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    BENZYL ACETATE

    First draft prepared by
    Dr P. Olsen
    Institute of Toxicology, National Food Agency of Denmark
    Ministry of Health, Soborg, Denmark

    1.  EXPLANATION

          This compound was previously evaluated at the eleventh,
    twenty-seventh, twenty-ninth, thirty-first, and thirty-fifth
    meetings (Annex 1, references 14, 62, 70, 77, and 88).  During some
    of these meetings the Committee also considered related substances,
    including benzyl alcohol, benzaldehyde, benzoic acid, and the
    benzoate salts.

          Benzyl acetate was first evaluated by the Committee at its
    eleventh meeting  (Annex 1, reference 14), when an ADI of 0-5 mg per
    kg of body weight was allocated in terms of benzoic acid,
    representing total benzoate from all food additive sources. At the
    twenty-seventh meeting (Annex 1, reference 62) the ADI for benzyl
    acetate was retained but made temporary because of concern raised by
    preliminary findings from screening tests for carcinogenicity.  At
    its twenty-ninth meeting (Annex 1, reference 70) the Committee
    considered new data on the metabolism of benzyl acetate and on the
    occurrence of tumours in rats and mice given benzyl acetate by
    gavage.  The Committee extended the temporary ADI of 0-5 mg per kg
    of body weight pending results from carcinogenicity studies with
    benzyl alcohol. The temporary ADI was again extended at the thirty-
    first meeting of the Committee (Annex 1, reference 77), as the
    expected data were not available.  At its thirty-fifth meeting
    (Annex 1, reference 89) the Committee reviewed a lifetime gavage
    studies with benzyl alcohol and found no evidence of a tumorigenic
    effect.  The Committee extended the temporary ADI of 0-5 mg per kg
    of body weight for benzyl acetate until 1993, pending the evaluation
    of results from ongoing long-term studies with benzyl acetate
    incorporated into the diet of mice and rats and requested data from
    and  in vivo test for chromosomal damage to bone marrow.

          Since the previous evaluation, additional toxicological data
    have become available and are summarized and discussed in the
    following monograph addendum.

    2.  BIOLOGICAL DATA

    2.1  Toxicological studies

    2.2.2  Short-term toxicity studies

    2.2.2.1  Mice

          Groups of 10 male and 10 female mice  (B6C3F1, average age at
    exposure 42 days; 13 days quarantine prior to test) received 0,
    3 130, 6 250, 12 500, 25 000, or 50 000 mg/kg feed (equivalent to
    0, 450, 900, 1 800, 3 600, or 7 200 mg/kg bw/day) of benzyl acetate
    (benzyl acetate, properties consistent with structure and literature
    references, purity: 99%; stability monitored periodically; no
    degradation of bulk chemical observed) in their diet for a period of
    13 weeks.  Feed [feed prepared weekly and stored in dark; dose
    formulation analyzed 4 times during study for benzyl acetate
    concentrations, stability, and homogeneity; contained low and
    biologically insignificant levels of aflatoxins, pesticides and
    heavy metals] and water were provided  ad libitum.  The feed
    consumption was recorded daily and the animals were weighed weekly. 
    Haematology, clinical chemistry (cholesterol and triglycerides), and
    pancreatic enzymes (amylase, lipase, carboxypetidase, chymotrypsin,
    ribonuclease), were performed at termination of the study. 
    Histopathology examinations were performed on all control, on 25 000
    mg/kg feed female, and all 50 000 mg/kg feed animals.

          Statistically significant (p<0.01) dose-related decreases in
    final body weights were observed in all treated animals compared to
    controls.  The mean feed consumption of all exposed mice was lower,
    but not statistically significantly lower, than that of the control
    groups.  Tremor was observed in female mice at a dose level of
    12 500 mg/kg feed and above.  At the dose level of 50 000 mg/kg feed
    one male died and one female mice was killed in extremis.  The
    absolute and relative organ weights in treated animals were
    influenced by the lowered terminal body weight and all significant
    differences between treated and control groups were attributed to
    treatment.  No dose-related effects in haematology, clinical
    chemistry, or pancreatic enzyme parameters were observed in treated
    animals.

          Histopathological examination revealed hippocampal necrosis,
    cerebellar haemorrhage of the brain and hepatocellular necrosis in 1
    male mouse receiving 50 000 mg/kg feed after 11 weeks of treatment. 
    At termination three female mice receiving 50 000 mg/kg feed showed
    hippocampal necrosis and depletion of the pyramidal layer cells in
    the brain (NTP, 1992).

    2.2.2.2  Rats

          Groups of 10 male and 10 female rats (F344, average age at
    exposure 43 days; 13 days quarantine prior to test) received 0,
    3 130, 6 250, 12 500, 25 000, or 50 000 mg/kg feed (equivalent to
    0, 210, 420, 840, 1 680, or 3 360 mg/kg bw/day) of benzyl acetate
    (benzyl acetate, properties consistent with structure and literature
    references, purity: 99%; stability monitored periodically, and no
    degradation of bulk chemical observed) in their diet for a period of
    13 weeks.  Feed: [feed prepared weekly and stored in dark; analyzed
    during study for benzyl acetate concentrations, stability, and
    homogeneity; contained low and biologically insignificant levels of
    aflatoxins, pesticides and heavy metals] and water 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 the 50 000 mg/kg feed group.  At termination
    liver peroxisomes morphometry were performed on female rats given 0,
    25 000, or 50 000 mg/kg feed benzyl acetate.  Histopathology
    examinations were performed on all control, 25 000 mg/kg feed and
    50 000 mg/kg feed rats.

          Nine male and female rats died or were killed moribund in the
    50 000 mg/kg feed group between weeks 2 and 8 of the study.  Final
    mean body weights of treated male and female rats were similar to or
    slightly lower than those of the controls.  Male rats given 25 000
    mg/kg feed showed 10% decreased (p<0.01) terminal mean body weight. 
    At the 50 000 mg/kg feed level the body weight of the one surviving
    male and female rats was less than half of the controls.  The
    average feed consumption was reduced in 25 000 mg/kg feed male rats
    and 50 000 mg/kg feed male and female rats.  Tremor, ataxia, and
    urine stains were observed in the 50 000 mg/kg feed group.  Serum
    cholesterol was significantly decreased in females in the 12 500
    mg/kg feed (p<0.01), 25 000 mg/kg feed (p<0.001), and 50 000 mg/kg
    feed (only one female rat alive after 11 weeks) groups.  No other
    dose-related effects were seen of the haematological, clinical
    chemical, or pancreatic enzyme parameters in treated rats.  The
    volume, surface, and numerical density of hepatic peroxisomes in
    female rats (25 000 mg/kg feed) were significantly (p<0.001)
    increased.  No differences in organ weights attributive to treatment
    were observed.  Histopathological examination of male and female
    rats receiving 50 000 mg/kg feed benzyl acetate revealed
    degeneration and necrosis of neurons and glia cells in cerebellum
    and hippocampus of the brain, 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 (NTP, 1992).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Mice

          Groups of 60 male and 60 female mice (B6C3F1, average age at
    initial exposure 40 days; 11 days quarantine prior to test) received
    0, 330, 1 000, or 3 000 mg/kg feed (equal to 0, 37, 112, or 346
    mg/kg bw/day for males, equal to 0, 42, 132, or 382 mg/kg bw/day for
    females) of benzyl acetate (benzyl acetate, properties consistent
    with structure and literature references, purity: 98-9%; stability
    monitored periodically, and no degradation of bulk chemical
    observed) in their diet for a period of 103 weeks.  Interim
    sacrifice was carried out on 10 mice of each sex from all groups
    after 15 months of exposure.  Feed [feed prepared weekly and stored
    in dark; analyzed during study for benzyl acetate concentrations,
    stability, and homogeneity; contained low and biologically
    insignificant levels of aflatoxins, pesticides and heavy metals] and
    water provided  ad libitum.  Feed consumption was measured daily
    per cage for 5 days once every 4 weeks.  The animals were weighed
    weekly during the first 13 weeks of the study and every 4 weeks
    thereafter.  Haematology and clinical chemistry (cholesterol
    triglycerides, alkaline phosphatase, creatinine kinase, and sorbitol
    dehydrogenase) were carried out on interim sacrifice mice after 15
    months.  Necropsy and a thorough histopathological examination were
    performed on all animals.  Organ weights included brain, right
    kidney, and liver.

          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 the 3 000 mg/kg
    feed group.  Almost all deaths occurred during the last 9 months of
    the study.  The average feed consumption of treated mice was similar
    to that of the control groups.  All treated mice, except 330 mg/kg
    feed females, showed decreased mean body weights compared to those
    of controls, weights were 13% and 9% lower at termination in males
    and females, respectively (statistics not reported).  A slight
    decrease (inconsistent dose-related significance at the p<0.05
    level) in cholesterol, triglyceride and (females only) alkaline
    phosphatase levels was observed for treated mice compared to the
    control groups.  No dose-related effects were seen in haematology. 
    Statistically significant (p<0.05 or lower) dose-related increased
    incidences and severities of non-neoplastic lesions of the nasal
    mucosa and glands occurred in all treated male and female mice
    compared to the control groups.  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 present in male and female
    mice at interim sacrifice after 15 months of exposure.  No neoplasms
    nor pre-neoplastic dose-related lesions occurred in the nose.  A
    dose-related negative trend in the incidence of hepatocellular

    carcinoma and hepatocellular adenoma, statistically significant
    (p<0.01) for hepatocellular adenoma in the 3 000 mg/kg feed group,
    occurred in male mice.  This effect was not seen in female mice
    (NTP, 1992).

    2.2.3.2  Rats

          Groups of 60 male and 60 female rats (F344; average age at
    initial exposure 41 days; 12 days quarantine prior to test) received
    0, 3 000, 6 000, or 12 000 mg/kg feed (equal to 0, 113, 225, and 550
    mg/kg bw/day) of benzyl acetate (benzyl acetate, properties
    consistent with structure and literature references, purity: 98-9%;
    stability monitored periodically, no degradation of bulk chemical
    was observed) in their diet for a period of 103 weeks.  Interim
    sacrifice was carried out on 10 rats of each sex from all groups
    after 15 months of exposure.  Feed [feed prepared weekly and stored
    in dark; analyzed during study for benzyl acetate concentrations,
    stability, and homogeneity; contained low and biologically
    insignificant levels of aflatoxins, pesticides and heavy metals] and
    water provided  ad libitum.  The feed consumption was measured
    daily per cage for 5 days once every 4 weeks.  The animals were
    weighed weekly during the first 13 weeks of the study and every 4
    weeks thereafter.  Haematology, clinical chemistry (cholesterol
    triglycerides, alkaline phosphatase, creatinine kinase, and sorbitol
    dehydrogenase) and (in males only) analysis of pancreatic enzymes
    (amylase, lipase, carboxypetidase) were carried out on interim-
    sacrificed rats after 15 months.  Necropsy and a thorough
    histopathological examination were performed on all animals.  Organ
    weights included brain, right kidney, and liver.

          No significant differences in the survival rate, average feed
    consumption, clinical findings, clinical chemistry, haematology,
    pancreatic enzyme assays, or incidences of neoplasms and non-
    neoplastic lesions were observed in treated male and female rats
    compared to those of the controls.  The mean body weights of the
    12 000 mg/kg feed males and treated females were approximately 5%
    lower than those of the control groups throughout most of the study
    (NTP, 1992).

    2.2.8  Special studies on genotoxicity

          Genotoxicity studies with benzyl acetate are summarized in
    Table 1.

    
    Table 1.  Results of genotoxicity tests on benzyl acetate

                                                                                                           

    Test system                  Test object          Concentration of        Result          Reference
                                                      benzyl acetate

                                                                                                           

    In vitro Bacterial           S.typhimurium        33-10 000 g/plate      Negative        NTP, 1992
    mutagenicity assay (1)       TA1535, TA1537,
                                 TA100, TA98

    Mammalian cell               Mouse lymphoma       0.25-1.75 l/ml         Positive (2)    NTP, 1992
    mutation assay (1)           cells (TK locus)     (3), 700-1 700
                                                      g/ml

    Sister chromatid             Chinese hamster      50-5 000 g/ml          Negative        NTP, 1992
    exchange assay (1)           ovary(CHO) cells

    Chromosomal aberration       Chinese hamster      160-1 600 g/ml         Negative (4)    NTP, 1992
    assay (1)                    ovary(CHO) cells

    In vivo Sex-linked           Drosophilia          300 ppm & 20 000        Negative        NTP, 1992
    recessive lethal             melanogaster         ppm (5)
    mutation test

    Sister chromatid             Mouse bone           312-1 250  mg/kg bw     Negative        NTP, 1992
    exchange test                marrow cells

    Chromosomal                  Mouse bone           325-1 700 mg/kg bw      Negative        NTP, 1992
    aberration test              marrow cells

    Micronucleus test            Mouse bone           312-1  250 mg/kg bw     Negative        NTP, 1992
                                 marrow cells
                                                                                                           

                                                                                                           

    Test system                  Test object          Concentration of        Result          Reference
                                                      benzyl acetate

                                                                                                           

    Micronucleus test            Mouse peripheral     3130-50 000 ppm         Negative        NTP, 1992
                                 blood
                                                                                                           

    (1)  In presence and absence of metabolic activation
    (2)  Laboratory 1: positive only in presence of metabolic activation
         Laboratory 2: positive in presence of metabolic activation, test
         in absence of metabolic activation not conducted
    (3)  Concentration unit equivocal, (laboratory 1)
    (4)  Negative in presence of metabolic activation.  Two out of three trials
         in a test showed P values of less than 0.05
         in absence of metabolic activation
    (5)  Feeding: 300 mg/kg feed, injection: 20 000 ppm
    

    3.  COMMENTS

          At its present meeting, the Committee reviewed data from short-
    term and long-term studies in rats and mice in which benzyl acetate
    had been incorporated into the diet.  These studies did not show any
    increase in the incidence of either hepatocellular or forestomach
    tumours in mice or of pancreatic tumours in rats, which had been
    observed previously in studies with benzyl acetate administered by
    gavage in corn oil.

          The Committee noted a documented association between the use of
    corn oil as a vehicle control and an increased incidence of
    pancreatic acinar cell hyperplasia and adenomas in male rats.  In
    addition, altered incidence of other site-specific neoplasms has
    been observed after administration of corn oil by gavage (see
    section 2.2.2).

          Considering the use of both modes of administration in long-
    term studies, the Committee concluded that the administration of
    benzyl acetate in the diet was more relevant to its safety
    assessment as a food additive than administration by gavage in corn
    oil.

          The Committee also reviewed data from new genotoxicity studies.
    These studies showed no evidence of  in vivo genotoxicity of benzyl
    acetate when tested for induction of sister chromatid exchange,
    chromosomal aberrations or micronuclei in mouse bone marrow cells.              
    The Committee noted the induction of necrosis of the brain involving
    the cerebellum and/or hippocampus in rats and mice treated with very
    high doses of benzyl acetate (50 000 mg/kg feed) for 13 weeks.  No
    such effect was observed in the long-term toxicity/carcinogenicity
    studies in mice or rats at lower doses (3 130-25 000 mg/kg feed). 
    In the long-term toxicity/carcinogenicity study mentioned above, of
    rats administered dietary benzyl acetate at levels up to 550 mg per
    kg of body weight per day, no adverse effects were observed.

          In the long-term toxicity/carcinogenicity study in mice given
    330, 1 000, or 3 000 mg benzyl acetate per kg diet (equal to 37,
    112, and 345 mg per kg body weight per day in males and 42, 132, and
    382 mg per kg body weight per day in females), dose-related
    degeneration and atrophy of the olfactory epithelium, cystic
    hyperplasia of the nasal submucosal glands, and pigmentation of the
    nasalmucosal epithelium were observed.  The Committee considered the
    changes of the nasal cavity to be a result of local irritant effects
    of the test compound and not toxicologically relevant to the
    assessment of food safety.

          At the end of the study, treated male and female mice showed
    lower mean body weights than controls.

    4.  EVALUATION

          In the absence of associated pathological lesions in the
    long-term toxicity/carcinogenicity study in mice and on the basis of
    the NOEL of 550 mg per kg of body weight per day in the long-term
    study in rats, the Committee included benzyl acetate in the group
    ADI of 0-5 mg per kg of body weight with benzyl alcohol,
    benzaldehyde and benzoic acid, and the benzoate salts.

          The Committee noted the absence of reproduction/teratogenicity
    studies for substances in this group, and recommended that a full
    review of benzoic acid and benzoates, benzaldehyde, benzyl alcohol,
    and benzyl acetate be performed in 1995 to determine whether these
    or other studies are required.

    5.  REFERENCE

    NTP (1992).  NTP (National Toxicology Program) technical report on
    the toxicology and carcinogenesis studies of benzyl acetate (CAS No.
    140-11-4) in F344 rats and B6C3F1 mice, (Feed studies).  NTP TR
    431. Board Draft.  NIH Publication 92-3162.  U.S.  Department of
    Health and Human Services, Public Health Service, National Institute
    of Health.  Research Triangle Park, NC, USA.


    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 37)
       BENZYL ACETATE (JECFA Evaluation)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 40, 1986)
       Benzyl Acetate  (IARC Summary & Evaluation, Volume 71, 1999)