INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF SOME FOOD COLOURS, EMULSIFIERS, STABILIZERS, ANTI-CAKING AGENTS AND CERTAIN OTHER SUBSTANCES FAO Nutrition Meetings Report Series No. 46A WHO/FOOD ADD/70.36 The content of this document is the result of the deliberations of the Joint FAO/WHO Expert Committee on Food Additives which met in Rome, 27 May - 4 June 19691 Food and Agriculture Organization of the United Nations World Health Organization 1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food Additives, FAO Nutrition Meetings Report Series, in press; Wld Hlth Org. techn. Rep. Ser., in press. POLYGLYCEROL ESTERS OF INTERESTERIFIED RICINOLEIC ACID Biological Data Biochemical aspects Twelve rats fed for 10 weeks a diet containing 1 per cent. or 10 per cent. groundnut oil or 1 per cent groundnut oil + 9 per cent. of the substance were divided into two groups. One was fed a fat-free diet for six days, the other group was fed test or control diets. No acetone or acetoacetic acid was detected in any urine, pointing to absence of interference with fat metabolism. Three groups of 10 male and six female rats fed 45 weeks on diets containing either 1 per cent. or 10 per cent. groundnut oil or 1 per cent. groundnut oil + 9 per cent. of the substance ware given three days later an additional 9 per cent. groundnut oil for seven days. No differences between tests and control groups were detected regarding oil absorption, protein digestibility or diet digestibility. Three groups of 10 male and six female rats were fed diets containing 9 per cent. or 18 per cent. groundnut oil or 9 per cent. groundnut oil + 9 per cent. of the substance. Clearing of lipaemic plasma by heparin was similar in all three groups and plasma turbidity was not increased. Chylomicron concentration in rats before and after feeding the substance in their diet showed absence of alimentary chylomicronaemia. Absorption does therefore not occur into intestinal lymphatics although a specific test to confirm absorption into portal capillaries was not done. Three groups of rats were fed four weeks on diets containing 1 per cent. or 10 per cent. groundnut oil or 1 per cent. groundnut oil + 9 per cent. of the substance. Digestibility of the substance was 98 per cent. Another three groups of two male and two female rats were fed eleven weeks on diets containing either 1 per cent. or 10 per cent. groundnut oil or 1 per cent. groundnut oil + 9 per cent. of the substance. No adverse influence was noted on fat storage, body composition and vitamin A content of the liver; the substance was not stored significantly in the body fat. The in vitro action of lipase on groundnut oil was not affected by the presence of the substance (Unilever Research Laboratories, 1966). Acute toxicity Animal Route LD50 Reference mg/kg body-weight rat oral > 18 500 Unilever Research Labs, 1966 rat i.p. > 12 000 " No mortality could be obtained, though animals received up to 47.5 g/kg daily for 5 days (Unilever Research Laboratories, 1966). Short-term studies Rat. Three groups of eight male and eight female rats were fed a diet containing either 1 per cent. or 10 per cent. groundnut oil or 1 per cent. groundnut oil + 9 per cent. of no substance for 30 weeks. No adverse effects on growth, food intake and faecal appearance were noted. During this test all three groups were restricted to 5 g fat free diet for 17 days and then realimented. No difference in weight gain was noticeable, nor in carcass and liver fat amounts or composition. The test group on the substance had a shorter Bromosulphophthalein retention time. The specific gravity of the urine was comparable in all groups. Organ weights of liver, kidney, adrenal, testes and spleen were similar in all groups. In view of the special design of the study, the statement on absence of significant changes in liver weight applies only to three groups of selected animals not participating in the food restriction experiment, i.e. two female and four male rats on 1 per cent. groundnut oil, four female and three male rats on 10 per cent. groundnut oil and two female and five male rats on 1 per cent. groundnut oil + 9 per cent. of the substance. Despite the small numbers the test may be taken to indicate that 9 per cent. of the substance was not causing liver enlargement when fed for 30 weeks. In another study three groups of 10 male and six female rats were kept on diets containing either 1 per cent. or 10 per cent. groundnut oil or 1 per cent. groundnut oil + 9 per cent. of the substance for 45 weeks. No adverse effect on growth or food intake was noted, The groups were comparable as regards Bromosulphophthalein retention, specific gravity of urine, haematological indices and red cell fragility. Organ weights of kidney, adrenal, testes and spleen were similar in all groups but the liver weights of rats fed of the substance (9 per cent. of the diet) were significantly higher than the controls. Similar effects were seen in rats fed castor oil. This was not due to an increased blood volume of the liver (Unilever Research Laboratories, 1966). Man. Nineteen male and female volunteers, aged 19-24 years, took 5 g of the substance per day for one week and 10 g per day for another week using constant dietary fat intake. Serum protein, albumin and globulin levels were unaffected. Thymol turbidity, serum bililubin, SGBT, serum cholesterol and serum cholinesterase were normal (Unilever Research Laboratories, 1963). Long-term studies Mouse. Four groups of 25 male and 25 female mice were fed a diet with 5 per cent. groundnut oil or the substance with no additional fat 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 groundnut 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 per cent.) 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. 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 groundnut 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. 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 (Unilever Research Laboratories, 1966). Rat. A three-generation test was carried out in two groups of 19 and 28 rats on diets containing 0 per cent. or 1.5 per cent. 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. In another experiment two groups of 30 male and 60 female rats each were fed diets containing five per cent. of the substance or five per cent. groundnut 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 five per cent. of the substance and the livers of females at five per cent. of the substance were significantly enlarged. There was no polyglycrol 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-methylcholanthrene. Thirty male and 30 female rats were injected s.c. with 0.5 ml twice weekly for 13 weeks at different sites, milk and groundnut 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 substance developed persistent nodules at injection sites. 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 (Unilever Research Laboratories, 1966). Comments The metabolic fate of this material has been studied by a number of indirect measurements. In-vivo studies, using labelled material, are not available. 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. No satisfactory explanation of the observed organ enlargements has been offered. EVALUATION Level causing no toxicological effect in the rat 1.5 per cent. (= 15 000 ppm) in the diet equivalent to 750 mg/kg bodyweight per day. Estimate of acceptable daily intake for man mg/kg body-weight Temporary acceptance 0 - 3 75 Further work required by June 1972 In-vivo metabolic studio in several species to determine more directly the fate of the major polymers in the material. A 90-day study in a non-rodent mammalian species to establish a no-effect level for organ enlargement. Investigation of the causes of the observed hepatic and renal enlargements. REFERENCES Unilever Research Laboratories (1966) Unpublished report submitted to WHO
See Also: Toxicological Abbreviations Polyglycerol esters of interesterified ricinoleic acid (WHO Food Additives Series 5) POLYGLYCEROL ESTERS OF INTERESTERIFIED RICINOLEIC ACID (JECFA Evaluation)