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    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 CARBOHYDRASE* (Aspergillus niger)

    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

         Studies on sensitizing effect was tested on 20 albino
    guinea-pigs, 10 males and 10 females, of the Pirbright White
    strain by a challenge injection of 0.05 ml intracutaneous two weeks
    after 10 sensitizing injections of 1% Ultrazym 100 in a 2%
    carboxymethylcellulose solution. The challenge injection produced a
    more intense reaction than the sensitizing injections, indicating a
    skin sensitizing activity (Sachsse, 1971).

    Acute toxicity

         A 70% suspension of Ultrazym 100 in a 2% carboxymethyl-cellulose
    solution was dispersed over the shaved back of 12 RAC/f rats for 24
    hours. Within 24 hours the rats of both dosage groups (1000 mg/kg and
    2150 mg/kg) showed dyspnoea and lachrymation, but no skin irritation.
    Recovering after five days, no substance-related gross organ changes
    were observed at autopsy (Sachsse & Bathe, 1971b).

              

    *    This enzyme preparation is prepared from some varieties of
    Aspergillus niger.

                                                                        

                            LD50
    Animal     Route        (mg/kg bw)           Reference
                                                                        

    Mouse      Oral            >3 200            Hunt & Garvin, 1963

                               >4 000            Hunt & Garvin, 1971

                               >3 200            Willard & Garvin, 1963

                                4 000            Garvin et al., 1966

    Rat        Oral            10 000            Gray, 1960

                               31 600            Kay & Calandra, 1962

                               >3 200            Willard & Garvin, 1968

                               >4 000            Garvin et al., 1966

                              >10 000            Gray, 1960

    Rabbit     Oral            >4 000            Garvin et al., 1966

    Dog        Oral            >4 000            Garvin et al., 1966

    Rat        Oral             7 750            Sachsse & Bathe (1971a)
                                                                        

    Short-term studies

    Rat

         Four groups of 10 male rats received in their diet for 30 days
    enzyme at 0, 0.5 and 5%. There were no adverse effects related to
    treatment regarding growth, appearance, behaviour, survival, food
    consumption, haematology, organ weights and gross pathology (Garvin et
    al., 1966).

         Two groups of 10 male and 10 female rats received daily for 91
    days in their diet either 0 or 5% enzyme. There was no difference from
    controls regarding appearance, behaviour, survival, weight gain,
    haematology, organ weights and gross pathology (Garvin & Merubia,
    1959).

         One group of 20 male and 20 female and four groups of 10 male and
    10 female ARS Sprague-Dawley rats were fed 0, 5 and 10% carbohydrase
    (Wallerstein Pectinase Evaporate) and 5 and 10% carbohydrase

    (Wallerstein Amylo glycosidase Evaporate) in the diet for 90 days
    (roughly equivalent to 3,5 and 7 g preparation/kg bw/day). Appearance
    and behaviour were normal, no deaths. Growth rate and food consumption
    were similar in all groups. No changes were seen in haematology and
    blood chemistry as compared with the respective controls. The relative
    weight of thyroid, liver and spleen in males and liver and spleen in
    females fed pectinase showed a dose-related decrease. The relative
    weight of liver in males and heart in females fed amyloglucosidase
    showed a tendency for dose-related decrease. Gross pathology and the
    only partly performed histopathology showed chronic pneumonia and
    renal tubular obstruction with hyalin casts, which did not seem to be
    dose related (Garvin et al., 1972).

         Three groups of 10 male and 10 female Charles River rats were fed
    0, 5 and 10% of Aspergillus niger mycelial preparation in their diet
    for 13 weeks. There were no overt signs of toxicity. Food consumption
    and growth were not affected. There was a drop in the efficiency of
    food utilization for both dosage groups and both sexes, which was
    significant for the high dose males. The haematological, the clinical
    and the ophthalmoscopic data, revealed no significant differences
    among the groups. There were significant increases in the relative
    kidney weight of females in both dosage groups. The gross and
    microscopic findings were mostly related to chronic respiratory
    disease and kidney lesions including hydropelvis or hydronephrosis,
    which did not appear to be treatment related (Swann & Cox, 1973).

    Duckling

         Groups of five ducklings received in their diet either 0, 1, 5 or
    10% of enzyme for 29 days. Growth, feed consumption, behaviour and
    development were comparable in all groups. No gross liver lesions were
    seen at autopsy and mean liver weights were similar to controls.
    Histopathology of the livers was normal. No toxic element was noted
    (FDRL., 1963).

    Mouse, Rat, Cat

         Enzyme preparations from Aspergillus niger, strain "Pectolytic"
    produced by different cultivation methods were in several different
    acute and short-term studies given to a total number of 300 mice,
    123 rats and 17 cats. The conclusion of the author is, that surface
    and deep culture preparations on media rich in sugars (sugar beet
    pulp, wheat bran) gave effects in some experimental animals after
    45-750 mg/kg bw/day, of the different preparations mainly given in the
    drinking water. The main effects were decreased weight gain, increased
    leucocyte count, accelerated sedimentation of erythrocytes and
    abscesses different places in the body. A preparation isolated from
    the same mould, but cultivated on grain husk had no similar effects on
    the animals. The preparation methods are not described, especially is
    not revealed whether the preparations were sterile (Magnova, 1968).

    Long-term studies

         None available.

    Comments:

         Aspergillus niger is a common contaminant of food. The
    available information indicates that it is not pathogenic to man.
    Duckling studies were done on two preparations and an adequate 90
    days' study in rats is now available showing no toxicological effects
    at 10% in the diet. This meets the requirements as laid down by the
    Committee.

    EVALUATION

         Acceptable daily intake not specified.*

    REFERENCES

    FDRL (1963) Unpublished report No. 84600f submitted by Miles Chem. Co

    Garvin, P. & Merubia, J. (1959) Unpublished report submitted by Baxter
         Laboratories Inc.

    Garvin, P. J. et al. (1966) Unpublished report submitted by Baxter
         Laboratories Inc.

    Garvin, P. J. (1972) Unpublished report No. R.D. 6 - 106 submitted by
         R. M. Gesler

    Gray, E. H. (1960) Unpublished report submitted by Miles Laboratories
         Inc.

    Hunt, R. F. & Garvin, P. J. (1963) Unpublished report submitted by
         Baxter Laboratories Inc.

    Hunt, R. F. & Garvin, P. J. (1971) Unpublished report submitted by
         Travenol Laboratories Ltd

              

    *    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 an acceptable
    daily intake (ADI) in mg per kg of body weight is not deemed
    necessary.

    Kay, J. H. & Calandra, J. C. (1962) Unpublished report submitted by
         Miles Chemical Co

    Magonova, N. B. (1968) Voprosy pitania, 27, 67-72

    Phillips, B. M., Hartnagel, R. E. & Thompson, D. (1967) Unpublished
         report submitted by Miles Chemical Co

    Sachsse, K. (1971) Unpublished report No. Siss 826 submitted by Ciba
         Geigy

    Sachsse, K. & Bathe, R. (1971) Unpublished report No. Siss 826
         submitted by Ciba-Geigy

    Swann, H. E. & Cox, G. E. (1973) Unpublished report laboratory No.
         1223 submitted by Searle Biochemicals

    Willard, R. L. & Garvin, P. J. (1968) Unpublished report submitted by
         Travenol Laboratories Ltd


    See Also:
       Toxicological Abbreviations