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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



    SATURATED ALIPHATIC ACYCLIC BRANCHED-CHAIN PRIMARY ALCOHOLS, 
    ALDEHYDES, AND 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 intake
            1.6 Conclusions
        2.  Relevant background information
            2.1 Biological data
                2.1.1   Absorption, distribution and excretion
                    2.1.1.1 Methyl-substituted aliphatic alcohols,
                            aldehydes and carboxylic acids
                    2.1.12  alpha-Ethyl-substituted aliphatic alcohols,
                            aldehydes and carboxylic acids
                2.1.2   Biotransformation
                    2.1.2.1 Methyl-substituted aliphatic alcohols,
                            aldehydes and carboxylic acids
                    2.1.2.2 alpha-Ethyl-substituted aliphatic alcohols,
                            aldehydes and carboxylic acids
                2.1.3   Toxicological studies
                    2.1.3.1 Acute toxicity
                    2.1.3.2 Short-term toxicity
                    2.1.3.3 Long-term toxicity/carcinogenicity
                    2.1.3.4 Genotoxicity
                    2.1.3.5 Reproductive toxicity
                    2.1.3.6 Developmental toxicity
                    2.1.3.7 Special studies on peroxisome proliferation
                    2.1.3.8 Special study on immunotoxicity
        3.  References

    1.  EVALUATION

    1.1  Introduction

        The Committee evaluated a group of 25 flavouring agents that
    includes selected saturated aliphatic acyclic branched-chain primary
    alcohols, aldehydes and acids using the Procedure for the Safety
    Evaluation of Flavouring Agents (the "Procedure") (see Figure 1 and
    Table 1).

        Twenty-two substances contain one or more methyl substituents and
    the three remaining have ethyl substituents in the alpha position.

        The Committee has evaluated two members of the group previously. 
    Isobutyl alcohol was evaluated at the twenty-third meeting, when an
    ADI was not allocated because of a lack of information (Annex 1,
    reference 50).  An ADI of 0-0.5 mg/kg bw was allocated to
    2-ethyl-1-hexanol at the forty-first meeting (Annex 1, reference 107).

    1.2  Estimated daily  per capita intake

        The total annual production volume of the 22 methyl-substituted
    saturated aliphatic branched-chain primary alcohols, aldehydes and
    acids from their use as flavouring substances in the USA is
    approximately 9.8 and 29 tonnes in Europe. In the USA seven substances
    (isobutyl alcohol, isobutyraldehyde, isobutyric acid,
    2-methylbutyraldehyde, 2-methylbutyric acid, 3-methylbutyraldehyde and
    isovaleric acid) constitute greater than 80% of the total volume of
    production. In Europe, greater than 85% of the total annual volume is
    accounted for by five substances, four of which are the same as in the
    USA (isobutyl alcohol, isobutyric acid, 2-methylbutyric acid,
    isovaleric acid and 2-methylvaleric acid). In the unlikely event that
    all 22 methyl-substituted alcohols, aldehydes and acids would be
    consumed simultaneously on a daily basis, the estimated total daily
     per capita intake of the 22 methyl-substituted substances from their
    use as flavouring agents would be 1900 g/person per day in the USA
    and 410 g/person per day in Europe.

        The total reported annual production volume of use of the three 2-
    ethyl substituted substances is 370 kg in the USA (NAS, 1987) and 1000
    kg in Europe (IOFI, 1995).  In the unlikely event that all three ethyl
    substituted alcohols, aldehydes and acids would be consumed
    simultaneously on a daily basis, the estimated total daily  per 
     capita intake of the three ethyl-substituted substances would be 
    < 71 g/person in the USA or < 145 g/person in Europe.

    Saturated aliphatic acyclic branched-chain primary alcohols, aldehydes
    and acids have been detected in a wide variety of foods such as
    cheese, fruits, vinegar and alcoholic beverages (CIVO-TNO, 1994).
    Quantitative data on the natural occurrence of these flavouring agents
    has been reported for 11 of the 25 substances in the group and
    corresponds to a total of 1500 tonnes/year consumed in food (Stofberg
    & Grundschober, 1987).



        Table 1.   Summary of results of the safety evaluations on saturated aliphatic acyclic branched-chain primary alcohols, aldehydes, and
               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 of the substances in this group are metabolized to innocuous products

                                                                                                                                             
    No.     Substance                Step A3                                          Comments                          Conclusion based on 
            (CAS No.)                Does intake exceed the                                                             current levels of 
                                     human intake threshold?1                                                           intake
                                     Intake Estimates
                                     (g/person per day) 
                                                                                                                                             

    Structural class I: methyl-substituted saturated aliphatic branched-chain primary alcohols, aldehydes, and acids

    0251    Isobutyl alcohol                       No
                                     USA:    290         Europe:  530                                                   No safety concern
    0252    Isobutyraldehyde                       No
                                     USA:    100         Europe:  130                                                   No safety concern
    0253    Isobutyric acid                        No
                                     USA:    140         Europe:  820                                                   No safety concern
    0254    2-Methylbutyraldehyde                  No
                                     USA:    370         Europe:  4.9                                                   No safety concern
    0255    2-Methylbutyric acid                   No
                                     USA:    480         Europe:  1200                                                  No safety concern
    0258    3-Methylbutyraldehyde                  No
                                     USA:    140         Europe:  110                                                   No safety concern
    0259    Isovaleric acid                        No
                                     USA:    96          Europe:  480                                                   No safety concern
    0260    2-Methylpentanal                       No
                                     USA:    8.5         Europe:  12                                                    No safety concern
    0261    2-Methylvaleric acid                   No
                                     USA:    2.3         Europe:  680                                                   No safety concern
    0262    3-Methylpentanoic acid               No
                                     USA:    8.8         Europe:  2.9                                                   No safety concern
    0263    3-Methyl-1-pentanol                    No
                                     USA:    4.2         Europe:  5.9                                                   No safety concern
    0264    4-Methylpentanoic acid                 No
                                     USA:    55          Europe:  1.6                                                   No safety concern

    Table 1.  Continued...

                                                                                                                                             
    No.     Substance                Step A3                                          Comments                          Conclusion based on 
            (CAS No.)                Does intake exceed the                                                             current levels of 
                                     human intake threshold?1                                                           intake
                                     Intake Estimates
                                     (g/person per day) 
                                                                                                                                             

    0265    2-Methylhexanoic acid                 No
                                     USA:   2.3         Europe:  15                                                     No safety concern
    0266    5-Methylhexanoic acid                 No
                                     USA:   8.6         Europe:  0.0                                                    No safety concern
    0268    3,5,5-Trimethyl-1-hexanol             No
                                     USA:   0.76        Europe:  13                                                     No safety concern
    0269    3,5,5-Trimethylhexanal                No
                                     USA:   150         Europe:  0.29                                                   No safety concern
    0270    2-Methyloctanal                       No
                                     USA:   0.95        Europe:  0.14                                                   No safety concern
    0271    4-Methyloctanoic acid                 No
                                     USA:   0.10        Europe:  11                                                     No safety concern
    0272    3,7-Dimethyl-1-octanol                No
                                     USA:   2.9         Europe:  94                                                     No safety concern
    0273    2,6-Dimethyloctanal                   No
                                     USA:   6.7         Europe:  0.01                                                   No safety concern
    0274    4-Methylnonanoic acid                 No
                                     USA:   1.5         Europe:  1.00                                                   No safety concern
    0275    2-Methylundecanal                     No 
                                     USA:   0.10        Europe:  0.61                                                   No safety concern

    Structural class II: ethyl-substituted saturated aliphatic acyclic branched-chain primary alcohols, aldehydes, and acids

    0256    2-Ethylbutyraldehyde                  No                       2-ethyl substituent inhibits the             No safety concern
                                     USA:   0.17        Europe:   0.57     -oxidation of aliphatic alcohols,
                                                                           aldehydes and carboxylic acids.  These
                                                                           compounds undergo omega and omega-1
                                                                           oxidation to yield polar metabolite
                                                                           primarily excreted in the urine.
    0257    2-Ethylbutyric acid                  No
                                     USA:   31          Europe:   60                                                    No safety concern

    Table 1.  Continued...

                                                                                                                                             
    No.     Substance                Step A3                                          Comments                          Conclusion based on 
            (CAS No.)                Does intake exceed the                                                             current levels of 
                                     human intake threshold?1                                                           intake
                                     Intake Estimates
                                     (g/person per day) 
                                                                                                                                             

    0267    2-Ethyl-1-hexanol                      No
                                     USA:    40         Europe:     86                                                  No safety concern
                                                                                                                                             

    1 The human intake threshold for class I is 1800 g per day; 540 g per day for class II; and 90 g per day for class III.
    

    1.3  Absorption, metabolism and elimination

        The metabolism of methyl and ethyl substituted saturated aliphatic
    acyclic branched-chain alcohols, aldehydes and carboxylic acids 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.  The 22 methyl-substituted saturated aliphatic acyclic
    branched-chain primary alcohols, aldehydes and acids were classified
    in structural class I (Cramer  et al., 1978). The three
    ethyl-substituted saturated aliphatic acyclic branched-chain primary
    alcohols, aldehydes and acids (2-ethylbutyraldehyde, 2-ethylbutyric
    acid and 2-ethyl-1-hexanol) contain sterically hindered functional
    groups and therefore were classified in structural class II.

        Step 2.  At current levels of intake from use as flavouring agents
    (see Table 1) the 22 methyl-substituted alcohols, aldehydes and
    carboxylic acids and the three 2-ethyl-substituted alcohols, aldehydes
    and carboxylic acids would not be expected to saturate the metabolic
    pathways and all the compounds were predicted to be metabolized to
    innocuous products.

        Step A3.  All 22 class I substances in this group have estimated
    USA and European daily  per capita intake levels less than the human
    intake threshold for this class (i.e. 1800 g/person per day).
    Therefore, the 22 methyl-substituted saturated aliphatic acyclic
    branched-chain primary alcohols, aldehydes and acids evaluated do not
    pose a safety concern when used at current levels of estimated intake
    as flavouring agents.

        The three ethyl-substituted substances in class II
    (2-ethylbutyraldehyde, 2-ethylbutyric acid and 2-ethyl-1-hexanol) have
    estimated intake levels (0.6, 58 and 86 g/person per day,
    respectively) in the USA and Europe that are below the human intake
    threshold for class II (i.e. 540 g/person per day). Therefore, the
    three ethyl-substituted saturated aliphatic acyclic branched-chain
    primary alcohols, aldehydes and acids evaluated do not pose a safety
    concern when used at current levels of estimated intake as flavouring
    agents.

    1.5  Consideration of combined intake

        The stepwise evaluations of the 25 saturated aliphatic acyclic
    branched-chain primary alcohols, aldehydes and carboxylic acids used
    as flavouring agents are summarized in Table 1.

        In the unlikely event that all 25 saturated aliphatic acyclic
    branched chain alcohols, aldehydes and carboxylic acids would be
    consumed simultaneously on a daily basis, the Committee judged that
    combined intake is of no safety concern, since all the substances in

    this group are expected to be efficiently metabolized and the combined
    intake level is not expected to saturate pathways.

    1.6  Conclusions

        The Committee concluded that the use of these substances as
    flavouring agents would not present a safety concern at current 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.  In cases where ADIs were previously established, these
    ADIs were maintained at the present meeting.

    2.  RELEVANT BACKGROUND INFORMATION

    2.1  Biological data

    2.1.1  Absorption, distribution and excretion

    2.1.1.1  Methyl-substituted aliphatic alcohols, aldehydes and
    carboxylic acids (22)

        In general, branched-chain aliphatic acyclic alcohols, aldehydes
    and acids are rapidly absorbed from the gastrointestinal tract
    (Gaillard & Derache, 1965; Dawson  et al., 1964).

     a) Branched-chain alcohols

        In a study designed to measure the absorption, distribution and
    excretion of alcohols and ketones, a mixture of isomers of amyl
    alcohol, including the structurally related branched-chain
    3-methylbutyl alcohol (isoamyl alcohol) and 2-methylbutyl alcohol, was
    administered to rats by intraperitoneal (i.p.) injection. Complete
    absorption of the primary alcohols from the peritoneum occurred within
    1 hour, and disappearance from the blood occurred within 3 to 9
    hours. 3-Methylbutyraldehyde was detected as an intermediate in the
    blood (Haggard  et al., 1945).

     b) Branched-chain carboxylic acids

        1-14C-Isobutyric acid was administered by gavage to male Charles
    River CD rats at doses of 4, 40 and 400 mg/kg bw and to female rats at
    a single dose of 400 mg/kg bw. Rapid elimination in the breath as
    expired 14CO2 was observed at all dose levels. Less than 5% of the
    radioactive dose was detected in the urine and faeces. Similar
    patterns of excretion were reported in all animals (DiVincenzo & 
    Hamilton, 1979). 1-14C-Isovaleric acid fed to rats was absorbed and
    utilized in the formation of acetyl groups, fatty acids and
    cholesterol (Zabin & Bloch, 1951).

    2.1.1.2  alpha-Ethyl-substituted aliphatic alcohols, aldehydes and
    carboxylic acids (3)

        2-Ethyl[1-14C]hexanol orally administered to male rats was
    readily absorbed. Within 28 hours, elimination of the radioactivity
    occurred in the urine (80-82%), faeces (8-9%) and expired CO2 (6-7%)
    (Albro, 1975).

    2.1.2  Biotransformation

    2.1.2.1  Methyl-substituted aliphatic alcohols, aldehydes and
    carboxylic acids (22)

        Following absorption, primary alcohols are successively oxidized
    to their corresponding aldehyde and carboxylic acid (Bosron &
    Ting-Kai, 1980; Levi & Hodgson, 1989). The resulting
    methyl-substituted carboxylic acids may undergo -oxidation
    predominantly in the longer branched chain to yield -hydroxyacids
    which may be further oxidized (-oxidation) and cleaved to yield
    short-chain acids that are completely metabolized via the fatty acid
    pathway or tricarboxylic acid cycles (Voet & Voet, 1990).

        The position of the methyl substituent plays a role in metabolism.
    Acids with a methyl substituent located at an even-numbered carbon
    (e.g., 2-methyl-pentanoic acid or 4-methyldecanoic acid) are
    extensively metabolized to CO2  via 
    -oxidative cleavage in the fatty acid pathway. If the methyl group is
    located at the 3-position, -oxidation is inhibited and
    omega-oxidation predominates, primarily leading to polar, acidic
    metabolites capable of being further oxidized or conjugated and
    excreted in the urine (Williams, 1959). As chain length and
    lipophilicity increase, omega-oxidation competes with -oxidative
    cleavage. Methyl substituted acids (e.g., 3-methylnonanoic,
    2-methyldodecanoic, 4-methyldo-decanoic acids) are, to some extent,
    omega-oxidized in animals to form diacids, which can be detected in
    the urine (Williams, 1959).

     a) Branched-chain alcohols and aldehydes

        In human isoenzyme mixtures, oxidation of primary aliphatic
    alcohols to aldehydes is catalysed by NAD+-dependent alcohol
    dehydrogenase (ADH) (Pietruszko  et al., 1973), and oxidation of
    aldehydes to carboxylic acids is catalysed by NAD+-dependent
    aldehyde dehydrogenase (ALD) (Weiner, 1980; Blair & Bodley, 1969).
    Isobutyl alcohol and isobutyraldehyde have been reported to be
    excellent substrates for ADH (Hedlund & Kiessling, 1969; Saito, 1975)
    and ALD, respectively (Prunonosa  et al., 1991). The rate of
    oxidation by ADH is greater for isobutyl alcohol than for ethanol
    (Lester & Benson, 1970), while the rate of oxidation by ALD is
    approximately the same for isobutyraldehyde and acetaldehyde
    (Walkenstein & Weinhouse, 1953). ALD-catalysed oxidation of low
    molecular weight aldehydes such as isobutyraldehyde requires

    glutathione (Eckfeldt & Yonetani, 1982), which implies that the
    substrate for oxidation may be the thiohemiacetal formed by rapid 
     in vivo conjugation of aldehydes with glutathione (Brabec, 1993).

        Isobutyraldehyde was oxidized by rat liver mitochondria  in 
     vitro and was found to compete with acetaldehyde for ALD (Hedlund &
    Kiessling, 1969). The metabolism of 3-methylbutyraldehyde and related
    aldehydes was studied in male and female rat liver homogenate and in
    rat liver  in situ. 3-Methylbutyraldehyde was shown to be oxidized to
    3-methylbutyric acid in an ADH-catalysed reaction at one half the rate
    of acetaldehyde (Hedlund & Kiessling, 1969). 3-Methylbutyraldehyde was
    reported to be oxidized to the corresponding acid in the mitochondria
    of rat liver and kidney (Walkenstein, 1953). 3-Methylbutyraldehyde was
    detected in the plasma of approximately 38 human subjects and was
    shown to be a metabolite of ingested protein (Goldberg  et al., 
    1979).

     b) Branched-chain carboxylic acids

        The resulting methyl-substituted carboxylic acids are substrates
    for -oxidation and cleavage in the amino acid and fatty acid pathways
    (Voet & Voet, 1990). Isobutyric acid, isovaleric acid and
    2-methylbutyric acid occur endogenously as intermediates in the human
    metabolism of the amino acids valine (Kinnory  et al., 1955), leucine
    (Henning & Hird, 1970) and isoleucine (Voet & Voet, 1990),
    respectively. For instance, in the leucine pathway isovaleryl CoA
    undergoes successive dehydrogenation and carboxylation 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).

        The metabolism of isobutyric acid has been established in rodents.
    Rats fed isobutyric acid excrete elevated levels of 2-methylmalonic
    acid, which is an intermediate in the conversion of propionyl CoA to
    succinyl CoA (Butenandt & Thomas, 1958).  -Oxidation of 2-
    methylbutyric acid has been observed in guinea-pigs  in vivo 
    (Stokke  et al., 1969). Isovaleric acid was identified as a urinary
    metabolite in rabbits following oral administration of isobutyl
    alcohol (Saito, 1975).

        2-Methylvaleric acid is an alpha-substituted acid and, therefore,
    undergoes -oxidation in the longer branched-chain followed by
    cleavage to yield two propionyl CoA fragments. In rabbits,
    2-methylvaleric acid is converted to propionyl CoA which is completely
    metabolized (Deuel, 1957). 4-Methylpentanoic acid was identified in
    the faeces of pigs fed a normal diet (Yasuhara  et al., 1982).

        5-Methylhexanoic acid was shown to conjugate with glucuronic acid
    in phenobarbital-induced rat liver microsomes (Hamdoune  et al., 
    1995). Rabbits have been shown to excrete methyl-substituted
    long-chain acids such as 2-methylnonanoic acid in the urine unchanged
    (Deuel, 1957).

        In conclusion, the metabolism of branched-chain alcohols,
    aldehydes and carboxylic acids containing one or more methyl
    substituents is determined primarily by the position of the methyl
    group on the branched-chain. Alcohols are successively oxidized to the
    corresponding aldehydes and carboxylic acids. The branched-chain acids
    are metabolized via -oxidation in the longer branched-chain followed
    by cleavage to yield linear acid fragments, which are completely
    metabolized in the fatty acid pathway or the tricarboxylic acid cycle.
    At high-dose levels, longer branched-chain acids may undergo
    omega-oxidation to yield diacids, which may undergo further oxidation
    and cleavage.

    2.1.2.2  alpha-Ethyl-substituted aliphatic alcohols, aldehydes and
    carboxylic acids (3)

        The presence of an ethyl or propyl substituent at the position,
    such as in 2-ethyl-1-hexanol, inhibits -oxidation (Deuel, 1957).
    Detoxication pathways of omega- and omega-1 oxidation compete with
    -oxidation of these sterically-hindered substances. In the principal
    detoxication pathway, the parent alcohol or corresponding carboxylic
    acid undergoes a combination of reactions including omega- or omega-1
    oxidation and functional group oxidation leading to polar, acidic
    metabolites capable of being excreted in the urine (Williams, 1959;
    Deisinger  et al., 1994). When the principal pathway is saturated,
    the corresponding carboxylic acid conjugates with glucuronic acid and
    is excreted primarily in the urine (Williams, 1959; Albro, 1975;
    Deisinger  et al., 1994).

        The metabolism of 2-ethyl-1-hexanol has been studied in rats and
    rabbits and it was shown that both species follow similar pathways.
    Rats and mice were administered 0, 140, 350, 700, 1050 or 1750 mg/kg
    bw/day 2-ethyl-1-hexanol by gavage for 14 days. As measured by the
    rate of palmitoyl CoA oxidation, 2-ethyl-1-hexanol administration
    resulted in a linear dose-related induction of peroxisomal -oxidation
    (Keith  et al., 1992).

        The major urinary metabolites of 2-ethyl-1-hexanol in rats
    include: the glucuronic acid conjugate of 2-ethylhexanoic acid; omega-
    and omega-1 oxidation metabolites, 2-ethyl-1,6-dihexanoic acid,
    5-hydroxy-2-ethylhexanoic acid and its corresponding delta-lactone;
    and 6-hydroxy-2-ethylhexanoic acid. Minute quantities of the
    omega-desaturation metabolite, 2-ethyl-5-hexenoic acid and metabolites
    formed from -oxidation and oxidative decarboxylation, 2- and
    4-heptanone, were also detected (Albro, 1975; Deisinger  et al., 
    1994).

        2-Ethyl[1-14C]hexanol was administered orally to male rats. Using
    acid extraction of the urine, the major urinary metabolite was
    determined to be 2-ethylhexanoic acid, which most likely was excreted
    as the glucuronic acid conjugate. According to the author,
    2-ethylhexanoic acid can undergo partial -oxidation and
    decarboxylation to produce CO2, and may also undergo omega- or

    omega-1-oxidation. 2-Ethyl-5-hydroxyhexanoic acid,
    2-ethyl-5-ketohexanoic acid, 2-ethyl-1,6-hexanedioic acid and 2- and
    4-heptanone were also identified as urinary metabolites of
    2-ethylhexanol. A small amount (3%) of the parent alcohol was excreted
    unchanged in the urine (Albro, 1975).

        Female Fischer rats were given doses of 50 or 500 mg/kg bw of
    [14C]-2-ethyl-1-hexanol. At single or repeated low dose levels (i.e.
    50 mg/kg bw) the omega-oxidation pathway predominated as observed in
    the excretion of the principal urinary metabolites, 2-ethyl-1,6-acid
    and its precursors. At the high-dose level of 2-ethyl-1-hexanol, the
    principal metabolite observed was the glucuronide of the corresponding
    acid, 2-ethylhexanoic acid (see Figure 1). The author indicated that
    the data suggest the occurrence of metabolic saturation of the
    omega-oxidation pathway at the high-dose level (Deisinger  et al., 
    1994).

    FIGURE 1

        When rats and rabbits were administered 2-ethylhexanol-C14 or
    2-ethylhexanol-C14 sulfate either orally or by intraperitoneal (i.p.)
    injection, the principal metabolites included unchanged alcohol and
    the sulfate ester, as well as hydroxylated 2-ethylhexanol and the
    glucuronide conjugate of 2-ethylhexanoic acid (Knaak  et al., 1966).

        2-Ethylbutyric acid was administered orally and by subcutaneous
    injection at a dose of approximately 1 gram to rabbits and 100 mg (as
    the sodium salt) to rats. The acid was primarily excreted unchanged in
    the urine as the glucuronic acid conjugate (Dziewiatowski  et al., 
    1949). In dogs, 2-ethylbutyric acid undergoes -oxidation and
    decarboxylation to yield 2-pentanone (Deuel, 1957).

        In conclusion, branched-chain alcohols, aldehydes and carboxylic
    acids with bulky (ethyl, propyl, etc.) alpha-alkyl substituents are
    metabolized by omega- and omega-1-oxidation to yield polar metabolites
    capable of excretion in the urine. At high-dose levels these oxidation
    pathways may be saturated, in which case, the corresponding acid may
    undergo conjugation with glucuronic acid.

    2.1.3  Toxicological studies

    2.1.3.1  Acute toxicity

        Acute toxicity studies have been reported for 20 of the 25
    saturated aliphatic branched-chain primary alcohols, aldehydes and
    acids in this group and are summarized in Table 2. The acute oral
    toxicity of the group is demonstrated by oral LD50 values of >2000
    mg/kg bw for the alcohols; >3200 mg/kg bw for the aldehydes; and
    >1000 mg/kg bw for the carboxylic acids, except for isobutyric and
    2-methylbutyric acid, which are 280 mg/kg bw, and 3-methylpentanoic
    acid which is >700 mg/kg bw. The rat oral LD50 values for the three
    alpha-ethyl-substituted substances 2-ethylbutyraldehdye,
    2-ethylbutyric acid and 2-ethyl-1-hexanol are 3980 mg/kg bw, 2200
    mg/kg bw and 2050-7100 mg/kg bw, respectively.

    2.1.3.2  Short-term toxicity

        The results of short-term toxicity studies with a
    methyl-substituted alcohol (isobutyl alcohol), a structurally related
    alcohol (isoamyl alcohol), and two methyl-substituted carboxylic acids
    (isovaleric acid and 2-methylhexanoic acid) are summarized in Table 3
    and described below. Short-term oral studies for 2-ethylbutyric acid
    and 2-ethyl-1-hexanol are summarized in the section on
    alpha-ethyl-substituted substances.



        Table 2.  Acute toxicity studies for saturated aliphatic branched-chain primary alcohols, aldehydes and carboxylic acids

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

    Isobutyl alcohol           rat         M/F       oral       2640-3100      Smyth et al., 1954; WHO, 1987
                                           M/F       gavage     2650-3100      Purchase, 1969
                               mouse                 oral       3500           WHO, 1987

    Isobutyraldehyde           rat         M/F       oral       3730           Smyth et al., 1954

    Isobutyric acid            rat         NR        oral       280            Lewis, 1989

    2-Methylbutyraldehyde      rat         NR        oral       8570           Opdyke & Leitizia, 1982

    2-Methylbutyric acid       rat         M/F       oral       280-2200       Moreno et al., 1982; Lewis, 1989;
                               mouse       M/F       oral       1238           Schafer & Bowles, 1985

    2-Ethylbutyraldehyde       rat         M/F       oral       3980           Smyth et al., 1951

    2-Ethylbutyric acid        rat         M/F       oral       2200           Smyth et al., 1954

    Isoamyl alcohol2           rat         M/F       oral       5700           Smyth et al., 1954
                                                     gavage     1300-4000      Purchase, 1969

    3-Methylbutyraldehyde      rat         M/F       oral       7166           Moreno, 1988

    Isovaleric acid            rat         NR        oral       2000; >3200    Fassett, 1963; NIOSH, 1976

    2-Methylvaleric acid       rat         M/F       oral       1600-3200      Smyth et al., 1954; ACGIH, 1989

    3-Methylpentanoic acid     rat         NR        oral       >700           Vollmuth et al., 1989

    3-Methyl-1-pentanol        rat         M/F       oral       >2000          Engler & Bahler, 1982

    2-Ethyl-1-hexanol          rat         M/F       oral       2050-7100      Hodge, 1943; Shaffer et al., 1945; 
                                                                               Smyth et al., 1969; Scala & Burtis, 1973; 
                                                                               Schmidt et al., 1973; Albro, 1975; Dave & Lidman, 1978

    Table 2.  Continued...

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

    3,5,5-Trimethyl-1-hexanol  rat         M/F       oral       2300           Moreno, 1977

    3,5,5-Trimethylhexanal     rat         M/F       oral       3240           Moreno, 1975; Opdyke & Leitizia, 1982

    2-Methyloctanal            rat         M/F       oral       >5000          Moreno, 1977

    3,7-Dimethyl-1-octanol     rat         M/F       oral       5000           Shelanski & Moldovan, 1973; Moreno, 1977

    4-Methylnonanoic acid      rat         NR        oral       3700           IFREB, 1975

    2-Methylundecanal          rat         M/F       oral       >5000          Owen & Meyer, 1971

    4-Methylpentanoic acid     rat         NR        oral       2050->3200     Smyth et al., 1954; Fassett, 1963; NIOSH, 1976;
                                                                               Union Carbide, 1991;
                               mouse       NR        oral       5000           NIOSH, 1991
                                                                                                                                     

    1  M = male; F = female; NR = Not reported.
    2  Structurally related substance.

    Table 3.  Short-term and long-term toxicity studies for saturated aliphatic acyclic branched-chain primary alcohols, 
    aldehydes and acids

                                                                                                                 
    Substance                  Species (Sex)     Route     Duration      NOEL              Reference
                                                                         (mg/kg bw
                                                                         per day)
                                                                                                                 
    Short-term studies
    Alcohols

    Isobutyl alcohol           rat (M/F)         oral      90 days       1450              BASF, 1992
                               rat               oral      4 months      >0.126 nmol/g     Hillbom et al., 1974
                                                                          bw/day3          Johannsen &
                               rat (M/F)         oral      53-56 weeks   2003              Purchase, 1969

    Isoamyl alcohol2           rat (M/F)         gavage    17 weeks      10003             Carpanini et al., 1973
                               rat (M/F)         oral      56 weeks      20003             Johannsen & Purchase, 1969

    2-Ethyl-1-hexanol          mouse             gavage    11 days       100               Astill et al., 1996a
                               mouse             gavage    90 days       125               Astill et al., 1996a
                               mouse             food      11 days       1150-4450         Astill et al., 1993
                               rat               gavage    14 days       1303              Rhodes et al., 1984
                                                                         NR                Lake et al., 1975
                               rat               oral      7 days        100               Astill et al., 1996a
                               rat               gavage    11 days       125               Astill et al., 1996a
                               rat               gavage    90 days       <500-540          Astill et al., 1993
                               rat               oral      11 days

    Carboxylic Acid                                                                        

    2-Ethylbutyric acid        rat               oral      90 days       3003              Amoore et al., 1978

    Isovaleric acid            rat               oral      90 days       25003             Amoore et al., 1978

    2-Methylhexanoic acid      rat (M/F)         oral      90 days       33                Posternak et al., 1969

    Long-term/ Carcinogenicity studies                                                     
    2-Ethyl-1-hexanol          mouse (M/F)       gavage    540 days      200               Astill et al., 1996b
                               rat (M/F)         gavage    730 days      50                Astill et al., 1996b
                                                                                                                 

    Table 3 (continued)

    1 M = Male; F = Female; NR = not reported.
    2 A structurally related branched-chain alcohol.
    3 The study was performed at a single dose level or multiple dose levels that produced no adverse effects and, therefore, a NOEL was not
    determined. The NOEL is probably higher than the reported dose level that produced no adverse effects.
    


     a) Methyl substituted aliphatic alcohols, aldehydes and carboxylic 
     acids

     i) 2-Methyl-1-propanol (isobutyl alcohol)

        Groups of eleven 4-month-old male Wistar rats were given 1 M
    solutions of isobutyl alcohol as their only drinking fluid for 4
    months. Control animals received tap water. An ordinary diet was
    provided for all animals. The level of isobutyl alcohol consumption
    was reported to be 0.126 nmole/g bw/day at 120 days. At necropsy,
    there was no evidence of hepatic steatosis, fibrosis or inflammation
    (Hillbom  et al., 1974).

        Groups of 20 male and 20 female Wistar albino rats were given a
    mixture of fusel oils including 0.2% isobutyl alcohol, which was
    calculated (FDA, 1993) to provide an approximate daily intake of 200
    mg/kg bw/day, as their sole drinking source for 53-56 weeks. Control
    animals were given tap water. There were no statistically significant
    differences in body or liver weights of treated animals as compared to
    controls. Liver enzymes were determined at 2- to 4-week intervals and
    revealed no significant differences in activity and protein content of
    the liver between test and control animals. Histological examination
    was performed on specimens of liver, kidney, heart, spleen and lung
    and revealed no significant abnormalities (Johannsen & Purchase,
    1969).

        Groups of 10 male and 10 female Wistar rats were given 2-methyl-1-
    propanol (i.e. 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 drinking-
    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 normal for all animals except for
    testicular atrophy, which was observed in two males in the high-dose
    group. Diffuse tubular generation and hyperplasia of the Leydig cells
    were reported in the two animals. The authors concluded that the
    absence of similar effects in other male rats indicated that the
    effect was most likely unrelated to the test material. The authors
    reported that the results of this study demonstrate a lack of toxicity
    associated with administration of 2-methyl-1-propanol in the drinking-
    water of rats, and that the NOEL is >1450 mg/kg bw per day (BASF,

    1992). The NOEL is >100 000 times the estimated daily  per capita
    intake1 ("eaters only") of 4.85 g/kg bw of isobutyl alcohol from
    its use as a flavouring substance in the USA and 8.79 g/kg bw of
    isobutyl alcohol in Europe.

     ii)    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 statistically significant 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. 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).

        Groups of 20 male and 20 female Wistar albino rats were given a 2%
    solution of isoamyl alcohol, which was calculated (FDA, 1993) to
    provide an approximate daily intake of 2000 mg/kg bw per day, as their
    sole drinking source for 53-56 weeks. Control animals were given tap
    water. There were no statistically significant differences in body or
    liver weights of treated animals as compared to controls. Liver
    enzymes were determined at 2- to 4-week intervals and revealed no
    significant differences in ADH, GOT or GPT activity and protein
    content of the liver. Histological examination was performed on
    specimens of liver, kidney, heart, spleen and lung and revealed no
    significant abnormalities (Johannsen & Purchase, 1969).

     iii) Isovaleric acid

        In a limited 90-day feeding study, Sprague-Dawley rats were
    administered doses of 0 or 5% isovaleric acid in the diet, which was
    calculated (FDA, 1993) to provide a daily intake of 0 or 2500 mg/kg
    bw. The rats were monitored for survival rates, body weight changes,
    food intake, blood and urine analysis, organ weights and histology. At
    2500 mg/kg bw per day, there were no observed changes in the rats
    (Amoore  et al., 1978).

                   

    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, 1995). Intake (mg/kg bw/day)
    calculated as follows: [(mg/day)/body weight], where body weight = 60
    kg. Slight variations may occur from rounding off.

     iv)    2-Methylhexanoic acid

        In a limited 90-day feeding study, 2-methylhexanoic acid was fed
    to male and female Charles River CD rats at a dose of approximately 3
    mg/kg bw/day. Observations included mortality rates, food intake, body
    weight, haematology, blood urea, organ weights and histopathology. The
    NOEL was 3 mg/kg bw dose level (Posternak  et al., 1969) which is
    >10 000 times the estimated daily  per capita intake ("eaters only")
    of 0.04 g/kg bw 2-methylhexanoic acid from use as a flavouring
    substance in the USA and 0.24 g/kg bw from its use in Europe.


     b) alpha-Ethyl substituted aliphatic alcohols, aldehydes and 
     carboxylic acids

     i) 2-Ethyl-1-hexanol

        Eleven (11-) day and 90-day studies were conducted in B6C3F1 mice
    as part of the dose selection process for an 18-month study (see
    section 2.1.3.3). In the 11-day gavage study, groups of ten B6C3F1
    mice of each sex were administrated nine applications of 0, 100, 330,
    1000 or 1500 mg 2-ethylhexanol/kg bw per day. Mice in the high-dose
    group exhibited effects such as ataxia, lethargy, loss of
    consciousness, death in four females, increased absolute and relative
    lever and stomach weights, macroscopic foci in the forestomach, and
    microscopic lesions in the forestomach, liver, testes and kidneys.
    These lesions consisted of hyperkeratosis and focal or multifocal
    acanthosis and inflammatory oedema in the submucosa in most mice,
    focal or multifocal ulceration of the mucous membrane in a few mice,
    hypertrophy of hepatocytes in all mice, focal necrosis of liver cells
    in one male and one female mouse, centrilobular fatty infiltration in
    female mice that died intercurrently, tubular giant cells in
    testicular tubules of two mice, and renal cortex tubular dilation and
    nephrosis in mice that died intercurrently. Mice receiving the dose of
    1000 mg/kg bw per day exhibited similar symptoms to those observed at
    the highest dose (1500 mg/kg bw per day), but to a lesser degree,
    while four mice (two male, two female) receiving 330 mg 2-ethyl
    hexanol/kg bw per day had acanthosis in the mucous membrane of the
    forestomach. No treatment-related adverse effects were observed in the
    mice administered 100 mg 2-ethylhexanol/kg bw per day (Astill 
     et al., 1996a).

        In the 90-day study, mice (number per group not reported) received
    doses of 0, 25, 125, 250 or 500 mg 2-ethylhexanol/kg bw per day. At
    the highest dose level, the treatment-related effects observed
    included increased relative stomach weights in male mice and focal or
    multifocal acanthosis of the forestomach mucosa in two male and one
    female mouse. Increased relative stomach weights were observed in male
    mice receiving 250 mg/kg bw per day. No treatment-related effects were
    reported in mice of the 125 or 25 mg/kg bw per day dose level. Based
    on the results of the 11- and 90-day studies, dose levels of 50, 200
    or 750 mg 2-ethylhexanol/kg bw per day were chosen for the 18-month
    mouse study (Astill  et al., 1996a).

        An 11-day mouse study was conducted using administration of
    2-ethylhexanol as microencapsulated material in the diet. Four dose
    groups of 10 male and 10 female B6C3F1 mice per group were used with
    concentrations of microencapsulated material in the diet of 0.48,
    0.96, 1.44 or 2.88%. The microcapsules contained 45.8% 2-ethylhexanol;
    therefore, 2-ethylhexanol concentrations in the diet were 0.22, 0.44,
    0.66 or 1.32%, respectively. These concentrations were reported to be
    equal to doses of 550, 1150, 1800 or 4450 mg/kg bw per day for males
    and 750, 1750, 2650 or 5750 mg/kg bw per day for females. The authors
    reported that diet spillage did occur, but this was not quantified in
    the treated groups of mice. As a result, the mg/kg bw per day dosage
    calculations would not account for spillage and may represent
    exaggerated 2-ethylhexanol intakes (Astill  et al., 1993).

        Significantly decreased body weight gains were observed at the
    high-dose level for females throughout the test period and for males
    at day 10 of the test period and at the 0.66% level in males at day 10
    of the test period. No other treatment-related findings were reported.
    The authors concluded the NOEL for male mice was in the range of 0.96
    to 1.44% microencapsulated material (0.44 to 0.66% 2-ethylhexanol or
    reportedly 1150 to 1800 mg/kg bw per day) and the NOEL for female mice
    was in the range of 1.44 to 2.88% microencapsulated material (0.66 to
    1.32% 2-ethylhexanol or reportedly 1800 to 4450 mg/kg bw per day)
    (Astill  et al., 1993). The NOELs reported in this study are higher
    than the NOEL of 100 to 330 mg 2-ethylhexanol/kg bw per day reported
    in the 11-day gavage study in B6C3F1 mice (Astill  et al., 1996a),
    most likely as a result of the different modes of administration. The
    lack of correction for diet spillage in the microencapsulation study
    may also have been a factor.

        To study the effect of 2-ethylhexanol on rat liver and testes,
    Rhodes  et al. (1984) administered 1 mmol 2-ethylhexanol/kg bw per
    day (approximately 130 mg/kg bw per day) to five Wistar rats by gavage
    for 14 days. 2-Ethylhexanol administration did not produce testicular
    atrophy, hepatomegaly, peroxisome proliferation or hypolipidaemia in
    the rats. Lake  et al. (1992) orally administered 1335 mg
    2-ethylhexanol/kg bw per day to six male Wistar rats for seven days.
    The authors reported significantly increased relative liver weights,
    increased microsomal biphenyl 4-hydroxylase activity, increased
    alcohol dehydrogenase activity in the centrilobular area of the liver
    lobule, increased number of microbodies, and dilatation of the smooth
    endoplasmic reticulum in the livers. No effect on the activities of
    aniline 4-hydroxylase or mitochondrial succinate dehydrogenase was
    observed.

        In an 11-day gavage study, groups of ten male and ten female
    Fischer F344 rats were administered doses of 0, 100, 330, 1000 or 1500
    mg 2-ethylhexanol/kg bw per day. Adverse effects, including reduction
    in food consumption and body weight gain, ataxia, lethargy, changes in
    blood chemistry, effects on absolute (increased liver and stomach,
    decreased spleen, brain, and adrenal) and relative (increased stomach,
    liver, kidney, brain, adrenal, and lung, decreased spleen) organ

    weights, gross lesions in the forestomach and microscopic findings in
    the forestomach, liver (high-dose only), spleen and thymus, were
    observed in the rats at the highest two dose levels. At the 330 mg/kg
    bw per day dose, female rats exhibited increased relative kidney
    weights, as well as microscopic lesions in the forestomach of one
    female rat and in the thymus of both sexes (it was not stated whether
    or not these lesions were treatment-related). No treatment-related
    adverse effects were observed at 100 mg/kg bw per day (Astill  et 
     al., 1996a).

        In the 90-day study, groups of ten male and ten female Fischer
    F344 rats were administered doses of 0, 25, 125, 250 or 500 mg
    2-ethylhexanol/kg bw per day by gavage. Reduced body weight and body
    weight gain, changes in blood chemistry (consisting of reduced
    cholesterol, glucose, alanine aminotransferase and alkaline
    phosphatase, levels and increased reticulocytes, total protein and
    albumin levels), increased absolute and relative liver and stomach
    weights and gross lesions in the forestomach were observed in the rats
    receiving 500 mg 2-ethylhexanol/kg bw per day. Also observed in the
    high-dose rats were microscopic changes consisting of focal or
    multifocal acanthosis in the mucosa of six rats (one male, five
    female) and whole mucose acanthosis and submucosa degeneration and
    oedema in one male rat. In addition, a number of high-dose rats with
    fatty infiltration in the liver, as well as a lower grade of fat
    deposition in the liver, were observed. At 250 mg/kg bw per day,
    changes in the blood chemistry (consisting of reduced glucose,
    alkaline phosphatase and alanine aminotransferase levels), increased
    relative liver and stomach weights, and a lower grade of fat
    deposition in the liver cells of the male rats were observed. No
    treatment-related adverse effects were observed at the 25 or 125 mg/kg
    bw per day dose levels. Based on the results of the 11- and 90-day
    studies, dose levels of 50, 150 or 500 mg 2-ethylhexanol/kg bw per day
    were chosen for a two-year study (Astill  et al., 1996a).

        An 11-day rat study was also conducted by administering
    2-ethylhexanol as microencapsulated material in the diet. Four dose
    groups of ten male and ten female Fischer rats per group were fed
    concentrations of microencapsulated material in the diet of 1, 2, 3 or
    6%. The microcapsules contained 45.8% 2-ethylhexanol; therefore,
    2-ethylhexanol concentrations in the diet were 0.46, 0.92, 1.38 or
    2.75%, respectively. These concentrations were reported to be equal to
    doses of 500, 980, 1430 or 2590 mg/kg bw per day for males and 540,
    1060, 1580 or 2820 mg/kg bw per day for females. A vehicle control
    group receiving placebo microcapsules was employed. Decreased food and
    water (top three doses only) consumption was observed at all dose
    levels at some point in the study period with decreased body weights
    reported for both sexes at the top dose and in females at day four in
    the second highest dose. Changes in blood chemistry, consisting of
    decreased cholesterol, triglycerides and alanine aminotransferase
    levels (all doses), decreased glucose, reticulocyte and mean
    corpuscular volume levels (high-dose only), decreased platelet level
    (top two doses only), increased total protein level (top three doses),
    increased erythrocyte level (high-dose only) and increased absolute

    and relative stomach and/or liver weights were observed to some extent
    in all dose groups. Varying degrees of hypertrophy of the hepatocytes
    were reported in the top three dose groups. The authors reported the
    NOEL to be less than 0.46% in the diet (500 or 540 mg/kg bw per day
    for male and female rats, respectively) (Astill  et al., 1993).

     ii)    2-Ethylbutyric acid

        In a limited 90-day feeding study, Sprague-Dawley rats were
    administered doses of 0, 0.62, 1.25 or 10% 2-ethylbutyric acid in the
    diet, equivalent to 0, 300, 625 or 5000 mg/kg bw per day,
    respectively. The rats were monitored for survival rates, body weight
    changes, food intake, blood and urine analysis, organ weights and
    histology. At the 300 mg/kg bw dose level, there were no observed
    changes in the rats. At both the 625 and 5000 mg/kg bw dose levels,
    slight body weight changes were observed within the first 14 days of
    the study (Amoore  et al., 1978).

    2.1.3.3  Long-term toxicity/carcinogenicity

        The results of long-term toxicity/carcinogenicity studies on
    2-ethyl-1-hexanol are summarized in Table 3 and described below.

     a) Mice

        Groups of 50 male and 50 female B6C3F1 mice were administered
    2-ethylhexanol by gavage at doses of 0, 50, 200 or 750 mg/kg bw per
    day, five days per week for 18 months. The vehicle consisted of doubly
    distilled water containing approximately 5 mg Polyoxyl 35 Castor Oil
    (Cremophor(R) EL) per 100 ml. Two control groups of 50 mice of each
    sex were employed, one receiving doubly distilled water containing
    approximately 5 mg Cremophor(R) EL/100 ml. The use of Cremophor(R)
    EL was reported to have no effect on the outcome of the study. Food
    consumption, body weights and haematological parameters were examined
    at specific intervals during the study. At the end of the study, gross
    and histopathological examinations were conducted.

        No treatment-related adverse effects were observed in the mice
    receiving 50 or 200 mg 2-ethylhexanol/kg bw per day. At the 750 mg/kg
    bw per day dose level, body weight gain reductions of approximately 26
    and 24% in males and females, respectively, were reported. These
    weight gain reductions were associated with a significant reduction in
    food consumption. Both male and female mice of the high-dose group
    showed increased mortality compared to the vehicle control mice (i.e.
    30% in the treated mice compared to 4 and 8% in the vehicle control
    group male and female mice, respectively). With respect to
    haematological parameters, both a slight increase in polymorphonuclear
    neutrophils and a slight decrease in lymphocytes were observed in the
    mice of the high-dose group. A slight, statistically insignificant,
    increase in focal hyperplasia of the forestomach epithelium was also
    observed in the high-dose group mice. A slight increase in
    hepatocellular carcinomas in the female mice of the high-dose group

    was determined to have no biological relevance. 2-Ethylhexanol showed
    no evidence of carcinogenicity in this study (Astill  et al., 1996b).

     b) Rats

        Groups of 50 male and 50 female Fischer F344 rats were
    administered 2-ethylhexanol by gavage at doses of 0, 50, 150 or 500
    mg/kg bw per day, 5 days per week for 2 years. The vehicle consisted
    of doubly distilled water containing approximately 5 mg Polyoxyl 35
    Castor Oil (Cremophor(R) EL) (as an emulsifier) per 100 ml. Two
    control groups of 50 mice of each sex were used, one receiving doubly
    distilled water containing 5 mg Cremophor(R) EL/100 ml. The use of
    Cremophor(R) EL was reported to have no effect on the outcome of the
    study. Food consumption, body weights, and haematological parameters
    were examined at specific intervals during the study. At the end of
    the study, gross and histopatho-logical examinations were conducted.

        No treatment-related adverse effects were observed at the 50 mg/kg
    bw per day dose level. At the 150 mg/kg bw per day dose level, rats
    exhibited a body weight gain reduction of approximately 16% in males
    and 12% in females. In addition, the rats also displayed a slightly
    increased incidence of clinical signs, such as poor general condition,
    laboured breathing, piloerection, and/or smearing of the genital
    region with urine. A definite increase in the number of rats and/or a
    definite increase in the incidence of clinical signs (same signs as
    mentioned for the mid-dose group) was observed in the high-dose group
    rats. Mortality was increased in the female rats of the high-dose
    group (52% compared to 28% in the vehicle control) and both male and
    female rats exhibited a reduction in the body weight gain (33% in
    males and 31% in females). With respect to haematological parameters,
    a slight increase in anisocytosis, predominantly microcytosis in
    males, was observed after 12 months, but not after 18 or 24 months.
    The incidence of bronchopneumonia was significantly increased in both
    sexes at the high-dose level. This effect was found to be due to the
    aspiration of the stomach contents, most likely resulting from the
    poor general condition of the animals. 2-Ethylhexanol showed no
    evidence of carcinogenicity in this study (Astill  et al., 1996b).

    2.1.3.4  Genotoxicity

        The results of genotoxicity studies on saturated aliphatic acyclic
    branched-chain primary alcohols, aldehydes, and carboxylic acids are
    summarized in Table 4.


        Table 4.  Genotoxicity studies of saturated aliphatic acyclic branched-chain primary alcohols, aldehydes and carboxylic acids

                                                                                                                                              
    Substance              Test system                 Test object                      Dose level                 Results     Reference
                                                                                                                                              

    Alcohols

    Isobutyl alcohol       Modified Ames test          S. typhimurium TA98, TA100,      100 to 10 000 g/plate1,2   Negative    Zeiger et
                                                       TA1535 TA97 and TA1537                                                  al., 1988
                           Modified Ames test          S. typhimurium TA100, TA98,      5 to 5000 mg/plate1        Negative    Shimizu et
                                                       TA1535, TA1537 and TA1538;                                              al., 1985
                                                       E. coli WP2 uvrA

    2-Ethyl-1-hexanol      8-azaguanine resistance     S. typhimurium TA100             0.5 to 1.5 mM              Positive3   Seed, 1982
                           assay
                           Ames test4                  S. typhimurium TA98, TA100,      up to 2 ml in urine/plate  Negative    Divincenzo
                                                       TA1535, TA1537, TA1538                                                  et al., 1985
                           Ames test                   S. typhimurium TA98, TA100,      0.01 to 1.0 l/plate1      Negative    Kirby et
                                                       TA1535, TA1537, TA1538                                                  al., 1983
                           Ames test                   S. typhimurium TA98, TA100,      33 to 220 g/plate1,2       Negative    Zeiger et
                                                       TA1535, TA1537                                                          al., 1985
                           Ames test                   S. typhimurium TA98, TA100,      100 to 2000 g/plate1       Negative    Agarwal et
                                                       TA1535, TA1537, TA1538,                                                 al., 1985
                                                       TA2637
                           L5178Y/TK+/- mouse          L5178Y/TK+/- mouse               0.01-0.3 l/ml1            Negative    Kirby et
                           lymphoma assay              lymphoma cells                                                          al., 1983
                           Rec-assay                   Bacillus subtilis                500 g/disk                 Negative    Tomita et al.,
                                                                                                                               1982
                           CHO mutation assay          Chinese hamster ovary cells      1.5-2.8 mM                 Negative    Phillips et al.,
                                                                                                                               1982
                           Unscheduled DNA synthesis   Primary rat hepatocytes          not specified              Negative    Hodgson et
                           assay                                                                                               al., 1982

    Aldehydes

    Isobutyraldehyde       Gradient plate technique    S. typhimurium G46, TA1535,      0.1 to 1000 g/ml           Positive5/  McMahon et
                                                       TA100, C3076, TA1537, D3052,                                Negative6   al., 1979
                                                       TA1538, and TA98; E. coli
                                                       WP2 and WP2 uvrA-


    Table 4.  Continued...

                                                                                                                                              
    Substance              Test system                 Test object                      Dose level                 Results     Reference
                                                                                                                                              

    Isobutyraldehyde       Ames test                   S. typhimurium                   3 mol/plate1              Negative    Florin et
    (cont'd)                                                                                                                   al., 1980
                           Spot test                   TA98, TA100, TA1535, TA1537
                           Sister chromatid exchange   adult human lymphocytes          0.002%                     Negative    Obe & Beek,
                                                                                                                               1979
                           Paper-disk method           E. coli Sd-4-73                  0.01- 0.025 ml/disk        Negative    Szybalski,
                                                                                                                               1958

    2-Methylbutyraldehyde  Ames test                   S. typhimurium TA98, TA100,      0.03 to 30 mol/plate1      Negative    Florin et
                                                       TA1535 and TA1537                                                       al., 1980
                           Ames test                   S. typhimurium TA98, TA100       9 nmol to 0.9 mmol/plate1  Negative    Aeschbacher
                                                       and TA102                                                               et al., 1989

    3-Methylbutyraldehyde  Sister chromatid exchange   Adult human lymphocytes          0.002 to 0.003%            Negative    Obe & Beek,
                                                                                                                               1979
                           Ames test                   S. typhimurium TA98, TA100       0.01 nmol to 1 mol/plate1  Negative    Aeschbacher
                                                       and TA102                                                               et al., 1989
                           Ames test                   S. typhimurium TA98, TA100,      3 mol/plate1               Negative    Florin et
                                                       TA1535 and TA1537                                                       al., 1980

    2-Methylpentanal       Ames test                   S. typhimurium TA98, TA100,      3 mol/plate1               Negative    Florin et
                           Spot test                   TA1535 and TA1537                                                       al., 1980

    2,6-Dimethyloctanal    Ames test                   S. typhimurium TA98, TA100,      up to 3.6 mg/plate         Negative    Wild et al.,
                                                       TA1535, TA1537, TA1538                                                  1983

    Carboxylic acids

    Isobutyric acid        Paper-disk method           E. coli Sd-4-73                  0.01 to 0.025 ml/disk      Negative    Szybalski,
                                                                                                                               1958
                           Cell mutagenesis assay      mouse lymphoma                   500 g/ml7,8                Positive    Heck et al,
                                                       L5178Y TK +/-                                               (weak)      1989
                                                                                        600 g/ml9,10               Negative


    Table 4.  Continued...

                                                                                                                                              
    Substance              Test system                 Test object                      Dose level                 Results     Reference
                                                                                                                                              

                           Unscheduled DNA synthesis   rat hepatocytes                  1000 g/ml9,10              Negative    Heck et al.,
                           assay                                                                                               1989
                           Plate incorporation assay   S. typhimurium TA98, 100,        75 000 g/plate9,10         Negative    Heck et al.,
                                                       1535, 1537, 1538                                                        1989
                           Rec-assay                   B. subtilis H17 rec+ and         19 g/disk                  Negative    Oda et al.,
                                                       M45 rec-                                                                1978

    2-Ethyl-1-hexanol      In vivo dominant lethal     ICR/SIM mice                     250, 500, 1000 mg/kg       Negative    Rushbrook et
                           assay                                                        bw/day for 5 days                      al., 1982
                           In vivo chromosomal         Fischer 344 rat bone marrow      16.7, 58.4 or 167 mg/kg    Negative    Putman et
                           aberration assay            cells                            bw/day for 5 days                      al., 1983

    Isobutyraldehyde       Sex linked recessive        Drosophila melanogaster          80 000 ppm (feeding)       Negative    Woodruff et
                           lethal mutation test                                         50 000 ppm (injection)                 al., 1985
                                                                                                                                              

    1  Both with and without metabolic activation
    2  Metabolic activation in the form of S9 derived from rat and hamster
    3  It is assumed that no S9 was used
    4  Test performed on the metabolites of 2-ethyl-1-hexanol
    5  Positive in G46, TA100, E. coli WP2, WP2 uvrA-
    6  Negative in TA1535, TA1537, TA1538, TA98, C3076 and D3052
    7  Without metabolic activation
    8  Lowest active dose tested
    9  With metabolic activation
    10 Highest inactive dose tested
    

     a) Methyl substituted aliphatic alcohols, aldehydes, and carboxylic 
     acids

     i) Isobutyl alcohol

        Isobutyl alcohol produced negative results in Ames tests with
     Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 at
    concentrations of 100 to 10 000 g/plate (Zeiger  et al., 1988).
    Negative mutagenicity results were also obtained by Shimizu  et al. 
    (1985) at dose levels of 5 to 5000 g/plate in  Salmonella 
     typhimurium strains TA98, TA100, TA1535, TA1537, TA1538, and 
     E. coli strain WP2 uvrA-.

     ii)    Branched-chain aldehydes

        In  Salmonella typhimurium, isobutyraldehyde (in strains TA98,
    TA100, TA1535, TA1537), 2-methylbutyraldehyde (TA98, TA100, TA1535,
    TA1537), 3-methylbutyraldehyde (TA98, TA100, TA1535, TA1537) and
    2-methylpentanal (TA100, TA1535, TA1537) were negative in spot tests
    at a concentration of 3 mol/plate with and without metabolic
    activation (Florin  et al., 1980). Similar results were reported for
    2-methylbutyraldehyde and 3-methylbutyraldehyde in  S. typhimurium 
    strains TA98, TA100 and TA102 at concentrations of 9.0 nmol/plate to
    0.9 mol/plate and 0.01 nmol/plate to 1 mol/plate, respectively, with
    and without metabolic activation (Aeschbacher, 1989). Florin  et al. 
    (1980) subsequently subjected 2-methylbutyraldehyde to a quantitative
    Ames test assessment at concentrations of 0.03 to 30 mol/plate, and
    found no evidence of mutagenicity in  S. typhimurium strains TA98,
    TA100 and TA1535, with and without metabolic activation.
    Isobutyraldehyde gave negative results for frame-shift mutations in
     S. typhimurium C3076, TA1537, D3052, TA1538 and TA98, and base
    substitution mutations in  S. typhimurium TA1535, and gave positive
    results for base substitution mutations in  S. typhimurium G46, TA100
    and  E. coli WP2 and WP2  uvrA-(McMahon  et al., 1979). It was
    non-mutagenic in  E. coli Sd-4-73 (Szybalski, 1958). In an  in vivo 
    study, isobutyraldehyde was negative for sex-linked recessive lethal
    mutation in germ cells of  Drosophila melanogaster by adult feeding
    at 80 000 ppm and adult injection at 50 000 ppm (Woodruff  et al., 
    1985).

     iii) Branched-chain carboxylic acids

        Isobutyric acid was non-mutagenic in the Rec-assay with  E. 
     coli Sd-4-73 at dose levels of 0.01 to 0.025 ml/disk (Szybalski,
    1958) and in  B. subtilis H17 and M45 at a dose of 19 g/ml (Oda,
    1978). In a mouse lymphoma L5178YTK +/- assay, isobutyric acid had
    negative results at a dose level of 600 g/ml with metabolic activation
    and had weakly positive results at a dose of 500 g/ml without
    metabolic activation (Heck  et al., 1989). Isobutyric acid had
    negative results in the Ames test plate incorporation assay with
     Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and
    TA1538 at doses ranging up to 75 000 g/ml (Heck  et al., 1989).

    Isobutyric acid did not induce unscheduled DNA synthesis in rat
    hepatocytes at doses of up to 1000 g/ml (Heck  et al., 1989).

        Based on a weight of evidence analysis, aliphatic acyclic
    branched-chain primary alcohols, aldehydes and carboxylic acids are
    not mutagenic or clastogenic in bacterial,  in vitro or  in vivo 
    systems. Sporadic positives were observed in some strains in three of
    the approximately 20 bacterial assays conducted on members of this
    group. Isobutyric acid was found in a single study to be mutagenic 
     in vitro in the presence of metabolic activation. However,
    isobutyric acid was found to be negative in the rat UDS assay. The
    mutagenic activity of isobutyric acid is not, therefore, likely to be
    related to a mechanism involving direct binding to DNA. Consistent
    with a lack of DNA damage, both isobutyraldehyde and
    3-methylbutyraldehyde were negative in the sister chromatid exchange
    assay. In addition, 2-ethyl-1-hexanol was found to be negative in two
    assays for  in vitro mutagenic activity (see 2-ethyl-1-hexanol
    below). There is no evidence of mutagenic or clastogenic activity 
     in vivo in any of the compounds tested.

     b) alpha-Ethyl-substituted aliphatic alcohols, aldehydes and 
     carboxylic acids

     i) 2-Ethyl-1-hexanol

        There are several reports in the literature concerning the  in 
     vitro testing of 2-ethylhexanol for mutagenicity in bacteria.
    2-Ethylhexanol has shown no evidence of mutagenicity in the Ames test
    with  Salmonella typhimurium strains TA98, TA100, TA1535, TA 1537,
    TA1538 or TA2637 with or without metabolic activation (Kirby  et 
     al., 1983; Zeiger  et al., 1985; Agarwal  et al., 1985). Agarwal
     et al. (1985) reported that 2-ethylhexanol was moderately cytotoxic
    in most of the cultures tested, thereby having the potential to mask a
    mutagenic effect. Weak dose-related mutagenic activity (maximum
    increase in mutant frequency was only 3.5 times background) was
    reported for 2-ethylhexanol in the  Salmonella 8-azaguanine
    resistance assay without activation at concentrations up to 1.5 mM.
    2-Ethylhexanol was highly cytotoxic at concentrations that were
    mutagenic (Seed, 1982).

        Further Ames test results on the urinary metabolites of 2-ethyl-1-
    hexanol, using  S. typhimurium strains TA98, TA100, TA1537, TA1538
    and TA1535, indicated no mutagenicity (Divincenzo  et al., 
    1985). The measurements were made with six male Sprague-Dawley rats
    that were administered 1000 mg 2-ethyl-1-hexanol/kg bw/day by gavage
    for 15 days. Urine samples were collected for use in the Ames assay at
    doses of up to 2 ml per plate.

        2-Ethylhexanol was not mutagenic in the L5178Y TK +/- mouse
    lymphoma cell mutagenicity assay, either with or without metabolic
    activation (Kirby  et al., 1983), and produced negative results in
    the rec-assay in  Bacillus subtilis at a concentration of 500 g/disk 

    (Tomita  et al., 1982). Negative results have also been reported for
    2-ethylhexanol in a clastogenicity test on cultured Chinese hamster
    ovary cells (Phillips  et al., 1982) and in an unscheduled DNA
    synthesis assay in primary rat hepatocytes (Hodgson  et al., 1982).

        In  in vivo assays, negative results were reported in a dominant
    lethal assay using oral doses of 250, 500 or 1000 mg 2-ethylhexanol/kg
    bw administered for five consecutive days to ICR/SIM mice (Rushbrook
     et al., 1982). 2-Ethylhexanol did not induce chromosomal aberrations
    in bone marrow cells after oral administration of 0.02, 0.07 or 0.2
    ml/kg bw per day (approximately 16.7, 58.4, 167 mg/kg bw per day
    assuming a density of 0.8344) to groups of five Fischer 344 male rats
    for five days (Putman  et al., 1983).

        The weight of evidence indicates that 2-ethylhexanol is not
    mutagenic or genotoxic. Additionally, the urinary metabolites of
    2-ethyl-1-hexanol were not mutagenic in the Ames tests. Negative 
     in vivo results were reported in a dominant lethal assay and in a
    chromosomal aberration assay.

    2.1.3.5  Reproductive toxicity

        Gray & Beamand (1984) and Sjoberg  et al. (1986) examined the
    effect of 2-ethylhexanol on  in vitro primary cultures of rat
    testicular cells. No effect on the rate of germ-cell detachment from
    Sertoli cells was observed at a 2-ethylhexanol concentration of 
    2x10-4 M.

        Sjoberg  et al. (1986) also examined the effect of 2-ethylhexanol
    on rat testes  in vivo. No testicular damage was observed in six
    young male Sprague-Dawley rats orally administered 2.7 mmol 2-
    ethylhexanol/kg bw per day (approximately 352 mg/kg bw per day) for 5
    days.

    2.1.3.6  Developmental toxicity

     a) Mice

        An oral developmental toxicity study was conducted using CD-1 mice
    (NTP, 1991). Groups of 28 plug-positive mice were exposed to
    microencapsulated 2-ethylhexanol at concentrations of 0, 0.009, 0.03
    or 0.09% in the diet throughout gestation (days 0 to 17). These
    concentrations were reported to be equal to 0, 17, 59 or 191 mg
    2-ethylhexanol/kg bw per day, respectively, based on food consumption.
    No evidence of maternal or developmental toxicity was observed in the
    study. There were no treatment-related effects with respect to the
    number of corpora lutea, uterine implantation sites, pre- and
    post-implantation sites, pre- and post-implantation loss, sex ratio or
    live fetal body weight, or to the incidence of individual, external,
    visceral, skeletal or total malformations or variations.

        Hardin  et al. (1987) examined the developmental toxicity of
    orally administered 2-ethylhexanol in pregnant mice. Fifty female CD-1
    mice were administered by gavage a dose of 1525 mg 2-ethylhexanol/kg
    bw per day on gestation days 6 to 13. A concurrent control group was
    employed. Maternal mortality in the study was 35%, with significant
    reductions in maternal weight gain and in the number of viable litters
    being observed. Significant reductions in the treated group were also
    reported for the following parameters: number of liveborn per litter,
    percentage of survival of the pups, and the birth weight and weight
    gain of the pups. The results of this study are of questionable
    significance due to the presence of maternal toxicity and are
    difficult to interpret due to the fact that only one dose level was
    employed.

     b) Rats

        Groups of ten pregnant Wistar rats were administered by gavage
    doses of 130, 650 or 1300 mg 2-ethylhexanol/kg bw per day on gestation
    days 6 to 15. Two control groups were included, one that received
    doubly distilled water and a second that received doubly distilled
    water containing the emulsifier (0.005% Cremophor(R) EL) used as the
    vehicle in the treated groups. On day 20 of gestation, the rats were
    killed.

        Significant maternal toxicity was observed in the high-dose rats.
    These rats exhibited significant reductions in food consumption, body
    weights and body weight gain. On days 6 to 10, a body weight loss was
    reported. Six of the rats in the high-dose group were found dead on
    days 9, 10 or 13. Other adverse effects reported in the high-dose rats
    included severe clinical symptoms (e.g., unsteady gait, apathy), light
    brown-gray discoloration of the liver in the rats that died during the
    study, lung oedema and emphysema, and markedly decreased mean gravid
    uterus weights. In the 650 mg/kg bw per day dose group, two dams were
    reported to exhibit piloerection. Significant embryotoxic and
    fetotoxic effects, such as an increase in the number of resorptions
    resulting in increased post-implantation loss, decreased mean fetal
    body weight, increased incidence of fetuses with dilated renal pelvis
    and/or hydroureter, and a higher number of fetuses with skeletal
    malformations, also occurred in the high-dose group. Variations and
    retardation were also reported in the high-dose group. A slight
    decrease in mean fetal body weight (statistically significant for all
    viable fetuses combined and male fetuses separately, but
    non-significant for female fetuses separately) and a statistically
    significant increase in the frequency of fetuses with skeletal
    variations and retardation were reported for the mid-dose group. The
    authors reported that the only indications of significant adverse
    effects on reproduction occurred in the high-dose group, which were
    associated with maternal toxicity (Anonymous, 1991).

        In an oral study, Ritter  et al. (1987) exposed groups of
    pregnant Wistar rats to doses of 6.25 or 12.5 mmoles 2-ethylhexanol/kg
    bw (approximately 814 or 1628 mg/kg bw) on day 12 of gestation, the
    rats being killed on day 20 of gestation. Several litters were

    examined from each of the two treatment groups. For the low- and
    high-dose groups of 2-ethylhexanol, the percentage of surviving pups
    with malformation was 2 and 22.2, respectively. The malformations
    observed in the high-dose group included tail and limb defects and
    hydronephrosis. As indicated from the results of tests following oral
    exposure (Anonymous, 1991) and dermal exposure (Tyl  et al., 1992) in
    rats, it would be expected that maternal toxicity could have occurred
    at both dose levels employed in the study of Ritter  et al. (1987),
    even though the effect of treatment on the mother rats was not
    discussed by the authors.

        Tyl  et al. (1992) conducted a range-finding study and a main
    study examining the developmental toxicity of dermally applied
    2-ethylhexanol in Fischer 344 rats. In the range-finding study, doses
    of 0, 0.5, 1.0, 2.0 or 3.0 ml 2-ethylhexanol/kg bw per day (stated by
    the authors to be equivalent to 0, 420, 840, 1680 or 2520 mg/kg bw per
    day, respectively) were applied by occluded dermal application for 6
    hours per day on gestation days 6 to 15 to groups of eight rats.
    Nave, sham (treated with 3.0 ml distilled water/kg bw per day) and
    positive (treated with 420 or 1260 mg methoxyethanol/kg bw per day)
    control groups were employed in the range-finding study. Valproic acid
    was administered orally to a group of eight rats at a dose of 400
    mg/kg bw per day as an oral reference compound.

        In the main study, doses of 0, 0.3, 1.0 or 3.0 ml
    2-ethylhexanol/kg bw per day (stated by authors to be equivalent to 0,
    252, 840 or 2520 mg/kg bw per day, respectively) were applied to
    groups of 25 rats by occluded dermal application for 6 hours per day
    on gestation days 6 to 15. A sham control group receiving 3.0 ml/kg bw
    per day distilled water and a positive control group receiving 840 mg
    2-methoxyethanol/kg bw per day were included (Tyl  et al., 1992).

        Both positive control groups (valproic acid and methoxyethanol)
    exhibited developmental toxicity and teratogenic effects. With respect
    to 2-ethylhexanol, maternal weight gain was significantly reduced
    during gestation days 6 to 15 at dose levels of 1680 and 2520 mg/kg bw
    per day in the range-finding study. At the highest dose level in the
    main study, maternal weight gain was significantly reduced during
    gestation days 6 to 9, but was not significantly different from sham
    controls for any other period examined (i.e. gestation days 0 to 6, 6
    to 15, 15 to 21, or 0 to 21). In both studies, exfoliation and
    encrustation at the site of application occurred at all dose levels.
    However, this effect was reported to be typical of the effect of
    alcohols on the skin. Erythema scores were mild to moderate at 840 and
    2520 mg 2-ethylhexanol/kg bw per day. Oedema was not observed at any
    treatment level. No treatment-related developmental or teratogenic
    effects were observed in the fetuses of any of the dose levels in
    either study. The authors reported no-observed-effect levels of 252 mg
    2-ethylhexanol/kg bw per day based in skin irritation and 840 mg/kg
    weight per day based on systemic toxicity (reduced maternal body
    weight gain) (Tyl  et al., 1992).

        In a study designed to evaluate the association between the
    pharma-cokinetics and teratogenicity of organic acids, pregnant
    Sprague-Dawley rats were administered 14.1 mmoles/kg bw methylhexanoic
    acid, or 12.5 or 15.625 mmoles/kg bw 2-ethylhexanoic acid undiluted by
    oral gavage on day 12 of gestation. At various post-treatment
    intervals (0.25-24 hours), embryos were dissected and exocoelomic
    fluid and yolk sacs were analysed for the presence of the test
    material. Samples of maternal blood and skeletal muscle were analysed
    to determine the concentration of the test substance. The mothers were
    killed on day 20 and fetuses were examined. Methylhexanoic acid
    exhibited no signs of embryotoxicity except for a slight reduction in
    fetal weight which was attributed to maternal toxicity. Ethylhexanoic
    acid was toxic to the embryos removed on day 12, causing increased
    death and malformation and a reduction of fetal weight. Both
    methylhexanoic acid and ethylhexanoic acid reached a high
    concentration in maternal plasma and embryo, but methylhexanoic acid
    was eliminated more rapidly (Scott  et al., 1994).

        An  in vivo developmental toxicity screen was conducted on 15
    aliphatic acids including three substances in this group. Groups of 9
    to 18 timed-pregnant Sprague-Dawley rats were treated by gavage once
    daily on gestation days 6 to 15 with 2-ethylbutyric acid (0, 150 or
    200 mg/kg bw per day), 2-methylvaleric acid (0, 187.5 or 250 mg/kg bw
    per day) and 5-methylhexanoic acid (0, 300 or 400 mg/kg bw per day).
    2-Ethylhexanoic acid, a metabolite of 2-ethylhexanol, was also tested
    at dose levels of 0, 900 or 1200 mg/kg bw per day.

        Maternal body weights were determined on gestational days 6, 8,
    10, 13, 16 and 20. Clinical observations were made on all animals
    throughout the study. Pups in each litter were examined and counted.
    Skeletal examination was performed on two postnatal day 6 survivors
    from each group. After postnatal day 6, the dams were killed and the
    number of uterine implantation sites was recorded. 2-Methylvaleric
    acid, 2-ethylbutyric acid and 5-methylhexanoic acid induced motor
    depression in some mothers, which was associated with severe
    respiratory effects (i.e. rales or dyspnoea). Perinatal mortality was
    noted for 2-ethylbutyric acid but only in litters of dams that
    exhibited severe respiratory effects. There was no delayed
    parturition, decreased progeny viability, lumbar ribs, or any other
    signs of developmental toxicity in pups of mothers treated with
    2-ethylbutyric acid, 2-methylvaleric acid, or 5-methylhexanoic acid.
    The highest dose levels of 200 mg/kg bw per day for 2-ethylbutyric
    acid, 250 mg/kg bw/day for 2-methylvaleric acid, and 400 mg/kg bw/day
    for 5-methylhexanoic that did not produce any signs of developmental
    toxicity are >10 000 times their respective daily  per capita 
    intakes ("eaters only") from use as flavouring substances in the USA
    and Europe (see Table 1).

        All dams treated with 2-ethylhexanoic acid exhibited motor
    depression. Developmental effects were observed in pups of mothers
    treated with 2-ethylhexanoic acid at both dose levels, including
    reduced progeny viability, full-litter resorptions, apparent
    stillbirths, postnatal deaths, reduced pup weights, external

    malformations, skeletal malformations, and lumbar ribs (Narotsky  et
    al., 1994). 2-Ethylhexanoic acid produced developmental toxicity only
    in the presence of maternal toxicity.

    2.1.3.7  Special studies on peroxisome proliferation

        Concentrations of 0.1 or 0.5 mM 2-ethylhexanol did not induce
    palmitoyl CoA oxidase activity (a marker enzyme for peroxisome
    proliferation) in rat hepatocytes  in vitro (Rhodes  et al., 1984).
    A concentration of 1 mM 2-ethylhexanol was reported to induce
    significantly (approximately 9 times the control level) the activity
    of carnitine acetyltransferase (a peroxisomal enzyme) in cultured rat
    hepatocytes  in vitro, while a concentration of 0.2 mM did not induce
    this enzyme (Gray  et al., 1982).

        In an  in vivo study, Rhodes  et al. (1984) reported that the
    administration of 1 mmol 2-ethylhexanol/kg bw per day (approximately
    130 mg/kg bw per day) to five male Wistar rats by gavage for 14 days
    did not result in hepatic peroxisome proliferation in the rats.

        Moody & Reddy (1978), however, reported that in five male F344
    rats fed 2% 2-ethylhexanol in the diet (approximately 1000 mg/kg bw
    per day) for 3 weeks, induction of hepatic peroxisome proliferation
    occurred. Significant increases (using a Student's t test) in
    activities of the enzymes hepatic catalase and carnitine
    acetyltransferase also were reported in the rats treated with
    2-ethylhexanol.

        Hodgson (1987) conducted a peroxisome induction study in groups of
    F344 male and female (five/sex/group) by administering doses of 100,
    320 or 950 mg 2-ethylhexanol/kg bw per day by gavage for 21 days. A
    significant reduction in body weight gain was observed in the
    high-dose group rats with no effect on food consumption. Both sexes of
    rats exhibited significant hepatomegaly at a dose of 950 mg
    2-ethylhexanol/kg bw per day. The activity of cyanide-insensitive
    palmitoyl CoA oxidation was increased in a dose-related manner in
    males, with a 1.6-fold increase above controls at 320 mg/kg bw per day
    and a 5.4-fold increase at 950 mg/kg bw per day. In females, a
    3.1-fold increase in palmitoyl CoA activity was reported at the
    high-dose level. Lauric acid hydroxylase activity of peroxisomes was
    observed (using electron microscopy) in the hepatocytes of the rats of
    the high-dose group.

        Keith  et al. (1992) examined the dose-response relationship for
    2-ethylhexanol with respect to peroxisome proliferation in rats and
    mice. Groups of five male and five female Alderley Park rats and mice
    were administered doses of 0, 140, 350, 700, 1050 or 1750 mg
    2-ethylhexanol/kg bw per day for 14 days by gavage. Rats in the
    high-dose group exhibited toxic effects, leading to death of the rats.
    A dose-related increase in the relative liver weight of both rats and
    mice was observed, with the increase being significant in rats at the
    700 and the 1050 mg/kg bw per day dose levels (rats in the high-dose
    group died or were killed during the study), in male mice at the three
    highest dose levels (700, 1050 and 1750 mg/kg bw per day), and in

    female mice at the highest dose level (1750 mg/kg bw per day).
    2-Ethylhexanol administration resulted in a virtually linear
    dose-related induction of peroxisomal -oxidation (measured as
    palmitoyl CoA oxidation activities) in both rats and mice. The level
    of induction was greatest in the male mice. The dose at which this
    effect became statistically significant was not stated. The increase
    in rats and female mice was approximately 4-fold at the highest dose,
    while for male mice, the increase was approximately 12-fold at the
    highest dose.

    2.1.3.8  Special study on immunotoxicity

        3-Methylpentanoic acid was administered to 6- to 8-week-old female
    CD-1 or B6C3F1 mice in corn oil by gavage for 5 days at dose levels
    of 0, 175, 350 or 700 mg/kg bw per day. Cell-mediated immunity was
    assessed by conducting a host resistance assay  (Listeria 
     monocytogenes bacterial challenge). Humoral immunity was measured by
    the antibody plaque-forming cell (PFC) response to sheep erythrocytes.
    Body weights, lymphoid organ weights and spleen cellularity were also
    measured and were normal as compared to controls. 3-Methylpentanoic
    acid did not modulate the cell-mediated or humoral immune response
    (Gaworski  et al., 1994).

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    See Also:
       Toxicological Abbreviations