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. POLYGLYCEROL ESTERS OF INTERESTERIFIED RICINOLEIC ACID Explanation These emulsifiers 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 BIOCHEMICAL ASPECTS The substance does not affect the hydrolysis of ground-nut oil by pancreatic lipase in vitro. The clearing of alimentary lipaemia by heparin was not affected in rats fed with 9% of the substance in the diet for 45 weeks. Ingestion of a diet containing 9% of the substance did not induce an increase in the chylomicron count in rats, whereas ingestion of a diet containing 10% of GNO (ground-nut oil) did. It was concluded that the substance was not absorbed into the intestinal lymphatic vessels, but was absorbed into blood capillaries and was cleared from the blood in the liver. A sample of the substance labelled with 14C in the polyglycerol moiety was mixed with ground rat food (approx. or equal 10%) and administered in a slurry by gastric intubation to three rats. Radioactivity was recovered in faeces (54%), urine (31%) and CO2 (8%). The faecal radioactivity was not in the lipid soluble fraction, suggesting that it was present as free polyglycerols. Tracer amounts of 14C-(1)-oleic and 14C-(1)-stearic acids were condensed with ricinoleic acid and coupled with polyglycerol. The products were administered to rats as a 50% aqueous emulsion by gastric intubation. With the 14C-stearyl marker, radioactivity was recovered in expired CO2 (18%), faeces (52%) and urine (1%): with 14C-oleyl marker, recoveries were: CO2 (30%), faeces (51%), urine (1%). 14C-stearyl marked material was also fed as a dietary component with recoveries in expired CO2 (11%), faeces (10%) and urine (1%); when intubated as a dietary slurry recoveries were: expired CO2 (27%), faeces (16%) and urine (2%). Although different modes of administration affect the pattern of turnover the findings indicate that the markers were hydrolyzed from esters with ricinoleic hydroxyl groups and were then utilized normally in metabolism. The substance was prepared with 3H-(12)-ricinoleic acid as a marker, and administered to rats by intubation in a dietary slurry containing a dose of 2 ml (14 µCi). After 24 hours, 5% of administered tritium was found in the faeces, and 1% was present in the remainder of the intestinal contents. The urine contained 10%. Epididymal fat contained 5% of which about one-third was incorporated in hydroxy fatty acids. Similar findings were reported with 3H-(9:10)-ricinoleic acid. Digestive enzymes in (i) the juice from duodenum and jejunum, (ii) mucosa scrapings, and (iii) pancreas homogenates prepared from rats liberated some free ricinoleic acid from the substance incorporating 3H-(12)-ricinoleic acid as a marker. The digestibility of the substance with 9% in diet (plus 1% of ground-nut oil) was 98.0% compared with 99.8% for 10% ground-nut oil, as calculated from intake and faecal excretion of fat (Howes & James, 1968; Jenkins & Philp, 1961, 1968; Rutherford & Jones, 1967; Watson & Gordon, 1962). TOXICOLOGICAL STUDIES Special studies on liver enlargement Mouse Groups of mice were fed diets containing 0%, 5%, 10% or 15% of the substance; control groups were fed corresponding levels of GNO. Each group was divided into two feeding regimes: ad libitum or restricted to seven hours per day. There were 10 male and 10 female mice on each regime. The trial continued for 14 days. Liver enlargement occurred in all groups fed with the substance, but the effect was reversible with return to normal after two further weeks on a normal diet. Kidney weights were not significantly affected. In a further similar trial the substance (or GNO) was fed at levels of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8%. Compared to diets containing equal levels of GNO, 3% or more of the substance significantly increased liver weight, but the degree of liver enlargement was not proportional to the feeding level (Wilson et al., 1967a, b). Rat The liver enlargement produced by the substance was studied in rats fed with 18% (+ 2% GNO) or 20% GNO for one week and was attributed to cytoplasmic hypertrophy of liver parenchymal cells rather than to hyperplasia since the liver weight:nucleus count ratio was increased and there was no increase in mitotic activity. There were no changes in the structural organization of the liver and no histopathological evidence of toxicity. The hypertrophy was regarded as a normal functional response to an increased hepatic work load. In another similar feeding trial, total liver DNA was not affected while liver RNA was increased by the substance, confirming that the increased weight was due to hypertrophy and not to hyperplasia (Gellatly & Jenkings, 1968; Wilson et al., 1968). Acute toxicity Animal Route LD50, mg/kg bw Reference Rat oral > 18 500 Jenkins & Philp, 1961 Rat i.p. > 12 000 Jenkins & Philp, 1961 Rabbit oral > 25 000 Jenkins & Edwards, 1967 Guinea-pig oral > 30 000 Jenkins & Edwards, 1967 Mouse oral > 100 000 Jenkins & Philp, 1961 Chicken oral > 30 000 Jenkins & Edwards, 1967 Slight diarrhoea was the only effect seen in 42 rats out of 55 given 20 ml/kg by gastric intubation and observed for 21 days subsequently; diarrhoea was seen in two of 22 control rats given ground-nut oil (Jenkins & Edwards, 1967). Twelve rats were given 10 ml/kg (approx. or equal 10 000 mg/kg) daily for five days without significant effect on weight gain or food intake and without note of toxicity during the period of administration or 14 days later at autopsy (Jenkins & Edwards, 1967). Short-term studies Rat Littermate trios of rats were fed on a diet containing 10% ground-nut oil, 1% ground-nut oil, or 1% ground-nut oil + 9% of the substance. Each trio was of the same sex and of similar weight, and its members were distributed to the three groups at random. Treatment of one lot of 16 trios was continued for 30 weeks and of another lot for 45 weeks during which there were no signs of toxicity and no significant effects of the substance on food or water intake or weight gain. The digestibility of the substance with 9% in diet (plus 1% of ground-nut oil) was 98.0% compared with 99.8% for 10% ground-nut oil, as calculated from intake and faecal excretion of fat. After 11 weeks, there were no significant differences in body fat weight, fatty acids, iodine value or hydroxyl value, or in liver content of vitamin A, fat or free fatty acids, but liver phosphorus was low. After 45 weeks, there were no differences in the digestibility of fat, protein or whole diet between the groups. Plasma clearance of bromsulphthalein was somewhat more rapid in rats treated with 9% of the substance in the diet for 17, 21 and 45 weeks than in rats on control diets containing ground-nut oil: there is no evidence, therefore, of impairment of liver function by the substance. The urine-concentrating power of the kidneys was not affected in rats treated with 9% of the substance in the diet for 21 and 45 weeks. There were no significant effects of 9% of the substance in the diet for 45 weeks on haemoglobin, erythrocyte count, haematocrit value, or prothrombin time. Erythrocyte fragility examined at 15 weeks was not affected (Jenkins & Philp, 1961). In the 30-week and 45-week trials, there were no significant effects on weight of kidney, spleen, testes or adrenals and all organs were macroscopically normal. The livers were significantly larger than control in rats of the 45-week trial, but not in those of the 20-week trial. Similar effects were seen in rats fed castor oil (Wilson & Jenkins, 1968),. The no-effect level for increased liver weight in a 13-week trial was determined by feeding a diet containing 0%, 1%, 2%, 4% and 8% of the substance using 10 rats in each group: there were significant increases in liver weight in the 4% and 8% groups, the effect being greater in females than males. In these rats (4% and 8% groups), acetone bodies were detected in the urine during the first six weeks of the trial, but the reaction was apparently due to a metabolite of the substance rather than to the usual acetone bodies of ketotic states (Wilson & Jenkins, 1968). Chicken Chickens in groups of 16 were used in a 90-day (13-week) trial. Groups were fed basic diet (two groups), 15% of the substance (two groups), 10%, 5% and 2% of the substance with 2% ground-nut oil (GNO) (one group at each level) and 15%, 10% and 5% GNO (one group at each level). Growth was adversely affected by the substances and by GNO at the 10% and 15% dietary levels and was apparently due to dietary imbalance and decreased food intake. Plasma levels of glutamate- pyruvate aminotransferase and glutamate-oxaloacetate amino-transferase were not significantly affected by any of the treatments. The haematocrit value, haemoglobin and erythrocyte sedimentation rates were not affected by the substance. Liver weight was significantly increased by 15% of the substance, but not with 10% or less. Kidney weight was significantly increased by 5%, 10% and 15% of the substance. Spleen and heart weights were not selectively affected by the substance, since GNO produced the same changes. Histological signs of toxicity in various organs were absent except for lymphoid infiltrations which were attributed to intercurrent subclinical virus disease (Marek's disease) (Kirby et al., 1969). Long-term studies Mouse Four groups of 25 male and 25 female mice were fed a diet with 10% ground-nut oil or 5% of the substance plus 5% GNO for 80 weeks. A positive control group received 0.5 mg, 9, 10-dimethyl-1, 2-benzanthracene once per week for 16 weeks. After 50 weeks there was no difference in growth between test and ground-nut oil groups but the basic diet and positive control group showed reduced growth. Survival was comparable for the three groups, also the haematological indices. Liver and kidney of the test group were significantly heavier than the controls. No specific histological lesion was seen. Carcass fat contained no polyglycerol but only a small amount (0.1%) of ricinoleic acid. Histopathology of all other major tissues showed no lesions specifically associated with the substance nor was there any significant difference in tumour incidence. The strain was sensitive to the carcinogen used in the positive control (Wilson et al., 1967). Groups of 20 male and 20 female mice were given repeated s.c. injections of 0.5 ml of the substance weekly for five weeks with milk or ground-nut oil as controls. After 80 weeks no adverse effects were noted on growth, survival, haematology, organ weights and histopathological findings. Tumour incidence was similar in test and control groups (Wilson et al., 1967). Seven groups of 20 male and 20 female mice were painted daily with 20 mg of the substance for 60 weeks with or without a single preceding application of 0.25 mg dimethylbenzanthracene and a positive control group was included. After 80 weeks no adverse effects were noted on growth, survival or tumour incidence in tests and controls involving the substance. There was no promoting or cutaneous carcinogenic effect (Wilson et al., 1967). Rat A three-generation test was carried out in two groups of 19 and 28 rats on diets containing 0% or 1.5% of the substance. No significant differences were seen between the two groups as regards fertility, pup weight, pup survival, litter number, etc. Each animal was observed for over one year. No consistent abnormalities or histopathological changes were seen in the third generation (Philp & Jenkins, 1961). In another experiment two groups of 30 male and 30 female rats each were fed diets containing 5% of the substance plus 5% ground-nut oil or 10% ground-nut oil for 104 weeks. No significant adverse effects were seen on growth, food consumption, liver function tests at weeks 89 and 103, specific gravity of the urine at weeks 89 and 103, or survival. The kidneys of males and females at 5% of the substance and the livers of females at 5% of the substance were significantly enlarged. There was no polyglycerol or ricinoleic acid accumulation in carcass fat. Histopathology of all organs showed no abnormalities related to the substance administration nor was there a rise in tumour incidence in the test group. The strain was sensitive to 20-methyl- cholanthrene (Philp et al., 1961). Thirty male and 30 female rats were injected s.c. with 0.5 ml twice weekly for 13 weeks at different sites, milk and ground-nut oil were controls. After two years there were no adverse effects on growth survival, haematological indices, organ weights or histopathology of all tissues. No increase in tumour incidence was found but most rats treated with the substances developed persistent nodules at injection sites. Subcutaneous injections produced 2/60 fibrosarcomata as did milk (2/60) but not ground-nut oil (Philp & Jenkins, 1961). Six groups of 10 male and 10 female rats were painted with 50 mg of the substance cutaneously daily for 60 weeks with or without a single preceding application of 0.25 mg benzanthracene and observed for two years. A positive control group was included. No deleterious effects were noted on growth and survival. No skin tumours were seen in the test group. The strain was sensitive to the carcinogen used as positive control (Philp & Jenkins, 1961). OBSERVATIONS IN MAN Nineteen volunteers took the substance in soups, cakes and toffees at the rate of 5 g/day for one week and 10 g/day for a second week. Otherwise, the diet was balanced and constant for the two weeks and an initial stabilization week. The group contained eight males and 11 females; except for two older females (64 and 66 years), all were university students (19 to 24 years). There were no significant adverse departures from control patterns attributable to the substance in serum proteins, thymol turbidity, serum bilirubin, blood cholesterol, serum glutamate-pyruvate animotransferase, serum cholinesterase, creatinine clearance faecal fat or faecal nitrogen (Groger et al., 1968). Comments: The metabolic fate of this material has been studied by a number of indirect measurements. The long-term studies in rats and mice did not show carcinogenic potential. The enlargement of liver and kidneys observed in long-term tests was not accompanied by any lesions detectable by histopathology. Only the rat study shows a no-effect level for liver enlargement. EVALUATION Level causing no toxicological effect Rat: 15,000 ppm (1.5%) in the diet equivalent to 750 mg/kg bw. Estimate of acceptable daily intake for man 0-7.5 mg/kg bw. REFERENCES Gellatly, J. B. M. & Jenkins, F. P. (1968) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Groger, W., Philp, J. McL.& Wilson, R. (1968) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Howes, D. & James, C. T. (1968) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Jenkins, F. P. & Edwards, K. (1967) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Jenkins, F. P. & Philp, J. McL. (1961) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Kirkby, W. et al. (1969) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Philp, J. McL. & Jenkins, F. P. (1961) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Philp, J. McL. et al. (1961) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Rutherford, T. & Jones, P. (1967) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Watson, W. C. & Gordon, R. S. (1962) Biochem. Pharmac., 11, 229 Wilson, R., Ashmole, R. & Gellatly, J. B. M. (1968) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Wilson, R., Kirkby, W.& Ashmole, R. (1967a) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Wilson, R., Kirkby, W. & Ashmole, R. (1967b) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Wilson, R. & Jenkins, F. P. (1968) Unpublished report of Unilever Res. Labs. submitted by Unilever Ltd. Wilson, R., Kirkby, W. & Philp, J. McL. (1967) Unpublished report of Unilever Res. Labs, submitted by Unilever Ltd.
See Also: Toxicological Abbreviations Polyglycerol esters of interesterified ricinoleic acid (FAO Nutrition Meetings Report Series 46a) POLYGLYCEROL ESTERS OF INTERESTERIFIED RICINOLEIC ACID (JECFA Evaluation)