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    BENZYL ACETATE, BENZYL ALCOHOL, BENZALDEHYDE, AND BENZOIC ACID 
    AND ITS SALTS

    First draft prepared by E. Vavasour, Chemical Health Hazard Assessment
    Division, Bureau of Chemical Safety, Food Directorate, Health
    Protection Branch, Health Canada, Ottawa, Ontario, Canada

    Explanation

    Biological data

         Biochemical aspects
              Absorption, distribution, and excretion
              Biotransformation
              Effects on enzymes and other biochemical parameters

         Toxicological studies
              Acute toxicity
              Short-term toxicity
              Long-term toxicity and carcinogenicity
              Reproductive toxicity
              Developmental toxicity
              Genotoxicity
              Special studies: Effects on the pancreas

         Observations in humans

    Comments

    Evaluation

    References

    1.  EXPLANATION

         All of these substances were evaluated previously by the
    Committee, most of them individually. Benzyl acetate was evaluated at
    the eleventh, twenty-seventh, twenty-ninth, thirty-first, thirty-
    fifth, and forty-first meetings (Annex 1, references 14, 62, 70, 77,
    88, and 107); monographs or monograph addenda were prepared at the
    eleventh, thirty-fifth, and forty-first meetings (Annex 1, references
    15, 89, and 108). Benzyl alcohol was evaluated at the twenty-third
    meeting (Annex 1, reference 50). Benzaldehyde was evaluated at the
    eleventh meeting, when a monograph was prepared (Annex 1, references
    14 and 15). Benzoic acid and its salts were evaluated at the sixth,
    ninth, seventeenth and twenty-seventh meetings (Annex 1, references 6,
    11, 32, and 62).

         With the exception of sodium benzoate, which is used as a food
    preservative, all of the other compounds are used in foods as
    flavouring ingredients (benzyl alcohol and benzyl benzoate are also
    used as carrier solvents in foods). In addition, further human
    exposure occurs due to the natural occurrence of these compounds in
    foods, endogenous formation of benzoate through the phenylalanine-
    tyrosine pathway, and a variety of uses such in soaps and cosmetics,
    pharmaceuticals, insect repellents, pesticides, and other industrial
    uses. Since these compounds are metabolized along a common pathway,
    they have been grouped under the same ADI of 0-5 mg/kg bw.

         At its forty-first meeting, the Committee recommended that a full
    review of these substances be conducted in order to determine whether
    additional studies would be required. In particular, the absence of
    studies of reproductive and developmental toxicity was noted. At the
    present meeting, studies on disposition and metabolism, short- and
    long-term treatment, genotoxicity, reproductive and developmental
    toxicity, and human observations were reviewed and evaluated. While
    few data pertaining specifically to benzyl benzoate were located, it
    was considered to be adequately represented by the existing database
    for the other compounds, since it is hydrolysed to benzyl alcohol and
    benzoic acid. The following monograph is a compilation of studies from
    the previous monographs and monograph addenda and those reviewed for
    the first time at the present meeting.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

    Benzyl acetate

         Benzyl acetate is absorbed from the gastrointestinal tract,
    through the lungs, and through the intact skin. It is hydrolysed in
    man to benzyl alcohol and acetate; the benzyl radical is oxidized to
    benzoic acid and excreted as hippuric acid (Snapper  et al., 1925).

         Benzyl acetate was readily hydrolysed  in vitro by a pancreatin
    preparation (Grundschoser, 1977). Benzylmercapturic acid and hippuric
    acid were isolated from the urine of rats that had been injected
    subcutaneously with benzyl acetate (Clapp & Young, 1970).

         Male B6C3F1 mice and Fischer 344 rats were treated either
    intravenously or orally with 14C-benzyl acetate. The intravenous
    dose was equivalent to 10 mg/kg bw for mice and 5 mg/kg bw for rats.
    For oral administration, benzyl acetate was dissolved in corn oil and
    administered at doses equivalent to 10, 100, or 1000 mg/kg bw for mice
    and 5, 50, or 500 mg/kg bw for rats. The compound was readily absorbed
    from the gastrointestinal tract of both species, and about 90% of the
    total dose was recovered as urinary metabolites after 24h. A small
    proportion (0.3-1.3%) of the total dose was excreted in the faeces
    after both intravenous and oral administration. Elimination of benzyl
    acetate as carbon dioxide or volatile substances was minimal after
    intravenous treatment and consequently was not determined after oral
    treatment. Analysis of tissues of animals sacrificed 24 h after
    intravenous or oral administration of labelled compound showed no
    14C activity, indicating that elimination of the label was virtually
    complete by this time. This clearance pattern indicates that benzyl
    acetate is readily absorbed and excreted after oral administration.
    The relative amounts of benzyl acetate absorbed, metabolized, and
    excreted were apparently unaffected by the size or number of doses
    administered. Repeated treatment of rats with benzyl acetate at
    500 mg/kg bw per day for 14 days, followed by a single dose of
    labelled compound did not change the clearance pattern. More than 90%
    of the radiolabel in the urine was present as hippuric acid, with
    minor amounts as benzyl alcohol and benzylmercapturic acid (up to 4%);
    no unchanged benzyl acetate was found, and the levels of benzoyl
    glucuronide were not measured. There was no evidence to suggest
    saturation or reduction of metabolic capacity in either species over
    the dose range tested (Matthews & Burka, 1984; Abdo  et al., 1985; US
    National Toxicology Program, 1986).

         Male Fischer 344 rats received [methylene-14C]benzyl acetate by
    gavage as the pure substance or in corn oil or propylene glycol at
    doses of 5, 250, or 500 mg/kg bw, and radiolabel was measured in
    faeces, urine, plasma, and tissues. Metabolites were identified in
    urine and plasma by high-performance liquid and thin-layer
    chromatography. Absorption of benzyl acetate was more rapid at the
    lower doses and in the absence of a vehicle. The bulk of the
    administered dose (70-89%) was excreted in the urine over the initial
    24-h period, and very little (about 4%) was found in the faeces after
    72 h. The elimination of benzyl acetate and metabolites was
    essentially complete by three days, as negligible residues were found
    in tissue, regardless of whether benzyl acetate was given pure or in
    corn oil. Since benzyl acetate was not found in the plasma or urine at
    any time after administration, the authors concluded that it was
    readily hydrolysed. Small amounts of benzyl alcohol, the initial
    product of ester cleavage, were detected only in plasma samples. At
    the higher doses, benzoic acid was the major metabolite in plasma,
    while at 5 mg/kg bw, hippuric acid (the glycine conjugate of benzoic
    acid) predominated. Hippuric acid was the major metabolite in urine,
    and the proportion of the original dose it represented was not
    significantly affected by dose. The proportion of the dose present as
    benzoyl glucuronide increased with dose, interpreted by the authors as
    a limited capacity for glycine conjugation. A small proportion of the
    total dose (1.0-3.6%) was excreted in the urine as benzoic acid and
    benzylmercapturic acid, and the proportion was not affected by dose or
    vehicle (Chidgey & Caldwell, 1986).

         [methylene-14C]Benzyl acetate was applied dermally to the
    shaved backs of male Fischer 344 rats at a dose of 100, 250, or
    500 mg/kg bw in order to study the metabolism and disposition of
    benzyl acetate applied by this route. After administration of the pure
    compound, 28-48% of the dose was recovered at the application site and
    26-46% was absorbed and excreted in the 0-24-h urine. Smaller amounts
    of the administered dose were recovered in the 24-48-h urine (< 2%),
    the 48-72-h urine (< 1%), the 0-72-h faeces (< 3%), and the carcass
    after 72 h (< 2%). The proportion absorbed was unaffected by dose,
    area of application, or use of ethanol as a vehicle. Excretion in the
    urine during the first 24 h after treatment accounted for about 95% of
    the absorbed dose. At all doses, the major urinary metabolite was
    hippuric acid (about 95%); benzoyl glucuronide, benzoic acid, and
    benzylmercapturic acid each accounted for 1-2% of the urinary
    radiolabel. Benzoyl glucuronide accounted for a larger proportion of
    the metabolites at 500 mg/kg bw than at 250 mg/kg bw (Chidgey
     et al., 1987).

         The effect of aging on the disposition of benzyl acetate was
    studied in male Fischer 344 rats and C57Bl/6N mice. Rats aged 3-4, 9,
    and 25 months, received a single oral dose of 5 or 500 mg/kg bw
    14C-benzyl acetate in corn oil or a single intravenous dose of
    5 mg/kg bw. The mice, aged 2, 13, or 25 months, received a single oral

    dose of 10 mg/kg bw in corn oil. Urine and faeces were collected for
    96 h. The major route of elimination in the rats was the urine, most
    of the radiolabel being excreted within 24 h in rats of all ages
    tested. More than 90% of the radiolabel in the urine was identified as
    hippuric acid and minor amounts as benzylmercapturic acid, the only
    other urinary metabolite. There was no dose- or age-related difference
    in the percentage excreted as hippuric acid at any time; however, the
    excretion of benzylmercapturic acid was affected by both dose and age:
    at the low dose, the proportion excreted in 25-month-old rats (2%) was
    significantly greater than that in 3-4-month and 9-month-old rats
    (1%); at the high dose, benzylmercapturic acid comprised 2% of
    metabolites in both 3-4-month- and 25-month-old rats. The percentage
    of the total dose excreted in the faeces was slightly affected by age,
    with a small but statistically significant decrease in the 25-month-
    old group, but not by dose. After intravenous administration of
    5 mg/kg bw benzyl acetate, four metabolites were identified in the
    plasma: benzyl alcohol, benzoic acid, hippuric acid, and benzyl
    glucuronide, and the plasma levels of hippuric acid and benzoyl
    glucuronide were significantly higher in 25-month-old rats than those
    at other ages. Most of the radiolabel in bile was associated with
    benzyl alcohol (90%), with minor amounts in benzoic acid (8%) and
    hippuric acid (2%). Comparison of the percentage of radioactivity
    excreted in bile with that retrieved in the faeces suggested that
    enterohepatic recirculation of benzyl acetate-derived radiolabel had
    occurred; the percentage of the total dose excreted in the bile was
    significantly lower in aged than young rats, confirming the decrease
    in faecal excretion and the higher plasma levels of benzyl acetate-
    derived radiolabel.

         The major route of excretion in mice receiving an oral dose of
    10 mg/kg bw benzyl acetate was the urine. Significantly less
    radiolabel was excreted in the 24-h urine of 25-month-old mice than in
    that of 2- or 13-month-old animals. Hippuric acid was the main urinary
    metabolite detected in all groups, constituting 93-96% of the total
    radiolabel. This was the only metabolite detected in the urine of
    2- and 25-month-old mice, while in 13-month-old mice, small amounts of
    benzaldehyde (3%) and benzylmercapturic acid (2%) were occasionally
    detected. Faecal excretion was a minor route and the amount was
    similar in all age groups, comprising about 1.0-1.5% of the total
    administered dose. The authors concluded that aging changes minor
    routes of metabolism and excretion of benzyl acetate occur but not the
    formation of hippuric acid from benzyl acetate (McMahon  et al.,
    1989).

         The effects of gavage and dietary administration on the
    toxicokinetics of benzyl acetate were compared in B6C3F1 mice and
    Fischer 344 rats. The plasma concentrations of benzoic acid, benzyl
    alcohol, and hippuric acid were measured at intervals of 24 h in
    groups of six rats and 12 mice given 500 and 1000 mg/kg bw benzyl
    acetate, respectively, in corn oil by gavage, and at intervals over a

    15-h period in 10 rats and 10 mice fed 10 800 ppm and 2700 ppm benzyl
    acetate, respectively, in the feed  ad libitum for seven days. The
    authors calculated that the latter doses represented about 615 and
    648 mg/kg bw in rats and 850 and 900 mg/kg bw in mice, which
    approximated the doses administered by gavage. [The calculation for
    the mouse appears to be incorrect, since, using a standard conversion
    factor, the dose would appear to be closer to 400 mg/kg bw.] The rate
    of hydrolysis of benzyl acetate in plasma was measured by adding pure
    compound to control rat plasma at room temperature. Hydrolysis was
    rapid under these conditions, with a half-life of about 4 min. Since
    partial inhibition was noted when sodium fluoride, an esterase
    inhibitor, was added, it was concluded that plasma esterases
    contributed to the rapid hydrolysis. After administration by gavage,
    benzyl acetate was not detected in plasma samples taken at any time,
    and benzyl alcohol was not detected in plasma after 5 min in mice and
    10min in rats. Benzoic acid and hippuric acid levels in the plasma
    rose rapidly after gavage, increasing to peak concentrations after
    about 3 h. The plasma concentrations of benzoic acid after
    administration in the feed were much lower than those after gavage--
    about 40-fold less in the rat and 300-fold less in the mouse--while
    the plasma concentrations of hippuric acid were similar after both
    routes of administration. The prolonged presence of hippuric acid in
    the plasma in both species after both routes of administration
    indicates a zero-order formation mechanism. The authors suggested that
    the difference in the plasma levels of metabolic products of benzyl
    acetate were the cause of the different carcinogenic responses after
    gavage and dietary administration in the long-term studies in rats and
    mice conducted by the US National Toxicology Program (see sections
    2.2.3.1 and 2.2.3.2) (Yuan  et al., 1993).

    Benzyl alcohol

         Blood and urine samples were collected from 14 term and nine
    pre-term infants who received similar intravenous doses of benzyl
    alcohol in medications, in order to estimate the levels of benzoic and
    hippuric acids. The mean peak concentrations of benzoic acid in the
    plasma of pre-term babies were almost 10times higher than those in the
    term newborns. The normalized area under the concentration-time curve
    (AUC) for benzoic acid in plasma was also significantly higher in the
    pre-term babies. In addition, larger percentages of benzyl alcohol
    doses were found as benzoic acid in the urine of pre-term babies and
    less as hippuric acid. These results suggest that hippuric acid
    formation is deficient in pre-term newborms (Lebel  et al., 1988).

    Benzoic acid and benzoate salts

         The liver is the main site of conjugation with glycine in man and
    in most experimental animals, except the dog, in which the kidney is
    the main site of biosyntheses (Friedmann & Tachau, 1911; Snapper
     et al., 1925). Sheep appear to have a reduced capacity to conjugate
    benzoic acid with glycine. Infusion of increasing amounts of benzoic

    acid into the rumen at levels up to 1.8 g/kg bw led to a progressive
    decrease in conjugation and increasing excretion of free benzoic acid
    in the urine. Doses of 1.1 and 1.8 g/kg bw were toxic, leading to
    death. Potassium deficiency also occurred, seen as the usual symptoms
    of severe muscular weakness and tremors (Martin, 1966). Benzoates
    exist in the free state in blood and are not bound to proteins. In the
    dog, kidney clearance was estimated to be 0.90-1.89% (Knoefel & Huang,
    1956).

         Since for many years a liver function test was used in man that
    was based on the urinary excretion of hippuric acid after a test dose
    of benzoic acid (6 g orally or 1.5-2.0 g intravenously), much
    information exists on the excretion of benzoic and hippuric acids in
    man. Normal urinary excretion of hippuric acid was estimated to be
    1.0-1.25 g/day, equivalent to 0.7-1.7 g of benzoic acid (Stein
     et al., 1954). Other determinations of the normal excretion in man
    and rat yielded values of 1-3 mg/kg bw (Armstrong  et al., 1955). The
    maximal rate of excretion after ingestion of benzoic acid was
    17 mg/min for hippuric acid and 0.67 mg/min for benzoylglucuronic
    acid, equivalent to 24 g/day as benzoic acid (Schachter, 1957). Up to
    10 g of benzoate are excreted quantitatively (Barnes, 1959). After
    high intakes of sodium benzoate, up to 3% is conjugated with
    glucuronic acid, and all metabolites are eliminated completely within
    14 h (Schachter, 1957). Up to 75-80% of an administered dose of
    benzoic acid is eliminated within 6 h. Sodium benzoate also decreases
    excretion of uric acid (Quick, 1931), urea, and ammonia in man (Lewis,
    1914).

         Benzoic acid is rapidly absorbed (Schanker  et al., 1958) and
    rapidly and completely excreted in the urine (Schachter, 1957; Barnes,
    1959). One healthy man given 6, 9, 13.9, 34.7, and 69.3 mmol sodium
    benzoate showed complete elimination of the drug within 10-14 h
    (Schachter, 1957). Sodium benzoate does not accumulate, as shown by
    experiments on the distribution and elimination of 1-14C-labelled
    compound administered intraperitoneally, orally, or subcutaneously to
    rats. Virtually quantitative excretion occurs in the urine within one
    to two days; less than 1% of radiolabel appears in the faeces and a
    few parts per million in organs. All of the radiolabel was identified
    as benzoic acid (Lang & Lang, 1956). Of an oral or subcutaneous dose
    of labelled benzoic acid, 90% appeared in the urine as hippuric acid,
    0.1% occurred in expired carbon dioxide, and 2% remained in the
    carcass (Bernard  et al., 1955).

         The maximal urinary excretory rate achieved depends on the dose
    of benzoate given. Limiting values of hippuric acid excretion were
    approached in man at a dose of 14 mmol (Schachter, 1957), due to
    limitations on the availability of glycine (Quick, 1933). In rats, the
    tolerance of large doses of benzoic acid depends on the addition of
    adequate amounts of glycine to the diet, leaving sufficient glycine
    for protein synthesis. Preformed glycine is usually used, although

    some is synthesized (Quick, 1931; Barnes, 1959). When rats were fed
    1.5% benzoic acid (as the sodium salt) in the diet, they excreted
    > 95% of the drug as hippuric acid in the urine. As the benzoate in
    the diet was increased to 3.75%, the ratio of hippuric acid to total
    benzoic acid in the urine decreased. Additional glycine increased
    elimination to 86-99%. The only other derivative found in significant
    amounts in the urine was benzoyl glucuronide (Griffith, 1929). Dogs
    and rabbits excrete hippuric acid independently of the route of
    administration of benzoic acid (Quick, 1931).

         The pharmacokinetics and metabolism of orally administered sodium
    benzoate in human subjects were studied by simultaneous measurement of
    concentration-time data for benzoic and hippuric acids in plasma and
    urine. After administration of doses of 40, 80, and 160 mg/kg bw, the
    AUC for benzoic acid increased disproportionately with dose, while
    that for hippuric acid increased proportionately with dose. While the
    peak concentrations of benzoic acid in plasma increased with
    increasing dose, the peak concentrations of its metabolite, hippuric
    acid, did not change. These results suggest that transformation of
    orally administered benzoic acid to hippuric acid is a saturable
    process in humans (Kubota  et al., 1988; Kubota & Ishizaki, 1991).

         The rate of excretion of hippuric acid in urine was studied in a
    person who received an oral dose of 250 mg (4.2 mg/kg bw) benzoic
    acid. The concentration of hippuric acid in the urine was maximal
    0.5-1 h after dosing, and excretion was almost complete within 4 h
    (Akira  et al., 1994). When the study was repeated with an oral dose
    of 10 mg (0.16 mg/kg bw), most of the hippuric acid was excreted
    within 0.5-1 h after dosing (Baba  et al., 1995).

    2.1.2  Biotransformation

    Benzyl acetate

         A study was conducted to ascertain the pathway by which benzyl
    acetate is metabolized to benzylmercapturic acid in the rat. Male
    Fischer 344 rats received 500 mg/kg bw benzyl acetate in corn oil by
    gavage, either alone or with intraperitoneal doses of 200 mg/kg bw
    pyrazole (an inhibitor of alcohol dehydrogenase), 10 mg/kg bw
    pentachlorophenol (an inhibitor of sulfotransferase activity), or
    both. Within 24 h, 92% of the dose was retrieved in the urine; a small
    proportion (3%) was recovered in the faeces over 72 h, and less than
    1% remained in the carcass after this time. Treatment with pyrazole or
    pentachlorophenol did not affect the rate or route of excretion, but
    treatment with both agents delayed the excretion of metabolites in the
    urine. Four urinary metabolites of benzyl acetate were identified:
    hippuric acid (74.6%), benzoyl glucuronide (12.9%, benzoic acid
    (3.0%), and benzylmercapturic acid (1.1%). Inhibition of the
    transformation of benzyl alcohol to benzaldehyde by co-administration
    of benzyl acetate and pyrazole increased the proportion of the dose

    excreted as benzylmercapturic acid from 1.1 to 12.0%, and the
    percentage of the dose excreted as benzoyl glucuronide was
    concomitantly reduced from 12.9 to 6.3%. Inhibition of the formation
    of the sulfate conjugate of benzyl alcohol by pentachlorophenol
    inhibited the formation of benzylmercapturic acid but had no effect on
    the other metabolites. The authors concluded that the formation of
    benzylmercapturic acid involves the formation of benzyl sulfate as an
    obligatory intermediate and that therefore it is unlikely that the
    formation of benzylmercapturic acid from benzyl acetate is associated
    with the formation of a reactive metabolite of toxicological
    significance (Chidgey  et al., 1986).

    Benzyl alcohol

         Benzyl alcohol and benzaldehyde were detected within 5 min in
    the plasma of CD-1 mice given single intraperitoneal doses of
    700-1100 mg/kg bw benzyl alcohol in peanut oil. Prior administration
    of pyrazole, an inhibitor of alcohol dehydrogenase, resulted in
    increased plasma levels of benzyl alcohol (203%), and prior
    administration of disulfiram, an inhibitor of aldehyde dehydrogenase,
    resulted in increased plasma levels of benzaldehyde (368%) (McCloskey
     et al., 1986).

         Benzyl alcohol generated by the metabolism of toluene in human
    and rat liver microsomes was further metabolized to benzoic acid and
    benzaldehyde by human, but not rat, microsomes. Human liver microsomes
    metabolized benzyl alcohol to benzaldehyde at a rate of 16.0 µmol/min
    per mg protein. The reaction was inhibited by the addition of carbon
    monoxide, and decreased by raising the pH of the incubation mixture
    from 7.4 to 10, the optimum for alcohol dehydrogenase. The addition
    of sodium azide, a catalase inhibitor, or ADP-ribose, an alcohol
    dehydrogenase inhibitor, had no effect on the reaction. These
    results suggest that cytochrome P450, but not catalase or alcohol
    dehydrogenase, is responsible for the metabolism of benzyl alcohol to
    benzaldehyde in human liver microsomes. Benzyl alcohol does not appear
    to be metabolized by microsomal enzymes in the rat (Chapman  et al.,
    1990).

    Benzaldehyde

         Benzaldehyde is hydrogenated in rabbits to benzoyl alcohol, which
    is further oxidized to benzoic acid and excreted as hippuric acid
    (Bray  et al., 1951).

         In a study of the effects of disulfiram on the oxidation of
    benzaldehyde in rat liver samples  in vitro, the Km values for
    aldehyde dehydrogenase activity were higher in microsomes than in
    mitochondria or cytosol, indicating that microsomal oxidation of
    benzaldehyde is unlikely to be important in the rat. Disulfiram was
    found to inhibit oxidation of benzaldehyde by aldehyde dehydrogenase

    in rat liver slices, by 24% at a benzaldehyde concentration of
    25 µmol/litre and by 13% at a concentration of 250 µmol/litre. At the
    latter concentration, only 12-16% of the benzaldehyde that disappeared
    was converted to benzyl alcohol by the action of aldehyde reductase
    (Hellstrom-Lindahl & Weiner, 1985).

         Evidence for the metabolism of benzaldehyde to benzylmercapturic
    acid was obtained in an experiment in which pure benzaldehyde was
    administered to groups of five male Sprague-Dawley rats at a dose of
    400, 750, or 1000 mg/kg bw per day by gavage for 13 consecutive days.
    Control rats received tap water. Urine was collected and analysed for
    the presence of benzylmercapturic acid after the second, eighth, and
    thirteenth doses; the metabolite was found in the urine of all treated
    animals but none of the controls. Since this metabolite is derived
    from conjugation of glutathione with the sulfate ester of benzyl
    alcohol, these results suggest that benzaldehyde can undergo reduction
    to form benzyl alcohol (Laham & Potvin, 1987).

         The urinary metabolites of benzaldehyde were identified and
    quantified in New Zealand white rabbits after administration of a
    single dose of 350 or 750 mg/kg bw by gavage; a control group received
    water. Urine was collected from all animals for 15 consecutive days.
    About 83% of the administered dose was excreted in the urine of both
    groups. The main urinary metabolite was hippuric acid, comprising 70%
    of the dose in the group receiving the low dose and 67% in the group
    at the high dose, indicating that benzaldehyde is predominantly
    oxidized to benzoic acid (of which hippuric acid is the glycine
    conjugate) in the rabbit. Small amounts of free benzoic acid were also
    detected (1.6 and 1.4%), as was the conjugate with glucuronic acid,
    benzoylglucuronic acid (8.8 and 11.2%). Other metabolites detected
    were benzyl glucuronide (2.9 and 3.0%) and traces of benzylmercapturic
    acid. Endogenously derived hippuric acid and free benzoic acid were
    detected in control rabbits at previously established baseline levels
    (Laham  et al., 1988).

    Benzoic acid and benzoate salts

         Phenylalanine and tyrosine are precursors of endogenous benzoate.
    Experiments with labelled phenylalanine showed that 1-2% is
    metabolized by this pathway. In a study in which humans were given
    14-28 mg/kg bw per day of deuterio-phenylalanine for 4-6 days, rabbits
    were given 50-400 mg/kg bw per day for 6-12 days, and guinea-pigs were
    given 300 mg/kg bw per day for 12 days, no labelled benzoic acid
    was seen in rabbits or guinea-pigs (Bernard  et al., 1955).
    [3-14C]-Phenylalanine given intraperitoneally to rats resulted in
    0.6-1% of the activity as urinary hippuric acid (Haberland  et al.,
    1954).

         1,3,4,5-Tetrahydroxycyclohexanoic acid (quinic acid) may also
    serve as a precursor of benzoic acid in intermediary metabolism
    (Dickens & Pearson, 1951). Several human subjects given 6 g quinic
    acid orally or 250 g of prunes excreted increased amounts of hippuric
    acid during the following 24 h (Quick, 1931). When deuterio-benzoic
    acid was administered to man and rats, it was excreted with its
    deuterium content unchanged. Feeding of hydro-aromatic compounds to
    guinea-pigs with body fluids enriched with deuterium led to urinary
    excretion of deuterio-benzoic acid with a high deuterium content. A
    similarly prepared rat fed 750 mg hydroxybenzoic acid over five days
    excreted urinary benzoic acid enriched in deuterium. When human
    subjects and guinea-pigs were given quinic acid over several days,
    47-72% was converted to benzoic acid and excreted in the urine
    (Bernard  et al., 1955). Four rats irradiated with 700R and four
    controls were given [carboxyl-14C]sodium benzoate intra-peritoneally
    and fasted. Irradiation had no effect on conjugating ability, but the
    irradiated rats excreted less labelled hippuric acid, owing to
    dilution by endogenously produced benzoic acid (Schreier  et al.,
    1954).

         The two known urinary metabolites of benzoic acid are hippuric
    and benzoylglucuronic acidc. Conjugation with glycine and glucuronic
    acid occurs in preference to oxidation because benzoic acid strongly
    inhibits fatty oxidation in the liver. In man, rabbit, and rat,
    benzoic acid is almost entirely excreted as hippuric acid, whereas
    dogs excrete more conjugated glucuronic acid (Williams, 1959). Sheep
    are less able to excrete hippuric acid and excrete large quantities of
    free benzoic acid in their urine (Martin, 1966). The urine of man,
    pig, rabbit, and sheep contains up to 10% benzoylglucuronic acid.

         Male volunteers received oral doses of 2 or 5 g sodium benzoate
    in aqueous solution; the 5-g dose was also given in conjunction with
    5 g glycine in aqueous solution 1 h before treatment with benzoate
    and with 2 g given every hour thereafter. Benzoate was excreted
    quantitatively in the urine as hippurate, with a minor fraction
    excreted as benzoyl glucuronide. No free benzoic acid was detected in
    the urine. Less benzoyl glucuronide was detected after the 2-g dose
    (1.8%) than the 5-g dose (3.3%), and concurrent administration of
    glycine with the 5-g dose reduced the excretion of benzoyl glucuronide
    (0.6%). Hippuric acid was excreted in the urine at an essentially
    constant rate from about 1 to 3 h after administration.
    Co-administration of glycine with benzoate increased the rate of
    excretion of hippurate. These results indicate that, in humans, the
    availability of glycine is rate-limiting for the formation of hippuric
    acid. Urinary excretion of hippuric acid in untreated individuals was
    estimated to be 15-30 mg benzoic acid equivalent per hour (Amsel &
    Levy, 1969).

         The urinary metabolites of benzoic acid were determined after
    oral administration of 14C-benzoic acid, mostly at a dose of
    50 mg/kg bw, to a large variety of species, including primates,
    rodents, carnivores, reptiles, and birds. Excretion of the label was
    rapid in all species except reptiles. The metabolites in most species
    were hippuric acid and benzoyl glucuronide. In most of the herbivorous
    and omnivorous species, including man, monkey, pig, and rodent,
    benzoic acid was excreted in the urine almost entirely as hippuric
    acid. Substantial amounts of the glucuronide of benzoic acid, in
    addition to hippuric acid, were detected in the urine of carnivores,
    except cats (Bridges  et al., 1970).

         The rate of conjugation of benzoic acid with glycine was measured
    in homogenates of 110 samples of human liver and 67 samples of human
    renal cortex. The rates of reaction were 254 ± 90 nmol/min per g liver
    and 321 ± 99 nmol/min per g kidney. The rate of conjugation in the
    liver decreased with age. While the rate of conjugation of benzoic
    acid was greater in the renal cortex than in the liver, the larger
    mass and strategic anatomical position of the liver were considered to
    make it quantitatively the more important organ with respect to
    glycine conjugation (Temellini  et al., 1993).

    2.1.3  Effects on enzymes and other biochemical parameters

    Benzyl acetate

         The possible role of glycine depletion in the toxicity induced by
    large doses of glycine was investigated in groups of male Fischer 344
    rats fed diets containing 0, 20 000, 35 000, or 50 000 ppm benzyl
    acetate for up to 28 days. Two additional groups received diets
    containing 50 000 ppm benzyl acetate and 27000 ppm glycine or
    50 000 ppm benzyl acetate and 32 000 ppm alanine. A third group
    received a diet containing 32 000 ppm alanine and served as an
    isonitrogenous control. Mortality was 0, 0, 10, and 100% in rats fed
    0, 20 000, 35000, and 50 000 ppm benzyl acetate alone. Supplementation
    of the high dose of benzyl acetate with glycine reduced the mortality
    rate from 100 to 10%, but supplementation with alanine had no effect.
    Administration of benzyl acetate induced a dose-related body-weight
    loss, which was alleviated by glycine but not alanine. Neuro-
    behavioural signs (ataxia or convulsions), which were observed in
    all groups receiving benzyl acetate alone or in combination with
    alanine, were not seen in the group receiving benzyl acetate and
    glycine. The authors concluded that supplementation with adequate
    levels of glycine protects against the toxicity of benzyl acetate and
    related compounds that are normally detoxified by conjugation with
    glycine (Abdo & Wenk, 1995).

    Benzyl alcohol

         In a study to evaluate the effects of short-term intake of benzyl
    alcohol on alcohol and aldehyde dehydrogenase activities, groups of
    four male and four female Sprague-Dawley rats received either a 2%
    (v/v) solution of benzyl alcohol as the sole drinking fluid  ad
     libitum or deionized water, for 12 consecutive days. Enzymatic
    activities were determined in the cytoplasmic and mitochondrial
    fractions of liver tissue obtained from the animals after treatment.
    Pretreatment with benzyl alcohol decreased cytoplasmic alcohol and
    aldehyde dehydrogenase activities when ethanol and acetaldehyde,
    respectively, were the substrates, but had no effect on alcohol
    dehydrogenase activity when benzyl alcohol was the substrate. The
    effect on alcohol dehydrogenase activity was seen only in female rats.
    Similarly, benzyl alcohol pretreatment resulted in decreased activity
    of aldehyde dehydrogenase in female rats when acetaldehyde was the
    substrate, but not when benzaldehyde was the substrate (Messiha 1991).

    Benzoic acid and benzoate salts

         Benzoic acid inhibits pepsin digestion, and sodium benzoate
    inhibits trypsin digestion of fibrin, but they have no effect on
    amylase or lipase. Trypsin digestion of casein is only initially
    depressed by sodium benzoate (Kluge, 1933). Benzoic acid is
    a specific, powerful inhibitor of D-amino acid oxidase, a
    10-4 mmol/litre solution resulting in 50% inhibition (Klein & Kamin,
    1964). Concentrations in the range of 10-3 mmol/litre have
    non-specific inhibitory effects on the metabolism of fatty acids,
    including acetoacetate formation (Avigan  et al., 1955).

         Mice fed 3 g sodium benzoate daily for 10 days had a 10%
    reduction in creatine output, probably due to depletion of the glycine
    pool (Polonowski & Boy, 1941).

         Large oral doses of benzoic acid used therapeutically to
    alleviate the effects of inherited urea cycle disorders were studied
    with respect to their effect on intermediary metabolism. Documented
    effects on the urea cycle, gluconeogenesis, fatty acid metabolism,
    carnitine status, and the tricarboxylic acid cycle were attributed to
    sequestering of coenzyme A by benzoate as a result of glycine
    depletion required for the formation of hippuric acid from benzoyl
    coenzyme A. In addition, binding of benzoate to albumin results in
    toxic reactions due to the displacement of other metabolites from
    albumin-binding sites; e.g. bilirubin toxicity and appetite
    suppression result from increased serum tryptophanlevels, which
    increase the turnover of brain serotonin (Tremblay & Qureshi, 1993).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         The results of studies of the acute toxicity of these compounds
    are summarized in Table 1.

        Table 1.  Acute toxicity of benzyl acetate, benzyl alcohol, benzaldehyde,
              benzoic acid, benzoate salts, and benzyl benzoate
                                                                                     

    Species        Route                    LD50           Reference
                                            (mg/kg bw)
                                                                                     

    Benzyl acetate
    Rat            Oral                     2490-3690      Jenner et al.( 1964);
                                                           von Oettingen (1960)
    Rabbit         Oral                     640            von Oettingen (1960)

    Benzyl alcohol
    Mouse          Intraperitoneal          650 (7 days)   McCloskey et al. (1986)
    Rabbit         Oral                     1040           Graham & Kuizenga, (1945)

    Benzaldehye
    Mouse          Intraperitoneal          1150           McCloskey et al. (1986)
    Rat            Subcutaneous             5000           Macht (1922)
    Rat            Oral                     1300           Taylor et al. (1964)
    Guinea-pig     Oral                     1000           Jenner et al. (1964)

    Benzoic acid and benzoate salts
    Rat            Oral (Na salt)           2700           Deuel et al. (1954)
    Rat            Intravenous (Na salt)    1714 ± 124     Spector (1956)
    Rat            Oral (acid)              2000-2500      Ignat'ev (1965)
    Rabbit         Oral (Na salt)           2000           Spector (1956)
    Rabbit         Subcutaneous (Na salt)   2000           Spector (1956)
    Dog            Oral (Na salt)           2000           Spector (1956)
                                                                                     
    
    Benzyl acetate

         Groups of five B6C3F1 mice or Fischer 344 rats of each sex were
    administered single doses of benzyl acetate (purity, 96.0-101.3%) at
    doses of 250, 500, 1000, 2000, or 4000 mg/kg bw in corn oil by gavage
    and were observed for 15 days. All mice receiving 4000 mg/kg bw and
    females receiving 2000 mg/kg bw became inactive immediately after

    treatment. All mice at the highest dose and one male and two females
    at 2000 mg/kg bw died on day 2 of the study. All rats receiving the
    highest dose became inactive 2 h after treatment, and four males and
    two females at this dose died on day 2. No other compound-related
    effects were reported (US National Toxicology Program, 1986).

    Benzyl alcohol

         Groups of 12-30 male CD-1 mice, weighing 23-28 g, were given
    benzyl alcohol as a 10% solution in peanut oil at intraperitoneal
    doses of 500-1500 mg/kg bw and were observed for up to two weeks for
    clinical signs. In animals at all doses between 500 and 1000 mg/kg bw,
    sedation, loss of motor function, and laboured respiration were
    observed. The lethal effect of benzyl alcohol occurred within two
    periods: an initial acute phase, with an LD50 of 1000 mg/kg bw, and
    a delayed lethal effect up to seven days after treatment with an
    LD50 of 650 mg/kg bw. Inhibition of the metabolism of benzyl alcohol
    to benzaldehyde by pretreatment with pyrazole markedly increased
    mortality (McCloskey  et al., 1986).

         Single intravenous doses of pure benzyl alcohol were administered
    to CD2F1, B6D2F1, and C57Bl/6 mice at doses of 0.05-0.2, 0.05-0.4, and
    0.025-0.1 ml/kg bw, respectively. The mice were observed for two
    weeks. Within the first 24 h of observation, convulsions were seen at
    the highest doses, and dyspnoea and reduced motility were seen at all
    doses except the lowest. A decrease in body-weight gain or a slight
    decrease in body weight was noted in B6D2F1 and C57Bl/6 mice at all
    doses except the lowest. Assessment of haemolytic and precipitation
    potential in blood samples from the mice indicated strong activity of
    benzyl alcohol at concentrations of 0.4-3.0 µl/ml (Montaguti  et al.,
    1994).

    Benzaldehyde

         Groups of three male and three female rats were given one-third
    of the LD50 intragastrically for four days and were then killed and
    the livers examined for gross changes. None were detected (Taylor
     et al., 1964).

         The toxicity of benzaldehyde was determined in a study similar to
    that described for benzyl alcohol above (McCloskey  et al., 1986).
    All deaths were observed within 4 h after treatment, with an LD50 of
    1150 mg/kg bw. The most prominent signs of toxicity were tremors,
    sedation, respiratory distress, and, in animals monitored for seven
    days, weight loss, averaging 2.25 g per animal.

    Benzoic acid and benzoate salts

         No LD50 values were available for potassium benzoate. Benzoic
    acid is not acutely toxic to man (Lehman, 1908) or to experimental
    animals at moderate doses (Rost  et al., 1913; Smyth & Carpenter,
    1948).

         An outbreak of poisoning affected 28 cats that had eaten meat
    containing 2.39% benzoic acid. The effects were nervousness,
    excitability, and loss of balance and vision. Convulsions occurred,
    and 17 cats died or were killed. Autopsies showed damage to the
    intestinal mucosa and liver. The sensitivity of cats may be due to
    their failure to form benzoyl glucuronide, and toxic effects may
    develop after ingestion of quantities > 0.45 g/kg bw as single doses
    or 0.2 g/kg bw as repeated doses (Bedford & Clarke, 1971).

    2.2.2  Short-term toxicity

    2.2.2.1  Mice

    Benzyl acetate

         Groups of five B63F/N mice of each sex, aged six weeks, received
    0, 125, 250, 500, 1000, or 2000 mg/kg bw benzyl acetate (purity, 96%)
    in corn oil by gavage daily for 14 days. On day 16, all surviving
    animals were killed and autopsied. All male mice at the highest dose
    had died by day 3 of the study. Weight changes were not dose-related.
    The only effect reported at autopsy was a roughening of the mucosa of
    the stomach in the cardiac region in two males and in all females at
    the highest dose and in one female at 1000 mg/kg bw per day (US
    National Toxicology Program, 1986).

         Groups of 10 B6C3F1 mice of each sex, about eight weeks of age,
    were given 0, 62.5, 125, 250, 500, or 1000 mg/kg bw per day (males) or
    0, 125, 250, 500, 1000, or 2000 mg/kg bw per day (females) of benzyl
    acetate (purity, 96%) in corn oil by gavage, five times a week for 13
    weeks. The animals were observed twice daily for signs of toxicity,
    and clinical examinations were conducted weekly. Necropsies were
    performed on all animals, and 35 tissues and organs, including brain,
    liver, kidney, and stomach, from mice in the control and high-dose
    groups were examined histologically. Seven of the females at the
    highest dose died. Compound-related clinical effects observed at the
    highest dose included trembling, inactivity, laboured breathing, and
    depressed body temperature. At autopsy, no gross or microscopic
    effects were seen (US National Toxicology Program, 1986). Subsequent
    histopathological examination of brain tissue revealed hippocampal
    necrosis in one female at 1000 mg/kg bw per day (US National
    Toxicology Program, 1993).

         Groups of 10 male and 10 female B6C3F1 mice with an average age
    at exposure of 42 days (13 days' quarantine before the test) received
    benzyl acetate (properties consistent with structure and literature
    references; purity, 99%; stability monitored periodically; no
    degradation of bulk chemical observed) in their diet at doses of
    0, 3130, 6250, 12 500, 25 000, or 50 000 mg/kg feed, equal to 0, 425,
    1000, 2000, 3700, and 7900 mg/kg bw per day for males and 0, 650,
    1280, 2980, 4300, and 9400 mg/kg bw per day for females, for 13 weeks.
    Feed was prepared weekly and stored in the dark, and the formulations
    were analysed four times during the study for benzyl acetate
    concentrations, stability, and homogeneity; it contained low,
    biologically insignificant levels of aflatoxins, pesticides, and
    heavy metals. Feed and water were provided  ad libitum; feed
    consumption was recorded daily, and the animals were weighed weekly.
    Haematological and clinical chemical tests (cholesterol and
    triglycerides) and assays for pancreatic enzymes (amylase, lipase,
    carboxypetidase, chymotrypsin, ribonuclease) were performed at
    termination of the study. Tissues from all control animals, females at
    25 000 mg/kg, and all animals at 50000 mg/kg feed were examined
    histologically.

         Statistically significant (P < 0.01), dose-related decreases in
    final body weights were observed in all treated animals in comparison
    with controls. The body-weight decrement exceeded 10% of control
    values in all treated groups, with the exception of males receiving
    the lowest dose. The mean feed consumption of all exposed mice was
    nonsignificantly lower than that of the control groups. Tremor was
    observed in female mice at > 12500 mg/kg feed. At 50000 mg/kg feed,
    one male died and one female mice was killed  in extremis. The
    absolute and relative organ weights of treated animals were influenced
    by the lowered terminal body weight, and all significant differences
    between treated and control groups were attributable to treatment. No
    dose-related effects were observed in haematological, clinical
    chemical, or pancreatic enzyme parameters.

         Histopathological examination revealed hippocampal necrosis,
    cerebellar haemorrhage of the brain, and hepatocellular necrosis in
    one male mouse receiving 50 000 mg/kg feed after 11 weeks of
    treatment. At termination, three female mice receiving 50000 mg/kg
    feed had hippocampal necrosis and depletion of cells of the pyramidal
    layer in the brain (US National Toxicology Program, 1993).

    Benzyl alcohol

         Benzyl alcohol (purity, 99%) was administered in corn oil to
    groups of five male and five female B6C3F1 mice by gavage at doses of
    0, 125, 250, 500, 1000, and 2000 mg/kg bw on five days a week for
    12 doses over a 16-day period. All mice receiving 2000 mg/kg bw and
    one male and two females receiving 1000 mg/kg bw died before the end
    of the study. Lethargy and rough coats were observed in male mice at

    the three highest doses and in female mice at the two highest doses.
    Mice at 1000 and 2000 mg/kg bw had blood in the urinary bladder at
    necropsy (US National Toxicology Program, 1989).

         Doses of 0, 50, 100, 200, 400, and 800 mg/kg bw benzyl alcohol
    (purity, 99%) in corn oil were administered to groups of 10 male and
    10 female B6C3F1 mice by gavage on five days a week for 13 weeks. The
    animals were observed twice daily, and their body weights were
    recorded at the beginning and end of the study. Necropsy was performed
    on all animals, and unspecified tissues from all vehicle controls and
    those at the highest dose were examined histologically. In addition,
    the brains of animals at 400 mg/kg bw per day and of all animals that
    died during the test were examined. Deaths occurred in most groups,
    with the exception of vehicle controls, but all except one of the
    deaths was attributable to the gavage procedure. The final mean body
    weight of males at 800 mg/kg bw per day was 5% lower than that of
    controls; the final mean body weight of female mice at this dose was
    8% lower and that of females at 400 mg/kg bw was 5% lower. Both male
    and female mice at the high dose showed staggering during the first
    and second weeks of the study. No treatment-related histopathological
    effects were observed (US National Toxicology Program, 1989).

    Benzaldehyde

         Benzaldehyde (purity, 99%) was administered by gavage in corn oil
    to groups of five male and five female B6C3F1 mice, aged eight weeks,
    at a dose of 0, 200, 400, 800, 1600, or 3200 mg/kg bw per day on five
    days per week for 16days (12 doses). The mice were observed twice
    daily, and their body weights were recorded weekly. Gross necropsy was
    performed on all animals. All mice at 1600 and 3200 mg/kg bw had died
    by day 3, and one male receiving 800 mg/kg bw had died by day 10. The
    mean body weights of the treated mice that survived to termination
    were comparable to those of the respective vehicle controls. No
    treatment-related gross lesions were observed (Kluwe  et al., 1983;
    US National Toxicology Program, 1990).

         In a 13-week study, benzaldehyde (purity, 99%) was administered
    to groups of 10 male and 10 female B6C3F1 mice, seven to eight weeks
    old, in corn oil by gavage at a dose of 0, 75, 150, 300, 600, or
    1200 mg/kg bw per day on five days per week. The animals were observed
    twice daily, and their body weights were recorded weekly. Gross
    necropsy was performed on all animals, and 35 tissues and organs from
    all control and high-dose animals and all male mice receiving
    600 mg/kg bw per day, including brain, liver, kidney, and stomach,
    were retained for histopathological examination. In addition, the
    spleen, stomach, and kidneys of all female mice at 600 mg/kg bw per
    day and the kidneys and livers of all males at 300 mg/kg bw per day
    were examined histologically. During the first week of the study, nine
    males and one female at the high dose died. By the end of the study,
    the mean body weight of male mice in the next highest dose group

    (600 mg/kg bw per day) was 9% less than that of the vehicle controls,
    while the mean body weight of females at the high dose was comparable
    to that of the respective controls. The only treatment-related effect
    reported was mild-to-moderate renal tubular degeneration in all male
    mice at 1200 mg/kg bw and in one male at 600 mg/kg bw per day. These
    lesions were not observed in males receiving lower doses of
    benzaldehyde or in the female mice (Kluwe  et al., 1983; US National
    Toxicology Program, 1990).

    Benzoic acid and benzoate salts

         Groups of 50 male and 50 female mice were given benzoic acid at a
    dose of 80 mg/kg bw per day, sodium bisulfite at 160 mg/kg bw per day,
    or a mixture of the two at the same levels by gavage. The highest
    mortality rate was observed in mice given the combination. A five-day
    period of food restriction at 2.5 months induced 85% mortality in both
    groups (Shtenberg & Ignat'ev, 1970).

    2.2.2.2  Rats

    Benzyl acetate

         Groups of 15 male and 15 female rats were fed a mixture of
    aromatic esters, including 15.8 mg/kg bw per day benzyl acetate, for
    12 weeks. No adverse effects were noted (Oser, 1967).

         Groups of five male and five female Fischer 344 rats, about six
    weeks old, were given 0, 250, 500, 1000, 2000, or 4000 mg/kg bw per
    day benzyl acetate (purity, 96%) by gavage in corn oil, daily for
    14 days. The surviving animals were killed and autopsied on day 16.
    None of the rats at 4000 mg/kg bw per day survived beyond two days,
    and all rats at 2000 mg/kg bw per day had died within five days. No
    other deaths were reported. Mean body-weight gain was > 10% lower
    than that of controls in males at 500 and 1000 mg/kg bw per day and in
    females at 1000 mg/kg bw per day. The only effect reported at autopsy
    was caecums that were redder than normal in three animals at
    4000 mg/kg bw per day (US National Toxicology Program, 1986).

         Groups of 10 Fischer 344/N rats of each sex, about eight weeks
    old, were given 0, 62.5, 125, 250, 500, or 1000 mg/kg bw per day
    benzyl acetate (purity, 96%) in corn oil on five days per week for
    13 weeks. The animals were observed twice daily for signs of toxicity,
    and clinical examinations were conducted weekly. Necropsies were
    performed on all animals, and 34 tissues and organs from rats in the
    control and high-dose groups, including brain, skeletal muscle,
    kidney, and pancreas, were examined histologically. Male rats at
    1000 mg/kg bw per day and females at 500 and 1000 mg/kg bw per
    day showed clinical symptoms including trembling, ataxia, and
    sluggishness. Two males and one female at the highest dose had died by
    day 86. Only male rats at 1000 mg/kg bw per day showed depressed mean

    body weight relative to controls (21%). At autopsy, thickened stomach
    walls were observed in two of nine surviving males and four females at
    the high dose (US National Toxicology Program, 1986). A subsequent
    microscopic re-examination of the brains revealed hippocampal necrosis
    in eight of eight surviving males and four females at 1000 mg/kg bw
    per day, the severity of the lesions being greater in the males than
    the females (US National Toxicology Program, 1993).

         Groups of 10 male and 10 female Fischer 344 rats, with an average
    age at exposure of 43 days (13 days' quarantine before the test),
    received benzyl acetate (properties consistent with structure and
    literature references; purity, 99%; stability monitored periodically,
    with no degradation of bulk chemical observed) in the diet at doses of
    0, 3130, 6250, 12 500, 25 000, or 50 000 mg/kg feed, equal to 0, 230,
    460, 900, 1750, or 3900 mg/kg bw per day for males and 0, 240, 480,
    930, 1870, or 4500 mg/kg bw per day for females, for 13 weeks. Feed
    was prepared weekly, stored in the dark, and analysed during the study
    for benzyl acetate concentrations, stability, and homogeneity; it
    contained low, biologically insignificant levels of aflatoxins,
    pesticides, and heavy metals. Feed and water were provided  ad
     libitum; the feed consumption was recorded daily, and the animals
    were weighed weekly. After 11 weeks of treatment, haematological and
    clinical chemical (cholesterol and triglycerides) parameters were
    determined; pancreatic enzymes (amylase, lipase, carboxypetidase,
    chymotrypsin, and ribonuclease) were determined in all treated male
    and female rats except those at 50 000 mg/kg feed. At termination,
    liver peroxisomes were examined morphometrically in female rats given
    0, 25000, or 50000 mg/kg feed. Histopathological examinations were
    performed on all control animals and those at 25 000 and 50 000 mg/kg
    feed.

         Nine male and female rats at 50 000 mg/kg feed died or were
    killed when moribund between weeks 2 and 8 of the study. Male rats
    given 25000 mg/kg feed showed a 10% decrease (P < 0.01) in mean
    terminal body weight. The body weights of the one surviving male and
    the one surviving female at 50000 mg/kg feed were less than half that
    of the controls. The final mean body weights of treated male and
    female rats in the other groups were similar to or slightly lower than
    those of the controls. The average feed consumption of males at
    25 000 mg/kg feed and males and females at 50000 mg/kg feed was
    reduced. Tremor, ataxia, and urine stains were observed in rats at
    50000 mg/kg feed. Serum cholesterol was significantly decreased in
    females at 12 500 mg/kg feed (P < 0.01), 25 000 mg/kg feed
    (P < 0.001), and 50000 mg/kg feed (only one female rat alive after
    11 weeks). No other dose-related effects were seen on haematological,
    clinical chemical, or pancreatic enzyme parameters in treated rats.
    The volume, surface, and numerical density of hepatic peroxisomes in
    female rats at 25 000 mg/kg feed were significantly (P < 0.001)
    increased. No differences in organ weights attributable to treatment
    were observed. Histopathological examination of male and female rats
    receiving 50 000 mg/kg benzyl acetate revealed degeneration and

    necrosis of neurons and glial cells in the cerebellum and hippocampus,
    renal tubular degeneration, and degeneration and sarcolemma nuclear
    hyperplasia in skeletal thigh muscles. Testicular tubular atrophy was
    seen in a few male rats receiving 12 500 mg/kg feed benzyl acetate or
    more (US National Toxicology Program, 1993).

    Benzyl alcohol

         Benzyl alcohol (purity, 99%) was administered to groups of five
    male and five female Fischer 344 rats in corn oil by gavage at a dose
    of 0, 125, 250, 500, 1000, or 2000 mg/kg bw on five days per week for
    12 doses over a 16-day period. All rats receiving 2000 mg/kg bw and
    two males and three females receiving 1000 mg/kg bw died before the
    end of the study. The final body weights of male rats at 1000 mg/kg bw
    were 18% lower than those of controls, while the females at this dose
    had a body-weight decrement of < 5%. Lethargy was observed in rats at
    the two highest doses, which also had blood around the mouth and
    nose, subcutaneous haemorrhages, and blood in the urinary and
    gastrointestinal tracts (US National Toxicology Program, 1989).

         Doses of 0, 50, 100, 200, 400, or 800 mg/kg bw benzyl alcohol
    (purity, 99%) in corn oil were administered to groups of 10 male and
    10 female Fischer 344 rats by gavage on five days per week for 13
    weeks. The animals were observed twice daily, and their body weights
    were recorded at the beginning and end of the study. Necropsy was
    performed on all animals, and histological examination (tissues
    unspecified) was performed on all vehicle controls and those at the
    highest dose; the brains of animals at 400 mg/kg bw per day dose group
    were also examined. Eight males and two females at 800 mg/kg bw per
    day, one female at 400 mg/kg bw per day, one male at 200 mg/kg bw per
    day, and one female in the vehicle control group died after treatment.
    Four of the deaths in males and one in females at the high dose group
    were attributed to gavage errors. Males and females at 800 mg/kg bw
    per day exhibited signs of neurotoxicity, which included staggering,
    laboured breathing, and lethargy. After eight weeks, eight males at
    800 mg/kg bw per day had blood around the nose and mouth. After 13
    weeks, the body weights of male and female rats at the high dose were
    7 and 5% lower than those of the respective controls. Treatment-
    related histopathological effects observed in rats at the high dose
    were: necrosis of the dentate gyrus of the hippocampus in nine of nine
    surviving males and seven of seven surviving females; skeletal muscle
    necrosis in five males; thymic congestion, haemorrhage, and atrophy in
    eight males; and nephrosis of the kidney in six of nine surviving
    males, consisting of degeneration and regeneration of the tubular
    epithelium (US National Toxicology Program, 1989).

    Benzaldehyde

         No tissue damage was seen grossly or histopathologically in
    groups of five male and five female rats given 0 or 0.1% benzaldehyde
    in their diet for 27-28 weeks or 1% for 16 weeks, (Hagan  et al.,
    1967).

         Benzaldehyde (purity, 99%) was administered by gavage in corn oil
    to groups of five male and five female Fischer 344 rats at a dose of
    0, 100, 200, 400, 800, or 1600 mg/kg bw per day on five days per week
    over 16 days (12 doses). The rats were observed twice daily, and their
    body weights were recorded weekly. Gross necropsy was performed on all
    animals. All rats that received 1600 mg/kg bw per day died on day 2 of
    the study, and two males and two females receiving 800 mg/kg bw per
    day died before the end of the study. The mean body weights of male
    rats at 800 mg/kg bw were 14% lower than those of controls, and those
    of females were 11% lower, at the end of the study. No treatment-
    related gross lesions were observed (Kluwe  et al., 1983; US National
    Toxicology Program, 1990).

         In a 13-week study, benzaldehyde (purity, 99%) was administered
    to groups of 10 male and 10 female Fischer 344 rats at a dose of 0,
    50, 100, 200, 400, or 800 mg/kg bw per day in corn oil by gavage on
    five days per week. The animals were observed twice daily, and their
    body weights were recorded weekly. Gross necropsy was performed on all
    animals, and 35 tissues and organs from all control animals and those
    at 400 and 800 mg/kg bw, including brain, kidney, and pancreas
    (skeletal muscle not mentioned), were retained for histopathological
    examination. Deaths occurred in six males and three females at the
    high dose and one female at 400 mg/kg bw before the end of the study.
    The terminal body weights of males at 800 mg/kg bw per day were 26%
    lower than those of controls, whereas the terminal body weights of
    treated female rats were comparable to those of controls. Treatment-
    related lesions of the brain, forestomach, liver, and kidney were
    observed in both male and female rats at 800 mg/kg bw per day, which
    included: degeneration and necrosis of the cerebellum and necrosis of
    the neurons of the hippocampus, hyperplasia and/or hyperkeratosis of
    the squamous epithelium of the forestomach, degeneration and necrosis
    (males only) of the liver, and degeneration or necrosis of the tubular
    epithelium of the kidney (Kluwe  et al., 1983; US National Toxicology
    Program, 1990).

    Benzoic acid and benzoate salts

         Groups of five male and five female rats were fed sodium benzoate
    for 30 days at levels of 16-1090 mg/kg bw. There were no effects on
    body weight, appetite, or mortality and no histological changes in
    organs (Smyth & Carpenter, 1948).

         Groups of three male and three female rats were fed diets
    containing 0, 2, or 5% sodium benzoate for 28 days. All animals
    at the 5% level died during the first two weeks after showing
    hyperexcitability, urinary incontinence, and convulsions. Male rats at
    the 2% level had a significant decrease in body weight, and the food
    intake of male and female animals at this dose was decreased in
    comparison with controls (Fanelli & Halliday, 1963). Four groups of
    15 rats were given sodium benzoate at a dietary level of 0 or 5%
    benzoate or 5% benzoate plus 1% glycine for three weeks. The body
    weights of animals fed benzoate were reduced, but less so when 1%
    glycine was added. The total cholesterol content of the liver was
    unaffected by treatment with benzoate, but that of phospholipids was
    significantly reduced. The potassium concentration of skeletal muscle
    was also low at this level. Supplementary glycine corrected the
    potassium and phospholipid deficiencies (Kowalewski, 1960). Twenty-
    eight young rats were given a diet containing 5% sodium benzoate for
    three weeks. Nineteen animals died within two weeks. Food consumption
    was significantly reduced, and most animals developed severe
    diarrhoea. The changes seen at autopsy were haemorrhage in the gut and
    nasal blood crust. Five adult rats on a similar diet died within five
    weeks with severe weight loss (Kieckebusch & Lang, 1960). Groups of
    four to 19 male rats were fed diets containing sodium benzoate at a
    level of 0, 1.5, 2.0, 2.5, 3, 3.25, or 3.75% for 40 days. The average
    growth of animals at all levels was reduced in comparison with that of
    controls, and mortality was high, food efficiency poor, and growth
    severely depressed at levels > 3%. Addition of glycine reduced the
    toxic effects. Animals died after showing incoordination, tremor, or
    convulsions and severe eye inflammation. Groups of 10-15 young male
    rats fed restricted diets containing 0, 1.5, 2.0, 2.5, or 3% sodium
    benzoate had no difference in weight gain, even at the 3% level.
    Supplementary glycine addition again alleviated the weight loss
    (Griffith, 1929).

         Groups of 10 male and 10 female rats, four to five weeks old and
    weighing 110-150 g, were fed diets containing sodium benzoate at a
    level of 0, 0.5, 1, 2, 4, or 8% for six weeks. All rats at 8% and 19
    at the 4% level died within four weeks, but 19 animals at 2%, 18 at
    1%, and 17 at 0.5% survived for six weeks. Significant reductions in
    body-weight gain were seen only in animals at 4 and 8%. The only acute
    toxic effect observed was hypersensitivity. No morphological change
    was seen at autopsy, except for atrophy of the spleen and lymph nodes
    in rats at 4 and 8% (Sodemoto & Enomoto, 1980).

         Groups of 8-10 rats were fed diets containing 1, 2, 4, or 8%
    sodium benzoate for 90 days. Four rats on the 8% diet died within an
    average of 13 days. The weight gain of the four survivors was two-
    thirds that of controls with an identical food intake. Kidney and
    liver weights were significantly higher than those of the control
    group (Deuel  et al., 1954).

    2.2.2.3  Guinea-pigs

    Benzoic acid and benzoate salts

         Groups of four guinea-pigs given 150 mg/kg bw benzoate plus
    benzoic acid daily for 65 days had no adverse effects. Scorbutic
    animals fed the same dose had a reduced lifespan (Kluge, 1933).

    2.2.2.4  Dogs

    Benzoic acid and benzoate salts

         Seventeen dogs given diets containing sodium benzoate or benzoic
    acid at 1000 mg/kg bw for 250 days showed no effect on growth,
    appetite, or well-being; but at higher doses ataxia, epileptic
    convulsions, and death occurred (Rost  et al., 1913).

    2.2.3  Long-term toxicity and carcinogenicity

    2.2.3.1  Mice

    Benzyl acetate

         Groups of 50 B6C3F1 mice of each sex, aged eight weeks, were
    given benzyl acetate (purity, 99-101%) in corn oil by gavage at a dose
    of 500 or 1000 mg/kg bw per day on five days per week for 103 weeks.
    The mice were observed twice daily for signs of toxicity, and body
    weights were recorded weekly for the first 13 weeks, monthly until
    week 91, and every two weeks until the end of the study. Vehicle
    control groups of 50 animals of each sex were given corn oil by
    gavage. Complete gross necropsies and histopathological examinations
    were performed on 40 tissues and organs, including brain, liver,
    kidney, and stomach, from animals found dead and those sacrificed at
    the end of the study.

         The mean body-weight gains of treated and control male mice were
    comparable throughout the study. Treated females had slightly higher
    mean body weights than controls after week 20. The survival of female
    mice was markedly lower in the control (30%) and low-dose groups (36%)
    in comparison with the high-dose group (60%), and high mortality was
    associated with infection, resulting in suppurative inflammation or
    abscesses of the ovaries, uterus, mesentery, peritoneum, or multiple
    organs (26/35 control, 14/32 low dose, and 8/20 high dose). In males,
    there were no significant differences in survival rates between
    controls and treated groups, although a greater number of control than
    treated males died before week 45.

         Hepatocellular adenomas occurred in 0/50 control, 5/49 low-dose,
    and 13/50 high-dose males and 0/50 control, 0/50 low-dose, and 6/50
    high-dose females. The incidence at the high dose was statistically
    significantly increased in both males (P < 0.001) and females

    (P < 0.05) and was also higher than that of historical controls
    treated by gavage: The cumulative historical incidences of
    hepatocellular adenomas in controls treated with corn oil by gavage in
    six studies conducted at the contract laboratory before 3 August 1984
    were 36/298 for male and 11/300 for female B6C3F1 mice. Hepatocellular
    carcinomas were observed in male mice and in females at the high dose,
    but the incidence did not increase with dose: 10/50 control 10/50,
    14/49 low-dose, and 12/50 high-dose males and 1/50 control, 0/50
    low-dose, and 4/50 high-dose females.

         Squamous-cell papillomas or carcinomas of the forestomach
    occurred in male mice and in females at the high dose. The incidence
    in males was 4/49 controls, 4/48 at the low dose, and 11/49 at the
    high dose, and that in female mice was 0/50 control, 0/50 at the low
    dose, and 4/48 at the high dose. Most of the tumours were papillomas.
    The incidence of combined papillomas and carcinomas in animals at the
    high dose, while not statistically significant, exceeded the values
    for historical controls gavaged with corn oil in six studies at
    the contract laboratory: 2/296 for males and 2/297 for females.
    Forestomach hyperplasia was also reported at a significantly higher
    incidence in mice at the high dose (P < 0.05) than in controls: 1/49
    control, 7/48 low-dose, and 22/49 high-dose males and 1/50 control,
    6/50 low-dose, and 17/48 high-dose females (Abdo  et al., 1985; US
    National Toxicology Program, 1986).

         Groups of 60 male and 60 female B6C3F1 mice, with an average age
    at initial exposure of 40 days (11 days of quarantine before test),
    received diets containing benzyl acetate (properties consistent with
    structure and literature references; purity, > 98%; stability
    monitored periodically, and no degradation of bulk chemical observed)
    at a dose of 0, 330, 1000, or 3000 mg/kg feed, equal to 0, 37, 112,
    and 346 mg/kg bw per day for males and 0, 42, 132, and 382 mg/kg bw
    per day for females, for 103 weeks. Feed was prepared weekly, stored
    in the dark, and analysed during the study for benzyl acetate
    concentrations, stability, and homogeneity; it contained low,
    biologically insignificant levels of aflatoxins, pesticides, and heavy
    metals. Feed and water were provided  ad libitum; feed consumption
    was measured daily per cage for five days once every four weeks. The
    animals were weighed weekly during the first 13 weeks of the study and
    every four weeks thereafter. Ten mice of each sex from each group were
    sacrificed after 15 months of exposure. Haematological and clinical
    chemical determinations (cholesterol, triglycerides, alkaline
    phosphatase, creatinine kinase, and sorbitol dehydrogenase) were
    carried out on mice killed at the interim sacrifice, and necropsy and
    a thorough histopathological examination were performed on all
    animals. The brain, right kidney, and liver were weighed.

         The survival rate of treated male mice was similar to that of the
    control group, while survival of treated female mice increased with
    dose, statistically significantly (P < 0.01) in those at 3000 mg/kg
    feed. Almost all of the deaths occurred during the last nine months of
    the study. The average feed consumption of treated mice was similar to
    that of the control groups. All treated mice except females at
    330 mg/kg feed had decreased mean body weights in comparison with
    controls, with weights 13 and 9% lower at termination in males and
    females, respectively (statistics not reported). The observation of
    decreased weight gain at such low levels of intake, with no apparent
    effect on food palatability, is inconsistent with the observations in
    the previous study (Abdo  et al., 1985; US National Toxicology
    Program, 1986), perhaps due to the unreliability of data on food
    consumption in studies conducted with organoleptic test materials.
    Slight, inconsistent, dose-related decreases (significant at the
    P < 0.05 level) in cholesterol, triglyceride, and (females only)
    alkaline phosphatase levels were observed; no dose-related effects
    were seen at haematology. Statistically significant (P < 0.05 or
    lower), dose-related increases in the incidence and severity of non-
    neoplastic lesions of the nasal mucosa and glands were found in all
    treated mice. The nasal lesions consisted of atrophy and degeneration,
    primarily of the olfactory epithelium, cystic hyperplasia of the nasal
    submucosal glands, and exudate and pigmentation of the nasal mucosal
    epithelium. The lesions were most pronounced in male mice and were
    already seen in male and female mice at interim sacrifice. No
    neoplasms or dose-related preneoplastic lesions occurred in the nose.
    A dose-related, negative trend in the incidences of hepatocellular
    carcinoma and hepatocellular adenoma, which was statistically
    significant (P < 0.01) for hepatocellular adenomas in animals at
    3000 mg/kg feed, was seen in male but not female mice (US National
    Toxicology Program, 1993).

    Benzyl alcohol

         Benzyl alcohol (purity, 99%) was given to groups of 50 B6C3F1
    mice of each sex, eight to nine weeks of age, at a dose of 0, 100, or
    200 mg/kg bw per day in corn oil by gavage on five days a week for 103
    weeks. The doses were selected on the basis of those found to induce
    neurotoxic effects (lethargy and staggering) in short-term studies.
    The mice were observed twice daily, and their body weights were
    recorded weekly for the first 12 weeks and once a month thereafter.
    Gross necropsy was performed on all animals, and 50 tissues and
    organs, including brain, liver, kidney, and stomach, from all vehicle
    controls, animals at the high dose, and animals at the other doses
    that died before 22 months or had gross lesions were examined
    histologically.

         The mean body weights of treated and control mice were comparable
    throughout the study. The survival of control females was
    significantly lower than that of animals at the high dose after week
    74, but no other differences in survival were seen: 68% of control,
    66% of low-dose, and 70% of high-dose males; and 50% of control, 62%
    of low-dose, and 72% of high-dose females. No significant treatment-
    related effects were noted at gross necropsy or histopathological
    examination. No increase was seen in the incidence of hepatocellular
    or forestomach neoplasia (US National Toxicology Program, 1989).

    Benzaldehyde

         Groups of 50 male and 50 female B6C3F1 mice, aged eight (male) or
    nine (female) weeks, received benzaldehyde (purity, 99%) at doses of
    0, 200, or 400 mg/kg bw per day (males) or 0, 300, or 600 mg/kg bw per
    day (females) in corn oil by gavage on five days per week for 103
    weeks. The mice were observed twice daily, and their body weights were
    recorded weekly for 13 weeks and monthly thereafter. Gross necropsy
    was performed on all animals, and 38 tissues and organs, including
    brain, kidney, stomach, liver, and tissues with gross lesions, from
    all mice at the high dose, vehicle controls, and all mice that died
    before the end of the study were examined histologically, as were
    gross lesions and stomachs from all mice at the low dose.

         Treatment had no effect on the body weights or survival of male
    or female mice, and no unusual clinical signs were observed. The
    incidence of focal hyperplasia of the forestomach was significantly
    increased in males at the high dose (P < 0.01) and in females at the
    low (P < 0.05) and high ( P < 0.01) doses (males: 7/50, 8/50, 16/50;
    females: 12/50, 23/50, 39/50), while the incidence of squamous-
    cell papillomas of the forestomach was increased significantly only in
    female mice (P < 0.05) (males: 1/50, 2/50, 5/50; females: 0/50, 5/50,
    6/50). Although the incidence of papillomas in male and female mice
    exceeded the historical control values for studies of the US National
    Toxicology Program (1.6% and 1.9%, respectively, for papillomas and
    carcinomas combined), they only slightly exceeded the highest
    incidence in the controls. No squamous-cell carcinomas were considered
    to have been identified in any of the groups. No treatment-related
    effects on the liver were detected (US National Toxicology Program,
    1990).

    Benzoic acid and benzoate salts

         Parenteral administration of benzoic acid has not been shown to
    cause tumours (Hosino, 1951). Groups of 25 male and 25 female mice
    were given benzoic acid at a dose of 40 mg/kg bw per day, sodium
    bisulfite at 80 mg/kg bw per day, or a mixture of the two for 17
    months. Mortality was greater in the groups receiving the mixture
    (62%) than in those receiving the individual substances (32%) at eight
    months. Mortality at 17 months and pathology were not reported
    (Shtenberg & Ignat'ev, 1970).

         Doses of 0.5, 1, 2, 4, and 8% sodium benzoate were administered
    in drinking-water to groups of four male and four female albino Swiss
    mice for 35 days. All of the mice receiving 8% died within three weeks
    of administration, three males and three females at the 4% level died
    during the study period, and the body weights of the surviving animals
    were substantially reduced. On the basis of survival, body weights,
    intake of test material, and histological changes, 2% sodium benzoate
    was chosen as the dose for a long-term study.

         In the main study, a 2% solution of sodium benzoate (purity, 99%)
    was administered in the drinking-water to groups of 50 male and 50
    female five-week-old mice for their lifetime. Groups of 100 males and
    100 females were used as untreated controls. Both treated and control
    animals were 'carefully checked'; their body weights were measured
    weekly, and gross pathological changes were recorded. The animals were
    either allowed to die or were sacrificed when moribund. Complete
    necropsies were performed on all animals, and the liver, spleen,
    kidneys, bladder, thyroid, heart, pancreas, testes, ovaries, brain,
    nasal turbinates, at least four lobes of the lungs, and organs with
    gross pathological changes were examined histologically. The average
    daily intake of sodium benzoate was 124.0 mg for males and 119.2 mg
    for females on the basis of daily water consumption of 6.2 and 5.9 ml,
    respectively. The dose of sodium benzoate was equivalent to 6200 mg/kg
    bw per day for males and 5960 mg/kg bw per day for females. Treatment
    had no effect on survival or the incidence of tumours in the
    limited numbers of tissues investigated. It is uncertain whether
    histopathological end-points other than neoplasmas were investigated
    (Toth, 1984).

    2.2.3.2  Rats

    Benzyl acetate

         Groups of 50 Fischer 344/N rats of each sex, seven weeks old,
    were given benzyl acetate (purity, 99-101%) in corn oil by gavage at
    a dose of 250 or 500 mg/kg bw per day on five days per week for 103
    weeks. The rats were observed twice daily for signs of toxicity, and
    body weights were recorded weekly for the first 13 weeks, monthly
    until week 91, and every two weeks until the end of the study. Vehicle
    control groups of 50 animals per sex were given corn oil by gavage.
    Complete gross necropsies and histopathological examinations of 39
    tissues and organs, including brain, kidney, pancreas, and skeletal
    muscle, were performed on animals found dead and on those sacrificed
    at the end of the study.

         Mean body-weight gains were comparable between treated and
    control groups throughout most of the study. No significant
    differences were found in the survival of treated rats as compared
    with controls. In male rats, 76% of the controls, 92% of the low-dose
    group, and 80% of the high-dose group survived to 104-106 weeks; the

    survival rates for females were 80% of the controls, 72% of the
    low-dose group, and 72% of the high-dose group.

         The incidence of all malignant epithelial tumours in the
    preputial gland (cystadenocarcinoma, adenocarcinoma, and carcinoma)
    was elevated in males at the high dose (control 1/50, low dose 1/50,
    high dose 6/50), but the increase was not statistically significant.
    The incidence of acinar-cell adenomas in the pancreas of male rats was
    22/50 in controls, 27/50 at the low dose, and 37/49 at the high dose,
    the last incidence being significantly greater than that in the
    vehicle controls (P = 0.001). Acinar-cell hyperplasia was also
    observed in male rats, but the incidence did not increase with dose
    (37/50 control, 34/50 low dose, and 36/49 high dose). As these
    incidences were calculated after examination of additional pancreatic
    tissue, the historical incidences of this lesion in vehicle controls
    were not relevant for comparison. When the slides of the pancreas were
    read initially, the incidences of acinar-cell adenomas in males were
    found to be 3/50, 8/50, and 8/49, which are within the range of the
    historical control values. No acinar-cell hyperplasia or adenoma of
    the pancreas was observed in females, and no acinar-cell carcinoma
    were observed in male or female rats. There were increased incidences
    of retinopathy (not specified) and cataracts in males at the high dose
    and females at the low dose, but the authors attributed this effect to
    the proximity of these rats to fluorescent light (Abdo  et al., 1985;
    US National Toxicology Program, 1986).

         Groups of 60 male and 60 female Fischer 344 rats, of an average
    of 41 days at initial exposure (12 days' quarantine before the test),
    received benzyl acetate (properties consistent with structure and
    literature references; purity, >98%; stability monitored
    periodically, and no degradation of bulk chemical observed) at doses
    of 0, 3000, 6000, or 12 000 mg/kg feed, equal to 0, 130, 260, and
    510 mg/kg bw per day for males and 0, 145, 290, and 575 mg/kg bw per
    day for females, for 103 weeks. Ten rats of each sex from each group
    were sacrificed after 15 months. Feed was prepared weekly, stored
    in the dark, and analysed during the study for benzyl acetate
    concentration, stability, and homogeneity; it contained low,
    biologically insignificant levels of aflatoxins, pesticides, and heavy
    metals. Feed and water were provided  ad libitum. The feed
    consumption was measured daily per cage for five days once every four
    weeks. The animals were weighed weekly during the first 13 weeks of
    the study and every four weeks thereafter. Haematological and clinical
    chemical determinations (cholesterol, triglycerides, alkaline
    phosphatase, creatinine kinase, and sorbitol dehydrogenase) and (in
    males only) assays for pancreatic enzymes (amylase, lipase, and
    carboxypeptidase) were carried out on rats at the interim sacrifice.
    Necropsy and a thorough histopathological examination were performed
    on all animals, and the brain, right kidney, and liver were weighed.

         The mean body weights of animals at 12 000 mg/kg feed were about
    5% lower than those of the control groups for most of the study. No
    significant differences in survival rate, average feed consumption,
    clinical findings, the results of clinical chemistry and haematology,
    pancreatic enzyme assays, or the incidences of neoplasms and
    non-neoplastic lesions were observed in treated rats in comparison
    with controls (US National Toxicology Program, 1993).

    Benzyl alcohol

         Benzyl alcohol was administered in corn oil by gavage to groups
    of 50 Fischer 344/N rats of each sex at a dose of 0, 200, or 400 mg/kg
    bw per day on five days a week for 103 weeks. The rats were observed
    twice daily, and body weights were recorded weekly for the first 12
    weeks and once a month thereafter. Gross necropsy was performed on all
    animals; and 49 tissues and organs, including brain, kidney, pancreas,
    and skeletal muscle, from all female rats and from male rats in the
    vehicle control and high-dose groups and those in the other groups
    that died before 22 months or which had gross lesions were examined
    histologically.

         The mean body weights of treated and control animals were
    comparable throughout the study. No compound-related clinical signs
    were observed, although a sialodacryoadenitis viral infection was
    widespread among the study animals in the third month. The survival of
    treated females was significantly lower than that of vehicle controls:
    70% of controls, 34% of low-dose females, and 34% of high-dose
    females; this was due to a much higher incidence of accidental deaths
    related to the gavage process. Survival among the male rats was
    comparable in all groups: 56% of controls, 54% at the low dose, and
    48% at the high dose.

         Cataracts and retinal atrophy were observed at increased
    incidences in rats at the high dose. The authors attributed this
    effect to the proximity of this group of animals to fluorescent light
    for most of the study. An increased incidence of hyperplasia of the
    forestomach epithelium was seen (not statistically significant) in
    male rats: control, 0/48; low dose, 0/19; high dose, 4/50. Haemorrhage
    and foreign material in the respiratory tract seen in treated rats
    that died before the end of the study were suggested by the authors to
    have been the result of either direct deposition of material into the
    lung during gavage 'accidents' or the anaesthetic properties of benzyl
    alcohol resulting in reflux of gavage material and aspiration into the
    lungs. No pancreatic acinar-cell adenomas were reported, and no other
    effects of treatment were seen at gross necropsy or histopathological
    examination (US National Toxicology Program, 1989).

    Benzaldehyde

         Groups of 50 male and 50 female Fischer 344 rats received a dose
    of 0, 200, or 400 mg/kg bw per day benzaldehyde (purity, 99%) in corn
    oil by gavage on five days per week for 103 weeks. The rats were
    observed twice daily, and their body weights were recorded weekly for
    13 weeks and monthly thereafter. Gross necropsy was performed on all
    animals, and 43 tissues and organs, including kidney, skeletal muscle,
    pancreas, and brain, from all male rats, vehicle controls, all females
    at the high dose, and all rats that died before the end of the study,
    were examined histologically, as were 13 tissues and organs from
    females at the low dose.

         Treatment had no effect on the body weights of male or female
    rats throughout the study. The survival of males at the high dose was
    significantly decreased in comparison with controls, and there was a
    significant, dose-related trend to reduced survival in the treated
    groups. The rate of survival among males at the end of the study was
    74% of controls, 58% at the low dose, and 42% at the high dose. There
    was no statistically significant difference in the survival rate of
    treated female rats in comparison with controls: 66% of controls, 66%
    at the low dose, and 58% at the high dose; however, survival tended to
    decrease at an earlier stage in animals at the high dose than in the
    other groups.

         An increased incidence of pancreatic acinar-cell nodular
    hyperplasia and adenomas was noted in males at the high dose
    (hyperplasia: 6/49, 6/49, 12/48; adenomas: 3/49, 2/49, 7/48 in
    control, low-dose, and high-dose animals, respectively). Although the
    incidence of pancreatic adenomas at the high dose was statistically
    significant (P = 0.038), it was within the range of the historical
    control incidences of pancreatic acinar-cell neoplasms at the study
    laboratory (0/49-11/50). No acinar-cell carcinomas were detected in
    any group. A number of other tumours were observed in treated groups,
    at statistically significantly increased incidences in comparison
    with controls, but these either were not dose-related (malignant
    mesotheliomas of the tunica vaginalis and/or mesentery in male rats)
    or were due to increases in early-stage but not advanced-stage
    neoplasms (mononuclear-cell leukaemia in males) (US National
    Toxicology Program, 1990).

    Benzoic acid and benzoate salts

         Three groups of 20 male and 20 female rats were pair-fed for
    eight weeks on diets containing 0, 0.5, or 1% benzoic acid and then
    fed  ad libitum over four generations. Two generations were fed for
    their lifespan, and the third and fourth generations were autopsied
    after 16 weeks. No effects were observed on growth, fertility,
    lactation, or lifespan. Examination  post mortem showed no
    abnormalities (Kieckebusch & Lang, 1960). In another experiment, 20

    male and 30 female rats were fed a diet containing 1.5% benzoic acid
    for 18 months; 13male and 12 female rats served as controls. Fifteen
    animals in the test group and three controls died, and the remaining
    treated animals had reduced body weight and food intake. Experiments
    with groups of 20 treated and 10 control animals of another strain
    gave similar findings (Marquardt, 1960).

         Groups of 10 male and female rats received benzoic acid at
    40 mg/kg bw per day, or sodium bisulfite at 80 mg/kg bw per day, or a
    mixture of the two in the diet for 18 months. Growth was slightly
    reduced, and the erythrocyte sedimentation rate was increased. Rats
    fed benzoic acid developed some tolerance to a lethal dose of the
    compound given terminally. No pathological analysis was reported
    (Shtenberg & Ignat'ev, 1970).

         Groups of 50 male and 52 female Fischer 344 rats, four to five
    weeks old, received diets containing 1% (500 mg/kg bw per day) or 2%
    (1000 mg/kg bw per day) sodium benzoate for 18-24 months. Controls,
    consisting of 25 male and 43 female rats, received basal diet. Food
    intake was adequately controlled to avoid an excess; tap water was
    available  ad libitum. Survival was very poor in all groups, due to
    intercurrent sialodacryoadenitis and mycoplasma infections. All
    surviving animals were sacrificed between 18 and 25 months, all were
    autopsied, and various tissues were examined histopathologically. No
    adverse clinical signs directly attributable to treatment were
    observed, and only negligible differences in average body weight and
    mortality rate were seen between the treated and control groups.
    Although a variety of tumours occurred among treated and control rats
    of each sex, they were of similar type and incidence (Sodemoto &
    Enomoto, 1980).

    2.2.4  Reproductive toxicity

    2.2.4.1  Mice

    Benzyl acetate

         The potential reproductive toxicity of benzyl acetate was
    assessed by examining sperm morphology, vaginal cytology, and the
    weights of male reproductive organs at the end of the 13-week feeding
    study (US National Toxicology Program, 1993). Dietary levels of
    3130-50 000 ppm benzyl acetate had no effect on the weights of the
    epididymis, cauda epididymis, or testis or on sperm motility or
    density or the percent of abnormal sperm. The mean length of the
    estrous cycle of mice at the high dose was significantly greater than
    that of the control group. This effect was associated with a
    significant decrease in body weight (Morrissey  et al., 1988).

    Benzyl alcohol

         Benzyl alcohol was one of several chemicals tested in a
    preliminary screening study in CD-1 mice. The study consisted of
    three phases: two to determine dose and the third phase to assess
    developmental toxicity. In the first phase, benzyl alcohol was
    administered by gavage at a dose of 10, 100, or 1000 mg/kg bw per day
    to groups of three virgin female mice for five consecutive days,
    followed by a seven-day observation period. All animals were observed
    daily for signs of toxicity and mortality; body weights were recorded
    on treatment days 1 and 5 and on days 3 and 7 after treatment. At each
    dose tested, signs of clinical toxicity, including salivation, stained
    fur, and rough coat, were reported. At 1000 mg/kg bw per day, one
    animal had a hunched posture, and another was sacrificed on day 4 of
    treatment after convulsing. The body weights did not appear to be
    affected by treatment. On the basis of the one death at the high dose,
    doses of 200, 380, 720, 1370, and 2605 mg/kg bw per day were selected
    for the second phase of the study.

         The aim of the second phase was to estimate the LD10 of benzyl
    alcohol in pregnant mice. Groups of four pregnant mice were given a
    dose of 200, 380, 720, 1370, or 2605 mgl/kg bw per day on days 6-15 of
    gestation. All surviving animals were sacrificed on day 17 of
    gestation and examined for the presence or absence of viable fetuses.
    All animals were observed daily for signs of clinical toxicity and
    mortality, and body weights were measured. Clinical signs were
    observed intermittently at the three lower doses but were not
    attributable to treatment; a single animal at 380 mg/kg bw per day was
    sacrificed after showing unsteady gait, languid behavior, and pale
    eyes, but similar symptoms were not observed at the next highest dose.
    At 1370 mg/kg bw per day, clinical signs of toxicity affecting all
    animals included unsteady gait, languid behavior, hunched posture,
    tremors, rapid respiration, squinted eyes, convulsions, hyperactivity,
    prostration, and laboured breathing, and two animals died (one was
    sacrificed and one was found dead) on day 4 of treatment. At
    2605 mg/kg bw per day, all animals died before the second day of
    treatment. Body-weight gains of 17.5, 11.1, 6.5, and 7.1 g were
    reported for the animals at 200, 380, 720, and 1370 mg/kg bw per day,
    respectively, between day 1 of treatment and day 2 after treatment,
    indicating a dose-dependent decrease (no statistical analysis was
    presented). All dams at 200 mg/kg bw per day, two of three at
    380 mg/kg bw per day, and two of four at 720 mg/kg bw per day had
    viable fetuses; the other surviving dams at these doses resorbed their
    litters. One dam at 1370 mg/kg bw per day had live fetuses, but the
    other showed no evidence of live pups or resorptions. The LD10 for
    benzyl alcohol was calculated to be 550 mg/kg bw per day, and this
    dose was used in the third phase of the study.

         In the phase aimed to study developmental toxicity, 50 female
    mice were given benzyl alcohol at 550 mg/kg bw per day by gavage on
    days 6-15 of gestation; a further 50 mice received the corn oil
    vehicle. All dams were allowed to deliver naturally, and pups and dams
    were observed until day 3  post partum, when the experiment was
    terminated. Body weight, clinical observations, and mortality were
    recorded daily throughout treatment and up to day 3  post partum.
    Mortality was not significantly increased in animals given benzyl
    alcohol over that in the control group. One treated mouse showing
    languid behaviour, laboured breathing, and a rough coat died, but no
    other deaths or clinical signs were reported. Maternal body weight and
    body-weight gain during treatment and up to day 3  post partum were
    virtually identical for treated and control animals. All other
    parameters examined, including gestation index, average number of live
    pups per litter, and postnatal survival and pup body weight on days 0
    and 3  post partum, were not significantly different from the control
    values. The authors concluded that, at the predicted LD10, benzyl
    alcohol had no significant effects on the development of CD-1 mice.
    The NOAEL was 550 mg/kg bw per day (York  et al., 1986).

         In another preliminary screening test for the reproductive hazard
    of benzyl alcohol in CD-1 mice, an initial eight-day evaluation was
    used to determine the maximum tolerated dose, which was used in the
    phase on developmental toxicity. Groups of 10 female mice were given
    benzyl alcohol at 0, 160, 325, 645, 1300, or 2595 mg/kg bw per day by
    gavage on eight consecutive days, followed by an eight-day observation
    period. Distilled water was used as the vehicle. The end-points
    evaluated included clinical toxicity, body-weight change, and
    mortality. During treatment, one animal in the control group and one
    each at 160 and 325 mg/kg bw per day died; no deaths occurred at
    645 mg/kg bw per day, but eight deaths occurred at 1300 mg/kg bw per
    day evenly over the 10-day treatment period. All animals at
    2595 mg/kg bw per day died on the first day of treatment. Clinical
    signs of toxicity, including hunched posture, tremors, piloerection,
    prostration, ataxia, and dyspnoea, were observed in the animals at
    1300 and 2595 mg/kg bw per day. Clinical signs were reported in at
    least one mouse at 645 mg/kg bw per day, with piloerection in four
    mice and all other signs in one mouse, suggesting that this dose
    approached the threshold for such effects. Body-weight gain was
    comparable to that of controls in animals at 160 and 320 mg/kg bw per
    day during both the treatment and observation periods. In animals at
    645 and 1300 mg/kg bw per day, body-weight gain during treatment was
    reduced by about 4% in comparison with that of the control group and
    remained at about 7% below control values until the end of the
    observation period. No statistical evaluation of the data was
    presented. The authors concluded that the maximum tolerated dose for
    benzyl alcohol was between 645 and 1300 mg/kg bw per day and chose
    750 mg/kg bw per day as the dose for use in the remainder of the
    study.

         Benzyl alcohol dissolved in distilled water was administered by
    gavage at a dose of 750 mg/kg bw per day to 50 mice on days 7-14 of
    gestation; evidence of copulation was considered the first day of
    gestation. A control group of 50 animals received distilled water
    only. All animals were allowed to deliver their litters and nurse
    their pups for three days, at which time necropsies were performed.
    Maternal body-weight gain and mortality, mating, gestation, numbers of
    live and dead pups per litter, total litter weight on days 1 and 2
     post partum, litter weight change between days 1 and 3  post partum,
    and pup survival on days 1 and 3  post partum were recorded.
    During the treatment period, 18 deaths were reported, all of
    which were attributed to treatment; a further death was reported on
    day 15 of gestation, the day after treatment was terminated. Clinical
    signs of toxicity, including hunched posture, tremors, inactivity,
    prostration, hypothermia, ataxia, dyspnoea, swollen or cyanotic
    abdomen, and piloerection, were reported in up to 20 mice during
    treatment. Piloerection was also reported in some animals up to day 3
     post partum, but no other clinical signs were seen after the period
    of administration. No differences were observed in the mating or
    gestation indices, the total number of resorptions, the mean length of
    gestation, or the number of live pups per litter between treated and
    control groups. Maternal body weight, measured on days 4 and 7 of
    gestation, was not significantly different from control values;
    however, statistically significant reductions were reported on day 18
    of gestation (P < 0.001) and on day 3  post partum (P < 0.05).
    Maternal body-weight gain during days 7-18 of gestation was
    significantly lower than that of controls (P < 0.001). Significant
    reductions in pup body weight were reported, including a lower mean
    pup weight per litter on days 1 (P < 0.01) and 3  post partum
    (P < 0.001), a mean litter weight change between day 1 and day 3
     post partum (P < 0.05), and a mean pup weight change between days 1
    and 3  post partum (P < 0.001). No differences in pup survival were
    observed by day 3  post partum. The authors concluded that benzyl
    alcohol may be a reproductive hazard, apparently on the basis of the
    reductions in pup body weights, an effect that was observed in
    conjunction with maternal toxicity evidenced by increased mortality,
    reduced body weights, and clinical toxicity during the period of
    administration. As effects were seen on the dams and fetuses at the
    only dose used in this study, there was no NOAEL. The LOAEL was
    750 mg/kg bw per day (US National Institute of Occupational Safety and
    Health, 1983; Hardin  et al., 1987).

    2.2.4.2  Rats

    Benzyl acetate

         The potential reproductive toxicity of benzyl acetate was
    assessed by examining sperm morphology, vaginal cytology, and the
    weights of male reproductive organs at the end of the 13-week feeding
    study (US National Toxicology Program, 1993). Dietary levels of

    3130-50 000 ppm benzyl acetate had no effect on the weights of the
    epididymis, cauda epididymis, or testis, on sperm motility, or on the
    density or percent of abnormal sperm.

    Benzaldehyde

         A single study was conducted to examine the potential
    reproductive toxicity of benzaldehyde, and the report was available as
    a translation from Romanian. A group of 10 rats of breeding age were
    given 2 mg benzaldehyde in oil (type not specified) by gavage every
    other day for 32 weeks, equivalent to about 5 mg/kg bw per day. Ten
    controls were used. Two pregnancies in each rat, one at 75 days and
    one at 180 days, were studied. The end-points examined included the
    number of pregnant females, number of offspring born, pup body weight
    at days 7 and 21  post partum, and pup viability. At the end of
    treatment, the body weights of control and treated rats were similar:
    265 g and 260 g, respectively. It was reported that fewer females in
    the group given benzaldehyde than in the control group became
    pregnant; however, no data or statistical analyses were presented. The
    authors concluded that treatment did not significantly modify any of
    the parameters studied. No further details were available. The NOAEL
    was about 5 mg/kg bw per day (Sporn  et al., 1967).

    2.2.5  Developmental toxicity

    2.2.5.1  Rodents

    Benzyl acetate

         A study of developmental toxicity was conducted with benzyl
    acetate in Wistar rats, 10-15 weeks old, which included an initial
    dose-finding phase. Groups of six pregnant rats were given 0, 250, or
    500 mg/kg bw per day on days 6-15 of gestation; the finding of sperm
    in a vaginal smear was considered to be day 0 of gestation. On day 20
    of gestation, all animals were examined and the state of the fetuses
    was determined. No significant effects on maternal weight gain or the
    fetuses were reported in this portion of the study; no other data were
    presented. On the basis of these results, doses of 0, 10, 100, 500,
    and 1000 mg/kg bw per day were selected for the next phase.

         In the study of developmental toxicity, groups of 20 rats were
    given benzyl acetate at 0, 10, 100, 500, or 1000 mg/kg bw per day in
    olive oil by gavage on days 6-15 of gestation; the finding of sperm in
    a vaginal smear was considered to be day 0 of pregnancy. A second,
    untreated control group was also included. During pregnancy, all
    animals were examined daily, and food intake and maternal body weights
    were recorded every other day. All pregnancies were terminated on day
    20 of gestation, and all maternal animals were examined for gross
    internal changes, and the numbers of implantations, corpora lutea,
    resorptions, and live and dead fetuses were recorded. All fetuses were

    examined for external malformations, sexed, and weighed. One-half of
    the fetuses from each litter were examined for skeletal malformations
    with alizarin red S and one-half for malformations of the internal
    organs by Wilson's method.

         During administration of benzyl acetate, no mortality or changes
    in maternal health were reported. Weight gain was comparable in all
    treated and control groups for the intervals days 0-6 of gestation
    (before treatment) and days 6-16 of gestation. Weight gain in dams
    given 500 or 1000 mg/kg bw per day was slightly but not significantly
    reduced on days 16-20 of gestation, but food intake was not
    significantly different from that of controls at any time during or
    after the period of administration. At termination of the pregnancies,
    no abnormalities were observed in the internal organs of the dams, nor
    were there any differences in the numbers of corpora lutea,
    implantations, live or dead fetuses, or resorptions, the implantation
    ratio, the sex ratio, or placental weight between treated and control
    groups. Fetal body weights were significantly reduced (P < 0.05) at
    the high dose and significantly elevated (P < 0.05) at 10 and
    100 mg/kg bw per day. Neither external nor internal malformations were
    reported in treated or control groups; however, the combined incidence
    of internal organ variations, which included slight dilatation of the
    lateral ventricle and renal pelvis, and the presence of a laevo-
    umbilical artery was significantly increased at 500 and 1000 mg/kg bw
    per day. When the incidence of individual variations was analysed,
    however, only dilatation of the renal pelvis at the high dose was
    significantly increased. Skeletal malformations were limited to a
    single fetus at the high dose, which had fused ribs; however, the
    incidence of fused ribs in this group was not statistically
    significantly increased over control values. The incidences of total
    and individual skeletal variations, including wavy ribs, dumb-bell-
    shaped vertebrae, the absence or splitting of thoracic vertebrae, the
    presence of lumbar ribs, and delayed ossification, in fetuses at the
    high dose were significantly increased over those in the control
    group. These variations were seen in multiple litters. The incidences
    of skeletal variations in the remaining groups treated with benzyl
    acetate were within the range seen in the control group. The authors
    concluded that benzyl acetate is not teratogenic and suggested that
    the increased incidences of skeletal and internal variations were due
    to slight maternal toxicity and to the significant decrease in fetal
    body weight. The NOAEL was 500 mg/kg bw per day on the basis of
    fetotoxic effects at the highest dose tested (Ishiguro  et al.,
    1993).

    Benzoic acid and benzoate salts

         Groups of Sprague-Dawley rats (number per group unspecified) were
    injected intraperitoneally with sodium benzoate at a dose of 100, 315,
    or 1000 mg/kg bw on days 9-11 or 12-14 of gestation; evidence of
    mating was considered day 1 of gestation. Control animals were treated

    with sodium chloride at doses of 90 or 100 mg/kg on the same days as
    the treated groups. Fetal body weight was reported to be reduced in
    groups given sodium benzoate at 1000 mg/kg bw per day on gestation
    days 12-14 and 9-11. The incidence of deaths  in utero was 12% in the
    group given the high dose on days 12-14 of gestation and 16% in the
    group given the high dose on days 9-11. These rates were reported to
    be greater than those in the sodium chloride control group; however,
    neither data on the number of deaths  in utero in the sodium chloride
    group nor statistical analyses were presented. No gross anomalies were
    observed in the groups given sodium benzoate on days 12-14 of
    gestation, whereas an increase in gross anomalies (unspecified) was
    reported in the animals given 1000 mg/kg bw per day on days 9-11 of
    gestation. The apparent NOAEL in this study was 315 mg/kg bw per day
    (Minor & Becker, 1971).

         A study of the developmental toxicity of sodium benzoate
    administered by gavage to multiple species was conducted by Food and
    Drug Research Labs, Inc. (1972). The species tested and the treatment
    protocol are presented in Table 2. The rationale for the choice of
    doses was not indicated. A positive control group was included for
    each species: control groups of rats and mice received aspirin at
    150 mg/kg bw per day, hamsters received aspirin at 250 mg/kg bw per
    day, and rabbits received 6-aminonicotinamide at 2.5 mg/kg bw per day;
    a vehicle control group treated by gavage was also available. Day 0 of
    gestation was taken as the day sperm was observed in a vaginal smear
    or, for rabbits, the day of artificial insemination. All animals were
    observed daily for adverse effects, and maternal body weights were
    recorded throughout pregnancy. The maternal and fetal end-points
    examined for each dose group in each study included the total number
    of pregnancies, maternal survival, body weight, total number of
    corpora lutea and implantation sites per pregnant female, total number
    of live litters, total number of resorptions, number of live fetuses
    per litter, sex ratio, total number of dead fetuses, number of dams
    with one or more dead fetuses, average fetal weights, and incidences
    of gross, skeletal, and soft-tissue abnormalities. No significant
    deviations from the control values and no dose-related effects were
    reported. There were no teratogenic effects of significance, and none
    occurred in excess over those seen in the negative control groups, but
    effects were seen in the respective positive control groups. As no
    adverse effects occurred with any of the treatment schedules, the
    NOAEL values were 175 mg/kg bw per day for mice and rats, 300 mg/kg bw
    per day for hamsters, and 250 mg/kg bw per day for rabbits.

        Table 2.  Species tested and protocol for study of the potential developmental
              toxicity of sodium benzoate
                                                                                 

    Species               No. of rats   Doses               Treatment    Time of
                          per dose      (mg/kg bw)          period       necropsy
                                                                                 

    CD-1 mice                20         1.75, 8, 38, 175    Days 6-15    Day 17
    Wistar rats              24         1.75, 8, 38, 175    Days 6-15    Day 20
    Golden hamsters          20         3, 14, 65, 300      Days 6-10    Day 14
    Dutch belted rabbits     10         2.5, 12, 54, 250    Days 6-18    Day 29
                                                                                 

    From Food and Drug Research Labs, Inc. (1972)
    
         Groups of 27-30 pregnant Wistar rats were fed diets containing 0,
    1, 2, 4, or 8% sodium benzoate on days 1-20 of gestation; the
    appearance of sperm in a vaginal smear was considered day 0 of
    gestation. All but five animals in each group were sacrificed on day
    20 of gestation, and the numbers of viable fetuses, dead fetuses,
    early and late resortions, and fetal, placental, and ovarian weights
    were measured; abnormalities of maternal organs and fetal appearance
    were also recorded. About 75% of the fetuses from treated animals were
    stained with alizarin red S for skeletal examination, and the
    remainder were fixed with Bouin's solution and examined for visceral
    anomalies by Wilson's method. The remaining five dams in each group
    were allowed to deliver naturally, and the number of offspring,
    survival, body weight, and abnormalities were recorded. Three weeks
    after birth, all surviving pups were weaned and examined for gross
    abnormalities, and one-half of the pups and all of the dams were
    necropsied. The remaining pups were necropsied at eight weeks of age,
    body weight and food intake being measured weekly until necropsy.

         During administration of sodium benzoate, maternal body weight
    and body-weight gain were comparable in the controls and in animals at
    1 and 2% in the diet; animals at the 4% level did not gain weight and
    those at 8% lost weight (statistical comparisons not presented). Feed
    intake was also comparable in the controls and animals at 1 and 2%
    dietary levels but was markedly reduced in those at 4 and 8%. The
    total intake of the compound over the period of administration was
    reported to be 14 g/kg bw, corresponding to 700 mg/kg bw per day for
    rats at the 1% level; 26.2 g/kg bw, corresponding to 1310 mg/kg bw per
    day for rats at the 2% level; 37.5 g/kg bw, corresponding to
    1875 mg/kg bw per day for rats at the 1% level; and 19.3 g/kg bw,
    corresponding to 965 mg/kg bw per day for the rats at 8%. Two dams at
    4% and three at 8% died after convulsions and depressed motor

    activity. No differences in the average number of implants per female,
    the numbers of dead, resorbed, or viable fetuses, or the average
    weight of viable fetuses were reported in the animals at 1 or 2%
    during examination on day 20 of gestation; in the groups at 4 and 8%,
    the number of dead or resorbed fetuses was significantly increased,
    and the average body weight of viable fetuses was significantly lower
    than that of controls. Significant abnormalities and pathological
    findings were seen only in the fetuses at 4 and 8%; these included
    mild systemic oedema, anophthalmia, pyelectasis, microphthalmia,
    hydrocephalus, pyelectasis, hydroplasia, and cerebral hypoplasia.
    Delayed ossification, lumbar or cervical ribs, and varied sternebrae
    were reported in animals in both the control and treated groups. The
    percentage of animals with these findings was comparable to that in
    controls among animals at 1 and 2% (each about 37%) but was increased
    in the groups at 4% (96.5%) and 8% (100%). Additional anomalies seen
    in the treated groups included a higher incidence of wavy ribs and
    abnormal vertebrae in the rats at 4%, but not at 8%. As the number of
    live fetuses examined was not reported, the toxicological significance
    of these findings is not clear.

         Among pups that were delivered naturally, no differences in the
    delivery rate, number of perinatal deaths, lactation rate, or survival
    up to week 8 were reported at the 1 and 2% dietary levels, but the
    groups at 4 and 8% were reported to have delivery rates reduced by
    50 and 8.2%, respectively, with complete loss of litters after
    parturition. The surviving pups in the control group and at 1 and 2%
    showed no significant differences in birth weight, weight at week 3 or
    week 8, incidence of abnormalities at week 3 or 8, or organ weights at
    week 8. The authors suggested that the effects on the dams and fetuses
    at the 4 and 8% dietary levels were due to reduced maternal feed
    intake in these groups, leading to malnutrition, since the actual
    compound intake of the animals at 8% was lower than that of the group
    at 2%, in which no adverse effects were seen. The authors concluded
    that the effects seen at 4 and 8% in the diet were not relevant to the
    experimental outcome. The NOAEL was 1310 mg/kg bw per day (Onodera
     et al., 1978).

    2.2.5.2  Chickens

         Sodium benzoate had no teratogenic effect on chicken embryos
    after injection into the air cell of eggs on day 4 of incubation at
    levels as high as 5 mg per egg (Verrett  et al., 1980).

    2.2.6  Genotoxicity

         The results of studies of the genotoxicity of benzyl acetate,
    benzyl alcohol, benzaldehyde, benzoic acid and benzoate salts, the
    metabolite, and hippuric acid are presented in Table 3.


        Table 3.  Results of tests for the genotoxicity of benzyl compounds
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzyl acetate

    In vitro

    Reverse                  S. typhimurium           0.03-30               Negativeb         Florin et al . (1980)
      mutation               TA98, TA100,             µmol/platea
                             TA1535, TA1537

    Reverse                  S. typhimurium           33-10 000             Negativeb         US National
      mutation               TA98, TA100,             µg/plate                                Toxicology Program
                             TA1535, TA1537                                                   (1986, 1993);
                                                                                              Mortelmans et al.
                                                                                              (1986)

    Gene mutation            Mouse lymphoma           0.25-1.75 µl/ml       Positiveb         US National
                             cells (L5178Y), tk       (260-1850 µg/ml);                       Toxicology Program
                             locus                    600-1700 µg/ml                          (1986, 1993);
                                                                                              Caspary et al.
                                                                                              (1988); McGregor et
                                                                                              al. (1988)

    Chromosomal              Chinese hamster          160-5000 µg/ml        Equivocalc        US National
      aberration             ovary cells                                                      Toxicology Program
                                                                                              (1986, 1993);
                                                                                              Galloway et al.
                                                                                              (1987)

    Mitotic chromosome       Saccharomyces            520-1817 µg/ml        Positived         Zimmerman et al.
      loss                   cerevisiae D61.M                                                 (1989)
                                                                                                                      

    Table 3.  (con't)
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzyl acetate (con't)

    Sister chromatid         Chinese hamster          50-5000 µg/ml         Negativeb         US National
      exchange               ovary cells                                                      Toxicology Program
                                                                                              (1986, 1993);
                                                                                              Galloway et al.
                                                                                              (1987)

    Unscheduled              Fischer 344 rat          NR                    Negative          Mirsalis et al. (1983)
      DNA repair             hepatocytes

    Unscheduled              Fischer 344 rat          50, 200, 1000         Negative          Mirsalis et al. (1983,
      DNA repair             hepatocytes              mg/kg bw                                1989)

    Unscheduled              Rat pancreatic           1000 mg/kg bw         Negative          Steinmetz &
      DNA repair             cells                    orally                                  Mirsalis (1984)

    In vivo

    Sex-linked               Drosophila               300 ppm (feed);       Negative          US National
      recessive lethal       melanogaster             20 000 ppm                              Toxicology Program
      mutation                                        (injection)                             (1993)

    Chromosomal              Mouse bone-              325-1700              Negative          US National
      aberration             marrow cells             mg/kg bw                                Toxicology Program
                                                      intraperitoneally                       (1993)
    Micronucleus             Mouse bone-              312-1250              Negative          US National
      formation              marrow cells             mg/kg bw                                Toxicology Program
                                                      intraperitoneally                       (1993)

    Micronucleus             Mouse peripheral         3130-50 000           Negative          US National
      ormation               blood                    ppm in diet                             Toxicology Program
                                                                                              (1993)
                                                                                                                      

    Table 3.  (con't)
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzyl acetate (con't)

    Sister chromatid         Mouse bone-              325-1700              Negative          US National
      exchange               marrow cells             mg/kg bw                                Toxicology Program
                                                      intraperitoneally                       (1993)

    Benzyl alcohol

    In vitro

    Reverse                  S. typhimurium           0.03-30               Negativeb         Florin et al. (1980)
      mutation               TA98, TA100,             µmol/platea
                             TA1535, TA1537

    Reverse                  S. typhimurium           0.1-5.0               Negative          Wissler et al. (1983)
      mutation               TA98, TA1535             µmol/platea

    Reverse                  S. typhimurium           < 10 000             Negativeb         Ishidate et al. (1984)
      mutation               TA92, TA94,              µg/plate
                             TA98, TA100,
                             TA1535, TA1537

    Reverse                  S. typhimurium           100-6666              Negativeb         Mortelmans et al.
      mutation               TA98, TA100,             µg/plate                                (1986); US National
                             TA1535, TA1537                                                   Toxicology Program
                                                                                              (1989); Zeiger
                                                                                              (1990)

    Gene mutation            Mouse lymphoma           156-5000 µg/ml        Positivee         McGregor et al.
                             cells (L5178Y), tk                                               (1988); US National
                             locus                                                            Toxicology Program
                                                                                              (1989)
                                                                                                                      

    Table 3.  (con't)
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzyl alcohol (con't)

    Chromosomal              Chinese hamster          < 1000 µg/ml         Negative          Ishidate et al. (1984)
      aberration             lung cells

    Chromosomal              Chinese hamster          50-5000 µg/ml         Positivef         US National
      aberration             ovary cells                                                      Toxicology Program
                                                                                              (1989); Anderson et
                                                                                              al. (1990)

    Sister chromatid         Chinese hamster          16-5000 µg/ml         Equivocalb        US National
      exchange               ovary cells                                                      Toxicology Program
                                                                                              (1989); Anderson et
                                                                                              al. (1990)

    In vivo

    Micronucleus             Mouse bone-              0, 50, 100, 200       Negative          Hayashi et al.
      formation              marrow cells             mg/kg bw                                (1988)
                                                      intraperitoneally

    Benzaldeyde

    In vitro

    Reverse                  S. typhimurium           0.03-30               Negativeb         Florin et al. (1980)
      mutation               TA98, TA100,             µmol/platea
                             TA1535, TA1537

    Reverse                  S. typhimurium           0.05-500              Negativeb         Kasamaki et al.
      mutation               TA98, TA100              µg/plateg                               (1982)
                                                                                                                      

    Table 3.  (con't)
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzaldeyde (con't)

    Reverse                  S. typhimurium           10-1000               Negativeb         Haworth et al.
      mutation               TA98, TA100,             µg/plate                                (1983)
                             TA1535, TA1537
    Reverse                  S. typhimurium           0.1-5.0               Negative          Wiessler et al.
      mutation               TA98, TA1535             µmol/platea                             (1983)

    Reverse                  S. typhimurium           33-3333               Negativeb         US National
      mutation               TA98, TA100,             µg/plate                                Toxicology Program
                             TA102, TA104,                                                    (1990)
                             TA1535, TA1537

    Gene mutation            Mouse lymphoma           50-800 µg/ml          Positivee         US National
                             cells (L5178Y), tk                                               Toxicology Program
                             locus                                                            (1990);
                                                                                              McGregor et al. (1991)

    Chromosomal              Chinese hamster          < 50 nmol/litre       Positive          Kasamaki et al.
      aberration             B241 cells                                                       (1982)

    Chromosomal              Chinese hamster          50-1600 µg/ml         Negativeb         Galloway et al.
      aberration             ovary cells                                                      (1987); US National
                                                                                              Toxicology Program
                                                                                              (1990)

    Sister chromatid         Chinese hamster          5-1600 µg/ml          Positiveb         Galloway et al.
      exchange               ovary cells                                                      (1987!; US National
                                                                                              Toxicology Program
                                                                                              (1990)
                                                                                                                      

    Table 3.  (con't)
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzaldeyde (con't)

    Sister chromatid         Human                    < 2.0 mmol/litre      Positivee         Jansson et al.
      exchange               lymphocytes                                                      (1988)

    In vivo

    Sex-linked               Drosophila               150 ppm (feed),       Negative          Woodruff et al.
     recessive               melanogaster             2500 ppm                                (1985); US National
     lethal mutation                                  (injection)                             Toxicology Program
                                                                                              (1990)

    Benzoic acid and benzoate salts

    In vitro

    Reverse                  S. typhimurium           < 10 000              Negativeb         Ishidate et al. (1984)
      mutation               TA92, TA94,              µg/plate (acid);
                             TA98, TA1535,            < 3000 µg/plate
                             TA1537                   (sodium salt)

    Reverse                  S. typhimurium           33-10 000             Negativeb         Zeiger et al. (1988)
      mutation               TA97, TA98,              µg/plate
                             TA100, TA1535,
                             TA1537

    Chromosomal              Chinese hamster          < 1500 µg/ml          Equivocal         Ishidate et al. (1984)
      aberration             lung cells               (acid); < 2000        (acid);
                                                      µg/ml (sodium         positive  
                                                      salt)                 (sodium salt)h
                                                                                                                      

    Table 3.  (con't)
                                                                                                                      

    End-point                Test object              Concentration         Result            Reference
                                                                                                                      

    Benzoic acid and benzoate salts (con't)

    Sister chromatid         Chinese hamster          1-10 mmol/litre       Equivocal         Oikawa et al. (1980)
      exchange               ovary cells

    Sister chromatid         Human lymphoblastoid     1-30 mmol/litre       Negativeb         Tohda et al. (1980)
      exchange               cells

    Sister chromatid         Human lymphocytes        < 2.0 mmol/litre      Negative          Jansson et al.
      exchange                                                                                (1988)

    Hippuric acid

    In vitro

    Reverse mutation         S. typhimurium           0.1-5.0               Negative          Wiessler et al.
                             TA98, TA1535             µmol/platea                             (1983)
                                                                                                                      

    a  Qualitative test only; not tested to limits of cytotoxicity or solubility
    b  In presence and absence of metabolic activation
    c  Negative in presence of metabolic activation; two of three trials showed P values <0.05 in absence of metabolic activation.
    d  Weak response at dose that induced about 80% toxicity
    e  In absence of metabolic activation
    f  In presence of metabolic activation
    g  No rationale provided for highest dose tested
    h  Tabulation of chromosomal aberrations included gaps
        Benzoic acid and benzoate salts

         Sodium benzoate at concentrations of 0.05 × 102 to 5 × 104 ppm
    induced an array of cytological effects in  Vicia faba root 
    mitotic cells, involving all the stages of the mitotic cycle. The
    most remarkable were inhibition of DNA synthesis and induction of
    anaphase bridges and subsequent micronuclei (Njagi & Gopalan, 1982).
    It induced chromosomal aberrations in rat cells  in vitro and was
    mutagenic in the recombination (rec) assay. Negative results were
    obtained in an assay for reverse mutation in  Salmonella typhimurium
    (Kawachi, 1975, cited by Sodemoto & Enomoto, 1980).

         A number of studies conducted in 1974 (Food and Drug Research
    Labs., Inc., 1974) were reviewed but were not included in this
    monograph, since they were considered not to contribute useful
    information.

    2.2.7  Special studies: Effects on the pancreas

    Benzyl acetate

         A series of experiments was conducted to assess the role of
    benzyl acetate in the induction of tumours of the pancreas in rats, as
    observed in the study of the US National Toxicology Program (1986) in
    which animals were treated by gavage. Male Fischer 344 rats were
    injected intraperitoneally with azaserine and fed diets containing
    benzyl acetate dissolved in corn oil at levels of 0, 0.4, or 0.8%,
    equivalent to 0, 200, and 400 mg/kg bw per day, respectively, for six
    months. Azaserine-induced atypical acinar-cell foci in pancreatic
    tissue were assessed for density, diameter, and volume percentage.
    Fewer lesions per cubic centimetre were seen in the groups fed benzyl
    acetate than in controls, but the mean diameter and volume of the foci
    were greater than in controls.

         In a second experiment, groups of 25 male rats were fed control
    diet or diet containing 0.8% benzyl acetate from weaning until autopsy
    at two years of age, at which time the pancreases were examined
    histologically. Benzyl acetate had no effect on the growth or survival
    of the rats, and the incidence of pancreatic acinar foci or adenomas
    was not increased over that in controls. A marginal increase in the
    number of carcinomas  in situ was observed (3/38 compared with 0/49),
    which was not statistically significant ( P = 0.0616; Fisher's exact
    test). The historical control incidence of this lesion was not
    provided. The authors in terpreted their result as indicating a weak
    promoting activity of benzyl acetate.

         A long-term study to assess the promoting effects of benzyl
    acetate in azaserine-pretreated rats had to be terminated after one
    year owing to poor survival, apparently due to chronic renal disease.
    In this study, the incidence of pancreatic carcinoma was lower in rats

    given dietary concentrations of 0.4 or 0.8% benzyl acetate than in
    controls, and the combined incidences of acinar adenoma, carcinoma
     in situ, and carcinoma were 89% in controls, 72% at 0.4% benzyl
    acetate, and 73% at 0.8%.

         Alkaline elution analysis of DNA from rats treated intra-
    peritoneally with 150, 500, or 1500 mg/kg bw benzyl acetate
    revealed no evidence of damage to DNA in pancreatic cells. The authors
    concluded that the results of the first two experiments suggest
    that benzyl acetate is a weak promoter of carcinogen-induced and
    spontaneously occurring preneoplastic foci in the pancreas (Longnecker
     et al., 1990).

    2.3  Observations in humans

    Benzyl alcohol

         Gasping respiration was observed in premature infants who had
    received benzyl alchohol in medications administered intravenously.
    The syndrome was characterized by central nervous system dysfunction
    with hypoactivity, hypotonia, and depression of the sensorium,
    followed by apnoea, seizure activity, and coma; severe metabolic
    acidosis; skin breakdown; haematological and hepatic disturbances,
    including thrombocytopenia, leukopenia, direct hyperbilirubinaemia,
    hyperammonaemia; hypotension and renal failure; cardiovascular
    collapse; and death at 6-46 days of age. Infants with this syndrome
    had received 99-234 mg/kg bw per day of benzyl alcohol, while a
    matched control group of infants who had received benzyl alcohol but
    who did not develop the syndrome had received doses of 27-99 mg/kg bw
    per day. (The median intravenous lethal dose in the rat is 314 mg/kg
    bw.) Benzyl alcohol was detected in the serum of these infants, and
    benzoic and hippuric acids were identified in urine samples at
    statistically significantly higher levels than in urine from infants
    who had not received benzyl alcohol. It should be noted that some
    endogenous formation of these compounds occurs. The authors suggested
    that the accumulation of benzyl alcohol was probably due to the large
    doses given relative to the size of the infants and/or to the reduced
    capacity of their metabolic systems to detoxify it (Gershanik
     et al., 1982).

         Contact allergy was diagnosed in a metal-grinder who developed an
    itchy, patchy rash on the fingers and hands after the introduction of
    a new cutting oil. There was no recurrence of the rash when use of the
    new oil was discontinued. Patch testing of the subject revealed that
    he was sensitive to a number of fragrances, including and/or
    containing benzaldehyde, and to benzyl alcohol, which was identified
    as a component of the cutting oil (Mitchell & Beck, 1988).

         A condition described as relapsing tinea affecting the anogenital
    region and adjacent thigh area was observed in a man after treatment
    with antimycotic lotions and ointments. The condition was resolved by
    treatment with zinc oxide and topical corticosteriods and after
    stopping use of perfumed soap and foam-bath preparations. Positive
    reactions to a number of therapeutic ointments were seen in patch
    testing, and benzyl alcohol was subsequently identified as the
    sensitizing agent (Wurbach  et al., 1993).

    Benzaldehyde

         The fatal dose in a case of acute poisoning was 50-60 ml (Dadlez,
    1928).

    Benzoic acid and benzoate salts

         Human tolerance appears to vary: marked gastrointestinal
    disturbances were induced by 5.7 g sodium benzoate (Meissner &
    Shepard, 1866), while other people tolerated 25-40 g (Bignami, 1924).

         Up to 12 g of sodium benzoate daily have been given
    therapeutically without ill effects (Senator, 1879), but the same dose
    of benzoic acid given over five days produced gastric burning and
    anorexia in 30% of other subjects (Waldo  et al., 1949). The toxic
    symptoms are local gastrointestinal mucosal irritation or effects on
    the central nervous system. Acute toxicity in man is readily
    reversible and is probably due to disturbance of the acid-base balance
    rather than to tissue damage (Barnes, 1959).

         In six men given 0.3-0.4 g benzoic acid in their diet for up to
    62 days, no abnormalities were seen in the blood picture, urine
    composition, nitrogen balance, or well-being (Chittenden  et al.,
    1909). Nine patients on penicillin treatment received 1200 mg benzoic
    acid daily, in eight doses, over five days for eight subjects and for
    14 days in one case. No effect was observed; in particular, no
    significant change was seen in endogenous creatinine clearance, and
    routine urine analysis showed no abnormality (Waldo  et al., 1949).

         Some patients who suffer from asthma, rhinitis, or urticaria
    experience exacerbation of symptoms after ingestion of foods or
    beverages containing benzoates (Freedman, 1977).

         Oral doses of 250 mg/kg bw sodium benzoate per day to infants,
    given to counteract the effects of inherited urea cycle disorders,
    were well tolerated, but boluses of the drug induced vomiting. Acute
    benzoate poisoning was observed after accidental treatment with
    800 mg/kg bw over 24 h. The clinical symptoms were similar to those
    observed in salicylate poisoning, with vomiting, hyperpnoea, and
    irritability. The plasma benzoate levels were elevated, and urinary
    excretion of hippuric acid was increased; plasma glycine levels were

    decreased. The plasma levels returned to normal and the symptoms were
    resolved within 24 h of discontinuation of benzoate treatment
    (Batshaw, 1983).

         Sodium benzoate was used therapeutically in three infants to
    alleviate the symptoms of non-ketotic hyperglycaemia, a metabolic
    disorder, at doses of 125-1000 mg/kg bw per day, given orally in four
    divided doses. All of them showed anorexia and vomiting while
    receiving sodium benzoate at the higher doses, and repeated vomiting
    was seen at 900 mg/kg bw day. In one patient, glycosuria,
    hypocalcaemia, and metabolic acidosis were observed after doses of
    1000 mg/kg bw per day, suggesting a causal relationship between
    ingestion of benzoate and the apparent renal tubular dysfunction
    (Wolff  et al., 1986).

         An episode of anaphylaxis in response to ingestion of sodium
    benzoate present in foodstuffs was described in a 19-year-old woman.
    The patient had experienced generalized itching one week after eating
    cheese. The symptoms consisted of flush, angioedema, dyspnoea, and
    severe hypotension; they were resolved by administration of adrenalin
    and corticosteroids. Adherence to a benzoate-free diet prevented any
    recurrence of the symptoms. An oral challenge with 20 mg sodium
    benzoate induced localized itching on the arms and generalized
    itching, suggesting intolerance to sodium benzoate. Resolution of an
    occult maxillary sinusitis resulted in better tolerance to sodium
    benzoate after a second oral challenge, with only mild localized
    itching after a 160-g dose (Michils  et al., 1991).

    3.  COMMENTS

         On the basis of the present review, a number of issues were
    identified that are important in the safety evaluation of benzyl
    acetate, benzyl alcohol, benzaldehyde, and benzoic acid and its salts.
    Since they are all metabolized to benzoic acid, it was considered
    reasonable to assume that the results of studies on one member of the
    group would apply to the others. The results of a number of studies in
    humans and experimental animals indicate that formation of hippuric
    acid from benzoic acid is a saturable process in which the
    availability of glycine is the rate-limiting step. This observation is
    particularly relevant to the interpretation of the toxic effects of
    these compounds in experimental animals, since supplementation of the
    diet with glycine was shown to alleviate the toxic effects induced by
    high doses of benzyl acetate and benzoic acid, including body-weight
    decrements and neurotoxic effects. Even with saturation of hippuric
    acid formation, however, clearance of compounds in the benzyl group is
    relatively rapid in experimental animals and humans. An extensive
    review of the toxicity of benzoate showed that at high doses it
    interferes with intermediary metabolism, including the urea cycle,
    gluconeo-genesis, fatty acid metabolism, and the tricarboxylic acid
    cycle, probably by sequestering coenzyme A before its conjugation with
    glycine. This observation is consistent with similar effects
    (metabolic acidosis, convulsions, and hyperpnoea) in experimental
    animals and humans given very high doses. Administration of benzyl
    acetate to rats by gavage was shown to result in higher peak plasma
    levels of benzoic acid in comparison with ingestion of a similar daily
    dose in the diet, while the plasma levels of hippuric acid were
    similar regardless of the method of administration. This finding is
    reflected in the greater toxicity of these compounds when administered
    by gavage. Since depletion of glycine may be a major factor in the
    effects observed at the high doses used in animals, the results of
    studies by gavage would appear to be inappropriate for extrapolating
    to human exposure. In addition, depletion of glycine might be of
    concern with respect to the developing fetus and neonate. It should be
    noted that benzoic and hippuric acids are generated as a result of
    phenylalanine-tyrosine metabolism.

         Long-term studies by feeding and gavage in mice and rats were
    available for review by the Committee. No definitive conclusion could
    be drawn from the results of carcinogenicity studies with sodium
    benzoate in mice and rats, as insufficient detail was provided and the
    survival in the study of rats was poor. The Committee reviewed the
    studies evaluated in the previous monographs and an additional study
    in which benzaldehyde was administered to rats and mice in corn oil by
    gavage and concurred with the conclusions that neither benzyl acetate
    nor benzyl alcohol is carcinogenic. As in the studies with mice and
    rats given benzyl acetate by gavage in corn oil, increased incidences
    of pancreatic acinar-cell adenomas in rats and of papillomas of the
    forestomach in mice were noted after administration of benzaldehyde.

    The Committee concluded that these results were of no relevance, as
    noted at the forty-first meeting, because of the use of corn oil as
    the vehicle and that the results of studies by dietary administration
    were more relevant to the assessment of food additives.

         Several studies addressing aspects of the potential developmental
    and reproductive toxicity of benzyl acetate, benzyl alcohol,
    benzaldehyde, and sodium benzoate were reviewed at the present
    meeting. Developmental delays and reduced fetal and postnatal pup body
    weights were observed only at doses that were maternally toxic. Doses
    that induced extreme maternal toxicity were associated with
    embryotoxic and fetotoxic effects and malformations in a study with
    sodium benzoate. A multigeneration study with rats showed no effect on
    growth, fertility, lactation, or survival.

         The genotoxicity database was also reviewed. None of the four
    compounds was mutagenic in assays for reverse mutation, either with or
    without metabolic activation. The compounds all induced gene mutation
    in the mouse lymphoma assay at the  tklocus (benzoate was not tested in
    this assay), although the requirement for metabolic activation varied.
    Some weak clastogenic activity was noted  in vitro but not in
     in vivo when the latter assays were performed.

    4.  EVALUATION

         The Committee was aware of reports of idiosyncratic human
    intolerance to benzoate. These data were not considered relevant to
    the establishment of an ADI for this group of compounds. The Committee
    endorsed the view expressed in the report of the twenty-seventh
    meeting that appropriate labelling is a feasible means of offering
    protection to susceptible individuals.

         The Committee was satisfied that the data reviewed on compounds
    in this group are sufficient to demonstrate lack of carcinogenic,
    developmental and reproductive potential. Consequently, it concluded
    that further studies were not required, and the group ADI of 0-5 mg
    per kg bw as benzoic acid equivalents was maintained.

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    See Also:
       Toxicological Abbreviations
       Benzyl acetate (ICSC)
       Benzyl acetate (FAO Nutrition Meetings Report Series 44a)
       Benzyl acetate (WHO Food Additives Series 26)
       Benzyl acetate (WHO Food Additives Series 32)
       BENZYL ACETATE (JECFA Evaluation)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 40, 1986)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 71, 1999)