748. Cellulase derived from Trichoderma longibrachiatum (WHO Food Additives Series 30)

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 µg/plate negative Crichton &
TA98, TA100, McGregor, 1977
TA1535, TA1537,
TA1538

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

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

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

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

Chromosomal¹ human 50-10 000 µg/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-ß-d-glucan 4-glucanohydrolase (EC 3.2.1.4),
1,4-ß-d-glucan glucohydrolase (EC 3.2.1.74), 1,4-ß-d-glucan
cellubiohydrolase (EC 3.2.1.91), and 1,3-ß-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