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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    IONONES AND STRUCTURALLY RELATED SUBSTANCES 

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

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


    1.  EVALUATION

    1.1  Introduction

         The Committee evaluated a group of 21 flavouring agents that
    includes alpha- and -ionone and structurally related substances
    (Table 1), using the procedure for the safety evaluation of flavouring
    agents (Figure 1, p. 222, and Annex 1, reference 131).

         Each of these substances has a cyclohexane ring with an allyl or
    alkyl side-chain containing a ketone or secondary alcohol functional
    group. With one exception, namely, 1,4-dimethyl-4-acetyl-1-cyclohexene
    (No. 402), each contains a 2,6,6-trimethylcylcohexyl carbon skeleton
    and an alkyl side chain of four to seven carbons located at the C-1
    position. With the exception of gamma-ionone, each of these substances
    has at least one endocyclic double bond. In the ionones, the carbonyl
    or hydroxyl group is positioned gamma to the ring, while in the
    damascones the carbonyl group is positioned alpha to the ring. 

         The Committee has previously evaluated three members of the
    group. alpha-Ionone and -ionone were both evaluated at the
    twenty-eighth meeting (Annex 1, reference 66), when an ADI of 0-0.1
    mg/kg bw was established for each. Allyl-alpha-ionone was evaluated at
    the twenty-fourth meeting (Annex I, reference 53), when the Committee
    concluded that the data were inadequate for setting an ADI.


        Table 1.  Summary of results of safety evaluations of ionones and structurally related substances used as flavouring agents

    Step 1: All of the substances are in structural class I.

                                                                                                                                           

    Substance                    No.     CAS No.        Estmated per     Step 2          Strep A3/B3     Step B4           Conclusion 
                                                        capita intake,   Metabolized     Intake exceed   Adequate NOEL     based on
                                                        Europe/USA       to innocuous    threshold of    for substance     current levels 
                                                        (g/day)         products?       concern?a       or related        of intake
                                                                                                         substance?
                                                                                                                                           

    -Damascone                  384     23726-92-3     43/10            No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    alpha-Damascone              385     43052-87-5     8/0.4            No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    delta-Damascone              386     57378-68-4     0.06/0.6         No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                           

    Substance                    No.     CAS No.        Estmated per     Step 2          Strep A3/B3     Step B4           Conclusion 
                                                        capita intake,   Metabolized     Intake exceed   Adequate NOEL     based on
                                                        Europe/USA       to innocuous    threshold of    for substance     current levels 
                                                        (g/day)         products?       concern?a       or related        of intake
                                                                                                         substance?
                                                                                                                                           

    Damascenone                  387     23696-85-7     86/5             No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    alpha-Ionone                 388     127-41-3       310/150          Yes             No              -                 No safety 
                                                                                                                           concernb
    CHEMICAL STRUCTURE 

    -lonone                     389     14901-07-6     150/100          Yes             No              -                 No safety 
                                                                                                                           concernb
    CHEMICAL STRUCTURE 

    gamma-Ionone                 390     79-76-5        0.01/15          No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                           

    Substance                    No.     CAS No.        Estmated per     Step 2          Strep A3/B3     Step B4           Conclusion 
                                                        capita intake,   Metabolized     Intake exceed   Adequate NOEL     based on
                                                        Europe/USA       to innocuous    threshold of    for substance     current levels 
                                                        (g/day)         products?       concern?a       or related        of intake
                                                                                                         substance?
                                                                                                                                           

    alpha-lonol                  391     25312-34-9     0.7/0.06         Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    -lonol                      392     22029-76-1     0.9/0.1          Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Dihydro-alpha-ionone         393     31499-72-6     0.7/0.02         Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Dihydro--ionone             394     17283-81-7     1/0.04           Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                           

    Substance                    No.     CAS No.        Estmated per     Step 2          Strep A3/B3     Step B4           Conclusion 
                                                        capita intake,   Metabolized     Intake exceed   Adequate NOEL     based on
                                                        Europe/USA       to innocuous    threshold of    for substance     current levels 
                                                        (g/day)         products?       concern?a       or related        of intake
                                                                                                         substance?
                                                                                                                                           

    Dihydro--ionol              395     3293-47-8      0.3/0.02         Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Dehydrodihydro-ionone        396     20483-36-7     0.1/0.08         No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Dehydrodihydro-ionol         397     57069-86-0     8/0.01           No              No              Yes               No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                           

    Substance                    No.     CAS No.        Estmated per     Step 2          Strep A3/B3     Step B4           Conclusion 
                                                        capita intake,   Metabolized     Intake exceed   Adequate NOEL     based on
                                                        Europe/USA       to innocuous    threshold of    for substance     current levels 
                                                        (g/day)         products?       concern?a       or related        of intake
                                                                                                         substance?
                                                                                                                                           

    Methyl-alpha-ionone          398     127-42-4       100/7            Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Methyl--ionone              399     127-43-5       6/0.2            Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Methyl-delta-ionone          400     7748-98-7      0.4/1            Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Allyl-alpha-ionone           401     79-78-7        35/25            Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    Table 1.  (continued)
                                                                                                                                           

    Substance                    No.     CAS No.        Estmated per     Step 2          Strep A3/B3     Step B4           Conclusion 
                                                        capita intake,   Metabolized     Intake exceed   Adequate NOEL     based on
                                                        Europe/USA       to innocuous    threshold of    for substance     current levels 
                                                        (g/day)         products?       concern?a       or related        of intake
                                                                                                         substance?
                                                                                                                                           

    alpha-Irone                  403     79-69-6        9/3              Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

    alpha-iso-Methylionone       404     127-51-5       6/1              Yes             No              -                 No safety 
                                                                                                                           concern
    CHEMICAL STRUCTURE 

                                                                                                                                           

    a  The threshold of concern for class I is 1800 g/day.
    b  The ADI values previously established for alpha-ionone and -ionone were maintained at the present meeting.
    

    1.2  Estimated daily per capita intake

          Per capita intake was estimated from data derived from 
    surveys in Europe (International organization of the Flavor 
    Industry, 1994) and the United States (US Academy of Sciences, 
    1989) (see Table 2). The estimated total daily  per capita 
    intake of all 21 ionones and related substances from their 
    use as flavouring agents is 0.76 mg/person in Europe and 0.33
    mg/person in the United States. In Europe, four substances, 
    4-(2,6,6-trimethylcyclohexa-1,3-dienyl)but-2-en-4-one (0.043
    mg/person), alpha-ionone (0.31 mg/person), -ionone (0.15 mg/person),
    and methyl-alpha-ionone (0.035 mg/person), account for approximately
    85% of the total annual  per capita intake of this group of
    substances when used as flavouring agents. In the United States, three
    substances, alpha-ionone (0.15 mg/person), -ionone (0.10 mg/person),
    and allyl-alpha-ionone (0.025 mg/person) account for about 85% of the
    total annual  per capita intake of this group of substances when used
    as flavouring agents. 

         Eleven of the substances in this group have been reported to
    occur naturally in foods including raspberries, carrots, roasted
    almonds, fruits, and herbs (Maarse et al., 1994). Quantitative data on
    natural occurrence and consumption ratios have been reported for seven
    substances (Nos 388, 389, 390, 391, 392, 393, and 394), which indicate
    that they are consumed predominantly in traditional foods, i.e. the
    consumption ratio is larger than 1 (Stofberg & Kirschman, 1985;
    Stofberg & Grundschober, 1987).

    1.3  Absorption, metabolism, and elimination

         The substances in this group are structurally related, in that
    each has a cyclohexane ring with an allyl side-chain containing a
    ketone or secondary alcohol functional group. The available metabolic
    data are derived largely from studies on -ionone and indicate at
    least two possible detoxification pathways:

    (i)  allylic hydroxylation of the ring at the 3 position, followed by 
         oxidation of the hydroxyl group to the 3-oxo derivative, and

    (ii) reduction of the ketone on the allyl side-chain to the
         corresponding secondary alcohol. 

    A combination of these reactions results in the formation of polar
    metabolites, which are excreted in the urine unchanged or conjugated
    with glucuronic acid. -Ionone can also be excreted unchanged in the
    urine.

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

                                                                         

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

    -Damascone (384)
     Europe                        300             43        0.7
     United States                 50              10        0.2
    alpha-Damascone (385)
     Europe                        57              8         0.1
     United States                 2               0.4       0.01
    delta-Damascone (386)
     Europe                        0.4             0.06      0.001
     United States                 3               0.6       0.01
    Damascenone (387)
     Europe                        600             86        1
     United States                 24              5         0.08
    alpha-Ionone (388)
     Europe                        2200            310       5
     United States                 770             150       2
    -lonone (389)
     Europe                        1100            150       3
     United States                 550             100       2
    gamma-Ionone (390)
     Europe                        0.1             0.01      0.0002
     United States                 80              15        0.3
    alpha-lonol (391)
     Europe                        5               0.7       0.01
     United States                 0.3             0.06      0.001
    -lonol (392)
     Europe                        6               0.9       0.01
     United States                 0.5             0.1       0.002
    Dihydro-alpha-ionone (393)
     Europe                        5               0.7       0.01
     United States                 0.1             0.02      0.0003
    Dihydro--ionone (394)
     Europe                        9               1         0.02
     United States                 0.2             0.04      0.0006
    Dihydro--ionol (395)
     Europe                        2.3             0.3       0.014
     United States                 0.1             0.02      0.0003
    Dehydrodihydroionone (396)
     Europe                        0.7             0.1       0.002
     United States                 0.4             0.08      0.001

    Table 2.  (continued)

                                                                         

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

    Dehydrodihydroionol (397)
     Europe                        0.1             0.01      0.0002
     United States                 40              8         0.1
    Methyl-alpha-ionone (398)
     Europe                        710             100       2
     United States                 35              7         0.1
    Methyl--ionone (399)
     Europe                        44              6         0.1
     United States                 0.9             0.2       0.003
    Methyl-delta-ionone (400)
     Europe                        3               0.4       0.01
     United States                 6               1         0.02
    Allyl-alpha-ionone (401)
     Europe                        250             35        0.6
     United States                 130             25        0.4
    alpha-Irone (403)
     Europe                        6.3             9         0.1
     United States                 15              3         0.05
    alpha-iso-Methylionone (404)
     Europe                        39              6         0.09
     United States                 5               1         0.02

    Total
     Europe                        5300            760       13
     United States                 1700            320       5
                                                                         

    a From US National Academy of Sciences (1989); International 
      Organization of the Flavor Industry (1995)


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

         The structure of 1,4-dimethyl-4-acetyl-1-cyclohexene (No. 402)
    was considered not to be sufficiently similar to that of the ionones
    to be included in the group, and it was therefore not included in the
    safety evaluation. The remaining 20 compounds were evaluated according
    to the procedure. 

    Step 1.   According to the decision-tree structural class
              classification (Cramer et al., 1978), all of the 20 ionones
              and related substances considered to be part of this group
              are in class I. 

    Step 2.   Data were available for -ionone that showed that it is
              metabolized by carbonyl reduction, hydroxylation of the
              alicyclic ring, and glucuronic acid conjugation of
              metabolites with alcohol groups. Only limited data were
              available on the fate of other compounds in the group.
              Although structural characteristics similar to that of
              -ionone were found, there were differences in the number
              and positions of the alicyclic double-bonds, the position of
              the carbonyl group within the side-chain, and its
              relationship to endocylic and exocyclic double-bonds. On the
              basis of the similarities of the functional groups present
              in the flavouring agents in this group, it was considered
              that alpha- and -ionones, their alcohol analogues,
              analogues with a single endocyclic double-bond, and
              analogues with saturated side-chains or more extended
              side-chains would be eliminated from the body by common
              metabolic processes, which would lead to innocuous products.

                   Members of the group with two endocyclic double-bonds,
              with the carboxyl group adjacent to the ring, or with an
              allyl double-bond attached to the ring might be eliminated
              more slowly, which might affect their toxic potency. The
              decision tree would take this into account to some extent by
              allocating compounds that are sterically hindered into class
              II rather than the class I. In consequence, the Committee
              was not able to conclude  a priori that the products of
              metabolism of such members of the group would be innocuous.

                   Substances that could be predicted to be metabolized to
              innocuous products fall into the following two groups:

                   Group 1: alpha-Ionone (No. 388), alpha-ionol (No. 391),
              dihydro-alpha-ionone (No. 393), methyl-alpha-ionone (No.
              398), methyl-delta-ionone (No. 400), alpha-irone (No. 403),
              2-iso-methylionone (No. 404), and allyl-alpha-ionone (No.
              401) were considered likely to share a common metabolic
              pathway to alpha-ionone, although the rate of metabolism for
              some of these substances may be slower than for
              alpha-ionone.

                   Group 2: -Ionol (No. 392), dihydro--ionone (No. 394),
              dihydro--ionol (No. 395), methyl--ionone (No. 399), and
              methyl-delta-ionone (No. 400) were considered likely to
              share common metabolic pathways with -ionone, although the
              rate of metabolism for some of these substances may be
              slower than for -ionone.

                   Evaluation of all of the substances in groups 1 and 2
              should proceed down the 'A' side of the scheme.

                   Substances that cannot be predicted to be metabolized
              to innocuous products are -damascone (No. 384),
              alpha-damascone (No. 385), delta-damascone (No. 386),
              4-(2,6,6-trimethylcyclohexa-1,3-dienyl)but-2-en-4-one (No.
              387), gamma-ionone (No. 390), dehydrodi-hydroionone (No.
              396), and dehydrodihydroionol (No. 397), and their
              evaluation should proceed down the 'B' side of the scheme.

    Step A3.  As the intakes in Europe and the United States of all 13
              substances on the 'A' side of the scheme are below the
              threshold of concern for class I (1800 g/person per day),
              these substances would not be expected to be of safety
              concern. 

    Step B3.  Intake of -damascone (No. 384), alpha-damascone (No. 385),
              delta-damascone (No. 386), 4-(2,6,6-trimethylcyclohexa-1,3-
              dienyl)but-2-en-4-one (No. 387), gamma-ionone (No. 390),
              dehydrodihydroionone (No. 396), and dehydrodihydroionol (No.
              397) in Europe and the United States is below the threshold
              of concern, and the safety evaluation proceeds to step B4.

    Step B4.  Information on each of the compounds considered at this step
              is given below:

     -Damascone (No. 384)

         The NOEL for this substance is > 2 mg/kg bw per day (90-day
    study in rats), and the margin of safety between this NOEL and the
    daily  per capita intake under the conditions of intended use is
    > 2500. 

     alpha-Damascone (No. 385), delta-damascone (No. 386), and 
     4-(2,6,6-trimethyl-cyclohexa-1,3-dienyl)but-2-en-4-one (No. 387)

         These substances were considered likely to share common metabolic
    pathways with -damascone (No. 384) for which the NOEL is > 2 mg/kg
    bw per day, although the rate of metabolism may be slower. Therefore
    the margin of safety between this NOEL and daily  per capita intake
    is > 1.5  104 for alpha-damascone, > 2  105 for
    delta-damascone, and > 14  103 for
    4-(2,6,6-trimethyl-cyclohexa-1,3-dienyl)but-2-en-4-one.

    gamma -Ionone (No. 390)

         This substance was considered likely to share a common metabolic
    pathway with alpha- and -ionone, for which the NOEL is 10 mg/kg bw
    per day (90-day study in rats). Therefore, the margin of safety
    between this NOEL and the daily  per capita intake of alpha-ionone is
    > 40  104. It was also considered likely to share a common
    metabolic pathway with carvone (No. 380), for which the NOEL is 93
    mg/kg bw per day (three-month study in rats). In this case, the margin
    of safety between the NOEL and the daily  per capita intake of
    gamma-ionone is > 4  105.

     Dehydrodihydroionone (No. 396)  and dehydrodihydroionol (No. 397)

         These substances were considered likely to share common pathways
    with alpha- and -ionone, for which the NOEL is 10 mg/kg bw per day.
    The margin of safety between this NOEL and the daily  per capita 
    intake is 6  106 for dehydrodihydroionone and > 7  105 for
    dehydrodihydroionol.

         Therefore, the seven substances considered on the 'B' side of the
    scheme would not be expected to be of safety concern. 

    1.5  Consideration of combined intakes from use as flavouring agents

         All of the 20 ionones and structurally related substances
    considered in this evaluation would be expected to have common
    metabolic pathways. In the unlikely event that all 20 substances were
    consumed simultaneously on a daily basis, the estimated daily
     per capita consumption in Europe and the United States would not
    exceed the human intake threshold for substances in class 1. 

    1.6  Conclusions

         In applying the procedure, the Committee concluded that use of
    any of the 20 ionones and related substances as flavouring agents
    would not present a safety concern at the current estimated intake
    levels. The Committee noted that all of the available data on toxicity
    are consistent with the results of the safety evaluation. The ADIs
    previously established for alpha-ionone and -ionone were maintained.


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Biological data

    2.1.1  Absorption, metabolism, and elimination 

         The substances in this group are structurally related, in that
    each has a cyclohexane ring with an allyl side-chain containing a
    ketone or secondary alcohol functional group. The available metabolic
    data are derived largely from studies on -ionone and indicate at
    least two possible detoxification pathways:

    (i)  allylic hydroxylation of the ring at the 3 position, followed by
         oxidation of the hydroxyl group to the 3-oxo derivative, and

    (ii) reduction of the ketone on the allyl side-chain to the
         corresponding secondary alcohol. 

    A combination of these reactions results in the formation of polar
    metabolites, which are excreted in the urine unchanged or conjugated
    with glucuronic acid. -Ionone can also be excreted unchanged in the
    urine. The data that support this conclusion are given below.

         Analysis of urine collected from two rabbits fed a total of 170 g
    alpha-ionone over an unspecified period revealed a hydroxylated
    derivative of alpha-ionone formed from allylic ring oxidation (Prelog
    & Wursch, 1951). 

         Urine was collected daily from one male rabbit fed a total of 23
    g -ionone over seven days at approximately 1000 mg/kg bw per day) and
    for four days after the final dose. 3-Oxo--ionone, 3-oxo--ionol,
    dihydro-3-oxo--ionol, and 3-hydroxy--ionol were identified, as were
    unchanged -ionone and the glucuronic acid conjugates of 3-oxo--ionol
    and dihydro-3-oxo--ionol (Ide & Toki, 1970). 

         The urinary metabolites of two rabbits that received a total of
    100 g -ionone by gavage over 18 days included 3-hydroxy--ionone,
    3-oxo--ionol, and 3-hydroxy--ionone. A hydroxyketone thought to be
    either 3-oxo--ionol or 3-hydroxy--ionone was also recovered (Fujii
    et al., 1972). After oral administration of -ionone to three rabbits
    at doses of 2000-5000 mg/day for two weeks, the urine contained
    -ionone, -ionol, and their exocyclic dihydro metabolites, namely,
    dihydro--ionol, 3-hydroxy--ionol, 3-hydroxydihydro--ionol,
    3-hydroxy--ionone, and 3-hydroxydihydro--ionone (Bielig & Hayasida,
    1940). (In these two studies, the urinary metabolites were identified
    by IUPAC nomenclature, but the metabolites were reported by a
    different naming system.)

         Two dogs fed a total of 100 g -ionone over 18 days also excreted
    3-oxo--ionone and 3-hydroxy--ionol in the urine (Prelog & Meier,
    1950).

         -Ionone has been found to induce biphenyl 4-hydroxylase,
    glucuronyl transferase, 4-nitrobenzoate reductase, and cytochrome P450
    in rats after a three-day intraperitoneal or oral administration
    (Parke & Rahman, 1969).

         Studies in humans of the metabolism of retinoids such as
     cis-13-retinoic acid (i.e. isotretinoin) and carotenoids such as
    -carotene, which possess ionone fragments, indicate that the
    metabolism of ionones is similar to that in animals. The primary
    metabolites in blood and bile after oral administration of
    isotretinoin to humans included the glucuronic acid conjugates of
    isotretinoin (Kraft et al., 1991) and the allylic oxidation product
    (Vane et al., 1990; Kraft et al., 1991). Both metabolites were found
    in the blood and bile of cynomolgus monkeys after oral administration
    of isotretinoin (Kraft et al., 1991). Allylic hydroxylation of the
    methyl-ring substituent and subsequent conjugation with glucuronic
    acid have also been shown to occur in humans (Vane et al., 1990).

         -Carotene is oxidized by carotenoid dioxygenase(s) and cleaved
    at the 15-15' (central) double bond to yield two molecules of vitamin
    A (retinal) (Simpson & Chichester, 1981), which may subsequently be
    cleaved at the 9'-10' double bond to yield -ionone and
    10'-apo--carotenals. The presence of 10'-apo--carotenal in rat liver

    after oral administration of -carotene suggests that oxidative
    cleavage of the 9'-10' double bond occurs in animals (Sharma et al.,
    1977).

    2.1.2  Toxicological studies

    2.1.2.1  Acute toxicity

         The results of studies of acute toxicity with ionone and
    structurally related substances are shown in Table 3.

    2.1.2  Short-term and long-term studies of toxicity

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

     -Damascone

         Groups of 16 male and 16 female Wistar CF/Gif Carworth strain
    rats were given -damascone in the diet at 0 or 2 mg/kg bw per day for
    90 days. Individual body weights were recorded weekly, but no
    difference was seen between treated and control groups. Food intake
    was slightly increased in treated females over that in controls but
    was associated with a slight decrease in food use. Haematological
    examination and blood urea determinations carried out on half of the
    rats at week 7 and on all animals at the end of the treatment period
    revealed no statistically significant changes, with no major
    difference between treated and control groups in haemoglobin
    concentration or total or differential leukocyte counts. At necropsy,
    a slight increase in the absolute and relative weights of the liver
    and kidneys of females was seen in comparison with controls, but these
    changes were not correlated with any histopathological lesions.
    Histological examination and clinical chemistry revealed no
    significant changes. Non-specific inflammatory changes were seen in
    the livers and kidneys of a few animals, but these changes were not
    considered to be related to treatment. The NOEL was 2 mg/kg bw per
    day, the only dose tested (Posternak et al., 1975).

     alpha-Ionone and -ionone 

         Studies on alpha- and -ionone were evaluated at the
    twenty-eighth meeting (Annex 1, reference 66).

     alpha-Irone

         As part of a study to examine the toxicity of a group of 23
    flavouring substances, groups of 15 FDRL strain rats of each sex
    received alpha-irone in their diets for 90 days at a daily
    concentration designed to provide 5 mg/kg bw for males and 6 mg/kg bw
    for females. Records of daily food and water consumption revealed no
    differences between test and control groups. Haematological and blood

    chemical parameters were measured in eight rats of each sex at week 6
    and in all rats at week 12; they were within normal ranges. At
    autopsy, no difference in liver or kidney weights was seen between
    test and control groups. Histological and gross pathological
    examinations revealed no statistically significant changes. Treated
    females had slightly higher haemoglobin and haematocrit values, but
    the findings were considered to be biologically insignificant because
    the mean erythrocyte count for the group was comparable to that of
    controls. Both test and control groups showed a slight degree of
    reactive lymphatic hyperplasia, but the findings were not related to
    treatment. The NOEL was 5 mg/kg bw per day (Oser et al., 1965). 

     alpha-iso-Methylionone

         As part of a study to examine the toxicity of a group of 23
    flavouring substances, groups of 15 FDRL strain rats of each sex
    received alpha- iso-methylionone in their diets for 90 days at a
    daily concentration designed to provide 4 mg/kg bw. Food and water
    consumption was similar for the test and control groups.
    Haematological and clinical chemical measurements conducted on eight
    rats of each sex at week 6 and on all rats at week 12 showed normal
    values. Liver and kidney weights recorded at necropsy were similar in
    test and control groups. Histological and gross pathological
    examinations revealed no changes related to treatment. The male rats
    had a slightly reduced haemoglobin level, but the haematocrit and
    erythrocyte counts were within the control ranges. The mean blood urea
    nitrogen concentration was slightly lower in the treated group than in
    controls at week 12, but this change was not accompanied by changes in
    kidney weight or histological appearance and was considered to be of
    no biological significance. The NOEL was > 4 mg/kg bw per day (Oser
    et al., 1965). 

    2.1.3  Genotoxicity

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


    4.  REFERENCES

    Bielig, H. & Hayasida, A. (1940) [On the release of -jonone in the
    animal (biochemical hydration VIII).]  Hoppe-Seyler's Z. Physiol. 
     Chem., 266, 99-111 (in German).

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

    Florin, I., Rutberg, L., Curvall, M. & Enzell, C.R. (1980) Screening
    of tobacco smoke constituents for mutagenicity using the Ames test.
     Toxicology, 15, 219-232.


        Table 3. Studies of the acute toxicity of ionone and related substances

                                                                                                                               

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

    4-[(2,6,6)-Trimethyl-cyclo-hex-1-enyl]but-2-en-4-one      384   Rat       NR     Oral     2920        Posternak et al.
                                                                                                          (1975)
      (-Damascone)                                                 Rat       M/F    Gavage   2000        Firmenich (1986)
    alpha-Damascone                                           385   Rat       M/F    Oral     1800        Piccirillo et al.
                                                                                                          (1979)
    delta-Damascone                                           386   Mouse     M/F    Gavage   1820        Moran et al. (1980)
    4-[(2,6,6)-Trimethylcyclo-hexa-1,3-dienyl]but-2-en-4-one  387   Rat       M/F    Gavage   > 2000      Firmenich (1986)
    alpha-Ionone                                              388   Rat       M/F    Oral     4590a       Jenner et al. (1964)
    -Ionone                                                  389   Rat       M/F    Oral     4590        Jenner et al. (1964)
    alpha-Ionol                                               391   Rat       NR     Oral     > 5000      Moreno (1980)
    alpha-Ionol                                               391   Mouse     NR     Oral     7400        Pellmont (1978)
    -Ionol                                                   392   Rat       NR     Oral     > 1220;     Moreno (1980)
                                                                                              < 5000
    -Ionol                                                   393   Mouse     NR     Oral     5700        Pellmont (1977)
    Dihydro-alpha-ionone                                      393   Rat       NR     Oral     > 5000      Moreno (1976)
    Dihydro--ionone                                          394   Mouse     NR     Oral     5700        Pellmont (1977)
    Dihydro--ionol                                           395   Mouse     NR     Oral     7400        Pellmont (1977)
    Methyl-alpha-ionone                                       398   Rat       NR     Oral     > 5000a     Moreno (1973)
    Methyl-delta-ionone                                       400   Rat       NR     Oral     > 5000      Moreno (1973)
    Allyl alpha-ionone                                        401   Mouse     NR     Gavage   8900        Givaudan (1955)
    alpha-Irone                                               403   Rat       M/F    Oral     > 5000      Shelanski &
                                                                                                          Moldovan (1972)
    alpha-iso-Methylionone                                    404   Rat       NR     Oral     > 5000b     Moreno (1973)
                                                                                                                               

    NR, not reported; M/F, male and female
    a  Mixture of alpha- and -ionone
    b  Mixture of methyl-alpha-ionone and alpha-iso-methylionone

    Table 4.  Short-term and long-term studies of toxicity with ionone and related substances in rats treated orally

                                                                                                                                              

    Substance                                                         No.    Sex    No. groups/   Duration       NOELa     Reference
                                                                                    no. per group                (mg/kg bw
                                                                                                                 per day)
                                                                                                                                              

    4-[(2,6,6)-Trimethylcyclohex-1-enyl]-but-2-en-4-one (-Damascone) 384    M/F    1/32          90 days        > 2       Posternak et al. 
                                                                                                                           (1975)
    alpha-Ionone                                                      388    M/F    1/30          90 days        > 11      Oser et al. (1965)
    alpha-Ionone                                                      388    M/F    2/30          90 days        10        Gaunt et al. (1983)
    Iononeb                                                           388    NR     3/8-10        7-8 weeks      > 10      Sporn et al. (1963)
    alpha-Iononec                                                     388    M/F    3/20          17 weeks       ND        Hagan et al. (1967)
    -Ionone                                                          389    M/F    2/15          90 days        > 11 (M)  Oser et al. (1965)
                                                                                                                 > 13 (F)
    -Ionone                                                          389    M/F    2/30          90 days        10        Gaunt et al. (1983)
    alpha-Irone                                                       403    M/F    2/15          90 days        > 5 (M)   Oser et al. (1965)
                                                                                                                 > 6 (F)
    alpha-iso-Methylionone                                            404    M/F    1/30          90 days        > 4       Oser et al. (1965)
                                                                                                                                              

    NR, not reported; M, male; F, female; ND, not determined
    a  NOEL given as 'greater than' (>) indicates that no adverse effects were observed at the highest dose in the study and, therefore, no NOEL
       was determined.
    b  Mixture of alpha-ionone and -ionone 
    c  Mixture of 60% alpha-ionone and 40% alpha-ionone


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

                                                                                                                                              

    Substance              No.    End-point                 Test object              Dose                Result       Reference
                                                                                                                                              

    alpha-Ionone           388    Chromosomal aberration    Chinese hamster B241     25 nmol/L           Positivea    Kasamaki et al. (1982)
                                                            cell line
                                  Gene mutation             S. typhimurium TA98,     0.01-50 mg/plate    Negativea    Kasamaki et al. (1982
                                                            TA100
                                  rec assay                 B.  subtilis H17 & M45   19 mg/disc          Negativeb    Oda et al. (1978)
    -Ionone               389    Gene mutation
                                  (preincubation)           S. typhimurium TA98,     1-180 mg/plate      Negativea    Mortlemans et al. (1986)
                                                            TA100, TA1535, 1537
                                  Gene mutation             S. typhimurium TA98,     3 mmol/plate        Negativea    Florin et al. (1980)
                                                            TA100, TA1535, TA1537
    Methyl-alpha-ionone    398    Gene mutation             S. typhimurium TA1535,   < 3600 mg/plate     Negativea    Wild et al. (1983)
                                                            TA1537, TA1538, TA98, 
                                                            TA100
    Methyl-alpha-ionone    398    Micronucleus formation    NMRI mice, male and      825-2063 mg/kg bw   Negative     Wild et al. (1983)
                                                             female, bone marrow
    Methyl-alpha-ionone    398    Gene mutation (basc)      Drosophila melanogaster  20 mmol/L           Negative     Wild et al. (1983)
    Methyl-delta-ionone    400    Gene mutation             S. typhimurium TA1535,   < 3600 mg/plate     Negativea    Wild et al. (1983)
                                                            TA1537, TA1538, TA98,
                                                            TA100
                                                                                                                                              

    a  With and without metabolic activation
    b  Activation status unknown
    

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    See Also:
       Toxicological Abbreviations