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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        SAFETY EVALUATION OF CERTAIN
        FOOD ADDITIVES AND CONTAMINANTS



        WHO FOOD ADDITIVES SERIES 40





        Prepared by:
          The forty-ninth meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998



    ESTERS DERIVED FROM BRANCHED-CHAIN TERPENOID ALCOHOLS AND ALIPHATIC
    ACYCLIC LINEAR AND BRANCHED-CHAIN CARBOXYLIC ACIDS

    First draft prepared by
    Dr G.J.A. Speijers and Ms M.F.A. Wouters
    National Institute of Public Health and
    Environment Protection (RIVM)
    Bilthoven, The Netherlands

        1.  Evaluation
            1.1 Introduction
            1.2 Estimated daily  per capita intake
            1.3 Absorption, metabolism and elimination
                1.3.1   Terpenoid alcohols
            1.4 Application of the Procedure for the Safety Evaluation of
                Flavouring Agents
            1.5 Consideration of combined intake
            1.6 Conclusions
        2.  Relevant background information
            2.1 Explanation
            2.2 Intake data
            2.3 Biological data
                2.3.1   Absorption, metabolism and elimination
                    2.3.1.1 Terpenoid alcohols
                    2.3.1.2 Aliphatic carboxylic acids
                2.3.2   Toxicological studies
                    2.3.2.1 Acute toxicity
                    2.3.2.2 Short-term toxicity
                    2.3.2.3 Long-term toxicity/carcinogenicity studies
                    2.3.2.4 Genotoxicity
                    2.3.2.5 Reproductive toxicity
                    2.3.2.6 Other relevant studies
            2.4 Observations in humans
                2.4.1   Geranyl acetate
                2.4.2   Neryl acetate
                2.4.3   Citronellyl acetate
                2.4.4   Rhodinyl acetate
                2.4.5   Neryl isobutyrate
        3.  References

    1.  EVALUATION

    1.1  Introduction

        A safety evaluation of a group of 26 terpenoid esters was
    conducted using the Procedure for Safety Evaluation of Flavouring
    Agents (the "Procedure") (see Figure 1 and Table 1).

    FIGURE 1


        Table 1.  Summary of results of the safety evaluations of esters of terpenoid alcohols
    and aliphatic carboxylic acids

    Step 1:  All of the substances in the group are in structural class I, the human intake
    threshold of which is 1800 g per day.
    Step 2:  All of the substances in the group are metabolized to innocuous products.

                                                                                              
    No.    Substance                  Step A3                           Conclusions based on
           (CAS No.)                  Does intake exceed intake         current intake
                                      threshold? Intake estimates
                                      (g/person per day1)
                                                                                              

    0053   Citronellyl formate                  No                      No safety concern
                                      USA:  2.5   Europe:  103
    0054   Geranyl formate                      No                      No safety concern
                                      USA:  48    Europe:  330
    0055   Neryl formate                        No                      No safety concern
                                      USA:  0.04  Europe:  0.01
    0056   Rhodinyl formate                     No                      No safety concern
                                      USA:  0.10  Europe:  unknown
    0057   Citronellyl acetate                  No                      No safety concern
                                      USA:  36    Europe:  217
    0058   Geranyl acetate                      No                      No safety concern
                                      USA:  205   Europe:  580
    0059   Neryl acetate                        No                      No safety concern
                                      USA:  63    Europe:  180
    0060   Rhodinyl acetate                     No                      No safety concern
                                      USA:  0.8   Europe:  1.1
    0061   Citronellyl propionate               No                      No safety concern
                                      USA:  1.5   Europe:  41
    0062   Geranyl propionate                   No                      No safety concern
                                      USA:  11    Europe:  81
    0063   Neryl propionate                     No                      No safety concern
                                      USA:  1.1   Europe:  4.3
    0064   Rhodinyl propionate                  No                      No safety concern
                                      USA:  0.02  Europe:  unknown
    0065   Citronellyl butyrate                 No                      No safety concern
                                      USA:  5     Europe:  32


    Table 1.  Continued...

                                                                                              
    No.    Substance                  Step A3                           Conclusions based on
           (CAS No.)                  Does intake exceed intake         current intake
                                      threshold? Intake estimates
                                      (g/person per day1)
                                                                                              

    0066   Geranyl butyrate                     No                      No safety concern
                                      USA:  25    Europe:  60
    0067   Neryl butyrate                       No                      No safety concern
                                      USA:  0.02  Europe:  0.4
    0068   Rhodinyl butyrate                    No                      No safety concern
                                      USA:  1.0   Europe:  unknown
    0069   Citronellyl valerate                 No                      No safety concern
                                      USA:  4.0   Europe:  0.7
    0070   Geranyl hexanoate                    No                      No safety concern
                                      USA:  0.5   Europe:  0.07
    0071   Citronellyl isobutyrate              No                      No safety concern
                                      USA:  1.3   Europe:  13
    0072   Geranyl isobutyrate                  No                      No safety concern
                                      USA:  3.0   Europe:  124
    0073   Neryl isobutyrate                    No                      No safety concern
                                      USA:  0.4   Europe:  2.0
    0074   Rhodinyl isobutyrate                 No                      No safety concern
                                      USA:  0.02  Europe:  0.03
    0075   Geranyl isoverate                    No                      No safety concern
                                      USA:  1.7   Europe:  43
    0076   Neryl isovalerate                    No                      No safety concern
                                      USA:  0.04  Europe:  0.03
    0077   Rhodinyl isovalerate                 No                      No safety concern
                                      USA:  0.02  Europe:  0.01
    0078   3,7-Dimethyl-2,6-
           octadien-1-yl 2-
           ethylbutanoate                       No                      No safety concern
                                      USA:  0     Europe:  0.6
                                                                                              

    1 The human intake threshold is 1800 g per day for Class 1, 540 g/day for Class 2 and 
    90 g/day for Class 3
    

        One member of the group, geranyl acetate, was previously evaluated
    at the twenty-third meeting of the Committee. It was evaluated as part
    of a group of other terpenoid flavouring substances, citral,
    citronellol, and linalool, which have close chemical, biochemical and
    toxicological relationships. The Committee allocated a group ADI of 0-
    0.05 mg/kg bw based on the clearly defined metabolism of these
    substances and their low toxicity in short-term toxicity studies
    (Annex 1, reference 50).

        Data relevant to the Procedure for the Safety Evaluation of 26
    esters formed from the esterification of the terpenoid alcohols, (for
    geraniol, nerol, ()-citronellol and rhodinol) and saturated aliphatic
    carboxylic acids (linear saturated aliphatic acids (C1-C6),
    isobutyric acid isovaleric acid and 2-ethylbutyric acid) are
    summarized.

    1.2  Estimated daily  per capita intake

        The total annual production volume of the 26 terpenoid esters used
    as flavouring agents is approximately 2100 kg in the USA and 12 600 kg
    in Europe. In the USA and in Europe, approximately 60% of the total
    annual volume is accounted for by the acetate and butyrate esters of
    citronellol, geraniol and nerol. Based on the annual volume reported
    in the USA and Europe, the total estimated daily  per 
     capita intake of the 26 terpenoid esters used as flavouring
    substances is 410 g in the USA and 1800 g in Europe. The total
    estimated daily  per capita intake of terpenoid alcohols (i.e.,
    citronellol, geraniol, nerol and rhodinol) formed via hydrolysis of
    these esters is 320 g in the USA and 1400 g in Europe.

        Terpenoid esters are principal flavour components of citrus and
    citrus peel oils, and have also been detected in wide variety of other
    fruits, spices and vegetables. The terpenoid esters are usually found
    at concentration of < 1 mg/kg in citrus fruit juices, < 20 000 mg/kg
    in citrus peel oils, and < 50 000 mg/kg in spices (CIVO-TNO, 1994).
    In the USA terpenoid esters are consumed predominantly as components
    of traditional foods (Stofberg & Kirschman, 1985). In the USA, the
    total annual consumption of seven of these terpenoid esters as natural
    components of food is estimated to be approximately 300 tonnes
    (Stofberg & Grundschober, 1987).

    1.3  Absorption, metabolism and elimination

        The terpenoid esters are hydrolysed to the corresponding terpenoid
    alcohols (geraniol, citronellol, nerol, and rhodinol) and aliphatic
    carboxylic acids (formic, acetic, propionic, butyric, valeric,
    hexanoic, isobutyric and isovaleric acids). Both the hydrolysis data
    and the metabolism of aliphatic carboxylic acids are discussed in the
    introduction to this chapter on flavouring agents.


    1.3.1  Terpenoid alcohols

        Following hydrolysis, the terpenoid alcohols undergo a complex
    pattern of alcohol oxidation, omega-oxidation, hydration, selective
    hydrogenation and subsequent conjugation to form oxygenated polar
    metabolites, which are excreted primarily in the urine. Geraniol,
    related terpenoid alcohols (citronellol and nerol), and the aldehydes
    (geranial and neral) exhibit similar pathways of metabolic
    detoxication in animals (Figure 1).

    1.4  Application of the Procedure for Safety Evaluation of Flavouring 
    Agents

        Step 1. All of the 26 terpenoid esters are in class I.

        Step 2. It is expected that the esters in this group will be
    readily hydrolysed to the component alcohols and carboxylic acids,
    which are considered to be innocuous. The terpenoid alcohols are
    expected to undergo omega-oxidation and functional group oxidation to
    yield polar metabolites which are excreted as the glucuronic acid
    conjugate in the urine. Eight of the 9 component carboxylic acids are
    endogenous in humans, participating in the fatty acid -oxidation
    pathway, amino acid pathways, the citric acid cycle, or the
    C1 tetrahydrofolate pathway and eventually yielding CO2 and H2O.
    The remaining carboxylic acid 2-ethylbutyric acid undergoes oxidation
    to polar metabolites that are conjugated with glucuronic acid and
    excreted. At current levels of intake these esters and their component
    terpenoid alcohols and aliphatic carboxylic acids would not be
    expected to saturate these metabolic pathways.

        Step A3. None of the 26 terpenoid esters has a USA or European
    daily  per capita intake that exceeds 1800 g/person per day.
    Therefore, results of the Procedure indicate that none of the 26
    terpenoid esters evaluated poses a safety concern when used at current
    levels of intake as flavouring substances.

        The stepwise evaluations of the 26 terpenoid esters used as
    flavouring substances are summarized in Table 1.

    1.5  Consideration of combined intake

        In the unlikely event that these 26 terpenoid esters were to be
    consumed simultaneously on a daily basis, the total daily intake would
    still be within the human intake threshold of Class I (1800 g/person
    per day). The Committee noted that the terpene alcohols, geranoil,
    citronellol and linalool are used as flavouring agents and that the
    combined intakes of these alcohols and esters would be less than the
    group ADI.


    1.6  Conclusions

        Applying the Procedure, the Committee concluded that for the
    esters derived from branched-chain terpenoid alcohols and aliphatic
    acyclic linear and branched-chain carboxylic acids there was no safety
    concern at current intake levels.

        No toxicity data were required for the application of the
    procedure. The Committee noted that the available toxicity data were
    consistent with the results of the safety evaluation using the
    procedure.

        The Committee noted that some of the esters are metabolized to
    alpha,-unsaturated carbonyl compounds, but concluded that these had
    been adequately evaluated previously (Annex 1, reference 50). The
    Committee maintained the group ADI of 0-0.05 mg/kg bw for geranyl
    acetate, citral, citronellol and linalool.

    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

        This monograph summarizes all the available data relevant to the
    safety evaluation of 26 esters formed from the esterification of the
    terpenoid alcohols: geraniol, nerol, ()-citronellol, and rhodinol and
    saturated aliphatic carboxylic acids (linear saturated aliphatic acids
    (C1-C6), isobutyric acid, isovaleric acid and 2-ethylbutyric acid).
    The four terpenoid alcohols from which the 26 esters are formed are
    close structural relatives. Geraniol and nerol are  cis-trans (E-Z)
    isomers of 3,7-dimethyl-2,6-octadien-1-ol; ()-citronellol is
    2,3-dihydrogeraniol (3,7-dimethyl-6-octen-1-ol); and rhodinol is a
    mixture containing mainly  l-citronellol and a small amount of the
    isomer 3,7-dimethyl-7-octen-1-ol. As a commercial product, geraniol or
    nerol may contain significant amounts of its (Z)- or (E)-isomer,
    respectively, the monounsaturated alcohol (citronellol) and saturated
    alcohol (3,7-dimethyl-1-octanol).

        The basic structures of esters derived from branched-chain
    terpenoid alcohols and saturated aliphatic acyclic carboxylic acids
    are as follows:


    CHEMICAL STRUCTURE 1


    2.2  Intake data

        The 26 esters formed from terpenoid primary alcohols (4) and
    aliphatic saturated carboxylic acids (9) are used as flavor
    ingredients in the USA at average maximum use levels less than 100
    mg/kg, except for geranyl isovalerate which is used up to 155 mg/kg.
    The total annual volume of the 26 terpenoid esters used as flavouring
    substances is appoximately 2.1 tonnes in the USA (NAS, 1987) and 12.6
    tonnes in Europe (IOFI, 1996) (Table 2). In the USA and in Europe,
    approximately 60% of the total annual volume (NAS, 1987; IOFI, 1996)
    is accounted for by the acetate and butyrate esters of citronellol,
    geraniol and nerol. Based on the annual volume reported in the USA
    (NAS, 1987) and Europe (IOFI, 1996), the total estimated daily 
     per capita intake ('eaters only') of the 26 terpenoid esters used as
    flavouring substances is 6.8 g/kg bw in the USA and 29.9 g/kg bw in
    Europe. The total daily  per capita intake ('eaters only') of
    terpenoid alcohols (i.e., citronellol, geraniol, nerol and rhodinol)
    formed via hydrolysis of these esters is 5.3 g/kg bw in the USA and
    13.3 g/kg bw in Europe.

    2.3  Biological data

    2.3.1  Absorption, metabolism and elimination

        In general, aliphatic esters are rapidly hydrolysed to their
    component alcohols and carboxylic acids. For example, geranyl esters
    are hydrolysed to geraniol and aliphatic carboxylic acids (see Figure
    2). Hydrolysis is catalysed by classes of enzymes recognized as
    carboxylesterases or esterases (Heymann, 1980), the most important of
    which are the -esterases. In mammals, these enzymes occur in most
    tissues throughout the body (Heymann, 1980; Anders, 1989) but
    predominate in the hepatocytes (Heymann, 1980).


    FIGURE 2

        Each terpenoid ester in this group is hydrolysed to its
    corresponding terpenoid alcohol (geraniol, citronellol, nerol and
    rhodinol) and aliphatic carboxylic acid (formic, acetic, propionic,
    butyric, valeric, hexanoic, isobutyric and isovaleric acids). A
    concentration of 15 l citronellyl acetate/litre was reported to be
    completely hydrolysed within 2 hours by simulated intestinal fluid
    containing pancreatin. A concentration <18 l citronellyl
    phenylacetate/litre was reported to be 60% hydrolysed within 2 hours
    (Grundschober, 1977). Terpenoid alcohols formed in the
    gastrointestinal tract are rapidly absorbed (Phillips  et al., 1976;
    Diliberto  et al., 1988).


        Table 2.  Most recent annual usage of terpenoid alcohol esters in the USA and Europe

                                                                                                      

    Substance               Most recent        Daily per capita intake2      Daily per capita
                            annual volume,     ("eaters only")               intake3 ("eaters only"),
                            (kg1)                                            alcohol equivalents,
                                              g/g         g/kg bw/day     (g/kg bw/day)
                                                                                                      
    Citronellyl formate
    USA                     13                2.47         0.04              0.03
    Europe                  718               102.46       1.71              1.45

    Geranyl formate
    USA                     250               47.56        0.79              0.67
    Europe                  2329              332.35       5.54              4.69

    Neryl formate
    USA                     0.2               0.04         0.0006            0.003
    Europe                  0.054             0.01         0.0001            0.00004

    Rhodinyl formate
    USA                     0.5               0.10         0.002             0.001

    Citronellyl acetate
    USA                     190               36.15        0.60              0.47
    Europe                  1522              217.19       3.62              2.85

    Geranyl acetate
    USA                     1070              203.58       3.39              2.66
    Europe                  3899              556.39       9.27              7.28

    Neryl acetate
    USA                     333               63.36        1.06              0.83
    Europe                  1264              180.37       3.01              2.36

    Rhodinyl acetate
    USA                     4                 0.76         0.013             0.007
    Europe                  8                 1.14         0.019             0.01
                                                                                                      


    Table 2.  Continued...

                                                                                                      

    Substance               Most recent        Daily per capita intake2      Daily per capita
                            annual volume,     ("eaters only")               intake3 ("eaters only"),
                            (kg1)                                            alcohol equivalents,
                                              g/g        g/kg bw/day      (g/kg bw/day)
                                                                                                      
    Citronellyl acetate
    USA                     8                 1.52         0.03              0.02
    Europe                  286               40.81        0.68              0.49

    Geranyl propionate
    USA                     60                11.42        0.19              0.14
    Europe                  565               80.63        1.34              0.98

    Neryl propionate
    USA                     0.5               0.10         0.002             0.001
    Europe                  30                4.28         0.07              0.05

    Rhodinyl propionate
    USA                     0.1               0.02         0.0003            0.0002

    Citronellyl butyrate
    USA                     25                4.76         0.08              0.06
    Europe                  224               31.96        0.53              0.37

    Geranyl butyrate
    USA                     130               24.73        0.41              0.28
    Europe                  423               60.36        1.01              0.69

    Neryl butyrate
    USA                     0.1               0.02         0.0003            0.0002
    Europe                  2.9               0.41         0.007             0.004

    Rhodinyl butyrate
    USA                     5                 0.95         0.02              0.001
    Europe                  0
                                                                                                      


    Table 2.  Continued...

                                                                                                      

    Substance               Most recent        Daily per capita intake2      Daily per capita
                            annual volume,     ("eaters only")               intake3 ("eaters only"),
                            (kg1)                                            alcohol equivalents,
                                              g/g        g/kg bw/day      (g/kg bw/day)
                                                                                                      

    Citronellyl valerate
    USA                     20                3.81         0.06              0.04
    Europe                  5                 0.71         0.01              0.01

    Geranyl hexanoate
    USA                     3                 0.57         0.01              0.01
    Europe                  0.5               0.07         0.001             0.0005

    Citronellyl isobutyrate
    USA                     7                 1.33         0.02              0.01
    Europe                  90                12.84        0.21              0.14

    Geranyl isobutyrate
    USA                     16                3.04         0.05              0.03
    Europe                  866               123.58       2.06              1.41

    Neryl isobutyrate
    USA                     2                 0.38         0.01              0.01
    Europe                  14                2.00         0.03              0.02

    Rhodinyl isobutyrate
    USA                     0.1               0.02         0.0003            0.002
    Europe                  0.2               0.03         0.0005            0.003

    Geranyl isoverate
    USA                     9                 1.71         0.03              0.02
    Europe                  339               48.38        0.81              0.52

    Neryl isovalerate
    USA                     0.2               0.04         0.001             0.0005
    Europe                  0.2               0.03         0.0005            0.0003
                                                                                                      


    Table 2.  Continued...

                                                                                                      

    Substance               Most recent        Daily per capita intake2      Daily per capita
                            annual volume,     ("eaters only")               intake3 ("eaters only"),
                            (kg1)                                            alcohol equivalents,
                                              g/g         g/kg bw/day      (g/kg bw/day)
                                                                                                      

    Rhodinyl isovalerate
    USA                     0.1               0.02         0.0003            0.0002
    Europe                  0.1               0.01         0.0002            0.0001

    3,7-dimethylocta-2,6-
    dienyl 2-ethylbutyrate
    USA                     0                 0.00         0.0               0.0
    Europe                  4                 0.57         0.010             0.005
                                                                                                      

    Total USA                2146.8            408.45       6.82              5.31
    Total Europe            12589.96          1798.59      29.94             13.33
                                                                                                      

    1  USA: National Academy of Science (NAS, 1987) Evaluating the safety of food chemicals.
    Washington DC.  Europe: International Organization of the Flavour Industry (IOFI, 1996)
    European Inquiry on volume of use. Private communication to FEMA
    2  Intake calculated as follows: [[(anual volume, kg)  (1 x 109 mg/kg)]/[population  0.6
     365 days]], where population (10%, "eaters only") = 24  105 for the USA and 32  105 for
    Europe; 0.6 represents the assumption that only 60% of the flavour volume was reported in
    the survey (NAS, 1987; IOFI, 1996). Intake (mg/kg bw/day) calculated as follows: [(mg/day)/body
    weight], where body weight = 60 kg. Slight variation may occur from rounding off.
    3  (Relative molecular mass of the component alcohol/relative molecular mass of the ester) x
    (daily per capita intake "eaters only" of the ester)
    

    2.3.1.1  Terpenoid alcohols

        Following hydrolysis, the terpenoid alcohols undergo a complex
    pattern of alcohol oxidation, omega-oxidation, hydration, selective
    hydrogenation and subsequent conjugation to form oxygenated polar
    metabolites which are excreted primarily in the urine. Alternately,
    the corresponding carboxylic acids formed by oxidation of the alcohol
    function may enter the -oxidation pathway and yield shorter chain
    carboxylic acids which are completely metabolized to carbon dioxide
    (Williams, 1959). Geraniol, related terpenoid alcohols (citronellol
    and nerol), and the aldehydes (geranial and neral) exhibit similar
    pathways of metabolic detoxication in animals (Figure 1).

        Male rats were given single oral doses of 800 mg
    [1-3H]-geraniol/kg bw by gavage daily for 20 days. Five urinary
    metabolites were identified via two primary pathways. In one pathway,
    the alcohol is oxidized to yield geranic acid 
    (3,7-dimethyl-2,6-octadienedioic acid), which is subsequently hydrated
    to yield 3,7-dimethyl-3-hydroxy-6-octenoic acid. In a second pathway,
    the alcohol undergoes omega-oxidation mediated by liver cytochrome
    P-450 to yield 8-hydroxygeraniol. Selective oxidation at C-8 yields
    8-carboxygeraniol, which undergoes further oxidation to the principal
    urinary metabolite 3,7-dimethyl-2,6-octadienedioic acid
    ("Hildebrandt's acid") (Chadha & Madyastha, 1984) (see Figure 1). In
    rat microsomes, the C-8 methyl group of geraniol or nerol utilizes
    NADP+ and O2 and undergoes stereoselective omega-hydroxylation to
    yield the (E)-isomer of the corresponding diol (Licht & Corsia, 1978).
    In rats, the corresponding aldehyde, geranial and its (Z)-isomer,
    neral, are metabolized via similar alcohol and omega-oxidation
    pathways (Diliberto  et al., 1990).

        Geraniol, citronellol and rhodinol, the latter of which contains
    mainly (-)-citronellol, exhibit a similar metabolic fate in rabbits.
    Geraniol orally administered to rabbits by gavage is metabolized to
    3,7-dimethyl-2,6-octadienedioic acid ("Hildebrandt's acid") and
    3,7-dimethyl-2-octendioic acid ("reduced Hildebrandt's acid) which are
    excreted in the urine (Fischer & Bielig, 1940). In rabbits,
    (+)-citronellol is also metabolized to 3,7-dimethyl-2-octendioic acid
    ("reduced Hildebrandt's acid) (Asano & Yamakawa, 1950). An alcohol
    precursor to "reduced Hildebrandt's acid 
    (8-hydroxy-3,7-dimethyl-6-octenoic acid) has been reported as a
    urinary metabolite in rabbits given citronellol by gavage (Fischer &
    Bielig, 1940). The corresponding aldehyde citronellal undergoes
    omega-oxidation mediated by liver cytochrome P-450 (Chadha &
    Madyastha, 1982) to yield "reduced Hildebrandt's acid" (Ishida 
     et al., 1989). Geraniol, as the pyrophosphate ester, is endogenous
    in humans as an intermediate in the synthesis of cholesterol (Voet & 
    Voet, 1990).


    2.3.1.2  Aliphatic carboxylic acids

        With the exception of 2-ethylbutyric acid (see below), the
    carboxylic acids formed via hydrolysis of the 26 terpenoid esters in
    this group are endogenous in humans as intermediates either in the
    fatty acid or amino acid pathways (Voet & Voet, 1990). In general, the
    component linear saturated carboxylic acids (C1 - C6) participate in
    fatty acid -oxidation and the citric acid cycle, or the C1
    tetrahydrofolate pathway to eventually yield CO2 and H2O. The
    component branched chain carboxylic acids (isobutyric acid and
    isovaleric acid) formed during the oxidative deamination of the amino
    acids valine and leucine, respectively, undergo oxidation,
    preferentially in the longer branched chain, to yield linear
    carboxylic acid fragments, which participate in the fatty acid pathway
    and tricarboxylic acid cycle (Voet & Voet, 1990).

        The non-endogenous substance 2-ethylbutyric acid contains an ethyl
    substituent in the alpha position, which inhibits -oxidation and
    complete metabolism to CO2 (Williams, 1959). Pathways of metabolic
    detoxication include direct conjugation of the acid with glucuronic
    acid, or -oxidation followed by conjugation. In rats and rabbits, 2-
    ethylbutyric acid (Dziewiatowski  et al., 1949) and 2-ethyl-1-butanol
    (Kamil  et al., 1953) are excreted unchanged in the urine principally
    as the glucuronic acid conjugates. 2-Ethylbutyric acid is, in part,
    metabolized by dogs to 2-pentanone, which is presumably derived from
    -oxidation and subsequent decarboxylation of 2-ethyl--ketobutyric
    acid (Williams, 1959) (Figure 3).

    FIGURE 3


    2.3.2  Toxicological studies

    2.3.2.1  Acute toxicity

        Acute toxicity studies have been conducted for 23 of the 26
    terpenoid esters and are summarized in Table 3.

    2.3.2.2  Short-term toxicity

        The results of short-term toxicity studies with three terpenoid
    esters (geranyl acetate, citronellyl acetate and citronellyl
    isobutyrate) and two component alcohols (geraniol and citronellol) are
    described below and are summarized in Table 4.

     a) Mixture of geranyl acetate and citronellyl acetate

        Groups of five male and five female B6C3F1 mice were administered
    0, 125, 250, 500, 1000 or 2000 mg/kg bw per day of a mixture of
    geranyl acetate (71% ) and citronellyl acetate (29% ) in corn oil by
    gavage daily for 14 consecutive days. The animals were observed twice
    daily and weighed on day 1, after one week, and at the end of the
    study. All animals were necropsied at the end of the study. Three
    female mice that received 2000 mg/kg bw per day died during the study.
    All the other animals survived to the end of the study. Three of five
    females receiving 2000 mg/kg bw per day exhibited a thickening of the
    cardiac stomach and one of five males exhibited thickening of the
    duodenal wall (NTP, 1987).


        Table 3.  Acute toxicity studies of terpenoid esters

                                                                                                 

    Substance                 Species    Sex1      Route       LD50          Reference
                                                               (mg/kg bw)
                                                                                                 

    Citronellyl formate         rat      M & F     gavage      8400          Calandra, 1973
    Geranyl formate             rat        NR      gavage      5460          Weir & Wong, 1971
    Neryl formate               rat        NR      oral        5000          Moreno, 1975
    Rhodinyl formate            rat        NR      oral        5000          Moreno, 1974
    Citronellyl acetate         rat      M & F     gavage      6800          Calandra, 1973
    Geranyl acetate             rat      M & F     gavage      6330          Jenner, 1964
    Neryl acetate               rat      M & F     gavage      4550          Opdyke, 1974
    Rhodinyl acetate            rat      M & F     gavage      5000          Opdyke, 1974
    Citronellyl propionate      rat        NR      oral        5000          Moreno, 1973
    Geranyl propionate          rat        NR      oral        5000          Russell, 1973
    Neryl propionate            rat        NR      oral        5000          Moreno, 1975
    Rhodinyl propionate         rat        NR      oral        5000          Moreno, 1976
    Citronellyl butyrate        rat        M       oral        5000          Moreno, 1972
    Geranyl butyrate            rat      M & F     gavage      10660         Jenner, 1964
    Rhodinyl butyrate           rat        NR      oral        5000          Moreno, 1975
    Geranyl hexanoate           rat        NR      oral        5000          Moreno, 1975
    Citronellyl isobutyrate     rat        NR      oral        5000          Denine & Palanker, 
                                                                             1973
    Geranyl isobutyrate         rat        NR      oral        5000          Shelanski & Moldovan, 
                                                                             1973
    Neryl isobutyrate           rat        NR      oral        5000          Moreno, 1980
    Rhodinyl isobutyrate        rat        NR      oral        5000          Moreno, 1975
    Geranyl isovalerate         rat        NR      oral        5000          Levenstein, 1975
    Neryl isovalerate           rat        NR      oral        5000          Moreno, 1976
    Geranyl 2-ethylbutanoate    mouse      NR      oral        >8000         Givaudan Roure, 1971
                                                                                                 

    1 M = males; F = females; NR = Not reported
    

        In a 13-week study, a mixture of geranyl acetate (71%) and
    citronellyl acetate (29%) was administered by gavage in corn oil to
    six groups of B6C3F1 mice (10/sex/group) at dose levels of 0, 125,
    250, 500, 1000 or 2000 mg/kg bw daily (5 days/week). Animals were
    checked twice daily for signs of morbidity, and body weight data were
    collected weekly. Histopathological examinations were performed on
    animals in the vehicle control group, animals in the 2000 mg/kg bw per
    day group, and on animals dying during the test. Seven of 10 males and
    9/10 females receiving 2000 mg/kg bw per day died during the study.
    Gavage errors resulted in the death of three females at lower dose
    levels. Mean body weights were comparable for dosed and control
    animals. Male and female mice in the 2000 mg/kg bw per day dose groups
    exhibited cytoplasmic vacuolization of the liver, kidney and
    myocardium. Vacuolization was the result of lipid droplets that were
    present throughout the liver lobule, particularly in the periportal
    region. No treatment-related histopathological lesions or other
    effects were observed in the 1000 mg/kg bw per day group (NTP, 1987).

        Groups of five male and five female F344/N rats were administered
    0, 62, 125, 250, 500 or 1000 mg/kg bw of a mixture of geranyl acetate
    (71%) and citronellyl acetate (29%) in corn oil by gavage daily for 14
    consecutive days. The animals were observed twice daily and weighed on
    day 1, after one week, and at the end of the study. All animals were
    necropsied at the end of the study. All animals survived to the end of
    the dosing period and no compound-related effects were observed at
    necropsy (NTP, 1987).

        In a 13-week study, a mixture of geranyl acetate (71%) and
    citronellyl acetate (29%) was administered by gavage in corn oil to
    six groups of F344/N rats (10/sex/group) at dose levels of 0, 250,
    500, 1000, 2000 or 4000 mg/kg bw daily (5 days/week). Animals were
    checked twice daily for signs of morbidity, and body weight data were
    collected weekly. Histopathological examinations were performed on
    animals in the vehicle control group, animals in the 2000 mg/kg bw per
    day group, and in animals dying during the test. Two out of ten males
    and one out of ten females receiving 4000 mg/kg bw per day died. Mean
    body weights were comparable for dosed and control animals, except for
    a decrease in mean body weight gain in males and females (19 % and 8%
    relative to controls, respectively) at the 4000 mg/kg bw per day dose
    level. No treatment-related histopathological effects were observed at
    necropsy (NTP, 1987).


        Table 4.  Short- and long-term toxicity studies for terpenoid esters, component terpenoid alcohols and 
    component aliphatic saturated carboxylic acids

                                                                                                        

    Substance                Species, sex1    Route   Duration     Repeated dose   Reference
                                                                   study, NOEL2
                                                                   (mg/kg bw
                                                                   per day)
                                                                                                        

    Terpenoid esters

    Geranyl acetate3         mouse, M & F     oral    13 weeks         1000        NTP, 1987
                             rat, M & F       oral    13 weeks         2000        NTP, 1987
                             rat, M & F       oral    17 weeks         5005        Hagan et al., 1967
    Citronellyl isobutyrate  rat, M & F       oral    90 days          14.75       Damske, 1980

    Component terpenoid
    alcohols

    Geraniol                 rat, M & F       oral    16 weeks         5005        Hagan et al., 1967
                             rat, M & F       oral    27-28 weeks      505         Hagan et al., 1967
    Citronellol4             rat, M & F       oral    12 weeks         505         Oser, 1967
                                                                                                        

    1 M = male; F = female.
    2 NOEL = No-observed-effect level.
    3 The test material was composed of 79% geranyl acetate and 29% citronellyl acetate.
    4 The test material was an equal mixture of citronellol and geraniol.
    5 The study was performed at a single dose level or multiple dose levels, none of which produced
    adverse effects. The reported dose level is, therefore, not an actual NOEL, but the highest level
    that produced no adverse effects.
    

     b) Geranyl acetate

        Groups of ten male and ten female Osborne-Mendel rats were
    provided geranyl acetate in the diet at concentrations of 0, 1000,
    2500 or 10 000 mg/kg (equivalent to an average daily intake of 0, 50,
    250 or 500 mg/kg bw) for 17 weeks. The diet was prepared weekly.
    Determination of the dietary concentration of geranyl acetate revealed
    a weekly loss of 4%. Measurement of body weight and food intake
    recorded weekly showed no significant difference between test and
    control animals at any intake level. At termination, haematological
    examinations revealed no difference from controls. At necropsy, no
    differences were reported between major organ weights of test and
    control animals. Gross examination of tissue of all animals was
    unremarkable and histopathological examination of 6-8 animals, equally
    represented by gender, for the high-dose group and the control group
    revealed no treatment-related lesions. The NOEL was 500 mg/kg bw per
    day (Hagan  et al., 1967).

     c) Citronellyl isobutyrate

        Citronellyl isobutyrate, prepared as a spray-dried material on gum
    arabic, was provided in the diet to a group of 24 male and female
    Charles River rats for 90 days at a level calculated to result in the
    average daily intake of 14.7 mg/kg bw. Measurements of body weight and
    food intake performed on a weekly basis showed no significant
    differences between test and control animals. Blood chemical
    determinations and urinalysis conducted at weeks 6 and 12 revealed
    normal values. At week 12, a significant decrease was recorded for
    haemoglobin and haematocrit values in males. Organ weights at necropsy
    revealed an increased relative heart weight in males, but there was no
    significant difference in absolute heart weight between test and
    control males. Gross examination and histopathology revealed no
    treatment-related effects (Damske  et al., 1980).

     d) Component alcohols

     i) Geraniol

        The component alcohol geraniol, formed by hydrolysis of the
    corresponding geranyl esters, was provided in the diet to groups of
    five male and five female Osborne-Mendel rats for 16 weeks as a
    mixture of 3,7-dimethyl-2,6-octadienol and 3,7-dimethyl-1,6-octadienol
    (no further details) at a dietary concentration of 10 000 mg/kg
    (equivalent to an average daily intake of 500 mg/kg bw). A second
    study was conducted for 27-28 weeks using a dietary concentration of
    1000 mg/kg (equivalent to an average daily intake of 50 mg/kg bw).
    Measurement of body weight and food intake recorded weekly showed no
    significant difference between test and control animals in either
    study. At termination, haematological examinations revealed normal
    values. At necropsy, no differences were reported between major organ
    weights of test and control animals. Gross examination of the tissue
    of all animals was unremarkable. Histopathological examination of 6-8


    animals, equally represented by gender, revealed no treatment-related
    lesions (Hagan  et al., 1967).

     ii) Citronellol

        The parent alcohol citronellol, as a mixture with an equal weight
    of linalool, was provided in the diet to groups of 10 male and 10
    female weanling rats (strain not specified) at a level calculated to
    result in the average daily intake of 50 mg/kg bw for a period of 12
    weeks. A significant depression in body weight and food intake in male
    rats was attributed to the impalatability of the test materials at the
    level administered. The efficiency of food utilization showed no
    significant difference between test and control animals. There were no
    significant changes in appearance, urinalysis parameters, blood
    haemoglobin, liver and kidney weights, or gross pathology (Oser,
    1967).

    2.3.2.3  Long-term toxicity/carcinogenicity studies

     a) Mice

        A carcinogenicity study was conducted in which groups of 50
    B6C3F1 mice of each sex were administered 0, 500 or 1000 mg/kg bw of
    a mixture of geranyl acetate (71%) and citronellyl acetate (29%) in
    corn oil by gavage daily, 5 days/week for 103 weeks. Body weights were
    recorded weekly for the first 12 weeks and monthly thereafter.
    Necropsies were performed on all animals at termination and those
    found dead during the study.

        Mean body weights of high-dose male and female mice were lower
    than those of control groups after week 18 of the study. Survival of
    male mice in the high-dose group was significantly reduced (controls,
    31/50; low dose 32/50; high dose, 0/50). Survival of the high- and
    low-dose groups of female mice was significantly less (p<0.001; low
    dose, 0.020) than that of the control group (controls, 28/50; low
    dose, 15/50; high dose, 0/50). The mortality in females appeared
    already from week 15 onwards. The probable cause of death of many
    females was a genital tract infection. Inflammation of the vagina,
    uterus, ovaries, or multiple organs occurred in 18 control, 14
    low-dose, and 2 high-dose female mice. Although the etiological agent
    was not isolated,  Klebsiella pneumoniae was isolated from similarly
    affected mice at this laboratory in subsequent chronic studies.
    Surviving male (36) and female (11) mice in the high-dose groups were
    killed in a moribund condition at week 91 after an inadvertent
    overdose of the test substance. Eleven other animals (3 control males,
    3 low-dose males, 3 low-dose females and 2 high-dose females) were
    killed by gavage accidents during the course of the study.

        There was no increase in the incidence of neoplastic lesions
    associated with administration of the test substance. The incidence of
    non-neoplastic lesions was significantly increased in high-dose male
    and female mice only, and there was an increased incidence of


    cytoplasmic vacuolization of the liver in male (control, 1/50; low
    dose, 7/50; high dose, 47/50) and female mice (control, 1/50; low
    dose, 27/50; high dose, 46/50) and the kidney or kidney tubule in male
    (control, 0/50; low dose, 0/50; high dose, 41/50) and female mice
    (control, 0/50; low dose, 24/49; high dose, 37/50). Under conditions
    of this study, the mixture of geranyl acetate and citronellyl acetate
    was not carcinogenic for either sex of B6C3F1 mice (NTP, 1987).

        Owing to the low survival rate in both sexes and the early
    mortality predominantly in female mice, this study should be
    considered as having limited value for a safety evaluation.

     b) Rats

        A carcinogenicity study was conducted in which groups of 50 F344/N
    rats of each sex were administered 0, 1000 or 2000 mg/kg bw of a
    mixture of geranyl acetate (71%) and citronellyl acetate (29%) in corn
    oil by gavage daily, 5 days/week for 103 weeks (2000 mg/kg bw
    corresponds to estimated daily dose levels of 1420 mg geranyl
    acetate/kg bw and 580 mg citronellyl acetate/kg bw). Body weights were
    recorded weekly for the first 12 weeks and monthly thereafter.
    Necropsies were performed on all animals at termination and on those
    found dead during the study.

        A statistically significant decrease in mean body weight was
    reported for high-dose male rats throughout the study and low- and
    high-dose female rats after week 40. Reduced mean body weight gains
    were dose-related. Survival of the high-dose group (18/50) of male
    rats was significantly less than those of the controls (34/50;
    p=0.001) and the low-dose group (29/50; p=0.003). There was no other
    significant difference in survival between any group of either sex.
    A positive trend (controls, 6/50; low dose, 8/50; high dose, 9/50) in
    the incidence of adrenal pheochromocytomas in male rats was not
    statistically significant. There was no significant increase in the
    incidence of any neoplasms in high-dose male or female rats compared
    to the control groups. The overall incidence of these commonly
    observed adrenal pheochromocytomas in paired control groups of male
    rats has been reported to be 25.1% (Haseman  et al., 1986).
    Under conditions of this study, geranyl acetate was not carcinogenic
    for either sex of F344/N rats. The NOEL based on body weight decreases
    was 1000 mg/kg bw per day (NTP, 1987).

        The toxicity of the component linear and branched-chain carboxylic
    acid is discussed in the monographs on  saturated 
     aliphatic  acyclic linear primary alcohols aldehydes and acids and
    on  saturated aliphatic acyclic branched-chain primary alcohols, 
     aldehydes and acids.

    2.3.2.4  Genotoxicity

        Results of  in vitro and  in vivo genotoxicity studies carried
    out on terpenoid esters, mainly geranyl acetate, are given in Table 5.


        Table 5.  Genotoxicity studies for terpenoid esters

                                                                                                                                  
    Test system          Substance name       Test object              Test concentration1    Results       Reference
                                                                                                                                  

    In vitro                                                                                                

    Ames test            Geranyl acetate      S. typhimurium TA98,       
                                              TA100, TA1535, TA1537,     2000 nl/plate2       negative3     Heck et al., 1989
                                              TA1538
    Ames test            Geranyl acetate      S. typhimurium TA98,
                                              TA100, TA1535, TA1537,     3333 g/plate        negative3     Mortelmans et al., 1986
                                              TA1538
    Rec assay            Geranyl acetate      B. subtilis                17 g/disk4          negative      Oda et al., 1978
    Rec assay            Geranyl acetate      B. subtilis                20 g/disk           negative      Yoo, 1986
    Rec assay            Geranyl formate      B. subtilis                18 g/disk4          negative      Oda et al., 1978
    HGPRT gene
    mutation assay       Geranyl acetate      CHO cells                  no details           negative3     Flowers & Li, 1988
    Gene mutation assay  Geranyl acetate      Human lymphoblast TK6      320 g/ml            negative5     Caspary et al., 1988
                                                                         500 g/ml            negative6
    Chromosomal
    aberrations          Geranyl acetate      Chinese hamster
                                              ovary cells                100 g/ml            negative5     Galloway et al., 1987;
                                                                         150 g/ml            negative6     Tennant et al., 1987
    Sister chromatid     Geranyl acetate      Chinese hamster
    exchange                                  ovary cells                 70 g/ml            positive5,7   Galloway et al., 1987;
                                                                         299 g/ml            positive6,7   Tennant et al., 1987
    Unscheduled DNA
    synthesis            Geranyl acetate      Rat primary hepatocytes    no details           negative      Mirsalis et al., 1983
    Unscheduled DNA
    synthesis            Geranyl acetate      Rat primary hepatocytes    100 nl/ml            negative      Heck et al., 1989
    Alkaline elution     Geranyl acetate      Rat primary hepatocytes    0.30 mM              negative      Storer et al., 1996
    PACE/PFGE8           Geranyl acetate      Rat primary hepatocytes    0.25 mM              positive9     Storer et al., 1996
                                                                                                                                  


    Table 5.  Continued...

                                                                                                                                  
    Test system          Substance name       Test object              Test concentration1    Results       Reference
                                                                                                                                  

    In vivo

    Ses linked 
    recessive lethal
    assay                Geranyl acetate      Drosophila melanogaster  feeding 250 ppm;       negative      Foureman et al., 1994
                                                                       injection 50 000 ppm   negative
    Micronucleus assay   Geranyl acetate      Marrow B6C3F1 mouse      1800 mg/kg bw/ day     negative      Shelby et al., 1993;
                                              bone cells               for 3 days, ip                       Shelby & Witt, 1995
    Chromosomal          Geranyl acetate      Marrow B6C3F1 mouse      no details, ip         negative      Shelby & Witt, 1995
    aberrations                               bone cells
    Unscheduled DNA
    synthesis            Geranyl acetate      Fisher F344 male rats    no details             negative      Mirsalis et al., 1983
                                                                                                                                  

    1 Highest ineffective dose (negative) or lowest affective dose (positive).
    2 Units based on information in Table 3.
    3 Both with and without metabolic activation.
    4 Single dose.
    5 Without metabolic activation.
    6 With metabolic activation.
    7 Positive results were obtained at concentrations that were cytotoxic.
    8 Programmed, autonomously-controlled electrode/pulsed-field gel electrophoresis.
    9 DNA fragmentation greater than that seen for 40 Gy of gamma radiation.
    
    2.3.2.5  Reproductive toxicity

        No studies on reproduction and teratogenicity have been reported.

    2.3.2.6  Other relevant studies

     a) Citronellyl acetate

        Citronellyl acetate applied full strength to intact and abraded
    rabbit skin was irritating (Calandra, 1973).

     b) Neryl acetate

        Neryl acetate applied full strength to intact and abraded rabbit
    skin for 24 hours under occlusion was not irritating (Opdyke, 1974).

     c) Rhodinyl acetate

        Rhodinyl acetate applied full strength to intact and abraded skin
    for 24 h under occlusion was mildly irritating (Opdyke, 1974).

     d) Neryl isobutyrate

        As part of an acute dermal LD50 study, the undiluted material
    produced slight irritant effects in rabbit patches tested for 24 h
    under occlusion at a dose of 5 g/kg (Moreno, 1980).

    2.4  Observations in humans

    2.4.1  Geranyl acetate

        A maximization test was carried out on 25 volunteers. Geranyl
    acetate was tested at a concentration of 4% in petrolatum and produced
    no sensitization reactions. Hypersensitivity occurred in some
    individuals (Opdyke, 1974).

    2.4.2  Neryl acetate

        A maximization test was carried out on 25 volunteers. Neryl
    acetate was tested at a concentration of 10% in petrolatum and
    produced no senstitization reactions (Kligman, 1972).

        When tested at 10% in petrolatum neryl acetate produced no
    irritation in a 48-h closed patch test on human subjects (Opdyke,
    1974).

    2.4.3  Citronellyl acetate

        When tested at a concentration of 4% in petrolatum, citronellyl
    acetate produced a mild irritation in a 48-h closed patch test in 25
    human subjects (Opdyke, 1974).


    2.4.4  Rhodinyl acetate

        When tested at 12% in petrolatum, rhodinyl acetate produced no
    irritation in a 48-h closed patch test on human subjects (Kligman,
    1974).

        A maximization test was carried out on 25 volunteers. The material
    was tested at 12% concentration in petrolatum and produced no
    sensitization reactions (Opdyke, 1974).

    2.4.5  Neryl isobutyrate

        A 48-h closed patch test at a concentration of 5% in petrolatum on
    the backs of 35 volunteers produced no irritation (Epstein, 1980).

        A maximization test was carried out on 35 volunteers. The material
    was tested at a concentration of 5% in petrolatum and produced no
    sensitization reactions (Epstein, 1980).

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    Moreno, O.M. (1980) Neryl isobutyrate. Cited in Ford, R.A., Letizia,
    C., & Api, A.M. (1988) Monographs on fragrance raw materials.  Food 
     Chem. Toxicol. , 26(4): 273-415.

    Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., &
    Zeiger, E. (1986) Salmonella mutagenicity tests: II. Results from the
    testing of 270 chemicals.  Environ. Mutagen., 8(7): 1-119.

    Munro, I.C., Ford, R.A., Kennepohl, E., & Spenger, J.G. (1996)
    Correlation of structural class with no-observed-effect levels: a
    proposal for establishing a threshold of concern.  Food Chem. 
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    Myhr, B.C. & Caspary, W.J. (1991) Chemical mutagenesis at the
    thymidine kinase locus in L5178Y mouse lymphoma cells: results for 31
    coded compounds in the National Toxicology Program.  Environ. Mol. 
     Mutagen., 18: 51-83.

    National Academy of Sciences (1987) Evaluating the safety of food
    chemicals. Washington, DC.

    National Toxicology Program (NTP) (1987) Carcinogenesis studies of
    food grade geranyl acetate (71%) and citronellyl acetate (29%).
    Research Triangle Park, NC, USA (NTP-TR-252; PB-88-2508).

    Oda, Y., Hamono, Y., Inoue, K., Yamamoto, H., Niihara, T., & Kunita,
    N. (1978) Mutagenicity of food flavors in bacteria.  Shokuhin Eisei 
     Hen, 9: 177-181.

    Opdyke, D.L.J. (1974) Priate communication to FEMA.

    Oser, B.L. (1967) Private communication to FEMA.

    Phillips, J.C. Kingsnorth, J., Gangolli, S.D., & Gaunt, I.F. (1976)
    Studies on the absorption, distribution and excretion of citral in the
    rat and the mouse.  Food  Chem. Toxicol., 14: 537-540.

    Russell, T. (1973) Unpublished report to FEMA.

    Shelby, M.D., Erexson, G.L., Hook, G.L., & Tice, R.R. (1993)
    Evaluation of a three-exposure mouse bone marrow micronucleus
    protocol: Results with 49 chemicals.  Environ. Mol. Mutagen., 21(2):
    160-179.

    Shelanski, M.V. & Moldovan, M. (1973) Unpublished report to RIVM.


    Shelby, M.D. & Witt, K.L. (1995) Comparison of results from mouse bone
    marrow chromosome aberration and micronucleus tests.  Environ. Mol. 
     Mutagen., 25: 302-313.

    Stanford, L.F. Jr., Aaron, C.S., Tuman, W.G., Godeh, E.G., Matthews,
    R.J., & Naismith, R.W. (1988a) Detection of mammalian cell mutagenesis
    in AS52 cells.  EMS Abstr., 101: 246.

    Stanford, L.F. Jnr., Godeh, E.G., Tuman, W.G., Biesczad, M.J., Miller,
    A, Stec, E.E., Matthews, R.J., & Naismith, R.W. (1988b) Evaluation of
    eight coded noncarcinogens in the CHO/WPRT and AS52/XPRT assays. 
     EMS Abstr., 101: 247.

    Stofberg, J. & Grundschober, F. (1987) Consumption ratio and food
    predominance of flavoring materials.  Perfum. Flavor., 12: 27.

    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.

    Storer, R.D., McKelvey, T.M., Kraynak, A.R., Elia, M.C., Barnum, J.E.,
    Harmon, L.S., Nichols, W.W., & DeLuca, J.G. (1996) Revalidation of the
     in vitro alkaline elution/rat hepatocyte assay for DNA damage:
    improved criteria for assessment of cytotoxicity and genotoxicity and
    results for 81 compounds.  Mutat. Res., 368: 59-101.

    Tennant, R.W., Margolin, B.H., Shelby, M.D., Zeiger, E., Haseman,
    J.K., Spalding, J., Caspary, W., Resnick, M., & Stasiewicz, S. (1987)
    Prediction of chemical carinogenicity in rodents from  in vitro 
    genetic toxicity assays.  Science, 236: 933-941. 

    Uno, Y., Takasawa, H., Miyagawa, M., Inoue, Y., Murata, T., &
    Yoshikawa, K. (1994) An  in vivo-in vitro replicative DNA sythesis
    (RDS) test using rat hepatocytes as an early prediction assay for
    non-genotoxic hepatocarcinogens screening of 22 known positives and 25
    noncarcinogens.  Mutat. Res., 320: 189-205.

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

    Weir, R.J. & Wong, L.C.K. (1971)  Private communication to FEMA.

    Williams, R.T. (1959) Detoxication mechanisms, 2nd ed.Chapman and Hall
    Ltd, London, pp. 88-113.

    Yoo, Y.S. (1986) Mutagenic and antimutagenic activities of flavoring
    agents used in foodstuffs . J. Osaka City Med. Center, 34(3-4):
    267-288.

    Yoshikawa, K. (1996) Anomalous nonidentity between  Salmonella 
    genotoxicants and rodent carcinogens: nongenotoxic carcinogens and
    genotoxic noncarcinogens.  Environ. Health Perspect., 104: 1, 40-46.

    


    See Also:
       Toxicological Abbreviations