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. 5. REFERENCES ABDO, K.M., HUFF, J.E., HASEMAN, J.K., DIETER, M.P., BOORMAN, G.A., HILDEBRANDT, P., PREJEAN, J.D. & FARNELL, D.R. (1983), Carcinogenesis bioassay of propyl gallate in F-344 rats and B6C3F1 mice, J. Am. College Toxicol., 3(6), 425-33 ALLEN, C.S. & de EDS, F.D. (1951), The chronic toxicity of lauryl gallate, J. Amer. Oil Chem. Soc., 28, 304 ARCHER, D., BUKOVIC-WESS, J. & SMITH, B. (1977), Suppression of macrophage-dependent T-lymphocyte function by gallic acid, a food additive, Proc. Soc. Exp. Bio. Med., 156, 465-9 de BIE, A.Th.H.J, and van OMMEN, B., (1992) Study on the in vitro biotransformation of propyl, octyl, and dodecyl gallate. TNO-report V92.101, TNO Toxicology and Nutrition Institute, Zeist, The Netherlands BLACKMORE, R.H. & VOELKER, R.W. (1969a), 13-week dietary administration, Rats, Octyl gallate, Final report (Project No. 458-115), unpublished report from Hazleton Labs., Inc., Falls Church, VA, USA, F. & M. Schaefer Brewing Co., Brooklyn, N.Y., USA BLACKMORE, R.H. & VOELKER, R.W. (1969b), 13-week dietary feedings, Dogs, Octyl gallate, Final report (Project No. 458-117), Unpublished report from Hazleton Labs., Inc., Falls Church, Va., F. & M. Schaefer Brewing Co., Brooklyn, N.Y., USA BLALOCK, J.E., ARCHER, D.L., & JOHNSON, H.M (1981) Anticellular and immunosuppressive activities of foodborne phenolic compounds (41185), Proc. Soc. Exp. Bio. Med., 167, 391-3 BOOTH, A.N., MASRI, M.S., ROBBINS, D.J., EMERSON, O.H., JONES, F.T. & de EDS, F. (1959), The metabolic fate of gallic acid and related compounds, J. Biol. Chem., 234, 3014-6 BRUN, R. (1970), Contact eczema due to an antioxidant of margarine (gallate) and change of occupation, Dermatologica, 140, 390-394 BUKHAN, N.D. (1962), The effect of propyl gallate antioxidant on the nutritional value of fats (In Russian), Vopr. Pitan., 21, 68 CALLE, L.M. & SULLIVAN, P.D. (1982), Screening of antioxidants and other compounds for antimutagenic properties towards benzo[ a] pyrene-induced mutagenicity in strain TA98 of Salmonella typhimurium, Mutat. Res., 101, 99-114 CARPENTER, M. (1981), Antioxidant effects on the prostaglandin endoperoxide synthetase product profile, Fed. Proc., 40, 189-194 DACRE, J.C. (1960), Metabolic pathways of phenolic antioxidants, J. N.A. Inst. Chem., 24, 161-171 DACRE, J.C. (1974), Long-term toxicity study of n-propyl gallate in mice, Fd. Cosmet. Toxicol., 12, 125-9 van ESCH, G.J. (1955), The toxicity of the antioxidants propyl-, octyl- and dodecyl gallate, Voeding, 16, 683-686 van ESCH, G.J. & van GENDEREN, H. (1954), Netherlands Institute of Public Health, Report No. 481 van ESCH, G.J. & van GENDEREN, H. (1954), Netherlands Institute of Public Health, Report No. 2, 49-52 JOHNSON, A.R. & HEWGILL, F.R. (1961), The effect of the antioxidants, butylated hydroxy anisole, butylated hydroxytoluene and propyl gallate on growth, liver and serum lipids and serum sodium levels of the rat, Aust. J. Exp. Biol. Med. Sci., 39, 353-360 KAMRA, O. & BHASKAR, G. (1978), Radiosensitization of mouse bone-marrow cells by a commonly used food additive, propyl gallate, Mutation Res., 53, 207 KING, M., MCCAY, P. (1980), DMBA-induced mammary tumour incidence: Effect of propyl gallate supplementation in purified diets containing different types and amounts of fat, Proc. Amer. Soc. Cancer Res., 21, 113 KING, M., MCCAY, P. (1981), Studies on liver microsomes of female rats fed purified diets varying in fat content with and without propyl gallate, Fd. Cosmet. Toxicol., 19, 13-17 KOZUMBO, W.J., SEED, J.L. & KENSLER, T.W. (1983), Inhibition by 2(3)- tert-butyl-4-hydroxyanisole and other antioxidants of epidermal ornithine decarboxylase activity induced by 12-0- tetradecanoylphorbolacetate, Cancer Res., 43, 2555-2559 LEHMAN, A.J., FITZHUGH, O.G., NELSON, A.A. & WOODARD, G. (1951), The pharmacological evaluation of antioxidants, Advanc. Food Res., 3, 197-208 LINDBERG, D.C., KEPLINGER, M.L. & FANCHER, O.E. (1970) Ninety-day subacute oral toxicity study with Cold-Pro, GA-8 in Beagle dogs (IBT No. C8472), Unpublished report from Industrial Bio-Test Labs., Inc., Northbrook, Ill., USA, submitted to the World Health Organization by Nutrico, Inc., Milwaukee, Wisconsin, USA MIKHAILOVA, Z. et al. (1985) Toxicological studies of the long term effects of the antioxidant dodecyl gallate on albino rats, Vopr. Pitan., No. 2, 49-52 MIRVISH, S., CARDESS, A., WALLCAVE, L. & SHUBIK, P. (1975), Induction of lung adenomas by amines or ureas plus nitrite and by n-nitroso compounds: Effect of ascorbate, gallic acid, thiocyanate and caffeine, J. Nat. Cancer Inst., 55(3), 633-636 NERA, E.A., LOK, E., IVERSON, F., ORMSBY, E., KARPINSKY, K.F. & CLAYSON, D.B. (1984), Short-term pathological and proliferative effects of BHA and other phenolic antioxidants in the forestomach of Fischer 344 rats, Toxicol., 32, 197-205 OLSON, W.A. & VOELKER, R.W. (1970) Modified two-generation reproduction study - Rats, Octyl Gallate, Final report (Project 458-116), Unpublished report from Hazleton Labs., Inc., Falls Church, Va., USA, submitted to the World Health Organization by F. & M. Schaefer Brewing Co., Brooklyn, N.Y., USA ORTEN, J.M., KUYPER, A.C. & SMITH, A.H. (1948), Studies on the toxicity of propyl gallate, Food Techn., 2, 308-316 PALAZZOLO, R.J. & FANCHER, O.E. (1971a) Human repeated insult patch test with n-octyl gallate (IBT No. F9309), Unpublished report from Industrial Bio-Test Labs., Inc., Northbrook, Ill., USA, submitted to the World Health Organization by Nutrico, Inc., Milwaukee, Wisconsin, USA PALAZZOLO, R.J. & FANCHER, O.E. (1971c) Oral mucosa irritation/sensitization test with treated beer (IBT No. F9655), Unpublished report from Industrial Bio-Test Labs., Inc., Northbrook, Ill., USA, submitted to the World Health Organization by Nutrico, Inc., Milwaukee, Wisconsin, USA PALAZZOLO, R.J. & FANCHER, O.E. (1971b) Oral mucosa irritation/sensitization test with treated beer (IBT No. F9310), Unpublished report from Industrial Bio-Test Labs., Inc., Northbrook, Ill., USA, submitted to the World Health Organization by Nutrico, Inc., Milwaukee, Wisconsin, USA PLANK, J.B., WRIGHT, P.L., KEPLINGER, M.L. & FANCHER, O.E. (1971) Three generation reproduction study with Cold-Pro, GA-8 in albino rats (IBT No. P84), Unpublished report from Industrial Bio-Test Labs., Inc., Northbrook, Ill., USA, submitted to the World Health Organization by Nutrico, Inc., Milwaukee, Wisconsin, USA RAHIMTULA, A., ZACHARIAH, P. & O'BRIEN, P. (1979), Differential effects of antioxidants, steroids and other compounds on Benzo(A)Pyrene 3-hydroxylase activity in various tissues of rats, Br. J. Cancer, 40, 105-112 ROSIN, M. & STICH, H. (1980), Enhancing and inhibiting effects of propyl gallate on carcinogen-induced mutagenesis, J. Environmental Path. Toxicol., 4, 159-167 de SESSO, J.M. (1981), Amelioration of teratogenesis, I. Modification of hydroxyurea-induced teratogenesis by the antioxidant propyl gallate, Teratol., 24, 19-35 SHELEF, L. & CHIN, B. (1980), Effect of phenolic anti-oxidants on the mutagenicity of aflatoxin B1, Applied Environ. Microbiol., 40, 1039-1043 SHIRAI, T., IKAWA, E., HIROSE, M., et al (1985). Modification by five antioxidants of 1,2-dimethylhydrazine-initiated colon carcinogenesis in F344 rats, Carcinogenesis 6:4, 637-9. van SLUIS, K.J.H (1951), The higher alkyl gallates as antioxidants, Food Manuf., 26, 99-101 SPEIJERS, G.J.A, JANSSEN, G.B., WALLBRINK-de DRIEU, Y., van LEEUWEN, F.X.R, van LOENEN, H.A., KRAJNC-FRANKEN, M.A.M., VAESSEN, H.A.M.G & WESTER, P.W (1993) Subchronic toxicity of propyl-gallate, RIVM Report No. 618311002 (Draft) Rijksinstituut voor Volksgezondheid en Milieuhygiëne, Bilthoven, The Netherlands STRIK, J.J.T.W.A. DANSE, L.H.J.C, HELLEMAN, P.W., van LEEUWEN, F.X.R, SPEIJERS, G.J.A, & VAESSEN, H.A.M.G (1986). Subacute toxicity of propyl gallate Report No. 618311 001 Rijksinstituut voor Volksgezondheid en Milieuhygiëne, Bilthoven, The Netherlands TAMANO, S., FUKUSHIMA, S., SHIRAI, T., HIROSE, M & ITO, N. (1987). Modification by O-tocopherol, propyl gallate and tertiary butylhydroquinone of urinary bladder carcinogenesis in Fischer 344 rats pretreated with N-butyl-N-(4-hydroxybutyl)nitrosamine. Cancer Letters, 35, 39 - 46. TOLLENAAR, F.D. (1957) (Pub. 1963) Prevention of rancidity in edible oils and fats with special reference to the use of antioxidants, 9th Proc. Pacific Sci. Congr., Bangkok, Thailand, 5, 92-103 TORRIELLI, M. & UGAZIO, G. (1975), Biochemical aspects of the protective action of propyl gallate on liver injury in rats poisoned with carbon tetrachloride, Toxicol. Appl. Pharmacol., 34, 151-169 WEIR, R.J. & BRUSICK, D. (1974), Mutagenic evaluation of Compound FDA 71-39, Propyl Gallate, Unpublished report from Litton Bionetics, Inc., Kensington, MD, USA, submitted to the World Health Organization by the US Food and Drug Administration YANG, C. & STRICKHART, F. (1974), Inhibition of hepatic mixed function oxidase activity by propyl gallate, Biochem. Pharmacol., 23, 3129-35.
See Also: Toxicological Abbreviations