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


    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

         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

         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).


         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.


    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.


    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)