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    BENZYL ACETATE

    1.  EXPLANATION

         This substance was evaluated for an acceptable daily intake for
    man (ADI) by the joint FAO/WHO Expert Committee on Food Additives at
    the eleventh, twenty-seventh, twenty-ninth and thirty-first meetings
    (Annex 1, references 14, 62, 70 and 77).  A toxicological monograph
    was issued in 1968 (Annex 1, reference 15).  The ADI of 0-5 mg/kg
    bw/day which was established at the eleventh Meeting was made
    temporary at the twenty-seventh meeting.

         Since the previous evaluation, additional data have become
    available and are summarized and discussed in the following monograph. 
    The previously published monograph has been expanded and is reproduced
    in its entirety below.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         This compound is absorbed from the gastrointestinal tract,
    through the lungs and through the intact skin.  It is hydrolyzed 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 hydrolyzed  in vitro with 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).

         Mice and rats were dosed either intravenously or orally with 14C
    labelled benzyl acetate.  The intravenous dose for mice was equivalent
    to 10 mg/kg bw and for rats 5 mg/kg bw.  For oral dosing, the benzyl
    acetate was dissolved in corn oil and administered at dose levels
    equivalent to 10, 100 or 1000 mg/kg bw for mice and 5, 50 and 500
    mg/kg bw for rats.  Elimination of benzyl acetate as CO2 or volatiles
    was minimal.  The elimination of the 14C label occurred mainly in the
    urine with less than 1% being detected in the feces.  More than 90% of
    the 14C in the urine was present as hippuric acid, with minor amounts
    present as benzyl alcohol and benzylmercapturic acid (up to 4%).  No
    unchanged benzyl acetate was present in urine.  The elimination was
    complete within 24 hours.  Neither route of administration or dose had
    any significant effect on the pattern of elimination.  Benzoyl
    glucuronide levels were not measured.  Repeated dosing of rats with
    500 mg/kg bw/day benzyl acetate  for 14 days, followed by a single
    dose of 14C benzyl acetate did not change the clearance pattern. 
    Analysis of tissues of animals, sacrificed 24 hours after
    administration of 14C either i.v. or orally, failed to detect the
    presence of 14C activity (Matthews & Burka, 1984).

         Male rats received [methylene-14C]-benzyl acetate by gavage
    either in corn oil or in propylene glycol at doses of 5, 250 or 500
    mg/kg bw.  Radioactivity was measured in feces, urine, plasma and
    tissues.  Metabolites in urine and plasma were identified by HPLC and
    TLC.  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-hr period with
    very little in the feces (approximately 4%) after 72 hours.  The
    elimination of benzyl acetate and metabolites was essentially complete
    by 3 days, as indicated by negligible residues in tissue, regardless
    of whether benzyl acetate was given neat or in corn oil.  Since benzyl
    acetate was not found in the plasma or urine at any time, the authors
    concluded that it was readily hydrolyzed.  Small amounts of benzyl
    alcohol, the initial product of ester cleavage, was detected only in
    plasma samples.  At the higher doses, benzoic acid was the major

    metabolite in plasma, while at the 5 mg/kg bw dose, 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 size.  The
    proportion of the dose present as benzoyl glucuronide increased with
    dosage, leading the authors to conclude that this demonstrated a
    limited capacity of the glycine conjugation mechanism (Chidgey &
    Caldwell, 1986).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

                                                                 

    Species    Sex    Route       LD50        Reference
                               (mg/kg bw)
                                                                 

    Rat        ?      oral     2490-3690     Jenner  et al., 1967
                                             von Oettingen, 1960

    Rabbit     ?      oral        2640       von Oettingen, 1960
                                                                 

    2.2.2  Short-term studies

    2.2.2.1  Mice

         Groups of 5 mice (B6C3F1) of each sex were administered single
    doses of benzyl acetate equivalent to 250, 500, 1000, 2000 or 4000
    mg/kg bw in corn oil by gavage.  The animals were observed for 15
    days.  All mice receiving 4000 mg/kg bw and females receiving 2000
    mg/kg bw became inactive immediately after dosing.  All mice in the
    highest dose group, and 1/5 males and 2/5 females dosed at 2000 mg/kg
    bw died by day 2 of the study.  No other compound-related effects were
    reported (NTP, 1986).

         Groups of 5 mice (B63F/N) of each sex were dosed with 0, 125,
    250, 500, 1000 or 2000 mg/kg bw benzyl acetate 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 level had died by day 3
    of the study.  Weight changes were not dose-related.  At autopsy the
    only effect reported was a roughening of the mucosa of the stomach in
    the cardiac region in 2/5 males and all females in the highest dose
    group, and 1/5 females in the 1000 mg/kg bw/day group (NTP, 1986).

         Groups of 10 mice (B6C3F1) of each sex were administered 0, 125,
    250, 500 or 12000 mg/kg bw/day benzyl acetate in corn oil by gavage,
    5 times a week for 13 weeks.  A total of 7/10 of the females in the
    highest dose group died.  Compound-related clinical effects were

    observed in the highest dose group, included trembling, inactivity,
    labored breathing and depressed body temperature.  At autopsy, no
    gross or microscopic effects were noted (NTP, 1986).

    2.2.2.2  Rats

         Groups of 15 males and 15 females were fed a mixture of aromatic
    esters, including 15.8 mg/kg bw/day of benzyl acetate for 12 weeks. 
    No adverse effects were noted (Oser, 1967).  Groups of 5 rats (F344/N)
    approximately 6 weeks old, were administered 0, 250, 500, 1000, 2000
    or 4000 mg/kg bw/day benzyl acetate by gavage in corn oil, daily for
    14 days.  On day 16, surviving animals were killed and autopsied. 
    None of the rats dosed at 4000 mg/kg bw/day survived beyond 2 days, at
    the 2000 mg/kg bw/day level all rats had died within 5 days.  No other
    deaths were reported.  There was a depression of mean body weight gain
    relative to controls of more than 10% in the 500 mg/kg bw/day male
    group, and in the 1000 mg/kg bw/day group in both males and females. 
    At autopsy the only effect reported was that the cecum was redder than
    normal in 3/5 of the animals in the 4000 mg/kg bw/day group (NTP,
    1986).

         Groups of 10 rats (F344/N) of each sex were administered 0, 62.5,
    125, 250, 500 or 1000 mg/kg bw/day benzyl acetate in corn oil, 5 days
    per week for 13 weeks.  Male and female rats in the 100 mg/kg bw/day
    group and females in the 500 mg/kg bw/day group showed clinical
    symptoms including trembling, ataxia and sluggishness.  A total of
    2/10 males and 1/10 females in the highest dose group had died by day
    86.  Only male rats in the 1000 mg/kg bw/day group showed a depression
    in mean body weight relative to controls (21%).  At autopsy a
    thickened stomach wall was observed in 2/9 males and 4/10 females in
    the high dose group (NTP, 1986).

    2.2.3  Long-term/carcinogenicity studies

         Lifetime studies in mice and rats with benzyl alcohol are
    included in this section since the initial step in metabolism of
    benzyl acetate is hydrolysis of the ester to benzyl alcohol (Abdo  et
     al., 1985; Chidgey  et al., 1986; NTP, 1986).

    2.2.3.1  Mice

         Groups of 50 (B6C3F1) mice of each sex were administered benzyl
    acetate in corn oil by gavage at doses equivalent to 500 or 1000 mg/kg
    bw/day, 5 days/week for 103 weeks.  Vehicle control groups of 50
    animals/sex were administered corn oil by gavage.  Complete gross
    necropsies and histopathological examinations were performed on
    animals found dead and on those sacrificed at the end of the study.

         Mean body weight gains of dosed and control male mice were
    comparable throughout the study.  In females, treated mice showed
    slightly higher mean body weights after week 20 than those of the
    controls.  Survival of female mice was markedly reduced in the control
    (30%) and low dose groups (36%) in comparison with the high dose group
    (60%).  The high mortality rate in female mice 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 between the survival rate of controls and
    either treatment group, although a greater number of control males
    died before week 45 than in the treated groups.

         Hepatocellular adenomas occurred in male mice in 0/50 control,
    5/49 low dose and 13/50 high dose animals, and in female mice in 0/50
    control, 0/50 low dose, and 6/50 high dose animals.  (The cumulative
    historical incidences of hepatocellular adenomas in corn oil gavage
    controls from 6 studies from the contract laboratory conducted prior
    to 3 August 1984, were 36/298 for male and 11/300 for female B6C3F1
    mice). Hepatocellular carcinomas were observed in male mice and in the
    high dose female group (Males: control 10/50, low dose 14/49 and high
    dose 12/50, and females: 1/50 control, 0/50 low dose, and 4/50 high
    dose).

         Squamous cell papillomas or carcinomas of the forestomach
    occurred in male mice and in the high dose female group.  The
    incidence in males was 4/49 control, 4/48 low dose, and 11/49 high
    dose.  For female mice the incidence was 0/50 control, 0/50 low dose
    and 4/48 high dose.  The historical incidences in corn oil gavage
    controls from 6 studies from the contract laboratory were 2/296 for
    males and 2/297 for females.  Forestomach hyperplasia was also
    reported in dosed mice (males: control 1/49, low dose 7/48 and high
    dose 22/49, and for females: control 1/50, low dose 6/50, high dose
    17/48) (NTP, 1986).

         Benzyl alcohol was administered by corn oil gavage to groups of
    50 mice (B6C3F1) of either sex, at dosages of 0, 100 or 200 mg/kg
    bw/day, 5 days a week for 103 weeks.  The neurotoxic effects of benzyl
    alcohol (lethargy and staggering) in short-term studies formed the
    basis for selection of the doses.  Complete gross necropsies and
    histopathological examinations were conducted on animals found dead
    and on those sacrificed at the end of the study.

         Mean body weights for dosed and control mice were comparable
    throughout the study.  The survival of control females was
    significantly lower than those in the high dose group after week 44,
    but no other differences in survival were noted between dosed groups
    and controls (male:  control 68%, low dose 66%, high dose 70%; 
    female:  control 50%, low dose 62%, high dose 72%).  No significant
    treatment-related effects were noted.  There was no association of
    treatment with the reported incidences of hepatocellular and

    forestomach neoplasias after administration of benzyl acetate (NTP,
    1987).

    2.2.3.2  Rats

         Groups of 50 rats (F344/N) of each sex were administered benzyl
    acetate in corn oil by gavage at doses equivalent to 150 or 500 mg/kg
    bw/day, 5 days/week for 103 weeks.  Vehicle control groups of 50
    animals per sex were administered corn oil by gavage.  Complete gross
    necropsies and histopathological examinations 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 male and female rats
    administered benzyl acetate 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.  Survival rates for females at
    104-106 weeks 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
    (cystadenocarcinoma, adenocarcinoma and carcinoma) in the preputial
    gland of male rats was elevated in the high dose group (control 1/50,
    low dose 1/50, high dose 6/50), but this was without statistical
    significance.

         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 incidence at the high dose was significantly greater
    than in the vehicle controls (p = 0.001).  Acinar cell hyperplasia was
    also observed in male rats (37/50 control, 34/50 low dose, and 36/49
    high dose).  No acinar cell hyperplasia or adenoma of the pancreas was
    observed in any of the female animals in the three experimental
    groups.  There was an increased incidence of retinopathy (not
    specified) and cataracts in the high dose males and low dose females. 
    However, this effect was attributed by the authors to the proximity of
    these rats to fluorescent light (NTP, 1986).

         Benzyl alcohol was administered by corn oil gavage to groups of
    50 F344/N rats of each sex at dosages of 0, 200 or 400 mg/kg bw/day,
    5 days a week for 103 weeks.  Complete gross necropsies and
    histopathological examinations were conducted on animals found dead
    and on those sacrificed at the end of the study.

         Mean body weights of dosed and vehicle control male and female
    rats were comparable throughout the study.  No compound-related
    clinical signs were observed, although at month 3 a
    sialodacryoadenitis virus infection was widespread among the study
    animals.  The survival of animals in both dosed female groups was
    significantly lower than that of vehicle controls (70% - control; 34%

    - low dose; 34% - high dose), which was the result of a much higher
    incidence of accidental gavage-related deaths in these groups. 
    Survival among male rats was comparable in all groups (control - 56%;
    low dose - 54%; high dose - 48%).

         Cataracts and retinal atrophy were observed at increased
    incidences in the high dose rats in this study.  This was again
    attributed by the authors to the proximity of this group of animals to
    fluorescent light for most of the study.  Hyperplasia of the
    forestomach epithelium was noted at an increased incidence in male
    rats (control 0/48, low dose 0/19, high dose 4/50).  Hemorrhage and
    foreign material in the respiratory tract were noted in dosed rats
    which died prior to the terminal kill.  The authors suggested this
    could have been the result either of direct deposition of material
    into the lung due to gavage "accidents" or of the anesthetic
    properties of benzyl alcohol resulting in reflux of gavage material,
    and aspiration into the lungs.  No pancreatic acinar cell adenomas
    were reported in any of the test or control groups in contrast to the
    results from the corn oil gavage study in rats with benzyl acetate. 
    No other effects of treatment were noted (NTP, 1987).



        2.2.5  Special studies on genotoxicity

    Results of genotoxicity assays on benzyl acetate

                                                                                                         

    Test System                Test Object        Concentration         Results      Reference
                                                  of benzyl acetate
                                                                                                         

    Ames test (1)              S.typhimurium     0-10,000 g/          Negative     NTP,1986;
                               TA100,TA1535,      plate                              Mortelmans
                               TA1537, TA98                                           et al., 1986

     In vitro                  Chinese            50-5,000 g/ml        Negative     NTP, 1986
    mammalian cytogenicity     hamster
    (SCE, chromosomal          ovary cells
    aberrations) (2)

     In vitro                  Mouse lymphoma     0.25-1.25 g/ml       Positive     NTP, 1986
    mammalian gene             cells                                    (3)
    mutation (2)               (L5178Y/Tk)

     In vitro                 Bacillus           21 g/disk            Negative     Oda  et al.,
    bacterial                  subtilis                                               1978
    gene mutation

    Unscheduled DNA            Rat hepatocytes                          Negative     Mirsalis  et al.,
    synthesis                                                                        1983
     in vivo and
     in vitro)
                                                                                                         

    (1)   in the presence or absence of induced rat or hamster liver S-9 fractions
    (2)   in the presence or absence of induced rat liver S-9 fraction
    (3)   positive in the presence of metabolic activation, negative in the absence of metabolic
          activation.
    

    3.  COMMENTS

         At the thirty-first meeting the Committee extended the temporary
    ADI of 0-5 mg per kg bw pending the evaluation of lifetime gavage
    studies with benzyl alcohol, a normal metabolite of benzyl acetate. 
    These studies did not reveal the increased incidence of either
    hepatocellular or forestomach tumours in mice or pancreatic tumours in
    rats which had previously been observed in studies with benzyl
    acetate.

         There are difficulties in interpreting the carcinogenicity
    studies with benzyl acetate that were conducted by gavage.  Since it
    is known that new long-term studies are underway with benzyl acetate
    incorporated into the diet of rats and mice, the present Committee
    decided to extend the temporary ADI of 0-5 mg/kg bw until 1993 pending
    the evaluation of the results of the new studies.

         In view of one positive report of mutagenic activity  in vitro,
    it would be desirable to ascertain whether results of an  in vivo
    study demonstrating the lack of the induction of unscheduled DNA
    synthesis can be confirmed by an  in vivo test for chromosome damage
    in bone marrow.

    4.  EVALUATION

         Level causing no toxicological effect
         Rat: 15.8 mg/kg bw/day.

         Estimate of temporary acceptable daily intake
         0-5 mg/kg bw.

         Further work or information
         Required (by 1993):
         Oncogenicity studies in rats and mice.
         Desired:
          In vivo study for chromosome damage in bone marrow.

    5. REFERENCES

    ABDO, K.M. HUFF, J.E., HASEMAN, J.K., BOORMAN, G.A., EUSTIS, S.L.,
    MATTHEWS, H.B., BURKA, L.T., PREJEAN, J.D. & THOMPSON, R.B. (1985).
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    344 rats and B6C3F1 mice.  Toxicology, 37, 159-170.

    CHIDGEY, M.A.J. & CALDWELL, J. (1986). Studies on benzyl acetate. I.
    Effect of dose size and vehicle on the plasma pharmacokinetics and
    metabolism of [methylene-14] benzyl acetate in the rat.  Food
     Chem.Toxic., 2, 1257-1265.

    CHIDGEY, M.A.J., KENNEDY, J.F. & CALDWELL, J. (1986). Studies on
    benzyl acetate. II. Use of specific metabolic inhibitors to define the
    pathway leading to the formation of benzylmercapturic acid in the rat.
     Food Chem.Toxicol., 24, 1267-1271.

    CLAPP, J.J. & YOUNG, L. (1970). Formation of mercaptic acid in rats
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    GRUNDSCHOSER, F. (1977). Toxicological assessment of flavouring
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    JENNER, P., HAGAN, E., TAYLOR, J., COOK, E. & FITZHUGH, O.G. (1967).
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    Toxicity.  Food Cosmet.Toxicol., 2, 327-343.

    MATTHEWS, H.B. & BURKA, L.T. (1984). Benzyl acetate metabolism and
    disposition in rats and mice, NTP (1984). NIH Publication No. 82-2506,
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    MIRSALIS, J., TYSON, K., BECK, J., LOH, F., STEINMETZ, K., CONTRERAS,
    C., AUSTERE, L., MARTIN, S. & SPALDING, J. (1983). Induction of
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    MORTELMANS, K., HAWORTH, S., LAWLOR, T., SPECK, W., TAINER, B. &
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    NTP (1986). NTP technical report on the toxicology and carcinogenesis
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    mice (gavage studies). NIH Publication No. 86-2506, U.S. Department of
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    of Health, Research Triangle Park, NC.

    NTP (1987). Board Draft. NTP technical report on the toxicology and
    carcinogenesis studies of benzyl alcohol (CAS NO. 100-51-6) in F344/N
    (gavage studies). NIH Publication No. 88-2599, U.S. Department of
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    of Health, Research Triangle Park, NC.

    ODA, Y., HAMANO, Y., INQUE, K., YAMAMOTO, H., NIIHARA, T. & KUNITA, N.
    (1978). Mutagenicity of food flavours in bacteria.  Osaka-furitsu
     Kosnu Eisei Kenkyu Hokoku snokunin eisein nem, 9, 177-181.

    OSER, B.L. (1967). Unpublished report.

    SNAPPER, I., GRUNBAUM, A. & STRUKOP, S. (1925).  Biochem.Z., 155,
    163.

    VON OETTINGEN, W.W. (1960).  AMA Arch.Ind. Health, 21, 28.


    See Also:
       Toxicological Abbreviations
       Benzyl acetate (ICSC)
       Benzyl acetate (FAO Nutrition Meetings Report Series 44a)
       Benzyl acetate (WHO Food Additives Series 32)
       Benzyl acetate (WHO Food Additives Series 37)
       BENZYL ACETATE (JECFA Evaluation)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 40, 1986)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 71, 1999)