FAO Nutrition Meetings
    Report Series No. 40A,B,C
    WHO/Food Add./67.29


    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met at Rome,
    13-20 December, 19651 Geneva, 11-18 October, 19662


    1 Ninth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, 1966 No. 40; 
    Wld Hlth Org. techn. Rep. Ser., 1966, 339

    2 Tenth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, 1967, in press; 

    Food and Agriculture Organization of the United Nations
    World Health Organization


    Chemical name                Diethyl pyrocarbonate, diethyl

    Empirical formula            C6H10O5

    Structural formula
                                      O   O
                                      "   "

    Molecular weight             162.14

    Definition                   Diethyl pyrocarbonate contains not less
                                 than 99 per cent. C6H10O5.

    Description                  Diethyl pyrocarbonate is a colourless
                                 liquid with a slightly fruity ester-like

    Caution                      Avoid inhalation of vapours and exposure
                                 to eyes, skin and mucous membranes.

    Use                          As a preservative in certain beverages,
                                 particularly effective against yeasts.

    Biological Date

    Biochemical aspects

         Diethyl pyrocarbonate is rapidly hydrolysed with the formation of
    carbon dioxide and ethanol. At pH 3 and 22-25°C, 99 per cent. is
    hydrolysed in 4 hours. A rise in pH somewhat increases the rate of
    hydrolysis. Diethyl pyrocarbonate reacts to a slight extent by
    carbethoxylation with the constituents of 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. Measurements using 14C-labelled diethyl
    pyrocarbonate showed the following residual radioactivity caused by
    the carbethoxylation or 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, 14 ppm: and in orange drink, 2 ppm (Bayer, 1965).

         With the exception of carbethoxylated ascorbic acid all the
    carbethoxylated derivatives of the beverage components examined 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: tricarbethoxygallic acid, dicarbethoxy
    chloregenic acid, mono-and di-carbethoxycatechin, carbethoxylactic
    acid, N-carbethoxy glycine, N-carbethoxy-L-proline, N
    carbethoxy-L-valine, N-carbethoxy-L-glutamic acid,
    a-N-carbethoxy-L-lysine, c-carbethoxy-L-lysine,
    N-carbethoxy-threonine, N-carbethoxy methionine,
    N-S-di-carbethoxycysteine and diethyl carbonate (Lang et al., 1966).
    Therefore, it seems 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 hydrolysed, but
    spontaneous decomposition occurs with a half-life of 5-10 days, to
    carbon dioxide, ascorbic acid, dehydroascorbic acid, diketogulonic
    acid and furfural (Bayer, 1965). In the reaction products resulting
    from treatment with diethyl pyrocarbonate, analytical studies did
    not reveal the presence of ethyl urethane (Bayer, 1965; Lang et al.

         Using 14C-labelled carbethoxyascorbic acid, balance studies
    showed that within 24 hours 18-22 per cent. of the orally given
    activity was eliminated in the faeces, 11-22 per cent. in the urine
    and 50-67 per cent. as CO2 in the breath; 0.4-1 per cent. was found
    in the content of the intestine and 1.271.35 per cent. in the organs
    and carcass of the rats (Lang et al., 1966), The same results ware
    also obtained in the laboratories of the Farbenfabriken Bayer (Bayer,
    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 per cent.
    of the activity was eliminated in the urine, 1 per cent. in the faeces
    and the remainder as CO2 in the breath with the exception of a small
    amount (1-3 per cent.) not eliminated within 48 hours. Less than 1 per
    cent. was eliminated in the bile. The loss of ascorbic acid in diethyl
    pyrocarbonate treated beverages is far less than that found on
    pasteurization (Bayer, 1965).

    Acute toxicity


    Animal   Route                      LD50            References

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

         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 1 hour inhalation of
    1 ppm (Hecht, 1961). Prolonged contact with the skin causes erythema
    which may lead to vesicle formation after contact for 1 hour or more.
    The substance is also irritant to the eyes and mucous membranes
    (Hecht, 1961).

         After a short time no unchanged pyrocarbonate is present in
    beverages treated with diethyl pyrocarbonate because of its rapid
    hydrolysis to carbon dioxide and ethanol. 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 300 mg/kg
    body-weight on oral administration and from 250 to 1000 mg/kg
    body-weight on intraperitoneal administration (Bayer, 1965).

    Short-term studies

         Rat. Twenty young male rats were given 0.25 ml/kg body-weight
    of diethyl pyrocarbonate in the form of an oily 10 per cent. solution
    13 times within 4 months. Twenty controls were treated in the same way
    with peanut oil without diethyl pyrocarbonate. No signs of poisoning
    were noticed. However, the test groups showed a decreased food intake
    and weight gain. During the experiment 6 animals of the test group and
    1 of the control group died. Two test animals were killed, after
    having been treated 10 times, for histological examination, which did
    not show any abnormalities (Hecht, 1961).

         In another experiment two groups of 15 male rats each were fed
    the same diet. Both groups received grape juice instead of drinking
    water. In the test group 0.5 per cent. diethyl pyrocarbonate was added
    to the juice every day for 59 days. The animals were observed for 24
    more days. No signs 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 received grape
    juice mixed with 0.5 per cent. diethyl pyrocarbonate, the mixture
    being permitted to stand 2 days before use so that the pyrocarbonate
    was completely hydrolysed. Another group was given a freshly prepared
    mixture of grape juice with 0.5 per cent. 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. Food intake was not measured in this experiment. Oxygen
    consumption and the respiratory quotient showed no differences between
    the groups. The same experiment was repeated with the same number of
    animals for 8 weeks. At the start the males had an average weight of
    165 g, the females 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, adrenals and ovaries (Bornmann & Loeser,

         Fifteen young male rats were fed for 4 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 per cent. of diethyl
    pyrocarbonate and then dried for a few bows at 9000. The controls were
    fed the same untreated diet. With the exception of a delayed weight
    gain no toxic signs 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 4 weeks. All rats tolerated the treatment
    without noticeable adverse effects on weight gain, blood picture,
    organ weight, gross and microscopic appearance of the organs, and
    urine composition (Bayer, 1965).


         In the case of diethyl pyrocarbonate the problem is to measure
    the toxicity of the reaction products of diethyl pyrocarbonate with
    food components. However, long-term feeding experiments with these
    reaction products are impractical as they occur in the foodstuffs In
    very minute quantities. Large quantities of fruit juice or wine
    treated with diethyl pyrocarbonate, when given to animals over long
    periods, may cause injuries which are unrelated to the substance under
    test. Therefore, evaluation is based on biochemical rather than
    long-term toxicity studies, as recommended in such cases by the Joint
    FAO/ WHO Expert Committee an Food Additives (FAO/WHO, 1958). Because
    of ready hydrolysis to carbon dioxide and the respective basic
    foodstuff components, it seems unlikely that the reaction products of
    diethyl pyrocarbonate are absorbed as such from the gastrointestinal
    tract and it seems even less likely that they accumulate in the body.

         No toxicological problems are raised by diethyl pyrocarbonate
    treatment of fruit juices, wine and other beverages containing no
    significant amounts of amino acids and proteins.


         It is not possible to give an evaluation by the usual method. The
    biochemical experiments and the short-term studies with representative
    reaction products permit an estimate of an acceptable level of
    beverage treatment for the use specified.

    Estimate of acceptable level of treatment

                       Beverages:    0-300 ppm

    The limitations of use are as follows:

         Beverages with pH greater than 4.5 and with significant content
    or amino acids and proteins, e.g. milk and milk products, should not
    be treated with diethyl pyrocarbonate.

         A minimum interval of 16 hours should be provided between the
    treatment of the beverages and their consumption.

         Where a new application is under consideration, safety evaluation
    will be necessary because the existing studies are applicable only to
    the uses already investigated.


    Bayer, A. G. (1965) Unpublished report

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

    FAO/WHO (1958) FAO Nutrition Meetings Report Series, No. 17; 
    Wld Hlth Org. techn. Rep. Ser., 144

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

    Lang, K., Fingerhut, M., Krug, E., Reinold, W. & Pauli O. (1966) 
    Z. Ernährunswiss., 6, 219

    See Also:
       Toxicological Abbreviations
       Diethyl pyrocarbonate (WHO Food Additives Series 4)
       Diethyl pyrocarbonate (WHO Food Additives Series 5)