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    GALLATES (PROPYL, OCTYL AND DODECYL)

    First draft prepared by
    Dr G.J.A. Speijers and Mrs M.E. van Apeldoorn
    National Institute of Public Health and Environmental Protection
    Laboratory for Toxicology
    Bilthoven, The Netherlands

    1.  EXPLANATION

         Propyl, octyl and dodecyl gallates have been evaluated for
    acceptable daily intake at the third, sixth, eighth, tenth, fifteenth,
    sixteenth, seventeenth, twentieth, twenty-fourth, and thirtieth
    meetings of the Committee (Annex 1, references 3, 6, 8, 13, 26, 30,
    32, 41, 53 and 73).  At the twenty-fourth meeting a group ADI of 0-0.2
    mg/kg bw was established, based on the supposed similarity in the
    biotransformation of these compounds.  The Committee used a higher
    than normal safety factor (250) because of concern for adverse effects
    shown in reproduction studies.  The gallates were again reviewed by
    the Committee at its thirtieth meeting.  Due to lack of adequate data,
    an ADI was not established for octyl or dodecyl gallate, and more
    information on the hydrolysis and the biotransformation (including
    lactating animals) of the different gallates was required.  For propyl
    gallate an ADI of 0-2.5 mg/kg bw was established.

         Since the last evaluation, additional data have become available. 
    These new data included four-week and 90-day toxicity studies in rats
    with propyl gallate and  in vitro studies on the hydrolysis of the
    gallates in different tissues.  These  data are summarized and
    discussed in this monograph, which also includes all data from
    previously published monographs.  Because this monograph covers the
    data on propyl, octyl and dodecyl gallates separately, a modified form
    of the general monograph format has been used.

    PROPYL GALLATE

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion.

         No information available.

    2.1.2  Biotransformation

         The available evidence indicates that the gallate esters are
    hydrolyzed in the body to gallic acid.  Most of the gallic acid is
    converted into 4-0-methyl gallic acid.  Free gallic acid or a
    conjugated derivative of 4-0-methyl gallic acid is excreted in the
    urine.  Conjugation of the 4-0-methyl gallic acid with glucuronic acid
    was demonstrated (Booth  et al., 1959).

         Detailed metabolic pathways for propyl gallate have been
    described (Dacre, 1960).

          In vitro incubations with propyl, octyl and dodecyl gallate
    were performed using homogenates of liver, mucosa of the small
    intestine, and contents of caecum/colon as a source of intestinal
    microflora.  The various homogenates were incubated at 37° C with the
    individual gallate esters.  At various time points up to 24 hours,
    samples were taken and analysed by HPLC in order to determine the
    concentration of gallic acid and residual ester.  From the time-course
    of gallic acid formation, as well as the disappearance of the specific
    esters, the rate of hydrolysis of the three esters was calculated.

         All test substances were extensively metabolized by the
    homogenate of the intestinal mucosa, which was demonstrated by the
    appearance of peaks in the chromatograms.  Furthermore, the caecum and
    colon contents also showed a high metabolic capacity, especially
    towards propyl gallate.  The amount of gallic acid detected in the
    incubations was always much smaller than the total decrease of the
    amount of ester.  It seems likely that apart from hydrolysis of the
    ester bond, other biotransformation routes (oxidation and/or
    conjugation) are of major importance for all three gallate esters.

         The three homogenates show quantitatively different structure-
    activity relationships for the three esters.  Homogenates of liver and
    of contents of caecum and colon metabolize propyl gallate most
    extensively, followed by octyl or dodecyl gallate.  Homogenate of the
    mucosa of the small intestine shows the highest rates with octyl
    gallate, lower rates with dodecyl gallate and propyl gallate.  For
    this homogenate, the rate of formation of gallic acid is inversely
    related to the chain length of the ester (de Bie & van Ommen, 1992).

    2.1.3  Effects on enzymes and other biochemical parameters

         Propyl gallate inhibited liver mixed function oxidase (MFO) and
    demethylase activity when added at concentrations of 50 to 500 µM to
    liver microsomal preparations obtained from male Sprague-Dawley rats. 
    Specifically, the compound inhibited benzpyrene hydroxylase activity
    and demethylase activity with ethyl morphine, aminopyrene or
    benzphetamine as substrate.  No induction of MFO activity was noted
    when propyl gallate was injected intraperitoneally at 300 mg/kg bw 24
    hours prior to sacrifice and assay.  The microsomes from the treated
    animals had lower demethylase activity, probably as a result of
    inhibitory amounts of the compound attached to the microsomes (Yang
    and Strickhart, 1974).

         Weanling female Sprague-Dawley rats were fed high polyunsaturated
    fat, high saturated fat or low fat diets for one month, with or
    without the addition of 0.3% propyl gallate.  Propyl gallate caused no
    measurable difference in body weights, relative liver weight or liver
    microsomal protein values.  Liver enzyme activity of aniline
    hydroxylase, aminopyrene N-demethylase and cytochrome-C-reductase were
    not affected by treatment, nor was the liver concentration of
    cytochrome P-450 and microsomal protein.  Rats dosed with BHT at 0.3%
    of the diet exhibited liver hypertrophy and induction of cytochrome P-
    450 microsomal protein and liver enzymes (King and McCay, 1981).

         Addition of 25 or 125 µmol of propyl gallate to an MFO assay
    system prepared from the tissues of male Sprague-Dawley rats (liver,
    kidneys, stomach, colon and small intestine) inhibited the oxidation
    of benzo (a)pyrene (Rahimtula  et al., 1979).

         Addition of 50 µmol of propyl gallate to a rat testis microsomal
    preparation in the presence of arachidonate stimulated the formation
    of prostaglandins PGF2.  Addition of 0.1 mmol vitamin E instead of
    propyl gallate did not result in increased production of any of the
    prostaglandins.  Addition of propyl gallate to similar preparations
    from vitamin E-deficient rats resulted in an increase in production of
    PGF2 only.  In the female rat both dietary lipid and dietary propyl
    gallate affect prostaglandin synthesis by mammary gland preparations. 
    These effects are concentration-dependent effects with stimulation of
    synthesis of some prostaglandins at one level of propyl gallate and
    inhibition at higher levels.  The effect of propyl gallate is also
    dependent upon the type of fat in the rat diet.  Propyl gallate
    stimulates formation of prostglandin PGF2 in mammary gland from rats
    fed polyunsaturated fats, but inhibits PGF2 synthesis in rats fed a
    saturated fat diet.  Stimulation of synthesis of different
    prostaglandins may occur in preparations to which propyl gallate was
    added exogenously as compared to a dietary source of the compound. 
    Changes in levels of PGF2 have been correlated with the
    susceptibility of rats to mammary tumours induced by 7,12-dimethyl-
    benzo (a)anthracene (Carpenter, 1981).

         Effects of propyl gallate on glutathione-S-transferase are
    described in discussion of the short-term toxicity study (Speijers  et
    al., 1993).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with propyl gallate are
    summarized in Table 1.

    Table 1.  Acute toxicity studies - propyl gallate

                                                                  

    Animal    Route     LD50 (mg/kg bw)    Reference

                                                                  

    Mouse     oral      2 - 3 000          Lehman  et al., 1951

    Rat       oral      3 800              Orten  et al., 1948

    Rat       oral      3 600              Lehman  et al., 1951

    Rat       i.p.      380                Orten  et al., 1948
                                                                  

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

         Levels of propyl gallate of 1.2% and 2.3% in the diet of rats
    caused a decreased weight gain, the bitter taste of the gallate
    apparently making the diet unpalatable.  The high dose level caused
    some deaths (about 40%) during the first month; the survivors
    continued to eat the diet for 10 - 16 months and showed retarded
    growth, but no pathological lesions.  The animals that died exhibited
    renal damage (Orten  et al., 1948).

         Weanling rats were fed diets which contained 20% lard and 0, 0.1,
    0.2, 0.3, 0.4 and 0.5% propyl gallate for six weeks.  There was no
    effect on body weight, liver weight, liver weight to body weight
    ratio, left adrenal weight, total liver lipid, composition of liver
    polyunsaturated fatty acids, liver cholesterol, adrenal cholesterol or
    serum sodium (Johnson and Hewgill, 1961).

         Propyl gallate was added to the dietary fat of weanling rats at
    levels of 0.02% in the fat for 13 weeks.  The fat content of the diet
    provided 30% of its caloric value.  There was a very slight inhibition
    of growth.  The same rats were then placed on a partial

    starvation diet and kept until they died.  The survival time of the
    animals which had received the propyl gallate was considerably reduced
    and the reduction in their total body protein was greater than control
    rats (Bukhan, 1962).

         Weight gain depression of more than 10% was observed in male rats
    receiving diets containing 12 500 or 25 000 and in females receiving
    25 000 mg propyl gallate/kg feed.  Dirty tails, indicating
    gastrointestinal disturbance, were noted in both sexes at 25 000 mg
    propyl gallate/kg feed.  In the 25 000 mg/kg feed groups, reddish
    duodenal mucosa was reported in both sexes, in addition to thickening
    of the stomach wall with necrosis and ulceration of the mucosal
    surface of the stomach, and moderate to severe granulomatous
    inflammatory response in the submucosa and muscular wall of the
    stomach.  No stomach nor duodenal lesions were noted in either sex at
    6 000 or 12 500 mg/kg feed (Abdo  et al., 1983).

         A 4-week oral toxicity study with propyl gallate was performed in
    rats (6 animals/group/sex) at dose levels of 0, 1 000, 5 000 and 25
    000 mg/kg feed.  Parameters studied comprised growth, food and water
    intake, biochemistry, haematology, organ weights and histopathology.

         In the high-dose group both females and males gained less weight
    than those in the control group.  Haemoglobin concentration, packed
    cell volume, red blood cell concentration, mean corpuscular volume and
    mean corpuscular haemoglobin were lowered in the high-dose group. 
    Consistent with the anaemia, an increased extramedullary
    haematopoiesis and slightly decreased haemosiderosis were noted in the
    spleen.  In kidneys hyperplastic tubuli in the outer medulla were
    detected.  In the liver of the animals of the 5 000 and 25 000 mg/kg
    feed groups increases in activity of aminopyrine-N-demethylase and
    glucuronyl-transferase and glutathione-s-transferase and an increase
    in cytochrome P-450 content were detected (Strik  et al., 1986).

         A toxicity study with propyl gallate in SPF-derived Wistar
    RIVM:Tox rats (10 animals/group/sex) was performed in which they were
    fed a semisynthetic diet containing 0, 490, 1 910 or 7 455 mg propyl
    gallate/kg feed for 13 weeks.  Body-weight gain was recorded weekly
    and food-intake twice weekly.  Other parameters included haematology,
    biochemical analyses in urine, serum and liver and complete
    histopathological examinations.

         Adverse effects of propyl gallate observed in the high-dose group
    were effects on the haematopoietic system reflected in the
    haematological parameters (Hb, Hct and RBC) and the morphological
    changes (extramedullary haematopoiesis) in the spleen.  The other
    effects of propyl gallate comprised a decreased incidence of
    nephrocalcinosis normally seen in female rats on semisynthetic diet,
    an increased activity of ethoxy-resorufin-o-deethylase (EROD) in the

    high-dose group and an increased activity of the conjugating enzymes
    glucuronyl-transferase and glutathion-s-transferase, in the mid and
    high-dose groups receiving propyl gallate.  The effects on the liver
    enzymes suggest that other biotransformation routes additional to
    hydrolysis of the gallate seem to be involved.  The effects on
    nephrocalcinosis and on the conjugating enzymes were not considered
    adverse.  Therefore the NOAEL was 1 910 mg propyl gallate/kg feed,
    equal to 135 mg propyl gallate/kg bw day (Speijers  et al., 1993).

    2.2.2.2  Guinea-pigs

         Propyl gallate fed to guinea-pigs in groups of 20 at a level of
    0.02% in the diet for 14 months caused no observed ill effects (Orten
     et al., 1948).

    2.2.2.3  Dogs

         A level of 0.01% propyl gallate in the diet was well tolerated by
    a group of seven dogs over a period of 14 months (Orten  et al.,
    1948).

    2.2.2.4  Pigs

         Diets containing 0.2% propyl gallate were fed to pigs without
    observed ill effect; no anaemia was observed (van Esch, 1955).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Mice

         Groups of 50 mice (University Animal Breeding Station closed
    strain colony) equally divided by sex were maintained on diets
    containing 0, 0.25 or 1.0% n-propyl gallate for a period of 21 months. 
    Water intake, food consumption and growth of test animals were
    comparable to controls.  Treated male mice showed a greater percentage
    survival than control mice at termination.  Haematologic measurements
    (haemoglobin, packed cell volume, differential white cell count) were
    similar for test and control animals.  At autopsy, a comparison of
    relative organ/body weight showed a reduction in the relative spleen
    weight of males on the 1% diet.  No compound-related histopathological
    changes were observed (Dacre, 1974).

         Abdo and coworkers maintained groups of B6C3F1 mice of each sex
    on diets containing 0, 6 000 or 12 000 mg propyl gallate/kg feed for
    105 - 107 weeks.  Lower body weights  compared to controls were
    observed throughout most of the duration of the study in both sexes
    and both dose groups.  At week 104, mean body weights of the male mice
    were 6% and 8% lower than controls in the high- and low-dose groups
    respectively.  In the females, both dose groups had about a 12% lower
    body weight than the controls at week 104.  Feed consumption in low
    and high-dose males was 91 and 100%,

    respectively, of that of controls while the corresponding figures for
    females were 109 and 106%, respectively.  No other compound-related
    clinical signs were observed.  There was no significant effect of
    treatment on survival.  The survival rate averaged 80% in males and
    75% in females.  Tumour incidences of the haematopoietic system and
    liver in the treated groups showed significant increases, as shown in
    Table 2.  Tumours at other sites were not significantly different from
    controls.

         There was a significant positive trend in the incidence of
    histiocytic lymphoma in male mice (8%) relative to controls.  The
    historical control rate for histiocytic lymphomas was 3.3% (21/640). 
    There was a significant positive trend in the incidence of all
    malignant lymphomas in male mice (1/3/8), and significantly increased
    incidence by a direct comparison between high-dose and control (p<
    0.028).  However the high dose incidence was not statistically
    significant when compared to the historical control rate at the
    performing laboratory for all malignant lymphomas of
    9.4 (60/640).  In females the highest incidence was noted in the
    control group.

         The number of male rats in which hepatic adenomas or carcinomas
    occurred showed a significant negative trend.  Hepatocellullar
    adenomas in female mice occurred with a positive trend (p < 0.022)
    and the incidence of adenoma in the high-dose females is significantly
    greater than in controls (p < 0.039).  However the incidence in the
    high-dose group was not different from the historical incidence of
    this tumour (94/3127; 3%).  Further, the combined incidence of
    hepatocellular adenomas or carcinomas was similar in dosed and control
    groups (Abdo  et al., 1983).

         A level of 5% propyl gallate in the diet in a two-year chronic
    toxicity study in rats and mice gave rise to patchy hyperplasia in the
    proventiculus.  At a level of 1%, no difference was noted between test
    and control animals (Lehman  et al., 1951).

    2.2.3.2  Rats

         Groups of 10 male and 10 female rats were fed for two years on
    diets containing 0, 0.001, 0.01, 0.12, 1.2 or 2.3% propyl gallate. 
    The groups receiving 1.17 and 2.34% propyl gallate showed stunted
    growth and evidence of renal damage.  In the other groups, there was
    no detectable effect on haemoglobin, erythrocyte or leucocyte levels
    in the blood, nor on the histopathological appearance of the organs
    examined (Orten  et al., 1948).

    
    Table 2:  Tumour incidence in treated mice, sites with significant increases

                                                                                     
                                                                Incidence
                                                                                     
                                                           Males          Females

                           Group - 50 animals/group        C    L    H    C    L    H

    Site                   Lesion                                                   

    Haematopoietic         Malignant lymphoma:                                      
    system

                           Histiocytic                     0    0    4    2    0    0

                           Mixed                           0    1    3    4    1    3

                           Lymphocytic                     1    2    0    2    1    3

                           Not Otherwise Specified         0    0    1    0    1    0

                           All                             1    3    8    8    3    6

    Liver                  Adenoma                         3    4    1    0    2    5

                           Carcinoma                       14   11   9    3    1    0

                           Both                            17   15   10   3    3    5
                                                                                     

    C: control group, L: low dose, H: high dose.
    
         Abdo and coworkers fed groups of 50 F-344 rats of each sex diets
    containing 0, 6 000 or 12 000 mg propyl gallate for 105 - 107 weeks,
    as shown in Table 3.  Throughout the study, there was a dose-related
    depression in body weights at both dose levels and in both sexes. 
    Mean feed consumption was 94% and 98% of the controls in the low and
    high-dose males, while the corresponding values for females were 95
    and 115% respectively.  In males 78% of the controls, 76% of the low-
    dose and 88% of the high-dose group lived to the end of the study.  In
    females the corresponding values were 78%, 76% and 84% respectively. 
    No treatment-related clinical signs were observed.  There were no
    significant differences in survival between the groups.  For males the
    survival was 78%, 76% and 88%, for control, low, and high-dose groups,
    respectively, and the corresponding  values for female rats were 78%,
    76% and 84%.

         As shown in Table 3, in male rats the incidence of three types of
    neoplasms was increased in the low-dose treatment compared to the
    control group, namely, phaechromocytoma of the adrenal medulla, islet
    cell neoplasms of the pancreas and neoplasm of preputial gland origin. 
    Equal or greater increases were not observed in the high-dose male
    groups.  The occurrence of these tumours was not considered to be
    treatment related.  The combined incidence of male rats with
    follicular cell adenomas or carcinomas of the thyroid was significant
    (p< 0.05) by the trend test, but the high-dose incidence was not
    statistically different in any tests in direct comparison with the
    control.  In the high-dose females there were 3 mammary adenomas while
    there were none in the other two groups.  The trend test was
    statistically significant but the incidence in the high-dose group was
    not significantly higher than control.  There was an increase in the
    incidence of females with endometrial stromal polyps of the uterus
    with a marginally significant trend.  The high-dose incidence falls
    within the overall historical control range (4-36%).

         Tumours of the brain (an astrocytoma and a glioma) were found in
    two low-dose female rats.  None of the high-dose female rats showed
    this tumour.  The incidence of these tumours in the brain of the low-
    dose females was not considered to be related to propyl gallate, since
    none of the high-dose females had this tumour (Abdo  et al., 1983).

        Table 3:  Tumour incidence at sites with significant variation in rats fed propyl gallate

                                                                                     
                                                                Incidence

                                                                                     

                                                           males          females

    Group                                                  C    L    H    C    L    H

                           Total Animals                   50   50   50   50   50   50

    Site                   Lesion                                                   

    Pancreas               Islet cell:                                              

                           Adenomas                        0    8    2    0    0    0

                           Carcinomas                      2    1    2              

                           Both                            2    9    4              

    Thyroid                Follicular cell:

                           carcinoma                       0    0    2    0    0    0

                           adenoma                         0    0    1    0    0    0

    Adrenal Gland          Phaeochromocytoma               4    12   8    4    1    3

    Preputial gland        Adenoma or carcinoma            1    8    0    2    1    3

    Mammary gland          Adenoma                         0    0    0    0    0    3

    Mammary gland          Fibroadenoma                    2    0    1    11   2    5
                                                                                     

    Table 3 (contd)

                                                                                     
                                                                Incidence

                                                                                     

                                                           males          females

    Group                                                  C    L    H    C    L    H


    Uterus                 Endometrial stromal                            6    8    3
                           polyp

    Brain                  Astrocytoma                     0    0    0    0    0    3

    Brain                  Glioma                          0    0    0    0    1    0

    Haematopoietic         Leukaemia or lymphoma           16   8    6    8    5    6
    system
                                                                                     

    C: control, L: low dose, H: high dose
    
    2.2.4  Reproduction studies

         Propyl gallate was fed to rats at concentrations of 0.035, 0.2 or
    0.5% in the diet for two successive generations.  Neither effects on
    reproduction performance nor on indices of reproduction were reported. 
    No abnormalities were observed in the organs or tissues of the rats at
    autopsy (van Esch, 1955).

    2.2.5  Special studies on genotoxicity

         Propyl gallate was investigated  in vitro at concentrations of
    0.5, 5.0 and 50 µg/ml employing WI-38 human embryonic lung cells for
    anaphase abnormalities.  It was also investigated  in vivo by the
    cytogenetic analysis of metaphase cells from the bone marrow of rats
    (Sprague-Dawley C-D strain).  The dosages employed were 5.0, 50.0  and
    500 mg/kg bw.  Propyl gallate was mutagenic in neither assay.

         The genotoxic effect of propyl gallate was studied using
     Salmonella typhimurium strains TA-1530 and G-46 and  Saccharomyces
    D-3 in presence or absence of metabolic activation.  A 0.25%
    concentration was tested.  Propyl gallate was non-mutagenic in all
    tests.

         In a host-mediated assay, propyl gallate was tested at dose
    levels equivalent to 5, 50, 500 and 2 000 mg/kg bw in ICR Swiss mice
    employing, as indicator organisms,  Salmonella G-46 and TA-1530 and
     Saccharomyces D-3.  Propyl gallate was non-mutagenic under the
    conditions of the test.

         In a dominant lethal test, Sprague-Dawley CD strain male rats
    were dosed at 5, 50 and 500 mg/kg bw.  In an acute study, a single
    dose was administered with subsequent mating for each of eight weeks. 
    Propyl gallate did not produce any significant dominant lethality.  In
    a subacute study, five daily doses were administered (5 x 5, 5 x 50,
    5 x 500 and 5 x 5 000 mg/kg bw).  Males were subsequently mated for
    each of seven weeks.  No dominant lethal effects were noted (Weir and
    Brusick, 1974).

    2.2.6  Special studies of the effect of propyl gallate on the
           forestomach

         Propyl gallate incorporated into the diet (0.52 and 2%) and fed
    to male F-344 rats, for 9 days neither affected the morphological
    appearance of the forestomach squamous epithelium nor induced changes
    in the (methyl-3H) thymidine labelling index in the fundic region of
    the forestomach (Nera  et al., 1984).

    2.2.7  Special studies on the effect of propyl gallate on toxicity
           of chemical agents

    2.2.7.1  Teratogenesis

         Pregnant New Zealand white rabbits (on gestation day 12) were
    injected s.c.  with propyl gallate (362 - 900 mg/kg bw) and
    hydroxyurea (600 - 750 mg/kg bw).  The materials were injected either
    simultaneously or mixed over periods of 45 minutes.  The extent of
    amelioration of the teratogenic effects of hydroxyurea was dependent
    on the dose of propyl gallate.  There was a significant linear
    decrease in both resorptions and specific malformations with
    increasing doses of propyl gallate (de Sesso, 1981).

    2.2.7.2  Genotoxicity

         Propyl gallate inhibited the genotoxicity of benzo( a)pyrene for
     Salmonella typhimurium (Strain TA 98), and moderately increased the
    mutagenicity of aflatoxin B1 for  Salmonella  typhimurium TA 100 and
    TA 98 (Calle and Sullivan, 1982).

         Four-week old random-bred ICR Swiss male mice were fed diets
    containing 0, 10, 100, 1 000 or 5 000 mg propyl gallate/kg feed for 3
    months.  They were exposed to 50 or 125 rad of whole gamma radiation
    from a 137Cs source.  Thirty hours after irradiation, animals were
    scored for micronuclei in polychromatic bone marrow erythrocytes.  As
    compared to controls not given propyl gallate, the propyl gallate-
    treated animals had an increased incidence of micronuclei of about 1.6
    - 2-fold.  However, there was no dose dependence, 10 mg/kg of propyl
    gallate was as effective in producing radio-sensitization as 5 000
    mg/kg feed (Kamra and Bhaskar, 1978).

          Propyl gallate itself was not mutagenic towards  Salmonella
     typhimurium strains TA 98 and TA 100 in the presence or absence of
    arochlor-induced rat liver homogenates.  Treatment of bacteria
    (evidently without activation) with propyl gallate and N-acetoxy-AAF
    or N-methyl-N-nitrosoguanidine (MNNG) resulted in a reduction of
    mutation rate compared to that observed in the presence of N-acetoxy-
    AAF or MNNG alone.  By contrast, mixtures of propyl gallates and 4-
    nitroquinoline oxide (4NQO) or N-hydroxy-AAF showed increased
    mutagenicity as compared to that observed with the compounds in the
    absence of the propyl gallate.  The proceeding studies were all done
    using  Salmonella strain TA 100, except that the propyl gallate-4NQO
    mixture was tested with both  Salmonella strains TA 100 and TA 98. 
    Propyl gallate was more efficient at enhancing mutagenesis for
     Salmonella TA 100 than TA 98.  A propyl gallate-aflatoxin B1
    mixture was also tested using liver activation from arochlor-treated
    rats; the addition of propyl gallate substantially reduced the
    mutagenic activity of aflatoxin B1  (Rosin and Stich, 1980).

         Propyl gallate was not mutagenic to  Salmonella strains TA-98 or
    TA-100 with or without activation by liver extracts from arochlor-
    induced rats.  In contrast to the results reported above these workers
    observed a small increase in aflatoxin B1 mutagenesis in TA 100 in
    the presence of propyl gallate (Shelef and Chin, 1980).

    2.2.8  Special studies on the effects of propyl gallate on the
           carcinogenic activity of carcinogens

         Lung adenomas were induced in strain A mice by chronic treatment
    with nitrite in drinking water and morpholine in food.  Addition of
    gallic acid to the diet resulted in an 86% inhibition of adenoma
    induction.  Dietary gallic acid reduced or did not affect the
    induction of adenomas by mononitrosopiperazine or nitrosomorpholine
    given in drinking water, and failed to induce lung adenomas when given
    alone (Mirvish  et al., 1975).

         Weanling (21 day old) female Sprague-Dawley rats were fed with
    one of three basal diets: polyunsaturated fat (20% corn oil, HPF);
    saturated fat (18% coconut oil and 2% linoleic acid, HSF); or low fat
    (2% linoleic acid, LF), with and without 0.3% propyl gallate (PG).  At
    50 days of age , one half of each group (30 rats/group) received 10 mg
    of DMBA in 1 ml corn oil p.o. as a tumour inducer.  Both the amount of
    fat and the degree of unsaturation were found to affect the extent of
    protection against tumorigenesis afforded by PG, with some protection
    seen in all three dietary groups.  Tumour incidences at 32 weeks of
    age were: HPF, 100%; HSF, 63%; LF, 29%; HPF+PG, 77%; HSF+PG, 11%. 
    Tumours grew most rapidly in the HPF group.  Propyl gallate did not
    alter the function of the hepatic mixed oxidase system by induction or
    inhibition under the dietary conditions used (King and McCay, 1980).

         The induction of epidermal ornithine decarboxylase by 12-0-
    tetradecanoylphorbyl-13 acetate in mouse epidemis was inhibited by the
    topical application of propyl gallate.  Its potency was approximately
    10% of that of BHA (Kozumbo, Seed and Kensler, 1983).

         Propyl gallate did not modify tumour development of
    1,2-dimethylhydrazine-initiated colon carcinogenesis in F344 rats 
    (Shirai  et al., 1985).

         The promoting activity of 3 antioxidants, alpha-tocopherol,
    propyl gallate and tertiary butylhydroquinone (TBHQ) in urinary
    bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)
    nitrosamine (BBN) in male Fischer 344 rats was examined.  Rats, 6
    weeks old, were treated with 0.05% BBN in the drinking water for 4
    weeks and then administered 1.50, 0.75 or 0.38% alpha-tocopherol, 1.0%
    propyl gallate or 2.0% TBHQ in the diet for 32 weeks.  The urinary
    bladder of each animal was examined histologically after the 36-week
    experimental period.  The incidence of papillary or nodular
    hyperplasia (PN hyperplasia) of the urinary bladder was

    significantly higher in the rats treated with BBN followed by 2.0%
    TBHQ than in controls initiated with 0.05% BBN followed by control
    diet.  This result indicates that TBHQ has weak promoting activity in
    urinary bladder carcinogenesis.  alpha-tocopherol and propyl gallate
    did not demonstrate a promoting effect for urinary bladder lesions
    (Tamano  et al., 1987).

    2.2.9  Special studies on the interactions of propyl gallate

         Propyl gallate, gallic acid and nordihydroguaiaretic acid were
    all potent inhibitors of the  in vitro mouse spleen cell antibody
    response as assayed by anti-sheep erythrocyte plaque-forming cell
    response.  These compounds also suppressed clone formation  in vitro
    by human WISH or mouse L-cells (Blalock  et al., 1981).

         Gallic acid was reported to suppress the  in vitro thymus-
    dependent plaque forming response of mouse C57B1/6 spleen cells to
    sheep red blood cells.  The compound also suppressed mitogen-induced
    DNA synthesis of 6 lymphocytes but had no effect on ß-lymphocyte
    function (Archer  et al., 1977).

    2.2.10  Special studies on sensitization

         Gallates have been shown to cause contact dermatitis in bakers
    and other workers handling gallates.  Patch tests with lauryl gallate
    at 0.2% showed a weak positive response in one sensitized individual. 
    Other individuals have suffered recurring episods of dermatitis,
    presumably caused by gallates in food products (Brun, 1970).

    2.2.11  Other special studies

         Partial protection against liver damage by single oral doses of
    2.5 or 0.25 ml/kg of chloroform was provided by i.p. injection of 150
    mg/kg bw of propyl gallate (Torrielli and Ugazio, 1975).

    2.3  Observations in humans

         No information available.

    OCTYL GALLATE

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         See propyl gallate.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with octyl gallate are
    summarized in Table 4.

    Table 4.  Acute toxicity studies - octyl gallate

                                                                

    Animal    Sex     Route    LD50 mg/kg bw    Reference

                                                                

    Rat        ?      oral     4 700            van Sluis, 1951

    Rat        ?      i.p.     60-80            van Esch, 1955

    Rat        M      oral     2 710            Brun, 1970

    Rat        F      oral     1 960            Brun, 1970

    Rat        M      oral     2 710            Brun, 1970

    Rat        F      oral     2 330            Brun, 1970
                                                                

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

         Groups each of 20 rats (equally divided by sex) were maintained
    on diets containing 0, 1 000, 2 500 or 5 000 ppm (0%, 0.1%, 0.25% and
    0.5%) octyl gallate for 13 weeks.  All groups showed normal weight
    gains and food consumption.  Haematology and blood chemistry and
    urinalyses revealed normal values.  A complete gross and
    histopathologic examination showed no compound-related effects
    (Blackmore and Voelker, 1969a).

    2.2.2.2  Dogs

         Groups of eight dogs, each equally divided by sex, were fed diets
    containing 0, 0.1, or 0.3% n-octyl gallate for 90 days, or 1.0% n-
    octyl gallate for four weeks; the 1.0% level was then reduced to 0.65%
    for the rest of the study.  All groups showed normal weight gain and
    food consumption, except the 1.0% group.  Haematology and blood
    chemistry and urinalyses were normal.  A complete gross and
    histopathologic examination showed no compound-related effects
    (Lindberg  et al., 1970).

         In another study groups of eight dogs, each equally divided by
    sex, were maintained on diets containing 0, 0.1, 0.25 or 0.5% octyl
    gallate for 13 weeks.  All animals showed normal food consumption and
    weight gain.  Haematology and urinanalyses were similar for test and
    control animals.  ASAT activity was slightly elevated in the 0.5%
    group.  Gross and histopathologic examination of tissues and organs
    showed no compound-related effects (Blackmore and Voelker, 1969b).

    2.2.2.3  Pigs

         Diets containing 0.2% octyl gallate were fed to pigs without
    demonstrable ill effect; no anaemia was observed (van Esch and van
    Genderen, 1955).

    2.2.3  Long-term toxicity/carcinogenicity studies

         No information available.

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         Young rats in groups of 12 males and 12 females were fed diets
    containing 7% fat and 0.2% octyl gallate.  There was no significant
    difference between test and control animals over three generations
    (van Sluis, 1951).

         Octyl gallate was fed to rats at concentrations of 0.035, 0.2 and
    0.5% in the diet for two successive generations.  No effects on
    reproductive performance or other indices of reproduction were
    reported.  A slight hypochromic anaemia was noticed in the 0.2% group. 
    No abnormalities were observed in organs or tissues of the rats at
    autopsy (van Esch, 1955).

         Groups, each of 10 male and 20 female rats, were maintained on
    diets containing 0, 1 000 or 5 000 mg/kg feed octyl gallate.  The
    animals were bred twice for the first generation, and three times for
    the second generation.  At the time of weaning of the F1B

    litters in the 5 000 mg/kg feed group, the level was reduced to
    2 500 mg/kg feed for the second generation.  In the second generation,
    approximately 24 hours after birth, selected litters were
    redistributed to female parents so that control females nursed pups
    from test animals, and test animals nursed pups from control and other
    test groups.  Half of the P2 females bred for the third time (F2c)
    were examined by Caesarean section at time of delivery and the number
    of implantation sites, corpora lutea and fetuses determined.  One half
    of the pups from each litter were examined for skeletal abnormalities,
    and the other half for visceral abnormalities.  The other parameters
    measured in this study were appearance, behaviour, growth of pups
    during the nursing and weaning process, fertility index, gestation
    index, live birth index, and weaning survival index.  Autopsies were
    carried out on F2b weanling pups, (control pups suckled by control
    dams, experimental group pups suckled by respective group dams), as
    well as microscopic examination of pituitary, thyroid, liver, spleen,
    kidneys, adrenals, stomach, pancreas, small intestine, large intestine
    and any unusual lesions of five males and five females from high-dose
    and control group.  Weanling survival index and body weight at weaning
    were considerably reduced in the 5 000 mg/kg feed group of the F1a and
    F1b generation.  Reduction of these indices was still apparent in the
    F2a and F2b generations, when the dietary level was reduced to 2 500
    mg/kg feed.  At 1 000 mg/kg feed, the indices were similar to control.

         Redistribution of F2b pups to females of control groups resulted
    in similar growth of all pups to weaning.  Allowing pups from high
    level group to be nursed by control dams resulted in a marked increase
    in survival indices, whereas when control pups were nursed by high
    level dams, there was a marked decrease in survival indices. 
    Examination of P2 parents following the third breeding indicated a
    dose-dependent reduction in implantation sites, as well as a reduction
    in number of corpora lutea.  The fertility index of high level P2
    females was depressed at the F2c stage.  Skeletal evaluation of F2c
    litters showed incomplete skull ossification in some pups in the test
    groups, but this was not considered remarkable for the size of the
    fetuses.  Necropsy of the pups indicated a higher incidence of gross
    kidney alterations than that observed in controls.  No compound-
    related histopathological effects were reported.  The NOEL was 1 000
    mg/kg feed/day, equal to 17.5 mg/kg bw/day (Olson and Voelker, 1970).

         n-Octyl gallate was fed in the diet to groups of eight male and
    16 female rats for two successive generations at levels of 0, 0.1 or
    0.3% (and 0.6% for one generation).  Rats were mated to produce two
    litters per generation with the next generation selected from
    weanlings of the second litter.  A dietary level of 0.1% had no effect
    on reproduction performance or the offspring.  At 0.3 and 0.6% dietary
    octyl gallate, there was no significant effect on the rat fetuses
    during pregnancy, but a marked effect was observed on

    survival through weaning.  In the case of the 0.6% group, return to
    normal diet for six weeks, prior to a third breeding, did not result
    in increased survival of offspring through weaning (Plank  et al.,
    1971).

    2.2.5  Special studies on sensitization

         Repeated insult patch tests with 0.1% n-octyl gallate solution
    showed reaction in 13/445, or 2.9%, of tested individuals.  Oral
    mucosa irritation/ sensitization tests conducted with beer containing
    20 mg/kg n-octyl gallate showed that the incidence and severity of
    erythema were greater with beer containing n-octyl gallate than with
    untreated beer.  Oedema was also greater with treated beer (Palazzolo
    & Fancher, 1971a,b,c).

    DODECYL GALLATE

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         See propyl gallate

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with dodecyl gallate are
    summarized in Table 5.

    Table 5.  Acute toxicity studies - dodecyl gallate
                                                              
    Animal   Sex     Route    LD50 mg/kg bw    Reference
                                                              
    Rat       ?      oral     6 500            van Sluis, 1951

    Rat       ?      i.p.     100-120          van Esch, 1955
                                                              

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

         Weanling rats were given diets containing 2.5 or 5% dodecyl
    gallate.  All animals fed the smaller quantity were dead within 10
    days, and all animals fed the larger quantity died within seven days
    (Allen & De Eds, 1951).

         Male Wistar rats were administered dodecyl gallate daily by
    gavage, at doses equivalent to 10, 50 or 250 mg/kg bw/dy for 150 days. 
    In the 250 mg/kg bw/dy group, numerous deaths occurred.  Both the 250
    and 50 mg/kg bw/dy dose caused changes in serum lipids and enzymes,
    reduction in weight of the spleen and pathological changes in the
    liver, kidney, and spleen.  The 10 mg/kg bw/dy level was considered to
    be the NOEL (Mikhailova  et al., 1985).

         Rats fed for 70 days on a diet containing 7% fat and 0.2% dodecyl
    gallate showed no effect on body weight (Tollenaar, 1957).

    2.2.2.2  Pigs

         Diets containing 0.2% of dodecyl gallate were fed to pigs without
    demonstrable ill effect; no anaemia was observed (van Esch and van
    Genderen, 1954).

    2.2.3  Long-term toxicity/carcinogenicity studies

         No information available.

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         Young rats in groups of 12 males and 12 females were fed diets
    containing 7% fat and 0.2% dodecyl gallate.  There was no significant
    difference between test and control animals over three generations
    (van Sluis, 1951).

         Dodecyl gallate was fed to rats at concentrations of 0.035, 0.2
    or 0.5% in the diet, for two successive generations.  There was
    significant retardation of growth in the 0.5% group.  Some litters in
    this group were lost in the second generation because they were not
    fed sufficiently by the mothers.  A slight hypochromic anaemia was
    observed in the 0.2% group.  No abnormalities were observed in the
    organs or tissues at autopsy (van Esch, 1955).

    2.2.5  Special studies on sensitization

         See propyl gallate.

    3.  COMMENTS

         Although there are similarities in the metabolism of the
    different gallates as evidenced by earlier limited data and a newly
    available  in vitro metabolism study, the Committee concluded that
    there was not enough evidence to allocate a group ADI for the gallates
    when  in vivo pharmacokinetic and metabolic studies were not
    available.  In addition, a 150-day gavage study with dodecyl gallate
    revealed a NOEL that was 10-fold lower than the dietary NOEL for
    propyl gallate.

    4.  EVALUATION

         In the 90-day toxicity study in rats at a high-dose level
    (7 450 mg/kg feed), changes in haematological parameters (decreased
    haemoglobin, haematocrit and red blood cell count), morphological
    changes (increased extra medullary haematopoeisis) in the spleen, and
    increased activity of hepatic ethoxy-resorufin-O-deethylase were
    observed.  The Committee allocated an ADI of 0-1.4 mg propyl
    gallate/kg bw, which was based on the NOEL in this study of 1 910 mg
    propyl gallate/kg feed, equal to 135 mg propyl gallate/kg bw/day to
    which a 100-fold safety factor was applied.

         The Committee concluded that it was unlikely that either octyl or
    dodecyl gallate is carcinogenic or genotoxic.  Therefore, the
    Committee allocated temporary ADIs to both gallates based on the NOELs
    observed in limited toxicological studies.

         With octyl gallate, a slight hypochromic anaemia was observed at
    100 mg/kg bw/day in a study in rats in which the substance was
    administered for two generations.  A temporary ADI of 0-0.1 mg octyl
    gallate/kg bw was allocated based on a NOEL of 17.5 mg/kg bw/day in a
    reproduction study with rats, to which a safety factor of 200 was
    applied.

         With dodecyl gallate, a reduction of spleen weight and
    pathological changes in the liver, kidney and spleen were observed in
    a 150-day gavage study with rats, with a NOEL of 10 mg/kg bw/day.  A
    temporary ADI of 0-0.05 mg/kg bw was allocated based on this study,
    using a 200-fold safety factor.

         The Committee concluded that additional information on the
    pharmacokinetic and metabolic behaviour of the different gallates may
    help to explain the differences in toxicological potency of the
    different gallates and required data from such studies by 1996.  If
    these studies do not satisfactorily resolve the issue with respect to
    similarity of octyl and dodecyl gallate to propyl gallate, further
    toxicological studies (including long-term toxicity/carcinogenicity
    studies and genotoxicity studies) on octyl and dodecyl gallate might
    be required.

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    See Also:
       Toxicological Abbreviations