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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 OF ALIPHATIC ACYCLIC PRIMARY ALCOHOLS WITH BRANCHED-CHAIN
    ALIPHATIC ACYCLIC ACIDS

    First draft prepared by
    Dr G. Semino,
    Institute of Pharmacological Sciences
    University of Milan
    Milan, Italy

        1.  Evaluation
            1.1 Introduction
            1.2 Estimated daily  per capita intake
            1.3 Absorption, metabolism and elimination
            1.4 Application of the procedure for the safety evaluation of
                flavouring agents
            1.5 Consideration of combined intakes
            1.6 Conclusions
        2.  Relevant background information
            2.1 Explanation
            2.2 Biological data
                2.2.1   Absorption, distribution and excretion
                2.2.2   Biotransformations
                    2.2.2.1 Branched-chain aliphatic acids
                    2.2.2.2 Aliphatic linear alcohols
                2.2.3   Toxicological studies
                    2.2.3.1 Acute toxicity
                    2.2.3.2 Short-term toxicity studies
                    2.2.3.3 Long-term toxicity/carcinogenicity studies
                    2.2.3.4 Genotoxicity studies
                    2.2.3.5 Reproductive toxicity studies
        3.  References

    1.  EVALUATION

    1.1  Introduction

        The Committee evaluated a group of 32 flavouring agents that
    includes selected esters of aliphatic acyclic primary alcohols with
    branched-chain aliphatic acyclic acids using the Procedure for the
    Safety Evaluation of Flavouring Agents (the "Procedure") (see Figure 1
    and Table 1).



        Table 1.  Summary of results of the safety evaluations of esters of aliphatic acyclic primary alcohols and branched-chain aliphatic
    acyclic 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 person per day
      Step 2: All substances in this group are metabolized to innocuous products

                                                                                                                                            
    No.     Substance                       Step A3                      Step A4            Comments                   Conclusion based on
                                       Does intake exceed             Endogenous or                                    current levels of
                                       intake threshold?              metabolized to                                   intake
                                       (g/person per day)       endogenous substances?1
                                                                                                                                            

    0185    Methyl isobutyrate                 No                         N/R                                          No safety concern
                                       USA: 270  Europe: 23
    0186    Ethyl isobutyrate                  No                         N/R                                          No safety concern
                                       USA: 470  Europe: 750
    0187    Propyl isobutyrate                 No                         N/R                                          No safety concern
                                       USA: .08  Europe: 15
    0188    Butyl isobutyrate                  No                         N/R                                          No safety concern
                                       USA: 1.9  Europe: 2.7
    0189    Hexyl isobutyrate                  No                         N/R                                          No safety concern
                                       USA: 57   Europe: 3.00
    0190    Heptyl isobutyrate                 No                         N/R                                          No safety concern
                                       USA: 3.0  Europe: 0.00
    0191    trans-3-Heptenyl 2-
            methylpropanoate                   No                         N/R                                          No safety concern
                                       USA: 2.3  Europe: 0.01
    0192    Octyl isobutyrate                  No                         N/R                                          No safety concern
                                       USA: 5.0  Europe: 11
    0193    Dodecyl isobutyrate                No                         N/R                                          No safety concern
                                       USA: 0.76 Europe: 50
    0194    Isobutyl isobutyrate               No                         N/R                                          No safety concern
                                       USA: 2.3  Europe: 65
    0195    Methyl isovalerate                 No                         N/R                                          No safety concern
                                       USA: 110  Europe: 7.8
    0196    Ethyl isovalerate                  No                         N/R                                          No safety concern
                                       USA: 540  Europe: 760

    Table 1.  Continued...

                                                                                                                                            
    No.     Substance                       Step A3                      Step A4            Comments                   Conclusion based on
                                       Does intake exceed             Endogenous or                                    current levels of
                                       intake threshold?              metabolized to                                   intake
                                       (g/person per day)       endogenous substances?1
                                                                                                                                            

    0197    Propyl isovalerate                 No                         N/R                                          No safety concern
                                       USA: 0.10 Europe: 2.00
    0198    Butyl isovaletate                  No                         N/R                                          No safety concern
                                       USA: 500  Europe: 94
    0199    Hexyl 3-methylbutanoate            No                         N/R                                          No safety concern
                                       USA: 3.1  Europe: 2.3
    0200    Octyl isovalerate                  No                         N/R                                          No safety concern
                                       USA: 0.57 Europe: 7.3
    0201    Nonyl isovalerate                  No                         N/R                                          No safety concern
                                       USA: 0.08 Europe: 0.01
    0202    3-Hexenyl 3-
            methylbutanoate                    No                         N/R                                          No safety concern
                                       USA: 30   Europe: 9.4
    0203    2-Methylpropyl 3-
            methylbutyrate                     No                         N/R                                          No safety concern
                                       USA: 130  Europe: 78
    0204    2-Methylbutyl 3-
            methylbutanoate                    No                         N/R                                          No safety concern
                                       USA: 0.95 Europe: 0.86
    0205    Methyl 2-methylbutyrate            No                         N/R                                          No safety concern
                                       USA: 69   Europe: 390
    0206    Ethyl 2-methylbutyrate            Yes                         Yes        The components ethyl alcohol      No safety concern
                                       USA: 560  Europe: 2200                        and 2-methylbutyric acid are
                                                                                     endogenous. The acid is as
                                                                                     an intermediate in the 
                                                                                     metabolism of the amino acid
                                                                                     isoleucine (Voet & Voet, 1990)
    0207    n-Butyl 2-methylbutyrate           No                         N/R                                          No safety concern
                                       USA: 0.02 Europe: 26
    0208    Hexyl 2-methylbutanoate            No                         N/R                                          No safety concern
                                       USA: 8.6  Europe: 4.9

    Table 1.  Continued...

                                                                                                                                            
    No.     Substance                       Step A3                      Step A4            Comments                   Conclusion based on
                                       Does intake exceed             Endogenous or                                    current levels of
                                       intake threshold?              metabolized to                                   intake
                                       (g/person per day)       endogenous substances?1
                                                                                                                                            

    0209    Octyl 2-methylbutyrate             No                         N/R                                          No safety concern
                                       USA: 0.10 Europe: 0.01
    0210    Isopropyl 2-methylbutyrate         No                         N/R                                          No safety concern
                                       USA: 0.10 Europe: 4.9
    0211    3-Hexenyl 2-
            methylbutanoate                    No                         N/R                                          No safety concern
                                       USA: 8.8  Europe: 5
    0212    2-Methylbutyl 2-
            methylbutyrate                     No                         N/R                                          No safety concern
                                       USA: 0.04 Europe: 3.6
    0213    Methyl 2-methylpentanoate          No                         N/R                                          No safety concern
                                       USA: 0.02 Europe: 0.17
    0214    Ethyl 2-methylpentaoate            No                         N/R                                          No safety concern
                                       USA: 320  Europe: 7.6
    0215    Ethyl 3-methylpentanoate           No                         N/R                                          No safety concern
                                       USA: 5.90 Europe: 0.31
    0216    Methyl 4-methylvalerate            No                         N/R                                          No safety concern
                                       USA: 0.10 Europe: 0.03
                                                                                                                                            

    1 N/R: Not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3
    of the Procedure
    


        The Committee had previously evaluated one member of the group,
    ethyl isovalerate, at its eleventh meeting (Annex 1, reference 14).
    Because of a lack of data, the Committee was unable to allocate an ADI
    to ethyl isovalerate.

    1.2  Estimated daily  per capita intake

        The total annual production volume of the 32 esters of aliphatic
    acyclic primary alcohols with branched-chain aliphatic acyclic acids
    from their use as flavouring substances is approximately 16 tonnes in
    the USA (NAS, 1987) and 32 tonnes in Europe (IOFI, 1995). In the USA,
    approximately 67% of the total annual volume (NAS, 1987) is accounted
    for by four esters: ethyl isobutyrate, ethyl isovalerate, butyl
    isovalerate and ethyl 2-methylbutyrate. In Europe, more than 90% of
    the total annual volume (IOFI, 1995) is accounted for by four esters:
    ethyl isobutyrate, ethyl isovalerate, ethyl 2-methylbutyrate and
    methyl 2-methylbutyrate. In the unlikely event that all of the
    substances in this group were simultaneously consumed on a daily
    basis, the estimated total daily  per capita intake is 3 mg per
    person per day in the USA and 4.6 mg/person per day in Europe. The
    daily  per capita intake of the branched-chain acids (i.e. isobutyric
    acid, isovaleric acid, and 2-methylbutyric acid) formed via hydrolysis
    of these esters is 2 mg/person per day in the USA and 3.1 mg/person
    per day in Europe.

        Esters of aliphatic acyclic primary alcohols and branched-chain
    aliphatic acyclic acids have been detected in a wide variety of foods.
    Quantitative data on the natural occurrence of these esters have been
    reported for seven substances in the group and corresponds to a total
    of 14 tonnes per year (CIVO-TNO, 1994). This estimate is approximately
    equal to the estimated intake from their use as flavouring substances.
    In the USA, the consumption of isobutyrate esters from natural
    occurrence in food is equivalent to their use as flavouring substances
    (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987). The
    consumption of isovalerate esters and 2-methylbutyrate esters from
    natural food sources is several orders of magnitude higher than from
    their use as flavouring agents.

    1.3  Absorption, metabolism and elimination

        It is expected that the esters in this group will be readily
    hydrolysed to their component alcohols and carboxylic acids in the
    intestinal tract, blood and liver. The metabolism of the hydrolysis
    products is discussed in the introduction to this chapter on
    flavouring agents.

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

        Step 1. All of the 32 esters of aliphatic acyclic primary alcohols
    and branched-chain aliphatic acyclic acids were classified in
    structural class I.

        Step 2. At current levels of intake (see Table 1) these esters
    would not be expected to saturate the metabolic pathways, and all the
    compounds were predicted to be metabolized to innocuous products.

        Step A3. All but one of the 32 esters of aliphatic acyclic primary
    alcohols with branched-chain aliphatic acyclic acids have USA and
    European estimated daily  per capita intakes (see Table 1) that fall
    below the human intake threshold for class I (1800 g/person per day),
    indicating that they pose no safety concern when used at current
    levels of estimated intake as flavouring agents. Only ethyl
    2-methylbutyrate has an estimated intake greater than 1800 g/person
    per day, which is 2200 g/person per day in Europe.

        Step A4. Ethyl 2-methylbutyrate is expected to be hydrolysed to
    ethyl alcohol and 2-methylbutyric acid, which is endogenous.
    Therefore, this substance was determined to be of no safety concern
    based on its structural class and known metabolism.

    1.5  Consideration of combined intakes

        The stepwise evaluations of the 32 esters of aliphatic acyclic
    primary alcohols with branched-chain aliphatic acyclic acids used as
    flavouring substances are summarized in Table 1.

        In the unlikely event that all 32 esters of aliphatic acyclic
    primary alcohols with branched-chain aliphatic acyclic acids would be
    consumed simultaneously on a daily basis, the estimated combined
    intake will exceed the human intake threshold for class I. Since all
    the 32 substances in this group are expected to be efficiently
    metabolized, the combined intake level is not expected to saturate
    metabolic pathways. On this basis of the evaluation of the collective
    data, the Committee concluded that there were no safety concerns from
    combined intake.

    1.6  Conclusions

        The Committee concluded that the substances in this group would
    not present safety concerns at currently estimated levels of intake.

        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.


    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Explanation

        The purpose of this monograph is to provide a safety evaluation of
    a group of aliphatic acyclic esters formed from simple saturated
    aliphatic branched-chain acids and linear and branched-chain aliphatic
    alcohols using the safety evaluation procedure for flavouring

    substances considered at the forty-fourth meeting and modified at the
    forty-fifth meeting of JECFA. JECFA has previously evaluated one
    member of the group, ethyl  iso-valerate, at the JECFA meeting in
    1967. Because of lack of data, the committee was unable to assign an
    ADI for ethyl  iso-valerate (Annex 1, reference 14).

        This monograph summarizes the key data relevant to the safety
    evaluation of 32 esters formed from the esterification of the
    saturated aliphatic acyclic branched-chain acids, isobutyric acid,
    isovaleric acid, 2-methylbutyric acid, and 2-, 3-, and
    4-methylpentanoic acids with 9 aliphatic acyclic linear primary
    alcohols (i.e., methanol, ethanol, propanol, butanol, hexanol,
    heptanol, octanol, nonanol, and dodecyl alcohol), 2 unsaturated linear
    primary alcohols (i.e., 3-hexenol and 3-heptenol), and 3 simple
    aliphatic branched-chain alcohols (i.e., isopropyl alcohol, isobutyl
    alcohol and 2-methylbutyl alcohol).

    2.2  Biological data

    2.2.1  Absorption, distribution and excretion

        In general, aliphatic esters are rapidly hydrolysed to their
    component alcohols and carboxylic acids (see Fig. 1). Hydrolysis is
    catalysed by classes of enzymes recognized as carboxylesterases or
    esterases. In mammals, these enzymes occur in most tissues throughout
    the body (Heymann, 1980; Anders, 1989) but predominate in the
    hepatocytes (Heymann, 1980). Select isoenzymes exhibit an increase in
    enzyme binding (lower Km) and maximum velocity (Vmax) as the carbon
    chain length of either the alcohol or carboxylic acid component of the
    substrate increases (Heymann, 1980).

    FIGURE 1

        Branched-chain esters in this group are hydrolysed  in vitro to
    their corresponding branched-chain acids (isobutyric acid, isovaleric
    acid or 2-methylbutyric acid) and corresponding aliphatic acyclic
    alcohols. Table 2 summarizes the hydrolysis data for the five esters
    containing a branched-chain alcohol and/or branched-chain acid (i.e.,
    ethyl  iso-valerate,  iso-amyl caproate, benzyl  iso-butyrate,
     iso-amyl butyrate and  iso-amyl  iso-valerate). Following
    hydrolysis, short-chain (<C6) branched-chain acids and alcohols
    are rapidly absorbed from the gastrointestinal tract (Dawson  et 
     al., 1964; Gaillard & Derache, 1965).



        Table 2. In vitro ester hydrolysis data

                                                                                                                      
    Ester                         Artificial         Artificial           Rat liver       Rat small     % hydrolysis
                                  gastric juice1     pancreatic juice1    preparation1    intestinal    after 2 hours
                                  t0.52 (min)        t0.5 (min)           t0.5 (sec)      mucosa1       
                                                                                          t0.5 (sec)
                                                                                                                      

    Ethyl iso-valerate               1390                  198               2.35          133            63; 344
    Isoamyl caproate7                146                   37.8              NR            NR             NR
    Benzyl isobutyrate7              577                   17.8              0.0422        0.0707         NR
    Isoamyl butyrate7                660                   11.3              0.492         0.0713         123; 1004
    Isoamyl isovalerate7             295                   10.2              NR            NR             NR
    Benzyl 2-methylbutanoate7        NR                    NR                NR            NR             1005
    Isoamyl acetate7                 NR                    NR                NR            NR             205; 1006
                                                                                                                      

    1  Longland et al., 1977
    2  t1/2 = Half-life
    3  In artificial gastric juice. Gangolli & Shilling, 1968
    4  In artificial pancreatic juice. Gangolli & Shilling, 1968
    5  By pancreatin. Leegwater & van Straten, 1974.
    6  In whole homogenate of pig jejunum. Grundschober, 1977.
    7  Structurally related ester
    

    2.2.2  Biotransformations

    2.2.2.1  Branched-chain aliphatic acids

        The saturated branched-chain aliphatic acids isobutyric acid,
    isovaleric acid and 2-methylbutyric acid formed via ester hydrolysis
    are endogenous in humans as intermediary products in the metabolism of
    the amino acids valine (Kinnory  et al., 1955), leucine (Henning &
    Hird, 1970), and isoleucine (Voet & Voet, 1990), respectively. These
    acids participate in the fatty acid pathway and the tricarboxylic acid
    cycle and are completely metabolized to CO2.

        Short (<C6) branched-chain acids undergo -oxidation,
    preferably in the longer chain. -Cleavage of the resulting acid
    yields linear acid fragments which are sources of carbon in the fatty
    acid pathway or tricarboxylic acid cycle (Voet & Voet, 1990). For
    example, isobutyric acid (DiVincenzo & Hamilton, 1979) and methacrylic
    acid (2-methylpropenoic acid) (Bratt & Hathway, 1977) given to rats by
    gavage are rapidly eliminated almost exclusively as CO2. The CO2 is
    presumed to arise from -oxidation and decarboxylation of isobutyric
    acid or methacrylic acid to yield propionyl CoA, which, after
    conversion to succinyl CoA, can participate in the tricarboxylic acid
    cycle (Saito, 1975). Studies of the metabolism of the amino acid
    leucine have shown that isovaleric acid is converted to acetyl
    coenzyme A and acetoacetate (Voet & Voet, 1990).

        The principal metabolic pathways utilized by the remaining 3
    branched-chain acids in this group (i.e., 2-, 3-, and
    4-methylpentanoic acid) are determined primarily by the position of
    the methyl substituent. Acids with a methyl substituent located at an
    even-numbered carbon (e.g., 2-methylpentanoic acid and
    4-methylpentanoic acid) are extensively metabolized to CO2 via
    -oxidation and cleavage in the fatty acid pathway. If the methyl
    group is located at the -position (e.g., 3-methylpentanoic acid),
    -oxidation is inhibited and alpha-oxidation predominates, primarily
    leading to short-chain acid fragments capable of being completely
    metabolized (Williams, 1959).

    a)  Isobutyric acid

        Rats administered (1-14C)-isobutyric acid by gavage rapidly
    eliminate 14CO2 (DiVincenzo & Hamilton, 1979). The CO2 is presumed
    to arise from -oxidation and decarboxylation of isobutyric acid to
    yield propionyl CoA which, after conversion to succinyl CoA, then
    participates in the tricarboxylic acid cycle (Voet & Voet, 1990). The
    formation of propionyl CoA is shown by the observation that rats fed
    isobutyric acid excrete elevated levels of 2-methylmalonic acid, an
    intermediate in the conversion of propionyl CoA to succinyl CoA (Voet
    & Voet, 1990). It is anticipated that humans would metabolize
    isobutyric acid by complete oxidation to CO2 via propionyl CoA and
    succinyl CoA, components of the tricarboxylic acid cycle (Fig. 2).

    FIGURE 2


    b)  Isovaleric acid

        Isovaleric acid as the CoA thioester undergoes successive
    dehydrogenation and carboxylation in the leucine pathway to yield
    -methylglutaconyl CoA. -Methylglutaconyl CoA is hydrated to yield
    -hydroxy -methylglutaryl CoA which is finally cleaved to
    acetoacetate and acetyl CoA (Voet & Voet, 1990) (Fig. 3).

    FIGURE 3

    c) 2-Methylbutyric acid, 2-methylpentanoic acid, and 4-methylpentanoic
    acid

        In general, branched-chain acids with an  alpha-methyl or
    4-methyl substituent are metabolized via oxidative cleavage to yield
    linear acid fragments (Deuel, 1957), which are completely oxidized to
    CO2 in the fatty acid pathway and tricarboxylic acid cycle. 2-
    Methylbutyric acid undergoes -oxidation and cleavage to yield acetyl
    CoA and propionyl CoA. Propionyl CoA is converted to succinyl CoA,
    which, together with acetyl CoA, can be completely metabolized to CO2
    in the tricarboxylic acid cycle (Deuel, 1957). -Oxidation of
    2-methylbutyric acid has been observed in guinea-pigs  in vivo 
    (Stokke  et al., 1969) (Fig. 4).

    FIGURE 4

        The metabolism of 2-methylpentanoic acid has been studied in
    mammals. 2-Methypentanoic acid undergoes -oxidation in the longer
    branched-chain followed by cleavage to yield two propionyl CoA
    fragments. In rabbits, 2-methylpentanoic acid is converted to
    propionyl CoA, which is completely metabolized (Deuel, 1957).
    4-Methylpentanoic acid, a gamma-methyl substituted acid, is expected
    to undergo -oxidative cleavage to acetyl CoA and isobutyl CoA which
    would be completely metabolized (Voet & Voet, 1990).

    d) 3-Methylpentanoic acid

        In general, -oxidation of branched-chain acids with a -methyl is
    inhibited. However, -substituted acids may undergo alpha-oxidative
    cleavage to yield acid fragments (Deuel, 1957; Williams, 1959) which
    are completely metabolized in the fatty acid and tricarboxylic acid
    cycles. In addition, they may undergo partial -oxidation to yield
    -hydroxy acids which are further oxidized (omega-oxidation) and
    fragmented to yield short-chain acids.

        The metabolism of 3-methylpentanoic acid has been studied in
    mammals. alpha-Oxidation and decarboxylation of 3-methylpentanoic acid
    would yield 2-methylbutyric acid followed by -oxidative cleavage to
    yield acetyl CoA and acetone. In guinea-pig liver homogenate,
    3-methylpentanoic acid is converted to 2-methylbutyric acid which is
    further metabolized via -oxidative cleavage (Stokke, 1969). In
    rabbits, 3-methylpentanoic acid is converted to -hydroxybutyric acid
    (Williams, 1959).

    2.2.2.2  Aliphatic linear alcohols

        Linear saturated and unsaturated alcohols are oxidized
    successively to their corresponding aldehydes and carboxylic acids,
    which enter the fatty acid -oxidation pathway. Branched-chain
    aliphatic alcohols are converted by similar oxidation reactions to
    their corresponding carboxylic acid, which undergoes metabolism via
    -oxidation and cleavage to yield CO2 in amino acid pathways, the
    fatty acid pathway, and the tricarboxylic acid cycle.

        Based on the above information, it is anticipated that the 32
    esters of aliphatic acyclic primary alcohols and branched-chain
    aliphatic acyclic acids will be hydrolysed in the intestinal tract,
    blood and liver to yield the corresponding branched-chain acids and
    aliphatic alcohols. The resulting branched-chain acids and aliphatic
    alcohols would be rapidly absorbed from the gastrointestinal tract and
    completely metabolized to CO2 in the tricarboxylic acid cycle and
    fatty acid pathway. The resulting linear alcohols would be
    successively oxidized to their corresponding aldehyde and acid. The
    resulting branched-chain aliphatic alcohols are converted by similar
    oxidation reactions to their corresponding carboxylic acid, which
    undergoes complete metabolism to CO2.

    2.2.3  Toxicological studies

    2.2.3.1  Acute toxicity

        The results of acute toxicity studies for 24 of the 32 esters of
    aliphatic acyclic primary alcohols and branched-chain aliphatic
    acyclic acids are summarized in Table 3. The low acute oral toxicity
    of the group is demonstrated by oral LD50 values >2300 mg/kg bw,
    with the majority being >5000 mg/kg bw.

    2.2.3.2  Short-term toxicity studies

        The results of short-term toxicity studies with 4 branched-chain
    esters (ethyl isobutyrate, isobutyl isobutyrate, ethyl isovalerate and
    isoamyl isovalerate), one component acid (isovaleric acid) and 7
    component alcohols (propyl alcohol, butyl alcohol, hexanol, octanol,
    3-hexen-1-ol, isobutanol and isoamyl alcohol) are summarized in Table
    4.

    a)  Branched-chain Esters

    i)  Ethyl isobutyrate

        Ethyl isobutyrate was added to the diet of 12 male rats and 12
    female rats at a level calculated to provide an average daily intake
    of 29.2 mg/kg bw for 90 days. Weekly measurement of body weights and
    food intake revealed no significant difference between test and
    control animals. Haematological examination, blood chemical
    determinations and urinalysis conducted at weeks 6 and 12 revealed no
    differences from controls. Determination of mean organ weights
    revealed a slight increase in both absolute and relative adrenal gland
    weights in females compared to controls, but these values were not
    statistically significant. Gross and histopathological examination
    failed to reveal any dose-related effects on the adrenal gland or any
    other organs (Mecler & Craig, 1980). The NOEL of 29.2 mg ethyl
    isobutyrate/kg bw per day in rats is >1000 times the estimated daily
     per capita intake1 ("eaters only") of 7.74 g/kg bw from use of
    ethyl isobutyrate as a flavouring substance in the USA and 12.44 g/kg
    bw from its use as a flavouring substance in Europe.

     ii)    Ethyl isovalerate

    For 90 days, ethyl isovalerate on gum arabic (10%) was added to the
    diet of 12 male rats and 12 female rats at a level calculated to
    provide an average daily intake of 12.1 mg/kg bw for males and 13.6
    mg/kg bw for females. Body weights and food intake were measured
    weekly. No significant differences between test and control animals
    were observed.

                   

    1 Intake calculated as follows: [[(annual volume, kg) x (1 x 109
    mg/kg)]/[population x 0.6 x 365 days]], where population (10%, "eaters
    only") = 24 x 106 for the USA  and 32 x 106 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 per
    day) calculated as follows: [(mg per day)/body weight], where body
    weight = 60 kg. Slight variations may occur from rounding off. 



        Table 3.  Acute toxicity studies for esters of aliphatic acyclic primary alcohols and branched-chain aliphatic acyclic acids

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

    iso-Butyrates

    Methyl iso-butyrate       Rats      NR           Oral      16 000        Sandmeyer & Kirwin, 1981
    Ethyl iso-butyrate        Rats      NR           Oral      >5000         Moreno, 1975
    Propyl iso-butyrate       Rats      NR           Oral      15 000        Jenner et al., 1964
    Butyl iso-butyrate        Rats      NR           Oral      >5000         Levenstein, 1974
    Hexyl iso-butyrate        Rats      NR           Oral      >5000         Moreno, 1977
    Trans-3-heptenyl 2-
    methylpropanoate          Rats      NR           Oral      >25 000       Food and Drug Research Labs, 1975
    Octyl iso-butyrate        Rats      NR           Oral      >5000         Levenstein, 1974
    iso-Butyl iso-butyrate    Rats 
                              and mice  NR           Oral      12 800         Sandmeyer & Kirwin, 1981

    iso-Valerates

    Methyl iso-valerate       Rabbits   NR           Oral      5690          Munch, 1972
    Ethyl iso-valerate        Rats      NR           Oral      >5000         Levenstein, 1976; BASF, 1980
                              Rabbits   NR           Oral      7030          Munch, 1972
    Propyl iso-valerate       Rabbits   NR           Oral      8220          Munch, 1972
    Butyl iso-valerate        Rats      NR           Oral      >5000         Moreno, 1978
                              Rabbits   NR           Oral      8230          Munch, 1972
    Hexyl 3-methylbutanoate   Rats      NR           Oral      >5000         Moreno et al., 1981

    2-Methylbutyrates

    2-Methylpropyl            Rabbits   NR           Oral      >5000         Munch, 1972
    3-methylbutyrate          Rats      NR           Oral      6970          Moreno, 1978
    2-Methylbutyl 
      3-methylbutanoate       Rats      NR           Oral      >5000         Moreno, 1978
    Methyl 2-methylbutyrate   Rats      NR           Oral      >5000         Moreno et al., 1982
    Hexyl 2-methylbutanoate   Rats      NR           Oral      >5000         Moreno, 1977
    iso-Propyl
    2-methylbutyrate          Rats      NR           Oral      >20 000       Griffiths & Giessinger, 1979

    Table 3.  Continued...

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

    3-Hexenyl
    2-methylbutanoate         Rats      NR           Oral      >5000         Moreno, 1977
    Ethyl 3-methylpentanoate  Rats      NR           Oral      >4000         Biosphere Research Center, Inc., 1981
    Methyl 2-methylpentanoate Rats      NR           Oral      >5000         Pharmakon Inc.,1980
                                                                                                               

    1  The study was performed at a single dose level or multiple dose levels that produced no effects and,
    therefore, a NOEL was not determined.
    The NOEL is probably much higher than the reported dose level that produced no adverse effects


    Table 4.  Short-term studies for branched-chain esters and their component alcohols and acids

                                                                                                                                   
    Substance                Species, sex         Route        Duration                 NOEL                  Reference
                                                                                 (mg/kg bw per day)
                                                                                                                                   
    Branched esters

    Isobutyl isobutyrate     Rats, M & F     Oral and gavage   18 weeks                1000                   Drake et al., 1978
    Ethyl isobutyrate        Rats, M & F          Oral         90 days                 29.21                  Mecler & Craig, 1980
    Ethyl isovalerate        Rats, M & F          Oral         13 weeks                12.11                  Mecler & Craig, 1980
    Isoamyl isovalerate2     Rats, M & F          Oral         90 days                 2201                   Damske et al., 1980

    Component acids

    Isovaleric acid          Rats                 Oral         90 days                 25001                  Amoore et al., 1978

    Component alcohols

    Propyl alcohol           Rat, male            Oral         4 months                 601                   Hillbom et al., 1974
    Butyl alcohol            Rats, male           Oral         13 weeks                 5.61                  Wakabayashi et al., 1984
                             Rats, M & F          Oral         14 days                 1380                   PPG, 1991
                             Rats, NR             Oral         28 days                  9401                  Bio-Fax, 1969
    Hexyl alcohol            Beagles, M & F       Oral         13 weeks                 230                   Eibert, 1992
                             Rat, M & F           Oral         13 weeks                 577                   Eibert, 1992
    Octanol                  Mice                 Oral         1 month                  1791                  Voskovofnikova, 1966
    3-Hexen-1-ol             Rats, M & F          Oral         90 days                  150                   Gaunt et al., 1969
    Isopropanol              Adult Human          Oral         6 weeks                  6.4                   Wills, 1969
    Isobutanol               Rats, M & F          Oral         90 days                 1450                   BASF, 1992
    Isoamyl alcohol2         Rats M & F          Gavage        17 weeks                1000                   Carpanini et al., 1973
                                                                                                                                   

    1  The study was performed at a single dose level or multiple dose levels that produced no effects and, therefore, a NOEL was not
    determined. The NOEL is probably much higher than the reported dose level that produced no adverse effects.
    2  Structurally related substance
    

    Haematological examination, blood chemical determinations and
    urinalysis conducted at weeks 6 and 12 revealed no difference from
    controls. At necropsy increases in absolute and relative mean adrenal
    gland and thyroid gland weights were observed, but the results were
    not statistically significant. Gross and histopathological examination
    did not support relative weight changes, and revealed no dose-related
    lesions (Mecler & Craig, 1980). The NOEL of 12.1 mg ethyl
    isovalerate/kg bw per day in rats is >1000 times the estimated daily
     per capita intake ("eaters only") of 9.01g/kg bw from use of ethyl
    isovalerate as a flavouring substance in the USA, and 12.75 g/kg bw
    from its use in Europe.

     iii) Isobutyl isobutyrate

        Groups of 15 male and 15 female rats were given daily doses of 0,
    10, 100 or 1000 mg isobutyl isobutyrate/kg bw by gavage for 18 weeks.
    Body weights, food consumption and water intake were measured and were
    not different from controls. Haematology, urine analyses and serum
    chemistry parameters were evaluated and did not reveal any
    treatment-related effects. At necropsy gross examinations were
    performed on all animals and organ weights were obtained. No
    significant effects were reported. Histological examination was
    conducted on all tissues from rats in the high-dose group and 50% of
    the control animals. Histopathological examination of the major organs
    was also performed on rats given 10 and 100 mg/kg bw per day. No
    evidence of histopathological effects was reported (Drake  et al., 
    1978). The highest level of 1000 mg isobutyl isobutyrate/kg bw per day
    which produced no adverse effects in rats is >100 000 times the
    estimated daily  per capita intake1 ("eaters only") of 0.04 g/kg bw
    per day from use of isobutyl isobutyrate as a flavouring substance in
    the USA and 1.09 g/kg bw per day from its use in Europe.

     iv) Isoamyl isovalerate

        Isoamyl isovalerate was added to the diet of male and female rats
    at levels calculated to provide average daily intakes of 22, 69 or 220
    mg/kg bw for 90 days. Body weights and food intake were measured
    weekly. No significant differences between test and control animals
    were observed. Haematological examinations, blood chemical
    determinations and urinalysis conducted at weeks 6 and 12 revealed no
    differences from controls. Organ weights at necropsy were normal
    compared to controls. Gross and histopathological examination revealed
    no dose-related lesions (Damske  et al., 1980). The intake level of
    220 mg isoamyl isovalerate/kg bw per day that produced no effects in
    rats is approximately 10 000 times the estimated daily  per capita 
    intake ("eaters only") of 13 g/kg bw from use of isoamyl isovalerate
    as a flavouring substance in the USA and 23.91 g/kg bw from its use
    as a flavouring substance in Europe (see footnote in section
    2.2.3.2a i).

     b) Component alcohols and acids

        Ester hydrolysis yields the component branched-chain acid and
    linear or branched-chain alcohol. The results of subchronic studies
    for the component acids and alcohols of esters of aliphatic acyclic
    primary alcohols and branched-chain aliphatic acyclic acids has
    revealed no evidence of toxicity at levels up to 2500 mg/kg bw per
    day. These short-term studies are described below and summarized in
    Table 4.

     i) 1-Propanol

        Four-month-old male Wistar rats were given a 1M solution of
    1-propanol, corresponding to 60 mg/kg bw per day, as the sole drinking
    fluid for 4 months. Control animals received tap water. Consumption of
    food and alcohol solution and the weight gain of each animal were
    recorded. A lower ratio of weight gain to caloric intake was observed
    in the rats given the 1-propanol, indicating that this group utilized
    food less efficiently than those untreated. However, this action is
    not indicative of toxicity. Histological examination of the livers of
    the experimental animals revealed no difference from controls. No
    hepatic steatosis was observed. Neither inflammation nor cirrhosis was
    seen in any of the livers (Hillbom  et al., 1974).

     ii)    Butanol

        No adverse effects were reported when 6.9% butanol with 25%
    sucrose was added to the drinking-water of male rats for 13 weeks
    (Wakabayashi  et al., 1984) at an estimated daily intake level of 5.6
    mg butanol/kg bw.

        A 14-day study was conducted using rats to evaluate the effects of
    butanol on gross and microscopic pathology, body weight, clinical
    signs and clinical chemistry. Levels of 1.38%, 2.75% or 5.5% butanol
    were administered orally to rats, which corresponds to daily intake
    levels of 1380-5500 mg butanol/kg bw. A statistically significant
    increase in the liver-to-body weight ratio resulted in males at all
    levels and females at the 5.5% level (PPG, 1991).

        No deaths or unusual symptoms were observed when rats (sex was not
    reported) were fed butyl alcohol in a 2% corn oil solution blended
    with the diet at concentrations of 0, 1000, 3500 or 10 000 mg/kg
    (equal to 0, 90, 300 or 940 mg/kg bw per day, respectively) for 28
    days. At necropsy, no significant gross lesions were observed. Liver
    and kidney weights showed no significant differences from those of the
    controls (Bio-Fax, 1969).

     iii) Hexyl alcohol

        Two groups of male and female rats were fed hexyl alcohol for 13
    weeks at dietary levels of 0.25 or 0.50%; a third group was fed 1% for
    weeks 1-10 and 2%, 4% and 6% for weeks 11, 12 and 13, respectively.
    Decreased food consumption was observed in females at the high-dose

    level, but body weights for all animals were normal. No significant
    haematological changes or differences in urine analyses were observed
    for the test and control groups. Gross pathology and microscopic
    evaluations were performed and revealed no treatment-related effects.
    The 1% level was reported to be equivalent to an intake of 577 mg/kg
    bw per day (Eibert, 1992).

        Three groups of 2 male and 2 female purebred beagle dogs were fed
    hexyl alcohol for 13 weeks at levels of 0.50%, 1.0% or 1000 mg/kg bw
    per day via gelatin capsules. A fourth group of 4 males and 4 females
    served as controls. Body weight, organ weight, and food consumption
    for the treated animals did not differ from controls. All animals in
    the high-dose group displayed gross signs of toxicity intermittently
    after treatment, including salivation, excitation, ataxia, tremors and
    anaesthesia. The animals generally returned to normal within 4 hours
    of treatment. One female dog died on the first day of treatment and
    was replaced by another female. Three of the four remaining animals
    died on either the 23rd or 38th day of the study. No signs of toxicity
    were observed in the other test animals. Haematology, serum chemistry
    and urinalysis was performed and showed no significant difference as
    compared to controls. All animals were necropsied at the end of the
    study.

        Animals in the high-dose group exhibited gastrointestinal
    inflammation and congestion of other visceral organs. Some gastric
    irritation was observed in the mid-dose group. Both males treated at
    the 1000 mg/kg bw per day level exhibited significant testicular
    atrophy (Eibert, 1992). Effects on the testes and other reproductive
    tissue have been observed with other aliphatic alcohols at high dose
    levels (Lington & Bevan, 1994). A finding of nodules on the lung
    surface of some animals was reported to be non-treatment-related. The
    1% NOEL corresponds to a daily intake level ranging from 230 to 695
    mg/kg bw (Eibert, 1992).

     iv) Octanol

        Seventeen groups of 10 mice each received oral doses of 1-octanol
    at a level of 179 mg/kg bw per day for one month. 1-Octanol was
    administered intragastrically in the form of a solution or suspension.
    No cumulative effects were observed (Voskovofnikova, 1966).

     v) Cis-3-hexen-1-ol

        Short-term toxicity studies have been reported for 
     cis-3-hexen-1-ol, which is the alcohol component for esters of
    unsaturated alcohols in this group. Groups of 15 male and 15 female
    weanling rats were given drinking-water containing 0, 310, 1250 or
    5000 mg/litre  cis-3-hexen-1-ol for 98 days. Body weight, food intake
    and water consumption were recorded weekly. A reduction in water
    intake was reported in male rats in the high-dose group only.
    Haematology studies were conducted on eight rats from each sex in the
    control, 1250 and 5000 mg/litre groups during the sixth week.
    Urinalysis, including pH, microscopic constituents and content of

    bile, blood and glucose, was performed for eight animals of each sex
    from each group during week 6, and from week 12 until the end of the
    study. Kidney function was assessed regularly. Necropsies were
    performed on all animals at the end of the study. Serum chemistry
    parameters were measured for all animals at necropsy. All tissues were
    examined for macroscopic abnormalities, and organ weights were
    obtained for the brain, pituitary, thyroid, heart, liver, spleen,
    kidneys, adrenals and gonads. Tissues from all rats in the high-dose
    group and from 50% of the controls were examined microscopically.
    Slightly increased relative kidney and adrenal gland weights observed
    in males only at the 5000 mg/litre level were not accompanied by any
    evidence of histopathology. These increases were not considered to be
    statistically significant. No treatment-related abnormalities were
    reported (Gaunt  et al., 1969).

     vi)    Isopropanol

        Daily doses of 2.6 or 6.4 mg isopropanol/kg bw were given to adult
    human males for a period of six weeks. Studies of the microscopic and
    chemical characteristics of blood and urine were repeated on the
    first, third and seventh days and at weekly intervals thereafter.
    Removal of BSP from blood was estimated once before initiation of
    ingestion of isopropyl alcohol and at the end of the experimental
    period. Retention of BSP in serum at the end of the experiment was not
    significantly different from that before the start in any of the men.
    No significant, persistent changes that are clearly attributable to
    the ingestion of isopropanol were evident (Wills, 1963).

     vii) Isobutanol

        Groups of 10 male and 10 female Wistar rats were given isobutyl
    alcohol in their drinking-water for 3 months. The test substance was
    administered in concentrations of 0, 1000, 4000 or 16 000 mg/litre,
    which was reported to correspond to approximate dose levels of 0, 60
    340 or 1450 mg/kg bw per day. Food and water consumption and body
    weight gain were not affected by the test substance. All animals
    tested were free from adverse clinical effects. Haematology and
    clinical chemistry parameters were measured and revealed no
    treatment-related adverse effects. Gross pathology and
    histopathological examinations were no different from controls. The
    authors concluded that results of this study demonstrate a lack of
    toxicity associated with administration of isobutyl alcohol in the
    drinking-water of rats, and that the NOEL is >1450 mg/kg bw per day
    (BASF, 1992).

     viii) Isoamyl alcohol

        Isoamyl alcohol was administered to groups of 15 male and 15
    female Ash/CSE rats in corn oil by gavage, providing daily dose levels
    of 0, 150 500, or 1000 mg/kg bw per day for 17 weeks. High-dose males
    exhibited a slight reduction in body-weight gain, which was associated
    with reduced food intake. Over the entire period of the study,
    however, there was no statistically significant reduction in mean food

    intake. Examination of haematology, serum analyses, urinalysis, renal
    concentration tests, and organ weights revealed no treatment-related
    effects. At necropsy, the animals were examined for macroscopic
    abnormalities, and the major organs were weighed. Microscopic
    examination was performed on several tissues of the control and
    high-dose animals. No treatment-related abnormalities were observed
    (Carpanini  et al., 1973).

     ix)    Isovaleric acid

        In a limited 90-day feeding study, Sprague-Dawley rats were
    administered doses of 5% isovaleric acid in the diet, which was
    calculated (FDA, 1993) to be equivalent to 2500 mg isovaleric acid/kg
    bw per day. The rats were monitored for survival rates, body weight
    changes, food intake, blood and urine analyses, organ weights and
    histology. At the 2500 mg/kg bw dose level, there were no
    treatment-related effects on the measured parameters (Amoore  et 
     al., 1978).

    2.2.3.3  Long-term toxicity/carcinogenicity studies

        The results of long-term toxicity/carcinogenicity studies with two
    of the component alcohols are described below and summarized in Table
    5.

     a) Isobutanol

        Twenty males and 20 female Wistar rats were given 0.2% isobutanol
    in drinking-water for 53 to 56 weeks. Clinical chemistry, including
    alcohol dehydrogenase, glutamic oxaloacetate transaminase glutamic
    pyruviuc transaminase, and protein content, were conducted and
    revealed no difference from controls. At necropsy, no abnormalities
    were observed during histological examination (Johannsen & Purchase,
    1969). The concentration of isobutanol that produced no adverse
    effects was calculated (FDA, 1993) to provide an approximate daily
    intake of 200 mg/kg bw. This is >10 000 times the daily  per capita 
    intake ("eaters only") of 4.85 g/kg bw isobutanol from use as a
    flavouring substance in the USA and of 8.79 g/kg bw isobutanol from
    its use in Europe (see footnote in section 2.2.3.2a i).



        Table 5. Long-term toxicity studies for esters of aliphatic acyclic primary alcohols and
    branched-chain aliphatic acyclic acids

                                                                                                                       
    Substance            Species,         Route          Duration           NOEL            Reference
                           sex                           (weeks)     (mg/kg bw per day)
                                                                                                                       

    Component Alcohols

    Isobutyl alcohol     Rats, M&F    Drinking-water      53-56            2001             Johannsen & Purchase, 1969
    Isoamyl alcohol2     Rats, M&F    Drinking-water      53-56           20001             Johannsen & Purchase, 1969
                                                                                                                       

    1  The study was performed at a single dose level or multiple dose levels that produced no
    effects and, therefore, a NOEL was not determined. The NOEL is probably much higher than
    the reported dose level that produced no effects

    2  A structually related branched-chain alcohol
    

     b) Isoamyl alcohol

        Twenty males and 20 female Wistar rats were given drinking-water
    containing 2% isoamyl alcohol for 53 to 56 weeks. Clinical chemistry,
    including alcohol dehydrogenase, glutamic oxaloacetate transaminase,
    glutamic pyruviuc transaminase and protein content, were conducted and
    revealed no difference from controls. At necropsy, no abnormalities
    were observed during histological examination (Johannsen & Purchase,
    1969). The concentration of isoamyl alcohol that produced no adverse
    effects was calculated (FDA, 1993) to provide an approximate daily
    intake of 2000 mg/kg bw. This is > 10 000 times the daily  per 
     capita intake ("eaters only") of 26.32 g isoamyl alcohol/kg bw from
    use as a flavouring substance in the USA and of 30.47 g isoamyl
    alcohol/kg bw from its use as a flavouring substance in Europe (see
    footnote in section 2.2.3.2a i).

    2.2.3.4  Genotoxicity studies

        The results of genotoxicity studies with the group of 32 branched-
    chain esters are summarized in Table 6. Mutagenicity testing of ethyl
    isovalerate  in vitro by means of the Ames test, at concentrations up
    to 10 mg/plate has shown no evidence of mutagenicity in  Salmonella
    typhimurium strains TA92, TA94, TA97, TA98, TA100, TA102, TA1535,
    TA1537 or in TA2637 with or without metabolic activation (Ishidate  et
    al., 1984; Fujita & Sasaki, 1987). There was no evidence of
    mutagenicity in Chinese hamster fibroblast cells when the chromosomal
    aberration test was performed with ethyl isovalerate at concentrations
    up to 2 mg/ml (Ishidate  et al., 1984). When incubated with  Bacillus
    subtilis, at concentrations up to 20 l/disk (Yoo, 1986), and at 17
    g/plate (Oda  et al., 1978), ethyl isovalerate was reported to be
    non-mutagenic. At high concentrations (100-200 l/ml) of ethyl
    isovalerate,  Bacillus subtilis was reported to show signs positive
    for mutagenicity in the rec assay (Koruda, 1984a). However, these
    results are not supported by the results of other rec assays using
    similar bacterial strains (Yoo, 1986; Oda  et al., 1978). Considering 
    the results of the rec assay, the Ames test and the chromosomal aberration
    test, it is concluded that the 32 branched-chain esters are not genotoxic.

    2.2.3.5  Reproductive toxicity studies

        No reproduction studies have been reported for esters of aliphatic
    acyclic primary alcohols and branched-chain aliphatic acyclic acids.



        Table 6. Genotoxicity studies for esters of aliphatic acyclic primary alcohols and branched-chain aliphatic acyclic acids

                                                                                                                                    
    Substance name           Test system         Test object            Concentration of    Results          Reference
                             in vitro                                   substance
                                                                                                                                    

    Ethyl isovalerate        Ames test           S. typhimurium TA92,   0.01-1 mg/plate     Negative1        Fujita & Sasaki, 1987
                                                 TA1535
                             Ames test           TA100, TA1537, TA94,   10 mg/plate         Negative1        Ishidate et al., 1984
                                                 TA98 and TA2637
                             Rec assay           B. subtilis            100-200 l/ml       Positive         Kuroda et al., 1984
                             Rec assay           B. subtilis M45(rec-)  up to 20 l/disk    Negative         Yoo, 1986
                                                 and H17(rec+)
                                                 S. typhimurium TA97
                                                 and TA102
                             Chromosomal
                             aberration test     Chinese hamster        up to 2 mg/ml       Negative         Ishidate et al., 1984
                                                 fibroblasts cells      
                             Rec assay           B. subtilis H17        17g/plate          Negative         Oda et al., 1978
                                                 and M45
                                                                                                                                    

    1  Both with and without S-9 activation
    

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    See Also:
       Toxicological Abbreviations