried out with  Salmonella typhimurium in
    the Ames test. Acetoin, diacetyl, and 1,2-cyclohexanedione showed some
    mtagenicity in strains TA100 and TA104. As the mutation frequencies
    were low and the positive results were always accompanied by negative
    results, the overall conclusion was that this group of substances does
    not induce gene mutation in bacteria  in vitro. Diacetyl did not
    induce mutation in  Saccharomyces cerevisiae (US Food & Drug
    Administration, 1974). Methylcyclopentenolone did not induce
    unscheduled DNA synthesis in rat hepatocytes (Heck et al., 1989).

    2.3.2.4  Other relevant studies

     3-Ethyl-2-hydroxy-2-cyclopenten-1-one (No. 419)

         In the three-generation, 23-month study of toxicity described
    above, treatment had no effect on clinical signs, weight gain, food
    consumption, copulation rates, or mating behaviour of males or
    females, fertility index, length of gestation, parturition, litter
    size, or incidence of stillbirth. The growth and survival rates of
    pups during lactation were normal. Gross examination of all offspring
    from birth to weaning revealed no significant treatment-related
    malformations or lesions (King et al., 1979).

     Diacetyl (No. 408)

         Groups of 25-27 Syrian golden hamsters, 21-24 CD-1 mice, and      
        21-23 albino Wistar rats were given a solution containing 90%
    diacetyl by gavage on days 6-10 of gestation for hamsters and days
    6-15 of gestation for mice and rats. The doses for all species were
    16, 74, 345, or 1600 mg/kg bw per day. No effects were seen on
    maternal survival, weight, or reproductive parameters or on fetal
    survival or microscopic appearance of external, skeletal, or soft
    tissues (US Food & Drug Administration, 1973).


    4.  REFERENCES

    Aeschbacher, H.U., Wolleb, U., Loliger, J., Spadone, J.C. & Liardon,
    R. (1989) Contribution of coffee aroma constituents to the
    mutagenicity of coffee.  Food Chem. Toxicol., 27, 227-232.

    Anders, M. W. (1989) Biotransformation and bioactivation of
    xenobiotics by the kidney. In: Hutson, D.H., Caldwell, J. & Paulson,
    G.D., eds,  Intermediary Xenobiotic Metabolism in Animals, New York,
    Taylor & Francis, pp. 81-97. 


        Table 4. Short-term and long-term studies of toxicity in rats with aliphatic acyclic and alicyclic alpha-diketones and related 
    alpha-hydroxyketones used as flavouring agents

                                                                                                                                        

    Substance                                No.   Sex   No. groups/     Route           Time     NOEL       Reference
                                                         no. per group                   (days)   (mg/kg bw
                                                                                                  per day)
                                                                                                                                        

    Acetoin                                  405   M/F   3/30            Drinking-water  90       330        Gaunt et al. (1972)
    Diacetyl                                 408   M/F   4/30            Gavage          90       90         Colley et al. (1969)
    3,4-Hexanedione                          413   M/F   2/10-16         Diet            90       17a        Posternak et al. (1969)
    Methylcyclopentenolone                   418   M/F   1/30            Diet            185      500a       Dow Chemical Co. (1953)
    3-Ethyl-2-hydroxy-2-cyclopenten-1-one    419   M/F   3/20            Diet            91       400a       King et al. (1979)
                                                   M/F   3/100           Diet            730      200        King et al. (1979)
    3,4-Dimethyl-1,2-cyclopentadione         420   M     3/10            Diet            90       645        Wheldon & Krajkeman (1967)
    3,5-Dimethyl-1,2-cyclopentadione         421   M     3/10            Diet            90       610a       Wheldon & Krajkeman (1967)
    2-Hydroxy-2-cyclohexen-1-one             424   M/F   1/30            Diet            90       5a         Morgareidge et al. (1974)
    1-Methyl-2,3-cyclohexadione              425   M     3/10            Diet            90       675-1350a  Wheldon & Krajkeman (1967)
                                                                                                                                        

    M, male; F, female
    a The study was performed at a single or multiple doses that had no adverse effects; therefore, no NOEL was determined. 
      The NOEL is probably higher than the dose reported to have no adverse effects.

    Table 5.  Results of assays for the genotoxicity of aliphatic acyclic and alicyclic alpha-diketones and related alpha-hydroxyketones 
    used as flavouring agents

                                                                                                                                              

    Substance                    No.   End-point         Test object             Dose                    Results      Reference
                                                                                                                                              

    Acetoin                      405   Reverse mutation  S. typhimurium TA100    < 4500 mg/plate         Negativea    Garst et al. (1983)
                                                                                                         Positiveb
                                       Reverse mutation  S. typhimurium TA100    420 mg/plate            Negativeb    Kim et al. (1987)

    Diacetyl                     408   Reverse mutation  S. typhimurium TA100,   Not reported            Positivea,c  Kato et al. (1989)
                                       (modified test)   TA104; E. coli
                                       Reverse mutation  S. typhimurium TA104    530 g/plate            Positived    Marnett et al. (1985)
                                       (modified test)
                                       Reverse mutation  S. typhimurium TA100    90 g/plate             Negativee    Kim et al. (1987)
                                       Reverse mutation  S. typhimurium TA104    5-500 g/platef         Positivea,c  Shane et al. (1988)
                                                                                                         Negativeb
                                       Reverse mutation  S. typhimurium TA102,   5-500 g/platef         Negativee    Shane et al. (1988)
                                                         TA100
                                       Reverse mutation  S. typhimurium TA100    152-950 g/plate        Negativeb    Dorado et al. (1992)
                                       Reverse mutation  S. typhimurium TA100    10, 100, 1000,          Positivee    Bjeldanes & Chew (1979)
                                                                                 10 000 g/plate
                                       Reverse mutation  S. typhimurium TA98     10, 100, 1000,          Negativee    Bjeldanes & Chew (1979)
                                                                                 10 000 g/plate
                                       Reverse mutation  S. typhimurium TA1535,  1%                      Negativee    US Food & Drug
                                       (suspension test) TA1537, TA1538                                               Administration (1974)
                                       Reverse mutation  S. typhimurium TA102    0.17-17 200 g/plate    Negativee    Aeschbacher et al. (1989)
                                       Reverse mutation  S. typhimurium TA98,    0.17-17 200 g/plate    Negativee    Aeschbacher et al. (1989)
                                                         TA100
                                       Reverse mutation  S. typhimurium TA100    2 or 4 mmol/plate       Positiveg    Suwa et al. (1982)
                                       Mutation          S. cerevisiae           Not reported            Negativee    US Food & Drug
                                                                                                                      Administration (1974)
    2,3-Pentanedione             410   Reverse mutation  S. typhimurium TA100    105 g/plate            Negativeb    Kim et al. (1987)
                                       Reverse mutation  S. typhimurium TA100,   0.9-90 000 g/plate     Negativee    Aeschbacher et al. (1989)
    3,4-Hexanedione              413   Reverse mutation  S. typhimurium TA100    228-4900 g/plate       Negativeb    Dorado et al. (1992)

    Table 5.  (continued)
                                                                                                                                              

    Substance                    No.   End-point         Test object             Dose                    Results      Reference
                                                                                                                                              

    Methylcyclopentenolone       418   Reverse mutation  S. typhimurium TA1535,  10 000 g/plate         Negativee    Heck et al. (1989)
                                                         TA1537, TA1538, TA98,
                                                         TA100
                                       Unscheduled DNA   Rat hepatocytes         500 g/plate            Negativea    Heck et al. (1989)
                                       synthesis
    2-Hydroxy-2-cyclohexen-1-one 424   Reverse mutation  S. typhimurium TA100    10, 100, 1000,          Positivee    Bjeldanes & Chew (1979)
                                                                                 10 000 g/plate
                                       Reverse mutation  S. typhimurium TA98     10, 100, 1000,          Negativee    Bjeldanes & Chew (1979)
                                                                                 10 000 g/plate
                                       Reverse mutation  S. typhimurium TA100    112-1000 g/plate       Negativeb    Dorado et al. (1992)
                                                                                                                                              

    a  With metabolic activation
    b  Without metabolic activation
    c Mutation frequency, 2-3
    d Result based on authors' criterion for significant mutagenic effect, i.e. mutation frequency > 1.5
    e With and without metabolic activation
    f Estimated from graph
    g Mutation frequency, < 2.5
    

    Bjeldanes, L.F. & Chew, H. (1979) Mutagenicity of 1,2-dicarbonyl
    compounds: Maltol, kojic acid, diacetyl and related substances.
     Mutat. Res., 67, 367-371.

    Bosron, W.F. & Li, T.-K. (1980) Alcohol dehydrogenase. In: Jacoby,
    W.B., ed.,  Enzymatic Basis of Detoxication, New York, Academic
    Press, Vol. I, pp. 231-248. 

    Colley, J., Gaunt, I.F., Lansdown, A.B.G., Grasso, P. & Gangolli, S.D.
    (1969) Acute and short-term toxicity of diacetyl in rats.  Food Chem.
     Toxicol., 7, 571-580.

    Cramer, G.M., Ford, R.A. & Hall, R.L. (1978) Estimation of toxic
    hazard: A decision tree approach.  Food Cosmet. Toxicol., 16,
    255-276.

    Dawson, J. & Hullin, R.P. (1954) Metabolism of acetoin. Formation and
    utilization of acetoin and 2,3-butanediol in the decerebrated cat.
     Biochem. J., 57, 177-180.

    Dix & Jeffcoat (1997) Disposition and excretion of
    [14C]2,3-butanedione in male rats following oral administration.
    Unpublished report from Research Triangle Institute (NIEHS contract
    No. NO1-ES-75407). Submitted to WHO by the Flavor and Extract
    Manufacturers' Association of the United States, Washington DC, United
    States.

    Dorado, L., Montoya, M.R. & Rodriguez-Mellado, J.M. (1992) A
    contribution to the study of the structure-mutagenicity relationship
    for alpha-dicarbonyl compounds using the Ames test.  Mutat. Res., 
    269, 301-306.

    Dow Chemical Co. (1953) Toxicity of cyclotene--Summary. Unpublished
    report from Biochemical Research Department, Midland, Michigan, 15
    July. Submitted to WHO by the Flavor and Extract Manufacturers'
    Association of the United States, Washington DC, United States.

    Gabriel, M.A., Jabara, H. & Al-Khalidi, A.A. (1971) Metabolism of
    acetoin in mammalian liver slices and extracts.  Biochem. J., 124,
    793-800.

    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.

    Garst, J., Stapleton, P. & Johnson, J. (1983) Mutagenicity of
    alpha-hydroxy ketones may involve superoxide anion radical.  Oxy 
     Radicals and Their Scavenger Systems, Amsterdam, Elsevier Science
    Publishing, Vol. 2, pp. 125-130.

    Gaunt, I.F., Branton, P.G., Kiss, I.S., Grasso, P. & Gangolli, S.D.
    (1972) Short-term toxicity of acetoin (acetyl methylcarbinol) in rats.
     Food Cosmet. Toxicol., 10, 131-141. 

    Givaudan Corp. (1952) Acute oral toxicity study of
    methylcyclopentenolone in mice. Unpublished report to the Research
    Institute of Fragrance Materials. Submitted to WHO by the Flavor and
    Extract Manufacturers' Association of the United States, Washington
    DC, United States.

    Gordon, A.J. & Ford, R.A. (1972)  The Chemist's Companion, New York,
    John Wiley & Sons, pp. 49-51. 

    Heck, J.D., Vollmuth, T.A., Cifone, M.A., Jagannath, D.R., Myhr, B. &
    Curren, R.D. (1989) An evaluation of food flavoring ingredients in a
    genetic toxicity screening battery.  Toxicologist, 9, 257.

    Heymann, E. (1980) Carboxylesterases and amidases. In: Jakoby, W.B.,
    ed.,  Enzymatic Basis of Detoxication, 2nd Ed., New York, Academic
    Press, pp. 291-323.

    International Organization of the Flavor Industry (1995) European
    inquiry on volume of use. Unpublished report. Submitted to WHO by the
    Flavor and Extract Manufacturers' Association of the United States,
    Washington DC, United States.

    Jrnefelt, J. (1955) Studies on the enzymatic synthesis and breakdown
    of acetoin in the animal organism.  Ann. Acad. Sci. Fenn. Ser. 
     A. V. Med Anthropol., 57, 7-78.

    Jenner, P.M., Hagan, E.C., Taylor, J.M., Cook, E.L. & Fitzhugh, O.G.
    (1964) Food flavorings and compounds of related structure I. Acute
    oral toxicity.  Food Cosmet. Toxicol., 2, 327-343.

    Juni, E. & Heym, G.A. (1956) A cyclic pathway for the bacterial
    dissimilation of 2,3-butanediol, acetyl methyl carbinol, and diacetyl.
     J. Bacteriol., 71, 425-432.

    Kato, F., Araki, A., Nozaki, K. & Matsushi, T. (1989) Mutagenicity of
    aldehydes and diketones.  Mutat. Res., 216, 366-367.

    Kawano, T. (1959) Relation of acetoin and pantothenic acid.  Fukuoka 
     Igaku Zasshi, 50, 2939-2953.

    Kim, S.B., Hayase, F. & Kato, H. (1987) Desmutagenic effect of
    alpha-dicarbonyl and alpha-hydroxycarbonyl compounds against mutagenic
    heterocyclic amines.  Mutat. Res., 177, 9-15.

    King, T., Faccini, J.M., Jachbaur, J., Perraud, J. & Monro, A.M.
    (1979) 3-Generation and chronic toxicity study in rats. Unpublished
    report from Pfizer Central Research. Submitted to WHO by the Flavor
    and Extract Manufacturers' Association of the United States,
    Washington DC, United States.

    Leberco Labs (1952) Acute toxicity of methylcyclopentenolone.
    Unpublished report. Submitted to WHO by the Flavor and Extract
    Manufacturers' Association of the United States, Washington DC, United
    States.

    Maarse, H., Visscher, C.A., Willimsens, L.C., Nijssen, L.M. & Boelens,
    M.H., eds (1994)  Volatile Components in Food: Qualitative and 
     Quantitative Data, 7th Ed., Zeist, Centraal Instituut voor
    Voedingsonderzoek TNO, Vol. III. 

    Marnett, L.J., Hurd, H.K., Hollstein, M.C., Levin, E., Esterbauer,
    H. & Ames, B.N. (1985) Naturally occurring carbonyl compounds are
    mutagens in Salmonella tester strain TA104.  Mutat. Res., 148, 25-34.

    Mills, G.A. & Walker, V. (1990) Urinary excretion of cyclohexanediol,
    a metabolite of the solvent cyclohexanone, by infants in a special
    care unit.  Clin. Chem., 36, 870-874.

    Moreno, O.M. (1976) Acute toxicity studies in rats, mice, rabbits and
    guinea pigs. Unpublished report to the Research Institute of Fragrance
    Materials. Submitted to WHO by the Flavor and Extract Manufacturers'
    Association of the United States, Washington DC, United States.

    Moreno, O.M. (1977) Acute toxicity studies in rats, rabbits and guinea
    pigs. Unpublished report to the Research Institute of Fragrance
    Materials. Submitted to WHO by the Flavor and Extract Manufacturers'
    Association of the United States, Washington DC, United States.

    Moreno, O.M. (1979) Acute toxicity studies in rats, rabbits and
    guinea-pigs. Unpublished report to the Research Institute of Fragrance
    Materials. Submitted to WHO by the Flavor and Extract Manufacturers'
    Association of the United States, Washington DC, United States.

    Morgareidge, K., Bailey, D.E. & Cox, G.E. (1974) 90 Day feeding study
    with 2-hydroxy-2-cyclohexen-1-one in rats. Unpublished report.
    Submitted to WHO by the Flavor and Extract Manufacturers' Association
    of the United States, Washington DC, United States.

    Ong, C.N., Sia, G.L., Chia, S.E., Phoon, W.H. & Tan, K.T. (1991)
    Determination of cyclohexanol in urine and its use in environmental
    monitoring of cyclohexanone exposure.  J. Anal. Toxicol., 15, 13-16.

    Otsuka, M., Mine, T., Ohuchi, K. & Ohmori, S. (1996) A detoxication
    route for acetaldehyde: Metabolism of diacetyl, acetoin, and
    2,3-butanediol in liver homogenate and perfused liver of rats.
     J. Biochem., 119, 246-251.

    Pellmont, D. (1969) Studies with rats and mice on substance No.
    R01-3801 (Toxikologisches Labor 256, Bau 69). Unpublished report to
    the Research Institute of Fragrance Materials. Submitted to WHO by the
    Flavor and Extract Manufacturers' Association of the United States,
    Washington DC, United States.

    Posternak, J.M., Linder, A. & Vodoz, C.A. (1969) Summaries of
    toxicological data. Toxicological test on flavoring matters.  Food 
     Cosmet. Toxicol., 7, 405-407.

    Shane, B.S., Troxclair, A.M., McMillin, D.J. & Henry, C.B. (1988)
    Comparative mutagenicity of nine brands of coffee to Salmonella
    typhimurium TA100, TA102 and TA104.  Environ. Mol. Mutag., 11,
    195-206.

    Stofberg, J. & Grundschober, F. (1987) The consumption ratio and food
    predominance of flavoring materials.  Perfum. Flavorist, 12, 27-56.

    Stofberg, J. & Kirschman, J.C. (1985) The consumption ratio of
    flavoring materials: A mechanism for setting priorities for safety
    evaluation.  Food Chem. Toxicol., 23, 857-860.

    Suwa, Y., Nagao, M., Kosugi, A. & Sugimura, T. (1982) Sulfite
    suppresses the mutagenic property of coffee.  Mutat. Res., 102,
    383-391.

    US Food & Drug Administration (1973)  Teratologic Evaluation of FDA 
     71-73 (Starter Distillate, Hansen) (NTIS PB-223-833;
    FDABF-GRAS-152), Washington DC, United States. 

    US Food & Drug Administration (1974)  Mutagenic Evaluation of 
     Compound FDA 71-73 (Starter Distillate) (NTIS PB-245-431;
    FDABF-GRAS-275), Washington DC, United States.

    US Food & Drug Administration (1993)  Priority-based Assessment of 
     Food Additives (PAFA) Database, Center for Food Safety and Applied
    Nutrition, Washington DC, United States, p. 58.

    US National Academy of Sciences (1970)  Evaluating the Safety of 
     Food Chemicals, Washington DC, United States.

    US National Academy of Sciences (1982)  Evaluating the Safety of 
     Food Chemicals, Washington DC, United States.

    US National Academy of Sciences (1987)  Evaluating the Safety of 
     Food Chemicals, Washington DC, United States. 

    Westerfeld, W.W. & Berg, R.L. (1943) Observations on the metabolism of
    acetoin.  J. Biol. Chem., 148, 523-528.

    Wheldon, G.H. & Krajkeman, A.J. (1967) The effects of ten
    food-flavoring additives administered to rats over a period of
    thirteen weeks. Unpublished report from Huntington Research Center.
    Submitted to WHO by the Flavor and Extract Manufacturers' Association
    of the United States, Washington DC, United States.

    Williams, T.R. (1959)  Detoxication Mechanisms: The Metabolism and 
     Detoxication of Drugs, Toxic Substances and Other Organic 
     Compounds, 2nd Ed., London, Chapman & Hall, pp. 62, 78.

    Zlatkis, A. & Sivetz, M. (1960) Analysis of coffee volatiles by gas
    chromatography.  Food Res., 25, 395-398.
    


    See Also:
       Toxicological Abbreviations