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    BENZYLPENICILLIN

    1.  EXPLANATION

         Benzylpenicillin is used to treat or prevent local and systemic
    infections caused by susceptible bacteria.  Intramammary
    administration to treat or prevent bovine mastitis is widespread. 
    Subtherapeutic concentrations in feed have been used for decades in
    some countries to improve growth and to prevent infections.

         Benzylpenicillin had been previously evaluated at the twelfth
    meeting of the FAO/WHO Joint Expert Committee on Food Additives (Annex
    1, Reference 17).

         Benzylpenicillin is a compound belonging to the class of -lactam
    antibiotics.  Its structure is shown in Figure 1.  The free acid (CAS
    registry number 61-33-6) is relatively unstable.  Therefore, the mono-
    sodium salt (CAS registry number 113-98-4), or other salts are
    normally used.  Benzylpenicillin is obtained from penicillium molds by
    fermentation.

         The international unit of penicillin is the specific penicillin
    activity contained in 0.5988 g of the international master standard.

    FIGURE 1

    2.  BIOLOGICAL DATA

    2.1  Biochemical Aspects

    2.1.1  Absorption, distribution, and excretion

         In humans, about one third of an orally administered dose of
    benzylpenicillin is absorbed from the intestinal tract under favorable
    conditions.  Only a small portion is absorbed from the stomach. 
    Absorption occurs mainly in the duodenum.  Ingestion of food
    interferes with enteric absorption.  Benzylpenicillin is widely
    distributed throughout the body.  Its apparent volume of distribution
    is in about 50% of total body water.  Benzylpenicillin is rapidly
    eliminated from the body (mainly by the kidney) (Mandell & Sande,
    1985).

    2.1.2  Biotransformation

         Gastric acid hydrolyzes the amide side chain and opens the lactam
    ring, with concomitant loss of antibacterial activity.  The
    biotransformation of benzylpenicillin is not well understood.  The
    drug is only partially metabolized and the major fraction is excreted
    unchanged (Huber, 1988).

    2.1.3  Effects on enzymes

         Benzylpenicillin selectively inhibits bacterial cell wall
    biosynthesis.   The linear polysaccharide ("glycan") of the cell wall
    is cross-linked by branched peptide chains to form a structure termed
    "peptidoglycan".  The primary transpeptidase reaction leading to
    covalent linkage of new chains to the pre-existing peptidoglycan
    network is highly sensitive to penicillin.  Several proteins,
    including transpeptidases, located in the bacterial cell membrane bind
    penicillin covalently in the form of a penicilloyl moiety via an ester
    linkage (cleavage of the lactam ring).  It appears that the antibiotic
    is recognized as a pseudo-substrate which subsequently acts as a
    strong acylating agent to form a covalent enzyme-inhibitor complex at
    the active site (Bycroft & Shute, 1985).

    2.1.4  Immunogenicity and antigenicity

         Many antigenic determinants may derive from benzylpenicillin. 
    The biochemical routes to the formation of some of these determinants
    may be multiple.  The benzylpenicilloyl (BPO) determinant has been
    designated the "major" antigenic determinant of penicillin allergy. 
    BPO haptenic groups can be formed by direct acylation of proteins or
    from benzylpenicillenic acid, either by direct reaction or via a
    postulated reactive intermediate, thiazolidinyl-oxazolidone.  The
    formation of BPO determinants from benzylpenicillin acid itself, the
    major product of hydrolysis of benzylpenicillin, has not yet been
    definitely established.

         All other determinants resulting from the binding of other
    metabolites of benzylpenicillin are collectively referred to as the
    "minor" determinants.  Many questions pertain to both.  The mechanisms
    of hapten formation and the immunogenicity of certain "minor"
    determinants remain open. Antibodies specifically directed to one or
    another of these determinants have, however, been found in patients
    (e.g., antibodies with penicillamine or penicillemyl specificity) or
    have been induced in experimental animals.  It cannot be ruled out
    that such determinants may be frequently involved in penicillin
    allergy.  Polymerization of benzylpenicillin, which can readily occur
    under certain conditions, has been described.  It is, however, not yet
    fully clear whether and under which circumstances such polymers are
    also capable of reacting as immunogens.

         Protein impurities in benzylpenicillin preparations, which are
    formed during the fermentation process and which could be sufficiently
    highly substituted to efficiently raise antibodies, could contribute
    to the overall immunogenicity.  The critical number of drug-related
    epitopes on carriers may depend on the nature of the carrier.  Low-
    substituted benzylpencilloyl-conjugates with human serum albumin were
    of very poor immunogenicity.  On the other hand, penicillin-autologous
    carrier protein conjugates have been shown to be immunogenic in a
    guinea pig model (De Weck, 1983).

         Penicilloylated proteins from milk or tissue as they may appear
    after high-dose treatment of animals  may possess antigenic properties
    if absorbed through the mucosa of the gut (Wal & Boris 1975; Wal,
    1980).

         Antibodies raised against benzylpenicillin-derived determinants
    may cross-react to some extent with certain antigens derived from
    other -lactams including cephalosporins.  Similarly, antigens
    carrying benzylpenicillin-derived haptenic groups may cross-react with
    antibodies to other -lactams (DeSwarte, 1985).

    2.1.5  Inhibitory activity on microorganisms used in industrial
           food-processing

         A variety of microorganisms (e.g., lactobacilli, streptococci,
    certain yeasts and fungi) are used as "starter cultures" in the
    manufacturing processes of milk products, such as sour milk, sour
    cream, yoghurt, butter, kefir, and many different kinds of cheese.

         It is well known that penicillin residues in milk can negatively
    influence both the quality and yield of such milk-products (Jakimov,
    1970; Cogan, 1972; Abo-Elnage et al., 1973; Dolezalek & Behavkova,
    1974; Bayer et al., 1978; Kondratenko et al., 1978; Loussouran,
    1982).

         Microbiologically active residues of benzylpenicillin can cause
    severe economic losses by inhibiting such microorganisms and thus
    interfering with biotechnological food processing.  Streptococcus
    thermophilus, for example, is partially inhibited at concentrations of
    penicillin as low as 0.0017 I.U./ml.  Total inhibition  occurs at
    0.025-0.05 I.U./ml (Terplan & Zaadhoff, 1967).

    2.2  Toxicological studies

         No experimental studies were available for review.  Information
    contained in the open literature does not meet minimum requirements
    for an evaluation of the toxicological properties of the drug.

    2.3  Observations in humans

         Benzylpenicillin may induce practically all possible clinical
    forms of allergic reactions depending on dose, route, frequency of
    exposure, genetic predisposition and other factors.  Penicillin will
    induce an immune response in practically every person who receives the
    drug.  Low titers of benzylpenicilloyl-specific IgM antibodies can be
    detected in virtually everyone.  However, the mere presence of
    antipencillin-antibodies of any class does not necessarily denote
    clinical sensitivity.  Hypersensitivity reactions are by far the most
    common adverse effects noted with benzylpenicillin (DeSwarte, 1985).

         The proportion of the general population that may be susceptible
    to the development of allergy is unknown.  Available data did not
    allow conclusions as to the true prevalence of penicillin-sensitized
    individuals in the population since all available studies were carried
    out in selected subpopulations using tests of limited diagnostic
    value.  The frequency of allergic side reactions has been reported to
    vary from 0.7% to 10% in different studies (Idso et al., 1968).

         The overall prevalence of penicillin allergy has been estimated
    to be between 3% and 10%, indicating that a substantial proportion of
    the population is at risk (Anderson & Adkinson, 1987).

         Frequencies of skin reactions to commonly used drugs were
    estimated from the records of the Boston Collaborative Drug
    Surveillance Program (data base from 1966 through May, 1975).  Fifty-
    one individuals out of a total of 3286 recipients showed
    benzylpenicillin-induced skin reactions corresponding to a rate of
    16/1000) (Arndt & Jick, 1976).

         In a second evaluation (data base: June 1975 through June 1982)
    17 recipients out of a total number of 918 gave a cutaneous reaction
    with benzylpenicillin (rate: 18.5/1000) (Bigby et al., 1986).

    2.3.1  Sensitizing capacity

    2.3.1.1  Qualitative aspects

         Using techniques such as skin testing and RAST (radio-
    allergosorbent test), antibodies with reactivities to the following
    determinants have been found in humans: benzylpenicilloyl-,
    benzylpenicillenate-, benzylopenicillenyl- and penicillamine-
    determinant (De Weck, 1983).

    2.3.1.2  Quantitative aspects

         It remains impossible to describe in quantitative terms in
    humans:

    -    the major pathways and kinetics (e.g., concentrations, rates,
         catalysts) of the in vivo formation of antigenic
         determinants,

    -    the nature of the biological carrier (e.g., soluble proteins,
         membranes of lymphoid cells).

    -    relative abundances and biological activities of the various
         haptenic groups/determinants derived from benzylpenicillin.

         There were no useful data available to calculate the minimum
    required dose of any potential hapten to produce the minimum required
    amount of complete immunogen with the appropriate number of haptenic
    epitopes per molecule to induce an immune response following oral
    administration.

         In a detailed analysis of data on penicillin-sensitive reactions
    in Taiwan, it has been reported that a 50-year old man who had taken
    a penicillin tablet of 50,000 units one year before the injection of
    a combination of benzylpenicillin and streptomycin died 20 minutes
    after the injection (Idso & Wang, 1958).  If one assumes that this was
    the patient's only exposure to the drug, this would suggest that a
    single dose of approximately 30 mg of benzylpenicillin could sensitize
    a human.

         However, this information is of very limited value since it is
    known that repeated administration of low doses of immunogen is the
    most effective way to produce an IgE antibody response (Levine & Vaz,
    1970; Marsh, 1975).  Virtually nothing about the immunogenicity of
    chronic low-level administration of benzylpenicillin in humans is
    known.

    2.3.2  Eliciting capacity

    2.3.2.1  Qualitative aspects

         The initial event in IgE-mediated reactions is the interaction of
    bivalent or polyvalent antigen with antibody bound to high affinity
    Fc-receptors for IgE on tissue mast cells and blood basophils followed
    by aggregation (at least dimerization) of Fc-receptors (Metzger,
    1988).

         The elicitation of reaction in already-sensitized individuals
    requires no macromolecular antigen.  The low molecular weight N6-N6-
    bis-benzylpencilloyl-diaminohexane, for example, can elicit
    anaphylactic reactions (Schneider, 1983).

         It is still not completely understood how benzylpenicillin itself
    and its active low molecular weight metabolites could so rapidly react
    in vivo to form such divalent or polyvalent antigens.  Positive
    skin tests have been obtained with the following haptens in patients
    allergic to penicillin:  benzylpenicillin, benzylpenicilloic acid,
    benzylpenicillin-oligomer, benzylpenicillin-polymers, and carrier-
    conjugates exhibiting the following haptenic groups/determinants:
    benzylpencilloyl, benzylpenicillenyl, and penicillamine (De Weck,
    1983).

         In consequence, the above substances, and at least all divalent
    antigens carrying benzylpenicilloyl- and/or benzylpenicillenyl- and/or
    penicillamine-determinants formed upon covalent binding of reactive
    haptens to tissue and/or milk proteins of target animals should be
    considered as potential eliciting substances.  It cannot be ruled out
    that some of these molecules (if they existed as residues) could reach
    at least the mast cells of the gut mucosa following oral ingestion.

    2.3.2.2  Quantitative aspects

         The overwhelming majority of penicillin preparations causing
    reactions were administered parenterally.  Severe reactions in
    sensitive individuals after skin tests with less than one unit of
    benzylpenicillin are documented including one exceptional case where
    only 3 x 10-7 units had been applied (Bierlein, 1956).  Such data,
    however, are inappropriate to evaluate the risk of orally ingested
    residues.

         In an analysis of 151 fatalities from anaphylactic penicillin
    reactions, it was reported that one patient died after an oral dose in
    the range of 0.1 to 0.5 mega-units of benzylpenicillin (Idso et
    al., 1968).

         Severe reactions following the use of penicillin tablets have
    also been reported.  A fatality occurred after administration of one
    tablet of 1000 units (Guthe et al., 1958).

         Siegel has described experiments in which sera obtained from
    patients with high IgE titers were used to sensitize skin sites on
    normal subjects maintained on a milk free diet.  Forty-eight to 72
    hours later, the recipients were given oral doses of benzylpenicillin
    in water.  In one experiment, the smallest oral dose of
    benzylpenicillin which could induce a whealing reaction was found to
    be 40 units with a time to occurrence of 50 minutes.  With penicillin
    administered in milk as a diluent, threshold levels were slightly
    elevated and time to occurrence was slightly prolonged.  The needed
    doses to elicit a similar reaction in allergic donors of such sera
    would probably be lower (by a factor of 100 to 1000 or even more)
    (Siegel, 1959).

         An acute allergic reaction in a patient who had ingested milk
    from a commercial supply which contained approximately 10 units/ml has
    been reported (Wicher et al., 1969).

         In a highly sensitized 25-year-old woman, less than 1 unit of
    daily orally administered penicillin was sufficient to provoke
    allergic symptoms.  This patient suffered from a moderately severe
    subacute eczematous eruption, which was traceable to penicillin-
    contaminated milk.  The patient's symptoms were relieved by addition
    of penicillinase to the milk she consumed (Borrie & Barret, 1961).

         A report documented a single case of acute angioedema and
    pruritus in a penicillin-allergic patient who ingested freshly
    processed meat from a pig injected with penicillin three days before
    slaughter.  The patient noted symptoms after the first bite of ground
    pork.  Analysis revealed a penicillin content of 0.3-0.45 units/gram
    of meat (Tscheuschner, 1972).

         Lindemayr and co-workers challenged nine penicillin-allergic
    volunteers with 150g of raw pork meat (content: 0.024-0.04 ug/g) from
    an animal treated with procaine-benzylpenicilline.  Two subjects
    reported itchy or local anesthetic sensations during the first 2
    hours.  However no objective symptoms of allergy could be observed
    (Lindemayr et al., 1981).

         Other cases (mainly anecdotal observations) have been reported. 
    There was, however, insufficient or even no evidence provided to
    support the view that penicillin was the causative agent (Dewdney &
    Edwards, 1984).

    3.  COMMENTS AND EVALUATION

         No toxicological studies were available for review.  Among the
    adverse reactions which had been reported in people consuming food
    containing benzylpenicillin residues, hypersensitivity reactions were
    the most common.  The overall prevalence of allergy to penicillin,
    taking into account various reports of allergic reactions in different
    populations and using a variety of test procedures, was estimated to
    be 3 - 10%.  There was no evidence of sensitization caused by
    benzylpenicillin residues in food.  The Committee evaluated the
    available data on allergic reactions caused by penicillin residues. 
    Only four cases were considered to be adequately documented to
    demonstrate that hypersensitivity reactions could be caused by
    ingestion of less than 40 g of the drug.

         Residues of benzylpenicillin can also inhibit starter cultures
    used in the production of yoghurt, cheese and other milk products.

         The Committee concluded that allergy was the determinating factor
    in the safety evaluation of residues of benzylpenicillin.  In the
    absence of adequate data to establish a no-effect level, the Committee
    recommended that the daily intake from food be kept as low as
    practicable, and in any case below 30 g of the parent drug.  The risk
    associated with the occurrence of mild hypersensitivity reactions at
    this level was considered to be insignificant.

    5.  REFERENCES

    ABO-ELNAGE, I.G. ABDEL-MOTTALEB, L., & MAHMOUD, M. (1973).
    Characteristics of Gouda cheese and yoghurt made from milk containing
    penicillin.   Scienza e Tecnica Lattiro-Casearia 24, 25-32.

    ANDERSON, J.A. & ADKINSON, N.F. (1987).  Allergic reactions to drugs
    and biologic agents.   JAMA 258, 2891-2899.

    ARNDT, K.A. & JICK, H. (1976).  Rates of cutaneous reactions to drugs. 
    A Report From the Boston Collaborative Drug Surveillance Program. 
     JAMA, 235, 918-922.

    BAYER, A.S., CHOW, A.W., CONCEPTION, N., & GUZE, L.B. (1978).
    Susceptibility of 40 lactobacilli to six antimicrobial agents with
    broad Grampositive anaerobic spectra.   Antimicrobial Agents and
     Chemotherapy, 14, 720-722.

    BIGBY, M., JICK, S., JICK, H., & ARNDT, K. (1986).  Drug induced
    cutaneous reactions. A report from the Boston Collaborative Drug
    Surveillance Program on 15,438 consecutive inpatients.  JAMA, 256,
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    BIERLEIN, K.J. (1956).  Repeated anaphylactic reactions in a patient
    highly sensitized to penicillin. A case report.   Ann. Allergy, 14,
    35-40.

    BORRIE. P., & BARRET, J. (1961).  Dermatitis caused by penicillin in
    bulked milk supplies.   Br. Med. J., 2, 1267.

    BYCROFT, B.W. & SHUTE, R.E. (1985).  The molecular basis for the mode
    of action of beta-lactam antibiotics and mechanisms of resistance. 
     Pharma. Res., 1985,3-14.

    COGAN, T.M. (1972).  Susceptibility of cheese and yoghurt starter
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    DESWARTE, R.D. (1985).  Drug Allergy. in: Patterson, R. (ed.) Allergic
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    DEWDNEY, J.M. & EDWARDS, R.G. (1984).  Penicillin hypersensitivity-is
    milk a significant hazard?  J. Roy. Soc. Med., 77, 866-877.

    DE WECK, A.L. (1983).  Penicillins and Cephalosporins. in: Allergic
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    DOLEZALEK, J. & BEHAVKOVA, A. (1974).  Effect of penicillin on the
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    GUTHE, T., IDSO, O., & WILLCOX, R.R. (1958).  Untoward penicillin
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    HUBER, W.G. (1988).  Penicillins. in: Booth, N.H. & McDonald, L.E.
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    IDSO, 0. & WANG, P.N. (1958).  Penicillin-sensitive reactions in
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    IDSO, O., GUTHE, T., WILLCOX, R.R., & DE WECK, A.L. (1968).  Nature
    and extent of penicillin side-reactions, with particular reference to
    fatalities from anaphylactic shock.   Bull. Wld. Hlth. Org., 38,
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    JAKIMOV, N. (1970). Antibiotics in milk and their effect on lactic
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    KONDRATENKO, M., SHISHKOVA, I., TSANEVA, K. & G'OSHEV, B. (1978).
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    LEVINE, B.B. & VAZ, N.M. (1970). Effect of combination of inbred
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    LINDEMAYR, H., KNOBLER, R., KRAFT, D., & BAUMGARTNER, W. (1981). 
    Challenge of penicillin-allergic volunteers with
    penicillin-contaminated meat.   Allergy, 36, 471-478.

    LOUSSOUARN, S. (1982).  Sensitivity of lactic cultures to certain
    antibiotics.  Technique Taitiere 965, 49-50.

    MANDELL, G.L. & SANDE, M.A. (1985). Antimicrobial agents. penicillins,
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    MARSH, D.G. (1975).  Allergens and the genetics of allergy. in: Sela,
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    METZGER, H. (1988).   Molecular aspects of receptors and binding
    factors for IgE.   Adv. in Immunol., 43, 277-312.

    OLSON, J.C., JR. & SANDERS, A.C. (1975).  Penicillin in milk and milk
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     Food Technol., 38, 630-633.

    SCHNEIDER, C.H. (1983).  Immunochemical basis of allergic reactions to
    drugs. in: Allergic reactions to drugs, De Weck, A.L. & Bundgaard, H.
    (eds.), Springer-Verlag, Berlin, pp. 3-35.

    SIEGEL, B.B. (1959).  Hidden contacts with penicillin.  Bull. Wld.
     Hlth Org. 21, 703-713.

    TERPLAN, G., & ZAADHOFF, K.J. (1967).  On the incidence and detection
    of inhibitory substances in milk - a short review (orig. in German).
     Milchwissenschaft, 22, 761-771.

    TSCHEUSCHNER, I. (1972).  [translation from German:] Anaphylactic
    reaction to penicillin after ingestion of pork.  Z. Haut
     Geschleantskr., 47, 591-592.

    WAL, J.M. (1980). Enzymatic unmasking for antibodies of penicilloyl
    residues bound to albumin.   Biochem. Pharmacol., 29, 195-199.

    WAL, J.M. & BORIS, G. (1975).  Elimination of free penicillin and
    penicilloyl-protein conjugates in the milk of cows following
    intramammary administration of penicillin G.   Annales biol. animale
     biochim. biophys., 15, 615-617.

    WICHER, K., REISMAN, R.E., & ARBESMAN, C. E. (1969).  Allergic
    reaction to penicillin present in milk.  JAMA, 208, 143-145


    See Also:
       Toxicological Abbreviations