Toxicological evaluation of some food
additives including anticaking agents,
antimicrobials, antioxidants, emulsifiers
and thickening agents
WHO FOOD ADDITIVES SERIES NO. 5
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
Geneva
1974
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
Explanation
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.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
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.,
1973).
TOXICOLOGICAL STUDIES
Special studies on reproduction
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 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).
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.
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
LD50
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
Rat
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).
Dog
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
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).
OBSERVATIONS IN MAN
No data are available.
Comments:
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
equivocally.
EVALUATION
No treatment level allocated.*
REFERENCES
Anon (1965) Unpublished report by Farbenfabriken Bayer submitted to
WHO
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
AG
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)
DIETHYL PYROCARBONATE (JECFA Evaluation)