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    ENZYMES DERIVED FROM ASPERGILLIS NIGER

    EXPLANATION

         A. niger is a contaminant of food and was not considered in the
    same light as those organisms regarded as normal constituents of food.
    It is necessary to show that the strains used in enzyme preparations
    do not produce mycotoxins.

         Microbial carbohydrases prepared from some varieties of
    A. niger were evaluated at the fifteenth meeting of the Committee,
    at which time a temporary ADI "not limited" was established (Annex 1,
    reference 26). A toxicological monograph was prepared (Annex 1,
    reference 27). An adequate 90-day study in rats was requested. Since
    the previous evaluation, additional data have become available on a
    number of carbohydrases, which are summarized and discussed in the
    following monograph. These enzymes were considered by the Committee to
    encompass the carbohydrases previously considered. The previously
    published monograph has been expanded and reproduced in its entirety
    below.

    AMYLOGLUCOSIDASES (E.C. 3.2.1.3)

    BIOLOGICAL DATA

    Biochemical aspects

    No information available.

    Toxicological studies

    Special studies on aflatoxin-related effects

    Ducklings

         Four groups of 5 ducklings received in their diet 0, 1, 5, or 10%
    enzyme preparation for 29 days. Growth, feed consumption, survival,
    behaviour, and mean liver weights were comparable, in all groups. No
    gross or histopathological lesions of the liver were seen
    (FDRL, 1963a).

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

    Acute toxicity1
                                                                        

    Species      Route      LD50                Reference
                            (mg/kg b.w.)
                                                                        

    Mouse        oral       > 3,200             Hunt & Garvin, 1963
                            > 4,000             Hunt & Garvin, 1971
                            > 3,200             Willard & Garvin, 1968
                            > 4,000             Garvin et al., 1966

    Rat          oral       10,000              Gray, 1960
                            31,600              Kay & Calendra, 1962
                            > 3,200             Willard & Garvin, 1968
                            > 4,000             Garvin et al., 1966
                            12,500 - 20,000     Kapiszka & Hartnage, 1978

    Rabbit       oral       > 4,000             Garvin et al., 1966

    Dog          oral       > 4,000             Garvin et al., 1966
                                                                        

    1    These data were obtained with several different commercial enzyme
         preparations.

    Short-term studies

    Rats

         Three groups of 10 male rats received 0, 0.5, or 5% enzyme
    preparation in their diets for 30 days. No adverse effects related to
    treatment were observed regarding growth, appearance, behaviour,
    survival, food consumption, haematology, organ weights, or gross
    pathology (Garvin et al., 1966).

         Two groups of 10 male and 10 female rats received either 0 or 5%
    enzyme preparation in their diets daily for 91 days. No differences
    from controls were observed regarding appearance, behaviour, survival,
    weight gain, haematology, organ weights, or gross pathology (Garvin &
    Merubia, 1959).

         Two groups of 10 male and 10 female ARS Sprague-Dawley rats were
    fed diets containing 5 or 10% of the test enzyme preparation
    (equivalent to 3.5 or 7 g enzyme preparation/kg b.w./day) for 90 to 94
    days. A control group of 20 male and 20 female rats were maintained on
    the diet alone. No signs of toxicity were observed during the test
    period. Body-weight gain and food consumption were similar between
    test and control groups. Differential blood counts were within the
    normal range at weeks 4 and 8 of the study in both test and control
    animals. At the end of the study serum clinical chemistry parameters,
    organ weight analyses, and gross and microscopic pathology showed no
    compound-related effects (Garvin et al, 1972).

    Long-term studies

         No information available.

    Observations in man

         No information available.

    COMMENTS

    Several short-term feeding studies  in rats on amyloglucosidase
    preparations from A. niger have been performed. One study, in which
    the preparation was fed at up to 10% of the diet, was considered to be
    acceptable by current standards. No compound-related effects were
    observed in this study or in duckling tests that were performed to
    investigate potential aflatoxin-related effects.

         The evaluations by the Committee of the carbohydrates and the
    protease from A. niger are summarized at the end of this section.

    REFERENCES

    FDRL (1963a). Unpublished report No. 84600e. Submitted to WHO by
    Laboratories, Inc., Elkhart, IN, USA.

    FDRL (1963b). Unpublished report No. 84600f. Submitted to WHO by
    Laboratories, Inc., Elkhart, IN, USA.

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

    Garvin, P.J., Willard, R., Merubia, J., Huszar, B., Chin, E., &
    Gilbert, C. (1966). Unpublished report. Submitted to WHO by Baxter
    Laboratories, Inc.

    Garvin, P.J., Ganote, C.E., Merubia, J., Delahany, E., Bowers, S.,
    Varnado, A., Jordan, L., Harley, G., DeSmet, C., & Porth, J. (1972).
    Unpublished report from Travenol Laboratories, Inc., Morton Grove,
    IL, USA. Submitted to WHO by Gist-brocades NV, Delft, Holland.

    Gray, E.H. (1960). Unpublished report. Submitted to WHO by Miles
    Laboratories, Inc., Elkhart, IN, USA.

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

    Hunt, R.F. & Garvin, P.J. (1971). Unpublished report. Submitted to WHO
    by Travenol Laboratories, Inc., Morton Grove, IL, USA.

    Kapiszka, E.L. & Hartnage, R.E. (1978). The acute oral toxicity of
    Diazyme concentrate and Diazyme 325 in the rat. Unpublished report
    No. 16 from Miles Laboratories, Inc., Elkhart, IN, USA. Submitted to
    WHO by Miles Laboratories, Inc., Elkhart, IN, USA.

    Kay, J.H. & Calendra, J.C. (1962). Unpublished report. Submitted to
    WHO by Miles Laboratories, Inc., Elkhart, IN, USA.

    Willard, R. & Garvin, P.J. (1968). Unpublished report. Submitted to
    WHO by Travenol Laboratories, Inc., Morton Grove, IL, USA.

    -GLUCANASE (E.C. 3.2.1.6)

    BIOLOGICAL DATA

    Biochemical aspects

         No information available.

    Toxicological studies

    (The TOS of the enzyme preparation used for toxicity studies was 49%).

    Special Studies on mutagenicity

         The enzyme preparation was tested for mutagenic activity using 5
    strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and
    TA1538 both with and without metabolic activation (S-9 fraction). The
    preparation was not mutagenic or toxic at concentrations up to
    40 mg/ml (McConville, 1980).

         A cytogenic bone marrow study was performed using adult male
    Chinese hamsters. Groups of adult male hamsters received up to
    5000 mg/kg b.w./day of the enzyme preparation for 5 consecutive days.
    Treatment did not result in an increased frequency of chromosomal
    aberrations in bone marrow (McGregor & Willins, 1981).

    Acute toxicity

                                                                        

    Species          Route      Sex       LD50             Reference
                                          (ml/kg b.w.)
                                                                        

    Mouse (NMRI)     oral       M & F     30               Novo, 1978a

    Rat (Wistar)     oral       -         28.1             Novo, 1978b
                                                                        

    Short-term studies

    Rats

         Three groups, each containing 5 male and 5 female Wistar/Mol SPF
    rats, were dosed orally by garage once a day for 14 days with enzyme
    preparation at dose levels equivalent to 2.5, 5.0, or 10 ml/kg b.w. No
    clinical changes were observed. Body-weight gains of test and control
    animals were similar. At termination of the study, measurements of
    organ weights showed no compound-related effects (Novo, 1978c).

         In another study, 4 groups, each containing 15 male and 15 female
    Wistar/Mol SPF rats, were dosed by gavage once a day for 90 days with
    enzyme preparation at dose levels equivalent to 0, 2.5, 5.0, or
    10 ml/kg b.w. Deaths, primarily in the high-dose group, appeared to be
    related to injury during dosing. No clinical signs were observed in
    the other test animals. Male rats in the high-dose group showed
    decreased weight gain and marked decrease in food intake. Haematology
    studies showed increased platelet counts and decreased clotting times
    in the high-dose group at week 6, but this effect was not apparent at
    week 12. No other effects were reported. Clinical chemistry and
    urinalysis values at weeks 6 and 12 were within the normal range. At
    termination of the study, organ weight analysis showed a marked
    increase in relative weights of the spleen and testes of the males in
    the high-dose group. Gross and histopathological examination of the
    principal organs and tissues showed no compound-related effects
    (Perry et al., 1979).

    Dogs

         Three groups, each containing one male and one female beagle dog,
    received single doses of 5, 10, or 15 ml/kg b.w. of the enzyme
    preparation over a 4-day period. Following a 7-day observation period
    the dogs were sacrificed and subjected to macroscopic post-mortem
    examination. No compound-related effects were observed, with the
    exception of vomiting during the first 4 days of the study. In another
    study, dogs were administered consecutive doses of 15 ml/kg b.w./day
    for 9 days, and 10 ml/kg b.w./day for 5 days. No deaths occurred
    during the course of the study. The only clinical sign noted was
    excessive salivation and emesis shortly after dosing. Body weights,
    electrocardiograms, haematological parameters, blood serum chemistry,
    organ weights, gross pathology, and histopathology showed no
    compound-related effects (Osborne et al., 1978).

         In another study, three groups, each containing 3 male and 3
    female beagle dogs, were dosed with the enzyme preparation by gavage
    once a day, seven days a week, for 13 weeks, at dose levels equivalent
    to 2, 5, or g ml/kg b.w./day. Two dogs in the high-dose group died
    during the course of the study, which the authors concluded was due to
    respiratory distress as a result of foreign material in the lungs.
    Vomiting was reported after dosing in the high-dose group.
    Haematological parameters at weeks 6 and 12 were within normal limits,
    with the exception of a significant increase in WBC count,
    specifically in the group mean neutrophil counts, in the high-dose
    group. Clinical chemistry values were within the normal range at weeks
    8 and 12, with the exception of slight increases in blood glucose and
    cholesterol in the high-dose group. Urinalysis showed no
    compound-related effects. At termination of the study, organ-weight
    analyses and gross and histopathological examination of the principal
    organs and tissues showed no compound-related effects (Greenough
    et al., 1980).

    Long-term studies

         No information available.

    Observations in man

         No information available.

    COMMENTS

         This enzyme preparation was not genotoxic in microbial or in
    mammalian test systems. Short-term studies in rats and dogs resulted
    in no observed compound-related effects at levels up to 5 ml/kg
    b.w./day of enzyme preparation.

         The evaluations by the Committee of the carbohydrases and the
    protease from A. niger are summarized at the end of this section.

    REFERENCES

    Greenough, R.J., Brown, J.C., Brown, M.G., Cowie, J.R., Maule, W.J., &
    Atken, R. (1980). -Glucanase 13 week oral toxicity study in dogs.
    Unpublished report No. 1630 from Inveresk Research International,
    Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    McConville, M. (1980). Testing for mutagenic activity with
    S. typhimurium strain TA98, TA100, TA1535, TA1537, and TA1538 of
    fungal -glucanase. Unpublished report No. 1751 from Inveresk Research
    International, Musselburgh, Scotland. Submitted to WHO by
    Novo Industri A/S, Bagsvaerd, Denmark.

    MGregor, D.B. & Willins, M.J. (1981). Cytogenic study in Chinese
    hamsters of fungal -glucanase. Unpublished report No. 2023 from
    Inveresk Research International, Musselburgh, Scotland. Submitted to
    WHO by Novo Industri A/S, Bagsvaerd, Denmark.

    Novo (1978a). Acute oral toxicity of -glucanase given to mice.
    Unpublished report No. 1978-06-30 RKH/PNi from Novo Industri A/S,
    Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    Novo (1978b). Acute oral toxicity of -glucanase given to rats.
    Unpublished report No. 1978-07-17 RICH/PNi from Novo Industri A/S,
    Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    Novo (1978c). Oral toxicity of -glucanase given daily to rats for 14
    days. Unpublished report No. 1978-08-21 RKH/PNi from Novo Industri
    A/S, Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    Osborne, B.E., Cockrill, J.B., Cowie, J.R., Maule, W., & Whitney, J.C.
    (1978). Beta-glucanase, dog acute and maximum tolerated dose study.
    Unpublished report No. 1208 from Inveresk Research International,
    Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    Perry, C.J., Everett, D.J., Cowie, J.R., Maule, W.J. & Spencer, A.
    (1979). -glucanase toxicity study in rats (oral administration by
    gavage for 90 days). Unpublished report No. 1310 from Inveresk
    Research International, Musselburgh, Scotland. Submitted to WHO by
    Novo Industri A/S, Bagsvaerd, Denmark.

    HEMI-CELLULASE

    BIOLOGICAL DATA

    Biochemical aspects

         No information available.

    Toxicological studies

    Special studies on mutagenicity

         The enzyme preparation was tested for mutagenic activity using
    Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 both
    with and without metabolic activation (S-9 fraction). The test
    substance was not mutagenic or toxic at concentrations up to
    5 mg/plate (Clausen & Kaufman, 1983).

         In an in vitro cytogenetic test using CHO-K1 cells, both with
    and without metabolic activation (S-9 fraction), the enzyme
    preparation at test levels up to 2.5 mg (dry wt)/ml did not induce
    chromosomal aberrations (Skovbro, 1984).

    Acute toxicity

         No information available.

    Short-term studies

    Rats

         Four groups, each containing 5 male and 5 female Wistar MOL/W
    rats, were dosed by garage once a day for 90 days with the enzyme
    preparation at doses equivalent to 0, 100, 333, or 1000 mg/kg
    b.w./day. No significant clinical changes were observed. Body-weight
    gain and food intake were similar among test and control animals.
    Haematologic and clinical chemistry measurements at termination of the
    study were within normal ranges. Post-mortem examinations,
    measurements of organ weights, and histopathology showed no
    compound-related effects. Slight increases in kidney and adrenal
    weights in the mid-dose group were not associated with
    histopathological effects, and did not show a dose response
    (Kallesen, 1982).

    Long-term studies

         No information available.

    Observations in man

         No information available.

    COMMENTS

         This enzyme preparation was not genotoxic in microbial or in
    mammalian test systems. In a limited 90-day study in rats, no effects
    were observed at the highest dose administered (1 g/kg b.w./day). This
    enzyme preparation contained high levels of pectinase. The pectinase
    enzyme preparation summarized below may be identical to this
    hemi-cellulase preparation, which provides added assurance of the
    safety of this preparation.

         The evaluations by the Committee of the carbohydrases and the
    protease from A. niger are summarized at the end of this section.

    REFERENCES

    Clausen, B. & Kaufman, U. (1983). Unpublished report from Obmutat
    Laboratiet. Submitted to WHO by Grinsted Products A/S,
    Brabrand, Denmark.

    Kallesen, T. (1982). A 90-day toxicity study. Unpublished report
    No. 10023 from Scantox Biological Laboratory Ltd., Denmark. Submitted
    to WHO by Grinsted Products A/S, Brabrand, Denmark.

    Skovbro, A. (1984). In vitro mammalian cytogenetic test (according
    to OECD Guideline No. 473). Unpublished report No. 10398 from Scantox
    Biological Laboratory Ltd., Denmark. Submitted to WHO by Grinsted
    Products A/S, Brabrand, Denmark.

    PECTINASE (E.C. 3.1.1.11; 3.2.1.15; 4.2.2.10)

    BIOLOGICAL DATA

    Biochemical aspects

    No information available.

    Toxicological studies (The TOS of the commercial
    preparation is approximately 5%).

    Acute toxicity
                                                                        

    Species     Route       LD50              Reference
                            (ml/kg b.w.)
                                                                        

    Rat         oral        18.8-22.1         Porter & Hartnagel, 1979
                                                                        

    Short-term studies

    Rats

         Two groups of 10 male and 10 female ARS Sprague-Dawley rats were
    fed diets containing 5 or 10% of the test enzyme preparation
    (equivalent to 3.5 or 7 g of the enzyme preparation/kg b.w./day), for
    90 to 94 days. A control group of 20 male and 20 female rats was
    maintained on the diet alone. No signs of toxicity were observed
    during the test period. Body-weight gain and food consumption were
    similar among test and control groups. Differential blood counts at
    weeks 4 and 8 of the study were within the normal range in test and
    control animals. At the end of the study serum clinical chemistry
    analyses, organ weight analyses, and gross and microscopic pathology
    showed no compound-related effects (Garvin et al., 1972).

    Long-term studies

    No information available.

    Observations in man

    No information available.

    COMMENTS

         In a short-term study in rats, no adverse effects were observed
    at dietary levels of the enzyme preparation up to the equivalent of
    7 mg/kg b.w./day. This enzyme preparation may be identical to the
    hemi-cellulase preparation summarized above. The hemi-cellulase enzyme
    preparation summarized above also contained high levels of pectinase,
    which provides added assurance of the safety of this preparation.

    REFERENCES

    Garvin, P.J., Ganore, C.E., Merubia, J., Delahany, E., Bowers, S.,
    Varnado, A., Jordan, L., Harley, G., DeSmet, C., & Porth, J. (1972).
    Carbohydrase from Aspergillus niger (pectinase, cellulase aud
    lactase). Unpublished report from Travenol Laboratories, Inc., Horton
    Grove, IL, USA. Submitted to WHO by Gist-brocades NV, Delft, Holland.

    Porter, M.C. & Hartnagel R.E. (1979). The acute oral toxicity of a new
    pectinase product in the rat. Unpublished report No. 11 from Miles
    Laboratories, Inc., Elkhart, IN, USA. Submitted to WHO by Enzyme
    Technical Association, Washington, DC, USA.

    PROTEASE

    No information available.

    GENERAL COMMENTS ON ENZYMES FROM A. NIGER

         Aspergillus niger is a contaminant of food. Although there may
    be posible strain differences in A. niger, and different cultural
    conditions might be used to prepare the various enzymes, the available
    toxicity data, which consist primarily of short-term feeding studies
    in rats and some studies in dogs, show that all the enzyme
    preparations tested were of a very low order of toxicity. The enzyme
    preparations tested were non-mutagenic in bacterial and mammalian cell
    systems. Studies on some strains of A. niger used to prepare
    carbohydrases showed no aflatoxin or related substance production.
    These studies provide the basis for evaluating the safety of enzyme
    preparations derived from A. niger. It was also noted that the
    enzyme preparations tested exhibit a number of enzyme activities, in
    addition to the major enzyme activity. Thus, there may be considerable
    overlap of the enzyme activities of the different enzyme preparations
    so that safety data from each preparation provides additional
    assurance of safety for the whole group of enzymes.

         Since the enzyme preparations tested were of different activities
    and forms, and most of the organic materials in the preparations are
    not the enzyme per se, the numerical ADI is expressed in terms of
    total organic solids (TOS) (see introduction to enzyme preparations
    section).

    EVALUATION

    Level causing no toxicological effect

         All enzyme preparations tested showed no-observed-effect levels
    greater than 100 mg TOS/kg b.w./day in 90-day studies in rats.

    Estimate of acceptable daily intake

         0-1 mg TOS/kg b.w. for each of the enzyme preparations.

    


    See Also:
       Toxicological Abbreviations