ENZYMES DERIVED FROM ASPERGILLIS NIGER
EXPLANATION
A. niger is a contaminant of food and was not considered in the
same light as those organisms regarded as normal constituents of food.
It is necessary to show that the strains used in enzyme preparations
do not produce mycotoxins.
Microbial carbohydrases prepared from some varieties of
A. niger were evaluated at the fifteenth meeting of the Committee,
at which time a temporary ADI "not limited" was established (Annex 1,
reference 26). A toxicological monograph was prepared (Annex 1,
reference 27). An adequate 90-day study in rats was requested. Since
the previous evaluation, additional data have become available on a
number of carbohydrases, which are summarized and discussed in the
following monograph. These enzymes were considered by the Committee to
encompass the carbohydrases previously considered. The previously
published monograph has been expanded and reproduced in its entirety
below.
AMYLOGLUCOSIDASES (E.C. 3.2.1.3)
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
Special studies on aflatoxin-related effects
Ducklings
Four groups of 5 ducklings received in their diet 0, 1, 5, or 10%
enzyme preparation for 29 days. Growth, feed consumption, survival,
behaviour, and mean liver weights were comparable, in all groups. No
gross or histopathological lesions of the liver were seen
(FDRL, 1963a).
Four groups of 5 ducklings received in their diet 0, 1, 5, or 10%
enzyme preparation for 29 days. Growth, feed consumption, survival,
behaviour, and development were comparable in all groups. No gross
liver lesions were seen at autopsy and mean liver weights of treated
animals were similar to those of controls. Histopathology of the
livers was normal. No toxic elements were noted (FDRL, 1963b).
Acute toxicity1
Species Route LD50 Reference
(mg/kg b.w.)
Mouse oral > 3,200 Hunt & Garvin, 1963
> 4,000 Hunt & Garvin, 1971
> 3,200 Willard & Garvin, 1968
> 4,000 Garvin et al., 1966
Rat oral 10,000 Gray, 1960
31,600 Kay & Calendra, 1962
> 3,200 Willard & Garvin, 1968
> 4,000 Garvin et al., 1966
12,500 - 20,000 Kapiszka & Hartnage, 1978
Rabbit oral > 4,000 Garvin et al., 1966
Dog oral > 4,000 Garvin et al., 1966
1 These data were obtained with several different commercial enzyme
preparations.
Short-term studies
Rats
Three groups of 10 male rats received 0, 0.5, or 5% enzyme
preparation in their diets for 30 days. No adverse effects related to
treatment were observed regarding growth, appearance, behaviour,
survival, food consumption, haematology, organ weights, or gross
pathology (Garvin et al., 1966).
Two groups of 10 male and 10 female rats received either 0 or 5%
enzyme preparation in their diets daily for 91 days. No differences
from controls were observed regarding appearance, behaviour, survival,
weight gain, haematology, organ weights, or gross pathology (Garvin &
Merubia, 1959).
Two groups of 10 male and 10 female ARS Sprague-Dawley rats were
fed diets containing 5 or 10% of the test enzyme preparation
(equivalent to 3.5 or 7 g enzyme preparation/kg b.w./day) for 90 to 94
days. A control group of 20 male and 20 female rats were maintained on
the diet alone. No signs of toxicity were observed during the test
period. Body-weight gain and food consumption were similar between
test and control groups. Differential blood counts were within the
normal range at weeks 4 and 8 of the study in both test and control
animals. At the end of the study serum clinical chemistry parameters,
organ weight analyses, and gross and microscopic pathology showed no
compound-related effects (Garvin et al, 1972).
Long-term studies
No information available.
Observations in man
No information available.
COMMENTS
Several short-term feeding studies in rats on amyloglucosidase
preparations from A. niger have been performed. One study, in which
the preparation was fed at up to 10% of the diet, was considered to be
acceptable by current standards. No compound-related effects were
observed in this study or in duckling tests that were performed to
investigate potential aflatoxin-related effects.
The evaluations by the Committee of the carbohydrates and the
protease from A. niger are summarized at the end of this section.
REFERENCES
FDRL (1963a). Unpublished report No. 84600e. Submitted to WHO by
Laboratories, Inc., Elkhart, IN, USA.
FDRL (1963b). Unpublished report No. 84600f. Submitted to WHO by
Laboratories, Inc., Elkhart, IN, USA.
Garvin, P.J. & Merubia, J. (1959). Unpublished report. Submitted to
WHO by Baxter Laboratories, Inc.
Garvin, P.J., Willard, R., Merubia, J., Huszar, B., Chin, E., &
Gilbert, C. (1966). Unpublished report. Submitted to WHO by Baxter
Laboratories, Inc.
Garvin, P.J., Ganote, C.E., Merubia, J., Delahany, E., Bowers, S.,
Varnado, A., Jordan, L., Harley, G., DeSmet, C., & Porth, J. (1972).
Unpublished report from Travenol Laboratories, Inc., Morton Grove,
IL, USA. Submitted to WHO by Gist-brocades NV, Delft, Holland.
Gray, E.H. (1960). Unpublished report. Submitted to WHO by Miles
Laboratories, Inc., Elkhart, IN, USA.
Hunt, R.F. & Garvin, P.J. (1963). Unpublished report. Submitted to WHO
by Baxter Laboratories, Inc.
Hunt, R.F. & Garvin, P.J. (1971). Unpublished report. Submitted to WHO
by Travenol Laboratories, Inc., Morton Grove, IL, USA.
Kapiszka, E.L. & Hartnage, R.E. (1978). The acute oral toxicity of
Diazyme concentrate and Diazyme 325 in the rat. Unpublished report
No. 16 from Miles Laboratories, Inc., Elkhart, IN, USA. Submitted to
WHO by Miles Laboratories, Inc., Elkhart, IN, USA.
Kay, J.H. & Calendra, J.C. (1962). Unpublished report. Submitted to
WHO by Miles Laboratories, Inc., Elkhart, IN, USA.
Willard, R. & Garvin, P.J. (1968). Unpublished report. Submitted to
WHO by Travenol Laboratories, Inc., Morton Grove, IL, USA.
ß-GLUCANASE (E.C. 3.2.1.6)
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
(The TOS of the enzyme preparation used for toxicity studies was 49%).
Special Studies on mutagenicity
The enzyme preparation was tested for mutagenic activity using 5
strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and
TA1538 both with and without metabolic activation (S-9 fraction). The
preparation was not mutagenic or toxic at concentrations up to
40 mg/ml (McConville, 1980).
A cytogenic 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 enzyme preparation for 5 consecutive days.
Treatment did not result in an increased frequency of chromosomal
aberrations in bone marrow (McGregor & Willins, 1981).
Acute toxicity
Species Route Sex LD50 Reference
(ml/kg b.w.)
Mouse (NMRI) oral M & F 30 Novo, 1978a
Rat (Wistar) oral - 28.1 Novo, 1978b
Short-term studies
Rats
Three groups, each containing 5 male and 5 female Wistar/Mol SPF
rats, were dosed orally by garage once a day for 14 days with enzyme
preparation at dose levels equivalent to 2.5, 5.0, or 10 ml/kg b.w. No
clinical changes were observed. Body-weight gains of test and control
animals were similar. At termination of the study, measurements of
organ weights showed no compound-related effects (Novo, 1978c).
In another study, 4 groups, each containing 15 male and 15 female
Wistar/Mol SPF rats, were dosed by gavage once a day for 90 days with
enzyme preparation at dose levels equivalent to 0, 2.5, 5.0, or
10 ml/kg b.w. Deaths, primarily in the high-dose group, appeared to be
related to injury during dosing. No clinical signs were observed in
the other test animals. Male rats in the high-dose group showed
decreased weight gain and marked decrease in food intake. Haematology
studies showed increased platelet counts and decreased clotting times
in the high-dose group at week 6, but this effect was not apparent at
week 12. No other effects were reported. Clinical chemistry and
urinalysis values at weeks 6 and 12 were within the normal range. At
termination of the study, organ weight analysis showed a marked
increase in relative weights of the spleen and testes of the males in
the high-dose group. Gross and histopathological examination of the
principal organs and tissues showed no compound-related effects
(Perry et al., 1979).
Dogs
Three groups, each containing one male and one female beagle dog,
received single doses of 5, 10, or 15 ml/kg b.w. of the enzyme
preparation over a 4-day period. Following a 7-day observation period
the dogs were sacrificed and subjected to macroscopic post-mortem
examination. No compound-related effects were observed, with the
exception of vomiting during the first 4 days of the study. In another
study, dogs were administered consecutive doses of 15 ml/kg b.w./day
for 9 days, and 10 ml/kg b.w./day for 5 days. No deaths occurred
during the course of the study. The only clinical sign noted was
excessive salivation and emesis shortly after dosing. Body weights,
electrocardiograms, haematological parameters, blood serum chemistry,
organ weights, gross pathology, and histopathology showed no
compound-related effects (Osborne et al., 1978).
In another study, three groups, each containing 3 male and 3
female beagle dogs, were dosed with the enzyme preparation by gavage
once a day, seven days a week, for 13 weeks, at dose levels equivalent
to 2, 5, or g ml/kg b.w./day. Two dogs in the high-dose group died
during the course of the study, which the authors concluded was due to
respiratory distress as a result of foreign material in the lungs.
Vomiting was reported after dosing in the high-dose group.
Haematological parameters at weeks 6 and 12 were within normal limits,
with the exception of a significant increase in WBC count,
specifically in the group mean neutrophil counts, in the high-dose
group. Clinical chemistry values were within the normal range at weeks
8 and 12, with the exception of slight increases in blood glucose and
cholesterol in the high-dose group. Urinalysis showed no
compound-related effects. At termination of the study, organ-weight
analyses and gross and histopathological examination of the principal
organs and tissues showed no compound-related effects (Greenough
et al., 1980).
Long-term studies
No information available.
Observations in man
No information available.
COMMENTS
This enzyme preparation was not genotoxic in microbial or in
mammalian test systems. Short-term studies in rats and dogs resulted
in no observed compound-related effects at levels up to 5 ml/kg
b.w./day of enzyme preparation.
The evaluations by the Committee of the carbohydrases and the
protease from A. niger are summarized at the end of this section.
REFERENCES
Greenough, R.J., Brown, J.C., Brown, M.G., Cowie, J.R., Maule, W.J., &
Atken, R. (1980). ß-Glucanase 13 week oral toxicity study in dogs.
Unpublished report No. 1630 from Inveresk Research International,
Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S,
Bagsvaerd, Denmark.
McConville, M. (1980). Testing for mutagenic activity with
S. typhimurium strain TA98, TA100, TA1535, TA1537, and TA1538 of
fungal ß-glucanase. Unpublished report No. 1751 from Inveresk Research
International, Musselburgh, Scotland. Submitted to WHO by
Novo Industri A/S, Bagsvaerd, Denmark.
MGregor, D.B. & Willins, M.J. (1981). Cytogenic study in Chinese
hamsters of fungal ß-glucanase. Unpublished report No. 2023 from
Inveresk Research International, Musselburgh, Scotland. Submitted to
WHO by Novo Industri A/S, Bagsvaerd, Denmark.
Novo (1978a). Acute oral toxicity of ß-glucanase given to mice.
Unpublished report No. 1978-06-30 RKH/PNi from Novo Industri A/S,
Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
Bagsvaerd, Denmark.
Novo (1978b). Acute oral toxicity of ß-glucanase given to rats.
Unpublished report No. 1978-07-17 RICH/PNi from Novo Industri A/S,
Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
Bagsvaerd, Denmark.
Novo (1978c). Oral toxicity of ß-glucanase given daily to rats for 14
days. Unpublished report No. 1978-08-21 RKH/PNi from Novo Industri
A/S, Bagsvaerd, Denmark. Submitted to WHO by Novo Industri A/S,
Bagsvaerd, Denmark.
Osborne, B.E., Cockrill, J.B., Cowie, J.R., Maule, W., & Whitney, J.C.
(1978). Beta-glucanase, dog acute and maximum tolerated dose study.
Unpublished report No. 1208 from Inveresk Research International,
Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S,
Bagsvaerd, Denmark.
Perry, C.J., Everett, D.J., Cowie, J.R., Maule, W.J. & Spencer, A.
(1979). ß-glucanase toxicity study in rats (oral administration by
gavage for 90 days). Unpublished report No. 1310 from Inveresk
Research International, Musselburgh, Scotland. Submitted to WHO by
Novo Industri A/S, Bagsvaerd, Denmark.
HEMI-CELLULASE
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 both
with and without metabolic activation (S-9 fraction). The test
substance was not mutagenic or toxic at concentrations up to
5 mg/plate (Clausen & Kaufman, 1983).
In an in vitro cytogenetic test using CHO-K1 cells, both with
and without metabolic activation (S-9 fraction), the enzyme
preparation at test levels up to 2.5 mg (dry wt)/ml did not induce
chromosomal aberrations (Skovbro, 1984).
Acute toxicity
No information available.
Short-term studies
Rats
Four groups, each containing 5 male and 5 female Wistar MOL/W
rats, were dosed by garage once a day for 90 days with the enzyme
preparation at doses equivalent to 0, 100, 333, or 1000 mg/kg
b.w./day. No significant clinical changes were observed. Body-weight
gain and food intake were similar among test and control animals.
Haematologic and clinical chemistry measurements at termination of the
study were within normal ranges. Post-mortem examinations,
measurements of organ weights, and histopathology showed no
compound-related effects. Slight increases in kidney and adrenal
weights in the mid-dose group were not associated with
histopathological effects, and did not show a dose response
(Kallesen, 1982).
Long-term studies
No information available.
Observations in man
No information available.
COMMENTS
This enzyme preparation was not genotoxic in microbial or in
mammalian test systems. In a limited 90-day study in rats, no effects
were observed at the highest dose administered (1 g/kg b.w./day). This
enzyme preparation contained high levels of pectinase. The pectinase
enzyme preparation summarized below may be identical to this
hemi-cellulase preparation, which provides added assurance of the
safety of this preparation.
The evaluations by the Committee of the carbohydrases and the
protease from A. niger are summarized at the end of this section.
REFERENCES
Clausen, B. & Kaufman, U. (1983). Unpublished report from Obmutat
Laboratiet. Submitted to WHO by Grinsted Products A/S,
Brabrand, Denmark.
Kallesen, T. (1982). A 90-day toxicity study. Unpublished report
No. 10023 from Scantox Biological Laboratory Ltd., Denmark. Submitted
to WHO by Grinsted Products A/S, Brabrand, Denmark.
Skovbro, A. (1984). In vitro mammalian cytogenetic test (according
to OECD Guideline No. 473). Unpublished report No. 10398 from Scantox
Biological Laboratory Ltd., Denmark. Submitted to WHO by Grinsted
Products A/S, Brabrand, Denmark.
PECTINASE (E.C. 3.1.1.11; 3.2.1.15; 4.2.2.10)
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies (The TOS of the commercial
preparation is approximately 5%).
Acute toxicity
Species Route LD50 Reference
(ml/kg b.w.)
Rat oral 18.8-22.1 Porter & Hartnagel, 1979
Short-term studies
Rats
Two groups of 10 male and 10 female ARS Sprague-Dawley rats were
fed diets containing 5 or 10% of the test enzyme preparation
(equivalent to 3.5 or 7 g of the enzyme preparation/kg b.w./day), for
90 to 94 days. A control group of 20 male and 20 female rats was
maintained on the diet alone. No signs of toxicity were observed
during the test period. Body-weight gain and food consumption were
similar among test and control groups. Differential blood counts at
weeks 4 and 8 of the study were within the normal range in test and
control animals. At the end of the study serum clinical chemistry
analyses, organ weight analyses, and gross and microscopic pathology
showed no compound-related effects (Garvin et al., 1972).
Long-term studies
No information available.
Observations in man
No information available.
COMMENTS
In a short-term study in rats, no adverse effects were observed
at dietary levels of the enzyme preparation up to the equivalent of
7 mg/kg b.w./day. This enzyme preparation may be identical to the
hemi-cellulase preparation summarized above. The hemi-cellulase enzyme
preparation summarized above also contained high levels of pectinase,
which provides added assurance of the safety of this preparation.
REFERENCES
Garvin, P.J., Ganore, C.E., Merubia, J., Delahany, E., Bowers, S.,
Varnado, A., Jordan, L., Harley, G., DeSmet, C., & Porth, J. (1972).
Carbohydrase from Aspergillus niger (pectinase, cellulase aud
lactase). Unpublished report from Travenol Laboratories, Inc., Horton
Grove, IL, USA. Submitted to WHO by Gist-brocades NV, Delft, Holland.
Porter, M.C. & Hartnagel R.E. (1979). The acute oral toxicity of a new
pectinase product in the rat. Unpublished report No. 11 from Miles
Laboratories, Inc., Elkhart, IN, USA. Submitted to WHO by Enzyme
Technical Association, Washington, DC, USA.
PROTEASE
No information available.
GENERAL COMMENTS ON ENZYMES FROM A. NIGER
Aspergillus niger is a contaminant of food. Although there may
be posible strain differences in A. niger, and different cultural
conditions might be used to prepare the various enzymes, the available
toxicity data, which consist primarily of short-term feeding studies
in rats and some studies in dogs, show that all the enzyme
preparations tested were of a very low order of toxicity. The enzyme
preparations tested were non-mutagenic in bacterial and mammalian cell
systems. Studies on some strains of A. niger used to prepare
carbohydrases showed no aflatoxin or related substance production.
These studies provide the basis for evaluating the safety of enzyme
preparations derived from A. niger. It was also noted that the
enzyme preparations tested exhibit a number of enzyme activities, in
addition to the major enzyme activity. Thus, there may be considerable
overlap of the enzyme activities of the different enzyme preparations
so that safety data from each preparation provides additional
assurance of safety for the whole group of enzymes.
Since the enzyme preparations tested were of different activities
and forms, and most of the organic materials in the preparations are
not the enzyme per se, the numerical ADI is expressed in terms of
total organic solids (TOS) (see introduction to enzyme preparations
section).
EVALUATION
Level causing no toxicological effect
All enzyme preparations tested showed no-observed-effect levels
greater than 100 mg TOS/kg b.w./day in 90-day studies in rats.
Estimate of acceptable daily intake
0-1 mg TOS/kg b.w. for each of the enzyme preparations.
See Also:
Toxicological Abbreviations