IPCS INCHEM Home


    CELLULASE FROM TRICHODERMA REESEI

    EXPLANATION

         This substance has not been evaluated previously by the Joint
    FAO/WHO Expert Committee on Food Additives. Cellulase is produced
    extracellularly by T. reesei (QM6a), a mutant of T. viride. The
    enzyme preparation is characterized by two activities,
    exo-cellobiohydrolase (E.C. 3.2.1.1) and 1,4-endoglucanase
    (E.C. 3.2.1.4).

         Tests have been performed to show that the strain of T. reesei
    used for the production of this enzyme preparation does not produce
    any antibiotics, and it can be regarded as non-pathogenic.

         The enzyme preparation used in the toxicological studies was a
    spray dried product derived from a cruder preparation than the
    commercial product. The TOS of the tested product was 31%.

         The available safety data have been summarized by Hjortkjer
    et al., 1986.

    BIOLOGICAL DATA

    Biochemical aspects

    No information available.

    Toxicological studies

    Special studies on mutagenicity

         The mutagenicity of the enzyme preparation was tested using
    Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and
    TA1538, both with and without metabolic activation by 5-9 fraction
    from rat liver. No mutagenic effects were observed at concentrations
    up to 10,0 mg/plate (Crichton & McGregor, 1977).

         A cytogenetic 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 cellulase preparation for 5 consecutive
    days. The cellulase preparation did not produce an increase in
    chromosome aberrations in bone marrow cells (McGregor, 1979).

         In a dominant lethal assay, adult male CD-1 mice were dosed
    orally by gavage for 5 consecutive days at dose levels up to
    5000 mg/kg b.w./day. No treatment-related effects were observed on
    implantation or fetal deaths (Cuthbert et al., 1980).

    Special study on reproduction

    Rats

         Four groups, each containing 20 male and 20 female 6-week old CD
    rats, were maintained on diets containing 0, 1, 2, or 5% of the enzyme
    preparation. The test diet was fed for 10 weeks prior to breeding and
    throughout mating, gestation, and lactation. Weanlings were maintained
    on the same diet as parents, until autopsied at 28 days of age.

         Parameters evaluated included body weights, feed consumption of
    F0 and F1 animals, and reproduction parameters (fertility index,
    gestation index, live birth index, litter size, viability index, and
    lactation index). Gross necropsies were conducted on all F0 and F1
    animals, except pups dying before day 12 of lactation. Organ weights
    from 10 male and 10 female F1 animals from each treatment group were
    measured, and the principal tissues and organs from a similar number
    of F1 animals from the high-dose and control groups were
    microscopically examined. Clinical chemistry, haematology, and
    urinalysis were not performed. Compound-related mortality was not
    reported in the F0 generation. There were no treatment-related

    clinical signs. Body weights of males in the high-dose group were
    lower than those of controls, which was associated with decreased food
    intake. There were no treatment-related effects on reproductive
    parameters. In the F1 generation, there was a trend to increased
    mean body weights during the early period of lactation, but this
    effect was not significant toward the end of the treatment period. No
    significant treatment-related effects on absolute or relative organ
    weights in F1 males and females were observed, and no
    treatment-related gross or microscopic adverse effects were reported
    (Hazelden et al., 1982).

    Special study on teratogenicity

         Three groups of 6 pregnant CD rats each were dosed by gavage with
    700, 2400, or 7000 mg/kg b.w./day of the enzyme preparation from days
    6 to 16 of gestation. The rats were killed on day 20 of gestation.
    Reduced body-weight gain was observed in the high-dose group, which
    was associated with decreased food consumption during the dosing
    period. No compound-related differences were observed in placental
    weights, number of corpora lutea, implantations, resorptions, or live
    fetuses. The small number of visceral and skeletal abnormalities
    showed no treatment associations or trends (Hazelden & Everett, 1980).

    Acute toxicity
                                                                        

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

    Mouse (NMRI)    oral      16               Modeweg-Hansen, 1978a
    Rat (Wistar)    oral      8                Modeweg-Hansen, 1978b
    Dog             oral      5                Osborne & Chambers, 1977
                                                                        

    Short-term studies

    Rats

         Four groups, each containing 15 male and 15 female CD rats 4
    weeks of age, were maintained for 13 weeks on diets containing 0, 1,
    2, or 5% of the enzyme preparation. Abnormal grooming in the high-dose
    groups was observed during the first 8 weeks of the study. Decreased
    weight gain from weeks 4 to 10 was observed in the high-dose group.
    Blood urea values were elevated in treated animals at weeks 4 and 13,
    but a consistent dose-response effect was not observed. There were no
    other compound-related effects on clinical chemistry or haematological
    measurements. No compound-related deaths were reported. At autopsy,
    male rats in the high-dose group had a significant increase in
    organ-to-body-weight ratios for the liver, prostate, and kidney when

    compared to control values, and females in the high-dose group had
    significantly higher spleen-to-body-weight ratios than controls. No
    compound-related gross or microscopic changes were observed, except in
    the case of the kidney of some rats in the high-dose group, where
    there was a small increase in the size of proteinaceous globules in
    the epithelial cells lining the renal convulated tubules
    (Ben-Dyke et al., 1977).

    Dogs

         Four groups, each containing 3 male and 3 female dogs, were dosed
    by gavage once a day, seven days a week, for 13 weeks with the enzyme
    preparation (30% dispersion in water) at doses equivalent to 0, 750,
    1500, or 3000 mg/kg b.w./day. Vomiting was reported after dosing in
    the high-dose groups during the first 3 to 4 weeks of the study, and
    diarrhea was observed in these groups during the first two weeks of
    the study. There were no significant differences in body weight and
    food consumption between dosed and control animals during the course
    of the study. Haematology, clinical chemistry, and urinalysis at weeks
    6 and 12 of the study showed no treatment-related effects.
    Ophthalmoscopic examinations at weeks 6 and 12 showed no
    treatment-related effects. At termination of the study, gross
    necropsies, organ weight analyses, and microscopic examinations of the
    principal organs and tissues showed no treatment-related effects
    (Osborne et al., 1977).

    Long-term studies

         No information available.

    Observations in man

         No information available.

    COMMENTS

         This cellulase preparation from T. reesei 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% in the
    diet, and it was not teratogenic in a rat study at doses up to 7 g/kg
    b.w./day. Short-term studies are available in dogs and rats, the
    no-adverse-effect levels being 3 g/kg b.w./day in dogs and 2 g/kg
    b.w./day in rats. The Committee was also informed that tests have been
    performed to show that the strain of T. reesei used for the
    production of this enzyme does not produce any antibiotics and it is
    not known to be a human pathogen. 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 the results of a long-term
    study in a rodent species.

    EVALUATION

    Level causing no toxicological effect

    Rat:      20,000 ppm in the diet, equivalent to 2000 mg/kg b.w./day
              (600 mg TOS/kg b.w./day).

    Estimate of temporary acceptable daily intake for man

    0-0.3 mg TOS/kg b.w.

    Further work or information

    Required (by 1992)

         Long-term feeding study in a rodent species.

    REFERENCES

    Ben-Dyke, R., Strachen, E., Kiss, I., & Finn, J.P. (1977). Cellulase:
    toxicity in dietary administration to rata for thirteen weeks.
    Unpublished report No. 77/NTL-33/382 from Life Science Research,
    Stock, England. Submitted to WHO by Novo Industri A/S, Bagsvaerd,
    Denmark.

    Crichton, C., & McGregor, D.B. (1977). Testing for mutagenic activity
    in cellulase(R) (SP-122). Unpublished IRI project No. 40849 from
    Inveresk Research International, Edinburgh, Scotland. Submitted to WHO
    by Novo Industri A/S, Bagsvaerd, Denmark.

    Cuthbert, J.A., McGregor, D.B., & Willins, M.J. (1980). Dominant
    lethal study in mice of acid cellulase. Unpublished IRI project
    No. 702021, report No. 1699, from Inveresk Research International,
    Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S,
    Bagsvaerd, Denmark.

    Hazelden, K.P. & Everett, A. (1980). Teratogenicity testing in rats of
    acid cellulase. Unpublished IRI project No. 702016 from Inveresk
    Research International, Edinburgh, Scotland. Submitted to WHO by Novo
    Industri A/S, Bagsvaerd, Denmark.

    Hazelden, K.P., Maddock, S.M., & Rushton, A.K.A. (1982). Acid
    cellulose dietary toxicity study in rats with in utero exposure.
    Unpublished IRI project No. 704851, report No. 2350, from Inveresk
    Research International, Musselburgh, Scotland. Submitted to WHO by
    Novo Industri A/S, Bagsvaerd, Denmark.

    Hjortkjer, R.K., Bille-Hansen, V., Hazelden, K.P., McConville, M.,
    McGregor, D.B., Cuthbert, J.A., Greenough, R.J., Chapman, E., Gardner,
    J.R., & Ashby, R. (1966). Safety of celluclast(R), an acid cellulase
    derived from Trichoderma reesei. Fd. Chem. Tox., 24, 53-63.

    McGregor, D.B. (1979). Cytogenetic study in chinese hamsters of acid
    cellulase. Unpublished IRI project No. 702042, report No. 1585, from
    Inveresk Research International, Edinburgh, Scotland. Submitted to WHO
    by Novo Industri A/S, Bagsvaerd, Denmark.

    Modeweg-Hansen, L. (1978a). Acute oral toxicity of cellulase SP-122 to
    rats. Unpublished report from Novo Industri A/S. Submitted to WHO by
    Novo Industri, Bagsvaerd, Denmark.

    Modeweg-Hansen, L. (1978b). Acute oral toxicity of cellulase SP-122 to
    mice. Unpublished report from Novo Industri A/S. Submitted to WHO by
    Novo Industri, Bagsvaerd, Denmark.

    Osborne, B.E. & Chambers, P.R. (1977). Cellulase SF-122, acute oral
    toxicity study in dogs. Unpublished IRI project No. 408473 from
    Inveresk Research International, Edinburgh, Scotland. Submitted to WHO
    by Novo Industri A/S, Bagsvaerd, Denmark.

    Osborne, B.E., Rushton, A.K.A., & Dent, N.J. (1977). Cellulase SP-122,
    toxicity study in beagle dogs (oral administration for 13 weeks).
    Unpublished IRI project No. 408489, report No. 919, from Inveresk
    Research International, Edinburgh, Scotland. Submitted to WHO by Novo
    Industri A/S, Bagsvaerd, Denmark.
    


    See Also:
       Toxicological Abbreviations