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        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 (g/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 (g/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 g/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 g/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 g/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