INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
Toxicological evaluation of some food colours, thickening
agents, and certain other substancse
WHO FOOD ADDITIVES SERIES NO. 8
The evaluations contained in this publication were prepared
by the Joint FAO/WHO Expert Committee on Food Additives which
met in Geneva, 14-23 April 19751
World Health Organization, Geneva 1975
1 Nineteenth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Wld Hlth Org. techn. Rep. Ser., 1975, No. 576;
FAO Nutrition Meetings Report Series, 1975, No. 55.
The monographs contained in the present volume are
also issued by the Food and Agriculture Organization
of the United Nations, Rome, as
FAO Nutrition Meetings Report Series, No. 55A
ISBN 92 4 166008 2
(C) FAO and WHO 1975
AMARANTH
Explanation
This substance was evaluated for acceptable daily intake for man
by the Joint FAO/WHO Expert Committee (see Annex 1, Refs No. 10 and
No. 30) in 1964 and 1972.
Since the previous evaluation, 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
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-amino-4-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 affected 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, 1962).
Rats fed amaranth showed no change in liver phosphatase,
glucose-6-phosphate dehydrogenase or gluconate-6-phosphate
dehydrogenase (Manchon & Albrecht, 1972) but an increase in catalase,
succinic dehydrogenase (Rubenchik, 1962) and azoreductase (Albrecht
et al., 1973).
The absorption and elimination of 1-amino-4-naphthalene-sulfonic
acid, one of the azo-reduction products in rats were examined after
dosing by gavage, in drinking-water and mixed into the diet. Little is
metabolised by mammalian liver azo-reductase systems, almost all
reduction occurring through gut microflora. 1-amino-2-hydroxy-3,
6-naphthalene sulfonic acid was not studied as it is not well
absorbed. After intravenous administration there is rapid clearance
from the serum within two hours, 42% being excreted in the urine
within 24 hours, only 11% in the faeces (accounting for 53%) orally
administered compound was rapidly absorbed but cleared within 24
hours. Fifteen per cent. appeared in the urine, 69% in the faeces
(accounting for 84%). Of FD & C Red No.2 administered orally 10%
appear in the urine and 43% in the faeces (accounting for 53%). Part
of the absorbed reduction product is further metabolised to unknown
products (Pritchard et al., 1974).
TOXICOLOGICAL STUDIES
Special studies on reproduction and teratogenicity
Mouse
Groups of eight to 10 pregnant mice were given by gavage once
daily either from day 0-7 or day 6-18 of pregnancy doses of amaranth
corresponding to 0, 7.5, 30 or 100 mg/kg body weight. Mothers were
sacrificed on day 18 and litter parameters, resorptions and
malformations examined. No significant effects were noted on
resorption incidence or foetal deaths and foetal growth. No specific
teratogenic effects related to amaranth administration were seen
(Larsson, 1974).
The administration of 27, 90, 300 or 1000 mg/kg body weight of
amaranth to pregnant CD-1 outbred mice for 10 consecutive days from
day six of gestation by gavage had no apparent effects on midation,
maternal or foetal survival. No difference in abnormalities were seen
in skeletal or soft tissues compared with controls (Anonymous, 1972a).
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 four to
five months, seven to eight 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, 1970).
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 noted on foetal sex (Collins et al.,
1972).
To elucidate the effect of gavage versus diet incorporation on
teratological findings a number of comparative studies using the same
protocol and material were carried out on Osborne-Mendel and Charles
River CD-1 rats in three different laboratories using a uniform
protocol. Six control and four experimental groups of 20 pregnant rats
were used. All experimental females were treated by gavage with
200 mg/kg amaranth/day either from days 0-19 or days 6-15 or days 7-9
and the fourth group were given 0.2% of the dye in drinking-water from
day 0-20 corresponding to an intake of 200 mg/kg. The controls
received by gavage a solution from days 0-19, days 6-15, days 7-9, by
intubation on days 0-19, distilled water gavage days 0-19 and a group
entirely untreated and unhandled. No significant differences in
embryotoxicity were noted between experimental rats and controls for
the Osborn-Mendal strain or the CD-1 strain. The observed significant
findings, reimplants, viable foetuses, percentage resorption/litter
and weights of male and female foetuses were overall negative. The
original gavage finding was not reproduced and the earlier study had
an unusually low incidence of resorptions compared with the normal
incidence (Anonymous, 1974a).
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 (Keplinger et al., 1974).
In another study five groups of four pregnant Charles River rats
received from gestation, days 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 et al.,
1974).
The administration of 27, 90, 300 or 1000 mg/kg body weight of
amaranth to pregnant rats for 10 consecutive days (6-15 of gestation)
had no clearly discernible effect on nidation or on maternal or foetal
survival. The incidence of abnormalities in soft and skeletal tissues
of the test groups did not differ significantly from those of control
animals (Anonymous, 1972).
Five groups of 10 male and 20 female rats (Fo) were fed 0, 1.5,
15, 45 or 150 mg colour in their diet for two weeks and then mated
twice to produce the F1 generation. This was mated three times, the
F2b litters being used for producing the F2 generation. The F2
parents were mated once to produce an F3a generation. No significant
findings were seen as regards growth of the parent generations or in
the litter, weaning and teratological observations on all three
generations (Haley et al., 1972; Smith et al., 1974a and 1974b).
In another three-generation reproduction and teratology study
groups of Osborne-Mendel rats were fed 0, 30, 300, 3000 or 30 000 ppm
colour from weaning to three months and then mated to produce an F1,
F2, and F3 generation. No significant effects were seen on parent or
offspring parameters, e.g. survival, weight gain, fertility, litter
size, viability of pups, weaning performance and offspring survival.
No dye effect could be linked distinctly to any adverse effects seen
except weanling weights were significantly decreased at the highest
dose level in females of the F1d generation and in males and females
of the F2a x F2c generation. No cumulative effects were seen (Collins
et al., 1975a).
Progeny from the F1a and F3b generation of Osborne-Mendel rats
treated with amaranth in the Fo generation at levels of 0, 30, 300,
3000 or 30 000 ppm were used for teratology studies. In the F1a
generation the number of corpora lutea was lower at 30 000 ppm but
preimplantation loss per litter did not differ from controls.
Resorption and decreased mean foetal weight of progeny in F1a were
not dose-related. Implantation and survival parameters in the F3b
generation were similar to controls. No specific skeletal or soft
tissue abnormality were seen which could be correlated to dose levels
of the colour (Collins et al., 1975b).
Samples of amaranth obtained from three manufacturers were
administered to rats by gavage, or mixed in a semi-synthetic diet to
provide in each case dosage levels of 0, 15, 30, 100 or 200 mg/kg body
weight/day, from days 1-19 of pregnancy. Prenatal values for corpora
lutea, live foetuses, deciduomas, dead foetuses and foetal weight gave
no evidence of any amaranth-related deleterious effect on implantation
or embryonic survival (Khera et al., 1974).
In a study on two metabolites (sodium naphthionate and the
R-amino salt) and one intermediate of amaranth, dose levels of 15, 30,
100 and 200 mg/kg/day were administered by stomach tube to pregnant
rats during days 0-19 of pregnancy. No adverse effects were noted on
implantation, but at the two highest dose levels of naphthionate and
R-salt the multiple resorption rate was significantly higher than that
among control litters. Sodium naphthionate at the 100 mg/kg dose level
induced a significant increase in percentage of foetuses with
sternebral abnormalities, but this was not observed with the R-salt.
The latter increased the number of skeletal abnormalities at the
30 mg/kg level (considered an aberrant finding) and adversely
influenced skeletal development at the higher dose levels (Collins et
al., 1973).
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 = 0.05) for the 1.5 and 15.0 mg/kg groups while for the 5.0 mg/kg
group the increase is significant (Keplinger et al., 1974).
The administration of 27, 90, 300 or 1000 mg/kg (body weight) of
amaranth to pregnant rabbits from days 6-18 of gestation had no
clearly discernible effect on nidation or on maternal or foetal
survival. The incidence of abnormalities in soft or skeletal tissues
of the test groups did not differ significantly from those of control
groups (Anonymous, 1972b).
Hamster
The administration of up to 1000 mg/kg (body weight) of amaranth
to pregnant hamsters from days 6-10 of gestation had no discernible
effect on nidation or on maternal or foetal survival. The incidence of
abnormalities seen in soft or skeletal tissues of the test groups did
not differ significantly from those of control groups (Anonymous,
1972c).
Cat
A teratogenic and reproduction study was performed in four groups
of 12 adult female shorthair cats given 0, 300, 900 and 3000 ppm
amaranth in their diet prior to and during breeding and gestation. All
females were bred with males fed a diet of 3000 ppm amaranth. Six
females were delivered by Caesarian section at approximately 60 days
gestation, the other six were allowed to queen litters normally.
Difficulties were experienced with standardizing the pregestation
feeding period because of uncertainty about natural oestrus onset. For
similar reasons the c.s. delivered kittens were at various stages of
gestation. Implantation and resorption sites, corpora lutea, stillborn
and viable progeny were examined for 24 hours after c.s. Normally
queened kittens were weaned after eight weeks and examined. The number
of resorption sites was increased at 3000 ppm and 24-hour viability
after c.s. was reduced in the 300 ppm group. The mean body weight at
queening was lower in the 900 ppm group only but no effects were seen
at eight weeks postnatally. None of the parameters examined could be
interpreted with certainty as evidence of adverse effects (Korinke et
al., 1974). Another study is under way in four groups of 20 female
cats using 0, 80, 160 and 300 mg/kg per day of amaranth in gelatin
capsules for 15-45 pregestation days. Oestrus was synchronized and
untreated males were used for mating (Khera, 1975).
Dog
A teratogenic and reproduction study was performed in four groups
of 12 adult female beagles given 0, 300, 900 and 3000 ppm amaranth in
their diet and mated with males treated with 3000 ppm. Pregestation
treatment varied from 45 to 382 days for the first litters and from
132 to 572 days for the second litters. Caesarian section was
performed on six females in each group after 60 days gestation. The
remaining females were allowed to deliver spontaneously for two
litters. Pups were weaned until eight weeks of age. Two consecutive
litters were studied. No significant effects were noted on breeding,
body weight, food consumption of dams or on litter size, viability,
pathology and skeletal development of pups (Mastalki et al., 1975).
Special studies on mutagenicity
The colour was tested for mutagenic action in a concentration of
0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
was found (Lück & Rickerl, 1960).
FD & C Red No.2 was shown to have a mutagenic response in the
host-mediated assay for the two strains of Salmonella typhimurium
G-46 and TA-1530, particularly when the compound was administered by
multiple injection over five days. Apparently this mutagenic response
is induced by some metabolite rather than by the compound itself,
since in vitro exposure of the compound to the bacteria did not
induce mutations. Similar results were obtained in recombination tests
with yeast (Legator, 1972; Newell & Maxwell, 1972a.
No consistent responses occurred to suggest that FD & C Red
No. 2 is mutagenic to the rat as a result of a dominant lethal test.
The positive reference control compound, TEM, produced mutagenic
responses from the second through the fifth weeks of the experiment,
as would be expected from this known mutagen (Newell & Maxwell,
1972b).
Other special studies
Chick
Chick embryo studies were performed with the dye and sodium
naphthionate being administered to the yolk and air cell of varying
doses. No definite dose/response relationship was seen the compound
being less toxic at intermediate levels than at low and high dose
levels (Winbush, 1972).
Guinea-pig
In experiments with guinea-pigs, it was found that this colour
had no sensitization activity (Bar & 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 (Anonymous,
1957).
Rabbit
Six groups of nine rabbits were used in dermal irritation and
percutaneous toxicity tests employing 0.1% × 1% dye in ointment or
water vehicle. No significant dermal or systemic toxicity related to
treatment was seen (Carson, 1962).
Acute toxicity
Animal Route LD50 References
per kg body weight
Rat I.p. >1 g Anonymous, 1957
Rat I.v. >1 g Anonymous, 1957
Mouse Oral >10 g Anonymous, 1959
C.p. >0.5 g Anonymous, 1952
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 oestrogenic activity (normal
uterine weight) was detected in comparison with a control group. No
other abnormalities were found (Graham & Allmark, 1959).
Six groups of six male and six female rats were fed either basal
stock diet, a highly purified basal diet, stock diet containing 2.5%
or 5% amaranth and purified basal diet containing 2.5% or 5% amaranth
for 21 days. As the 5% level amaranth had practically no adverse
effect on weight gain or appearance but in the purified diet growth
ceased and animals died within two weeks. Vitamin supplements had no
protective effect but 10% cellulose, 10% alfalfa meal or watercress
powder protected against this dietary effect (Ershoff & Thurston,
1974).
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 C57B1 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
(Anonymous, 1964a).
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-benzopyrene 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).
Fifty male and 50 female Swiss-Webster albino mice were treated
weekly for 18 months with 0.1 ml 1% colour suspension in water. One
hundred males x 100 females were controls. No carcinogenic effects on
skin were noted (Carson, 1963, 1966).
Rat
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, histopathology 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 (Anonymous,
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 (Anonymous, 1957).
At a level of 4% in the diet the colour was fed to five male and
five female rats for periods up to 18 months. Gross staining of the
flandular 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).
Rats fed the dye at 20 mg/day for 78 weeks showed 68% mortality
compared with 13% for controls, a lowering of the vitamin A content of
liver and vacuolar dystrophy with eventual fatty degeneration of liver
cells (Galea et al., 1972).
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 three,
three, six and four 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 Sprague-Dawley rats at 0.0,
1.0, and 2.0% with 50 male and 50 female animals of each strain. There
was no statistically significant influence on the formation of
tumours. One hundred males and 100 females were used as controls
(Anonymous, 1964b).
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 out of 48 survivors) included three lymphosarcomas,
four sarcomas, one adenofibroma, three intestinal carcinomas, one
hepatoma and three 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).
When amaranth was fed to rats for up to 18 months at a dietary
level of 0.12% growth was markedly depressed, mortality was increased
and liver damage was evident. The level of vitamin A was decreased by
about 50% within a few days and continued to decrease progressively
throughout the study. A rise in serum albumin and gamma-globulin
was noted, but there were no marked increases in serum or liver
glutamic-oxaloatic or glutamic pyruvic transaminases (Galea et al.,
1962).
However, feeding of 1200, 3000, 10 000 and 20 000 ppm of amaranth
in the diet did not confirm the reduction of vitamin A reserves in the
rat liver in another experiment (Truhaut & Ferrando, 1975).
In another study unmated rats of both sexes were given by gavage
either 1.5 or 15 mg/kg amaranth for 12 months. Females showed
inhibition of oestrus cycle, increased foetal deaths, impaired
lactation. Males had decreased length of sperm life and mortality and
reduced resistance (Shtenberg & Gavrilenko, 1972).
Groups of animals from the F2a generation of a multi-generation
study using dose levels of 0, 30, 300, 3000 or 30 000 ppm in the diet
are being used for a two-year long-term study. No results are yet
available (Anonymous, 1975).
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
(Anonymous, 1974b).
OBSERVATIONS IN MAN
Of seven patients with recurrent urticaria or angio-oedema
suspected to be sensitive to azo-dyes such as amaranth, one reacted
with urticaria to amaranth provocation. Objective signs of a reaction
were observed in one further case and in three other patients
subjective symptoms were noted (Michaelson & Juhlin, 1973).
Comments:
Many long-term studies have been carried out in rats, mice and
dogs. Only two of the long-term studies indicated a carcinogenic
potential not seen in any of the other studies. These studies were
also evaluated in a report of the International Agency for Research on
Cancer (Lyon). This Committee, as well as the IARC, concluded that
because of the uncertainty about the impurity content of the amaranth
employed in these two studies, the carcinogenicity of this compound
could not be evaluated.
Several new studies on reproduction and teratology were available
for evaluation. These gave some conflicting results with regard to
foetotoxicity, although none of them produced any evidence of
teratogenic effects related to amaranth administration. Further
studies to elucidate the observations generating concern over
reproductive effects have shown that, in retrospect, a study showed
apparent adverse effects because of the unexpectedly low foetal
resorption in control animals compared with non-contemporary controls.
An extensive comparative study has failed to reproduce these effects
in the same strain. The single positive teratogenic study also
suffered from an inadequate specification for the dye employed. The
Committee took account of the fact that the new data in addition to
the new information on the deficiency of the specification of amaranth
in the positive studies previously reviewed, allowed for more precise
assessment of the ADI.
EVALUATION
Level causing no toxicological effect
Rat: 0.3% in the diet equivalent to 150 mg/kg body weight.
Estimate of acceptable daily intake for man
0-0.75 mg/kg body weight.*
FURTHER WORK OR INFORMATION
Required by 1978.
The result of long-term feeding study on the progeny of rats that
were fed amaranth during the gestation and lactation period.
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