FAO Nutrition Meetings
Report Series No. 40A,B,C
WHO/Food Add./67.29
TOXICOLOGICAL EVALUATION OF SOME
ANTIMICROBIALS, ANTIOXIDANTS, EMULSIFIERS,
STABILIZERS, FLOUR-TREATMENT AGENTS, ACIDS AND BASES
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
1967
DIETHYL PYROCARBONATE
Chemical name Diethyl pyrocarbonate, diethyl
dicarbonate
Empirical formula C6H10O5
Structural formula
O O
" "
C2H5OC-O-COC2H5
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
odour
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.
1966).
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
(mg/kg
body-weight)
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,
1961).
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).
Comments
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.
Evaluation
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.
REFERENCES
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