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. MICROBIAL RENNET* (Mucor pusillus) Explanation This enzyme preparation has been evaluated for acceptable daily intake by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1, Ref. No. 27) in 1971. 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 aflatoxins production Duckling Six groups of 18-20-day-old ducklings were given 0, 2 mg, 10 mg, 50 mg and 250 mg material by gavage as well as a positive control group receiving 12.5 mg aflatoxin. Deaths occurred only in the aflatoxin and 250 mg groups. There was some weight loss throughout the test in the 250 mg group and less so at lower test levels. Liver weight was increased on the eighth day at all levels. Histologically no lesions were seen which pointed to the presence of aflatoxin (van Logten & Kroes, 1968). Special studies on reproduction Rat A four-generation study in rats was performed on the same compound as in the long-term study by Til et al., 1972a. Four groups of 10 males and 20 females were fed 0, 0.04, 0.2 and 1% compound in the diet. Each generation was mated in week 12 and week 20 to get two litters a and b. Only the b litters were raised to the next * This enzyme preparation is prepared from the species Mucor pusillus. generation. Ten males and 10 females in each group of F3b were kept for a five-week feeding study. The F2-generation rats showed relatively low body weights in the 1% group in both sexes, but this phenomenon did not occur in the F3-generation. The number of litters born was high in all groups, and the number of youngsters per litter was high in all groups and all generations. No consistent differences in body weight and mortality were observed during the lactation period. In F3b no distinct differences in body weight, food consumption and food efficiency were found. The relative organ weight of the spleen of males fed 1% was decreased. Gross and microscopic examination revealed no abnormalities related to feeding the compound (Til et al., 1972b). Acute toxicity LD50 Animal Route (mg/kg bw) Reference Mouse Oral 4 500 Hara et al., 1963 i.v. 358 Ito et al., 1964 Rat Oral > 5 000 Hara et al., 1963 20% aqueous suspension was nonirritant to rabbit conjunctiva (Ito et al., 1964). Short-term studies Rat A 10-week study in groups of male and female rats receiving 0 (0%), 2 (0.0002%), 20 (0.002%) and 1000 (0.1%) ppm in their diet showed a dose-related weight increase compared with controls. No data are available on organ weights or haematology but LFTs showed no deleterious effect. Histopathology showed nothing abnormal (Ito et al., 1964). A 90-day study was carried out on groups of rats using 0 (0%), 10 (0.001%), 1000 (0.1%) and 10 000 (1%) ppm in their diet. Some reduction in growth was observed which was not dose-dependent and statistically not significant. However, the WBC showed increases in lymphocytes with increasing dosage and changes in monocyte and neutrophil counts which were unrelated to dose. Organ weights and histopathology were normal (Hara et al., 1963). Another 90-day study was carried out in three groups of 10 male and 10 female rats receiving 0 (0%), 1250 (0.125%) or 12 500 (1.25%) ppm in their diet. No abnormalities were detected as regards behaviour, appearance, food consumption, growth, haematology, organ weights, gross and histopathology (van Logten & Kroes, 1968a). A 90-day toxicity study was performed in three groups of rats, each consisting of 10 male and 10 female fed 0 (0%), 1250 (0.125%) and 12 500 (1.25%) ppm enzyme preparation from Mucor pusillus Lindt in the diet. The fungus was cultured on wheat bran, the preparation purified by series of partial precipitation with ethanol and vacuum- dried. Compared with controls the food intake and growth were slightly increased. A slight, not significant dose-dependent increase in the number of lymphocytes and increase in the relative weight of thyroid and the prostate (P<0.05) were found. No gross or histopathological changes were observed (van Logten et al., 1972). Dog Groups each comprising four male and four female dogs were fed dietary levels of 0, 0.5, 1.0 or 2.0% of fungal rennet (from the same batch used in the rat study described below) for two years. There was only one death unrelated to compound administration. General behaviour and appearance, body weight gain and food intake were all comparable between groups. Blood pressures and heart rates were generally within normal limits and electrocardiograms revealed no gross abnormalities. Slight ophthalmological abnormalities observed in two control dogs, three dogs fed 0.5% and five fed 2.0% were considered not related to compound administration. Haemograms, clinical chemistry and qualitative urinary analyses were comparable for all groups. There were no gross or histopathological abnormalities that could be related to feeding fungal rennet (Hollingsworth & Woodard, 1968b). Long-term studies Rat Groups each comprising 20 male and 20 female rats were fed dietary levels of 0, 0.5, 1.0 or 2.0% of fungal rennet (identified as microbial rennet from Meito Sangyo Co. Ltd, Lot No. R2G7801). General appearance and behaviour were comparable in test and control groups. Incidence of mortality appeared unrelated to feeding the test compound. Body weight gain and food intake were normal between the groups. Periodic haemograms including haemoglobin, microhaematocrit, coagulation time and general clinical chemistry determination as well as absolute and relative organ weights and gross and histopathology revealed no abnormalities related to feeding the rennet (Hollingsworth & Woodard, 1968a). In a two-year study four groups of Wistar rats, each group 30 males and 30 females, received in the diet respectively, 0, 0.04, 0.2, and 1.0% rennet from Mucor pusillus Lindt (strain: University of Tokyo, No. F-27). The diets were prepared once a fortnight and stored at room temperature. Mean body weight and food consumption showed no significant differences. Biochemical blood parameters and urine analyses showed no dose-related differences. The relative weights of testes were significantly increased in all the test groups. Gross and histopathological findings were related to aging and infectious diseases. At autopsy of about 90% of the animals, the tumour rate in the control group was 80% and 82% in the 1% group (Til et al., 1972a). Comments: This preparation has been tested by short-term studies in rats and dogs. A duckling study was done and the results of a further 90-day study and two-year study in rats and a four generation reproduction study showed no significant abnormalities at the 1% level, the highest level tested. This meets the requirements laid down by the Committee. EVALUATION Acceptable daily intake not specified.* REFERENCES Hara, S. et al. (1963) Report of the Department of Pharmacology, Tokyo Medical College submitted to WHO Hollingsworth, R. C. & Woodard, G. (1968a) Unpublished Report dated 7 November 1968 from Woodard Research Corporation submitted by Noury & van der Lande, N. V. Hollingsworth, R. C. & Woodard, G. (1968b) Unpublished report dated 6 November 1968 from Woodard Research Corporation submitted by Noury & van der Lande, N. W. * The statement "ADI not specified" means that, on the basis of the available data (toxicological, biochemical, and other), the total daily intake of the substance, arising from its use or uses at the levels necessary to achieve the desired effect and from its acceptable background in food, does not, in the opinion of the Committee, represent a hazard to health. For this reason, and for the reasons stated in individual evaluations, the establishment of any acceptable daily intake (ADI) in mg per kg of body weight is not deemed necessary. Ito, H., Kond, H. & Tokunaga, Y. (1964) Fd. Hyg. Mag., Japan, 5(1), 1 Nakamura, K. (1966) Report submitted by the National Institute of Hygienic Sciences, Tokyo to WHO Til, H. P., van der Meulen, H. C. & de Groot, A. P. (1972a) Unpublished report dated December 1972 from CIVO/TNO submitted By Noury & van der Lande, N. V. Til, H. P., Spanjers, M. T. & Willems, M. I. (1972b) Unpublished report dated December 1972 from CIVO/TNO submitted by Noury & van der Lande, N. V. van Logten, M. J. & Kroes, R. (1968) Report 82/68 Tox submitted by Ryksinstituut, Utrecht van Logten, M. J. & Kroes, R. (1968a) Report 88/68 Tox submitted by Ryksinstituut, Utrecht van Logten, M. J. et al. (1972) Fd. Cosmet Toxicol., 10, 649 Woodard Research Co. (1968) Unpublished report dated 6 November 1968 submitted to WHO Woodard Research Co. (1968a) Unpublished report dated 7 November 1968 submitted to WHO
See Also: Toxicological Abbreviations