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    BETA-GLUCANASE FROM TRICHODERMA HARZIANUM

    EXPLANATION

         This enzyme preparation has not been evaluated previously by the
    Joint FAO/WHO Expert Comittee on Food Additives. The preparation used
    in the toxicological studies were obtained by spray drying the enzyme
    preparation; the preparation contained 80 - 95% TOS.

    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 and
    Escherichia coli WP 2uvra pmK 101 (CM891), both with and without
    metabolic activation (S-9 fraction). No dose-related increases in
    revertants were obtained at test levels up to 10 mg/ml of the
    incubation mixture (Pedersen, 1984).

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

         The enzyme preparation was not mutagenic in mouse lymphoma L5178Y
    cells at concentrations up to 3.5 mg/ml, with or without metabolic
    activation (McGregor & Riach, 1984).

    Special study on reproduction and teratogenicity

    Rats

         Groups of 8 pregnant Sprague-Dawley CD rats were used to
    establish the maternal embryonic maximum tolerated dose. The test
    animals were dosed daily from day 6 through day 16 of gestation and
    killed on day 20 of gestation; the number of corpora lutea
    graviditatis in each ovary and the number of and location of all
    implantations in the uterus were recorded. The maternal embryonic
    tolerated dose was established at 5 g/k.g, b.w. For the teratology
    study, four groups each containing 24 pregnant Sprague-Dawley rats,
    were dosed daily from day 6 through day 16 (day 17 for controls) with
    0, 1, 3, or 8 g/kg b.w. of the enzyme preparation. The rats were
    killed on day 20 of gestation. Maternal weight gain was reduced at all
    dose levels of the test compound, and this was accompanied by reduced
    food consumption in the high-dose group. There were slight reductions
    in mean litter fetal weight and in mean litter placental weight, which
    were dose-related. There were no significant dose-related trends in
    pregnancy data (number of corpora lutea, implantations, resorptions,
    or live fetuses) or in skeletal abnormalities. However, both the 3 and

    8 g/kg b.w. groups showed slight increases in the incidence of
    hydronephrosis, and the incidence of hydroureter was marginally
    increased in a dose-related manner. The incidences of these effects,
    although not significantly different from those in concurrent
    controls, exceeded the usual background in historical controls
    (Hazelden & Maddock, 1982).

    Acute toxicity

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

    Mouse (Novo)        oral      > 20              Novo, 1975
    Rat (Mollegard)     oral      > 10              Novo, 1982
                                                                        

    Short-term studies

    Rats

         Four groups of 15 male and 15 female Charles River CD rats, 4
    weeks of age, were maintained for 13 weeks on diet containing 0, 500,
    1,500, or 5,000 mg/kg b.w./day of the enzyme preparation. No
    compound-related deaths were reported. Food intake and body-weight
    gain were similar in test and control groups, with a tenancy for
    increased weight gain in females in the high-dose group during the
    last weeks of the test. Opthalmoscopic examinations at weeks 0 and 12
    showed no abnormalities. Haematologic, clinical chemistry, and
    standard urinalysis values were within normal ranges. However, urinary
    alkaline phosphatase levels were increased in male rats in the two
    high-dose level groups at week 12 of the study. Liver cytochrome P-450
    measurements showed no evidence of enzyme induction. At termination of
    the study, no compound-related changes were observed after organ
    weight analysis and gross and microscopic examination of the principal
    organs and tissues, with the exception of a slight increase in the
    incidence of inflammatory reactions in the kidney cortex in the
    high-dose groups (Warwick et al., 1976).

    Dogs

         Four groups of 3 male and 3 female dogs were dosed with the
    enzyme preparation by gavage once a day 7 days a week for 13 weeks at
    dose levels equivalent to 0, 300, 1000, or 3000 mg/kg b.w./day.
    Vomiting was reported in the high-dose group. No other clinical signs
    were observed. Decreased weight gain was observed in female dogs in
    the two high-dose groups. Although food consumption was also decreased
    in these groups, the reduced body weight in the high-dose group was

    greater than expected from the decreased food intake. Haematologic,
    clinical chemistry, and urinalysis values at weeks 6 and 12 showed no
    compound-related effects, with the exception of increased urinary
    alkaline phosphatase at week 12 of the study. At termination, gross
    necropsy, organ weight analysis, and microscopic examination of the
    principal organs and tissues showed no treatment-related effects.
    Liver cytochrome P-450 measurements showed no evidence of enzyme
    induction (Edwards et al., 1976).

    Long-term studies

    No information available.

    Observations in man

    No information available.

    COMMENTS

         The beta-glucanase preparation was not mutagenic in bacterial or
    in mammalian systems. The preparation caused no adverse effects in a
    reproduction study in rats at levels up to 5%, and it was not
    teratogenic in a rat study at doses up to 1 g/kg b.w./day. Short-term
    studies showed no adverse effects at 3 g/kg b.w./day in dogs or at
    2 g/kg b.w./day in rats. Based on the available information, the
    Committee established a temporary ADI for this enzyme preparation.

         Because this enzyme is derived from a microorganism that is
    neither a normal constituent of food nor a common contaminant in food,
    in accordance with Annex III of "Principles for the Safety Assessment
    of Food Additives and Contaminants in Food" (Annex 1, reference 76),
    this preparation requires the submission of results of a long-term
    study in a rodent species as well as specifications to show that the
    organism does not produce antibiotics and is non-pathogenic to man.

    EVALUATION

    Level causing no toxicological effect

    Rat:      1000 mg/kg b.w./day (teratogenicity study).

    Estimate of temporary acceptable daily intake

         0-0.5 mg TOS/kg b.w.

    Further work or information

    Required (by 1992)

         1. Long-term feeding study in a rodent species.

         2. Additional information to show that this organism does not
    produce antibiotics and is non-pathogenic to man.

    REFERENCES

    Edwards, D.B., Osborne, B.E., Kinch, D.A., & Dent, N.J. (1976).
    Mutanase toxicity study in beagle dogs (oral administration for 13
    weeks). Unpublished report No. 433 from Inveresk Research
    International, Musselburgh, Scotland. Submitted to WHO by Novo
    Industri A/S, Bagsvaerd, Denmark.

    Hazelden, K. & Maddock, S. (1982). Teratogenicity study in rats.
    Unpublished report No. 2253 from Inveresk Research International,
    Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    McGregor, D.B. & Holmstrom, L.M. (1981). Cytogenetic study in Chinese
    Hamsters of SP 116. Unpublished report No. 220B from Inveresk Research
    International, Musselburgh, Scotland. Submitted to WHO by Novo
    Industri A/S, Bagsvaerd, Denmark.

    McGregor, D.B. & Riach, C.G. (1984). SP 116 batch 1531, mouse lymphoma
    mutation assay. Unpublished report No. 2894 from Inveresk Research
    International, Musselburgh, Scotland. Submitted to WHO by Novo
    Industri A/S, Bagsvaerd, Denmark.

    Novo (1975). Acute oral toxicity of mutanase to mice. Unpublished
    report No. F-751886.1 from Novo Industri A/S, Bagsvaerd, Denmark.
    Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark.

    Novo (1982). Acute toxicity of SP 116 (Batch PPM 1216) given once
    orally to rats. Unpublished report No. 5181 from Novo Industri A/S,
    Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    Pedersen, P.B. (1984). Glucanex (batch No. PPM 1531), testing for
    mutagenic activity with Salmonella typhimurium and Escherichia
     coli WP 2uvrA (pK M 101) in liquid culture assay. Unpublished report
    No. E.0184 from Novo Industri A/S, Bagsvaerd, Denmark. Submitted to
    WHO by Novo Industri A/S, Bagsvaerd, Denmark.

    Warwick, M.H., Osborne, B.E., Collings, A.J., Kinch, D.A., & Dent, N.J.
    (1976). Mutanase toxicity study in rats (oral administration for 13
    weeks). Unpublished report No. 461 from Inveresk Research International,
    Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd,
    Denmark.
    


    See Also:
       Toxicological Abbreviations