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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN FOOD
    ADDITIVES AND CONTAMINANTS



    WHO FOOD ADDITIVES SERIES: 44





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





    World Health Organization, Geneva, 2000
    IPCS - International Programme on Chemical Safety


    POTENTIAL ALLERGENICITY OF REFINED FOOD PRODUCTS

    PEANUT OILS AND SOYA BEAN OILS

    First draft prepared by Dr J.B. Greig

    Joint Food Safety & Standards Group, Department of Health, London,
    United Kingdom

    Explanation
    Biological data
       Protein content and immunoreactivity of crude and refined oils
       Observations in humans
           Peanut oil
           Soya bean oil
    Comments
    Evaluation
    References

    1.  EXPLANATION

         The allergenicity of foodstuffs has not previously been
    considered by the Committee. The Codex Committee on Food Labelling
    considered allergens in food on a number of occasions after 1993 and
    issued a list of foods and food ingredients known to cause allergy.
    That list, with modifications, was revised at an FAO Technical
    Consultation in 1995 (FAO, 1995). After debate in the Codex Committee
    (FAO, 1998), the list was forwarded at Step 8 for adoption by the
    Codex Alimentarius Commission. The Joint FAO/WHO Expert Committee on
    Food Additives was asked by the Codex Committee on Food Labelling
    (FAO, 1998) to provide scientific advice on this issue, including the
    development of criteria for identifying products of foods on the list
    for which labelling of the food source is not necessary. An Expert
    Panel was convened to assist the Committee (see Annex 6 of the report
    of the present meeting: Annex 1, reference  143).

         In its report, the Panel developed criteria for excluding a
    product from the list, which included: (i) evidence that a clinical
    study in which neither the investigators not the participants were
    aware of who received the test substance and who received placebo
    (double-blind) had been conducted in which challenge with the specific
    product did not elicit allergic reactions in a group of patients with
    clinical allergy to the parent foodstuff, and (ii) the existence of
    specifications for the product and its manufacturing process that
    demonstrate that the process can yield a consistently safe product.
    The Panel identified only two foodstuffs that it considered may
    currently fulfil the criteria for inclusion on a list of products of
    foodstuffs for which labelling of the allergen-containing food source
    is unnecessary: refined peanut oil and refined soya bean oil. The
    Panel recommended that these be considered by the Committee at its
    present meeting.

    2.  BIOLOGICAL DATA

    2.1  Protein content and immunoreactivity of crude and refined oils

         Peanut oil is commonly marketed as a refined oil or in a crude
    form, sometimes called 'gourmet' oil, which may be used for flavouring
    purposes. The refined oil is produced by a process which involves
    degumming, neutralization, bleaching, filtration, and deodorization to
    achieve a concentration of free fatty acids of < 0.1%. Two
    alternative processes can be used to achieve this specification, one
    physical and the other chemical. A code of practice for the production
    and labelling of peanut oil in connection with peanut allergy was
    adopted in the United Kingdom and was subsequently adopted by the
    European Association FEDIOL, to be implemented no later than 1 January
    1998. All refined vegetable oils undergo the same degree of processing
    (VCH Veriagsgesellschaft mbH, 1987; Seed Crushers' and Oil Processors'
    Association, 1999).

         The mean protein content of peanut oil is reduced at each stage
    of the refining process described above. Although the results differed
    depending on whether the Lowry assay or the Pierce Micro bicinchoninic
    acid assay was used for protein analysis, there was a consistent
    downward trend with each analytical procedure. The mean protein
    content in extracts of oil prepared by two separate procedures and
    analysed by each assay were 187 g/ml of crude peanut oil; 60 g/ml of
    oil after alkali refining, neutralization, and washing; 15 g/ml of
    oil after bleaching and filtering; and 2.2 g/ml of oil after
    deodorization (Skinner & Haynes, 1998).

         The presence of soya bean proteins in phosphate-buffered saline
    (pH 7.2) extracts of eight soya bean oils of unspecified origin was
    tested by enzyme-linked immunosorbent assay. Three of the oil extracts
    showed measurable quantities, two contained less than 50% of the
    maximum concentration tested, and three were considered to contain no
    protein in comparison with negative controls (Porras et al., 1985).

         The protein in a refined, deodorized, alkali-refined, and
    steam-refined peanut oil, a refined, deodorized, alkali-refined, and
    steam-deodorized soya bean oil, and an alkali-refined and bleached
    soya bean oil was extracted by chromatography on a diethylaminoethyl
    cellulose column. After elution of non-polar, polar, and acid lipids,
    the proteins were eluted with 1 mol/L aqueous sodium chloride and 0.2
    mol/L aqueous sodium hydroxide. Only an acid hydrolysate of the sodium
    hydroxide extract of the alkali-refined and bleached soya bean oil had
    a detectable content of amino acids, estimated as being equivalent to
    0.96 g/g of oil (Tattrie & Yaguchi, 1973).

         Extracts of soya bean oil, crude and processed by unspecified
    means, and of five types of processed peanut oil from various sources
    were extracted with 0.15 mol/L phosphate-buffered saline (pH 7.4). The
    protein content of the extracts was measured with Bradford dye-binding
    reagent, and the lectin-like activity was assayed by agglutination of

    human erythrocytes. The crude and processed soya bean oils had protein
    contents of 1900 and 720 g/kg and lectin-like activities of 860 and
    45 g/kg, respectively, while the protein content and lectin-like
    activity of the peanut oils ranged from 120 to 580 g/kg and 26 to 55
    g/kg, respectively (Klurfeld & Kritchevsky, 1987).

         Oils from walnuts, almonds, hazelnuts, and macadamia nuts and
    refined and unrefined peanut oil were each extracted with 0.2 mol/L
    ammonium bicarbonate solution. The protein concentrations of the
    aqueous extracts were measured and binding to immunoglobulin (Ig) E
    was assayed by slot-blot and western immunoblotting. The IgE was
    derived from atopic sera in pooled samples from a serum bank
    consituted from persons with food allergies. Standard extracts of the
    nut and legume proteins were prepared from nuts and peanut oils from
    various sources. The analytical results indicated that those oils that
    had undergone the least processing at lower maximum temperatures had
    higher protein concentrations. Of the peanut oils, those that were
    unrefined elicited immunoreactive bands in the western immunoblotting
    assay, whereas such bands were absent from the lanes corresponding to
    the refined and deodorized oils. Confirmation of this observation was
    provided by the slot-blot immunoassay (Teuber et al., 1997).

         Various commercially available refined peanut oils from the
    European market were extracted with 0.1 mol/L sodium bicarbonate (pH
    8.0), and the protein content of the extract was analysed by the
    Pierce bicinchoninic acid method after further complex purification
    steps. Whereas the crude, cold-pressed oil had a protein content of
    3.4 g/g, a neutralized oil contained  0.2 g/g and the protein
    content of five commercial refined oils ranged from 0.1 to 0.2 g/g.
    Although protein extracts released histamine from the leukocytes of
    peanut-allergic patients, as did a micellar casein-peanut oil
    preparation in most instances, the results were not totally concordant
    and did not completely correlate with the presence of a positive
    reaction to a double-blind challenge with peanut oil. An
    immunoblotting experiment with protein extracted from two of the oils
    revealed that the extract could inhibit binding of IgE from a patient
    allergic to peanuts. Western blot analysis of the protein extract of
    refined oil showed that the reactive species was an 18-kDa protein
    with a relative molecular mass similar to that of one of the major
    peanut allergens (Olszewski et al., 1998).

         Since the Lowry and bicinchoninic acid methods have been found to
    be subject to interference, fully refined peanut and soya bean oils
    were assayed for protein content by aqueous extraction and amino acid
    analysis of an acid hydrolysate. The peanut oil had a protein content
    of 0.83 mg/kg, and three samples of soya bean oil had protein contents
    of 0.08-0.22 mg/kg (Institute of Shortening and Edible Oils, 1999).

    2.2  Observations in humans

         The allergenicity of edible oils has been reviewed (Hefle &
    Taylor, 1999).

    2.2.1  Peanut oil

         Seven male and three female patients aged 17-45 years with a
    known history of immediate hypersensitivity reaction to peanuts were
    recruited into a double-blind, placebo-controlled food challenge study
    in which a commercial peanut oil containing no detectable protein and
    olive oil (the placebo) were administered in a cross-over design. All
    the patients gave an immediate response in skin-prick tests with two
    crude peanut extracts, and all the patients had elevated serum IgE
    antibody levels to peanut allergens in a radioallergo-sorbent test
    with a crude peanut extract. The serum from nine of the 10 patients
    showed two to 11 times more binding than negative control serum to the
    major peanut allergen, peanut-1 or  Ara h I, by the
    radioallergosorbent test.

         Neither peanut oil nor olive oil elicited any reaction in
    skin-prick tests. In food challenge tests performed on two separate
    occasions at least 14 days apart, the patients being randomly
    allocated to receive peanut oil or olive oil on the first day, none of
    the patients experienced any adverse immediate or delayed reaction
    when challenged with sequential doses of 1, 2, or 5 ml of peanut or
    olive oil in gelatin capsules (Taylor et al., 1981).

         In a double-blind, placebo-controlled food challenge study in
    which four patients who were sensitive to peanuts were exposed to 30
    ml of commercial peanut oil, no reaction was recorded (Bock & Atkins,
    1989), but no additional details were given.

         Vitamin D supplementation may be administered in an oil
    preparation in early infancy. After skin-prick testing of 122 children
    aged 7-60 months who had been referred to an allergy clinic, the
    children were classified according to whether they had received a
    vitamin D preparation without peanut oil, one containing peanut oil
    that had been administered monthly, or one containing peanut oil that
    had been administered daily. Although the groups did not differ in
    respect of allergic status, statistically significantly children more
    showed a positive reaction to peanut if they had been exposed to a
    peanut oil-containing vitamin preparation. The peanut oil used in the
    vitamin preparations was not specified (de Montis et al., 1993).

         In a study in France, two male and two female infants aged 4-13
    months who had received a diagnosis of atopic dermatitis were found to
    react to peanut allergens during skin-prick testing or labial
    challenge with peanut extract, peanut butter, or peanut oil. In a
    single blind oral challenge test with peanut oil, the infants reacted
    with a rash to doses of 1 or 5 ml of peanut oil. In each case, the
    infant was receiving a formula containing peanut oil in such an amount
    that it contributed 67 or 80% of the lipids. In general, the condition
    of the children improved when they were placed on an exclusion diet,
    but the posssibility that concurrent medication contributed to the
    recovery was not ruled out (Moneret-Vautrin et al., 1994). An earlier
    letter from the same group may have concerned two of these infants
    (Moneret-Vautrin et al., 1991).

         The same group reported the results of studies in a group of six
    male and five female allergy patients aged 2-17 years. Positive
    results in skin-prick tests were found for 10/10 patients challenged
    with native peanut, 9/9 with commercially available peanut protein
    extracts, 0/9 with refined peanut oil, and 5/7 with protein extracts
    of crude or refined peanut oil. Only four of the 11 patients reacted
    adversely in a double-blind food challenge with peanut oil (not
    specified whether crude or refined) (Olszewski et al., 1998).

         A randomized, double-blind, cross-over challenge study was
    conducted to determine the allergenicity of crude and refined peanut
    oil in a group of 15 men and 54 women of a mean age of 26 years
    (range, 14-48 years) who had participated in a questionnaire study of
    peanut allergy. Each individual was subjected to skin-prick tests with
    peanut extract and with the crude and refined peanut oils. Those 62
    individuals who gave positive reactions in the skin-prick test with
    peanut (weal diameter equal to or greater than that elicited by 1%
    histamine) were tested on the same day by oral challenge with the
    oils, which were administered in a random order determined by a person
    who was not involved in assessing the participants' reactions, at
    increasing doses of 1, 5, and 10 ml. The flavour was disguised, six
    subjects being offered the oil with bread, one with soya milk, and the
    remainder with rice pudding. The onset of symptoms was monitored
    during an observation period of 10-15 min between doses. If a reaction
    occurred, at least 1 h was allowed to elapse before administration of
    the next dose.

         None of the subjects reacted to the refined oil, but six subjects
    reacted to the crude peanut oil. One subject reacted with wheeze to a
    dose of 1 ml of crude oil, four subjects reacted with oral itch,
    throat itch, or lip swelling to a dose of 5 ml of crude oil, and one
    subject reacted with oral itch to the 10-ml dose of crude oil. The
    reactions of four of these six subjects were subjective, with no
    observable or measurable sign of reaction. When the 58 individuals who
    showed reactions on skin-prick testing were challenged with peanuts,
    two ate a cumulative dose of 32 peanuts with no evidence of reaction.
    The other 56 subjects all responded to the challenge with reactions
    ranging from severe (after labial challenge) to mild (after
    consumption of four nuts). The study authors suggested that refined
    peanut oil does not induce an allergic reaction and that the true
    incidence of measurable reaction to crude peanut oil among patients
    who are allergic to peanuts may be 3.3% rather than the 10% suggested
    by the reactions of six individuals, which in four instances were
    possibly psychologically mediated (Hourihane et al., 1997).

    2.2.2  Soya bean oil

         A person who worked and lived near a soya bean mill experienced
    asthma which was dependent on the wind direction. He gave a positive
    reaction to skin scratch tests with soya bean products, including soya
    bean oil of unspecified grade. The report stated that when the oil was
    'filtered through stone' it ceased to cause a reaction (Duke, 1933).

         A study of the allergenicity of soya bean food products,
    including two brands of soya bean oil, was carried out in children who
    were passively sensitized by injection of serum from a patient
    allergic to soya beans. The patient, a woman, had experienced shock
    and collapse after eating food containing a soya bean filler. She was
    presumed to have been sensitized by inhalation of dust from a soya
    bean processing plant opposite her home. The presence of antibodies to
    soya bean in her serum was confirmed by passive sensitization of eight
    adult volunteers who were challenged one to seven days later with soya
    bean extract at the site of sensitization. All gave a positive
    reaction.

         Eight children (sex and age unspecified) were injected
    intradermally with the antibody-containing serum at two sites on the
    forearm. Before breakfast 24 and 72 h later, they drank 40-55 g of a
    soya bean oil. One brand of the oil was described as crude, but
    details of the other brand and the numbers of children ingesting each
    oil were not specified. No reactions were observed at the sensitized
    skin sites over the next 1-24 h. All of the children had positive
    reactions after ingestion of a suspension of soya bean flour in water
    (Ratner et al., 1955).

         Three men and four women aged 18-63 years who were sensitive to
    soya beans were recruited into a double-blind placebo-controlled
    cross-over study of the allergenicity of soya bean oil. The time since
    the last exposure of the subjects that had resulted in an allergic
    reaction ranged from < 1 to 10 years. The oils tested were partially
    hydrogenated, unhydrogenated, and cold-pressed soya bean oils; the
    placebo was an olive oil. The sequence of administration of the oils
    during the study was randomized. Before the start of the study, all
    the subjects reacted to a skin-prick test with soya bean extract, but
    none gave a positive reaction to a skin-prick test with the test oils.
    The percent binding of serum IgE antibody to soya bean allergens,
    assessed in a radioallergosorbent test in six of the seven subjects,
    was 230-2800% that of a pooled control serum. On the second day of the
    study, the subjects were challenged with 2, 5, or 8 ml of the assigned
    oil administered in gelatin capsules, these doses being equivalent to
    the amount that might be ingested during a meal. Each dose was
    followed by a 30-min observation period. Challenges to each of the
    other oils were made after intervals of at least six days. None of the
    subjects experienced an immediate or delayed adverse reaction, whether
    typical or atypical of an allergic reaction, to any of the soya bean
    oils (Bush et al., 1985).

    3.  COMMENTS

         The Committee recognized that the allergenicity of vegetable oils
    is highly dependent on the processes used to extract and then refine
    the oils. It was aware that several steps are involved in the refining
    process and that different producers may use variations of the basic
    procedures. In addition, in any clinical trial of the oils, the mode

    of administration, the allergic sensitivity of the subjects to the
    source material, and use of double-blind protocols can affect the
    outcome of the trial.

         The Committee was aware of studies of challenges of
    peanut-sensitive individuals with various grades of peanut oil, all
    involving a double-blind procedure. In a study from the United States
    involving 10 male and female patients with known sensitivity to
    peanuts, all gave a positive reaction in skin-prick tests with peanut
    extracts and had elevated serum titres of antibodies to peanut
    allergens. A cross-over challenge with commercial peanut oil and olive
    oil did not elicit adverse reactions, although the Committee noted
    that use of gelatin capsules to administer the oils may have masked
    any reactions of the lips and oral cavity.

         In a study in France, 11 children with symptoms possibly due to
    allergies were found to react to skin-prick tests with peanut or
    peanut protein extracts. Four of the patients reacted to a
    double-blind oral challenge with peanut oil. The origin or grade of
    the peanut oil used was not defined, and the Committee recognized that
    it may have been obtained before adoption of a revised code of
    practice for the refining of vegetable oils by the continental
    European industry. Earlier studies of French infants suggested that
    peanut oils used as a vitamin carrier or in infant formulas may have
    contained allergenic proteins.

         A randomized, double-blind, cross-over challenge study with crude
    and refined peanut oils involved a group of 62 patients in the United
    Kingdom who had reacted to skin-prick tests with peanut suspensions.
    None of the subjects reacted to challenge with refined peanut oil,
    although six reacted to the crude oil. Sixty of the 62 also reacted to
    an open oral challenge with peanuts. The Committee considered that the
    study was well designed and had good statistical power and recognized
    the value of the confirmation of the sensitivity of the subjects to
    peanuts after the double-blind challenge had been completed. Although
    the study provided adequate evidence for lack of allergenicity of the
    oil used, appropriate descriptions of the manufacturing process and
    the consequent specifications of the oil were not provided and the
    results could not be extrapolated to other oils.

         A double-blind, placebo-controlled, cross-over challenge study of
    the allergenicity of hydrogenated, partially hydrogenated, and
    cold-pressed soya bean oils was conducted in a group of seven
    individuals who had experienced allergic reactions after exposures
    that had occurred up to 10 years previously. All had positive
    reactions to a skin-prick test with soya bean extract. The titres of
    serum immunoglobulin E binding to soya bean proteins were increased in
    six of the seven patients. None of the subjects reacted to increasing
    volumes of any of the oils, although the Committee noted that use of
    gelatin capsules to administer the oils may have masked any reactions
    of the lips and oral cavity. Although the study provided some evidence

    that the oil used was not allergenic, appropriate descriptions of the
    manufacturing process and the consequent specifications of the oil
    were not provided, and the results could not be extrapolated to other
    oils.

    4.  EVALUATION

         The Committee noted the absence of clear descriptions of the
    processes that had been used to refine the peanut and soya bean oils
    tested. Additionally, comparable data on the protein content of those
    oils that were clinically tested were not available. Furthermore, the
    Committee expressed reservations about the quality of the analytical
    procedures used and the lack of validation of the methods to determine
    the concentrations of residual protein in the oils. In view of these
    considerations, it concluded that distinct processes that would
    consistently yield safe products have not been defined.

         The Committee therefore indicated that the results of studies of
    representative refined peanut and soya bean oils would be required for
    a full evaluation. Such studies should provide extensive information
    on a wide range of oils representing refining procedures throughout
    the world. Full descriptions of the refining process used and evidence
    for lack of allergenicity of these oils as determined by appropriately
    designed clinical studies should be provided. Evidence for the nature
    and quantities of protein in the oils would be essential for ensuring
    the representative nature of the oils tested.

    5.  REFERENCES

    Bock, S.A. & Atkins, F.M. (1989) The natural history of peanut
    allergy.  J. Allergy Clin. Immunol., 83, 900-904.

    Bush, R.K., Taylor, S.L., Nordlee, J.A. & Busse, W.W. (1985) Soybean
    oil is not allergenic to soybean-sensitive individuals.  J. Allergy
     Clin. Immunol., 76, 242-245.

    Duke, W.W. (1933) Soy bean as a possible important source of allergy.
     J. Allergy, 5, 300-302.

    FAO (1995)  Report of the FAO Technical Consultation on Food
    Allergies,  Rome, Italym 13-14 November 1995, Rome, Food and
    Agricultural Organization of the United Nations.

    FAO (1998)  Report of the Twenty-sixth Session of the Codex Committee
     on Food Labelling, Ottawa, 26-29 May 1998 (unpublished FAO document
    ALINORM 99/22), Rome, Food and Agricultural Organization of the United
    Nations.

    Hefle, S.L. & Taylor, S.L. (1999) Allergenicity of edible oils.
     Food Technol., 53, 62-70.

    Hourihane, J.O'B., Bedwani, S.J., Dean, T.P. & Warner, J.O. (1997)
    Randomised, double blind, crossover challenge study of allergenicity
    of peanut oils in subjects allergic to peanuts.  Br. Med. J., 314,
    1084-1088.

    Institute of Shortening and Edible Oils (1999) The protein content of
    peanut and soybean oils. Unpublished study. Submitted to WHO by R.M.
    Reeves, Institute of Shortening and Edible Oils, Inc., Washington DC.

    Klurfeld, D.M. & Kritchevsky, D. (1987) Isolation and characterisation
    of lectins from vegetable oils.  Lipids, 22, 667-668.

    Moneret-Vautrin, D.A., Hatahet, R., Kanny, G. & Ait-Djafer, Z. (1991)
    Allergenic peanut oil in milk formulas.  Lancet, 338, 1149.

    Moneret-Vautrin, D.A., Hatahet, R. & Kanny, G. (1994) Risks of milk
    formulas containing peanut oil contaminated with peanut allergens in
    infants with atopic dermatitis.  Pediatr. Allergy Immunol., 5,
    184-188.

    de Montis, G., Gendrel, D., Chemillier-Truong, M. & Dupont, C. (1993)
    Sensitisation to peanut and vitamin D oily preparations.  Lancet,
    341, 1411.

    Olszewski, A., Pons, L., Moutt, F., Aimone-Gastin, I., Kanny, G.,
    Moneret-Vautrin, D.A. & Guant, J.L. (1998) Isolation and
    characterization of proteic allergens in refined peanut oil.  Clin.
     Exp. Allergy, 28, 850-859.

    Porras, O., Carlsson, B., Fllstrm, S.P. & Hanson, L.. (1985)  Int.
     Arch. Allergy Appl. Immunol., 78, 30-32.

    Ratner, B., Untracht, S., Crawford, L.V., Malone, H.J. & Retsina, M.
    (1955) Allergenicity of modified and processed foodstuffs. V. Soybean;
    influence of heat on its allergenicity; use of soybean preparations as
    milk substitutes.  Am. J. Dis. Child., 89, 187-193.

    Seed Crushers' and Oil Processors' Association (1999) Submission to
    WHO dated 4 March 1999.

    Skinner, J.M. & Haynes, C. (1998) Measurement of protein content in
    various oils during stages of refining. Unpublished report H6672 from
    Leatherhead Food Research Association, Leatherhead, United Kingdom.
    Submitted to WHO by the Seed Crushers' and Oil Processors'
    Association, London.

    Tattrie, N.H. & Yaguchi, M. (1973) Protein content of various
    processed edible oils.  J. Inst. Can. Sci. Technol. Aliment., 6,
    289-290.

    Taylor, S.L., Busse, W.W., Sachs, M.I., Parker, J.L. & Yunginger, J.W.
    (1981) Peanut oil is not allergenic to peanut-sensitive individuals.
     J. Allergy Clin. Immunol., 68, 372-375.

    Teuber, S.S., Brown, R.L. & Haapanen, L.A.D. (1997) Allergenicity of
    gourmet nut oils processed by different methods.  J. Allergy Clin.
     Immunol., 99, 502-507.

    VCH Veriagsgesellschaft mbH (1987) Fats and fatty oils. In:  Ullman's
     Encyclopedia of Industrial Chemistry, 5th Ed., Weinheim, Vol. A10,
    pp. 199-206.
    


    See Also:
       Toxicological Abbreviations