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