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.