Toxicological evaluation of some food
    additives including anticaking agents,
    antimicrobials, antioxidants, emulsifiers
    and thickening agents


    The evaluations contained in this publication
    were prepared by the Joint FAO/WHO Expert
    Committee on Food Additives which met in Geneva,
    25 June - 4 July 19731

    World Health Organization


    1    Seventeenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
    FAO Nutrition Meetings Report Series, 1974, No. 53.



         Diethyl pyrocarbonate has been evaluated by the Joint FAO/WHO
    Expert Committee on Food Additives (see Annex 1, Refs No. 13 and
    No. 30) in 1965 and 1972.

         Since the previous evaluations additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monographs have been expanded and are
    reproduced in their entirety below.



         Diethyl pyrocarbonate is rapidly hydrolyzed by water with the
    formation of CO2 and ethanol. At pH 3 and 22-25°C 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 into 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 (0.0001%).
    Measurements using 14C-labelled diethyl pyrocarbonate showed the
    following residual radioactivity caused by the carbethoxylation of
    constituents of the beverages, when 100 mg diethyl pyrocarbonate was
    added to one litre of beverage: in apple juice, 2 ppm (0.0002%); in
    red grape juice, 4 ppm (0.0004%); in lemon juice, 6 ppm (0.0006%); in
    orange juice, 48 ppm (0.0048%); in orange drink, 2 ppm (0. 0002%); in
    blackcurrant juice, 13 ppm (0.0013%); wine, 15 ppm (0.0015%); beer,
    48 ppm (0.0048%) (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-carbethoxycathechin, carbethoxylactic acid,
    N-carbethoxy glycine, N-carbethoxy-L-proline, N-carbethoxy-L-valine,
    N-carbethoxy-L-glutamic acid, alpha,N-carbethoxy L-lysine,
    epsilon-carbethoxy-L-lysine, N-carbethoxy-threonine, N-carbethoxy
    methionine, N-S-di-carbethoxycysteine and diethyl carbonate (Lang et
    al., 1966). Therefore, it seems very unlikely that the carbethoxy

    derivatives are absorbed from the gut as such or are accumulated in
    the body. Mono- and di-carbethoxy 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).

         Using 14C-labelled carbethoxy ascorbic acid balance studies
    showed that within 24 hours 18 to 22% of the orally given activity was
    eliminated in the faeces, 11 to 22% in the urine and 50 to 67% as CO2
    in the breath. 0.4 to 1% was found in the content of the intestine and
    1.27 to 1.35% in the organs and carcass of the rats (Lang et al.,
    1966). The same results were also obtained in the laboratories of the
    Farbenfabriken Bayer (Anon., 1965). The intravenous administration
    into rats of 10 mg/kg bw 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 to 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).

         In the reaction products resulting from treatment with DEPC, a
    number of studies did not reveal the presence of (ethyl) urethan
    (Anon., 1965; Lang et al., 1966), although DEPC is known to react with
    ammonia to form urethan (C2 H5. O.CO.NH2) at neutral or alkaline pH
    and theoretically also at acid pH. The earliest experiments using high
    levels of DEPC (1900 ppm (0.19%)) at pH 3-5 and 100 ppm (0.01%) NH3
    produced 30 to 250 ppb urethan as determined by gas chromatography.
    Extrapolation to treatment at pH 4 with 200 ppm (0.02%) DEPC and
    50 ppm (0.005%) NH3 predicted less than 10 ppb. Further
    investigations using model systems with a method sensitive to 5 ppb
    urethan pointed to possible levels of 100 ppb being formed when
    230 ppm (0.023) DEPC and 100 ppm (0.01%) NH4+/NH3 were reached at
    pH 4. A repeat of model studies using gas chromatography and levels of
    340 to 680 ppm (0.034% to 0.068%) NH4+/NH3 and 800 to 1600 ppm
    (0.08% to 0.16%) DEPC from pH 4-10 showed urethan to be present at
    levels of 22 to 53 ppm (0.0022% to 0.0053%). Using tritium-labelled
    DEPC it was claimed that 0.2 to 3 ppm (0.00002% to 0.0003%) urethan
    were formed in orange juice, white wine and beer treated with 250 to
    100 ppm (0.025% to 0.01%) DEPC at pH 418 and 2 to 20 ppm (0.0002% to
    0.002%) NH3. Specifically 1.3 ppm (0.00013%) urethan was reported in
    beer treated with 500 ppm (0.05%) labelled DEPC at an NH3+ content of
    2 ppm (0.0002%) (Löfroth & Gejvall, 1971).

         More recent data on typical soft drinks and juices treated with
    DEPC up to 280 ppm (0.028%) and with NH4+ range of 1 to 20 ppm
    (0.0001% to 0.002%) at pH 2.9-3.4 showed presence of urethan ranging
    from less than 5 to 10 ppb (Anon,, 1973). An improved method using

    isotope dilution of 14C-labelled DEPC sensitive to 1 ppb gave for 
    fruit juices and soft drinks, at treatment levels of 25 to 300 ppm 
    (0.0025% to 0.03%) DEPC, urethan levels from 0.7 to 14 ppb (Fischer, 
    1972), when freshly squeezed grapefruit juice was treated with DEPC 
    at 300 ppm (0.03%), urethan was detected using GLC/MS methodology, 
    the method having a sensitivity below 10 ppb. Similar investigations
    were done on wine using treatment levels of 110 to 200 ppm (0.011% to
    0.02%) DEPC, using a NH4+ range of 13 to 27 ppm (0.0013% to 0.0027%)
    and pH 3.0-3.8. Urethan levels ranging from 9 to 25 ppb were found
    (Anon., 1973). Further studies using 14C-labelled DEPC produced a
    maximum value of 40 ppb (Fischer, 1972). Natural occurrence of urethan
    was studied in untreated alcoholic beverages, juices, fermented
    cabbage, cheese, yoghurt and pickled meat. Only in some untreated
    wines was there evidence of urethan at levels of up to 10 ppb (Anon.,


    Special studies on reproduction


         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 age 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, fetal or placental weight and
    malformations between controls and test animals (Lorke, 1969).


         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.

    Special studies on the reaction products

         Diethyl carbonate is a reaction product of alcohol and DEPC. The
    available acute toxicity studies are summarized:

    Animal    Route          mg/kg bw            Reference

    Rat       Oral           15 000              Bornmann & Loeser, 1961

    Acute toxicity

    Animal    Route                    (mg/kg bw)          References

    Rat       Oral (oily solution)     1 200               Hecht, 1961
              Oral (aqueous emulsion)  1 390-1 570         Bornmann &
                                                            Loeser, 1961
              i.p. (oily solution)     100 approx.         Hecht, 1961

    Mouse     Oral                     1 558               Anon., 1966

    Rat       Oral                     850                 Anon., 1966
              i.p.                     47                  Anon., 1966

    Cat       Oral                     100-250             Anon., 1966

    Rabbit    Oral                     500-750             Anon., 1966

    Dog       Oral                     500                 Anon., 1966

         Toxicity on inhalation was tested on rabbits, guinea-pigs, rats 
    and mice. One hour exposure at a concentration of 10 ppm (0.001%) was
    lethal. Chronic respiratory symptoms were produced after one hour
    inhalation of 1 ppm (0.0001%). 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 bw on oral administration and from 250 to
    >1000 mg/kg bw on i.p. administration (Anon., 1966).

    Short-term studies


         Twenty young male rats were given 0.25 ml/kg bw 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 grape 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
    juice instead of drinking-water for 28 days. One group drank grape
    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 (Bormann & Loeser, 1961).

         Fifteen young male rats were fed for four weeks a diet consisting
    of 7 parts of wheat flour and 3 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 90°C. 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 bw 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 (0.02%)
    DEPC, beer, beer + 150 DEPC, orange juice, orange juice + 4000 ppm
    (0.4%) DEPC, blackcurrant juice or blackcurrant juice + 4000 ppm

    (0.4%) DEPC. Five animals were killed at two weeks, six weeks and the
    rest at 13 weeks. There were no substance-related abnormalities as
    regards behaviour, body weight gain, food intake, haematology, kidney
    function, gross and histopathology (Sharratt et al., 1971).


         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 and
    four times a week for a further six weeks. No adverse effects were
    noted on weight gain, haematology and urinalysis. No histopathological
    examination was performed (Bornmann & Loeser, 1966).

    Long-term studies


         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).


         No data are available.


         In the case of diethyl pyrocarbonate 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 pyrocarbonate, 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 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 possible presence of urethan, a
    known carcinogen. Earlier claims stated that at treatment levels of
    300 ppm (0.03%) DEPC a pH below 4.5 and low amounts of ammonia less
    than 10 ppb urethan were formed. More recent claims put this figure at
    200-1000 ppb but these have not been substantiated and the latest
    studies show that except in wines, levels do not exceed 10 ppb. In
    treated wine, however, levels of 20-40 ppb have been found, some of
    which may have arisen from natural or other sources. Urethan is
    carcinogenic in animals if relatively high doses are used, e.g.
    0.1-0.3 g/kg or 0.3-1 g/kg orally. A wide variety of tumours is
    induced in several aspects but not in guinea-pigs, monkeys and hens
    after oral administration. Mice are the most sensitive species. The
    lowest effective level found was 50 mg/kg/day for only 14 weeks. Adult
    animals excrete or degrade more than 90% of orally administered
    urethan within 24 hours but new-born 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 less than
    50 mg/kg for new-born mice in conventionally sized groups of animals.

         Recent evidence has been provided about the formation of ethyl
    urethan in beverages treated with DEPC. Urethan is carcinogenic,
    therefore, the previous acceptance of this treatment has to be
    revoked. Natural occurrence of ethylurethan has not been demonstrated


         No treatment level allocated.*


    Anon (1965) Unpublished report by Farbenfabriken Bayer submitted to

    Anon (1966) Unpublished report (dated 19/10/1966) by Farbenfabriken
         Bayer submitted to WHO

    Anon (1973) Unpublished report (dated 25 April 1973) by Farbenfabriken
         Bayer submitted to WHO

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

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

    Fischer, E. (1972) Ztsch. Lebensmitt. Untersuch. Forsch., 148, 221


    *    The treatment level allocated in previous evaluation has been
    withdrawn. This additive should not be used.

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

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

    Lang, K. et al. (1966) Z. Ernährungswiss., 6, 213

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

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

    Sharratt, M. et al. (1958) Wld Hlth Org. techn. Rep. Ser., No. 144, 13

    See Also:
       Toxicological Abbreviations
       Diethyl pyrocarbonate (FAO Nutrition Meetings Report Series 40abc)
       Diethyl pyrocarbonate (WHO Food Additives Series 4)