INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
SAFETY EVALUATION OF CERTAIN
FOOD ADDITIVES AND CONTAMINANTS
WHO FOOD ADDITIVES SERIES 40
Prepared by:
The forty-ninth meeting of the Joint FAO/WHO Expert
Committee on Food Additives (JECFA)
World Health Organization, Geneva 1998
alpha-ACETOLACTATE DECARBOXYLASE
First draft prepared by
Dr. P. Abbott,
Australia New Zealand Food Authority (ANZFA)
Canberra, ACT, Australia
1. Explanation
2. Biological data
2.1 Biological properties
2.2 Toxicological studies
2.2.1 Acute toxicity of the host organism
2.2.2 Short-term toxicity studies
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.4 Reproductive toxicity studies
2.2.5 Special studies on teratogenicity
2.2.6 Special studies on genotoxicity
2.3 Observations in humans
3. Comments
4. Evaluation
5. References
1. EXPLANATION
alpha-Acetolactate decarboxylase is produced by submerged
fermentation of Bacillus subtilis carrying the gene (AldB) coding
for alpha-acetolactate decarboxylase from Bacillus brevis and is
used as a processing aid in the brewing and alcohol industry. Two
forms of alpha-acetolactate decarboxylase have been used in the
toxicity studies, namely, an unstabilized form, referred to as ALDC,
and a glutaraldehyde-stabilized form, referred to as d-ALDC, which is
the form used in the final commercial product. alpha-Acetolactate
decarboxylase has not been previously evaluated by the Committee.
Construction of the recombinant B. subtilis strain, UW226, which
contains the AldB gene was performed by a series of steps using
recombinant DNA technology. Firstly, the structural gene for ALDC,
AldB, was isolated from B. brevis and cloned into the plasmid
pUC19 in Escherichia. coli K-12. The fragment containing AldB was
then cloned on a B. subtilis plasmid pDN2801 and transformed into
B. subtilis strain DN1885, giving plasmid pUW102. Into this plasmid
was inserted a kanomycin-resistance gene from pUB110, the resulting
plasmid being pUW160. This plasmid was then transformed into the B.
subtilis host strain UW168, producing plasmid pUW199. This plasmid
contains kanB from plasmid pUB110 which confers kanomycin
resistance. This plasmid was then transformed into B. subtilis UW193
to give strain UW226. Deletion of the kanomycin-resistance gene from
UW193 gave strain UW277, which was used for the production of ALDC.
2. BIOLOGICAL DATA
2.1 Biological properties
alpha-Acetolactate decarboxylase is used to avoid formation of the
unpleasant tasting alpha-diacetyl from alpha-acetolactate during
fermentation. In the traditional brewing process, the alpha-diacetyl
formed from alpha-acetolactate is further reduced to acetoin over a 2-
to 4-week maturation period.
alpha-Acetolactate decarboxylase causes direct decarboxylation of
alpha-acetolactate to acetoin, thus avoiding the need for this
maturation period. The enzyme can similarly be used in the
fermentation of alcohol, where diacetyl is otherwise formed and
requires a maturation step before distillation.
2.2 Toxicological studies
2.2.1 Acute toxicity of the host organism
The pathogenicity of the source organism, Bacillus subtilis,
and the donor organism, Bacillus brevis, have been evaluated,
firstly, by investigating cases of human infections and a
consideration of the history of use of these organisms in relation to
food and, secondly, by a specific study on the pathogenicity of
Bacillus subtilis in mice.
The source organism, Bacillus subtilis, is considered to be a
non-pathogenic species, and has a history of safe use in food enzyme
manufacturing. Similarly, in an examination of reviews dealing with
infections caused by Bacillus spp., the donor organism, Bacillus
brevis, was found in only one report to have caused infection (in
one patient). No other cases of infection by B. brevis were noted in
these reviews. B. brevis is therefore regarded as a non-pathogenic
organism. The vector, pUB 110, is a plasmid commonly used in the
construction of recombinant microorganisms for use in the production
of enzymes.
In a study to investigate the pathogenicity of four Bacillus
subtilis strains involved in either the construction of the
ALDC-producing strain or in producing ALDC, three separate groups of
five mice each were treated intraperitoneally with a particular strain
of B. subtilis at varying dose levels between 2-7.6 × 105 and
2-7.6 × 108 cells/kg bw. The strains used were UW 226, UW 277, the
recipient strain, and DN 297. A control group received a buffer
solution. The mice were observed for 30 min after dosing for clinical
symptoms associated with treatment and then daily for 14 days. At the
end of the 14-day period, all mice were sacrificed and a macroscopic
pathological examination performed. There were no clinical symptoms
related to treatment and no pathological changes noted at the end of
the study that could be associated with treatment (Sietski de Boer,
1990a).
2.2.2 Short-term toxicity studies
Groups of Wistar rats (5/sex per dose) were administered a diet
containing either ALDC (92.9% TOS) at concentrations of 0, 6000, 17
000 or 50 000 mg/kg (equivalent to 300, 850 or 2500 mg/kg bw per day)
or a diet containing d-ALDC (glutaraldehyde stabilized, 92.8% TOS) at
a concentration of 50 000 mg/kg (equivalent to 2500 mg/kg bw per day)
for 14 days. At the end of the test period, all animals were killed
and subject to necropsy. There were no deaths or clinical signs of
toxicity. There were no significant differences between the control
and the treated groups with respect to food consumption, body weight
gain or food conversion ratio. Macroscopic examination did not reveal
any treatment-related effects on tissues. There was no
treatment-related organ weight changes in liver or kidney.
Haematological examination revealed a significant decrease in red
blood cell (RBC) count in females in all treated groups. Corresponding
to this, the mean cell volume in females at 17 000 and 50 000 mg/kg
ALDC was significantly higher than in controls and also higher than in
the 50 000 mg/kg d-ALDC group. For males, there was also a decrease in
RBC count in the treated group, which was marginally significant only
at the 6000 mg/kg level. There was no corresponding change in mean
cell volume in treated males. Viral infection was considered a
possible cause of these effects. There were no treatment-related
changes in histopathology in the liver, kidney or jejunum (Sietski de
Boer, 1990b).
Groups of CD rats (20/sex per dose) were administered a diet
containing either ALDC (92.9% TOS) at a concentration of 0, 200, 1400
or 10 000 mg/kg (equivalent to 0, 10, 70 or 500 mg/kg bw per day) or a
diet containing d-ALDC (glutaraldehyde stabilized, 92.8% TOS) at a
concentration of 10 000 mg/kg (equivalent to 500 mg/kg bw per day) for
13 weeks. At the end of the study period, all animals were killed and
subject to necropsy. There were no clinical signs of toxicity although
one control male was killed accidently during collection of blood at
week 13. There were no significant differences between the control and
the treated groups with respect to food consumption, body weight gain
or food conversion ratio. No treatment-related ocular lesions were
detected by ophthalmoscopy. In males only, there was a slight increase
in platelet counts in the two groups receiving 10 000 mg/kg ALDC or
d-ALDC. No other haematological changes were observed. There were
sporadic differences between treated and control groups with respect
to blood chemistry parameters, but these could not be clearly
attributed to treatment. There was no treatment-related effect on
urinalysis parameters. There was a marginal increase in liver weight
in males at the 10 000 mg/kg d-ALDC dose level, although there was no
statistical difference between males receiving 10 000 mg/kg d-ALDC and
those receiving 10 000 mg/kg ALDC. There were no treatment-related
macroscopic or microscopic pathological changes and no significant
toxicological changes at any of the dose levels tested (Broadmeadow,
1990).
2.2.3 Long-term toxicity/carcinogenicity studies
No information was available.
2.2.4 Reproductive toxicity studies
No information was available.
2.2.5 Special studies on teratogenicity
No information was available.
2.2.6 Special studies on genotoxicity
The results with ALDC and d-ALDC are summarized in Tables 1 and 2,
respectively.
Table 1. Results of genotoxicity assays using alpha-acetolactate decarboxylase (ALDC) from Bacillus brevis
Test system Test object Concentration of Result Reference
enzyme (µg/ml)
Bacterial gene mutation S. typhimurium TA1535, 33-10 000 negative Pedersen, 1990
TA1537, TA98, TA100
(liquid culture)
Mammalian gene mutation Mouse lymphoma L5178Y 1.58-5000 ± S9 negative Clare, 1990a
Chromosome aberrations Human lymphocytes 44-5000 ± S9 negative Marshall, 1990a
Table 2. Results of genotoxicity assays using stabilized alpha-acetolactate decarboxylase
(d-ALDC) from Bacillus brevis
Test system Test object Concentration of Result Reference
enzyme (µg/ml)
Mammalian gene mutation Mouse lymphoma L5178Y 1.58-5000 ± S9 negative Clare, 1990b
Chromosome aberrations Human lymphocytes 44-5000 ± S9 negative Marshall, 1990b
In a bacterial gene mutation assay, ALDC (92.9% TOS) was tested
for its ability to induce reverse mutations in a liquid culture assay
with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100.
Bacteria were exposed to ALDC with or without S9 metabolic activation
at dose levels between 33 and 10000 µg/ml for 3 hours before plating
and scoring. There was no treatment-related increase in revertants
compared to the negative controls (Pedersen, 1990).
In a mammalian gene mutation assay, ALDC (92.9% TOS) or d-ALDC
(92.8% TOS) were tested for their ability to induce mutations at the
HGPRT locus (6-thioguanine resistance) in mouse lymphoma cells. Cells
were exposed to ALDC or d-ALDC with or without S9 metabolic activation
at dose levels between 1.58 and 5000 µg/ml for 2 hours before being
transfered to flasks for growth during the expression period. There
was no treatment-related increase in mutants with either ALDC or
d-ALDC compared to the negative controls (Clare, 1990a,b).
In a chromosome aberration assay, ALDC (92.9% TOS) or d-ALDC
(92.8% TOS) were tested for their ability to induce chromosome
aberrations in cultured human lymphocytes. Cells were exposed to ALDC
or d-ALDC with or without S9 metabolic activation at dose levels
between 43.75 and 5000 µg/ml for either 20 or 44 hours before
harvesting and analysis. There was no treatment-related increase in
mutants with either ALDC or d-ALDC compared to the negative controls
(Marshall, 1990a,b).
2.3 Observations in humans
No information was available.
3. COMMENTS
alpha-Acetolactate decarboxylase is an enzyme preparation of
reasonably high purity derived from a genetically modified organism.
The available data indicate that both the source organism,
B. subtilis, and the donor organism, B. brevis, are considered to
be non-pathogenic species.
In both 14-day and 13-week feeding studies in rats, there was no
indication of toxicity at dietary levels equivalent to 2500 mg/kg bw
per day (14-day study) or 500 mg/kg bw per day (13-week study) for
either the unstabilized or stabilized ALDC. No long-term studies were
available. In the genotoxicity studies, negative results were obtained
with both stabilized and unstabilized ALDC in both the bacterial and
mammalian gene mutation assays and in a chromosome aberration assay in
human lymphocytes.
4. EVALUATION
On the basis of the available data, the Committee concluded that
alpha-acetolactate decarboxylate is an enzyme of low toxicity and that
no further studies are required to assess its safety. The Committee
established a temporary ADI "not specified" for alpha-acetolactate
decarboxylase from this recombinant strain of B. subtilis when the
preparation is used in accordance with good manufacturing practice. A
temporary ADI was allocated because the specifications are temporary.
5. REFERENCES
Broadmeadow, A. (1990) ALDC: Toxicity study by dietary administration
to CD rats for 13-weeks. Unpublished report No. 90/0691 from Life
Science Research Ltd (Submitted to WHO by Novo NordiskA/S, Denmark).
Clare, C.B. (1990a) Study to determine the ability of ALDC to induce
mutations to 6-thioguanine resistance in mouse lymphoma L5178Y cells
using a fluctuation assay. Unpublished report No. NOD 19/ML from
Hazleton Microtest, York, UK (Submitted to WHO by Novo Nordisk A/S,
Denmark).
Clare, C.B. (1990b) Study to determine the ability of d-ALDC to induce
mutations to 6-thioguanine resistance in mouse lymphoma L5178Y cells
using a fluctuation assay. Unpublished report No. NOD 20/ML from
Hazleton Microtest, York, UK (Submitted to WHO by Novo Nordisk A/S,
Denmark).
Marshall, R. (1990a) Study to evaluate the chromosome damaging
potential of ALDC by its effects on cultured human lymphocytes using
an in vitro cytogenetics assay. Unpublished report No. NOD 19/HLC
from Hazleton Microtest, York, UK (Submitted to WHO by Novo Nordisk
A/S, Denmark).
Marshall, R. (1990b) Study to evaluate the chromosome damaging
potential of d-ALDC by its effects on cultured human lymphocytes using
an in vitro cytogenetics assay. Unpublished report No. NOD 20/HLC
from Hazleton Microtest, York, UK (Submitted to WHO by Novo Nordisk
A/S, Denmark).
Pedersen, P.B. (1990) ALDC: Testing for mutagenic activity with
Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 in a
liquid culture assay. Unpublished report No. 90001 from Novo Nordisk
A/S (Submitted to WHO by Novo Nordisk A/S, Denmark).
Sietske de Boer, A. (1990a) Pathogenicity in mice of three Bacillus
subtilis strains either taking part in the construction of the ALDC
producing strains or producing ALDC. Unpublished report No. 90011 from
Enzyme Toxicology Laboratory, Novo Nordisk A/S (Submitted to WHO by
Novo NordiskA/S, Denmark).
Sietske de Boer, A. (1990b) Fourteen day oral dose range finding study
with ALDC and d-ALDC in rats. Unpublished report No. 89089 from Enzyme
Toxicology Laboratory, Novo Nordisk A/S (Submitted to WHO by Novo
Nordisk A/S, Denmark).
See Also:
Toxicological Abbreviations