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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    RIBOFLAVIN DERIVED BY FERMENTATION WITH GENETICALLY MODIFIED BACILLUS 
    SUBTILIS

    First draft prepared by
    M.E.V. Apeldoorn, H.V. Steeg, and G.J.A. Speijers
    National Institute of Public Health and the Environment, Center of
    Substances and Risk Assessment, Bilthoven, The Netherlands

          Explanation
          Biological data
                Toxicological studies
                      Acute toxicity
                      Short-term studies of toxicity
                      Genotoxicity
          Methods of genetic modification
                Molecular genetic methods used to clone and 
                      express the riboflavin  (rib) operon from 
                       Bacillus subtilis
                Genetic stability of the production strain
                Foreign DNA present in the end-product
          Comments
          Evaluation
          References


    1.  EXPLANATION

          Riboflavin derived by a fermentation process with genetically
    modified  Bacillus subtilis has not been previously evaluated by the
    Committee. Synthetic riboflavin was evaluated by the Committee at its
    thirteenth meeting (Annex 1, reference 19), when an ADI of 0-0.5 mg/kg
    bw was allocated on the basis of limited data. At the twenty-fifth
    meeting (Annex 1, reference 56), a group ADI of 0-0.5 mg/kg bw was
    allocated to riboflavin and riboflavin-5'-phosphate, expressed as
    riboflavin, on the basis of a more extensive database including
    information on reproductive toxicity.

          Riboflavin (vitamin B2) is a water-soluble vitamin. It is
    synthesized by all plants and many microorganisms, but it is not
    produced by higher animals. Because it is a precursor of coenzymes
    that are required for the enzymatic oxidation of carbohydrates,
    riboflavin is essential to basic metabolism. Its chemical structure is
    given in Figure 1.

          In higher animals, insufficient riboflavin can cause loss of
    hair, inflammation of the skin, vision deterioration, and growth
    failure. Riboflavin is therefore widely used as an additive to
    foodstuffs and feedstuffs. It is used in foods predominantly for
    fortification; however, quantities accounting for approximately 2% of
    the total sales are used for colouring, e.g. for sweets. The foods to
    which it is added for fortification or restoration are breakfast
    cereals, soft drinks, slimming diet products, and baby food products.
    These account, for example, for 82% of the riboflavin sold in the

    United Kingdom for food use; a further 16% is used in ice cream, meat
    and fish, sauces and soups, and food supplements.

    FIGURE 1

          Although riboflavin can be produced by both synthetic and
    fermentation processes, the latter was not found to be competitive for
    production of very pure material. Recent developments in biotechnology
    have resulted in microbiological processes that compete successfully
    with the synthetic chemical process. Fermentation-produced riboflavin
    is prepared by the controlled, submerged growth of a selected strain
    of  B. subtilis that has been genetically modified to produce
    riboflavin. Manufacture involves fermentation resulting in a broth
    containg riboflavin, recovery of riboflavin from the broth, and
    further purification to a final product with a purity of 96 or 98%,
    which is used in animal feed and human food, respectively (Pfister et
    al., 1995).

    *     Fermentation is performed in vessels by aseptic, large-scale
          microbiological techniques under controlled conditions of
          temperature, aeration, agitation, and pH. The cultural purity of
          the fermentation is routinely tested by checking growth behaviour
          and colony identity. When the fermentation has proceeded to the
          desired point, the broth is delivered for recovery.

    *     Riboflavin is recovered from the broth by centrifugation after
          inactivation of the microorganisms by heat. Pasteurization of the
          broth ensures that no viable cells of the production organism are
          present in the final product. Differential centrifugation leads
          to separation of cells and riboflavin crystals because of
          differences in size and sedimentation behaviour.

    *     Mineral acid treatment (about 2% hydrochloric or sulfuric acid)
          yields 96% riboflavin, which, after drying and packaging, is
          either directly sold for application in feed or used for further
          processing into a formulation. The acid treatment also ensures
          destruction (via depurination) of DNA from the processing
          organism. Recrystallization of the 96% pure material from
          concentrated hydrochloric acid (> 25%) results in a food-quality
          material containing 98% riboflavin. This last purification step
          is identical to that currently used for purification of
          riboflavin derived from chemical synthesis.

          Three lots of 98% pure riboflavin derived by fermentation, four
    lots of 96% pure material derived by fermentation, and one lot of 98%
    pure material derived by synthesis were analysed by two separate
    high-performance liquid chromatography (HPLC) methods in order to
    identify and quantify any impurities. The riboflavin content of the
    98% pure products derived by fermentation and synthesis ranged from 99
    to 101%, and that of the 96% pure products derived by fermentation
    from 97 to 99%. All of the peaks detected in riboflavin from
    fermentation could be identified and appeared also to be present in
    synthesized riboflavin, indicating that the impurities probably occur
    during the purification steps (acidic treatment and crystallization)
    (Simon & Vogt, 1989; Ludwig et al., 1994). Four impurities were
    identified in the fermentation-derived material (for chemical
    structures, see Figure 2). Lumiflavin, a known degradation product of
    riboflavin after basic treatment (Meinrad & Gerber, 1991), was present
    in the synthesized product but not in the fermentation-derived
    material. 

    FIGURE 2

          The presence of amino acids originating from fermentation was
    analysed by the method of Spackman et al. (1958), and DNA was analysed
    by polymerase chain reaction (PCR) techniques (Schurter & Hermann,
    1995). The amounts of impurities in the different products are given
    in Table 1. The concentration of 8alpha-hydroxymethylriboflavin, which
    is a known degradation product after acid treatment of riboflavin, was
    slightly increased in the fermentation-derived product, but only in
    that of 98% purity. Since this product is derived from the 96% pure
    material by crystallization from acidic media, this is to be expected;
    however lumiflavin, which is a known degradation product after basic
    treatment, was present in the synthesized riboflavin but not in the
    fermentation-derived material.

        Table 1. Composition of riboflavin products derived by fermentation and by synthesis 
             (average % weighed samples)

                                                                                                  

    Component                          96% ex              98% ex              96% ex synthesis
                                       fermentation        fermentation
                                       HPLC method         HPLC method         HPLC method

                                       I         II        I         II        I         II
                                                                                                  

    Riboflavin                         98.4      98.3      100.1     99.7      98.8      98.6
    Ribityl-oxo-chinoxalic acid        Trace     ND        ND        ND        Trace     Trace
    8alpha-Hydroxy riboflavin          Trace     Trace     0.45      0.2       Trace     Trace
    Formylmethyl-flavin acetal         Trace     ND        0.11      Trace     0.52      ND
    Lumichrome                         0.14      0.11      ND        Trace     0.18      Trace
    Lumiflavin                         ND        ND        ND        ND        0.56      0.5
    Amino acids                        0.9       0.9       0.06      0.06      -         -
    DNAa                               ND        ND        ND        ND        -         -

    Total                              99.44     99.31     100.72    99.96     100.1     99.3
                                                                                                  

    Trace, below limit of detection; ND, below limit for quantitative determination, 0.1%
    a See also section 3.3, 'Foreign DNA present in the end product'
    
    2.  BIOLOGICAL DATA

    2.1  Toxicological studies

    2.1.1  Acute toxicity

          No studies of the acute toxicity of riboflavin derived by
    fermentation with genetically modified  B. subtilis are available.
    Table 2 gives the LD50 values for riboflavin (not derived from
    fermentation), riboflavin phosphate, and some of the impurities
    present in riboflavin. Except for the study with

    8alpha-hydroxy-riboflavin, no full reports were available. The data in
    Table 2 give an indication of the acute toxicity of synthetic
    riboflavin (phosphate) and some of its impurities but are not relevant
    for evaluating riboflavin derived by fermentation.

    2.1.2  Short-term studies of toxicity

     Rats

          Groups of 10 male and 10 female six-week-old Wistar rats (the
    males weighing 160-170 g and the females, 130-140 g) received diets
    providing 20, 50, or 200 mg/kg bw of riboflavin -- either 99.4% pure
    derived by fermentation from 96% pure riboflavin, 100.2% pure derived
    by fermentation from 98% pure riboflavin, or 99.8% pure derived by
    sunthesis from 98% pure riboflavin -- daily, on seven days per week
    for 13 weeks. The study was performed according to good laboratory
    practice (GLP) and OECD Guideline 408; a quality assurance (QA)
    statement was included. A control group of 30 male and 30 female rats
    was used. A satellite group of six male and six female animals was
    attached to each group for determination of riboflavin concentrations
    in blood and urine and for investigation of the reversibility of any
    toxicological parameter. Feed and water were provided  ad libitum. 
    The different assays were performed at one-week intervals. Samples of
    the diet were taken at the start of treatment and at weeks 6 and 13 to
    determine the content, homogeneity, and stability of the test compound
    in the diet. 

          All animals were checked twice daily (once daily at weekends) for
    behaviour and general condition. Ophthalmoscopy was performed on all
    animals in the main control group and those at the three highest doses
    before the start and at the end of the study. Body weight and food
    intake were determined weekly. Water consumption was determined over
    five consecutive days in weeks 1, 5, and 12. Haematological parameters
    (haemoglobin concentration; haematocrit; erythrocyte, leukocyte, and
    differential cell counts; mean corpuscular volume, haemoglobin, and
    haemoglobin concentration; reticulocyte and thrombocyte counts; and
    prothrombin time) were measured at week 6 and at autopsy in 10 rats of
    each sex in the main control and treated groups. Haemoglobin
    concentration; haematocrit; erythrocyte and leukocyte counts; mean
    corpuscular volume, haemoglobin, and haemoglobin concentration; and
    thrombocyte count were measured in all rats in the satellite groups at
    the end of the recovery period. Clinical chemical parameters (alanine
    and aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl
    transferase, and lactic dehydrogenase activities; bilirubin, total
    protein, albumin, albumin:globulin ratio, urea, creatinine, Na, K, Ca,
    Cl, inorganic phosphate, cholesterol, triglycerides, and
    phospholipids) were determined in all rats in the main treated groups
    and in 15 rats of each sex in the main control group in weeks 7 and 13
    after one night of fasting; the same parameters were measured in all
    rats in the satellite groups at the end of the recovery period. In
    weeks 7 and 13, all rats in the main treated groups and 15 rats of
    each sex in the main control group underwent a renal concentration
    test and urinalysis (appearance, pH, glucose, occult blood, ketones,

    proteins, bilirubin, urobilinogen, sediment), except that sediment was
    measured only in rats at the three highest doses. The sediment of
    urine from all rats in the satellite groups was examined
    microscopically for the presence of sperm at the end of the recovery
    period. The weights (absolute and relative to body weight) of the
    adrenals, kidneys, liver, spleen, brain, thymus, heart, and testes of
    all rats were determined at the end of the treatment period in all of
    the main groups and at the end of the recovery period in all satellite
    groups. All rats in the main groups were examined macroscopically at
    the end of treatment, and about 30 tissues from all animals in the
    main control group and in the main three highest-dose groups were
    examined microscopically The testes, epididymides, kidneys, liver,
    lungs, and all gross lesions from all rats in the main groups were
    examined microscopically. The riboflavin concentration in blood was
    determined in all rats in the satellite groups before the start of the
    study, at weeks 2, 6, and 13 of treatment, and at the end of the
    recovery period. The riboflavin content of the urine was determined in
    all rats in the satellite control group and in all rats in the
    satellite groups treated with 96 and 98% pure riboflavin derived by
    fermentation, before the study and at week 2 and 6. At the end of the
    treatment period and at the end of the recovery period, the riboflavin
    content of the urine was determined in all rats in all satellite
    groups. 

          The test substances were homogeneously distributed in the diets
    and showed storage stability under simulated experimental conditions.
    The actual measured concentrations in the diet were within the
    tolerable range of ± 10% of the nominal concentration. Alopecic areas
    were seen frequently in various groups, but these findings were
    transient and were not related to dose. Ophthalmoscopy showed no
    abnormalities. Excrement collected from rats at the highest doses of
    the three test substances was yellowish throughout the study. No
    dose-related difference in food and water intake was seen. 

          During treatment, females at 200 mg/kg bw per day 98% pure
    riboflavin ex fermentation showed a slightly but usually statistically
    significant decrease in growth of about 6%. Males and females at
    50 mg/kg bw per day 98% pure riboflavin ex synthesis also had a slight
    but statistically significant decrease in growth. These effects were
    not considered to be toxicologically relevant because they were small
    (< 10%) and food conversion was not affected. During the recovery
    period, body weights were similar in all groups. No changes in
    haematological parameters were seen after six weeks of treatment, but
    at the end of treatment (week 13) females at 200 mg/kg bw per day 98%
    pure riboflavin ex fermentation had a slightly but statistically
    significantly lower mean haemoglobin value and erythrocyte count,
    whereas the mean reticulocyte count was significantly increased. The
    individual reticulocyte counts in these animals were markedly
    increased in two of 10 females and moderately increased in another
    two. Slight but statistically significant increases in mean
    thrombocyte counts in females at the highest dose of 98% pure
    riboflavin ex fermentation and in males at the highest dose of
    riboflavin 98% ex synthesis were within the range of normal variation

    and therefore considered not to be toxicologically relevant. At six
    and 13 weeks, the total leukocyte counts were statistically
    significantly decreased in several treated groups but with no
    dose-response relationship and inconsistently within groups; these
    changes were also not considered to be related to treatment.

          Clinical chemical and urinary analyses showed no
    treatment-related changes. At the end of treatment, a slight but
    statistically significant increase in relative liver weight was seen
    in females at 200 mg/kg bw per day 98% pure riboflavin ex
    fermentation, and a slight but statistically significant increase in
    relative spleen weight in males at 50 and 200 mg/kg bw per day 96%
    pure riboflavin (ex fermentation), but with no dose-response
    relationship. Macroscopic and microscopic examination did not reveal
    any treatment-related abnormality. The changes in organ weights were
    considered to be of questionable biological relevance because there
    was no dose-response relationship and they were not accompanied by
    histopathological changes or changes in clinical chemistry. At the end
    of the recovery period, all of the haematological parameters were
    normal, except for a statistically significant decrease in mean
    thrombocyte counts in females at 200 mg/kg bw per day 98% pure
    riboflavin ex fermentation, and there were no notable changes in organ
    weights.

          The riboflavin concentrations in blood and urine were not
    reported.

          The authors concluded that the NOEL was 200 mg/kg bw per day for
    all three test substances. The decreases in mean haemoglobin
    concentration and erythrocyte count in females at 200 mg/kg bw per day
    98% pure riboflavin ex fermentation were considered to be fortuitous
    findings because, except in two females, there was no overt
    association between the low erythrocyte counts and the high
    reticulocyte counts. Moreover, according to the authors, the measured
    values did not exceed the expected range, and there was also no clear
    dose-response relationship in any group. In addition, there were no
    related changes (in e.g. haemolysis or weight and histopathology of
    haematopoietic organs) that could be associated with variations in the
    red blood cell profile (Buser et al., 1995). 

          The Committee concluded that the NOEL for 98% pure riboflavin ex
    fermentation was 50 mg/kg bw per day, on the basis of the significant
    decreases in mean haemoglobin concentration and erythrocyte count and
    the significant increase in mean reticulocyte count in females at
    200 mg/kg bw. For 96% pure riboflavin ex fermentation and 98% pure
    riboflavin ex synthesis, the NOEL was 200 mg/kg bw per day, the
    highest dose tested.


        Table 2. Acute toxicity of synthetic riboflavin phosphate and some of its impurities

                                                                                                                                           

    Compound                       Species     Sex    Route              LD50           Remarks                          Reference
                                                                         (mg/kg bw)
                                                                                                                                           

    Riboflavin                     Mouse       NR     Oral               > 40 000       Observation period, 10 days      Bächtold (1980)
    Riboflavin                     Rat         NR     Intravenous             560                                        Bächtold (1980)
    Riboflavin                     Rat         NR     Subcutaneous          5 000                                        Bächtold (1980)

    Riboflavin phosphate sodium    Mouse       NR     Oral               > 40 000       Observation period, 10 days      Bächtold (1980) 
    Riboflavin phosphate sodium    Mouse       NR     Intraperitoneal         890       Observation period, 10 days      Bächtold (1980)
    Riboflavin phosphate sodium    Mouse       NR     Intravenous             780       Observation period, 10 days      Bächtold (1980)
    Riboflavin phosphate sodium    Rat         NR     Oral               > 20 000       Observation period, 10 days      Bächtold (1980)
    Riboflavin phosphate sodium    Rat         NR     Intraperitoneal       1 030       Observation period, 24 h         Bächtold (1980)
    Riboflavin phosphate sodium    Rat         NR     Intraperitoneal         560                                        Bächtold (1980)
    Riboflavin phosphate sodium    Rat         NR     Intravenous             710       Observation period, 10 days      Bächtold (1980)
    Riboflavin phosphate sodium    Rat         NR     Subcutaneous            790                                        Bächtold (1980)

    8alpha-Hydroxyriboflavin       Rat         M&F    Oral                > 2 000       Limit test, by gavagea           Schöni (1989)
       (purity > 95%)

    Lumichrome                     Mouse       NR     Oral                > 9 000       Observation period, 10 days      Bächtold (1980)
    Lumichrome                     Mouse       NR     Intraperitoneal      11 000       Observation period, 10 days      Bächtold (1980)
    Lumiflavin                     Mouse       NR     Oral                > 6 000       Observation period, 10 days      Bächtold (1980)
                                                                                                                                           

    NR, not reported; M&F, male and female
    a Study performed according to OECD Guideline 401; no GLP statement or QA declaration was included.

    Table 3. Results of assays for the genotoxicity of riboflavin derived by fermentation from B. subtilis and of 8alpha-hydroxyriboflavin

                                                                                                                                               

    End-point           Test object          Concentration           Results             Reference
                                                                                                                                               

    Riboflavin 96%      Reverse mutationa    S. typhimurium TA97,    50-5000 µg/plate    Negativeb                        Albertini (1995a)
    ex fermentation                          TA98, TA100, TA102,     in DMSO             No toxicity; increasingly 
    (purity, 99.4%)                          TA1535                                      milky suspensions at
                                                                                         > 50 µg/plate. At 1666 and 
                                                                                         5000 µg/plate, precipitation 
                                                                                         prevented colony counting 
                                                                                         in preincubation assay.

    Riboflavin 98%      Reverse mutationa    S. typhimurium TA 97,   50-5000 µg/plate    Negativeb                        Albertini (1995b)
    ex fermentation                          TA98, TA100, TA102,     in DMSO             No toxicity; increasingly 
    (purity, 100.2%)                         TA1535                                      milky suspensions at
                                                                                         > 50 µg/plate. At 1666 and 
                                                                                         5000 µg/plate, precipitation 
                                                                                         prevented colony counting 
                                                                                         in preincubation assay.

    8alpha-Hydroxy-     Reverse mutationc    S. typhimurium TA97,    100-5000 µg/plate   Negativeb                        Albertini (1989)
    riboflavin (purity                       TA98, TA100, TA102,      in DMSO            No toxicity; yellow-to-orange
    not given)                               TA1535, TA1537, TA1538                      precipitate at 2500 and 5000
                                                                                         µg/plate
                                                                                                                                               

    DMSO, dimethyl sulfoxide
    a   Ames test; standard plate incorporation, preincubation modification assay. Study performed according to GLP; protocol resembled OECD
        Guideline 471 except for the performance of an independent repeat experiment; a QA statement was included.
    b   With and without exogenous metabolic activation
    c   Ames test; standard plate incorporation, preincubation modification assay. Study not performed according to GLP; protocol resembled OECD
        Guideline 471 except for the performance of an independent repeat assay; no QA statement was included.
    

    2.1.3  Genotoxicity

          Studies on the genotoxic potential of riboflavin derived by
    fermentation from  B. subtilis and of 8alpha-hydroxyriboflavin, an
    impurity of the fermentation-derived material, are summarized in Table
    3.

    3.  METHODS OF GENETIC MODIFICATION

    3.1  Molecular genetic methods used to clone and express the riboflavin 
    (rib) operon from Bacillus subtilis

          The riboflavin operon of  B. subtilis containing the genes
     ribT, ribH,  ribA, and  ribG was cloned into pUC19 as a 6.4-kb
     NcoI/XbaI DNA fragment. In order to enhance expression of the  rib 
    genes, a strong constitutive  B. subtilis bacteriophage SPO1 promoter
    was included. Two versions were made: (i) the SPO1-15 promoter was
    inserted in front of the endogenous  ribP2 promoter in a construct
    later designated as pRF93 and (ii) the endogenous  ribP1 promoter was
    replaced by SPO1-15 in a construct later designated as pRF69. In order
    to make the constructs selectable after integration into the  B. 
     subtilis genome (the  ß-lactamase gene of pUC19 is not expressed in
     B. subtilis), the two constructs were provided with different
    selectable genes, i.e. the tetracycline-resistant  (tet) gene derived
    from  B. cereus was inserted into pRF93, and the chloramphenicol-
    resistant  (cat) gene derived from pUC194, origin  S. aureus/ 
     B. subtilis, into pRF69. Both constructs were integrated into the
    genome of recipient  B. subtilis strain RB50. The integration sites
    were mapped for pRF69 at 209° and for pRF93 at 139° in the  B. 
     subtilis genome.

          RB50, a strain of  B. subtilis that overproduces riboflavin, was
    obtained by classical mutagenesis and selection procedures. With
    various unknown mutations, RB50 contains a mutation that maps at the
     ribC locus, which is present at 147° of the  B. subtilis genome. 

          In order to further increase riboflavin production, the
    RB50::[pRF69]: :[pRF93] was subjected to increasing concentrations of
    the antibiotics tetracycline and chloramphenicol, resulting in
    amplification of pRF69 and pRF93. In general, pRF69 was multiplied
    20-30 times whereas pRF93 was multiplied 8-10 times, resulting in the
    production strain RB50::[pRF69]n: :[pRF93]m. Although neither
    antibiotic was present during the production stage, there was no
    detectable loss of either pRF (Maruo & Yoshikawa, 1989; Kreneva &
    Perumov, 1990; Mironov et al., 1994; Schurter, 1994). All of the
    procedures were extensively and well described.

    3.2  Genetic stability of the production strain

          The production strain obtained after amplification was
    RB50::[pRF69]n: :[pRF93]m. Samples were taken from the fermentor
    during the process, and the numbers of copies of pRF69 and pRF93 were
    determined by Southern blotting. There was no apparent loss of the
    foreign DNA present in RB50: :[pRF69]n::[pRF93]m during the

    process, which took 48 h, with samples taken at 6-h intervals
    (Hümbelin & Hermann, 1995). As all of the experiments were well
    described and appeared to be sound and convincing, it can be concluded
    that the production strain is genetically stable during the
    fermentation process.

    3.3  Foreign DNA present in the end-product

          One of the purification steps involved treatment of the product
    with diluted hydrochloric acid for 60 min at 90°C. Under these
    conditions, DNA is heavily depurinated and degraded (Hermann &
    Schurter, 1994). A sensitive PCR method was used to detect any
    residual transforming DNA (monitored as a 557-bp pRF69 PCR  cat 
    fragment), with a detection limit of 0.5 ppb DNA per mg riboflavin. No
    foreign DNA was detected. Furthermore, no transforming activity was
    detected after only 5 min of hot acid treatment of crude riboflavin
    (Schurter & Hermann, 1995). As all of the experiments were described
    in detail, and the results are convincing, it can be concluded that
    the riboflavin samples were free of any detectable transforming DNA.


    4.  COMMENTS

          The strain used in riboflavin production has been genetically
    modified to overproduce riboflavin by amplification of the chromosomal
    region of the riboflavin operon containing suitable promoters and
    flanked by pUC19 and antibiotic-resistance marker genes. The lack of
    pathogenicity and toxicity of the strain of microorganism
     (B. subtilis) from which the genetic information encoding riboflavin
    was cloned is well documented, and the methods for genetic
    modification have been well described. The production strain of
     B. subtilis was shown to be genetically stable during fermentation.
    It was convincingly established that the final product is riboflavin
    of a purity comparable to, or greater than, that produced by
    conventional methods and is free of DNA from the production organism.
    Thus, antibiotic resistance marker genes are not present. 

          In a 90-day study of toxicity, rats were fed diets containing
    food-grade riboflavin (98% pure) produced by fermentation from
    genetically modified  B. subtilis or riboflavin derived by chemical
    synthesis, also of 98% purity. The doses tested were 0, 20, 50, or 200
    mg/kg bw per day. Some animals at the high dose had reduced weight
    gain, but generally by less than 10%, and food conversion efficiency
    was not affected. In the group fed the high dose of
    fermentation-derived riboflavin of 98% purity, some minor changes in
    red blood cell parameters were observed, but they were not considered
    relevant. The NOEL for 98% pure riboflavin produced by fermentation
    was 200 mg/kg bw per day, which was the same as that for chemically
    synthesized riboflavin.

          Fermentation-derived riboflavin and the degradation product
    8alpha-hydroxyriboflavin did not induce gene mutations in bacteria
     in vitro in either the absence or the presence of metabolic
    activation. 


    5.  EVALUATION

          The Committee concluded that the recombinant DNA techniques used
    to derive the production strain of  B. subtilis were well
    characterized, providing assurance that no DNA is present in the
    end-product. On the basis of molecular biological data and chemical
    analytical research, it can be concluded that fermentation-derived
    riboflavin from genetically modified  B. subtilis is substantially
    equivalent to synthetic riboflavin.

          For 98% pure fermentation-derived riboflavin for use in food, the
    NOEL in the 90-day study of toxicity in rats was 200 mg/kg bw per day,
    the highest dose tested. Fermentation-derived riboflavin was evaluated
    on the basis of its substantial equivalence to synthetic riboflavin.
    Therefore, the Committee included riboflavin derived from a production
    strain of genetically modified  B. subtilis in the previously
    established group ADI of 0-0.5 mg/kg bw for synthetic riboflavin and
    riboflavin-5'-phosphate.


    6.  REFERENCES

    Albertini, S. (1989) Mutagenicity evaluation of Ro 42-7491/000
    (8alpha-hydroxy-riboflavin) in the Ames test (preincubation version).
    Unpublished report from F. Hoffmann La Roche Ltd.dated 20 February
    1989. Submitted to WHO by F. Hoffmann La Roche Ltd, Basel,
    Switzerland.

    Albertini, S. (1995a) Mutagenicity evaluation of Ro 01-3131/055
    (riboflavin 96% ex fermentation) in the Ames test (Study No. 139M95).
    Unpublished report from F. Hoffmann La Roche Ltd. dated 8 September
    1995. Submitted to WHO by F.Hoffmann La Roche Ltd, Basel, Switzerland.

    Albertini, S. (1995b) Mutagenicity evaluation of Ro 01-3131/054
    (riboflavin 98% ex fermentation) in the Ames test (Study No. 138M95).
    Unpublished report from F. Hoffmann La Roche Ltd. dated 8 Sepember
    1995. Submitted to WHO by F.Hoffmann La Roche Ltd, Basel, Switzerland.

    Bächtold, H. (1980) Acute toxicity of riboflavine, some intermediates
    and by-products of the synthesis, degradation products and
    metabolites. Unpublished report No. 9024 from F. Hoffmann La Roche
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    See Also:
       Toxicological Abbreviations