Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants, emulsifiers and thickening agents WHO FOOD ADDITIVES SERIES NO. 5 The evaluations contained in this publication were prepared by the Joint FAO/WHO Expert Committee on Food Additives which met in Geneva, 25 June - 4 July 19731 World Health Organization Geneva 1974 1 Seventeenth Report of the Joint FAO/WHO Expert Committee on Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539; FAO Nutrition Meetings Report Series, 1974, No. 53. SUCROSE ESTERS OF FATTY ACIDS, AND SUCROGLYCERIDES Explanation These substances have been evaluated for acceptable daily intake by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1, Ref. No. 20) in 1969. Since the previous evaluation, additional data have become available and are summarized and discussed in the following monograph. The previously published monograph has been expanded and is reproduced in its entirety below. BIOLOGICAL DATA SUCROSE MONOPALMITATE BIOCHEMICAL ASPECTS The ester linkage is only hydrolyzed with great difficulty by pancreatic or intestinal enzymes in vitro (Berry & Turner, 1960). The presence of sucrose monopalmitate had no effect on the absorption of Ca45 from aqueous CaCl2 solution when given intragastrically to six dogs. Estimation of blood activity up to six hours later showed only a delayed peak in the activity/time curve compared with plain aqueous CaCl2. Repetition in five dogs with induced impaired fat absorption secondary to ligation of the pancreatic duct showed higher Ca45 absorption postoperatively from the control solution and test solution containing sucrose monopalmitate. Giving intramuscular 300 000 USP units of vitamin D decreased the blood levels in both cases but less so for the solution containing sucrose monopalmitate (Tudisco, 1961a, b). TOXICOLOGICAL STUDIES Acute toxicity Intragastric administration of 0.1 g to rats had no effect on the osmotic fragility of erythrocytes but intravenous administration to mice of 0.5 g/kg bw produced considerable haemolysis (Tudisco, 1965a,b). An oral dose totalling 20 000 mg/kg, administered in 10 equally divided doses at 30 to 60 minute intervals produced no deaths in five rats (Tokita, 1958). An oral dose totalling 20 000 mg/kg, in 10 equally divided doses at 30 to 60 minute intervals, produced no deaths in 10 mice (Tokita, 1958). Short-term studies Rat In another experiment groups of 10 male or female rats were given 0, 1, 2, 3, 5, 10, and 25% sucrose monopalmitate for 100 days. At the 2 and 3% levels (female rats used only) there was marginal reduction in body weight gain of the test groups. At the 5% level there were several deaths preceded by diarrhoea, weight gain was reduced but food efficiency high compared with controls. The 10% and 25% groups did not survive the first week and had white semi-soft faeces. Histopathology showed nothing of note (Tudisco; 1961a,b). Rats given daily oral doses of 100, 200, 1000, 2000 mg/kg for 60 days grew normally and had normal organ weights (brain, heart, stomach, liver, spleen and kidney) at necroscopy (Hara, 1959). Dog Twelve male and 12 female young beagles were divided in four groups of three male and three female animals and fed a diet containing 0, 0.3, 1.0 and 3.0% sucrose monopalmitate for two years. Appearance, behaviour, appetite, elimination, body weight gains, urinalysis, organ weights, histopathology and organ function studies revealed no gross abnormalities or differences between test and control groups related to the compound (Hazelton Lab., 1966a). Long-term studies Rat Groups of 25 male and 25 female albino rats were fed dietary levels of 0.3, 1 and 3% sucrose monopalmitate for two years. The control groups consisted of 50 males and 50 females. There was one death in the first six months, a male in the 3% test group, apparently caused by bilateral pyelonephritis. Periodic records of body weight and food consumption disclosed a suggestion of lowered body weight gains in the 3% test groups. The first half-year growth rate and the 78-week mean body weights of the 3% group females were significantly lowered; food consumption in the 3% group males was significantly lower at 26 weeks and slightly below that of controls in the first year; and the food efficiency of the 3% group females was slightly lowered in the first quarter year. No other differences were noted between the groups on the basis of observation of general appearance and behaviour, mortality and tumour incidence, haematology and urinalysis of pooled samples at 1, 3, 6, 12, 18 and 24 months, and sacrifice with necropsy, organ weight measurement and histological examination of tissues on five males and five females from each group at three months and of all survivors at 24 months (Hazelton Lab., 1966b). A concurrent reproduction study over three generations was carried out on groups of eight male and 16 female rats over 22 months using sucrose monopalmitate at 0 or 1% of the diet. The parent generation (P) was kept for the whole study. Mean body weight gain, survival and food consumption of the P generation showed no significant differences between controls and test group. The P generation was mated twice to give F1A and F1B filial generations. Of the F1A generation 10 male and 10 female pups were sacrificed for autopsy and the rest discarded. No significant findings were detected. The fertility index of the F1B test groups was significantly lower than that for the control groups due to adverse environment and unrelated to the test substance. Gestation, live birth and lactation indices were comparable between test and control groups. The F1B litter had slightly more frequent small weak pups with blue marks on the body but all survivors later developed normally. Sixteen F1B females and eight F1B males were mated twice to produce F2A and F2B filial generations, all other F1B animals were discarded including the 24 F1B animals used for mating. The F2A and F2B generation developed normally both in control and the test group. Growth of the males in the test group was slightly higher and for the females slightly lower otherwise control and test group showed no significant abnormalities. The F2A litter and all animals of the F2B litter except 16 females and eight males were discarded, the latter being mated twice to produce the F3A and F3B filial generations. Five males and five females of these 24 F2B animals were sacrificed. Gross and histopathology of all major organs revealed no significant abnormalities. The F3A and F3B litters showed no difference from previous generations or between the test group and controls. Mean litter size, physical appearance and growth of litter were comparable among test and control groups for each generation and among the three filial generations. The autopsies and histological examinations were normal (Hazelton Lab., 1965). SUCROSE MONOSTEARATE BIOCHEMICAL ASPECTS Surviving intestine has been shown capable of hydrolyzing the glucosidic linkage of sucrose monostearate to glucose and fructose at one-quarter of the rate of sucrose hydrolysis. alpha-amylase and gluco- or fructo-invertase were unable To hydrolyze sucrose mono-stearate. Liver homogenate, but not intestinal mucosa homogenates, could oxidize sucrose monostearate. Only pancreatic juice was able to hydrolyze the ester linkage to a moderate degree (Berry & Turner, 1960). TOXICOLOGICAL STUDIES Acute toxicity Mouse An oral dose of 20 000 mg/kg, administered in 10 equally divided doses at intervals of 30 to 60 minutes, produced no deaths in 10 mice (Tokita, 1958). Rat An oral dose totalling 20 000 mg/kg administered in 10 equally divided doses at intervals of 30 to 60 minutes produced no deaths in 10 rats (Tokita, 1958). Short-term studies Rat Rats were given daily oral doses of 100, 200, 1000 and 2000 mg/kg for 60 days without deleterious effect on weight gain or relative organ weights (brain, heart, stomach, liver, spleen, kidney) (Hara, 1959). Long-term studies None available. MIXED PALMITIC AND STEARIC ACID ESTERS OF SUCROSE TOXICOLOGICAL STUDIES Short-term studies Rat Daily doses of 3000, 4000 and 6000 mg/kg were administered orally to rats in groups of six for 33 days. There were no effects on weight gain and no histopathology (Tokita, 1959). SUCROSE MONOOLEATE BIOCHEMICAL ASPECTS Rat liver homogenates hydrolyzed the ester bond almost completely in 60 minutes without attacking the glucosidic linkage. alpha-amylase, lipase and dog pancreatin are similarly effective. Invertases only released 3 to 6% of the existing glucosidic links. Intestinal mucosa and liver homogenates were able to oxidize 0.1% of the substrate (Berry & Turner, 1960). TOXICOLOGICAL STUDIES Acute toxicity No data available. Short-term studies Rat Groups (unstated number) of rats received 0, 2, 5, 10 and 20% of ester in their diet for six months. On]y at the 20% level (0.09% dimethylformamide) was there retardation of growth compared with controls. Soft faeces or mild diarrhoea occurred initially with 10% and 20% test diets but animals later become tolerant. On return to normal diet there was rapid regaining of full body weight (Oshima & Kajiwara, 1960). Long-term studies None available. SUCROSE MONOLINOLENATE BIOCHEMICAL ASPECTS Five rats with either their thoracic duct cannulated or having a bile and lymph fistula were given intragastrically an aqueous suspension of sucrose ester or linolenic acid. Linolenic acid increased in the lymph within 24 hours. No linolenic acid was found in the bile of animals given the ester during 48 hours and it appeared only slowly after 72 hours in controls given linolenic acid. Rats with both bile and thoracic duct fistula showed a rise in the lymph content of linolenic acid within 24 hours and none in the bile even after 72 hours. Thus lymphatic absorption of the linolenic acid moiety was demonstrated. The percentage linolenic acid absorbed was the same whether the sucroester or the free acid was administered (Tudisco & Turner, 1963). TOXICOLOGICAL STUDIES Acute toxicity None available. Short-term studies None available. Long-term studies None available. PALM OIL SUCROSE ESTERS BIOCHEMICAL ASPECTS When aqueous suspensions of palm oil sucrose esters were given intragastrically to rats after pyloric ligation there was no evidence of digestive hydrolysis up to eight hours. In vitro use of intestinal juice on a substrate containing 1% palm oil sucrose esters for six hours produced only negligible hydrolysis of the ester and glucosidic linkage. A metabolic balance study on 35 male and female rats using food containing 5% palm oil sucrose esters and determining the amounts of ester given, the residue in stomach, gut, excreta and left over in food showed some 75% as unaccounted and, therefore, metabolized. Skeletal development as a measure of calcium absorption was determined by measuring growth of rat tails in two groups of 10 rats receiving daily 1 ml of 50% palm oil sucrose esters suspension for 10 weeks. Tail growth was more rapid in the test group during the first four weeks but later was not significantly different from controls (Balea et al., 1966). TOXICOLOGICAL STUDIES Acute toxicity Animal Route LD50 mg/kg bw Reference Rat Oral > 30 000 Balea, 1963 Mice receiving intravenous doses of 1-2 g/kg bw showed no haemolysis (Tudisco, 1965a, b). Short-term studies Rat Groups of eight rats received 5% or 10% palm oil sucrose esters in their diet for 150 days without showing any deleterious effect on body weight gain (Tudisco & Chiancone, 1965). Three groups of 10 rats were fed diets containing 10 g/kg bw lard with either 0, 50 ml/kg bw 10% palm oil sucrose esters and 10 ml/kg bw 50% palm oil sucrose esters for six weeks. No gastrointestinal disturbances were seen. Test groups showed a slightly greater body weight gain and slightly larger fat deposits in their carcass compared with controls (Balea et al., 1966). When the compound was fed to groups of eight males and eight female rats at 0, 3, 5, and 10% levels of their diet for 100 days no difference was noted between tests and controls regarding general condition. Test groups showed a slightly greater weight increase. Haematology, organ weights and gross autopsy were comparable in tests and controls, while histopathology revealed no abnormalities in the test group (Balea, 1963). In a similar experiment with 8-10 female rats per group, these received 0, 5 and 10% of the compound in their diet for 100 days. No abnormalities related to the test substance were detected in general appearance, food consumption, body weight gain, food efficiency, haematology, gross autopsy findings, histology (thyroid, liver, adrenal) and biochemistry (plasma and liver cholesterol, phospholipids, total lipids) only the liver lipids of test animals were significantly raised (Tudisco, 1963a). Two groups of 10 rats received 0 or 2% palm oil sucrose esters in their diet for three-and-one-half months. No deleterious effect was noted on mating, reproduction, litter number and litter size. Pups gained weight satisfactorily during weaning with better performance in the test group. Lactation was unaffected and no external fetal abnormalities were seen (Fernando, 1964). Long-term studies Rat Groups of 15 male or female rats were fed diets containing 0 or 0.5% of the compound for 14 months. No tumours were seen at the end of 13 months nor any growth abnormalities in male rats. Female rats showed a temporary lowering of growth rate during the eighth to tenth week. Three animals were examined histologically and no evidence of abnormality attributable to palm oil sucrose esters was found. Haematologic findings and blood proteins were comparable in test and control groups (Chiancone et al., 1963). Groups of 12 male rats were fed 0 or 10% of the ester as only source of lipids in their diet for 25 to 28 months. No abnormalities were found with respect to body weight, general health, mortality, haematology, plasma proteins and food efficiency. Tumour incidence was similar in test groups and controls. Visceral abnormalities were equally distributed (Tudisco & Chiancone, 1965). A two-generation study was carried out on 15 male and 15 female rats given 0 and 5 g/kg bw palm oil sucrose esters containing 68 ppm (0.0068%) of dimethyl formamide daily, The parent generation (P) was observed for two years. Body weight gain, external appearance and incidence of tumours were identical to controls. Three male animals died from intercurrent respiratory disease, three female animals either died or were sacrificed, the remaining 12 animals survived 24 months. No haematological abnormalities were detected and blood proteins had a normal electrophoretic pattern. At autopsy no significant lesions were found in the test group which were not also found in the controls. Histology was similarly comparable for tests and controls. Five male and five female rats (P generation) were mated after nine months' treatment to produce 23 F1 generation animals. The F1 generation was observed for 21 months. Body weight gain was similar to controls and no spontaneous deaths or illnesses occurred. Five male and five female survivors were autopsied after 21 months. No evidence of malignant tumours or other significant abnormalities was found which differed from the controls. Five male and five female F1 animals were mated after nine months to produce 30 F2 generation animals. The F2 generation was observed for 14 months. All litter mates gained weight identical to controls. Five male and five female F2 animals were autopsied after 14 months. No malignant tumours or other pathological conditions were found which did not also occur in the controls. No other significant abnormalities were seen. Seven from 50 animals examined showed hepatic hyperfunction. No evidence of any adverse effects on fertility and fetal development was seen (Mosinger, 1964b). LARD AND TALLOW SUCROSE ESTERS BIOCHEMICAL ASPECTS Each of four dogs aged five to six years were given intragastrically l131-labelled triolein in olive oil, oil/water emulsion and oil/water emulsion containing lard sucrose esters and the plasma activity determined as a measure of absorption. Each animal served as its own control, the administrations being separated by seven-day intervals. With olive oil peak absorption occurred after six hours with a fall between six and eight hours. The oil/water emulsion gave inconsistent patterns while oil/water emulsion with lard sucrose esters had a peak absorption of four hours and less abrupt fall during four to eight hours. Lard sucrose esters did not appear to interfere with intestinal fat absorption (Tudisco, 1961a, b). Calcium absorption using Ca45Cl2 was tested in eight dogs using aqueous CaCl2 solution. CaCl2 in oil/water emulsion and CaCl2 in oil/water emulsion with lard sucrose ester. All doses were administered intragastrically and blood activity determined subsequently. Each dog acted as its own control. The presence of lard sucrose ester had no consistent effect on Ca45 absorption (Tudisco, 1961a. b). Each of four dogs aged one year received simultaneous l131-labelled triolein and CA45Cl2 either in oil/water emulsion and oil/water emulsion with lard sucrose esters. All doses were given intragastrically. Lard sucrose esters had no effect on the pattern of absorption. Experiments were repeated in young and old dogs with ligated pancreatic duct and administering intragastrically l131-labelled triolein in oil/water emulsions or oil/water emulsions with lard sucrose ester. Pre-and post-operative blood samples showed that no significant effect was exercised by the ester (Tudisco, 1961a, b). As much as 100 g fat as lard sucrose ester may be administered to dogs and human subjects without producing plasma turbidity or an increase in excreted faecal fat (Berry & Turner, 1960). Humans have been maintained for short periods on this material as the sole source of dietary lipid (Tudisco, 1965a, b). TOXICOLOGICAL STUDIES Acute toxicity Rats tolerated doses of 5 g/kg bw lard sucrose ester every two hours up to a total of 15 g; 10 g/kg bw every two hours up to a total of 30 g produced diarrhoea and death from overdistension. Single doses of 3.8 g/kg bw in dogs had no effect. Rabbits tolerated 2.0 g/kg bw single doses without deleterious effects (Tudisco & Chiancone, 1965). Intragastric administration of 1 g to rats or 20% of the ester in their diet produced no osmotic fragility of erythrocytes. Rabbits given 3-6 g intragastrically also showed no osmotic fragility rise. Intravenous administration of 1-2 g/kg bw to mice had no haemolytic effect (Tudisco, 1965a, b). Single oral doses of lard sucrose ester (48% sucrose ester, 12% triglycerides, 21% diglycerides, 19% monoglycerides) were administered to four adult dogs aged four to six years as follows: 56 g lard sucrose ester, after a long interval 40 g refined lard, after a long interval 56 g lard sucrose ester and 40 g refined lard. Thus each animal served as its own control. No gastrointestinal disturbances, toxic symptoms or abnormal findings were discovered when blood sugar, total lipid, glyceride, cholesterol, phospholipid and percentage fatty acid composition were estimated. Plasma turbidity was lowest in the dog receiving the ester alone (Tudisco, 1963b). Short-term studies Rat Groups of 12 animals received 0, 5%, 10% and 25% lard sucrose ester daily in their diet for 15 days without adverse effects except that the test group at the 25% level showed reduced body weight gain (Tudisco & Chiancone, 1965). In another experiment groups of 12 rats each received for 200 days 5%, 10% and 25% lard sucrose ester in their diet while controls received 3.6%, 7.7% and 18% lard. Body weight gain was reduced at the 10% and especially the 25% levels. Food consumption and food efficiency were reduced at both the 10% and 25% level. No abnormal findings were seen in haematology, and clinical tests (blood glucose, plasma lipids, plasma proteins, liver lipids). There were no undue gastrointestinal symptoms. Only test animals at the 25% level were autopsied, there were no significant abnormalities in organ weights or histology of kidney, spleen, thyroid and adrenals. The liver showed frequent "steatosis" (Lepetit Lab., 1961). When male rats were given a diet containing 25% lard sucrose ester or 18% lard and 7% sucrose for 200 days there was no adverse effect noticeable in body weight gain, food consumption, survival. Clinical findings which could not be ascribed to high fat diet were absent. Only the total liver fat of tests and controls was raised while the blood cholesterol of controls was also higher. Autopsy revealed no significant organ changes (Tudisco, 1967). Long-term studies Rat Groups of nine to 11 male rats were fed 3.6% lard, 5% and 10% lard sucrose ester in their diet as only source of lipids for 24 to 28 months. No abnormalities were found with respects to body weight, general health, mortality or haematology. Tumour incidence was similar in test groups and controls. Visceral abnormalities were equally distributed (Chiancone et al., 1963, Tudisco & Chiancone, 1965). A two-generation study was carried out starting with 15 male and 15 female rats receiving 0 and 5 g/kg bw lard sucrose ester containing 52 ppm (0.0052%) of dimethyl formamide for two years. The parent generation was observed for two years. Four females died or were sacrificed for reasons unrelated to the test substance while 11 animals survived. Three males died from respiratory disease. After 24 months autopsies showed no malignant tumours or other conditions to be present which did not also occur to a similar extent in controls. Haematological findings were normal and electrophoresis or plasma proteins showed nothing unusual. Five males and five females were mated after nine months to produce 35 F1 generation pups. All F1 animals gained weight satisfactorily without spontaneous deaths and were kept for 21 months. Five males and five females were examined after 21 months without showing any malignant or other tumours not also present equally in the controls. No abnormal findings were seen. Five males and five females of the F1 generation were mated after nine months to produce 32 F2 generation animals. The F2 generation survived for 14 months without showing any abnormalities as regards body weight gain or mortality. Five male and five female F2 animals were autopsied at 14 months without showing any unusual tumours of benign lesion or other condition not occurring equally frequently in the controls. No adverse effects were seen on fertility or fetal development (Mosinger, 1964a). Groups of 15 male or female rats were fed diets containing 0 or 0.5% tallow sucrose ester for 14 months. No tumours were seen at the end of 13 months nor any growth abnormalities in male rats. Female rats showed a temporary lowering of growth rate during the eighth to tenth week. Three animals were examined histopathologically and no evidence of abnormality attributable to the ester was found. Haematology and blood protein investigation showed comparable results in tests and controls. No animal died (Oshima & Kajiwara, 1960). Comments: It seems reasonable, in evaluating these compounds, to consider mainly their metabolic fate in the animals studied provided that it is similar to their metabolic behaviour in man. However, the available metabolic studies are not adequate to reveal the pathways of individual products. The long-term feeding studies in rats on some representative compounds are adequate. A two-year study has been reported on sucrose monopalmitate. The presence of dimethyl formamide at levels up to 50 ppm (0.005%) has not introduced an adverse effect in the long-term toxicity tests on sucrose esters. However, most of the short-term and all long-term tests have been performed in the rat only. EVALUATION Level causing no toxicological effect Rat: 10 000 ppm (1%) in the diet equivalent to 500 mg/kg bw. Estimate of acceptable daily intake for man 0-2.5* mg/kg bw.** FURTHER WORK OR INFORMATION Required by June 1976. Metabolic studies on representative individual sucrose esters. Two-year studies on another sucrose ester in a non-rodent mammalian species. REFERENCES Balea, T. (1963) Unpublished report from LARAC Laboratories Balea, T., Cariou, J. & Snozzi, C. (1966) Revue Française des Corps Gras, No. 2, 3 * This applies to material the dimethyl formamide content of which does not exceed 50 ppm (0.005%). ** Temporary Berry, J. F. & Turner, A. D. (1960) J. Amer. Oil Chem. Soc., 37, 302 Chiancone, F. M. et al. (1963) Ann. Fals. Exp. Chim., 56, 193 Fernando, R. (1964) Unpublished report Hara, S. (1959) Submission to Japanese Ministry of Health and Welfare Hazelton Laboratories (1965) Report to Sucro-Chemical Division dated 3 June 1965 Hazelton Laboratories (1966a) Report to Sucro-Chemical Division dated 25 March 1966 Hazelton Laboratories (1966b) Report to Sucro-Chemical Division dated 20 April 1966 Lepetit Laboratories (1961) Unpublished report dated October Mosinger, M. (1964a) Unpublished report Mosinger, M. (1964b) Unpublished report Oshima, M. & Kajiwara, M. (1960) Takeda Kenkyusho Nempo, 19, 172 Tokita, K. (1958 & 1959) Submissions to Japanese Ministry of Health and Welfare Tudisco, R. (1961) Unpublished report of Biochemistry Research Division, Dept. Medicine, Sinai Hospital, Baltimore Tudisco, R. (1961) Unpublished report to Lepetit Laboratories Tudisco, R. (1963a) Boll. Soc. ital. Biol. sper., 39, 1037 Tudisco, R. (1963b) Boll. Soc. ital. Biol. sper., 39, 1914 Tudisco, R. (1965) Il Farmaco, 20, 372 Tudisco, R. (1965), Rass. Med. Sper., 12, 139 Tudisco, R. (1967) Arzneimittel-Forsch. 17, 350 Tudisco, R. & Chiancone, F. M. (1965) Third Congr. Naz. Stud. Alim., Bologna Tudisco, R. & Turner, A. D. (1963) Boll, Soc. ital. Biol. sper., 39, 1041
See Also: Toxicological Abbreviations