AMARANTH*
Explanation
Amaranth was last reviewed by the JECFA in 1982 (Annex I,
Ref. 59) when new studies on metabolism and mutagenicity were
considered; no evidence of potential toxicity was revealed by these
studies. The temporary ADI of 0-0.75 mg/kg body weight established by
the 22nd meeting of JECFA (Annex I, Ref. 47) was extended to 1984
pending the submission of data from long-term feeding studies.
Since the previous evaluation, additional data has become
available and is summarized and discussed in the following monograph
addendum.
BIOLOGICAL DATA
Special studies on renal calcification
Groups of 25 mature (11 week old) male and female Wistar rats
were fed diets designed to provide intakes of 0, 20, 40, 80, or
1250 mg amaranth/kg body weight per day for either 28 or 90 days. Over
the 90-day period, the actual intakes were close to the target figures
but in the 28-day study, the actual mean daily intakes were 0, 15, 30,
63 or 1005 mg/kg body weight per day for males and 0, 17, 33, 69 or
1046 mg/kg body weight per day for females. As a positive control,
similar groups were fed diet containing 50% lactose for 28 days.
After lactose treatment for 28-day, there was a reduced weight
gain and an increased incidence of microscopically visible
calcification and hyperplasia of the renal pelvis.
There were no statistically significant differences in body
weight between controls and the groups given up to 80 mg amaranth/kg
body weight per day for either 28 or 90-day. In males, there was a
tendency toward reduced weight gain in the groups given 1250 mg
amaranth/kg body weight per day resulting in a statistically
significant (P<0.05) difference in body weight after 28-day but not
after 90-day. Both sexes given the highest dose level consumed more
water than controls.
* Monograph addendum
Relative kidney weights and the renal concentrations of calcium,
magnesium and phosphorus were not affected by treatment with amaranth
at any dose level and for either period of time. The overall incidence
of histopathological findings in the kidney was low but there was a
small increase in the number of high dose male animals with renal
pelvic hyperplasia and calcification after 90-day, but not after
28-day.
It was concluded that the calcification occurs only in animals
with developing senile nephrosis. The no-effect-level in this study
was 80 mg amaranth/kg body weight per day for 90-day (Ford, Butler &
Gaunt, 1983).
Long-term studies
A long-term study in rats including in utero exposure to
amaranth was performed as follows:
Amaranth was added to the diet of groups of 114 (control) or 66
(treated) Wistar-derived outbred rats of each sex to provide daily
intakes of 0 (control), 50, 250 or 1250 mg amaranth/kg body weight for
60 days prior to mating (Fo generation). The rats were then mated
monogamously, avoiding brother-sister mating, and the females allowed
to produce and rear their young with treatment continuing throughout;
the offspring were weaned on the same diet as that received by their
respective dams. When the last litter had been weaned, offspring were
selected (one of each sex only from a litter) to provide group sizes
of 90 (control) or 54 (treated) rats of each group of long-term study
(F1 generation). Treatment was continued for 3 weeks (males) or 112
weeks (female) after the F1 generation had been selected at 3-5 weeks
old. General condition, body weight, food and water intake were
monitored at frequent intervals in both generations. Haematological
examinations were carried out on blood samples from the tail vein from
20 animals of each sex from the F1 groups at 3, 6, 12 and 18 months
and on blood collected from the aorta of all survivors at termination;
the examination included PCV, haemoglobin, methaemoglobin, erythrocyte
count, total leucocyte count, differential leucocyte count and
reticulocyte count. Renal function tests were performed on 20 animals
of each sex from the F1 groups at 3, 6, 9, 12, 18 and 24 months.
Serum chemistry was investigated at termination of the F1 phase,
including urea, glucose, albumin and total protein, and the activities
of glutamate-oxaloacetate transaminase, glutamate-pyruvate
transaminase, lactate dehydrogenase and alkaline phosphatase.
Detailed post-mortem examinations were carried out on any animals
which died or were killed because of poor conditions during the study
as well as on 15 rats of each sex from each group of the f0
generation and on all survivors of the F1 generation. The organs
collected at autopsy were: adrenals, aorta, bladder, brain, caecum,
colon, epididymis, eye, gonads, Harderian gland, kidneys, liver,
lungs, lymph nodes, mammary glands, muscle (skeletal), nasal bones,
nerve (sciatic), oesophagus, pancreas, prostate, salivary glands,
seminal vesicles, skin, small intestine, spinal cord, spleen, stomach,
thymus, thyroid, tongue, trachea, uterus, vagina, vein. A film of
femoral bone marrow was also prepared. Histopathological examinations
were performed on all the tissues collected from all animals of the
F1 generation except the nasal bones and spinal cord where the
examination was confined to thise from the control and highest dose
groups.
During the observation period, the only indications that could be
associated with treatment were an external red contamination, a red
colouration of the faeces and, at the highest dose, less well formed
faecal pellets. Over 90% of the Fo generation females produced
litters irrespective of treatment and the number of young was greater
in the treated groups. At the highest dose the mean pup weight was
lower than the control although, due to the larger numbers, the total
litter weight was not reduced.
In the F1 generation, both sexes given 1250 mg amaranth/kg body
weight were slightly lighter than controls despite a small increase in
food consumption and it was concluded that the efficiency of food
utilization was reduced. There was a small (10-12%) increase in water
intake at this highest dose level but a tendency to lower volumes of
more concentrated urine in the renal function test on males. It was
concluded that the increased water intake was compensatory for
increased water loss in faeces.
There were no consistent findings in the haematological or serum
chemistry studies that could be related to treatment. Male and female
rats in the highest dose group showed slightly higher haemoglobin
concentrations in the blood obtained at autopsy but this was only
statistically significant in the females. The number of cells in the
urine was increased in both sexes at the highest doses at 18 months
and the females of this group tended to excrete more protein in urine
after 12 months. Although these were isolated observations they may be
indicative of kidney damage developing earlier in the high dose than
in the control animals.
There was no significant difference in mortality between control
and treated animals, and the incidence and distribution of tumours
seen in the F1 generation were considered to be those expected in the
strain of rat used and not to have been influenced by treatment. The
only finding in terminal organ weight analysis that was related to
treatment was an increased caecal weight seen at the two highest doses
in males in both generations, at the highest dose in females of both
generations, and at the 1250 mg/kg body weight dose level in Fo
generation females.
At all dose levels, there was an increase in the number of female
rats with calcification of the kidneys and a pelvic epithelial
hyperplasia but no significant differences were observed in the
incidence of these lesions in males even at the highest dose level. A
wide range of age-related degenerative changes were observed
histopathologically at termination but were not considered to be
related to treatment except where secondary to the kidney injury in
females of the highest dose group.
It was concluded that the exposure of rats to doses of up to
1250 mg amaranth/kg body weight and during pregnancy and lactation
followed by exposure of the offspring for over 2 years did not lead to
any carcinogenic effect. However, because of the effects on the
kidneys of the females at all the dose levels used, it was not
possible to establish a no-untoward-effect level in this study (Clode,
Hooson, Butler & Conning, 1982).
The initial histological assessment in the above study was not
performed in a random order and it was considered that this may have
influenced the assessment of the degree of changes. Consequently, the
kidney and adrenal tissues were re-evaluated in random order and
without prior knowledge of the treatment groups. The re-evaluation
confirmed that there was a dose-related trend for increased
calcification and epithelial hyperplasia in the renal pelvis of the
female F1 rats; the hyperplasia usually was present in animals with
pelvic calcification. Statistical analysis (one-tailed Fisher
exact test) showed the low dose (50 mg/kg) incidences of pelvic
calcification and hyperplasia not to be significantly different from
the control. The only finding in the males was a dose-related trend
for pelvic calcification but none of the treatment groups were
statistically significantly different from the control. In both sexes,
the senile glomerulonephrosis and the adrenal pathology for the F1
rats were not affected by treatment. There was no significant increase
in calcification at any site nor in pelvic hyperplasia in either sex
of Fo rats and glomerulonephrosis was seldom present in these younger
animals (Butler & Conning, 1983).
Comments
These studies indicate that amaranth is not carcinogenic to rats
exposed in utero and subsequently for more than 2 years at doses up
to 1250 mg/kg body weight.
The pelvic nephrocalcinosis observed in the F1 female rats in
the long-term study was not seen in the Fo generation nor in the
short-term study and may be dependent on the age-related development
of glomerulonephrosis. Amaranth caused caecal enlargement at the two
highest dose levels and nephrocalcinosis is commonly associated with
this, possibly due to effects on mineral absorption and urine
concentration consequent on increased faecal water loss.
The re-evaluation of the long-term study would indicate that the
no-effect level in respect of renal calcification and hyperplasia was
50 mg amaranth/kg body weight per day.
EVALUATION
Level causing no toxicological effect
Rat: 50 mg/kg bw in the diet.
Estimate of acceptable daily intake for man
0 - 0.5 mg/kg bw.
REFERENCES
BUTLER, W.H. & CONNING, D.M. (1983) Further investigation of the
pathology of tissues from rats treated with Amaranth. BIBRA Report
No. 452/1/82. Unpublished report from the European Colours Steering
Group submitted to WHO.
CLODE, S.A., HOOSON, J., BUTLER, W.H., & CONNING, D.M. (1982) Long-
term study in rats with Amaranth using animals exposed in utero.
BIBRA Report No. 242/1/82. Unpublished report from the European
Colours Steering Group submitted to WHO.
FORD, G.P., BUTLER, W.H., & GAUNT, K.F. (1983) Report of the effects
of Amaranth on renal calcification in the mature rat - a 28 and 90-day
study. BIBRA Report No. 453/2/83. Unpublished report from the European
Colours Steering Group submitted to WHO.
See Also:
Toxicological Abbreviations
Amaranth (WHO Food Additives Series 4)
Amaranth (WHO Food Additives Series 8)
Amaranth (WHO Food Additives Series 13)
AMARANTH (JECFA Evaluation)
Amaranth (IARC Summary & Evaluation, Volume 8, 1975)