INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION TOXICOLOGICAL EVALUATION OF SOME FOOD COLOURS, ENZYMES, FLAVOUR ENHANCERS, THICKENING AGENTS, AND CERTAIN FOOD ADDITIVES WHO FOOD ADDITIVES SERIES 6 The evaluations contained in this publication were prepared by the Joint FAO/WHO Expert Committee on Food Additives which met in Rome, 4-13 June 19741 World Health Organization Geneva 1975 1 Eighteenth Report of the Joint FAO/WHO Expert Committee on Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557. FAO Nutrition Meetings Report Series, 1974, No. 54. ANNATTO EXTRACTS Explanation These compounds 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 No information available. TOXICOLOGICAL STUDIES Special studies on mutagenicity Mutagenic action was tested in a concentration of 0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect was found (Lück & Rickerl, 1960). Acute toxicity LD50 Animal Route Extract type (mg/kg bw) Reference Mouse i.p. water soluble 700 Durham & Allard, 1960 Rat oral fat soluble > 50 ml Van Esch et al., 1959 Rat oral fat soluble > 25 ml Van Esch et al., 1959 Rat oral water soluble > 35 ml Van Esch et al., 1959 Administration of an aqueous extract of bixa root depressed spontaneous motor activity in the mouse, the intraperitoneal ED50 being 21 mg/kg bw. The extract also affects the volume of gastric secretion but not its pH (400 mg/kg intraduodenally). It has some antispasmodic (1 mg//ml isolated guinea-pig ileum) and hypotensive properties (i.v. 50 mg/kg rat) (Durham & Allard, 1960). Short-term studies Mouse Seventy male and 30 female mice were injected s.c. with 0.1 ml annatto. Occasionally a sarcoma was produced at the site of injection. No definite effect was seen on distant tumoric development either as regards time of appearance or number (Engelbreth-Holm & Iverson, 1955). Fifty male and 50 female mice were painted twice a week for three months at the interscapular region with 0.05 ml 50% annatto in benzene. No skin papillomas or other tumours were encountered (Engelbreth-Holm & Iverson, 1955). Rat Three groups of 10 male and 10 female rats were fed 0% and 2% of fat soluble annatto and 2% water soluble annatto for 13 weeks. Food intake, growth, haematological examination, organ weights and histopathology of major organs showed no abnormalities (Van Esch et al., 1959). Two groups of 10 male and 10 female rats were given 0 and 1000 mg/kg bw of annatto orally for 100 days. No abnormalities were seen (Zbinden & Studer, 1958). Two groups of 10 male and 10 female rats were injected s.c. at the same site for 36 weeks, three times per week, with 0.05 ml corn oil and fat soluble annatto. After observation for 24 months there were no local tumours (Van Esch et al., 1959). Dog Two groups of three male and three female beagles were fed 0 or 2.7% on the diet of fat soluble extract of annatto seed for nine weeks, then fed normal diet for five weeks and then fed only 1.35% in the diet of fat soluble extract in capsules for 38 weeks. No abnormalities were found as regards growth, food intake, mortality, liver and kidney function, haematology or histopathology. One female dog died in the test group. The liver of this animal showed hepatocellular degeneration (Kay & Calandra, 1961a). Four groups of three male and three female beagles were fed in their diet 0, 5% and 10% aqueous extract of annatto seed for one year. The fourth group received 20% aqueous extract for 16 weeks in their diet and then half of the extract in the diet and half in gelatine capsules for 36 weeks. Controls received 0.48% potassium chloride. Growth inhibition and reduced food intake occurred at the 20% dietary level. Mortality rate, liver and kidney function tests, haematology and histopathology of all major tissues showed no abnormalities attributable to the test substance (Kay & Calandra, 1961b). Pig Three groups of two male and one female pigs were fed 0% and 1% fat soluble annatto and 1% water soluble annatto for 21 weeks. One animal in the test group died from a cause unrelated to the test substance. Food intake, growth, haematology, organ weights and histopathology of all major tissues were normal (Van Esch et al., 1959). Long-term studies Mouse Fifty female and 50 male mice were fed daily, one drop of a 10% solution of annatto in soy oil for 24 months. There was no significant difference from a similar control group on normal diet (Engelbreth- Holm & Iverson, 1955). Two groups of 50 male and 50 female mice were fed either 0.5% corn oil or 0.5% fat soluble annatto for their life span. The same animals also received s.c. 0.1 ml oil three times per week for 17 months. Two other groups of 25 males and 24 females were fed for their life span 0% or 0.05% concentrated fat soluble annatto and the same animals were also injected 0.001 ml s.c. for 10-1/2 months. Cyst formation with local necrosis was seen at the site of injection. Most animals died between 15 and 21 months due to inter- current infection. No statistically significant increase in tumour production was observed (Van Esch et al., 1959). Rat Two groups of 100 female rats were given daily 26 mg annatto in soy oil for 26 months. No effect was noted on the pathological experience of the two groups (Engelbreth-Holm & Iverson, 1955). Three groups of 10 male and 10 female rats received corn oil with 0, 0.05% fat soluble annatto and 0.5% water soluble annatto for their life span. Those extracts varied in total bixin content from 0.2-2.6%. Two daughter generations were bred each being fed similar diets for seven and eight-and-a-half months. No deleterious effect was observed on growth and reproduction. No teratogenic effects were seen. No consistent effect on mortality was noted in the three generations. Organ weights and tumoric incidence were comparable in all groups (Van Esch et al., 1959). Two groups of 10 male and 10 female rats were fed 0 or 0.05% of concentrated fat soluble annatto for 32 months. A first filial generation received the same diet for seven months. No deleterious effects were seen on growth and reproduction, mortality, organ weights and tumoric production (Van Esch et al., 1959). Comments: Adequate long-term tests in two species have been performed on a well-defined type of extract containing 0.2-2.6% of carotenoid expressed as bixin. Short-term tests in two other species suggest a lack of cumulative action even at levels of 15% carotenoid in vegetable oil or 10% in water. The long-term study in the rat provides a basis for evaluation. However, information on the metabolism is lacking while the dog studies indicate that the high levels of carotenoids do not produce adverse effects. Studies on the metabolism of the carotenoids are in progress. EVALUATION Level causing no toxicological effect Rat: 0.5% (= 5000 ppm) in the diet equivalent to 250 mg/kg bw. Estimate of acceptable daily intake for man 0-1.25* mg/kg bw** FURTHER WORK OR INFORMATION Required by June 1978. Completion of metabolic studies presently in progress. REFERENCES Durham, N. W. & Allard, R. K. (1960) J. Amer. pharm. Assoc., 49, 218 Engelbreth-Holm, J. & Iverson, S. (1955) Acta. Path. Microb. Scand., 37, 483 van Esch, G. J., van Genderen, H. & Vink, H. H. (1959) Z. Lebensm. Untersuch., 111, 93 Kay, J. H. & Calandra, J. C. (1961a) Unpublished report by Industrial Bio-Test Laboratories Inc., 25/7/61 to Marshall Dairy Lab. Inc. Kay, J. H. & Calandra, J. C. (1961b) Unpublished report by Industrial Bio-Test Laboratories Inc., 6/3/61 to Marshall Dairy Lab. Inc. Lück, H. & Rickerl, E. (1960) Z. Lebensm.-Untersuch., 112, 157 Zbinden, G. & Studer, A. (1958) Z. Lebensm.-Untersuch., 108, 113 * Temporary. ** Expressed as bixin.
See Also: Toxicological Abbreviations Annatto Extracts (WHO Food Additives Series 52) Annatto extracts (FAO Nutrition Meetings Report Series 46a) Annatto extracts (WHO Food Additives Series 17) Annatto extracts (WHO Food Additives Series 44) ANNATTO EXTRACTS (JECFA Evaluation)