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
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 is rapidly hydrolyzed by water with the
formation of carbon dioxide 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 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 &
Löfroth, 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 (Löfroth & 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 µg/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 µg/litre
urethane. Further investigation using model systems with a method
sensitive to 5 µg/litre urethane pointed to possible levels of 100
µg/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 µg/iitre has shown the presence of urethane at the 190 and 160
µg/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 µg/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 µg/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 µg/litre at
treatment levels up to 300 ppm DEPC. These values have been found
using a method giving reasonably reliable results at 25 µg/litre.
Tests on original beverages only produced reliable results if the
amounts of 50 µg/litre and higher were being determined. Analysis of
orange juice (NH4 content 17 ppm, pH 3.4) gave values of 25 µg/litre
urethane compared with 180 µg/litre claimed by Löfroth and Gejvall.
Other commercial fruit juices (pH 2.5-3.2, NH4+ 1-4 ppm) showed
urethane levels of <10 µg/litre. Further tests on wine (pH 3.2-3.6,
NH4+ 17-67 ppm) produced values for urethane of <5O µg/litre
compared with 2600 µg/litre claimed by Löfroth and Gejvall. Tests on
beer (pH 3.9, NH4 1.5 ppm) treated with 100 ppm DEPC showed <5
µg/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 µg/litre and in wine at 40 µg/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 µg/litre (U.S., FDA,
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).
Animal Route LD50 References
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,
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
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 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 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.
Animal Route LD50 References
Rat Oral >15 000 Bornmann & Loeser, 1966
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,
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).
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).
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 µg/litre urethane was formed. More recent
claims put this figure at 200-1000 µg/litre but these have not been
substantiated and the latest studies show, that except in wine, levels
do not exceed 10 µg/litre. In treated wine, however, up to 50
µg/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.
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 µg/litre. When arriving at their previous
decision the Committee had already recognized the presence of urethane
at this level. However, amounts above 10 µg/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
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.
Anon. (1965) Unpublished report by Farbenfabriken Bayer submitted to
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
Fischer, E. (1972) Ztsch. Lebensm. - Unters. Forsch., 148 (4) (in
Gangolii, S. D. (1971) Unpublished report by BIBRA submitted to WHO
Gejvall, T. & Löfroth, G. (1971) EMS Newsletter No. 5, November 1971
Hecht, G. (1961) Z. Lebensmitt. Untersuch., 114, 292
Lang, K. (1965) Unpublished report to the Deutsche
Löfroth, 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
U.S. Food and Drug Administration, Unpublished data 1972
World Health Organization (1958) Wld Hlth Org. techn. Rep. Ser., No.
144, p. 13