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.

    (Mucor pusillus)


         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.



         No information available.


    Special studies on aflatoxins production


         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


         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

    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

    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


         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).


         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


         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).


         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.


         Acceptable daily intake not specified.*


    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

    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