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

    Biological data
         Biochemical aspeects
         Absorption, distribution, and excretion
    Toxicological studies
         Acute toxicity studies
         Short-term toxicity studies
         Long-term toxicity/carcinogenicity studies
         Reproductive toxicity studies
         Special studies on genotoxicity
         Observations in humans


         Ethyl vanillin was first evaluated at the eleventh meeting of the
    Committee (Annex 1, reference 14), when an ADI of 0-10 mg/kg bw was
    allocated on the basis of a long-term study in rats. At that time, the
    Committee noted that few metabolism studies had been carried out on
    ethyl vanillin and concluded that further studies of that type were
    desirable. Ethyl vanillin was re-evaluated at the thirty-fifth meeting
    of the Committee (Annex 1, reference 88) on the basis of the partial
    application of the procedure for setting priorities for the safety
    review of food flavouring ingredients (Annex 1, reference 83). At that
    time, the Committee noted that none of the previously evaluated
    long-term toxicity or carcinogenicity studies met modern standards in
    that fewer animals per group had been used than would be the present
    norm, and it therefore reduced the ADI to 0-5 mg/kg bw and made it
    temporary. A consolidated monograph was prepared (Annex 1, reference
    89). The Committee requested submission of the results of adequate
    short-term toxicity and metabolism studies in rats for evaluation in
    1992. At the thirty-ninth meeting (Annex 1, reference 101) the
    Committee was informed that the studies requested had been initiated,
    and that preliminary results did not indicate any cause for concern.
    On the basis of this information, the Committee extended the
    previously allocated temporary ADI of 0-5 mg/kg bw, pending the
    submission of the final results of the ongoing short-term toxicity
    and metabolism studies in rats for evaluation by 1994.

         At the present meeting, the Committee reviewed the studies
    requested. Relevant information from the previous monographs and
    information received since the previous evaluation are summarized and
    discussed in the following monograph addendum.


    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         Early reports indicated that ethyl vanillin was probably
    metabolized to glucuroethyl vanillin and ethyl vanillic acid, of which
    some was conjugated with glucuronic and sulfuric acids (Williams,

         Ethyl 14C-vanillin was administered to male and female
    Sprague-Dawley CD rats by gavage in polyethylene glycol solution at
    single doses of 50, 100, or 200 mg/kg bw. Ethyl vanillin was rapidly
    absorbed and peak plasma radioactivity occurred within 2 h after
    dosing at all dose levels, falling rapidly to undetectable levels
    within 96 h. Plasma radioactivity tended to be higher in female than
    male rats and it was postulated that this might reflect a lower
    metabolic capacity of female rats.

         Urinary excretion of radioactivity was rapid and more than 94%
    of the dose was excreted by this route within 24 h. Only 1-5% of the
    dose was excreted in faeces. After 5 days, more than 99% of the
    administered dose was excreted. No radioactivity was detected in
    expired air, indicating that the aromatic ring was in a metabolically
    stable position (Hawkins  et al., 1992).

    2.1.2  Biotransformation

         Ethyl 14C-vanillin was administered to male and female Sprague
    Dawley CD rats at single oral doses of 50, 100, or 200 mg/kg bw. Rapid
    metabolism occurred and the principal metabolite at all dose levels
    was ethyl vanillic acid.

         Analysis of urine after hydrolysis with glucuronidase and/or
    sulfatase indicated that the major metabolites were glucuronide or
    sulfate conjugates of ethyl vanillic acid (56-62%), ethyl vanillyl
    alcohol (15-20%), and ethyl vanillin (7-12%). A minor proportion of
    the dose (2-8%) was excreted as the glycine conjugate of vanillic acid
    (ethyl vanilloyl glycine) (Hawkins  et al., 1992). The major
    metabolic pathways of ethyl vanillin in rats are shown in Figure 1.

         Ethyl vanillic acid was also the major metabolite after dietary
    administration of ethyl vanillin to rats at doses of 500, 1000 or
    2000 mg/kg bw (Hooks  et al., 1992a).

         During urinary organic acid profiling in human subjects, several
    patients excreted high concentrations of ethyl vanillic acid
    (3-ethoxy-4-hydroxybenzoic acid) and traces of 3-ethoxy-4-hydroxy-
    mandelic acid.


         Ethyl vanillic acid was identified by GC/MS in the urine
    of a 9-year old female patient who had received liquid dietary
    supplementation flavoured with vanilla. Other patients excreting this
    acid were also known to have consumed foodstuffs flavoured with ethyl
    vanillin. Eight different urine samples containing more than 50 mg
    ethyl vanillic acid/g creatinine were also found to contain small
    amounts of vanillylmandelic acid. Unchanged ethyl vanillin was not
    detected in any of the urine samples.

         A healthy adult male volunteer drank a 235 ml aliquot of a liquid
    dietary supplement containing an unknown quantity of ethyl vanillin. A
    concentration of 13 mg ethyl vanillic acid/g creatinine was found in a
    12-hour urine sample. The compound was not present in urine collected
    before exposure (Mamer  et al., 1985).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with ethyl vanillin are
    summarized in Table 1.

         The lowest lethal dermal dose in rats was reported to be
    1800 mg/kg bw (RTECS, 1990).

         When groups of 6 rabbits were given ethyl vanillin by gavage, a
    dose of 150 mg/kg bw caused no adverse effects. At 2500 mg/kg bw, only
    a transient increase in respiration rate was observed. The minimum
    oral lethal dose was reported to be 3000 mg/kg bw (Deichmann &
    Kitzmuller, 1940).

    Table 1.  Acute toxicity studies with ethyl vanillin

    Species        Route      LD50          Reference
                              mg/kg bw

    Mouse          i.p.       7501          Caujolle & Meynier, 1954a

    Rat            oral       1590          Sporn, 1960
                   oral       > 2000        Jenner et al., 1964
                   s.c.       1800          Deichmann & Kitzmuller, 1940

    Guinea-pig     i.p.       1140          Caujolle & Meynier, 1954b

    Dog            i.v.       760           Caujolle et al., 1953

    1    Maximum non-lethal dose, 450 mg/kg bw; Lethal dose: 950 mg/kg bw

    2.2.2  Short-term toxicity studies  Rats

         Doses of 300 mg ethyl vanillin/kg bw were administered to rats by
    gavage twice weekly for 14 weeks without any adverse affects. In
    another experiment, groups of 16 rats were fed ethyl vanillin at a
    dose of 20 mg/kg bw/day for 18 weeks without adverse effect. However,
    64 mg/kg bw/day for 10 weeks reduced growth rate and caused
    myocardial, renal, hepatic, lung, spleen and stomach injuries (nature
    not specified) (Deichmann & Kitzmuller, 1940).

         Sixteen rats were given 30 mg ethyl vanillin weekly for 7 weeks
    without adverse effect on growth, food intake or protein utilization
    (Spore, 1960).

         Groups of 5 male rats were fed 0, 2%, or 5% ethyl vanillin in the
    diet for 1 year without any adverse effects (Hagan  et al., 1967).

         Groups of CD Sprague-Dawley BR rats (20/sex/group) were fed ethyl
    vanillin of > 99.9% purity (nature of diet e.g., semi-synthetic/chow
    diet, not specified) at dose levels of 0, 500, 1000 or 2000 mg/kg
    bw/day for 13 weeks. The study was designed in accordance with
    toxicological principles for the safety assessment of food additives
    established by the US FDA (FDA, 1982). The diet was prepared weekly
    and showed stability for up to 18 days at room temperature. The
    achieved mean dose over the 13-week period was within 1.5% of the
    nominal value. Food consumption and body weight were recorded weekly.
    Ophthalmoscopy was done before treatment and at termination of the
    study. Detailed haematological and clinical chemical examinations were
    carried out at week 6 and 13. At termination, all animals were
    necropsied and organ weights recorded (adrenals, brain, heart,
    kidneys, liver, lungs, ovaries, pituitary gland, prostate, spleen,
    testes, thyroids gland, uterus). A complete histological examination
    was performed on rats in the control and top-dose groups (adrenals,
    alimentary tract, aorta, brain, eyes, femur, Harderian gland, heart,
    kidneys, larynx and pharynx, liver, lungs, cervical and mesenteric
    lymph nodes, mammary gland, ovaries, pancreas, pituitary gland,
    prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal
    muscle, skin, spleen, sternum, testes, thymus, thyroid gland, tongue,
    trachea, urinary bladder, uterus, vagina). The examination was
    extended to the low and intermediate dosage groups where
    treatment-related effects were suspected.

         No clinical signs or treatment-related deaths of toxicological
    significance were observed in treated animals during the study. Food
    intake was statistically significantly reduced in females at the
    highest dose group at week 1, and in treated male groups at weeks 1-4;
    thereafter there were no significant differences in food intake
    between controls and treated animals. Water intake, measured
    accurately during week 12 of treatment, did not differ notably from
    controls. Body-weight gain in males and females in the high-dose group
    was significantly reduced compared to control throughout the study;
    significant lower body-weight gain was also apparent in males of the
    intermediate- and low-dose groups during the first 4 weeks of
    treatment. The authors considered these differences from control not
    to be treatment-related since the differences were not dosage-related
    in magnitude, and because of intra-group variability noted in feeding
    patterns of all groups of male rats. Impaired food efficiency was
    noted for both male and female rats at the highest dose level.

         There were no treatment-related differences from control in
    haematological parameters at week 6 or at termination. Clinical
    biochemical analyses showed statistically significant higher values in
    the high-dose group compared to control for ALAT, ALP, cholesterol and
    total plasma protein. Cholesterol levels were significantly increased

    in males at the intermediate-dose group at week 6 only. The authors
    considered the alteration of the clinical biochemical parameters
    secondary to the hepatic changes seen histologically. Other sporadic
    differences from control values were generally within normal ranges
    for the strain and were not considered of toxicological significance.

         At autopsy, enlarged cervical lymph nodes were noted in males at
    the intermediate-dose group, and in both sexes at the highest dose
    group. In addition, there was a reduction in adipose tissue in rats of
    both sexes at the highest dose group. Absolute liver weights were
    similar to controls but relative liver weights were increased in the
    intermediate- and high-dose animals. Absolute and relative spleen
    weights were increased in the intermediate- and high-dose groups.
    Although relative spleen weights were increased in the low-dose males,
    the absolute organ weights were unaffected, and in the absence of
    histopathological changes this observation was considered by the
    authors to be of no toxicological significance.

         Histological examination revealed a dose-related increased
    incidence of hepatic peribiliary inflammatory change in both males and
    females of the intermediate- and high-dose groups, and minor bile duct
    hyperplasia affecting 1/20 intermediate- and 4/20 high-dose males.
    There were no changes observed in the liver parenchyma and no
    degenerative or inflammatory changes of the bile duct epithelium.
    Increased white pulp cellularity and prominence of germinal centres in
    the spleen, and increased prominence of germinal centres and lymphoid
    proliferation in cervical lymph nodes were seen in the intermediate-
    and high-dose groups. The authors considered the findings of the
    lymphoid tissue to be associated reactive changes to the hepatic
    peribiliary inflammatory observations.

         The authors concluded that no treatment-related changes were
    observed at 500 mg/kg bw/day which was considered to be the NOEL in
    this study (Hooks  et al., 1992b).  Rabbits

         Single rabbits were given ethyl vanillin orally in 10% aqueous
    glycerine at doses of 15 mg/kg bw/day for 13 or 26 days; 32 mg/kg
    bw/day for 15 days; 41 mg/kg bw/day for 26 days; or 49 mg/kg bw/day
    for 43 days. At the highest close level, anaemia, diarrhoea and lack
    of weight gain were observed but no toxic signs were reported at any
    of the lower doses (Deichmann & Kitzmuller, 1940).

         Subcutaneous injection of ethyl vanillin to rabbits at doses of
    148-154 mg/kg bw/day for 6 days did not elicit any observed adverse
    effects. Similarly, oral intubation of ethyl vanillin in a milk
    vehicle at a dose of 240 mg/kg bw during 25 days (observation period
    56 days), or during 54 days (observation period 126 days) did not
    produce any observed effects (the parameters observed were not
    specified in any of these studies) (Deichmann & Kitzmuller, 1940).

    2.2.3  Long-term toxicity/carcinogenicity studies  Mice

         The maximum tolerated dose for ethyl vanillin in strain A mice
    when administered i.p. 3 times/week for 2 weeks was reported to be
    75 mg/kg bw. Administration of ethyl vanillin i.p. at doses of 15 or
    75 mg/kg bw, 3 times/week for 8 weeks resulted in mortalities of 8/20
    and 10/20 animals, respectively. Control animals receiving i.p. 
    injections of the vehicle tricaprylin, had survival rates of 77/80
    males and 77/80 females. In the control group, 28% of males and 23% of
    females developed lung tumours whereas in the treated groups only one
    animal, in the higher dose group, exhibited a single lung nodule. It
    was concluded that ethyl vanillin did not potentiate the pulmonary
    tumour response in strain A mice (Stoner  et al., 1973).  Rats

         Groups of Osborne-Mendel rats (12/sex/group) were fed diets
    containing 0, 0.5, 1 or 2% ethyl vanillin for 2 years, and 2% or 5%
    for 1 year. Haematological examinations (RBC, WBC, haemoglobin and
    haematocrit) were performed at 3, 6, 12 and 22 months and at
    termination in the 2-year study. All animals were necropsied and
    liver, kidney, spleen, heart and testes weights recorded. Histological
    examinations were performed on these organs and remaining thoracic and
    abdominal viscera, bone and bone marrow, and muscle. No adverse
    effects on growth, haematology, organ weights or histology of major
    tissues were reported (Hagan  et al., 1967).

    2.2.4  Reproductive toxicity studies

         No reproductive toxicity or teratogenicity studies have been
    reported on ethyl vanillin.

    2.2.5  Special studies on genotoxicity

         The results of genotoxicity studies with ethyl vanillin are
    summarized in Table 2.

         From the SCE studies with human lymphocytes the authors concluded
    that benzaldehyde derivatives, including ethyl vanillin, were probably
    direct acting SCE inducers and the aldehyde moiety was of primary
    importance (Jansson  et al., 1988). This contrasts with the negative
    effect in CHO cells (Sasaki  et al., 1987).

         In a study on the anti-mutagenic potential of flavourings, ethyl
    vanillin was reported to show marked anti-mutagenic activity against
    mutagenicity induced by 4-nitroquinoline 1-oxide, furylfuramide,
    captan or methylglyoxal in  Escherichia coli WP2s but was ineffective

    against mutations induced by Trp-P-2 or IQ in  Salmonella typhimurium
    TA98. It was proposed that the anti-mutagenic activity was due to
    enhancement of an error-free recombinant repair system (Ohta  et al.,
    1986; Watanabe  et al., 1988).

    2.3  Observations in humans

         In a 24-hour closed patch test in 25 subjects, ethyl vanillin
    tested at 2% in petrolatum produced a mild irritation. No
    sensitization reactions occurred when ethyl vanillin was used at 2% in
    petrolatum in a maximization test on 25 volunteers (Kligman, 1970).

         People previously sensitized to balsam of Peru, benzoin, rosin,
    benzoic acid, orange peel, cinnamon and cloves have been reported to
    cross-react with hydroxybenzaldehydes such as vanillin or ethyl
    vanillin. A patient with contact dermatitis showed strong reactions
    to balsam of Peru, cassia oil and ethyl vanillin, it was not known
    whether the dermatitis was a response to occupational exposure to
    ethyl vanillin in a candy factory or to rubber (Rudzki & Grzwa, 1976).

        Table 2.  Results of genotoxicity assays on ethyl vanillin

    Test system         Test object        Concentration of    Results      Reference
                                           ethyl vanillin

    Micronucleus        Mouse              2  0-1000          Negative     Wild et al., 1983
    test                                   mg/kg bw

    Ames test1          Salmonella         0-10 mg/plate       Negative     Ishidate et al.,
                        typhimurium                                         1984
                        TA92, TA94,
                        TA98, TA100

    Ames test2          S. typhimurium     0-10 mg/plate       Negative     Mortelmans et al.,
                        TA98, TA100                                         1986

    Ames test1          S. typhimurium     0-3.6 mg/plate      Negative     Wild et al., 1983
                        TA98, TA100

    Chromosomal         Chinese hamster    0-0.25 mg/ml        Negative3    Ishidate et al.,
    aberrations         ovary (CHO)                                         1984
                        cells in vitro

    Sister chromatid    Chinese            0-100 M             Negative4    Sasaki et al., 1987
    exchange (SCE)      hamsterd ovary
                        cells in vitro

    Sister chromatid    Human              0-2 M               Positive     Jansson et al.,
    exchange (SCE)      lymphocytes in                                      1988

    Table 2.  Results of genotoxicity assays on ethyl vanillin (cont'd).

    Test system         Test object        Concentration of    Results      Reference
                                           ethyl vanillin

    Heritable           Drosophila         50 mM               Negative     Wild et al., 1983
    mutations           melanogaster

    1     with or without metabolic activation using rat liver S9 fractions
    2     with or without metabolic activation using rat or hamster liver S9 fractions
    3     ethyl vanillin did not induce chromosomal aberrations but did cause an increase
          in polyploid cells, however the significance of this was unclear and similar
          polyploidy was induced by riboflavin
    4     ethyl vanillin did not induce sister chromatid exchanges in cultured CHO cells
          in vitro but was reported to enhance the ability of mitomycin C to cause sister
          chromatid exchanges.

         The metabolism studies indicated that ethyl vanillin was rapidly
    absorbed, metabolized and excreted in the rat. The principal
    metabolite identified was ethyl vanillic acid (3-ethoxy-4-hydroxy-
    benzoic acid). This compound, which is not a normal constituent of
    human urine, has also been identified in the urine of humans known to
    have ingested vanilla-flavoured foodstuffs.

         In the recent 13-week toxicity study in which rats were fed ethyl
    vanillin at 500, 1000 or 2000 mg/kg bw/day, treated males showed a
    transient reduction in body-weight gain compared with controls during
    the first 4 weeks of treatment. Since this effect was only transient
    and associated with reduced food intake, probably due to impaired
    palatability, the Committee concluded that the NOEL was 500 mg/kg

         The Committee considered ethyl vanillin not to be genotoxic on
    the basis of negative results in a large number of studies, although
    one assay for sister chromatid exchange was positive.


         The Committee concluded that, in the light of the information
    showing daily intakes to be in the range of 0.06-7 mg/person/day, the
    safety evaluation could be based on the principles applicable to
    materials occurring in foods in small amounts. In view of the limited
    toxicological information available, the Committee withdrew the
    previous temporary ADI and allocated an ADI of 0-3 mg/kg bw for ethyl
    vanillin, based on a NOEL of 500 mg/kg bw/day in the 13-week toxicity
    study in rats and a safety factor of 200.


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     Ann. Pharm. Fr., 12: 42-49.

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    See Also:
       Toxicological Abbreviations
       Ethyl vanillin (FAO Nutrition Meetings Report Series 44a)
       Ethyl vanillin (WHO Food Additives Series 26)
       ETHYL VANILLIN (JECFA Evaluation)