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
Geneva
1972
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.
AMARANTH
Biological data
Biochemical aspects
Adult rats were given intravenous injections of the colour. The bile
was collected for six hours and an average of 53% (43-79%) of the
quantity of the colour administered was found (Ryan & Wright, 1961).
Six adult rats were given a single oral dose of 100 mg as aqueous
solution per animal. Only 0.45% of the dose administered was found in
the faeces collected over a period of 48 hours. A single oral dose of
50 mg per animal was administered to four rats. Only 2.8% was
absorbed from the gastro-intestinal tract; the metabolites in the
urine and bile were predominantly products resulting from the
reductive fission of the azo-linkage, such as 1-amino4-naphthalene
sulfonic acid and 1-amino-2-hydroxy-3, 6-naphthalene disulfonic acid.
The former compound was found also in the faeces. The liver enzyme
that reduces azo-linkages plays little part in the metabolism, as was
shown in experiments in which the colour was given by intrasplenic
infusion. Reduction of the compound is therefore most probably
effected by the intestinal bacteria (Radomski & Mellinger, 1962).
After a prolonged administration of a daily dose of 50 mg/rat the
vitamin A content of the liver showed a threefold to fourfold
decrease. The glutathione content of the liver and spleen was
increased (Galea et al., 1962).
Acute toxicity
Animal Route LD50 References
per kg body-weight
Rat I.p. 1 g Deutsche Forsch., 1957
Rat I.v. 1 g Deutsche Forsch., 1957
Special studies
The colour was tested for mutagenic action in a concentration of
0.5 g/100 mi in cultures of Escherichia coli. No mutagenic effect
was found (Rück & Rickerl, 1960).
Short-term studies
Rat
A group of five young rats were given the colour subcutaneously twice
daily for three days. The rats were killed on the fourth day. The
colour was administered in aqueous solution at a level of 250 mg per
kg body-weight each injection. No estrogenic activity (normal uterine
weight) was detected in comparison with a control group. No other
abnormalities were found (Graham & Allmark, 1959).
Guinea-pig
In experiments with guinea-pigs, it was found that this colour had no
sensitization activity (Bär & Griepentrog, 1960).
Cat
The test for Heinz bodies was negative after injecting four cats with
a 5% aqueous solution of this colour at a level of 1 g on the first
day and 0.1 g on the ninth and on the eighteenth day. (Deutsche
Forsch., 1957).
Long-term studies
Mouse
Twenty mice were fed 15-20 mg of this colour five days a week for
periods up to 477 days. Autopsies were conducted on 18 of the mice.
No lesions were observed in the one liver examined (Cook et al.,
1940).
Feeding studies were-conducted with C3Hf and C57Bl mice; 100 of each
strain were fed at 1.0 and 2.0% and 200 of each strain served as
controls. No tumours were observed in the mice of either strain
(U.S., FDA, 1964).
Two groups of 100 mice received either 0 or 0.01 g amaranth paste
(= 0.004 g amaranth) by gavage daily. One drop of either
9,10-dimethyl-2-benzanthracene or 3,4 benzypyrene was applied once per
week to interscapular skin. Papillomata appeared 3.5 weeks earlier in
test animals and in a greater number of animals. A larger percentage
became malignant in test animals (Baigusheva, 1968).
Three groups of 15 male and 15 female rats were given diets containing
0.03%, 0.3% and 1.5% of the colour for 64 weeks. The mortality of the
rats was the same as that in a similar control group. At a level of
1.5% a significant decrease in growth rate was found in female rats
but not in male rats. This was attributed to an effect on food
utilization rather than on food consumption. Female rats fed the
colour at 0.3% and 1.5% showed an increase in liver weight. At the
higher concentration there was also an increase in kidney weight. No
influence on food intake, histo pathology and blood-picture and no
significant difference in tumour incidence was found (Mannel et al.,
1958).
Ten rats were fed the colour at a level of 0.2% in the diet. Each
animal received an average of 0.1 g/kg body-weight per day for 417
days. The total intake of the colour was 11 g/animal. The
observation period was 830 days. One intestinal carcinoma was
observed (Deutsche Forsch., 1957).
No tumours were noted in 11 rats subjected to the subcutaneous
injection of 0.5 ml of 1% solution twice weekly for 365 days. The
observation period was 879 days. The total dose administered was
0.5 g/animal (Deutsche Forsch., 1957).
At a level of 11% in the diet the colour was fed to five male and five
female rats for periods up to 18 months. Gross staining of the
glandular stomach and small intestine was observed. Granular deposits
were noted in the stomach, small intestine and in some cases in the
colon. A lymphosarcoma was observed. No tumours occurred in 50
control animals surviving 20 months or more (Willheim & Ivy, 1953).
This colour was injected subcutaneously into 18 rats of both sexes for
94 to 99 weeks. In general 1 ml of a 2-3% solution was injected
weekly for 693 days. No tumours were observed (Nelson & Hagan, 1953).
Groups of 24 weanling rats were fed the colour of 0.5%, 1.0%, 2.0% and
5.0% in the diet. A similar group served as controls. The animals on
5.0% showed slight growth inhibition. Gross and microscopic
examinations revealed a questionable increase in the number of mammary
tumours; two tumours were observed in the control group and 3, 3, 6
and 4 tumours respectively in the groups receiving the colour.
Additional feeding studies for two years were carried out to repeat
this result with Osborne-Mendel and Spragul-Dawley rats at 0.0, 1.0,
and 2.0% with 100 animals of each strain. There was no statistically
significant influence on the formation of tumours (U.S., FDA, 1964).
One group of 50 outbred rats was fed a diet containing amaranth paste
(40% amaranth) for 25 months. A control group of 35 rats was used.
Dietary levels ranged from 0.8% to 1.6% amaranth. Tumours of the
peritoneum and intestine began to appear in 18 survivors at 19 months.
By 25 months a total of 11 tumours was found. No tumours were seen in
controls. All tumours were histologically malignant (Baigusheva,
1968).
Two groups of 50 outbred male rats were fed on a diet containing
either 0% or 2% amaranth for 33 months, that is until all animals
died. A small depression of body-weight, compared with controls was
observed but this was not statistically significant. The 15 tumours
found (in 13 rats) included 3 lymphosarcomas, 4 sarcomas, 1
adenofibroma, 3 intestinal carcinomas, 1 hepatoma and 3 skin
carcinomas. The first tumour appeared after six months, most of the
others at 21-23 months. The author reported that not one of the 50
control rats developed any kind of tumour during the whole course of
the experiment (Andrianova, 1970).
Dog
A seven year toxicity study was carried out on female beagles. Five
dogs were fed 2% of the colour in the diet, three dogs were used as
controls. No histopathological or other abnormalities were found
(U.S., FDA, 1964).
Reproduction and teratogenicity studies
Rat
Groups of one male and four female rats were fed diets supplying 1.5
and 15 mg/kg body-weight/day of amaranth. There were two control
groups of a similar size. Each group was mated three times, at 4-5
months, 7-8 months and 10-12 months after the start of the study. The
following were studied: female fertility, gestation period, numbers of
live and dead pups born and numbers surviving for four days and one
month. Abnormalities in the pups were noted. Similar observations
were carried out on first (F1) and second (F2) generations of rats
fed on the same diets as their parents. The authors considered that
their results showed amaranth decreased fertility, increased the
number of stillbirths, produced deformities in the young and reduced
survival of the young (Shtenberg & Gavrilenko, 1971).
Groups of 13-15 pregnant rats were given 0, 7.5, 15, 30, 100 or 200
mg/kg/day amaranth by stomach tube from day 0-19 and sacrificed on day
20. No adverse effects were noted on implantation. The percentage of
dead foetuses increased in a dose-related manner. At 200 mg/kg/day
there was a foetotoxic effect. Resorptions increased from 15 mg/kg
upwards with total resorption of occasional litters at 100 and 200
mg/kg/day. No teratogenic abnormality related to amaranth was seen.
No obvious effect was notod on foetal sex (Collins & McLaughlin,
1972).
Eight groups of 16-22 pregnant rats were given 0, 15, 50 or 150
mg/kg/day of amaranth by stomach tube on days 6-15 of pregnancy and
Caesarean section was performed on day 20. No compound-related
adverse effects were noted in terms of implantation, foetal mortality,
foetal weight or reproductive performance in any of the experimental
groups as compared to the control animals. No evidence of
compound-induced teratogenic effects was noted (Industrial BioTest
Laboratories, 1972a).
In another study five groups of four pregnant Charles River rats
received from gestation day 6-15 inclusive, 0, 15, 150, 450 and 1500
mg/kg body-weight amaranth per day by gavage. Females were killed on
day 20. No abnormal effects were seen as regards maternal weight
gain, litter size, average foetal weight and number of resorptions.
No gross abnormalities related to the compound were seen (Burnett,
1972).
Rabbit
Eight groups of 10-14 pregnant rabbits were given 0, 1.5, 5.0 and 15.0
mg/kg/day of amaranth by capsule on days 6-16 of pregnancy and
sacrificed on day 29 by Caesarean section. No evidence of
compound-induced teratogenic effects were noted, nor were significant
effects seen in terms of implantation, pup weight, young born alive
and total numbers of resorptions. Average numbers of early
resorptions per litter revealed an increase which approaches
statistical significance (P =.05) for the 1.5 and 15.0 mg/kg groups
while for the 5.0 mg/kg group the increase is significant (Industrial
Bio-Test Laboratories, 1972b).
Comments on experimental studies reported
Several long-term studies have been carried out in rats but only two
studies extended over their lifespan. Long-term studies have also
been done in mice and dogs. Many aspects of the newer studies were
difficult to interpret particuarly with regard to the carcinogenic
potential of amaranth and its adverse effects on the rat foetus and
reproduction. However, the most recent studies gave somewhat
conflicting results with regard to feototoxicity although none of them
produced any evidence of teratogenic effects related to amaranth
administration. It was not possible to establish the reason for this
difference as there were divergencies in the conduct of these tests in
relation to time and route of administration. Considerable
controversy still exists over the correct route of administration in
teratogenicity tests and there is evidence that variations in this
parameter could lead to observable effects which are not
compound-related. Such factors made the interpretation of the
experimental data very difficult. The teratogenic study in rabbits
was similarly not clear-cut in its results.
EVALUATION
The new information from reproduction and other studies was considered
in conjunction with the fact that further relevant studies are in
progress at present. The Committee was unable to make a final
re-evaluation of the colour at this stage. However, the unconditional
ADI previously established has been changed to a temporary ADI, with
appropriate adjustment.
mg/kg body-weight
Temporary acceptable daily intake for man 0-0.75
Further work required
Results of various studies now in progress to be submitted within
three years.
REFERENCES
Andrianova, M. M. (1970) Vop. Pitan., 29 (5), 61
Baigusheva, M. M. (1968) Vop. Pitan., 27 (4), 46
Bär, F. & Griepentrog, F. (1960) Med. und Ernähr., 1, 99
Burnett, C. (1972) Unpublished report submitted to WHO
Collies, T. F. X. & McLaughlin, J. R. (1972) Unpublished report to
FDA submitted to WHO
Cook, J. W. et al. (1940) Amer. J. Cancer, 40, 62
Deutsche Forschungsgemeinschaft, Bad-Godesberg, Federal Republic of
Germany, Farbstoff Kommission (1957) Mitteilung 6
Galea, V., Ariesan, M. & Luputiu, G. (1962) Parmacia (Buc), 10, 531
Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol., 1,
144
Industrial Biotest Labs. Inc. (1972a) Report No. B700 submitted to WHO
Industrial Biotest Labs. Inc. (1972b) Report No. J701 submitted to WHO
Mannel, W. A. et al. (1958) J. Pharm. Pharmacol., 10, 625
Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397
Radomski, J. L. & Mellinger, T. J. (1962) J. Pharmacol. exp. Ther.,
136, 259
Rück, H. & Rickerl, E. (1960) Z. Lebensm.-Untersuch., 112, 157
Ryan, A. J. & Wright, S. E. (1961) J. Pharm. Pharmacol., 13, 492
Shtenberg, A. I. & Gavrilenko, E. V. (1970) Vop. Pitan., 29 (2), 66
U.S. Food and Drug Administration, Unpublished report submitted to
WHO, April 1964
Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1