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    CELLULASE FROM TRICHODERMA LONGIBRACHIATUM

    First draft prepared by
    Dr D.L. Grant and Dr E. Vasavour
    Health and Welfare Canada
    Ottawa, Ontario, Canada

    1.  EXPLANATION

          This substance was evaluated previously under the name
     Trichoderma reesei at the thirty-first meeting of the Joint
    FAO/WHO Expert Committee on Food Additives (Annex 1, reference 77).
    Since that time, the International Commission of the Taxonomy of
    Fungi has recommended that the source organism for this cellulase
    enzyme preparation be referred to as  Trichoderma longibrachiatum
    instead of  T. reesei. It should also be pointed out that the total
    organic solids (TOS) content of the commercial (31%), rather than
    that of the tested product (87.8%) was used to calculate the ADI in
    the previous review.

          Some additional studies on cellulase from  T. longibrachiatum
     (reesei) have been submitted and are presented in this monograph
    together with the previously-reviewed studies. A long-term study,
    which had been requested, has not been conducted.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

          No information available.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

        Table 1. Results of acute toxicity studies on cellulase from T. longibrachiatum
                                                                                       

    Species        Sex       Route     LD50        Reference
                                       (g/kg bw)
                                                                                       

    Mouse (NMRI)   M&F       oral     >16          Modeweg-Hansen,
                                                   1978a

    Rat (Wistar)   M&F       oral     >8           Modeweg-Hansen,
                                                   1978b

    Rat (Wistar)   M&F       oral     >10          Komulainen, 1988

    Dog            M&F       oral     >5           Osborne & Chambers,
                                                   1977
                                                                                       
    
    2.2  Short-term studies

    2.2.2.1  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,
    prostrate, 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).

          Groups of 20 male and 20 female CD rats, 6 weeks old, were fed
    carbohydrase from  T. longibrachiatum in the diet at concentrations
    of 0, 1, 2 or 5% for 13 weeks equal to a mean intake of 0, 670,
    1330, or 3350 mg/kg bw/day in the males and 0, 820, 1640, or
    4050 mg/kg bw/day in the females. Intakes were corrected for TOS. No
    treatment-related effect on mortality was reported. Dietary
    administration of the test material had no effect on body weight or
    food consumption in the female rats, but was associated with a 4.5%
    decrease in the body weight gain of the high-dose males which was
    not statistically significant. Measurement of haematology, clinical
    chemistry and urine pH at termination of the study indicated no
    effect of treatment except for a decrease in the neutrophil
    leucocyte count in the high-dose males which the authors stated was
    within the range commonly seen for this parameter (data were not
    provided to support this claim). Ophthalmological examinations at
    week 13 did not reveal any effect of treatment. At termination,
    increases in liver and kidney weights of mid- and high-dose males
    and high-dose females were noted. However, no histopathological
    changes were detected in the liver or kidneys, and thus the organ
    weight changes were considered to have been toxicologically
    insignificant (Weisenburger, 1990).

          Four groups of 10 male and 10 female CD rats, 3-4 weeks old,
    were fed 0, 1, 2 or 5% of cellulase preparation as TOS in the diet
    for 13 weeks corresponding to doses of 0, 815, 1620 or 4181 mg/kg
    bw/day in the males and 0, 933, 1775 or 4514 mg/kg bw/day in the
    females. No treatment-related effects were detected for body weight
    or food consumption during the course of the study. Measurement of
    haematological, clinical chemistry and urinalysis parameters at 13
    weeks did not indicate an effect of treatment. No compound-related
    gross or microscopic changes or organ weight differences were noted
    at the end of the study (Stewart, 1991).

    2.2.2.2  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 bw/day. Vomiting was reported after
    dosing in the high-dose groups during the first 3 to 4 weeks of the
    study, and diarrhoea 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 micro-scopic
    examinations of the principal organs and tissues showed no
    treatment-related effects (Osborne  et al., 1977).

    2.2.3  Long-term/carcinogenicity studies

          No information available.

    2.2.4  Reproduction studies

    2.2.4.1  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).

    2.2.5  Special study on teratogenicity

          Three groups of 6 pregnant CD rats each were dosed by gavage
    with 700, 2400, or 7000 mg/kg bw/day of the same preparation from
    days 6 to 16 to 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).

    2.2.6  Special studies on genotoxicity

        Table 2. Results of genotoxicity assays on cellulase from T. longibrachiatum
                                                                                               

    Test system       Test object          Concentration of        Results       Reference
                                           cellulose enzyme
                                           preparation
                                                                                               

    Ames test1        S. typhimurium       30-10 000 g/plate      negative      Crichton &
                      TA98, TA100,                                               McGregor, 1977
                      TA1535, TA1537,
                      TA1538

    Ames test1        S. typhimurium       100-10 000 g/plate     negative      Kiesvara &
                      TA98, TA100,                                               Tikkanen, 1987
                      TA1535, TA1537,
                      TA1538

    Ames test1        S. typhimurium       1.56-200 l/plate       positive      Nylund &
                      TA98, TA100,                                 (weak)        Linnainmaa,
                      TA1535                                                     1987

    Ames test1        S. typhimurium       8-5000 g/plate         negative      Asquith, 1989a
                      TA98, TA100,
                      TA1535, TA1537,
                      TA1538

    Chromosomal1      Chinese hamster      25-200 l/ml            negative      Norppa &
    aberration        ovary cells                                                Jarventaus,
                                                                                 1988

    Chromosomal1      human                50-10 000 g/ml         negative      Asquith, 1989b
    aberration        lymphocytes

    Chromosomal       bone                 0,1.0, 5.0              negative      McGregor, 1979
    aberration        marrow-Chinese       g/kg bw/day
                      hamsters
                                                                                               

    Table 2. Results of genotoxicity assays on cellulase from T. longibrachiatum
                                                                                               

    Test system       Test object          Concentration of        Results       Reference
                                           cellulose enzyme
                                           preparation
                                                                                               

    Dominant lethal   mouse                0, 0.5, 1.6,            negative      Cuthbert
                                           5.0 g/kg bw/day                       et al., 1980

    Mouse             mouse                0, 0.5, 1,              negative      Asquith,
    micro-nucleus                          1.5, 2.0 g/kg                         1989c
                                           bw/day
                                                                                               

    1 Both with and without rat liver S-9 fraction
    
    2.3  Observations in humans

          No information available

    3.  COMMENTS

          Cellulase derived from  Trichoderma longibrachiatum earlier
    referred to as  T. reesei was previously reviewed at the
    thirty-first meeting of the Committee (Annex 1, reference 77). The
    enzyme preparation is characterized by four primary enzyme
    activities: 1,4--d-glucan 4-glucanohydrolase (EC 3.2.1.4),
    1,4--d-glucan glucohydrolase (EC 3.2.1.74), 1,4--d-glucan
    cellubiohydrolase (EC 3.2.1.91), and 1,3--d-glucan
    3-glucanohydrolase (EC 3.2.1.6).

          At the thirty-first meeting, the Committee reviewed 13-week
    studies in rats and dogs, reproduction and teratogenicity studies in
    rats, and studies on the mutagenicity of the enzyme preparation. A
    temporary ADI of 0-0.3 mg TOS per kg of body weight was established
    by the application of a 2000-fold safety factor to the 2% NOEL
    (after conversion to TOS) in the 13-week study in rats. In
    accordance with Annex III of "Principles for the safety assessment
    of food additives and contaminants in food" (Annex 1, reference 76),
    the Committee requested a long-term study in a rodent species.
    Inadvertently, a TOS of 31% instead of 87.8% was used when
    converting the 2% NOEL for the enzyme test preparation to TOS; thus
    the temporary ADI was determined to be 0-0.3 mg TOS per kg of body
    weight, whereas the true figure should have been 0-0.85 TOS per kg
    of body weight.

    4.  EVALUATION

          At its present meeting, the Committee reviewed two additional
    13-week studies in rats in which no adverse effects were observed up
    to the highest dietary level (5%) tested. Since the potential intake
    was minimal and following a reconsideration of its guidelines for
    evaluating enzymes derived from miroorganisms, the Committee
    concluded that it had been provided with sufficient information to
    establish an ADI "not specified" when the preparation is used in
    accordance with good manufacturing practice.

    5.  REFERENCES

    ASQUITH, J.C. (1989a). Bacterial reverse mutation assay. Cellulase
    enzyme preparation derived from  Trichoderma longibrachiatum.
    Unpublished report from Toxicol Laboratories Ltd., Ledbury, England.
    Submitted to WHO by Genencor International, Helsinki, Finland.

    ASQUITH, J.C. (1989b). Metaphase analysis of human lymphocytes
    treated with cellulase enzyme preparation derived from  Trichoderma
     longibrachiatum. Unpublished report from Toxicol Laboratories
    Ltd., Ledbury, England. Submitted to WHO by Genencor International,
    Helsinki, Finland.

    ASQUITH, J.C. (1989c). Mouse micronucleus test. Cellulase enzyme
    preparation derived from  Trichoderma longibrachiatum. Unpublished
    report from Toxicol Laboratories Ltd., Ledbury, England. Submitted
    to WHO by Genencor International, Helsinki, Finland.

    BEN-DYKE, R., STRACHEN, E., KISS, I. & FINN, J.P. (1977) Cellulase:
    toxicity in dietary administration to rats 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 (SP-122). Unpublished IRI Project 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
    702021, report 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.

    KIESVAARA, M. & TIKKANEN, L. (1987). Ames mutagenicity test.
    Unpublished report from the Technical Research Centre of Finland,
    Espoo, Finland. Submitted to WHO by ALKO Ab, Helsinki, Finland.

    KOMULAINEN, H. (1988). Acute oral toxicity of the enzyme preparation
    Econase CEP in rats. Unpublished report from the National Public
    Health Institute, Kuopio, Finland. Submitted to WHO by ALKO Ab,
    Helsinki, Finland.

    McGREGOR, D.B. (1979). Cytogenetic study in Chinese hamsters of acid
    cellulase. Unpublished IRI Project 702042, report 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.

    NORPPA, H. & JARVENTAUS, H. (1990) Econase CE15:  In vitro test for
    the induction of chromosomal aberrations. Unpublished report from
    the Institute of Occupational Health, Helsinki, Finland. Submitted
    to WHO by ALKO Ab, Helsinki, Finland.

    NYLUND, L. & LINNAINMAA, K. (1987). Testing of two enzyme
    preparations labelled Econase CF15 and Econase EP431 using the
     Salmonella/mammalian microsome assay. Unpublished report from the
    Institute of Occupational Health, Helsinki, Finland. Submitted to
    WHO by ALKO Ab, Helsinki, Finland.

    OSBORNE, B.E. & CHAMBERS, P.R. (1977). Cellulase SP-122, acute oral
    toxicity study in dogs. Unpublished IRI Project 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. (19777) 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.

    STEWART, J.S. (1991). Cellulase: toxicity study by dietary
    administration to CD rats for 13 weeks. Unpublished report by Life
    Science Research Ltd., Suffolk, England. Submitted to WHO by
    Genencor International, Helsinki, Finland.

    WEISENBURGER, W.P. (1990). 13-week dietary toxicity study in rats.
    Unpublished report from International Research and Development
    Corporation, Mattawa, Michigan. Submitted to WHO by Genencor
    International, Helsinki, Finland.


    See Also:
       Toxicological Abbreviations