WHO Food Additives Series, 1972, No. 4 EVALUATION OF MERCURY, LEAD, CADMIUM AND THE FOOD ADDITIVES AMARANTH, DIETHYLPYROCARBONATE, AND OCTYL GALLATE The evaluations contained in this publication were prepared by the Joint FAO/WHO Expert Committee on Food Additives which met in Geneva, 4-12 April 19721 World Health Organization Geneva 1972 1 Sixteenth Report of the Joint FAO/WHO Expert Committee on Food Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 505; FAO Nutrition Meetings Report Series, 1972, No. 51. AMARANTH Biological data Biochemical aspects Adult rats were given intravenous injections of the colour. The bile was collected for six hours and an average of 53% (43-79%) of the quantity of the colour administered was found (Ryan & Wright, 1961). Six adult rats were given a single oral dose of 100 mg as aqueous solution per animal. Only 0.45% of the dose administered was found in the faeces collected over a period of 48 hours. A single oral dose of 50 mg per animal was administered to four rats. Only 2.8% was absorbed from the gastro-intestinal tract; the metabolites in the urine and bile were predominantly products resulting from the reductive fission of the azo-linkage, such as 1-amino4-naphthalene sulfonic acid and 1-amino-2-hydroxy-3, 6-naphthalene disulfonic acid. The former compound was found also in the faeces. The liver enzyme that reduces azo-linkages plays little part in the metabolism, as was shown in experiments in which the colour was given by intrasplenic infusion. Reduction of the compound is therefore most probably effected by the intestinal bacteria (Radomski & Mellinger, 1962). After a prolonged administration of a daily dose of 50 mg/rat the vitamin A content of the liver showed a threefold to fourfold decrease. The glutathione content of the liver and spleen was increased (Galea et al., 1962). Acute toxicity Animal Route LD50 References per kg body-weight Rat I.p. 1 g Deutsche Forsch., 1957 Rat I.v. 1 g Deutsche Forsch., 1957 Special studies The colour was tested for mutagenic action in a concentration of 0.5 g/100 mi in cultures of Escherichia coli. No mutagenic effect was found (Rück & Rickerl, 1960). Short-term studies Rat A group of five young rats were given the colour subcutaneously twice daily for three days. The rats were killed on the fourth day. The colour was administered in aqueous solution at a level of 250 mg per kg body-weight each injection. No estrogenic activity (normal uterine weight) was detected in comparison with a control group. No other abnormalities were found (Graham & Allmark, 1959). Guinea-pig In experiments with guinea-pigs, it was found that this colour had no sensitization activity (Bär & Griepentrog, 1960). Cat The test for Heinz bodies was negative after injecting four cats with a 5% aqueous solution of this colour at a level of 1 g on the first day and 0.1 g on the ninth and on the eighteenth day. (Deutsche Forsch., 1957). Long-term studies Mouse Twenty mice were fed 15-20 mg of this colour five days a week for periods up to 477 days. Autopsies were conducted on 18 of the mice. No lesions were observed in the one liver examined (Cook et al., 1940). Feeding studies were-conducted with C3Hf and C57Bl mice; 100 of each strain were fed at 1.0 and 2.0% and 200 of each strain served as controls. No tumours were observed in the mice of either strain (U.S., FDA, 1964). Two groups of 100 mice received either 0 or 0.01 g amaranth paste (= 0.004 g amaranth) by gavage daily. One drop of either 9,10-dimethyl-2-benzanthracene or 3,4 benzypyrene was applied once per week to interscapular skin. Papillomata appeared 3.5 weeks earlier in test animals and in a greater number of animals. A larger percentage became malignant in test animals (Baigusheva, 1968). Three groups of 15 male and 15 female rats were given diets containing 0.03%, 0.3% and 1.5% of the colour for 64 weeks. The mortality of the rats was the same as that in a similar control group. At a level of 1.5% a significant decrease in growth rate was found in female rats but not in male rats. This was attributed to an effect on food utilization rather than on food consumption. Female rats fed the colour at 0.3% and 1.5% showed an increase in liver weight. At the higher concentration there was also an increase in kidney weight. No influence on food intake, histo pathology and blood-picture and no significant difference in tumour incidence was found (Mannel et al., 1958). Ten rats were fed the colour at a level of 0.2% in the diet. Each animal received an average of 0.1 g/kg body-weight per day for 417 days. The total intake of the colour was 11 g/animal. The observation period was 830 days. One intestinal carcinoma was observed (Deutsche Forsch., 1957). No tumours were noted in 11 rats subjected to the subcutaneous injection of 0.5 ml of 1% solution twice weekly for 365 days. The observation period was 879 days. The total dose administered was 0.5 g/animal (Deutsche Forsch., 1957). At a level of 11% in the diet the colour was fed to five male and five female rats for periods up to 18 months. Gross staining of the glandular stomach and small intestine was observed. Granular deposits were noted in the stomach, small intestine and in some cases in the colon. A lymphosarcoma was observed. No tumours occurred in 50 control animals surviving 20 months or more (Willheim & Ivy, 1953). This colour was injected subcutaneously into 18 rats of both sexes for 94 to 99 weeks. In general 1 ml of a 2-3% solution was injected weekly for 693 days. No tumours were observed (Nelson & Hagan, 1953). Groups of 24 weanling rats were fed the colour of 0.5%, 1.0%, 2.0% and 5.0% in the diet. A similar group served as controls. The animals on 5.0% showed slight growth inhibition. Gross and microscopic examinations revealed a questionable increase in the number of mammary tumours; two tumours were observed in the control group and 3, 3, 6 and 4 tumours respectively in the groups receiving the colour. Additional feeding studies for two years were carried out to repeat this result with Osborne-Mendel and Spragul-Dawley rats at 0.0, 1.0, and 2.0% with 100 animals of each strain. There was no statistically significant influence on the formation of tumours (U.S., FDA, 1964). One group of 50 outbred rats was fed a diet containing amaranth paste (40% amaranth) for 25 months. A control group of 35 rats was used. Dietary levels ranged from 0.8% to 1.6% amaranth. Tumours of the peritoneum and intestine began to appear in 18 survivors at 19 months. By 25 months a total of 11 tumours was found. No tumours were seen in controls. All tumours were histologically malignant (Baigusheva, 1968). Two groups of 50 outbred male rats were fed on a diet containing either 0% or 2% amaranth for 33 months, that is until all animals died. A small depression of body-weight, compared with controls was observed but this was not statistically significant. The 15 tumours found (in 13 rats) included 3 lymphosarcomas, 4 sarcomas, 1 adenofibroma, 3 intestinal carcinomas, 1 hepatoma and 3 skin carcinomas. The first tumour appeared after six months, most of the others at 21-23 months. The author reported that not one of the 50 control rats developed any kind of tumour during the whole course of the experiment (Andrianova, 1970). Dog A seven year toxicity study was carried out on female beagles. Five dogs were fed 2% of the colour in the diet, three dogs were used as controls. No histopathological or other abnormalities were found (U.S., FDA, 1964). Reproduction and teratogenicity studies Rat Groups of one male and four female rats were fed diets supplying 1.5 and 15 mg/kg body-weight/day of amaranth. There were two control groups of a similar size. Each group was mated three times, at 4-5 months, 7-8 months and 10-12 months after the start of the study. The following were studied: female fertility, gestation period, numbers of live and dead pups born and numbers surviving for four days and one month. Abnormalities in the pups were noted. Similar observations were carried out on first (F1) and second (F2) generations of rats fed on the same diets as their parents. The authors considered that their results showed amaranth decreased fertility, increased the number of stillbirths, produced deformities in the young and reduced survival of the young (Shtenberg & Gavrilenko, 1971). Groups of 13-15 pregnant rats were given 0, 7.5, 15, 30, 100 or 200 mg/kg/day amaranth by stomach tube from day 0-19 and sacrificed on day 20. No adverse effects were noted on implantation. The percentage of dead foetuses increased in a dose-related manner. At 200 mg/kg/day there was a foetotoxic effect. Resorptions increased from 15 mg/kg upwards with total resorption of occasional litters at 100 and 200 mg/kg/day. No teratogenic abnormality related to amaranth was seen. No obvious effect was notod on foetal sex (Collins & McLaughlin, 1972). Eight groups of 16-22 pregnant rats were given 0, 15, 50 or 150 mg/kg/day of amaranth by stomach tube on days 6-15 of pregnancy and Caesarean section was performed on day 20. No compound-related adverse effects were noted in terms of implantation, foetal mortality, foetal weight or reproductive performance in any of the experimental groups as compared to the control animals. No evidence of compound-induced teratogenic effects was noted (Industrial BioTest Laboratories, 1972a). In another study five groups of four pregnant Charles River rats received from gestation day 6-15 inclusive, 0, 15, 150, 450 and 1500 mg/kg body-weight amaranth per day by gavage. Females were killed on day 20. No abnormal effects were seen as regards maternal weight gain, litter size, average foetal weight and number of resorptions. No gross abnormalities related to the compound were seen (Burnett, 1972). Rabbit Eight groups of 10-14 pregnant rabbits were given 0, 1.5, 5.0 and 15.0 mg/kg/day of amaranth by capsule on days 6-16 of pregnancy and sacrificed on day 29 by Caesarean section. No evidence of compound-induced teratogenic effects were noted, nor were significant effects seen in terms of implantation, pup weight, young born alive and total numbers of resorptions. Average numbers of early resorptions per litter revealed an increase which approaches statistical significance (P =.05) for the 1.5 and 15.0 mg/kg groups while for the 5.0 mg/kg group the increase is significant (Industrial Bio-Test Laboratories, 1972b). Comments on experimental studies reported Several long-term studies have been carried out in rats but only two studies extended over their lifespan. Long-term studies have also been done in mice and dogs. Many aspects of the newer studies were difficult to interpret particuarly with regard to the carcinogenic potential of amaranth and its adverse effects on the rat foetus and reproduction. However, the most recent studies gave somewhat conflicting results with regard to feototoxicity although none of them produced any evidence of teratogenic effects related to amaranth administration. It was not possible to establish the reason for this difference as there were divergencies in the conduct of these tests in relation to time and route of administration. Considerable controversy still exists over the correct route of administration in teratogenicity tests and there is evidence that variations in this parameter could lead to observable effects which are not compound-related. Such factors made the interpretation of the experimental data very difficult. The teratogenic study in rabbits was similarly not clear-cut in its results. EVALUATION The new information from reproduction and other studies was considered in conjunction with the fact that further relevant studies are in progress at present. The Committee was unable to make a final re-evaluation of the colour at this stage. However, the unconditional ADI previously established has been changed to a temporary ADI, with appropriate adjustment. mg/kg body-weight Temporary acceptable daily intake for man 0-0.75 Further work required Results of various studies now in progress to be submitted within three years. REFERENCES Andrianova, M. M. (1970) Vop. Pitan., 29 (5), 61 Baigusheva, M. M. (1968) Vop. Pitan., 27 (4), 46 Bär, F. & Griepentrog, F. (1960) Med. und Ernähr., 1, 99 Burnett, C. (1972) Unpublished report submitted to WHO Collies, T. F. X. & McLaughlin, J. R. (1972) Unpublished report to FDA submitted to WHO Cook, J. W. et al. (1940) Amer. J. Cancer, 40, 62 Deutsche Forschungsgemeinschaft, Bad-Godesberg, Federal Republic of Germany, Farbstoff Kommission (1957) Mitteilung 6 Galea, V., Ariesan, M. & Luputiu, G. (1962) Parmacia (Buc), 10, 531 Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., 1, 144 Industrial Biotest Labs. Inc. (1972a) Report No. B700 submitted to WHO Industrial Biotest Labs. Inc. (1972b) Report No. J701 submitted to WHO Mannel, W. A. et al. (1958) J. Pharm. Pharmacol., 10, 625 Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397 Radomski, J. L. & Mellinger, T. J. (1962) J. Pharmacol. exp. Ther., 136, 259 Rück, H. & Rickerl, E. (1960) Z. Lebensm.-Untersuch., 112, 157 Ryan, A. J. & Wright, S. E. (1961) J. Pharm. Pharmacol., 13, 492 Shtenberg, A. I. & Gavrilenko, E. V. (1970) Vop. Pitan., 29 (2), 66 U.S. Food and Drug Administration, Unpublished report submitted to WHO, April 1964 Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1
See Also: Toxicological Abbreviations Amaranth (WHO Food Additives Series 8) Amaranth (WHO Food Additives Series 13) Amaranth (WHO Food Additives Series 19) AMARANTH (JECFA Evaluation) Amaranth (IARC Summary & Evaluation, Volume 8, 1975)