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    WHO Food Additives Series, 1972, No. 4




    EVALUATION OF MERCURY, LEAD, CADMIUM
    AND THE FOOD ADDITIVES AMARANTH,
    DIETHYLPYROCARBONATE, AND OCTYL GALLATE




    The evaluations contained in this publication were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives which met in Geneva,
    4-12 April 19721






    World Health Organization
    Geneva
    1972





                   

    1 Sixteenth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 505; FAO
    Nutrition Meetings Report Series, 1972, No. 51.


    DIETHYL PYROCARBONATE

    Biological data

    Biochemical aspects

    Diethyl pyrocarbonate is rapidly hydrolyzed by water with the
    formation of carbon dioxide and ethanol.  At pH 3 and 22-25C it is
    completed in four hours.  A rise in pH somewhat increases the rate of
    hydrolysis.  Diethyl pyrocarbonate reacts to a slight extent by
    carbethoxylation with the constituents of the beverages.
    Investigations using labelled diethyl pyrocarbonate revealed that it
    reacts principally with amino acids, polyphenols, hydroxy acids,
    ascorbic acid and ethanol.  The predominant reaction, however, remains
    the normal hydrolysis to CO2 and ethanol.  The reaction products
    formed with individual ingredients of the beverages are present only
    in very small amounts of the order of a few ppm and frequently at a
    level of less than 1 ppm.  Measurements using 14C-labelled diethyl
    carboxylation of constituents of the beverages, when 100 mg diethyl
    pyrocarbonate was added to one litre of beverage: in apple juice, 2
    ppm; in red grape juice, 4 ppm; in lemon juice, 6 ppm; in orange
    juice, 48 ppm; in orange drink, 2 ppm; in blackcurrant juice, 13 ppm;
    wine, 15 ppm; beer, 48 ppm (Anon., 1965).

    With the exception of carbethoxylated ascorbic acid all the
    carbethoxylated derivatives of the beverage components are hydrolyzed
    by enzymes of the intestine, pancreas and liver to carbon dioxide and
    the basic substances.  The following substances were investigated in
    this respect; tricarbethoxy gallic acid, dicarbethoxy chlorogenic
    acid, mono- and di-carbetboxycathechin, carbethoxylactic acid,
    N-carbethosy glycine, N-carbethoxy-L-proline, N-carbethoxy-L-valine,
    N-carbethoxy-L-glutamic acid, N-carbethoxy L-lysine,
    E-carbethoxy-L-lisine, N-carbethoxy-threonine, N-carbethoxy
    methionine, N-S-dicarbethoxycysteine and diethyl carbonate (Lang,
    1965). Therefore, it seems very unlikely that the carbethoxy
    derivatives are absorbed from the gut as such or are accumulated in
    the body.  Mono- and dicarbethoxy ascorbic acid are not enzymatically
    hydrolyzed, but spontaneous decomposition occurs (with a half-life
    time of five to 10 days) to carbon dioxide, ascorbic acid,
    dehydroascorbic acid, diketogulonic acid and furfural (Anon., 1965).

    In the reaction products resulting from treatment with diethyl
    pyrocarbonate, early analytical studies did not reveal the presence of
    ethyl urethane (Anon., 1965; Lang, 1965).  However, a repeat of model
    studies using gas chromatography and levels of 340-680 ppm NH4+/NH3
    and 800-1600 ppm DEPC from pH 4 to 10 showed urethane to be present,
    with a 5-12% (22-53 ppm) conversion to urethane at pH 4 (Gejvall &
    Lfroth, 1971).  A recent investigation using isotopic dilution
    analysis claimed the formation of 0.2-3.0 ppm urethane in orange
    juice, white wine and beer when treated with 250-1000 ppm of
    tritium-labelled DEPC. Specifically, 1.3 ppm of urethane was reported
    in beer with an ammonium content of 2 ppm, when treated with 500 ppm

    of the labelled DEPC (Lfroth & Gejvall, 1971).  DEPC reacts with
    ammonia at neutral or alkaline (preferably) pH to form urethane.  The
    reaction may also theoretically occur at acid pH where NH4+/NH3
    equilibria exists.  Model experiments using high levels of DEPC (1800
    ppm) and 100 NH4+/NH3 at pH 3.5 were shown to produce 30-250 g/litre
    urethane as determined by gas chromatography.  From these results it
    was concluded that normal use of DEPC (100-200 ppm), if 50 ppm
    NH4+/NH3 was present, would give rise to less than 10 g/litre
    urethane.  Further investigation using model systems with a method
    sensitive to 5 g/litre urethane pointed to possible levels of 100
    g/litre being formed when 230 ppm DEPC and 100 ppm NH4+/NH3 were
    reacted at pH 4.  However, since natural fruit juices were found to
    contain less than 30 ppm of NH4+/NH3 at pH 3.3 (Anon., 1971), the
    formation of urethane in DEPC treated beverages would be expected to
    be much lower.  Moreover, a gas chromatographic method sensitive to
    100 g/iitre has shown the presence of urethane at the 190 and 160
    g/litre level when beer, with a natural ammonium content of 2.4 ppm,
    was treated with 400 and 500 ppm of DEPC, respectively.  Upon
    treatment of the beer with an additional 100 ppm NH4+/NH3 and 500
    ppm DEPC, 410 g/litre urethane was reported to be formed.  However,
    no urethane was observed, at the sensitivity of the method, when 300
    ppm DEPC was substituted in the aforementioned experiment.  A further
    study using combined GLG/MS has shown the absence of urethane at a
    sensitivity of 50 g/litre in a sample of orange drink treated with
    600 ppm of DEPC (Coltec, unpublished results, 20 December, 1971).

    Recent work carried out with model beverages containing ammonium ions
    (0-60 ppm) and with a pH range 2.8-4.5 showed that the maximum amount
    of urethane (ethyl carbamate) likely to be formed was 50 g/litre at
    treatment levels up to 300 ppm DEPC.  These values have been found
    using a method giving reasonably reliable results at 25 g/litre.
    Tests on original beverages only produced reliable results if the
    amounts of 50 g/litre and higher were being determined.  Analysis of
    orange juice (NH4 content 17 ppm, pH 3.4) gave values of 25 g/litre
    urethane compared with 180 g/litre claimed by Lfroth and Gejvall.
    Other commercial fruit juices (pH 2.5-3.2, NH4+ 1-4 ppm) showed
    urethane levels of <10 g/litre.  Further tests on wine (pH 3.2-3.6,
    NH4+ 17-67 ppm) produced values for urethane of <5O g/litre
    compared with 2600 g/litre claimed by Lfroth and Gejvall.  Tests on
    beer (pH 3.9, NH4 1.5 ppm) treated with 100 ppm DEPC showed <5
    g/litre methane (Farbenfabriken Bayer, AG, 1972).  Determination of
    urethane formation was carried out using 14C-labelled DEPC in orange
    juice, grapefruit juice (pH 3.0-3.2, NH4+ 6-22 ppm), in wine (pH 3.2,
    NH4+ 25 ppm) treated with 300 ppm DEPC.  Urethane was present in
    fruit juice at levels of 14 g/litre and in wine at 40 g/litre
    (Fischer, 1972).  When freshly squeezed grapefruit juice was treated
    with DEPC at 300 ppm, urethane was detected using GLC with
    confirmation by MS.  The DEPC itself was devoid of any urethane.  The
    method used had a sensitivity somewhat below 10 g/litre (U.S., FDA,
    1972).

    Using 14C-labelled carbethoxy ascorbic acid balance studies showed
    that within 24 hours 18-22% of the orally given activity was
    eliminated in the faeces, 11-22% in the urine and 50-67% as CO2 in
    the breath. 0.4-1% was found in the content of the intestine and
    1.27-1.35% in the organs and carcass of the rats (Lang, 1965).  The
    same results were also obtained in the laboratories of the
    Farbenfabriken Bayer (Anon., 1965).  The intravenous administration
    into rats of 10 mg/kg body-weight of the ascorbic acid derivative
    showed a different pattern of elimination as compared to oral
    administration.  About 60% of the activity was eliminated in the
    urine, 1% in the faeces and the remainder as CO2 in the breath with
    the exception of a small amount (1-3%) not eliminated within 48 hours. 
    In the bile less than 1% was eliminated.  The loss of ascorbic acid in
    diethyl pyrocarbonate treated beverages is far less than that found on
    pasteurization (Anon., 1965).

    Acute toxicity

                                                                

    Animal    Route          LD50                  References
                        mg/kg body-weight
                                                                

    Mouse     oral           1 558                 Anon., 1966

    Rat       oral             850                    "
              i.p.              47                    "

    Cat       oral           100-250                  "

    Rabbit    oral           500-750                  "

    Dog       oral           > 500                    "
                                                                

    Toxicity on inhalation was tested on rabbits, guinea-pigs, rats and
    mice.  One hour exposure at a concentration of 10 ppm was lethal.
    Chronic respiratory symptoms were produced after one hour inhalation
    of 1 ppm.  Prolonged contact with the skin causes erythema which may
    lead to vesicle formation after contact for one hour or more.  The
    substance is also irritant to the eyes and mucous membranes (Hecht,
    1961).

    After a short time, in the beverages treated with diethyl
    pyrocarbonate no unchanged pyrocarbonate is present because of its
    rapid hydrolysis to carbon dioxide and water.  However, very small
    amounts react with the components of the beverages yielding
    carbethoxylated derivatives.  The LD50 of representative
    carbethoxylated compounds was, therefore, estimated.  The values
    ranged from >1000 to >3000 mg/kg body-weight on oral administration
    from 250 to >1000 mg/kg body-weight on intraperitoneal
    administration.

    Short-term studies

    Rat

    Twenty young male rats were given 0.25 ml/kg body-weight of diethyl
    pyrocarbonate in form of an oily 10% solution 13 times within four
    months.  Twenty controls were treated in the same way with peanut oil
    without diethyl pyrocarbonate.  Poisoning symptoms were not noticed.
    However, the test groups showed a decreased food intake and weight
    gain.  During the experiment six animals of the test group and one of
    the control group died.  Two test animals were killed, after having
    been treated 10 times, for histological examination which did not show
    any abnormal picture (Hecht, 1961).

    In another experiment two groups of 15 male animals each were fed the
    same diet.  Both groups received grapefruit juice instead of drinking
    water.  In the test group 0.5% diethyl pyrocarbonate was added to the
    juice every day for 59 days.  The animals were observed for 24 more
    days.  No symptoms of poisoning were observed (Hecht, 1961).

    Four groups of 25 male and 25 female rats each received grape fruit
    juice instead of drinking water for 28 days. One group drank the juice
    mixed with 0.5% diethyl pyrocarbonate, the mixture being permitted to
    stand two days before use so that the pyrocarbonate was completely
    hydrolyzed.  Another group was given a freshly prepared mixture of
    grape juice with 0.5% diethyl pyrocarbonate. Two groups served as
    controls.  In the test groups there was some delay in weight gain.  It
    seems likely that this was due to a diminished food intake.
    Unfortunately, food intake was not measured in this experiment. 
    Oxygen consumption and the respiratory quotient showed no differences
    between the groups (Bornmann & Loeser, 1961).  The same experiment was
    repeated with the same number of animals for eight weeks.  At the
    start the males had an average weight of 165 g, the females of 140 g.
    In this experiment no influence of the diethyl pyrocarbonate treated
    grape juice was seen on weight gain, reproduction, blood picture,
    histopathology of the organs and weight of pituitary gland, thyroid,
    suprarenals and ovaries (Bornmann & Loeser, 1961).

    Fifteen young male rats were fed for four weeks a diet consisting of
    seven parts of wheat flour and three parts of whole milk powder
    stirred into a paste with a little water, mixed with 2% of diethyl
    pyrocarbonate and then dried for a few hours at 90C.  The controls
    were fed the same untreated diet.  With the exception of a delayed
    weight gain no toxic symptoms were observed (Hecht, 1961).

    Four groups of 12 young male rats each were fed 0, 100, 200 and 500
    mg/kg body-weight of the reaction product of ascorbic acid and diethyl
    pyrocarbonate for four weeks.  All rats tolerated the treatment
    without noticeable adverse effects on weight gain, blood picture,
    organ weight, macroscopic and microscopic appearance of the organs,
    and urine composition (Anon., 1965).

    Nine groups of 25 male and 25 female rats were given in their drinking
    water either sucrose (11%), wine, wine + 200 ppm DEPC, beer, beer +
    150 ppm DEPC, orange juice, orange juice + 4000 ppm DEPC, blackcurrant
    juice or blackcurrant juice + 4000 ppm DEPC.  Five animals were killed
    after two, six weeks and the rest after 13 weeks.  There were no
    substance-related abnormalities as regard behaviour, body-weight gain,
    food intake, haematology, kidney function, gross and histopathology
    (Sharratt et al., 1971).

    Studies on diethylcarbonate

    As this is a reaction product of alcohol and DEPC the available
    studies are summarized.

    Acute toxicity

                                                                     
    Animal    Route          LD50                References
                        mg/kg body-weight
                                                                     

    Rat       Oral           >15 000        Bornmann & Loeser, 1966
                                                                     

    Short-term studies

    Two groups of one male and two female mongrel dogs received 0 or 1 ml
    of a 60% solution of DEC by gavage twice weekly for six weeks.  No
    adverse effects were noted on weight gain, haematology and urinalysis.
    No histopathological examination was performed (Bornmann & Loeser,
    1966).

    Long-term studies

    Rat

    Four groups of 30 male and 30 female rats received, 0, 0.015% 0.075%
    or 0.3% DEC in their drinking water for 100 weeks.  No adverse
    substance-related effects were noted on survival, growth, haematology,
    clinical and biochemical parameters, gross and histopathology
    (Bornmann & Loeser, 1966).

    Reproduction studies

    Rat

    Ten male and 20 female rats were used to produce P, F1, F2, and
    F3 generations.  After being kept on 0, 0.015%, 0.075% or 0.3% DEC
    from the age of four weeks.  No effects were noted on fertility,
    postnatal development nor were any teratogenic effects observed. 
    Litter size was normal in all groups (Bornmann & Loeser, 1966).

    In another experiment groups of 10 pregnant rats received 0, 0.01%,
    0.1% or 1% of DEC in their drinking water from day 6 to day 15 of
    pregnancy.  At Caesarean section on day-20 no differences were seen as
    regards implantation, resorption, foetal or placental weight and
    malformations between controls and test animals (Lorke, 1969).

    Hamster

    Three pregnant hamsters were injected i.p. on day-8 of pregnancy with
    0.4 or 0.9 g/kg DEC.  Examination on day-13 showed some increased
    resorption and malformations at the highest level tested.

    Comments on the experimental studies reported

    In the case of diethyl pyrocabonate the problem is to measure the
    toxicity of the reaction products of diethyl pyrocarbonate with food
    components, as some 8% of added DEPC reacts in this way.  However
    long-term feeding experiments with these reaction products are
    impractical to carry out as the reaction products occur in the
    foodstuffs in very minute quantities.  One such reaction product,
    diethyl carbonate has been shown not to be carcinogenic or teratogenic
    when given orally to rats.

    Large quantities of fruit juice or wine treated with diethyl
    pyrocarbonate, when given to the animals over long periods, may cause
    injuries, which are unrelated to the substance under test, e.g. teeth
    erosions and their consequences due to a prolonged administration of
    acid fruit juice or ethanol.  Therefore, evaluation can be based on
    biochemical rather than long-term toxicity studies as recommended in
    such cases by the FAO/WHO Joint Expert Committee (WHO, 1958).  Because
    of ready hydrolysis into carbon dioxide and the respective basic
    foodstuff component, it seems unlikely that the reaction products of
    diethyl pyrocarbonate are absorbed as such from the intestinal tract.
    It seems even less likely that they accumulate in the body.  The only
    toxicological problem raised by diethyl pyrocarbonate treatment of
    fruit juices, wine and other beverages containing small amounts of
    ammonia, amino acids and proteins are those arising from the formation
    of urethane, a known carcinogen.  Earlier claims stated that at
    treatment levels of 300 ppm DEPC, at pH below 4.5 and low amounts of
    ammonia, less than 10 g/litre urethane was formed.  More recent
    claims put this figure at 200-1000 g/litre but these have not been
    substantiated and the latest studies show, that except in wine, levels
    do not exceed 10 g/litre.  In treated wine, however, up to 50
    g/litre may be present, some of which may have arisen from natural or
    other sources.  Urethane is carcinogenic in animals, if relatively
    high doses are used, e.g. 0.1-0.3 g/kg i.p. or 0.3-1 g/kg orally.
    Tumours are induced in rats, mice, dogs and rabbits but not in
    guinea-pigs, monkeys and hens after oral administration.  Mice are the
    most sensitive species.  Adult animals excrete or degrade more than
    90% of orally administered urethane within 24 hours but newborn
    animals are less able to metabolize it.  Long-term injection
    experiments suggest that about 100 mg/kg represents the no-effect

    level for adult mice and 50 mg/kg for newborn mice in conventionally
    sized groups of animals.

    EVALUATION

    The use of diethyl pyrocarbonate has been reconsidered in view of the
    suspicion raised concerning the presence of urethane in beverages
    treated with 300 ppm DEPC at pH 4.5 or less.  The most recent
    analytical work has shown that detectable levels of urethane in soft
    drinks do not exceed 10 g/litre.  When arriving at their previous
    decision the Committee had already recognized the presence of urethane
    at this level.  However, amounts above 10 g/litre, arising from the
    action of DEPC, are not regarded by the Committee as admissible.  For
    these reasons the Committee decided to reduce the previously accepted
    level of treatment and to limit its use to certain beverages at pH 4
    or less.

    MAXIMUM LEVEL OF TREATMENT OF SOFT DRINKS, CARBONATED OR NOT: 250 ppm

    Limitation of use

    Beverages with pH above 4.0 and with significant content of ammonia,
    amino acids and proteins, e.g. milk and milk products, should not be
    treated with DEPC.  The use of DEPC in other beverages such as beer,
    fruit juices and fruit "nectars" is not technologically justified.  A
    minimum time interval of 24 hours should be provided between the
    treatment of the beverages and their consumption.

    Although there may be a natural low level of urethane in fermented
    products like wine, DEPC should not be used in wine.  There may be
    circumstances when the residual amount of total sulfur dioxide can be
    considerably reduced as a result of the use of DEPC but the high level
    of urethane produced in wine by DEPC is unacceptable.

    REFERENCES

    Anon. (1965) Unpublished report by Farbenfabriken Bayer submitted to
    WHO

    Anon. (1966) Unpublished report by Farbenfabriken Bayer, 19 October
    1966, submitted to WHO

    Anon. (1971) Unpublished report by Farbenfabriken Bayer, 1 July 1971,
    submitted to WHO

    Anon. (1972) Unpublished report, 15 March 1972, submitted to WHO

    Bornmann G. & Loeser, A. (1961) Arch. Toxikol., 19, 69

    Bornmann, G. & Loeser, A. (1966) Arch. Toxikol., 22, (2), 98

    Coltec, Unpublished data, 20 December 1971

    Farbenfabriken Bayer, A.G. (1972) Report dated 15 March 1972 submitted
    to WHO

    Fischer, E. (1972) Ztsch. Lebensm. - Unters. Forsch., 148 (4) (in
    press)

    Gangolii, S. D. (1971) Unpublished report by BIBRA submitted to WHO

    Gejvall, T. & Lfroth, G. (1971) EMS Newsletter No. 5, November 1971

    Hecht, G. (1961) Z. Lebensmitt. Untersuch., 114, 292

    Lang, K. (1965) Unpublished report to the Deutsche
    Forschungsgemeinschaft

    Lfroth, G. & Gejvall, T. (1971) Science, 174, 1248

    Lorke, D. (1969) Report No. 1203 submitted by Farbenfabriken Bayer AG

    Sharratt, M. et al. (1971) Unpublished report by BIBRA submitted to
    WHO

    U.S. Food and Drug Administration, Unpublished data 1972

    World Health Organization (1958) Wld Hlth Org. techn. Rep. Ser., No.
    144, p. 13
    


    See Also:
       Toxicological Abbreviations
       Diethyl pyrocarbonate (FAO Nutrition Meetings Report Series 40abc)
       Diethyl pyrocarbonate (WHO Food Additives Series 5)
       DIETHYL PYROCARBONATE (JECFA Evaluation)